# Increased processing of SINE B2 ncRNAs unveils a novel type of transcriptome deregulation in amyloid beta neuropathology

1. Department of Chemistry and Biochemistry, University of Lethbridge, Canada
2. Southern Alberta Genome Sciences Centre, University of Lethbridge, Canada
4. Alberta RNA Research and Training Institute, University of Lethbridge, Canada
Research Article

## Abstract

The functional importance of many non-coding RNAs (ncRNAs) generated by repetitive elements and their connection with pathologic processes remains elusive. B2 RNAs, a class of ncRNAs of the B2 family of SINE repeats, mediate through their processing the transcriptional activation of various genes in response to stress. Here, we show that this response is dysfunctional during amyloid beta toxicity and pathology in the mouse hippocampus due to increased levels of B2 RNA processing, leading to constitutively elevated B2 RNA target gene expression and high Trp53 levels. Evidence indicates that Hsf1, a master regulator of stress response, mediates B2 RNA processing in hippocampal cells and is activated during amyloid toxicity, accelerating the processing of SINE RNAs and gene hyper-activation. Our study reveals that in mouse, SINE RNAs constitute a novel pathway deregulated in amyloid beta pathology, with potential implications for similar cases in the human brain, such as Alzheimer’s disease (AD).

## Introduction

The number of patients with Alzheimer’s disease (AD) is expected to skyrocket in the upcoming years (Cornutiu, 2015). The exact molecular mechanisms underlying AD are not fully understood, a fact that is underlined by the inauspicious results of recent clinical trials for potential therapeutic agents (Hane et al., 2017; Cummings et al., 2019). Amyloid pathology, and particularly, amyloid beta peptides and their aggregated forms have been connected with AD pathogenesis (Bloom, 2014) as well as with neurotoxicity in mouse models of amyloid pathology (Ittner et al., 2010). Nevertheless, the transcriptome changes involved in cell stress response to amyloid toxicity in brains with extensive amyloid beta pathology are still not entirely clear. The hippocampus is a primary target of amyloid pathology in humans. In healthy hippocampi, among the genes that have been implicated in transcriptome-environment interactions are stress response genes (SRGs) (Gallo et al., 2018). These genes have been initially described in other biological contexts, such as thermal and oxidative stress, as pro-survival genes activated early after the application of a stress stimulus, such as heat shock, that help the cell overcome the stress condition (Mahat et al., 2016). However, in addition to the cellular response to stress, many SRGs were shown to have a central role in the function of the mouse hippocampus, by mediating cell signaling and genome-environment interactions. In particular, we and others have shown that in the healthy hippocampus during neural response to environmental stimuli, SRGs, such as those of the MAPK pathway, are transiently activated during various hippocampal processes including learning and response to cellular stress (Peleg et al., 2010; Yutsudo et al., 2013; Sananbenesi et al., 2003). Activation of SRGs is followed by a transient upregulation of the pro-apoptotic factor Trp53 (Peleg et al., 2010) and, subsequently, of a pro-apoptotic miRNA, Mir34c, which are transiently induced by and as a response to the activation of pro-survival SRGs (Zovoilis et al., 2011; Yamakuchi and Lowenstein, 2009). Trp53 activates the expression of genes engaged in promoting cell death in response to multiple forms of cellular stress including Mir34c (Yamakuchi and Lowenstein, 2009). This miRNA acts transiently as a guard and fine tuner of the expression of many SRGs by targeting them, thus, creating a negative feedback regulatory loop that keeps SRG expression in healthy cells under strict control. This facilitates the return to the pro-stimulation state in approximately 3 hr after the application of the stimulus (Zovoilis et al., 2011). In contrast, hippocampi of mouse models of amyloid pathology and postmortem brains of human patients of AD are characterized by abnormally high Mir34c levels that subsequently can lead to prolonged high Trp53 levels and neural death (Zovoilis et al., 2011; Yamakuchi and Lowenstein, 2009). Given that many SRGs are upstream regulators of Trp53-Mir34c activation (Gao et al., 2010), high Trp53 and Mir34c levels in amyloid pathology implied a possible transcriptome deregulation of the pathways that involve SRGs but whether such a deregulation exists, and which is the mechanism underlying this, it remained unknown. Interestingly, in a recent publication, we showed that expression of a number of SRGs is regulated by a class of non-protein coding (non-coding) RNAs called B2 SINE RNAs (Zovoilis et al., 2016), raising the possibility that these non-coding RNAs may be a missing link in the pathways connecting amyloid beta toxicity with transcriptome changes in mouse hippocampus during amyloid pathology.

SINE non-coding RNAs are transcribed by repetitive small interspersed nuclear elements (SINEs), with the subclass B2-repetitive elements being one of the most frequent in mouse (Kramerov and Vassetzky, 2011). SINE B2 elements, which are retrotransposons present in hundreds of thousands of copies, are part of the non-protein coding genome, and over the long haul, they have been regarded as genomic parasites and ‘junk DNA’ with no function (Karijolich et al., 2017). However, SINE B2 elements can be transcribed by RNA Polymerase III into SINE B2 RNAs (Kramerov and Vassetzky, 2011) and a number of recent studies have revealed a key role for SINE B2 RNAs in cellular response to stress. In particular, studies from the J. Kugel and J. Goodrich labs have shown that during response to cellular stress, levels of SINE B2 RNAs increase and suppress the transcription of a number of housekeeping genes through binding of RNA Polymerase II (RNA Pol II), potentially facilitating the redirection of cell resources to pro-survival pathways (Yakovchuk et al., 2009). In addition, we have recently shown that SINE B2 RNAs mediate cellular response to stress through the regulation of pro-survival stress response genes by acting as transcriptional switches. In particular, in the pro-cellular stress state SINE B2 RNAs bind RNA Pol II at several SRGs and suppress their transcription. In this way, stalled or delayed RNA Pol II remains poised for a fast activation and ramp-up of transcription when needed. Upon application of a stress stimulus, SINE B2 RNAs, which have a self-cleavage activity that is accelerated by their interaction with a protein (Ezh2) (Hernandez et al., 2020), are processed into unstable fragments that lack the ability to bind and suppress RNA Pol II. This event releases the delayed or stalled RNA Pol II and enables fast transcriptional activation of stress response genes (Zovoilis et al., 2016).

The above findings have revealed a novel role in cellular function for processing of SINE B2 RNAs (hereafter referred simply as B2 RNAs) through the activation of a number of SRGs regulated by them (hereafter called B2-SRGs). However, an association of the processing and destabilization of B2 RNAs with any pathological cellular process remains unknown. Here, given the importance of SRGs in hippocampal neuronal function, we examine whether this newly described B2 RNA-SRG regulatory mechanism is linked with pathological processes, focusing on transcriptome response to amyloid beta toxicity and pathology. To this end, we investigate whether B2-SRGs are indeed deregulated during amyloid pathology, which would imply a role for B2 RNAs in this condition and we examine whether amyloid toxicity is connected with changes in B2 RNA processing. Subsequently, we investigate the further upstream molecular mechanisms underlying any potential deregulation of B2 RNA processing in response to amyloid toxicity in hippocampal neural cells.

## Results

### B2 RNA regulated SRGs (B2-SRGs) are enriched in neural functions

In a previous study, we have identified genomic locations that are subject to regulation by SINE B2 non-coding RNAs during response to thermal stress (heat shock) through binding and suppression of transcription by the RNA Pol II at the pre-stimulus state. Upon induction of cellular stress through the application of a stimulus, SRGs in these locations become activated through B2 processing and release of RNA Pol II suppression (Figure 1A; Zovoilis et al., 2016). A list of B2-SRGs at these locations is available in Supplementary file 1. Response to thermal stress (heat shock) has been used for years as a basic study model of cellular response to stress. Proteins and gene pathways initially identified in heat shock have been subsequently shown to play identical pro-survival roles in other biological systems. Thus, we questioned whether there are other known cellular functions, beyond response to heat shock, that are connected with B2-SRGs.

Figure 1

As shown in Figure 1B, after performing a tissue enrichment analysis to identify tissue terms that are over-represented in the list of our SRGs, we found a significant enrichment of neural tissue terms compared to other tissues in our list. Similarly, during Gene Ontology term enrichment analysis, cellular compartments closely related to neural functions top the list of enriched terms in these genes, including among the first 10 entries terms such as synapse, postsynaptic density and membrane, dendrites, neural projections, and pre-synaptic membrane (Figure 1C, Supplementary file 1). Most importantly, after performing the same analysis for Biological Processes GO terms, B2-SRGs were found to be enriched considerably in neural-function-related terms. GO terms such as learning, nervous system development, synaptic transmission, synapse receptor localization, and neurotransmitter transport were among the first 10 entries with the highest adjusted p value scores enriched in B2-SRGs (Figure 1D, Supplementary file 1).

Among the biological processes potentially affected by B2 RNAs that were identified above was learning. We and others have already shown that learning processes in the mouse hippocampus are connected with the transient activation of a number of learning-associated genes and pathways, including many known SRGs (Peleg et al., 2010). For this reason, we examined whether any learning-associated genes are among the binding targets of B2 RNAs. To this end, we compared the distribution of B2 RNA binding sites in the genome between learning-associated genes and all genes. Indeed, as shown in Figure 1E, B2 RNA-binding sites were found to be enriched in learning-associated genes compared to other genes. In particular, based on learning-associated genes previously identified in mouse hippocampus (Peleg et al., 2010), among the B2-SRGs (1684 genes), 102 genes (Figure 1E, Supplementary file 1) are associated with learning. In addition, biological process terms enriched in B2-SRGs included, among others, pathways implicated with response to cellular stress in neural cells, and various genes implicated with synaptic function (Figure 1E, Supplementary file 1).

This data suggests that B2-SRGs could potentially affect a wide spectrum of functions beyond heat shock including response to cellular stress in neural cells, synaptic function, and learning. All these biological processes are heavily impaired in AD. Given the role of hippocampus as a primary target of amyloid pathology in AD, we decided to expand our investigation towards the potential role of B2-SRGs regulation in this pathological process. We have previously shown that in hippocampus the pro-apoptotic miRNA Mir34c, which is induced among others by Trp53 and controls it in a negative feedback loop manner (Yamakuchi and Lowenstein, 2009), targets many learning-associated genes in order to help to restore their expression and Trp53 expression levels after application of a stimulus (Figure 1F; Zovoilis et al., 2011; Yamakuchi and Lowenstein, 2009). In both mouse models of amyloid beta pathology and AD patient brains, we found persistently high levels of Mir34c. High Mir34c levels in hippocampi with amyloid pathology are indicative of a transcriptome-wide deregulation of Trp53 and associated genes, as many of these genes are either direct or indirect upstream regulators of Mir34c (Figure 1F; Rokavec et al., 2014). Since, as shown above, B2-SRGs include many learning genes as well as other genes involved in neural function, we questioned whether such transcriptome changes in response to amyloid pathology involve B2-SRGs.

### A number of B2-SRGs get hyper-activated during the neurodegeneration phase of amyloid pathology

To test whether B2-SRGs are indeed deregulated in amyloid pathology, we employed a transgenic mouse model of amyloid pathology, APPNL-G-F (Saito et al., 2014), and the respective wild-type control (C57BL/6J). The same animal cohorts that were previously characterized through a battery of immunohistochemistry (IHC) and behavioral tests (Mehla et al., 2019) were used to isolate whole hippocampi for the transcriptome analysis conducted in the current study. Figure 2A depicts our experimental design while Figure 2B depicts the amyloid plaque deposition in the brains. The behavioral tests in these mouse cohorts are presented in our previous study (Mehla et al., 2019).

Figure 2 with 3 supplements see all

We have focused on three different mouse ages that correspond to different phases of amyloid beta pathology and represent the: (i) pre-symptomatic stage with undetectable (very low) amyloid plaque load (3 months - 3 m, Figure 2B left panels), (ii) stage of symptom manifestation (6 months - 6 m) that coincides with the active neurodegeneration phase and appearance of amyloid plaques (Figure 2B, middle-panels), and (iii) terminal stage of the pathology (12 months - 12 m, Figure 2B right panels) when mice have already acquired the extensive brain atrophy due to neural cell death. Whole hippocampi from mice of these three groups were isolated and the extracted RNA was subjected to next-generation sequencing. We performed directional RNA sequencing for these samples and subsequently quantified gene expression levels. Sequenced samples depicted high expression levels for known hippocampal markers such as Gad1, Gad2, Slc17a7, and Mbp (Figure 2—figure supplement 1).

In accordance with increased cell death during the active neurodegeneration phase at 6 months, levels of Trp53 were elevated in APP mice of this age compared to controls of the same age (t-test, p<0.05, n = 3/group) (Figure 2C). Differential expression analysis at this time point revealed a number of genes that are upregulated in 6-month-old APP mice (Supplementary file 1), including 72 B2-SRGs among them (Supplementary file 1), of which 13 are learning associated (Supplementary file 1). These 72 B2-SRGs were enriched in neural-function-related terms, such as neural development, learning, synapse function, and calcium signaling (Supplementary file 1). Consistent with a hypothesis of a transcriptome-wide deregulation that involves B2-SRGs, levels of the 72 B2-SRGs were found to be strongly upregulated during the active phase of neurodegeneration at 6 months (Figure 2D–F). In particular, in healthy hippocampi (WT control animals), levels of these genes are normally downregulated in 6-month and 12-month-old mice compared to 3-month-old mice (p=0.02 and p=0.05, respectively) (Figure 2E). This is in accordance with the higher level of neural synaptic activity and plasticity that younger mice have (Lilja et al., 2013). In contrast, levels of B2-SRGs in APP mice remain abnormally high in 6-month-old APP mice compared to the 6-month-old control mice (p<0.05) (Figure 2E and F). A validation of our RNA-seq data through RT-qPCR for selected genes that we identified by RNA-seq to be upregulated in 6-month-old APP mice, is presented in Figure 2—figure supplement 2. In Figure 2, we only tested the expression of those B2-SRGs that overlapped with the upregulated genes revealed by DESeq in 6-month APP mice. When expression dynamics for all B2-SRGs are tested (Figure 2—figure supplement 3), these are similar to the ones observed for genes in Figure 2, while such dynamics are not observed when a random set of non-B2 RNA regulated genes is tested (Figure 2—figure supplement 3). Thus, as explained in Figure 2—figure supplement 3, it cannot be excluded that our findings may extend to additional B2-SRGs beyond the 72 identified through our differential gene expression analysis.

These findings show a transcriptome wide hyper-activation of certain B2-SRGs during the active neurodegeneration phase of amyloid pathology.

### B2 RNA processing ratio increases during the neurodegeneration phase of in vivo amyloid pathology

Given the role of B2 RNAs in the regulation of SRGs, we hypothesized that the observed hyperactivation of B2-SRGs in the hippocampus of 6-month-old APP mice may reflect a similar upstream deregulation at the level of the B2 RNAs.

To test this, we employed a customized version of RNA sequencing and analysis used in our previous study (Zovoilis et al., 2016) that allows for enrichment and sequencing of the short SINE RNA fragments (<100 nt) produced by B2 RNA processing (short-RNA-seq). In contrast to standard long-RNA-seq protocols that include RNA fragmentation and, thus, may introduce bias, this approach circumvents this problem. Moreover, the long-RNA-seq protocols exclude short RNA fragments of <100 nt making the identification of B2 short fragments challenging. After short-RNA-seq (Figure 3A), mapping of the 5′ ends of the sequenced fragments across the B2 loci enables the determination of processing points at B2 RNA (depicted as ‘X’ in Figure 3A), including those at the critical RNA Pol II binding region (depicted as a rectangle in Figure 3A). Figure 3B depicts an example of these fragments from one of the samples. As shown in Figure 3A, B2 RNA is extensively processed in hippocampi with amyloid pathology. In particular, this data revealed a increased number of B2 RNA fragments in 6-month-old mice compared to controls (Figure 3A), a difference that cannot be explained by full-length B2 RNA levels between these groups of mice using long-RNA-seq (Figure 3C).

Figure 3 with 2 supplements see all

This data suggests that the processing ratio of B2 RNAs may be higher in APP mice of this age. To estimate this ratio, we used the short RNA-seq data in combination with standard long-RNA-seq. As in our previous study, for normalization of short RNA 5′ end values we used a class of short RNAs that is not affected by B2 RNAs, the RNA Pol III-transcribed tRNAs and estimated the levels of B2 RNA processing fragments (Figure 3E). Moreover, the absolute numbers of fragmented B2 RNA may vary according to the underlying expression of full-length B2 RNA transcripts. Therefore, in order to factor in any differences in the amount of fragments due to basal expression levels of the full-length B2 RNA, full-length B2 RNA levels were calculated by the directional long-RNA-seq (Figure 3F). The long-RNA-seq approach excludes short fragments, and subsequently, B2 RNA fragment values from short RNA-seq were normalized to the levels of the full-length B2 RNAs to calculate the processing ratio. Consistent with our hypothesis and Figure 3A findings, 6-month-old APP mice were found to have substantially increased ratio of B2 RNA processing compared to control mice of the same age (Figure 3G) (p<0.05, n = 3/group). This increase was observed only in the 6-month-old mice (Figure 3G, Figure 3—figure supplement 2) and coincides with the increase in Trp53 levels and B2-SRGs levels observed in this age.

Thus, APP mice at the active neurodegeneration phase are characterized by higher destabilization and processing ratio of B2 RNAs, consistent with the observed increase in B2-SRG levels in these same animals.

### Hsf1 accelerates B2 RNA processing

We then focused on the molecular mechanism underlying the increased B2 RNA processing during response to amyloid toxicity. When we had previously examined the mechanism of B2 RNA processing in non-neural cells (NIH/3T3 cells during heat shock), a member of the PRC2 protein complex, Ezh2, was reported as being responsible for the B2-RNA-accelerated destabilization and processing during response to stress (Zovoilis et al., 2016). However, as shown in Figure 4—figure supplement 1A–B, scant expression levels of Ezh2 levels in neural cells indicate that Ezh2 is not a key factor in B2 RNA processing in brain. Thus, the factors that may mediate destabilization for B2 RNAs during stress in neural cells remain elusive.

In our earlier study that described the induction of B2 RNA processing by Ezh2, it remained unclear how Ezh2 exerted its impact on B2 RNAs, since Ezh2 lacked any known RNAse activity (Zovoilis et al., 2016). However, in subsequent experiments (Hernandez et al., 2020), we showed that instability is in fact inherent to the B2 RNA molecule while interaction with Ezh2 only accelerates this destabilization and Ezh2 does not cleave B2 RNA by itself. This finding suggests that other proteins may have a similar effect on B2 RNA stability. Therefore, we started searching for stress-related candidate proteins that could affect the B2 RNA processing.

We showed before that, during response to stress in NIH/3T3 cells, B2 RNA binding is enriched near stalled RNA polymerase genomic sites. These areas are known to be highly enriched in binding sites of various stress related proteins, among which Hsf1, a master regulator of stress response for various types of cellular stress (Pandey et al., 2011). Hsf1 has been previously connected with activation of SRGs through both transcriptional factor (TF) activities as well as other yet unknown TF-independent processes (Inouye et al., 2007). Interestingly, when we examined the proximity of Hsf1 binding sites, identified by the Lis lab (Mahat et al., 2016), to genes with B2 RNA binding sites (Supplementary file 1; Zovoilis et al., 2016), we found that increased number of Hsf1-binding sites were found near B2-SRGs (Figure 4—figure supplement 1C) and were further enriched after application of a heat-shock stimulus (KS-test <0.05). Moreover, as shown in Figure 4—figure supplement 1A–B, in contrast to Ezh2, Hsf1 is expressed in neural tissues and especially in hippocampus. These findings provided an indication that Hsf1 may be implicated in B2 RNA biology and that it is also expressed in neural cells, which urged us to test further its expression pattern in our biological context. Hsf1 levels were found to be upregulated in APP 6-month-old mice compared to control group of the same age (p<0.05, n = 3/group) (Figure 4A) in the RNA-seq data, a result confirmed also through RT-qPCR (Figure 4B). As mentioned above, at the same time, 6-month-old APP mice have increased B2 RNA processing ratio. Thus, this data suggests that Hsf1 may be a good candidate for accelerating B2 RNA processing in the context of amyloid pathology.

Figure 4 with 1 supplement see all

To check this, we investigated whether the interaction between B2 RNA and Hsf1 can accelerate B2 RNA destabilization. In particular, we incubated full-length B2 RNA with the Hsf1 protein in vitro. As shown in Figure 5A, in the presence of Hsf1, the destabilization of full-length B2 RNA was accelerated in contrast to the control protein (PNK) or in the absence of protein (no protein). No processing was observed in an RNA fragment co-incubated with B2 RNA and the protein (marked with an asterisk across Figure 5). We then questioned whether the fragments observed during B2 RNA processing in vitro are the same with those we observed in NIH/3T3 cells in our previous study and hippocampal cells in our current study (Figure 3A). To test this, we subjected the in vitro processed B2 RNA fragments to short RNA-sequencing as with the in vivo samples and compared the processing patterns between the two cases. As shown in Figure 5—figure supplement 1A, the in vitro processing points were the same for most of the positions identified in vivo including the most prominent ones around 99 and 33. The two processing patterns differed only at positions 90 and 47, suggesting that in vivo B2 RNAs may be protected or processed in these two positions by yet unidentified mechanisms.

Figure 5 with 1 supplement see all

Figure 5B shows B2 RNA destabilization in time. B2 RNA destabilization in the presence of Hsf1 was accelerated compared to no protein (Figure 5C and Figure 5—source datas 1 and 2). Consistent with the above finding, the rate of processing of B2 RNA was dependent on Hsf1 concentration in the reaction (Figure 5D and E) and increased upon increase of Hsf1 concentration. In Figure 5D and our analysis in Figure 5E, as a zero concentration point (no Hsf1 protein) we used RNA incubated in the same buffer and for the same time as the samples with the Hsf1 protein to take into account any RNA destabilization due to non-specific degradation, hydrolysis or B2 RNA endogenous self-cleavage. In order to confirm further the Hsf1-mediated acceleration of B2 RNA we transcribed a mutant B2 RNA that harbors a 6nt deletion in the hairpin at position 124 (Figure 5F). Then, we compared B2 RNA processing during incubation in the presence and absence of Hsf1 and observed an impairment in the ability of Hsf1 to accelerate B2 RNA processing (Figure 5G–H). Similar negative results were obtained when other control RNAs were tested (Figure 5H, control RNA #1, 143nt; control RNA #2, 163nt). Subsequently, we tested the impact on acceleration of B2 RNA processing by Hsf1 in the case of heat denaturation of Hsf1. As shown in Figure 5I, heat denaturation of Hsf1 resulted in abrogation of its ability to accelerate B2 RNA processing (Figure 5I). Similar results were obtained with the incubation of B2 RNA with another RNA binding protein of similar size to Hsf1 (Hsf1 ~60 KDa, Poly A polymerase ~56 KDa) (Figure 5I).

This data shows that Hsf1 has the potential to accelerate B2 RNA processing in vitro.

### Hsf1 mediates increased B2 RNA processing in response to amyloid beta toxicity

The increased levels of Hsf1/B2 RNA processing in APP 6-month-old mice raised the question whether response to amyloid toxicity in hippocampal cells is connected with an Hsf1-mediated increase in B2 RNA processing. In order to test this, we employed a hippocampal cell culture model using the a HT-22 cell line, which has been used extensively in the past as hippocampal cell stress model (Davis and Maher, 1994; Liu et al., 2009). We incubated these cells with amyloid beta peptides (1–42 aa) and compared their transcriptome to the one of cells incubated with an inverted sequence control peptide (R, reverse 42–1) (Figure 6A). Incubation of these cells with 1–42 amyloid beta peptides results in upregulation of a number of genes (Supplementary file 1), 25 of which are also found upregulated in amyloid beta pathology (6-month-old APP mice) (Supplementary file 1). The increase in expression levels of genes associated with amyloid pathology (Figure 6B and C) suggests that this cellular model simulates to a certain extend the amyloid toxicity effect on the transcriptome of hippocampal cells and their response to cellular stress. However, as any cell culture system, it has limitations regarding tissue level functions such as learning.

Figure 6 with 1 supplement see all

Genes upregulated in our amyloid toxicity model included 25 B2-SRGs (Supplementary file 1). When testing for enriched terms in these 25 genes, biological processes related to apoptosis, such as regulation of apoptotic process and programmed cell death were at the top of the list (Supplementary file 1) and included, among others, genes such as Fosb and Mitf that have been connected with AD (Solé-Domènech et al., 2016; Gupta et al., 1986). Validation of our RNA-seq data in HT-22 cells through RT-qPCR for selected B2-SRGs identified to be upregulated in 42 vs. R is presented in Figure 6D. In the same figure, we also present the expression levels of these genes during recovery, 24 hr after application of the amyloid beta toxicity stimulus, confirming the return to pre-stimulus levels and the specificity of the treatment with amyloid beta peptides (Figure 6D).

Out of the 25 genes that are upregulated in both mice and our cell culture system, six are B2-SRGs (4932438A13Rik, Fosb, Pag1, Ptprs, Sema5a, and Sgms1) and include a well-known immediate early gene (Fosb), genes associated with sensitivity to amyloid toxicity (Pag1, Sema5a, Sgms1, Fosb) (Hadar et al., 2016; Lin et al., 2009; Hsiao et al., 2013), as well as genes associated with Trp53 (Ptprs, Fosb)(Motiwala and Jacob, 2006; Liu et al., 2018).

We then questioned whether by inducing an artificial degradation of B2 RNA, we would be able to induce expression of B2-SRGs in our model system of HT22 cells in the absence of any stimulus such as amyloid beta. To achieve this, we employed a similar approach and the same LNAs against B2 RNA that we used in our previous study (NIH/3T3, heat shock). Application of the LNA against B2 (Figure 7A) was able to reduce levels of B2 RNA compared to the control LNA (Figure 7B). Similarly to NIH/3T3 cells, targeting of the B2 RNA in HT22 cells, resulted in the increase of the expression levels of selected B2-SRGs that are upregulated in amyloid beta pathology (see Figure 2—figure supplement 2, the first 10 genes and Figure 6D). The increase in gene expression occurred in the absence of the stress stimulus, in this case amyloid beta, suggesting that these genes are under the suppressive control of B2 RNAs in HT22 cells (Figure 7C). At the same time, B2 RNA destabilization did not affect expression of five non-B2-SRGs that were used as negative controls, including example genes, such as Adcy1 and Kalrn, that are nevertheless upregulated in amyloid beta pathology (Figure 2—figure supplement 2, the last two genes).

Figure 7

Subsequently, in order to test the impact of amyloid toxicity to Hsf1-mediated B2 RNA processing we treated HT22 cells with either an LNA against Hsf1 or a scramble LNA (control) followed by incubation with the 1–42 peptides, that subject the cells to amyloid toxicity stress, or the respective control peptide (Figure 8A, same experimental design and cells as in Figure 6). As shown in Figure 8B, treatment with the anti-Hsf1 LNA suppressed any increase in Hsf1 levels between 42-anti-Hsf1 and R-anti-Hsf1 cells, while this increase is observed between non-Hsf1 LNA-treated cells upon application of the amyloid beta peptides (42-ctrl vs R-ctrl). Treatment with with the anti-Hsf1 LNA during transfection with the amyloid beta peptides also led to a decrease in protein levels of Hsf1 (Figure 5—figure supplement 1B). As observed in the APP mice, amyloid beta peptides resulted in increased B2 RNA processing only in cells without Hsf1 knock down, suggesting that it is the toxicity of these peptides that induces the SINE B2 RNA transcriptome changes (Figure 8C, Figure 8—figure supplement 1). In contrast, under anti-Hsf1 LNA, application of the 1–42 peptides was unable to increase B2 RNA processing in the cells compared to the control cells (Figure 8C). A similar effect was observed in B2-SRGs that are upregulated during amyloid toxicity (Supplementary file 1, Figure 8D–F). A selected number of genes was also tested with RT-qPCR to confirm the patterns observed in the RNA-seq data (Figure 8E). As in case of amyloid pathology, it cannot be excluded that our findings may extend to additional B2-SRGs (Figure 8—figure supplement 2).

Figure 8 with 2 supplements see all

These results suggest that amyloid beta toxicity induces B2 RNA processing also in vitro and Hsf1 comprises a necessary component in the upstream activation pathway of B2 RNA processing and, thus, of the genes regulated by B2 RNA.

## Discussion

Fewer than five ribozymes have been identified in mammals, including our previous work on RNAs made from two retrotransposon families, murine SINE B2 RNAs and human SINE ALU RNAs, which are self-cleaving RNAs (Hernandez et al., 2020). We previously showed that cleavage in SINE B2 RNAs controls response to cellular stress through activation of stress response genes in heat shock. However, no connection between this novel molecular mechanism and pathologic processes was until now known. Moreover, since B2 RNAs are intrinsically reactive, and contact with Ezh2 only accelerates cleavage, it remained plausible that other stress-related proteins may also have a similar effect on accelerating B2 RNA processing, which would link this ribozyme-like property to stress response through pathways other than Ezh2. This is especially relevant in mouse tissues, such as the brain, where Ezh2 expression is limited, an expression pattern observed also in human (Human Protein Atlas) (Uhlén et al., 2015).

Here, we unveil increased processing of SINE B2 RNAs as a novel type of transcriptome deregulation underlying amyloid beta neuro-pathology. Our data provides a new link in the murine hippocampal pathways connecting amyloid beta toxicity with transcriptome changes in SRGs through processing of B2 RNAs. In particular, the B2 RNA processing ratio increases upon progression of amyloid pathology both in mouse hippocampus and a hippocampal cell culture model, and B2-SRGs become hyperactivated. Consistent with the spatial proximity between B2-SRGs and Hsf1 binding sites, Hsf1 proved to be key for mediating B2 RNA processing in response to amyloid toxicity. This correlation is observed throughout all sequencing experiments performed in this study (Figure 8—figure supplement 1). Our work assigns to Hsf1 a new function that is independent of its long-established transcription factor function and includes the interaction with and processing of SINE B2 RNAs. The high levels of Hsf1 trigger a downstream cascade of events which are orchestrated into a cell-wide, SRG-mediated response to stress conditions. This axis is mediated by the ability of B2 RNA to get processed and, thus, act as a molecular switch. Although healthy cells and animals are able to restore the expression levels of Hsf1, SRGs and their regulating B2 RNAs upon removal of the stress-generating stimulus, the amyloid beta load in our biological models acts as a continuous stimulus that causes the Hsf1 - B2 RNA - SRG axis to ‘lock’ into an activated mode. Upregulation of SRGs results in increased Trp53 levels that induce neuronal cell death (Figure 9).

Figure 9

Stress response genes, that constitute the basis of response to heat shock, have been shown in various studies from us and others to play a critical role also in hippocampal function (Peleg et al., 2010; Zovoilis et al., 2011). Thus, our findings here on a potential role of B2 RNAs in the context of neural response to stress constitute a natural continuation of our previous study in heat shock (Zovoilis et al., 2016). The fact that B2-SRGs identified in our previous study were found in the current study to be highly enriched in neuronal tissue related biological processes terms and compartments was what has compelled us to investigate further a potential role of B2 RNAs in neural tissue-associated pathologies.

In this study, we employed a mouse model of amyloid pathology in order to test the impact of increased amyloid beta load on B2 RNA processing in vivo. This mouse model has the NL- G-F mutations in the amyloid precursor protein knocked-in to a C57BL genomic background, with each mutation contributing to an increased severity and speed manifestation of the disease (Mehla et al., 2019). The combined effect of the APPNL?G?F mutations results in mice that experience rapid onset of AD-like symptoms at approximately 6 months (Mehla et al., 2019). This is exactly the time point that we observed the massive hyper-activation of B2 RNA SRGs, Trp53, and B2 RNA processing, suggesting that these changes constitute a molecular signature for the active neurodegenerative phase months, before the end state of 12 months that the mice develop terminal AD-like pathology and symptoms.

Interestingly, symptoms and molecular changes are not observed in younger APP mice suggesting the existence of a yet unknown protective mechanism against increased neuronal activity of SRGs in 3-month-old APP mice. This may be attributed to the increased neuronal plasticity observed in younger brains (Lilja et al., 2013), which suggest that mechanisms in the younger brain may exist for counter-acting this excessive activity. In particular, stress response genes such as Fosb have been shown to participate in specification of cell-type-specific activity-dependent gene programs early in development (Yap and Greenberg, 2018). In contrast, during aging, B2-SRG activity in WT mice ramps down, which is not the case in APP mice (Figure 2), explaining at the molecular level the increased Trp53 levels and subsequent activation of cell death (Figure 1F). Our findings for B2 RNA SRG upregulation and B2 RNA increased processing coincide with the active neurodegeneration phase of amyloid pathology but in later stages the effect is not that prominent. This suggests that the initial active phase differs from the terminal stage regarding role of B2 RNA. As in the terminal stage a large number of cells have already died, B2 processing activation appears to be more connected with the initial response of cells to amyloid toxicity.

The B2-RNA-mediated regulation of gene expression during stress identified in one biological context may be relevant to a broad range of cellular types and disease pathophysiology. In the current study, to identify examples of genes that are subject to B2 RNA regulation in hippocampus molecular pathology we utilized the gene list of B2-SRGs identified in our previous study in heat shock. Of the previously identified 1684 B2-SRGs, we found here 72 genes that are deregulated specifically during the active neurodegeneration phase in hippocampal cells. However, this does not exclude the possibility that beyond these 72 genes, B2 RNA processing may also affect expression of additional B2-SRGs that are associated with other hippocampal functions that are independent of amyloid beta pathology. To get a better insight into this possibility, we tested the correlation between the gene expression of other B2-SRGs and the B2 RNA processing ratio, independently of amyloid beta status. In particular, for each of these genes we have calculated the Pearson correlation coefficient between gene expression levels and B2 RNA processing ratio in the hippocampal samples of the current study. As shown in Figure 8—figure supplement 1E, we have been able to calculate a statistically significant correlation coefficient for at least 659 genes (the rest where either not sufficiently expressed in all samples or returned a p value > 0.05). From these 659 genes, expression of 344 genes (52.2%) showed a strong correlation with B2 RNA processing ratio (r ≥ 0.5), while an additional 75 genes (11.3%) showed a weak correlation (0.5 > r ≥ 0.25), and only 240 genes (36.4%) had an r < 0.25 and no correlation. This data suggests that for a large number of the B2-SRGs previously identified in the context of heat shock, correlation between gene expression and B2 RNA processing ratio holds true also in the context of hippocampal cells.

During our amyloid toxicity experiments, cells inoculated with the reverse peptide and the anti-Hsf1 LNA did not show any reduction in Hsf1 levels in contrast to those inoculated with amyloid beta (Figure 8B). This could be attributed to compensation during non-stress conditions. In contrast, under stress conditions, when Hsf1 is heavily used due to stress response, cellular needs surpass the available Hsf1 transcripts that are continuously depleted by the LNA. This is also in agreement with levels of B2-SRGs in this condition (Figure 8E), which are minimal in both R-ctrl and R-anti-Hsf1 conditions and only get activated during stress response to the amyloid beta in the 42-ctrl condition.

Our study leaves a number of open questions. Particularly, our study raises the question whether a similar mode of regulation of SRGs by SINE RNAs may exist also in human and which SINE RNAs could play such a role. Similarly to B2 RNA, such SINE RNAs would be able to bind and inhibit RNA Pol II and would be subject to a similar RNA processing mechanism enabling the release of RNA Pol II. A number of studies have described that in human, repetitive SINE RNAs of the Alu class are also upregulated during cellular stress and can bind RNA Pol II inhibiting the transcription of target genes (Yakovchuk et al., 2009). Alu RNAs are widely regarded as the equivalent in human of B2 RNA. Most importantly, as we showed before, human Alu RNAs, alike B2 RNAs, are self-cleaving RNAs and can become destabilized in vitro (Hernandez et al., 2020). It remains unknown whether SINE RNAs and Hsf1 play a similar role in amyloid pathology in the case of humans and whether we can extrapolate the generated conclusions in murine models to deduce that SINE RNAs are key components of the pathophysiological mechanisms underlying debilitating diseases such as AD. One major limitation compared to human pathophysiology is that the phenotype of amyloid pathology is not observed in mice even during aging. Nonetheless, a stress-central role of Alu RNAs, the human counterpart of B2 RNAs is plausible and, thus, future studies need to elucidate whether Alu RNA processing is also hyperactivated in the brain of patients with amyloid pathology in the context of AD.

Moreover, in the RNA-seq data one cannot distinguish between Pol III transcribed B2 RNA and Pol II transcribed B2 RNA (typically embedded within introns and UTRs of mRNAs). To get an indication whether such transcripts may contribute to our data, we have separated the B2 elements against which we map the RNA fragments into two categories, those that fall within exonic/genic regions and those outside these regions (Figure 3—figure supplement 2). Although B2 RNAs are produced by multiple copies in the genome, each copy does harbor multiple SNPs, insertions and deletions, which means that each B2 RNA fragment is mapped to a specific set of B2 elements and not to all of them. Thus, despite multiple mapping of the reads, a level of spatial specificity is maintained. If the B2 RNAs we map were coming exclusively from either only Pol III B2 elements or mRNA-embedded B2 elements, we would expect at least some difference in the distribution of fragments between B2 elements of these two categories, as the second one overlaps with mRNAs. However, as shown in Figure 3—figure supplement 2, the fact that distribution models are very similar between the two categories supports the hypothesis that both types of B2 elements may contribute to B2 RNA processing. Thus, it cannot be excluded whether the regulatory role of B2 RNAs may extend from Pol III transcribed B2 RNAs into B2 RNAs embedded into mRNAs (likely nascent ones) that may be also under the same endogenous ribozyme activity of this sequence, may suppress Pol II and get processed in response to stimuli.

Our results suggest that B2 RNA regulation is a new process implicated in response to stress in amyloid pathology but it is definitely not the only one. Since B2-SRGs are highly interconnected and interweaved into various pathways, the impact of SRG hyperactivation by B2 RNA processing may extend to pathways that lie downstream of SRGs and affect various gene programs without binding directly B2 RNA. However, here it should be noted that high levels of Hsf1 are certainly expected to affect transcription also through Hsf1’s conventional transcriptional factor function while there are still many SRGs that are not directly regulated by B2 RNA. Thus, B2 RNA processing described here does not constitute the only one but just one of the parameters in the equation of SRG regulation. It remains unclear which is the interplay between Hsf1 traditional transcription factor activities and its ability to affect B2 RNA processing and whether there is any overlap or synergies. For example, it remains unclear what percentage of activated genes are activated though Hsf1 DNA binding and what through binding of B2 RNA and subsequent release of suppressed Pol II activity.

Moreover, given how easily B2 RNA is processed in the presence of certain proteins, Hsf1 may be only one of the factors accelerating B2 processing in mouse hippocampus as we are just beginning to understand the implications of this form of SINE RNA regulation in cells. A broader role of SINE RNA processing in brain physiology and pathophysiology constitutes, thus, a significant possibility that could further revise our understanding of these RNAs as something more than just transcriptional noise and ‘junk DNA’ products.

## Materials and methods

Key resources table

### Amyloid beta peptide preparations

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The amyloid beta 1–42 peptides and the respective control peptides (having the reverse aa sequence compared to 1–42 peptides) were synthesized by Sigma Aldrich’s custom synthesis service using the following sequences: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (peptide 1-42) and AIVVGGVMLGIIAGKNSGVDEAFFVLKQHHVEYGSDHRFEAD (Reverse). Upon receiving, the peptides were dissolved in 10% NH4OH at a concentration of 2.1 mg/mL, sonicated for 5 min, aliquoted, dried, and stored at ?80°C for further use (see below).

### Animals and behavioral measurement

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Immunohistochemistry was done as described in our previous study (Mehla et al., 2019) for the same mice cohorts for which behavioral studies were performed in that study (Mehla et al., 2019). In brief, mouse pairs of APP-KI mice carrying Arctic, Swedish, and Beyreuther/Iberian mutations (APPNL-G-F/NL-G-F) were gifted by RIKEN Center for Brain Science, Japan, and the colony of these mice was maintained at Canadian Center for Behavioral Neuroscience vivarium. C57BL/6J mice were used as a WT control and all animals were housed in groups of four mice in each cage in a controlled environment (22°C–25°C, 50% humidity and a 12 hr light:dark cycle). All experimental procedures were approved by the institutional animal care committee and performed in accordance with the standards set out by the Canadian Council for Animal Care. In total, we extracted RNA from hippocampi of three different mice per age group per condition. During the hippocampal RNA extractions, the RNA of one of the three 3-month-old wild-type mice had very low RIN scores, which could be a confounding factor for the short-RNA-seq. As this happened some months after the hippocampal extractions, we did not have available other 3-month mice of the same cohort used in our previous study of these mice for the behavioral and IHC studies. Thus, we decided to include only two replicates in this condition. Since the results presented in the current study involve mainly 6-month-old mice we expect the impact to be minimal.

### Cell culture and transfections

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HT22 cells, from an immortalized mouse hippocampal cell line (Davis and Maher, 1994) (Millipore Cat# SCC129, RRID:CVCL_0321). Cells were provided by the vendor tested negative for infectious diseases by a Mouse Essential CLEAR panel by Charles River Animal Diagnostic Services and verified to be of mouse origin and negative for inter-species contamination from rat, chinese hamster, Golden Syrian hamster, human, and non-human primate (NHP) (as assessed by a Contamination Clear panel by Charles River Animal Diagnostic Services) as well as negative for mycoplasma contamination. Cells were cultured in DMEM (Sigma) and 1% Penicillin/Streptomycin (Gibco). Cells were thawed and passaged at least 3 days before the transfection date in order to allow sufficient time for cells to recover from the stress of cryopreservation and not interfere with the assessment of cellular response to stress in subsequent experiments. For knocking down of Hsf1 mRNA levels, we used an LNA long RNA GapmeR against Hsf1 (Exiqon/Qiagen) with the following sequence: 5′-′GAAGGATGGAGTCAA-3′ and an LNA long RNA GapmeR with the following scramble sequence: 5′-CCTCAATTTTATCAC-3′. For knocking down B2 RNA transcripts, we used an anti-B2 LNA pool consisting of five LNAs, synthesized using the K and E DNA and RNA synthesizer: 1. 5' - G*T*T*A*CGGATGGTT*G*T*G* - 3′; 2. 5′- A*G*A*T*CTCATTACA*G*A*T*- 3′; 3. 5′ - A*G*A*T*CTCATTACG*G*A*T* - 3′’; 4. 5′ - A*G*A*T*CCCATTACA*G*A*T* - 3′; 5. 5′ - A*G*A*T*CCCATTACG*G*A*T* - 3′; 6. 5′-T*G*TAGCTGTCTTCA*G*?3′. Nucleotides were ordered from Sigma Aldrich as DMT phosphoramidites. The day of LNA transfections, following 5-min incubation with TrypLE Express Enzyme (Gibco) (1x), cells were passaged and transferred to a six-well plate at a 100,000 cells/well density and LNA transfections were performed simultaneously, using the HiPerfect reagent (Qiagen). Transfection was performed as follows: Firstly, LNAs were reconstituted in nuclease-free water to 50 μM. Subsequently, 3 μL 50 μM LNA were mixed and incubated with 4 μL Hiperfect reagent and 30 μL nuclease-free water, at room temperature for 20 min, and then added drop-wise to cells that had just been plated in 1 ml of medium/well (still not attached) to a final LNA concentration of 150 nM. For anti-Hsf1 LNAs transfections, incubation with amyloid beta (peptide 1-42) and control peptides (Reverse) was performed 24 hr after transfection with LNAs. Peptides were initially dissolved in DMSO for incubation at 37°C for 1 hr and then added to cells to a final concentration of 30 μM for 6 hr before treating with 0.5 mL TrypLE (Gibco), pelleting cells at 1000 rpm for 5 min and resuspending the pellet in 1 mL Trizol reagent (Thermofisher) for RNA extraction based on Manufacturer’s instructions. For anti-B2 LNA, transfection and incubation lasted 6 hr before RNA extraction.

### Reverse transcription and quantitative PCR

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RNA was extracted using Trizol reagent as described and reverse transcribed using Superscript III (18080093, Invitrogen) by the following method: 50 ng total RNA was mixed with 100 ng random primers (C1181, Promega) and 1 μL of 10 mM dNTP mix. The mixture was incubated for 5 min at 65°C and placed immediately on ice. The mixture was then incubated with 4 μL of 5x First strand buffer, 1 μL of 0.1M DTT and 0.4 μL Superscript III (18080093, Invitrogen) for 5 min at 25°C, 60 min at 55°C and 15 min at 70°C. cDNA was analyzed by qPCR using 2 μL of 1:20 diluted cDNA, 0.5 μL of 10 μM of each gene-specific primer, 2 μL H2O and 5 μL of Luna Universal qPCR Master Mix (M3003E, NEB). Thermocycler conditions are as follows: 3 min at 95°C (15 s at 95°C, 30 s at 54°C, 30 s at 66°C) × 40 cycles. Fluorometer readings were taken during extension and qPCR was performed using the Bio-Rad CFX384 Real-time detection system. Standard curves were prepared for relative expression and the analysis of PCR efficiency by pooling 2 μL of each cDNA sample and standard diluting SD1: 1:5, SD2: 1:10, SD3: 1:20, SD4: 1:40. Samples were either analyzed by standard curve relative expression or $2-??CT$ fold change analysis. Student’s T-test were used to study significance as described in the respective figure legends. Primers were ordered from IDT as custom oligos and are listed in Appendix 1—table 1.

### RNA in vitro transcription and RNA-protein incubations

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B2 template for in vitro RNA transcription was ordered as IDT g-block (lower case denotes the T7 promoter sequence): 5′- taatacgactcactata GGGGCTGGTGAGATGGCTCAGTGGGTAAGAGCACCCGACTGCTCTTCCGAAGGTCCGGAGTTCAAATCCCAGCAACCACATGGTGGCTCACAACCATCCGTAACGAGATCTGACTCCCTCTTCTGGAGTGTCTGAAGACAGCTACAGTGTACTTACATATAATAAATAAATAAATCTTTAAAAAAAAA - 3′. The template was amplified by PCR using a T7 promoter sequence as the forward primer: 5′-TAATACGACTCACTATAG and the following sequence as reverse primer: 5′-TTTTTTTTTAAAGATTTATTTATTTATTATATGTAAGTACA. B2mut4b was transcribed from the following template (same primers): taatacgactcactataGGGCTGGTGAGATGGCTCAGTGGGTAAGAGCACCCGACTGCTCTTCCGAAGGTCCGGAGTTCAAATCCCAGCAACCACATGGTGGCTCACAACCATCCGTAACGAGATCTGACTCCCTCTTCTTCTGAAGACAGCTACAGTGTACTTACATATAATAAATAAATAAATCTTTAAAAAAAAA. Primers were diluted to 10 mM and PCR was performed using the NEB Q5 polymerase, Q5 reaction buffer (10x), Q5 high GC enhancer (10x). The reaction proceeded at hot start 98°C – 30 s (98°C – 5 s, 58°C – 10 s, 72°C – 10 s) X35 cycles, 72°C – 10 min. The samples were then analyzed by agarose gel electrophoresis (Bio-Rad, 1613100EDU) and the bands were gel extracted at the desired size and purified using the BioBasic EZ-10 gel extraction kit (BS353). A subsequent PCR was then repeated and 1 μg of the amplified g-block was then in vitro transcribed by T7 RNA polymerase (NEB, M0251) for 2 hr at 37°C. The reaction was buffered using the T7 RNA polymerase buffer in addition to 10 mM NTPs (ATP: P1132, CTP: P1142, GTP: P1152, UTP: P1162) in a final 20 μL reaction. RNA was purified using the Zymo Research RNA Clean and Concentrator - 25 kit. Sequences of RNA controls used were as follows: Control #1(G-44U): 5′GCCCCGUUGCAAUGGAAUGACAGCGGGUAUGUUAAACAACCCCAUCCGUCAUGGAGACAGGUGGACGUUAAAUAUAAACCUGAAGAUUAAACAUGACUGAAUCUUUUGCUACUAGAAUGGUGAGCAAGGGCGAGGAGCUGUUC 3′, control #2:(5′ Zika UTR scramble):5′ UACAAUCACGAAAGUCAAUUAUAGUUUCGAGUCGUAACGAGAACAUUUCCCGCGGACCAAUUUAAGGAGUAACUAAAGUGUGAAAUGAUUCCGGAAUACUGUUGAAAUUGCGGAUCGAGCUUGCAGCCGUUAAAUUACCGGACGUUAGUGAAGUGCAGAUAUG 3′.

B2 RNA re-folding and B2 RNA – Hsf1 incubations were performed as described previously (14). In brief, in vitro-transcribed B2 RNA was folded with 300 mM NaCl through incubation for 1 min at 50°C and cooling at a rate of 1°C/10 s until 4°C. Subsequently, B2 RNAs were incubated at a final concentration of 0.4 μM unless otherwise stated with the addition of Hsf1 diluted in TAP buffer (final reaction concentrations: 5 nM Tris pH 7.9, 0.5 mM MgCl2, 0.02 mM EDTA, 0.01% NP-40, 1% glycerol, 0.2 mM DTT). Hsf1 protein incubations were performed with phosphorylated, recombinant, His-tagged Hsf1 (Enzo Life Sciences: ADI-SPP-902-F; ~60KDa). Hsf1 working concentrations were 250 nM unless otherwise specified, diluted in TAP buffer. PNK (NEB, M0201; ~35KDa) was used as a control protein because it is an RNA-binding protein that is used in the construction of our short RNA libraries and we wanted show that short RNA seq data are free of such confounding factors that could potentially generate artificial fragments. PolyA (NEB, M0276; ~56KDa) was used as control protein because it is an RNA-binding protein that should not process B2 RNAs. Denatured Hsf1 was prepared by diluting in TAP buffer as above and heating at 95°C for 3 min; denatured Hsf1 was used as a control to ensure buffer components of Hsf1 themselves were not responsible for B2 RNA processing.

Fragmentation of B2 RNA was analyzed on 8M urea 10% PAGE gels stained by SYBR II (Invitrogen, S7564). Gel analysis occurred on Amersham Typhoon instruments. Band absorbance was analyzed using ImageJ area under the curve software and normalized by the ratio of experimental over initial as described previously (Zovoilis et al., 2016).

### Short-RNA-seq and long-RNA-seq

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Using the miRvana miRNA size selection kit (Thermo Fisher) as described before, 1.5 μg total RNA was size separated into short and long fractions (Zovoilis et al., 2016). In brief, following addition of the lysis/binding buffer and the homogenate additive solution to the RNA, 1/3 of the volume 100% EtOH was added and the mix was passed through the column for binding long RNAs. 100% EtOH at 2/3 of the flow through volume was subsequently added to the flow through and passed through a second column for binding short RNAs. Eluted RNAs were tested for size and quality using the Agilent Bioanalyzer RNA pico-kit. For long-RNA-seq, the long RNA fractions were cleaned and concentrated using the RNeasy Minelute kit (Qiagen) and ribodepleted using the rRNA depletion kit (NEB). The library was then prepared using the NEBNext Ultra II direction Library preparation kit (NEB, E7760), and sample cleanups were performed using the Omega NGS Total Pure Mag Beads (Omega, SKU: M1378-01) 0.5X and 1.2X before library amplification and 0.9X following amplification. nine cycles were used during amplification. For short-RNA-seq, the short RNA fractions were subjected to 3′-phosphoryl removal for 1 hr at 37°C, treated with the T4 PNK enzyme (NEB, M0201), using exclusively the 10X PNK buffer (NEB). The short fractions were cleaned and concentrated using the RNeasy Minelute kit and the library was prepared using the NEBNext small RNA library prep set (E7330) as described before (Zovoilis et al., 2016). Sample cleanups were performed using again the Omega NGS Total Pure Mag Beads (Omega) 1.2X following library amplification. Library amplification used 15 cycles. Quantification of libraries was done by qPCR using the NEBNext library quant kit for Illumina (NEB, E7630) and library sizes were analyzed using the Agilent bioanalyzer 2100 HS DNA kit. Equimolar amounts were prepared for sequencing. Libraries were sequenced on an Illumina HiSeq platform using 150nt read lengths. For B2 RNA in vitro sequencing, a 10X reaction as described of B2 RNA in solution with Hsf1 was incubated for 60 min at 37°C. The RNA was prepared as described above at twice the concentration of the recommended kit components. RNA was cleaned and concentrated using the modified RNeasy minElute kit.

### Western blotting

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HT22 cells were seeded at 100,000 cells per well and transfected with LNA in a six-well plate as described. Cells were harvested and lysed using RIPA lysis buffer with protease and phosphatase inhibitors added. Next, the cell suspension was centrifuged at 13,000 rpm for 20 min at 4°C, supernatant was aspirated and pellet discarded. Protein concentration was determined using a Bradford assay (bio-rad; 500–0205). Equal amounts of soluble protein were loaded (25 μg) for resolving with SDS-PAGE. Proteins were transferred onto nitrocellulose membrane (GE healthcare), blocked for 1 hr with 1% milk in 0.02% PBST at room temperature. Individual proteins were detected using polyclonal rabbit HSF1 antibody (1:1000; Enzo Life Sciences; ADI-SPA-901; RRID:AB_10616511). After incubating primary antibody, blots were washed using 0.1% PBST and reprobed with anti-rabbit-HRP conjugate secondary antibody (1:5000; abcam; ab97051). Proteins were visualized using Pierce enhanced chemiluminescence detection system (Thermo Fisher Scientific; 32106). Blots were imaged in an AI600 imager (GE Healthcare) and densitometry performed using ImageJ.

### Bioinformatics analysis

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For the tissue enrichment and GO term analysis of B2-SRGs (Supplementary file 1), we used the DAVID function annotation platform (DAVID 6.8, February 2020) with default parameters (EASE score 0.1, max. 1000 entries), and a reporting EASE score threshold of 0.05 and p-adjusted values calculated based on the Benjamini method (Huang et al., 2009a; Huang et al., 2009b). For both GO Biological process and Cellular Compartment, we selected the BP- or CC-direct options.

For the analysis of the short-RNA-seq and long-RNA-seq data, initially FastQC (Babraham Bioinformatics, https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was run for quality control of generated reads in fastq format. Subsequently, standard Illumina adaptor sequences were trimmed off using cutadapt-1.18 (https://doi.org/10.14806/ej.17.1.200). Short-RNA-seq reads were mapped to mouse reference genome (UCSC mm10) (November 2017) using bwa-0.7.17 in single-end mode with default aln parameters (Li and Durbin, 2009). Long-RNA-seq reads for each sample were mapped to reference genome ensembl GRCm38 (November 2018) primary assembly using hisat2-2.1.0, in single-end mode, with the following parameters: Report alignments tailored for transcript assemblers including StringTie, searches for at most one distinct, primary alignments for each read (Kim et al., 2019). SAM format files generated from mapping were converted to BAM format files using samtools-1.6 (Li et al., 2009), and to files in BED format with bamToBed utility from BEDTools-2.26.0 (Quinlan and Hall, 2010).

In long-RNA-seq, FPKM (Fragments Per Kilobase of transcript per Million) and TPM (Transcripts Per Million) for genes were generated using StringTie-1.3.4d (Pertea et al., 2015) with the following annotation: ensembl GRCm38 patch 94 gff3 file, and parameters limiting the processing of read alignments to only estimate and output the assembled transcripts matching the reference transcripts given in annotation and excluding non-regular chromosomes. Because TPM already includes scaling of the data it is unsuitable for the averaging of the gene expression levels of multiple genes (B2-SRGs) used in the boxplots of Figure 2. This does not apply in case of single genes as in Figure 2C (Trp53) or in the heatmap of the same figure, where each gene is presented in a separate row, and for which TPM values are used. For data visualization, statistics and differential expression analysis, we employed R (version 3.4.3) (https://www.R-project.org/) and the package DESeq2 (Love et al., 2014). Differential expression analysis was implemented on transcript count data for 6-month-old mice between APP and wild type. Boxplots central line represents median and t-test was applied on the group numbers mentioned in the text. PCA plots for samples used and read count correlation matrix between 6-month-old mice samples are presented in Figure 6—figure supplement 1.

For Hsf1 metagene analysis, we used peak.txt files of Hsf1 peaks for ChIP-seq from Mahat et al., 2016. Peaks were analyzed with Seqmonk around Transcription start sites of genes (TSS) based on the Eponine annotation (Down and Hubbard, 2002), and filtered based on their overlap with B2 RNA regulated genes (Zovoilis et al., 2016). Also, B2 RNA-binding (CHART-seq) peaks were analyzed with Seqmonk around TSS (Eponine), then filtered by overlapping with learning-associated SRGs or all genes (Peleg et al., 2010). Relative density metagene plots of the distribution of the above peaks were generated using Seqmonk.

### Data access

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Short- and long-RNA-seq raw data have been deposited to GEO with access number GSE149243.

## Appendix 1

Appendix 1—table 1

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## Decision letter

1. Joaquín M Espinosa
Reviewing Editor; University of Colorado Anschutz Medical Campus, United States
2. James L Manley
Senior Editor; Columbia University, United States
3. Leo Kurian
Reviewer; Center for Molecular Medicine Cologne/ CECAD; Cologne Cluster of Excellence in Cellular Stress Responses in Ageing-associated Diseases

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

[Editors' note: this paper was reviewed by Review Commons.]

Acceptance summary:

The revised manuscript addresses the reviewer's comments and includes new data supporting the discovery that amyloid beta neuropathology involves deregulation of SINE B2 ncRNAs. The burden of Alzheimer's disease continues to increase, and novel insights into the mechanisms by which the pathogenic amyloid peptide exerts its toxic effects on neuronal tissues are appreciated and necessary. This manuscript reveals a previously unreported phenomenon elicited by the amyloid peptide leading to activation of cell death pathways, which supported by results from animal models and in vitro cell cultures.

Decision letter after peer review:

Thank you for submitting your article "Increased processing of SINE B2 non coding RNAs unveils a novel type of transcriptome de-regulation underlying amyloid beta neuro-pathology" for consideration by eLife. Your article has been reviewed by two peer reviewers at Review Commons, and the evaluation has been overseen by a Reviewing Editor and James Manley as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

After thorough discussion, the reviewers agreed that the manuscript by Zovoilis et al. is much improved from the original submission to Review Commons and that the authors have addressed many of the original concerns raised by reviewers. However, two major concerns remain, and reviewers agreed to encourage a resubmission addressing these.

B2 RNAs encoded from SINE B2 elements have been directly implicated in stress response by its inherent ability to bind RNA Pol II and suppress stress response genes (SRG) in homeostatic conditions. However, upon stimuli, B2 RNAs are cleaved and degraded, resulting in the release of RNA polymerase II (RNAPII) and upregulation of SRGs. Previous work from the senior author of this manuscript identified the Polycomb repressive complex 2 (PRC2) component EZH2 to be the B2 RNA processing factor, cleaving B2 and releasing RNAPII. SRGs are upregulated upon stress, for example in age associated neuropathologies like Alzheimer's disease (AD). Considering that hippocampus is a primary target of amyloid pathologies and given that SRGs are suggested to be key for the function of a healthy hippocampus, the authors set out to understand the role of B2 RNAs that are linked to SRG regulation in the mouse hippocampus with amyloid pathology. They use disease relevant in vivo and in vitro models combined with unbiased RNA seq data analysis for this endeavor, which indicate the potential relevance of B2 RNAs in APP-mediated neuronal pathologies in mice while also identifying Hsf1 as the factor cleaving B2 RNAs in the hippocampus. The work is deemed interesting and identification of Hsf1 as the processing factor for B2 RNAs in the hippocampus is significant.

1) Reviewers remain concerned that the bulk of the analyses relies on genes that were identified as B2-regulated SRGs in a prior experimental system (heat-shocked NIH3T3 cells) that is completely different from the amyloid pathology models used here. The authors sought to address this issue in the revised manuscript, but questions remain. Indeed, the new Supplementary tables 4 and 5 in Supplementary file 1 show that of the ~1600 B2-SRGs identified in NIH3T3 cells, only 72 show expression patterns consistent with the regulatory model proposed; moreover, this was using FDR<0.2. How many genes would be left with FDR<0.05? The authors did include important new data in the HT22 cell model showing that mRNA levels for four genes increase after treatment with a B2-targeted LNA. These data are compelling, but a few additional controls are needed. Figure 6F needs negative controls showing qRT-PCR of genes that are not thought to be B2-SRGs. In Figure 6E, it is important that the full length B2 RNA is being detected (i.e. a PCR product ~180 nt) since their model states that these genes are repressed by full length B2 RNA prior to its degradation. The data in Figure 6 support the model that these four genes are under B2 control, but they don't show the relationship with amyloid pathology. What do the expression patterns of the 4 genes in Figure 6F look like in the mouse RNA-seq data (3m, 6m, 12m, WT vs APP)? Does the amyloid beta peptide treatment of the HT22 cells no longer induce expression of these four genes in the presence of the B2 LNA?

2) Reviewers remain concerned about the strength of the data regarding the role of Hsf1 in B2 RNA processing (although it seems like the authors are currently working on important control experiments, which might alleviate reviewer's concerns.) The negative controls with recombinant proteins prepared similarly to Hsf1, and with similarly sized control RNAs are critical. In addition, the full gels for these experiments need to be shown so the formation of short B2 RNA products can be evaluated in conjunction with the loss of the full length B2 RNA. This will help distinguish between specific processing controlled by the B2 ribozyme/Hsf1 activity versus non-specific breakdown. Moreover, the sizes of the in vitro processed products should correlate with the 5' end peaks from Figure 3A if the cellular and in vitro processing indeed arises from the same mechanism.

https://doi.org/10.7554/eLife.61265.sa1

## Author response

Reviewer 1:

Reviewer #1 (Evidence, reproducibility and clarity Required):

B2 RNAs, encoded from SINE B2 elements has been directly implicated in stress response by its inherent ability to bind RNA Pol II and suppress stress response genes (SRG) in homeostatic conditions. However, upon stimuli, B2 RNAs are cleaved and degraded, resulting in the release of RNA pol II and upregulation of SRGs. Previous work from the senior author identified PRC2 component EZH2 to be the B2 RNA processing factor, cleaving B2, and releasing POL2. SRGs are upregulated upon stress, for example in age-associated neuropathologies like Alzheimer's disease (AD). Considering that the hippocampus is a primary target of amyloid pathologies as well as since SRGs are suggested to be key for the function of a healthy hippocampus, the authors set to understand the role of B2 RNAs that are linked to SRG regulation in the mouse hippocampus with amyloid pathology. They use disease-relevant in vivo and in vitro models combined with unbiased RNA seq data analysis for this endeavor, which indicates the potential relevance of B2 RNAs in APP mediated neuronal pathologies in mice as well as identifies Hsf1 as the factor cleaving B2 RNAs in the hippocampus.

This reviewer generally remarks that “The work is interesting and identification of Hsf1 as the processing factor for B2 RNAs in the hippocampus is significant. I would like to credit the authors for their elegant in vivo experimental design in Figure 2.”

General comment: The reviewer finds “some of the conclusions to be overstated” and has brought a number of concerns to our attention. Indeed, we agree that provision of additional data and details is needed to avoid any confusion about the gene pathways to which our findings apply. In the initial manuscript, (Figures 2 D, F and 6 D, F), we presented the gene expression levels of all B2 RNA regulated SRGs identified in our previous study (Zovoilis et al., 2016), referred as B2 RNA regulated SRGs or B2-SRGs throughout the manuscript. To this end, we performed the respective statistical tests between the different conditions considering these genes, in order to show the transcription dynamics of these genes in either amyloid beta pathology (APP mice /Figures 2D, F) or amyloid beta toxicity (HT22 cells / Figures 6D, F). Since we were not looking for new candidate genes upregulated in APP mice or in our HT22 cell culture system, we did not narrow our analysis only to genes delivered by a general-purpose differential gene expression approach such as DESeq but tested all B2-SRGs. However, based on the reviewer’s comments below, we realize that the paper would benefit by presenting in the main figures only those B2 RNA regulated SRGs that overlap with differentially expressed genes identified by DEseq in each experimental system. This will help to avoid confusion and any misunderstanding that all B2 RNA regulated genes are equally affected in our system, which is not the case and would be an overstatement. We are now presenting in new Figure 2 (2E, 2F) only those B2-SRGs that overlap with upregulated genes identified by DESeq in 6m old APP mice (listed in new Supplementary table 5 in Supplementary file 1) and in new Figure 7 (7D, F) we are now presenting only those B2-SRGs that overlap with upregulated genes identified by DESeq in HT22 cells treated with amyloid beta (listed in new Supplementary table 11 in Supplementary file 1). The conclusions drawn by the new figures remain the same as with the old ones and we believe that this new way of presentation of this data will prevent confusion and potential over-statements. We thank the reviewer for bringing this to our attention. Based also on this reviewer’s minor point 3, we recommend that the old figures that included all B2-SRGs (and not only the differentially expressed ones identified by DESeq) are moved to the Supplement as new Figure 2—figure supplement 3 and Figure 8—figure supplement 2, respectively, so that readers can still get a view of all the data and the transcription dynamics of all B2-SRGs, while we provide both in text and the supplement an explanation about the value as well as limitations of these figures.

1) In Figure 1, the authors indicate a strong connection between B2 RNA regulated SRGs and learning and memory. In Figure 2, they identify the SRGs in the hippocampus, please provide a direct comparison of learning and memory associated SRGs and the SRGs they identify in Figure 2 that are significantly upregulated in APP mice in 6 months.

In the revised version of the manuscript we now provide:

i) As a new figure panel (lower panel in new Figure 1E), the number of B2 RNA regulated SRGs that are associated with learning based on our Peleg et al., 2010 paper and as a new Supplementary table 3 in Supplementary file 1, the exact list of these genes.

ii) As a new Supplementary table 4 in Supplementary file 1, the list of all genes that are significantly upregulated in APP mice (6 months).

iii) As a new Supplementary table 5 in Supplementary file 1, the list of those genes upregulated in amyloid pathology (APP 6 months) that are B2-SRGs (expression levels of these genes are presented in new Figure 2E,F).

Per reviewer’s question, we now provide as a new Supplementary table 6 in Supplementary file 1, the list of B2 RNA regulated SRGs that are both learning associated genes and upregulated in 6 month old APP mice. In the text (first two sections of the Results), we provide direct comparisons of the number of genes in each category and their overlap.

2) To better understand the data in the context of hippocampal function, please include functional annotation of SRGs they identified in Figure 2F as they do it in Figure 1 (desirably for each time point, at least for 6M). How many of the SRGs they identify in Figure 1 are part of Figure 2F? Please include functional annotation of significantly upregulated B2 regulated SRGs in Figure 2 and compare them with that of Figure 1.

The number of B2 RNA regulated SRGs in Figure 1 that are part of Figure 2 (in particular Figures 2E,F) is now presented in the new Supplementary table 5 in Supplementary file 1 and also in the text. We now provide as a new Supplementary table 7 in Supplementary file 1 the functional annotation of these genes (see also general comment for this reviewer) and discuss the findings in the text.

We recommend to include only the 6M old mice as this is the time point in which B2 RNA processing was found to differ between WT and APP mice. However, if the reviewer thinks that this is necessary we will add also differential expression lists of other ages as additional supplementary tables.

3) In Figure 3, the authors report that the B2 processing rates are high at the 6M time point at in hippocampi of the APP mice. Please include the levels of unprocessed and processed B2 RNAs in these samples along with this figure, without which it is difficult to gauge the significance of its correlation with SRGs in Figure 2.

We now provide as new figure panels 3E and 3F the levels of processed B2 RNA fragments and unprocessed (full length) B2 RNAs in these samples, respectively, along with the processing ratio which is now labeled as subfigure 3G.

4) What is the % of B2 regulated SRGs that are hsf1 bound in Figure 4C? What is there dynamics in the wild type and APP hippocampi?

Old Figure 4C is now Figure 4A. The exact number of B2 RNA regulated SRGs that are close to Hsf1 binding sites is now presented as a new figure (Figure 4C) and discussed in the text. A list of these genes is provided as new Supplementary table 8 in Supplementary file 1. For genes that are upregulated in APP mice compared to wild type, the difference in Hsf1 binding dynamics between B2 RNA regulated and not regulated genes is now presented as Figure 4—figure supplement 1.

5) What is the distribution of Hsf1 binding sites on (a) non-B2 regulated SRGs and (b) non-SRG genes in hippocampi?

This point is related with point 4. We now present a new panel (Figure 4B) for non B2 RNA regulated genes (listed in Supplementary table 13 in Supplementary file 1) along with the distribution we have in the initial manuscript for all B2 RNA regulated SRGs (now presented as Figure 4A). The direct comparison of these genes is presented in the new Figure 4—figure supplement 1 together with a similar comparison only for genes upregulated in APP mice.

6) In Figure 4D, the 3months old Wt HSF1 levels are high, yet B2 processing (Figure 3E) is low. Please comment.

The reviewer’s comment made us realize that we should include a plot that describes the correlation between Hsf1 levels and B2 RNA processing ration across all sequenced samples. This should reveal whether differences such as those observed by the reviewer affect our conclusion regarding the relationship between these two parameters. We now provide this in the new Figure 8—figure supplement 1, where we found a strong positive correlation between Hsf1 levels and B2 RNA processing ratio. We thank the reviewer for this comment which helped us to substantiate further this relationship.

7) While the authors show in vitro cleavage of B2 RNA by Hsf1, the experiment lacks controls to be conclusive. At least, please include a similar size protein as HSF1 with no-known RNA binding activity and a similar size protein with RNA binding activity as controls in 5A. Please justify the use of PNK as the control protein. Please include the use domain-based deletions of Hsf1 to map the region of HSF1 that is binding and potentially cleaving the B2 RNA. Please include an RNA of similar size and Antisense-B2 RNA to show the specificity of the Hsf1 based cleavage of B2 RNA. Without these controls, the conclusions in Figure 5 cannot be substantiated.

The endogenous ribozyme activity of B2 RNA compared to other control RNAs has already been shown in two previous works but we will also include the relative controls here by providing control incubations with other RNAs. We will also include the incubations with additional control proteins as suggested by the reviewer. We are currently performing these experiments and will include them in the revised version. PNK is used as a control protein because it is an RNA binding protein that is used in the construction of our short RNA libraries and we wanted show that short RNA seq data are free of such confounding factors that could potentially generate artificial fragments. We now include this information in the text.

We feel that the application of domain based deletions for Hsf1, while it would add additional information on the exact biochemistry underlying B2 RNA processing though Hsf1, is beyond the scope of this manuscript. In the current manuscript we are just focusing on the fact that Hsf1 can accelerate B2 RNA processing in vitro and not on the mechanism how this happens. This should be addressed in our opinion on a separate manuscript.

8) The authors should show that the incubated APP peptides are taken up by the cells (experiments in Figure 5F and Figure 6).

These figures are now labelled as Figure 6C and Figure 7, respectively.

That’s a very interesting point and we thank the reviewer for this comment. Multiple studies have shown that toxicity after incubation by amyloid beta is mediated mainly by cell surface receptors, which through cell signalling leads to the response to cellular toxicity that induces stress genes such as Hsf1.

Nevertheless, APP peptides may enter the cell, and the reviewer’s questions raised the possibility that oligomers entering the cell could have a direct impact on the stability of the B2 RNA. In that case, providing evidence that the amyloid enters the cell would be important if we had indications that amyloid beta interacts directly with B2 RNA. We did test this and we found no direct effect of amyloid beta on B2 RNA, so the processing in our case is not induced by oligomers that may have entered the cell. We were planning to present this information in a different manuscript, but if the reviewer or editor thinks that it would be beneficial for the paper, we could present this as supplement figure that shows that amyloid beta incubations with B2 RNA do not induce further processing beyond what Hsf1 causes. For the moment we just present this in Author response image 1:

Author response image 1

9) Please provide the list, functional annotation, and % of the SRGs upregulated upon incubation with APP in HT22 cells in comparison to 6month old APP mice. Comment on learning-related Genes.In the revised version, we now provide and mention in the text the following data:

i) a list of genes upregulated in HT22 cells during amyloid toxicity upon incubation with amyloid beta (new Supplement table 9 in Supplementary file 1),

ii) a list of genes according to point (i) that are common with genes upregulated in APP mice (new Supplementary table 10 in Supplementary file 1),

iii) the list and number of B2-SRGs that are upregulated in HT22 cells during amyloid toxicity (the reviewer’s question) (new Supplementary table 10 in Supplementary file 1). We mention in the text the gene numbers and also the genes that are common in all three lists.

iv) Functional annotation of genes of point (iii) (new Supplementary table 12 in Supplementary file 1),

We also mention in the text the limitations of our comparisons between the in vivo model of amyloid pathology (APP mice) and the in vitro cell culture model of amyloid toxicity (HT 22 cells) and we clarify that the cell culture model is used just as a simulation of the effect of amyloid beta in gene pathways associated with response to cellular stress and the role of Hsf1 on B2 RNA processing.

10) The authors should show the efficient downregulation of Hsf1 (protein) upon anti-Hsf1 LNA transfection.

In the revised version, in addition to the RNA-seq data we provide a second confirmation at the mRNA level with an independent method (RT-qPCR) in new figures 4E and 7B (lower panel). We are currently performing the protein extractions and will provide a WB or an Elisa in the revised version.

11) Please present the total B2 RNA levels for conditions in Figure 6C.

We now provide as new supplementary figure (Figure 8—figure supplement 1B and C) the levels of processed B2 RNA fragments and the total levels of unprocessed full length B2 RNAs of these samples that relate to old Figure 6C (now labeled as Figure 7C)

12) Hsf1 levels are not significantly downregulated in Control cells which were inoculated with the reverse APP peptide. Please comment.

We assume that the reviewer here refers to the lack of reduction in Hsf1 levels in the cells inoculated with the reverse peptide and the anti-Hsf1 LNA. Indeed, this lack of reduction is confirmed also by the new qPCR we performed (new Figure 7B, lower panel, R-ctrl vs R-anti-Hsf1). This should likely be attributed to compensation during non-stress conditions. In contrast, under stress conditions, Hsf1 is heavily used in stress response, which could explain the differences we see as cellular needs surpass the available Hsf1 transcripts due to degradation by the LNA. This is also supported by the new RT-qPCR experiments we have performed for B2-SRGs (new Figure 7E). In agreement with what is known for stress response genes such as immediately early genes (for example FosB), levels of these genes are minimal in both Rctrl and R-anti-Hsf1 conditions and only become activated during stress response. We now discuss this in the text of the revised manuscript.

13) Please compare and contrast the % of genes, the overlap, and the functional distinctions in 6F to that of 5G and Figure 1. What are the genes that are common between Figure 1, and that are specifically upregulated upon Anti-Hsf1 LNA transfection along with 1-42 APP. What is % of the occurrence of B2 binding sites in those genes? What are their functional annotations and what is their connection to learning, memory, and cell survival?

Old Figure 6F is now Figure 7F, while old Figure 5G is now Figure 6C.

This point is discussed in the response to points 1 and 9 of this reviewer. In summary, genes upregulated in our amyloid toxicity model included 25 B2-SRGs (new Supplementary table 11). When testing for enriched terms in these 25 genes, biological processes related with apoptosis, such as regulation of apoptotic process and programmed cell death were at the top of the list (new Supplementary table 12) and included, among others, genes such as FosB and Mitf that have been connected with Alzheimer’s disease. Out of the 25 genes that are up-regulated in both mice and our cell culture system, six are B2-SRGs (4932438A13Rik, Fosb, Pag1, Ptprs, Sema5a, and Sgms1) and include a well-known immediate early gene (Fosb), genes associated with sensitivity to amyloid toxicity (Pag1, Sema5a, Sgms1, Fosb), as well as genes associated with p53 (Ptprs, Fosb). All these genes get upregulated in amyloid toxicity (42-Ctrl vs R-Ctrl) but are not upregulated when Hsf1 LNA is applied (42-anti-Hsf1 vs R-anti-Hsf1, no significant difference). This information is now included in the text.

1) Please include TPM/ FPKM values for hippocampal markers as control in Figure 2 to do justice to the hippocampus specific RNA seq conducted by the Authors.

To our understanding, the reviewer here suggests the testing of well-known hippocampal markers in our mouse data as controls to confirm that they are indeed hippocampus specific. We have selected as reference markers, the genes employed by the Allen Brain Atlas RNA-sequencing project and we provide a comparison of their data in hippocampal cells with our data from mouse hippocampus. This is now presented as new Figure 2—figure supplement 1.

2) In Figure 2D the authors show that B2 RNA regulated SRGs in the 3 months' wild type mice are significantly high. P53 has been reported to be high in young wild types hippocampus, but not SRGs in my opinion. The authors should comment on this.

Old Figure 2D is now Figure 2E. We now mention the reviewer’s comment particularly in the Discussion and cite a landmark review article in Neuron journal by Michael Greenberg regarding the role of stress response genes, such as FosB, early during development.

As to prevent any confusion, we have also replaced SRGs with B2-SRGs since we tested only B2-SRGS in our study.

3) In Figure 2F, under the 6m APP condition, the replicate 3 looks substantially different from the other replicate. This can significantly impact the analysis and conclusions made. Either remove that replicate and present the analysis without it or please provide a valid explanation. To make the data more valid, please provide hierarchical clustering of the entire data, the non-B2 regulated genes and the B2 regulated SRGs.

We now provide in the new Figure 6—figure supplement 1C a PCA plot, which includes 6m APP mice vs. their WT counterparts and HT22 cells, and shows that this variability is within the biological replicate variability we can expect in these models. To substantiate this further, we have constructed the correlation matrix of the RNA-seq data of both WT and APP 6 month old mice in the new Figure 6—figure supplement 1D. As shown in this matrix, all APP mice clearly correlate with each other and not with their WT counterparts.

In the initial manuscript the heatmaps of former Figure 2 were indeed provided with hierarchical clustering of the entire data and also included non-B2 RNA regulated genes. This data is included now as Figure 2—figure supplement 1.

In Figure 2C RNA seq data is represented in TPM while its FPKM in Figure 2D.

Figure 2D is now Figure 2E, while Figure 2C remains labelled with the same number.

Given that TPM already includes scaling of the data, it is unsuitable for the averaging of the gene expression levels of multiple genes (B2-SRGs) used in the boxplots of Figure 2. This does not apply in the case of single genes as in Figure 2C (p53) or in the heatmap where each gene is presented in a separate row. This explanation is now included in the Materials and methods section.

Figure 2: the number of replicates in the case of 3-month-old wild types only 2. Please specifically denote it and comment why only 2 replicates are provided.

During the hippocampal RNA extractions, the RNA of one of the three 3m old mice had very low RIN scores, which could be a confounding factor for the short-RNA-seq. As this happened some months after the hippocampal extractions, we did not have any other 3 month mice of the same cohort used for the behavioral and IHC studies. Thus, we decided to include only two replicates in this condition. Since the results presented in the current study focus mainly on 6 month old mice, we expect the impact to be minimal. We include this note in the Materials and methods section.

4) Considering that p53 and SRGs are significantly upregulated in 6months in the APP model, it would be great if (allowing that these samples are still available) the authors can include a staining for apoptotic markers, for example, Active Casp3 or similar. This will allow us to better gauge the gene expression changes presented by the authors especially regarding SRGs.

Unfortunately, we do not have these slides but in the revised version we will provide qPCR data for some of these markers.

5) Under subheading: Hsf1 accelerates B2 RNA processing, 3rd paragraph when the authors comment on known hsf1 binding sites on SRG genes, please correct from: Increased Hsf1-binding was found…. "To the increased number of hsf1 binding sites were found", unless the authors would like to show increased Hsf1 binding by performing ChIP-seq for Hsf1 in the hippocampus at least at the 6-month time point between Wt and APP mice.

We have changed the text accordingly.

Reviewer #1 (Significance (Required)):

B2 RNAs, encoded from SINE B2 elements has been directly implicated in stress response by its inherent ability to bind RNA Pol II and suppress stress response genes (SRG) in homeostatic conditions. However, upon stimuli, B2 RNAs are cleaved and degraded, resulting in the release of RNA pol II and upregulation of SRGs. Previous work from the senior author identified PRC2 component EZH2 to be the B2 RNA processing factor, cleaving B2, and releasing POL2. SRGs are upregulated upon stress, for example in age-associated neuropathologies like Alzheimer's disease (AD). Considering that the hippocampus is a primary target of amyloid pathologies as well as since SRGs are suggested to be key for the function of a healthy hippocampus, the authors set to understand the role of B2 RNAs that are linked to SRG regulation in the mouse hippocampus with amyloid pathology. They use disease-relevant in vivo and in vitro models combined with unbiased RNA seq data analysis for this endeavor, which indicates the potential relevance of B2 RNAs in APP mediated neuronal pathologies in mice as well as identifies Hsf1 as the factor cleaving B2 RNAs in the hippocampus.

The work is interesting and identification of Hsf1 as the processing factor for B2 RNAs in the hippocampus is significant. I would like to credit the authors for their elegant in vivo experimental design in Figure 2.

Reviewer 2:

Reviewer #2 (Evidence, reproducibility and clarity Required):

Summary:

This manuscript follows from previous work by the corresponding author showing that SINE-encoded B2 RNAs function as regulators of the expression of stress response genes (SRGs). Specifically, stimulus triggers the processing of repressive B2 RNAs that are bound at the SRGs, thereby activating SRG transcription. In this work, the authors investigate whether a similar mechanism might be controlling the expression of genes in models of amyloid beta neuropathology (i.e. mouse hippocampi from an amyloid precursor protein knock-in mouse model, and a cell culture model of amyloid beta toxicity). They performed RNA-seq in these models. Their data show a correlation between the progression of amyloid pathology, expression of genes thought to be regulated by B2 RNA, and the processing of B2 RNA. In addition, they show biochemical data supporting a role for Hsf1 in enhancing the processing of B2 RNA. Knockdown of Hsf1 also reduced B2 RNA processing and the expression of SRGs.

1) In the RNA-seq data one cannot distinguish between Pol III transcribed B2 RNA and Pol II transcribed B2 RNA (typically embedded within introns and UTRs of mRNAs). The models they present, and the structures they show, clearly imply regulation by Pol III transcribed B2 RNA. However, there is no way to know that the short B2 RNAs they sequence aren't coming from degraded mRNAs. This needs to addressed. Minimally, in writing as a caveat of their model. Ideally, it would be addressed experimentally.

That’s a very interesting point, as it implies that the regulatory role of B2 RNAs may extend from PolIII transcribed B2 RNAs into B2 RNAs embedded into mRNAs (likely nascent ones) that may be also under the same endogenous ribozyme activity of this sequence, suppress PolII and are processed in response to stimuli. The RNA RIN values of our samples were pretty high except one 3m old mouse sample which was for this reason excluded from further analysis. Moreover, during the library construction shorter and longer RNAs have been separated. Thus, any generation of B2 RNA fragment that may have originated from mRNA should be biologically but not technically related and must have happened in the cell before our RNA extraction. To address this point, we now provide a new supplementary figure (Figure 3—figure supplement 2), where we have separated the B2 elements against which we map the RNA fragments into two categories, those that fall within exonic/genic regions and those outside of these regions. Although B2 RNAs are produced by multiple copies in the genome, each copy does harbor multiple SNPs, insertions and deletions, which means that each B2 RNA fragment is mapped to a specific set of B2 elements and not to all of them. In other words, despite multiple mapping a level of spatial specificity is maintained. If the B2 RNAs we map were coming exclusively from either only Pol III B2 elements or mRNA embedded B2 elements, we would expect at least some difference in the distribution of fragments between B2 elements of these two categories, as the second one overlaps with mRNAs. As shown in the new figure 3—figure supplement 2, the fact that distribution models are very similar between the two categories indeed supports the hypothesis that both types of B2 elements may contribute to B2 RNA processing. Most importantly, the profile of B2 RNAs in genic regions shows that B2 RNA processing is not random but follows the same processing rules as B2 RNAs from Pol III promoters. Given the limitations posed by the repetitive nature of B2 RNAs, it remains difficult though to provide an exact number regarding the portion of B2 RNA fragments produced by each category and this is clearly noted in our revised discussion part. However, even the indication that B2 RNAs embedded in mRNAs may also play an important role in our model provides a new perspective that should be investigated further in future studies.

2) The direct regulation of SRGs by B2 RNA was not shown in their model systems for amyloid beta neuropathology. Rather, the authors' used the genes identified in their prior studies as B2 RNA-regulated, which I believe were in the NIH3T3 cell line. Given that transcription is highly cell-type specific, these genes might not be regulated by B2 RNA in mouse hippocampi or their cell culture model, despite the correlations shown. This needs to be addressed. Ideally, a targeted approach to show that transcription of even a couple genes in their system is indeed regulated by B2 RNA would provide stronger support for their conclusions.

We agree with the reviewer and we now provide a new figure (Figure 6D-F) with the targeted approach that this reviewer proposed. In particular, we have tested whether fragmentation of full length B2 RNAs is in connection with activation of target genes also in our biological system (HT22 cells) as it did in NIH/3T3 cells in our Cell paper. We now show in new Figure 6 that this is indeed the case.

3) The following bioinformatics analyses would strengthen their conclusions. This should be straightforward to do because it involves data they already have, and perhaps analyses they have already have performed.

a) Regarding the plot in Figure 3A (lower panel). The same plot should be shown for the 3m old and the 12m old APP mice (i.e. not just the 6m data). This would show the specificity of processing B2 RNA and that it indeed correlates with disease progression.

We now provide this plot as new supplementary figure (Figure 3—figure supplement 1). It shows that increased B2 RNA processing coincides only with the active neurodegeneration phase at 6 months and not the terminal stage.

b) Regarding the plots of B2 RNA processing rate. This value could increase either due to more short RNAs or less full length RNA. Which is it for the 3m, 6m, and 12m APP mice? Showing the short and long B2 RNAs as boxplots (as opposed to only the processing rate) would address this and also provide additional insight into the regulation involved. The same applies to the data in Figure 6. (As an aside… do the authors mean processing ratio as opposed to rate? I'm not clear where the time component is coming into play to call this a rate.)

Old Figure 6 is now Figure 7.

We now provide all these figures that show that increase in processing ratio at 6 months is mainly due to increase in the processed fragments and not a decrease in full length B2 RNAs. For APP mice these are new Figures 3E and F, and for HT22 cells , these are new Figure 8—figure supplement 1B and C.

c) The random genes in Figures 2E and 6E are plotted as heat maps, but statistical significance is hard to see. What do boxplots of the random genes look like, and is the significant difference between 6m old APP and 6m old WT then lost?

Old Figure 2E is now new Figure 2—figure supplement 3C, while old Figure 6E is now new Figure 8—figure supplement 2C. We now provide these boxplots in new Figure 2—figure supplement 3B and Figure 8—figure supplement 2B.

4) It is interesting that B2 RNA self-processing is enhanced by both Ezh2 and also Hsf1. It would strengthen the data to perform a control with a protein prepared more similarly to the Hsf1 (rather than PNK) to confirm that the enhanced B2 RNA breakdown is indeed attributable to Hsf1 and not a contaminant in the protein prep. Similarly, the authors should provide information on which RNA was added as the negative control for Hsf1-stimulated breakdown (i.e. the ~80 nt RNA).

This point is also discussed in reviewer 1 point 7. The ribozyme endogenous activity of B2 RNA has been shown already in two previous studies that performed incubations with control RNAs and proteins. We are currently preparing and will provide these additional incubations as anew supplementary figure in the revised manuscript.

1) Regarding the GO analyses in Figure 1 (panels B, C, and D). I wasn't clear whether the authors are showing all statistically enriched terms, or only those relevant to neuronal processes and learning. I recommend showing a supplemental table with all terms that have an adjusted p value below a specified cut-off (e.g. 0.05).

The statistical threshold used was an EASE score of 0.05 and all presented terms were above this threshold. In the initial manuscript we filtered only the top 5 terms in tissue enrichment and the top 10 terms for GO Biol process and Cell Compartment that had passed the threshold. We now provide all the terms that passed the threshold as a new Supplementary table 2 in Supplementary file 1, including gene counts, exact gene numbers and related statistics.

2) The authors show several figures that are not new data (2B, 4A, 4B, Supplementary figures 1 and 2). I think it would be more clear if these data were summarized and referenced in the Results, rather than shown.

Old Supplementary figures 1 and 2 that were results of previous studies or web resources directly available (such as Human Protein Atlas) have been now removed and they are now just referenced in the text. Old Figures 4A and 4B have been removed from the main figures but may be helpful to the readers if they are still available in the Supplement (currently as Figure 4—figure supplement 1A and B), as not all users are familiar with the RNA-seq browsing tools of Allen Brain Atlas resources.

Regarding Figure 2B that contains data from our previous study on this exact cohort of mice: If the reviewer and the editor agree we recommend that it remains in the main figure (with the appropriate image credit citations), as it provides in an efficient way the clear connection between amyloid load and our results at the molecular level, and, most importantly, it clearly draws a line in amyloid pathology progression between 3m old and 6m old, that agrees with our findings in the RNA-seq data of these mice.

3) In Figure 3A the schematic shows that B2 is 155 nt, the plots in Figures 3A,B,C show B2 RNA is 120 nt, and Figure 5 shows the RNA is 188 nt. Can the authors please clarify these differences?

The full length of B2 consensus sequence is 188nt and this is the one we use for the in vitro experiments. However, the structure of the B2 RNA has been resolved only for the first 155nt by the Kugel lab, and this is the only publicly available structure that we can reference in our figures.

For the mapping of 5’ends of short fragments in Figure 3A we have used the same range tested in our Cell paper to maintain consistency of the results. The reason why this 120nt threshold was selected in the Cell paper was to exclude artifacts from short RNAs mapping partially in our metagene as well as downstream of those B2 elements that are shorter from the consensus sequence. We now explain in Materials and methods section these differences.

4) In the Materials and methods section, the sequence of the g block template didn't contain the T7 promoter sequence that was used as the forward primer for PCR amplification?

We have now included this sequence in lower case.

5) In Figure 6B, why were Hsf1 levels not decreased in the R treated cells after treatment with the LNA?

Old Figure 6B is now new Figure 7B.

Please see response to reviewer 1, major point 12.

Reviewer #2 (Significance (Required)):

The models presented for the regulation of stress response genes (SRGs) in amyloid beta neuropathologies are compelling. As are the correlations they found between the progression of amyloid pathology, expression of genes thought to be regulated by B2 RNA, and the processing of B2 RNA. This is a unique direction of research for brain disease and represents an interesting conceptual advance. Most prior studies in this area use common model cell lines, and this lab seems well-positioned to unravel the proposed molecular mechanisms in neuronal systems.

Reviewer 3:

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

This manuscript describes a regulatory mechanism involving Hsf1 and B2 RNAs in the control of stress response genes (SRGs) during amyloid induced toxicity. In particular Hsf1, upregulated in 6m old APP mice and in HT22 cells treated with beta amyloid peptides, is shown to stimulate the B2 RNA destabilization leading to SRGs activation. While in healthy cells this upregulation can be reverted once the stimulus is removed, the pathological condition fuels the circuitry leading to p53 upregulation and neuronal cell death. The authors previously described the same mechanism acting during cellular heath shock response but in this case the protein identified as trigger of B2 RNA destabilization and SRGs activation was EZH2 (Zovoilis et al., 2016).

Indeed, the first part of the manuscript describes additional analyses of the previous data that prompts further investigation on the potential role of B2 RNA in AD condition. Nevertheless, it is not clear how the prior findings obtained in not biologically related cellular models might be used to obtain helpful indication of B2 RNA neuronal activity.

We thank the reviewer for this comment. Indeed, the current study’s main aim was to expand the findings of our previous work on the role of B2 RNA in cellular response to thermal stress in NIH/3T3 cells to other types of cellular response to stress, in our case to amyloid toxicity and the resulting amyloid pathology in neural cells. Response to thermal stress (Heat Shock) has been used for years as a basic study model for cellular response to stress. Proteins and gene pathways initially identified in heat shock have been subsequently shown to play identical pro-survival roles in other biological systems and there are studies showing the role of Hsf1, heat shock related proteins and cell stress response pathways in neural cells and the mammalian brain (we will provide these references in the revised version). For example, pathways such as the MAPK pathway and early response genes, that constitute the basis of response to heat shock, have been shown in studies by us and others to be activated and play a critical role in hippocampal function. Thus, examining the role of B2 RNA in the context of neural response to stress constituted a natural continuation of our previous study in NIH/3T3 cells. The fact that the list of B2 RNA regulated SRGs was found to be highly enriched in neuronal tissue terms and cellular compartments related to neuronal functions plainly confirms the close relationship among cellular response pathways in the two biological systems. Due to these facts we were compelled to investigate in more detail our previous findings also in a neural cell model. However, as discussed in point 2 of reviewer 2, the initial manuscript did not confirm the direct control of B2 RNA on expression of target genes also in our cellular model. This information is now part of the new Figure 6 and we thank both reviewers for bringing this to our attention.

The research fields of non-coding RNAs and neurodegeneration are attractive and challenging and, in my opinion, the molecular circuitry involving B2 RNAs might add important insights for understanding beta amyloid toxicity and neuronal death; however, the data provided are not in the shape making the manuscript suitable for publication: some controls are missing, the way the experiments are presented is not easy to follow and more importantly the authors does not provide any data (tables or lists) of the NGS experiments and the study lacks validation of them. Therefore, in my opinion the manuscript needs a profound revision before to be considered for publication in Review Commons.

Based on this reviewer’s and the other reviewers’ suggestions we now provide additional controls, detailed tables and gene lists, and qPCR validation of these results. We have also substantially revised the text in the first section of the Results and beginning of the Discussion, to make our rational for testing B2SRGs more clear and easier to follow.

Major concerns:

1) The first paragraph of the Results is entirely dedicated to reanalyze the data previously published by the same group (Zovoilis et al., 2016). However, this is not adequately explained. In line with this, the Table 1 is not required since the data are already provided by Zovoilis et al., 2016, unless the authors handled the data using additional new criteria that have to be explained.

We now explain our rational for using this data in more detail in the text. Please see also response to the general comment of this reviewer and response to the next point.

In the Zovoilis et al., 2016, study, the data presented did not include the list of regulated genes in a direct way but as part of the annotation of the B2 CHART peaks. This may pose difficulty to non-experts to extract the gene list from that data and we thought to include them as separate gene list here so that readers can directly use it for their analysis. Nevertheless, if the reviewer or the editor think that the list is redundant, we can surely omit it.

Moreover, Zovoilis and colleagues (2016) focused on SRGs regulated upon heat shock and using NIH/3T3 and HeLa cell lines, therefore, it is difficult to me understand how, searching for "cellular function connected with B2 RNA regulated SRGs", the list resulted enriched of neuronal tissue terms or cellular compartments related to neuronal functions. Please clarify this point since the following analyses are based on these findings.

Neural pathologies, such as amyloid pathology in brain, are often connected with cellular stress due to proteotoxicity. The ability of neural cells to respond to proteotoxicity challenges is connected with various molecular mechanisms, including stress related proteins that were firstly described in the context of heat shock. Thus, both contexts (heat shock and amyloid toxicity) refer to cellular response to stress, which explains why genes identified to be regulated during stress response in NIH/3T3 cells constitute part of the basic stress response toolbox that neural cells have also been described to possess. We have now modified the text accordingly to make our rational more clear.

2) In Figure 1F there is no arrow indicating that some of the SRGs regulate directly miR-34 as stated in the main text. Moreover, it is more appropriate to replace SRGs with learning-associated genes both in the figure and in text (second paragraph of the Results) since Zovoilis and colleagues focused on them. Finally, they did not show in their manuscript the rescue of p53 expression mediated by mir-34; indeed, for miR-34-p53 regulatory axis Zovoilis and colleagues referred to Peleg et al., 2010 and Yamakuchi and Lowenstein, 2009. Please fix all these concerns.

We have restructured the figure as suggested by the reviewer and made clear the distinction between learning genes and B2 RNA regulated SRGs (B2-SRGs) from the two different studies. In connection with point 1 of reviewer 1, we believe that new Figure 1E, that includes the exact number of B2-SRGs that are learning associated, will represent more efficiently and accurately the data. We have also corrected in the text the citation regarding miR-34c and p53 in both the Introduction and first section of the Results (last paragraph).

-The Figure 1A and Figure 1F are wrongly indicated at the end of the sentence "….levels of these genes are normally downregulated in 6m and 12m old mice compared to 3m old mice (p=0.02 and p=0.04, respectively)"; please correct this point.

The error has been corrected.

3) Regarding Figure 2:

a) Since three mice for each condition have been used for the RNA seq analyses, please provide a blot with the Principal Component Analysis (PCA).

Please see also response to minor point 3 of reviewer 1. We provide the PCA plots for WT and APP mice in the new Figure 6—figure supplement 1 and we also provide a comparison of the six month old mice with the HT cell samples as well as a correlation matrix for 6 month old mice in the same figure.

b) Figure 2F comes first of Figure 2E in the text, however, I suggest to move this latter to supplementary material.

Old Figure 2E has now been moved to supplementary material as new Figure 2—figure supplement 1C and we also provide in a boxplot the exact gene expression levels as new Figure 2—figure supplement 1B.

c) In general, this study lacks validation of the RNA-seq results. Western blot and/or qRTR-PCR to verify the variation of p53 and of some selected SRGs have to be provided.

In the current revised version we already provide qPCRs for p53 and Hsf1 in APP mice and we will include additional genes in the final version.

d) It is also not clear how the authors defined SRGs in the hippocampus: do they correspond to learning associated genes described by in Zovoilis et al., 2011 or to B2 RNA H/S regulated genes by Zovoilis et al., 2016?

The way we presented B2 RNA SRGs in the results with regard to learning associated genes was indeed unclear. We now present the distinction between the two gene categories and their relationship as a new Figure 1E panel and we also provide detailed gene lists of common genes and the exact numbers (please see also response to Review 1, major point 1).

APP 12 month old mice show the sever phenotype of the terminal AD-like pathology, however this does not correlate with significant SRGs and B2 processing increase. Can the author make a comment on this?

That’s a very important point and we thank the reviewer for raising this point. We now comment on this in the Discussion part explaining how our findings are characteristic of the initial active neurodegeneration phase of amyloid pathology rather than more terminal stages.

4) Regarding Figure 5:

a) A gel with no-protein control for the time course of panel B was cited in the text but missing among the panels. Moreover, the time course shown in the graph in 5C does not correspond to the one in 5B.

Indeed, the no-protein control time line should refer only to panel C and not to B, we have now corrected the text. Nevertheless, we now present in the new Supplementary Figure 5 the gels, based on which the graph in panel C was calculated, including also the gel with no protein timeline.

The time course shown in the initial 5C had been mislabeled. It has now been corrected. We apologize for this and we thank the reviewer for bringing this to our attention.

b) 5G indicates that four samples for each condition have been analysed by RNA-seq, since they do not seem to be homogeneous please provide a PCA analysis together with the validation by qRT-PCR of a selected group of deregulated genes.

Old Figure 5G is new Figure 6C. PCA analysis for these samples is now provided in Figure 6—figure supplement 1 and qPCR validation of a number of these genes is provided in new Figure 7E.

Moreover, it is not clear whether all the genes shown in the heatmap or a number of them, as stated in the text, were found upregulated in 6m old APP mice. Please clarify this point and modify the figure and the text accordingly. A Venn diagram showing the overlap between genes upregulated in 42vsR treatment and those upregulated in 6m old APP mice might help the comprehension of the experiment.

Please see response to reviewer 1, point 9. We now provide as new supplementary tables the exact overlapping lists and mention these numbers in the text.

5) Regarding Figure 6 (now labeled as Figure 7):

a) The evaluation of the levels of Hsf1 mRNA and protein upon LNA transfection is missing for both R and 42 treated HT22 cells. From TPM in panel B, Hsf1 downregulation seems to have been more effective in 42 than in R condition. This would mess up the interpretation of the data.

We now provide qPCR data for Hsf1 gene expression levels which confirm the ones from the RNAseq. The reason why Hsf1 downregulation seems not to affect the R condition is discussed in our response to reviewer 1, major point 12, and the respective explanation is provided in the revised text.

b) Again, in this case any validation of the RNA seq data is provided (any B2 regulated SRGs).

Now, we provide qPCR data for these genes in Figure 7B and new Figure 7E

c) Panels E and F should be swapped or panel E moved to supplementary material.

Panel E is now moved to supplementary material as new Figure 8—figure supplement 2C.

6) In a previous paper the authors discovered B2 RNAs as a class of transcripts bound to EZH2 and this interaction leads to B2 RNA destabilization in heath shock (H/S) condition. The authors also conclude that the genes controlled by B2 RNAs may not overlap with the ones controlled by Hsf1 during H/S. The author should make a comment on this explaining why during H/S B2 RNAs work independently from Hsf1 and on different target SRGs while, during beta amyloid stress ,the two act together on the same SRGs. Moreover, as shown for EZH2, Hsf1-RIP experiment should be performed in order to confirm the direct involvement of Hsf1 in the SRGs-B2 destabilization.

In the last two paragraphs of our Discussion we indicate that B2 RNA regulation is a new process implicated in the response to stress in amyloid pathology but certainly not the only one. We have revised the text in this part accordingly in the revised version to prevent any confusion. We are currently performing a series of RIP-seq experiments with various antibodies. As, to our knowledge, there is no prior published study performing RIP-seq or CLIP-seq for any tissue using Hsf1 antibodies, the success of this experiment is not guaranteed and depends on the existence of appropriate antibodies.

7) There is any table listing the results of the RNA seq experiments performed in this paper: control vs APP 3-6-12 m old mice and in R vs 42 treated HT22 cells in presence or absence of LNA against Hsf1. Please provide these data.

We now provide these lists as new supplementary tables. Please see response to major points 1 and 9 of reviewer 1.

8) In the Discussion the authors claim that healthy cells are able to restore the expression of Hsf1, SRGs and B2 RNA upon removal of the stress. Since there are evidence for the rescue of SRGs and B2 RNA expression post H/S, no data are available for Hsf1, SRGs and B2 RNA upon the removal of 1-42 beta amyloid peptide. This might be a nice information to add to the manuscript.

This would indeed substantiate further our results in our HT22 cell model. We have now performed this experiment, in which HT-22 cells were removed from the amyloid 42 (and the respective R peptide control) and left to recover for 12 hours before estimating through RT-qPCR the Hsf1 levels ( see graph in Author response image 2, REC corresponds to recovered HT-22 cells). Hsf1 levels in 42-REC have returned to the same levels as in R, p< 0.05 for the difference between 42 and 42-REC, y axis=rel expression levels (A.U.).

Author response image 2

We currently perform the RT-qPCRs of these samples also for B2-SRGs and will include them in the final version as a supplementary figure.

Minor criticisms:

– In the Introduction the reference Yamakuchi and Lowenstein 2009, should be added in the sentence: "In contrast, hippocampi of mouse models of amyloid pathology and post- mortem brains of human patients of AD … and neural death (Zovoilis et al., 2011)."

We have now changed the text at that point accordingly and also updated the legend of Figure 1F that also refers to this same study.

– Authors refer to Hernandez et al., 2020 to state that B2 self-cleavage is stimulated by some proteins however, Hernandez and colleagues studied only the effect of EZH2 protein. Please rephrase the sentence accordingly.

Text has been modified accordingly.

– Indicate a reference for the sentence: "……Ezh2, was reported as being responsible for the B2 RNA accelerated destabilization and processing during response to stress."

– The format of many references is not consistent and has to be revised.

We have switched to the Vancouver style. Some references in the legend and Materials and methods sections are referred independently from EndNote in case these text sections have to be moved to supplement in the final version in order to not create inconsistencies with endnote.

Reviewer #3 (Significance (Required)):

The research fields of non-coding RNAs and neurodegeneration are attractive and challenging and, in my opinion, the molecular circuitry involving B2 RNAs might add important insights for understanding beta amyloid toxicity and neuronal death. However, this manuscript does not really add technical advances since the authors employed experimental approaches and bioinformatic analyses previously published by Zovoilis and colleagues in 2011 and 2016.

Our aim in the current manuscript was not to introduce a new method or experimental approach but rather to study the mechanisms behind B2 RNA regulation of gene expression in neural cells and particularly in amyloid pathology. Nevertheless, the current study constitutes the first reported short-RNA seq in this tissue and offers for the first time the ability to study B2 RNA processing in this tissue which is not possible with standard small and long RNA-seq.

The reported findings might of interest of an audience of experts in non coding RNAs and neurodegeneration. The area of my expertise almost regards the biology of non coding RNAs from biogenesis to function manly focusing on neuronal and muscular systems both in physiological and pathological conditions.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

After thorough discussion, the reviewers agreed that the manuscript by Zovoilis et al. is much improved from the original submission to Review Commons and that the authors have addressed many of the original concerns raised by reviewers. However, two major concerns remain, and reviewers agreed to encourage a resubmission addressing these.

B2 RNAs encoded from SINE B2 elements have been directly implicated in stress response by its inherent ability to bind RNA Pol II and suppress stress response genes (SRG) in homeostatic conditions. However, upon stimuli, B2 RNAs are cleaved and degraded, resulting in the release of RNA polymerase II (RNAPII) and upregulation of SRGs. Previous work from the senior author of this manuscript identified the Polycomb repressive complex 2 (PRC2) component EZH2 to be the B2 RNA processing factor, cleaving B2 and releasing RNAPII. SRGs are upregulated upon stress, for example in age associated neuropathologies like Alzheimer's disease (AD). Considering that hippocampus is a primary target of amyloid pathologies and given that SRGs are suggested to be key for the function of a healthy hippocampus, the authors set out to understand the role of B2 RNAs that are linked to SRG regulation in the mouse hippocampus with amyloid pathology. They use disease relevant in vivo and in vitro models combined with unbiased RNA seq data analysis for this endeavor, which indicate the potential relevance of B2 RNAs in APP-mediated neuronal pathologies in mice while also identifying Hsf1 as the factor cleaving B2 RNAs in the hippocampus. The work is deemed interesting and identification of Hsf1 as the processing factor for B2 RNAs in the hippocampus is significant.

1) Reviewers remain concerned that the bulk of the analyses relies on genes that were identified as B2-regulated SRGs in a prior experimental system (heat-shocked NIH3T3 cells) that is completely different from the amyloid pathology models used here. The authors sought to address this issue in the revised manuscript, but questions remain. Indeed, the new Supplementary tables 4 and 5 in Supplementary file 1 show that of the ~1600 B2-SRGs identified in NIH3T3 cells, only 72 show expression patterns consistent with the regulatory model proposed; moreover, this was using FDR<0.2. How many genes would be left with FDR<0.05? The authors did include important new data in the HT22 cell model showing that mRNA levels for four genes increase after treatment with a B2-targeted LNA. These data are compelling, but a few additional controls are needed. Figure 6F needs negative controls showing qRT-PCR of genes that are not thought to be B2-SRGs. In Figure 6E, it is important that the full length B2 RNA is being detected (i.e. a PCR product ~180 nt) since their model states that these genes are repressed by full length B2 RNA prior to its degradation. The data in Figure 6 support the model that these four genes are under B2 control, but they don't show the relationship with amyloid pathology. What do the expression patterns of the 4 genes in Figure 6F look like in the mouse RNA-seq data (3m, 6m, 12m, WT vs APP)? Does the amyloid beta peptide treatment of the HT22 cells no longer induce expression of these four genes in the presence of the B2 LNA?

We thank the reviewers for this comment. We now provide more clarity and supporting data to address this comment. In particular:

a) Our model does not propose that all B2-SRGs are deregulated in APP mice or that all deregulated genes in APP mice are B2-SRGs. Instead, it shows that a subset of the B2 SRGs is deregulated in amyloid pathology. This may not have been clear in the model described in our initial manuscript (old Figure 8, now Figure 9), in which we have used the general term “Stress response genes” to describe genes under this regulatory mechanism. In the revised Figure 9 model we have now substituted this term with the more accurate term “Target genes” to prevent any confusion and denote that there may be a variety of B2 RNA-targeted genes in different biological contexts. Moreover, our current data addresses B2 RNA-mediated regulation only in the context of amyloid beta pathology and focuses on differentially expressed B2 SRGs based on a statistical modeling approach (DESeq2, a total of 72 genes). Beyond this set of differentially expressed genes, we searched for additional SRGs that are expressed in the hippocampus, yet, they may be associated with other hippocampal functions that are independent from amyloid beta pathology. The expression of hippocampal B2-SRGs would be expected to correlate with B2 RNA processing. As shown in the new Figure 8—figure supplement 1E and the respective table (new Supplementary table 12 in Supplementary file 1) a detailed breakdown of these B2-SRGs with regard to expression in hippocampal cells and correlation with B2 RNA processing reveals 659 B2-SRGs that are sufficiently expressed in our tested hippocampal samples and, of those, more than 63% shows a weak (11%) or strong (52%) correlation between expression levels and B2 RNA processing ratio. This data suggests that, for a large number of the B2-SRGs previously identified in the context of heat shock, correlation between gene expression and B2 RNA processing ratio holds true also in the context of hippocampal cells, independently of the presence of amyloid beta pathology.

b) By applying DESeq2 to statistically model the expression changes observed between 6m old WT and APP mice we obtained 72 B2-SRGs upregulated in 6m old APP mice (listed in Supplementary table 5 in Supplementary file 1). All of those B2-SRGs have a p-value < 0.05, with 34 of them belonging to the group with an FDR value threshold below 5% and the rest 38 to the group with an FDR value at least below 20%. We used a more flexible FDR threshold since expression dynamics of the 72 differentially expressed genes (shown in Figure 2) show an almost identical pattern with those ones of several other B2-SRGs that may have been missed by the DESeq2 (shown in Figure 2—figure supplement 3). This strongly suggests that use of DESEq2 is rather conservative in our case for reasons listed in the respective note in the legend of Figure 2—figure supplement 3. In order to mitigate any concerns for an increased number of false positives in our data due to selection of a higher FDR threshold, in the new Figure 2—figure supplement 2, we now provide validation through RT-qPCR of the DESeq2 results. We have selected 12 of the differentially expressed genes (Figure 2—figure supplement 2A) and tested them through RT-qPCR (Figure 2—figure supplement 2B). The majority of the genes tested have been intentionally selected from the group with the higher FDR threshold (8 out of 12 have an FDR more than 5% and less than 20%). The resulting RT-qPCR data presented in Figure 2—figure supplement 2B are in alignment with the RNA-seq results for the same genes presented in Figure 2—figure supplement 2A. Thus, through the use of an orthogonal validation technique, the differentially expressed genes identified by DESeq2 in 6m APP mice indeed correspond to true positive findings. This data, together with data in Figure 2—figure supplement 3, support further that application of a standard RNA-seq differential expression statistical model such as DEseq2 in our case may indeed be a rather conservative approach. Nevertheless, given the widespread use of DESeq2, we still suggest the use of this approach in the main body of the study in order to facilitate comparison of our data with those from other groups in the future.

c) In order to substantiate further the connection between B2-SRGs previously identified in heat shocked NIH/3T3 cells and B2 RNA destabilization we have increased the number of B2-SRGs tested through RT-qPCR in the B2 KD assay in hippocampal cells (initially 4 genes, now 11 genes). In the new Figure 7C, we now show that, as previously observed in NIH/3T3 cells, also in hippocampal cells, destabilizing B2 RNA leads to increase in the expression of these genes. In addition to these 11 genes, we now include 5 negative controls (non-B2 SRGs) that showed no change during treatment. Among the negative controls we have also intentionally included non-B2-SRGs such as Adcyl1 and Kalrn, both of which are upregulated in amyloid beta pathology, to support further the specificity of our findings (i.e. being upregulated in amyloid beta pathology does not mean regulation by B2 RNA unless being a B2-SRG).

d) Indeed, in Figure 7A we test the unprocessed B2 RNA encompassing the full Pol II binding region (80-132) including all cutting points. The reverse primer is located downstream at position 142. Making a reverse primer beyond the position 142 is not possible as it overlaps with B2 RNAs poly-A repeats. The primers are now listed in the Materials and methods.

e) Data for the expression patterns of all genes in the new Figure 7C (old Figure 6F) that are differentially expressed in mouse APP and WT mice are now presented in Figure 2—figure supplement 2A for all three ages and conditions.

f) “Does the amyloid beta peptide treatment of the HT22 cells no longer induce expression of these four genes in the presence of the B2 LNA?”. To our understanding, we are asked whether treating the HT22 cells with amyloid beta and B2 LNA induces expression of these genes. Based on the data presented in the manuscript the amyloid beta treatment induces expression of these four genes (see new Figure 6D) as does the B2 LNA independently (Figure 7). Thus, we are a little unsure on the scope of such as experiment as we feel that inoculating B2 LNA together with amyloid beta would not add any additional information regarding our model. Such an experiment would have been informative if B2 LNA treatment was inhibiting SRG activation as in case of Hsf1 LNA (Figure 8), however, since both the amyloid and B2 LNA activate SRGs, we did not see any reason for combining them in one experiment as we did in case of Hsf1 in Figure 8, in which we combined LNA treatment with amyloid beta treatment.

2) Reviewers remain concerned about the strength of the data regarding the role of Hsf1 in B2 RNA processing (although it seems like the authors are currently working on important control experiments, which might alleviate reviewer's concerns.) The negative controls with recombinant proteins prepared similarly to Hsf1, and with similarly sized control RNAs are critical. In addition, the full gels for these experiments need to be shown so the formation of short B2 RNA products can be evaluated in conjunction with the loss of the full length B2 RNA. This will help distinguish between specific processing controlled by the B2 ribozyme/Hsf1 activity versus non-specific breakdown. Moreover, the sizes of the in vitro processed products should correlate with the 5' end peaks from Figure 3A if the cellular and in vitro processing indeed arises from the same mechanism.

We are grateful to the reviewers for this comment. Although some of these controls had already been presented in our previous studies, including control proteins not inducing B2 RNA processing and control RNAs not destabilized as rapidly as B2 RNA, we fully understand that it is due to the proposed biochemistry of B2 RNA as a potential ribozyme, and the difficulty to distinguish from non-specific RNA degradation, that additional data are required. To this end we respond below to the reviewer’s questions, and additionally we further test the effect of (i) changes in B2 RNA sequence itself and, (ii) denaturation of the Hsf1 protein on B2 RNA processing. In detail:

a) In new Figure 5F-I we now provide the following:

Incubation of a mutant B2 RNA in the presence and absence of Hsf1 and comparison with the same incubations with the original B2 RNA, that showed that acceleration of B2 RNA processing by Hsf1 is sequence-specific and, thus, not the result of non-specific RNA degradation.

Incubation of two control RNAs in the presence and absence of Hsf1 and comparison with same incubations with the B2 RNA, that also confirmed that Hsf1-induced processing is limited to B2 RNA.

Incubation of B2 RNA in the presence of a denatured Hsf1 and a control protein of similar size, that failed to accelerate B2 RNA processing as the native Hsf1 did.

b) In order to exclude non-specific RNA destabilization due to degradation, hydrolysis or B2 RNA endogenous self-cleavage, we have included in Figure 5 a number of control RNA incubations without protein in the same buffer (TAP) and for the same time as the samples treated with Hsf1 or control proteins. Full gels are also included now as two source files.

c) We have now performed short RNA-seq of the in vitro processed B2 RNA and compared the resulted fragments with those observed in vivo, now presented in the new Figure 5—figure supplement 1 A. This data reveals a similar processing pattern between the two conditions.

https://doi.org/10.7554/eLife.61265.sa2

## Article and author information

### Author details

1. #### Yubo Cheng

1. Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Canada
2. Southern Alberta Genome Sciences Centre, University of Lethbridge, Lethbridge, Canada
4. Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Canada
##### Contribution
Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
##### Contributed equally with
Luke Saville, Babita Gollen and Christopher Isaac
##### Competing interests
No competing interests declared
2. #### Luke Saville

1. Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Canada
2. Southern Alberta Genome Sciences Centre, University of Lethbridge, Lethbridge, Canada
4. Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Canada
##### Contribution
Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing
##### Contributed equally with
Yubo Cheng, Babita Gollen and Christopher Isaac
##### Competing interests
No competing interests declared
3. #### Babita Gollen

1. Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Canada
2. Southern Alberta Genome Sciences Centre, University of Lethbridge, Lethbridge, Canada
4. Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Canada
##### Contributed equally with
Yubo Cheng, Luke Saville and Christopher Isaac
##### Competing interests
No competing interests declared
4. #### Christopher Isaac

1. Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Canada
2. Southern Alberta Genome Sciences Centre, University of Lethbridge, Lethbridge, Canada
4. Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Canada
##### Contribution
Data curation, Formal analysis, Investigation, Methodology
##### Contributed equally with
Yubo Cheng, Luke Saville and Babita Gollen
##### Competing interests
No competing interests declared
5. #### Abel Belay

1. Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Canada
2. Southern Alberta Genome Sciences Centre, University of Lethbridge, Lethbridge, Canada
4. Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Canada
##### Contribution
Investigation, qPCR assays
##### Competing interests
No competing interests declared
6. #### Jogender Mehla

##### Contribution
Resources, Methodology
##### Competing interests
No competing interests declared
7. #### Kush Patel

1. Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Canada
2. Southern Alberta Genome Sciences Centre, University of Lethbridge, Lethbridge, Canada
WB assays
##### Competing interests
No competing interests declared
8. #### Nehal Thakor

1. Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Canada
2. Southern Alberta Genome Sciences Centre, University of Lethbridge, Lethbridge, Canada
##### Contribution
Resources, WB assays
##### Competing interests
No competing interests declared
9. #### Majid H Mohajerani

##### Competing interests
No competing interests declared
10. #### Athanasios Zovoilis

1. Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Canada
2. Southern Alberta Genome Sciences Centre, University of Lethbridge, Lethbridge, Canada
4. Alberta RNA Research and Training Institute, University of Lethbridge, Lethbridge, Canada
##### Contribution
Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing
##### For correspondence
athanasios.zovoilis@uleth.ca
##### Competing interests
No competing interests declared

### Funding

#### Alberta Innovates (201700011)

• Majid H Mohajerani
• Athanasios Zovoilis

#### Alberta Prion Research Institute (201700011)

• Majid H Mohajerani
• Athanasios Zovoilis

#### Alzheimer Society of Alberta and Northwest Territories (201900003)

• Majid H Mohajerani
• Athanasios Zovoilis

#### Alberta Prion Research Institute (201900003)

• Majid H Mohajerani
• Athanasios Zovoilis

#### Natural Sciences and Engineering Research Council of Canada (RGPIN-2018–05955)

• Athanasios Zovoilis

#### Genome Canada (BioNet Alberta grant)

• Athanasios Zovoilis

#### Compute Canada (Resource Allocation Grant)

• Athanasios Zovoilis

• Athanasios Zovoilis

• Athanasios Zovoilis

• Yubo Cheng

#### Government of Alberta (AMR One Health grant)

• Luke Saville

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

### Acknowledgements

This work has been supported by an Explorations Grant # 201700011 to AZ and MM from Alberta Innovates and the Alberta Prion Research Institute, a Grant # 201900003 to AZ and MM from the Alzheimer Society of Alberta and Northwest Territories and the Alberta Prion Research Institute, a Discovery Grant # RGPIN-2018–05955 to AZ from NSERC, the BioNet Alberta grant to AZ from Genome Canada and a Compute Canada Resource Allocation Grant to AZ. AZ is supported by the Canada Research Chairs Program and the Canada Foundation for Innovation and is a former EMBO and DFG long-term fellow. YC is supported by an Alberta Innovates (AITF) fellowship. LS is supported by the AMR One Health grant by the Government of Alberta. We are grateful to Dr. Angeliki Pantazi for extensively reviewing, editing and commenting on the manuscript. We are grateful to the Patel Lab (Trushar Patel, Darren Gemmill) and Wieden Lab (Justin Vigar, Hans Joachim Wieden) at the University of Lethbridge for providing us with the RNA controls #1 and #2.

### Ethics

Animal experimentation: All experimental procedures were approved by the institutional animal care committee protocol number1404 and performed in accordance with the standards set out by the Canadian Council for Animal Care.

### Senior Editor

1. James L Manley, Columbia University, United States

### Reviewing Editor

1. Joaquín M Espinosa, University of Colorado Anschutz Medical Campus, United States

### Reviewer

1. Leo Kurian, Center for Molecular Medicine Cologne/ CECAD; Cologne Cluster of Excellence in Cellular Stress Responses in Ageing-associated Diseases

### Publication history

2. Accepted: November 15, 2020
3. Accepted Manuscript published: November 16, 2020 (version 1)
4. Version of Record published: December 4, 2020 (version 2)

? 2020, Cheng et al.

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### Open citations (links to open the citations from this article in various online reference manager services)

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All organisms on Earth are exposed to low doses of natural radioactivity but some habitats are more radioactive than others. Yet, documenting the influence of natural radioactivity on the evolution of biodiversity is challenging. Here, we addressed whether organisms living in naturally more radioactive habitats accumulate more mutations across generations using 14 species of waterlice living in subterranean habitats with contrasted levels of radioactivity. We found that the mitochondrial and nuclear mutation rates across a waterlouse species’ genome increased on average by 60% and 30%, respectively, when radioactivity increased by a factor of three. We also found a positive correlation between the level of radioactivity and the probability of G to T (and complementary C to A) mutations, a hallmark of oxidative stress. We conclude that even low doses of natural bedrock radioactivity influence the mutation rate possibly through the accumulation of oxidative damage, in particular in the mitochondrial genome.

1. Evolutionary Biology
2. Genetics and Genomics

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Analysis of the smallest known arthropod genome reveals a mechanism for genome reduction that appears to be driven by a specialized ecological interaction with plants.