key: cord-0800572-66xb2ln2
authors: Burke, James M.; Ripin, Nina; Ferretti, Max B.; St Clair, Laura A.; Worden-Sapper, Emma R.; Sawyer, Sara L.; Perera, Rushika; Lynch, Kristen W.; Parker, Roy
title: RNase L-mediated RNA decay alters 3’ end formation and splicing of host mRNAs
date: 2022-01-28
journal: bioRxiv
DOI: 10.1101/2022.01.28.478180
sha: ab40a0206e8130d991448a6f48a63512ec6b82c5
doc_id: 800572
cord_uid: 66xb2ln2

The antiviral endoribonuclease, RNase L, is a vital component of the mammalian innate immune response that destroys host and viral RNA to reduce viral gene expression. Herein, we show that a consequence of RNase L-mediated decay of cytoplasmic host RNAs is the widespread re-localization of RNA-binding proteins (RBPs) from the cytoplasm to the nucleus, due to the presence of nuclear RNA. Concurrently, we observe global alterations to host RNA processing in the nucleus, including alterations of splicing and 3’ end formation, with the latter leading to downstream of gene (DoG) transcripts. While affecting many host mRNAs, these alterations are pronounced in mRNAs encoding type I and type III interferons and coincide with the retention of their mRNAs in the nucleus. Similar RNA processing defects also occur during infection with either dengue virus or SARS-CoV-2 when RNase L is activated. These findings reveal that the distribution of RBPs between the nucleus and cytosol is fundamentally dictated by the availability of RNA in each compartment and thus viral infections that trigger cytoplasmic RNA degradation alter RNA processing due to the nuclear influx of RNA binding proteins.

Herein, we demonstrate that RNase L-mediated degradation of host mRNAs primarily occurs in (SARS-CoV-2) infection. These findings show that RNase L-mediated mRNA decay alters the balance of 74 RNA-binding protein subcellular localization, host RNA processing events, and antiviral gene expression.

Since RBPs can also regulate transcription (Xiao et al., 2019) , this work strongly implies that widespread 76 RNA degradation in the cytosol will also lead to changes in transcription of multiple genes. One mechanism by which RNase L could alter the localization of RBPs between the nucleus and 97 cytoplasm is differential RNA degradation between the cytoplasm and nucleus, whereby higher RNA decay 98 in the cytoplasm relative to the nucleus would lead to disassociation of RBPs from cytoplasmic RNA more 99 rapidly than from nuclear RNA. To assess this, we quantified poly(A)+ RNA in the cytoplasm and nucleus 100 of WT and RL-KO cells lipofected with or without post-poly(I:C) ( Fig. 2A) . We also stained cells for

While previous studies have shown that RNase L can localize to both the cytoplasm and nucleus 113 (Bayard and Gabrion, 1993 ; Al-Ahmadi et al., 2009), we observed that endogenous RNase L is almost 114 exclusively localized to the cytoplasm in A549 cells via cellular fractionation followed by immunoblot 115 analysis (Fig. 2C) . Combined, these data indicate that RNase L-mediated RNA decay primarily occurs in 116 the cytoplasm.

Nuclear influx of RBPs is dependent on nuclear RNA 119 120

In principle, RNase L activation could alter RBP localization in two manners. First, RBPs may be 121 subject to post-translational modifications, indirectly promoted by RNase L activation, that increase their 122 nuclear accumulation. Alternatively, free RBPs may shuttle between the nucleus and cytosol faster that 123 RBPs bound to RNA, and therefore the degradation of bulk cytoplasmic RNA would allow RBPs to shuttle 124 to the nucleus, where binding to nuclear RNA would retain the RBPs in the nucleus. A prediction of this 125 latter model is that the accumulation of nuclear RBPs will be dependent on the presence of a pool of nuclear 126 RNA to bind the RBPs and increase their dwell time in the nucleus (Fig. 2D ).

To test whether nuclear RNA is required for PABP accumulation in the nucleus, we assayed PABP 129 localization when nuclear RNAs are degraded concurrently with cytoplasmic RNAs in response to RNase 130 L activation (Fig 2D) . To do this, we used A549 cells that constitutively express RNase L tagged with a 

These data demonstrate that RNase L activation increases the nuclear localization of several RBPs 148 by increasing the relative number of RBP-binding sites in the nucleus relative to the cytoplasm as a result 149 of degradation of cytoplasmic but not nuclear mRNAs. 

This analysis identified 140 splicing events, across 136 genes, that showed differential splicing 166 either due to poly(I:C) treatment in the WT cells, or were different between the WT and RL KO cell lines 167 post-poly(I:C). These changes are calculated as the difference in PSI between two conditions, the ∆PSI.

The changes in splicing in both cases were generally correlated, which indicates that the majority of changes 169 in alternative splicing observed are due to activation of RNase L ( Figure 3A ). Splicing alteration in 60 170 genes were statistically significant under both comparisons ( Figure 3B ). Strikingly, 14 of these 60 genes 171 encode RBPs involved in pre-mRNA splicing. This is notable since many RBPs autoregulate their own 

To validate the analysis of RNA-Seq data, we prepared RNA from mock and poly(I:C) treated WT poly(I:C) treatment that was dependent on RNase L ( Figure 3C ). This demonstrates that RNase L activation 179 can lead to changes in alternative splicing patterns.

Another alteration of splicing can be overall decreased splicing rates and the increased retention of 

We note that for upregulated genes, a shift towards higher intron/exon ratio is visible in wild-type 194 cells compared to RL-KO cells, consistent with an increase in intron retention. However, an additional shift 195 is also visible in both unstressed conditions (Fig. S2C ). This is caused by multiple factors. First, a few genes 196 showed high intron retention in RL-KO cells even without stress. Second, transcriptional read-through from 197 upstream genes (see below), causes increased ratios. The majority of such transcripts is filtered out by initial 198 filtering steps (material and methods), however, especially for upregulated genes, some transcripts remain 199 and can cause false positive increased ratios. Third, wrong gene annotations (e.g. an exon with increased 200 reads is counted within intronic region), can make increase in ratio for upregulated genes more pronounced.

And lastly, despite an overall increase in IR in WT cells upon poly(I:C) (Fig. S1F ), the intron/exon ratio 202 decreases due to the increased expression and higher exon counts over the intron counts, especially for 203 genes with short introns and large exons.

These data argue that RNase L activation also decreases the efficiency of intron removal, which is 206 supported by smFISH data of selected targets (see below).

We also observed that RNase L activation perturbs transcription termination. Specifically, we 249 or IFNL1 expression in response to poly(I:C), as determined by co-smFISH for the CDS regions of these 250 genes ( Fig. S4A ,B,C,D,E), we observed abundant and disseminated DoG smFISH foci (Fig. 5B ,C,D). We 251 note that we did not observe abundant smFISH foci targeting the DoG-2 region, which is further 252 downstream of the DoG 1 region of IFNB in WT cells (Fig S5A) . This correlates with lower reads mapping 253 to this region and consistent with lower abundance of these transcripts as assessed by RNA-seq. We did 254 observe staining for the DoG-2 region of IFNL1, but note that it was predominantly localized to the sites 255 of transcription (Fig. S5B ). 277 that have exported IFNB mRNAs that only stain for the CDS region to the cytosol (Fig. 6A,B ; red arrows).

Second, in WT cells with abundant and disseminated IFNB1-DoG foci, the DoG foci co-localize with 282 IFNB1-CDS foci that are retained in the nucleus ( Fig. 6A ; red arrows). Third, IFNB1-CDS foci located in 283 the cytoplasm only contain DoGs in very rare cases ( Fig. 6A ; red arrows). Lastly, we observed that the 284 abundance of DoG foci positively correlates with the ratio (nuclear/total) of IFNB-CDS or the absolute 285 number of nuclear IFNB-CDS foci (Fig. 6C,D) . We observed similar effects eight hours post-transfection 286 with poly (I:C) (Fig. S6A ,B,C,D,E). These data argue that IFNB1 RNA transcripts containing the DoG 287 RNA are not exported to the cytoplasm and argue that DoG formation contributes to the inhibition of export 288 of IFNB mRNAs.

Our data suggests that additional mechanisms can inhibit mRNA export. For IFNB mRNA, we 291 also observed cells in which most nuclear-retained IFNB1-CDS RNAs did not contain IFNB-DoG RNA 292 ( Fig. 6A , yellow arrow). The accumulation of IFNB mRNAs in the nucleus that do not hybridize to DoG 293 probes suggests two possibilities. First, there could be a mechanism independent of DoG transcriptional 294 read-through that inhibits IFNB1 mRNA export, which is supported by our analysis of IFNL1 mRNA (see 295 below). However, we cannot rule out the formal possibility that all the nuclear retained IFNB RNAs could 296 contain DoGs, but with some being too short to hybridize to the DoG probes. However, since the vast 297 majority of RNA-seq reads end at the normal 3' end of IFNB mRNAs (Fig. 5A) , we consider this possibility 298 unlikely.

The examination of IFNL1 mRNAs provides additional evidence for a block to mRNA export that 301 is independent of RNA processing defects. Specifically, we observed that most of the nuclear-retained 302 IFNL1 mRNA did not hybridize to smFISH probes for IFN1L introns or DoGs and were much more 303 abundant than the DoG and intron foci (Fig. 6E ,F,G). We note that we did observe that IFN1L mRNAs 304 that contained DoGs or intron sequences were mostly nuclear-retained (Fig. 6E,H) , consistent with defects 305 in RNA processing limiting RNA export.

Taken together, these observations demonstrate the defects in RNA processing can contribute to 308 nuclear retention of mRNAs after RNase L activation, but also provide evidence for a block to mRNA 309 export independent of DoG-RNA inclusion and intron retention. 

CoV-2 infection was higher in WT but not RL-KO cells (Fig. 7 D,E) . Notably, IFNB1 transcripts containing 329 DoG RNAs were largely localized in the nucleus (Fig. 7D) . Fig. 8A and Fig. S7B ). However, we observed many DENV2-infected WT cells assembled RLBs, whereas 335 many DENV2-infected RL-KO cells assembled SGs (Fig. 8A ). This allowed us to identify cells that 336 activated the dsRNA immune response. We then calculated the nuclear to cytoplasmic PABP in DENV-337 infected WT cells that activated RNase L (RLB+) or RL-KO cells that activated PKR (SG+) in comparison 338 to cells that did not activate dsRNA response (RL-for WT cells or SG-for RL-KO cells). These analyses 339 revealed a substantial increase in nuclear PABP specifically in DENV2-infected WT cells that activated 340 RNase L (Fig. 8B,C) .

Examination of RNA processing defects in DENV-infected cells via smFISH revealed that RNase 343 L-dependent RNA processing alterations occurred during these infections. Specifically, we observed that Taken together, the analysis of IFN mRNAs documents that RNase L activation either due to 348 poly(I:C) transfection or viral infection triggers the accumulation of RBPs in the nucleus and affects nuclear 349 RNA processing of antiviral mRNAs, with a stronger effect on transcriptional termination leading to the 350 production of DoGs for these mRNAs.

Several observations support that RNase L-mediated RNA decay results in re-localization of RBPs 354 from the cytoplasm to the nucleus, which in turn alters nuclear RNA processing (Fig. 9) . First, we observed 355 that several RBPs concentrate in the nucleus following RNase L activation (Fig. 1A,B) . Second, RNase L 356 and RNase L-mediated RNA decay are localized the cytoplasm ( Fig. 2A,B,C) , sparing nuclear RNA from 357 degradation. Moreover, intact nuclear RNA is required for the accumulation of RBPs to the nucleus,

suggesting RBPs associate with nuclear RNA upon influx into the nucleus (Fig. 2E,F,G) . Third, our RNA- RBPs. Indeed, a number of RBPs known to act in mRNA processing are themselves differentially spliced 378 upon exposure to poly(I:C) in an RNase L-dependent manner (Fig 3) . Interestingly, some RBPs are less 379 affected by cytosolic RNA decay with respect to their localization. For example, G3BP1 shows only a small 380 increase in nuclear accumulation following RNase L activation (Fig. 1B,C) . We suspect that this is due to 381 their association with protein substrates that regulate their localization, or a slower intrinsic rate of protein 382 import into the nucleus. 

Our data establish that RNase L activation promotes the formation of DoG transcription read-throughout the nucleus (Fig. 6A,B) . Moreover, the IFNB1 transcripts with DoG RNA were almost 419 exclusively localized to the nucleus, whereas IFNB1 transcripts in the same cells without the DoG RNA 420 were localized to the cytoplasm. These observations suggest that the DoG RNA on IFNB1 transcripts 421 inhibits their mRNA export, even after their release from the site of transcription. The inclusion of 422 downstream elements on the IFNB1 mRNA transcript may present a new RNase L-dependent regulatory 423 mechanism, which will be a focus of future work.

While much lower in abundance and more localized to the IFNB1 transcription site in comparison 426 to WT cells, the IFNB1 DoG also formed in RL-KO cells (Fig. 5B ). This indicates that IFNB1 DoG 427 formation is a normal aspect of IFNB1 gene induction. However, unlike IFNB1, we did not observe any 428 IFNL1 DoG RNA in RL-KO cells. Thus, DoG transcriptional read-through is differential with respect to 429 different dsRNA-induced genes. Understanding this difference may reveal key aspects for transcriptional 430 and RNA processing regulatory mechanisms of these genes.

The RNA processing defects promoted by RNase L activation, such as DoG read-through 

Future work will examine these potential functions and address the specific mechanism by which RNase L 440 activation alters nuclear RNA processing and transcription. 

Intron retention analysis was performed on all RNAs with the Deseq2 baseMean cutoff > 30, to 500 remove RNAs with too low counts. Intron/exon ratios were derived by diving the TPM normalized intron 501 counts by TPM normalized exon counts. Intron/exon ratios > 8.5 were a result from "towers" in intronic 502 regions, counts in non-annotated regions, noise due to low counts or read-through from an upstream gene.

Therefore, all intron/exon ratios >8.5 were removed from analysis.

DoG formation was estimated similarly, by generating TPM normalized counts over the first 5000 505 pb following the exon annotation. These TPM normalized DoG1-5000 counts were divided by the TPM 506 normalized exon counts to derive and a DoG/exon ratio. For the same reasons as describe for intron/exon 507 ratios, DoG/exon ratios > 5 were removed from analysis. Moreover, small RNAs (exon size < 300bp) were 508 removed from analysis.

In our ratio analysis, genes were not filtered for "clean" genes, genes that have counts due to 

However, many genes that were not expression but showed read-through transcription from up-coming 512 gene were filtered out by the intron/exon ratios >15 and DoG/exon ratios > 5 step. We do anticipate false-513 positives left in our analysis, especially in the upregulated group.

Figures were made using ggplot2/3.3.3.

For the splicing analysis, raw fastq reads were trimmed to remove adapters and low quality reads Pol II read-through Association with nuclear RNA Figure 9 nucleus

RNase L downmodulation of the 569

RNA-binding protein, HuR, and cellular growth

Ribosomal protein mRNAs are 572 primary targets of regulation in RNase-L-induced senescence

2',5'-Oligoadenylate-dependent RNAse located in nuclei: biochemical 579 characterization and subcellular distribution of the nuclease in human and murine cells

RNase L limits host and viral protein synthesis via 583 inhibition of mRNA export. Sci Adv

RNase L promotes the formation of unique ribonucleoprotein 586 granules distinct from stress granules

RNase L Reprograms Translation by Widespread mRNA 589

Turnover Escaped by Antiviral mRNAs

SARS-CoV-2 infection triggers widespread host mRNA 592 decay leading to an mRNA export block

DUSP11 -An RNA phosphatase that regulates host and viral non-coding RNAs 595 in mammalian cells

BBMap: A Fast, Accurate, Splice-Aware Aligner

A specific subset of SR proteins shuttles continuously between the 600 nucleus and the cytoplasm

RNA is required for the 603 maintenance of multiple cytoplasmic and nuclear membrane-less organelles

RNase L targets distinct sites in influenza A virus RNAs

609 STAR: ultrafast universal RNA-seq aligner

Rapid RNase L-driven arrest of protein synthesis 612 in the dsRNA response without degradation of translation machinery

Multiple Posttranscriptional 618

Strategies To Regulate the Herpes Simplex Virus 1 vhs Endoribonuclease

Interferon action: RNA cleavage pattern of a (2'-5')oligoadenylate-621 -dependent endonuclease

Enzymatic production of single-molecule FISH and RNA capture 624 probes

Changes in mRNA abundance drive 627 shuttling of RNA binding proteins, linking cytoplasmic RNA degradation to transcription

The exonuclease and host shutoff functions of the SOX protein of 630

Kaposi's sarcoma-associated herpesvirus are genetically separable

A phylogenetically conserved 634 RNA structure in the poliovirus open reading frame inhibits the antiviral endoribonuclease RNase 635

GTFtools: a Python package for analyzing various modes of gene models

RNA-binding proteins TIA-1 and TIAR link the 640 phosphorylation of eIF-2 alpha to the assembly of mammalian stress granules

The Stress Granule Transcriptome 643 Reveals Principles of mRNA Accumulation in Stress Granules

The oligoadenylate synthetase 647 family: an ancient protein family with multiple antiviral activities

Nuclear import of cytoplasmic poly(A) binding protein restricts gene 650 expression via hyperadenylation and nuclear retention of mRNA

The Subread aligner: fast, accurate and scalable read mapping by seed-and-654 vote

Genome Project Data Processing Subgroup. The Sequence Alignment/Map format and 658

RNase L is dependent on OAS3 expression during infection with diverse human viruses

mediated innate immune responses in respiratory epithelial-derived cells and cardiomyocytes

Diverse roles of host RNA binding proteins in RNA virus replication

A model system for activation-induced alternative splicing of CD45 pre-mRNA in 675 T cells implicates protein kinase C and Ras

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Auto-regulatory feedback by RNA-binding 686 proteins

Dom34 mediates targeting of exogenous RNA in the antiviral OAS/RNase L 690 pathway

Critical role of an antiviral stress 694 granule containing RIG-I and PKR in viral detection and innate immunity

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The stress granule protein G3BP1 recruits protein kinase R to promote multiple 704 innate immune antiviral responses

Integrative 707 genomics viewer

Hyperosmotic stress 710 alters the RNA polymerase II interactome and induces readthrough transcription despite 711 widespread transcriptional repression

Widespread disruption of host transcription termination in 715 HSV-1 infection

Viral encounters with 2',5'-oligoadenylate synthetase and RNase L during the interferon 718 antiviral response

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DHX36 enhances RIG-I signaling by facilitating PKR-744 mediated antiviral stress granule formation

Quantification of poly(A)+ RNA signal in the nucleus (nuc.) or cytoplasm (cyto.) in WT and RL-KO 762 cells with or without RLBs or SGs, respectively, as represented in (A). (C) Immunoblot analysis of 763

nuclear fraction (n), or cytoplasmic fraction (c) from A549-764 WT cells. (D) Schematic showing RBP re-localization following either cytoplasmic RNase L activation 765 (left) or activation of nuclear-localized RNase L (RL-NLS). (E) Immunoblot for RNase L

and cytoplasmic (c) crude fractions showing nuclear localization of the RNase L-NLS

A)+ RNA in parental A549 cells or A549 that 768 express RNase L-NLS construct following mock or poly(I:C) lipofection. (G) Quantification of PABP and 769 poly(A)+ RNA signal from (F)

RNase L activation results in alterations to alternative splicing

(I:C) treated cells on the x axis. The 140 splicing events shown are significant in 774 at least one comparison. Genes validated in panel C are labeled. (B) List of genes significant in both 775 comparisons from panel A. RBPs known to regulate splicing are shown in bold. (C) Validation of splicing 776 events by low cycle radiolabeled RT-PCR

Bar graphs show quantification of 4 biological replicates. * is p < 0.05, ** is p < 0.01, *** is p < 0.001, (A) Distribution of intron/exon ratios of host RNAs in WT and RL-KO cells following mock or poly(I:C)

Distribution of DoG1-5000bp/exon ratios of host RNAs in WT and RL-KO cells following 783 mock or poly(I:C) lipofection. (C) IGV traces mapping to an example gene. Intron retention and DoG 784 formation is

RNase L promotes DoG transcriptional read-through and intron retention in type I and type 787 III interferon RNAs

Below shows the regions targeted by smFISH probes. (C) smFISH for IFNB1-DoG 790 sixteen hours post-lipofection of poly(I:C) in WT and RL-KO cells. The cells that induced IFNB1, as 791 determined by smFISH for the CDS of IFNB1 (Fig. S4A,B), are demarcated by a white line. IFNB1 DoG 792 smFISH foci are quantified in WT an RL-KO cells in the graph below

IFNL1-DoG-1 RNA sixteen hours post-lipofection of poly(I:C) or (E) IFNL1-intron RNA twelve hours 794 post-lipofection of poly(I:C). Staining and quantification of IFNL1 CDS is shown in Fig

DoG RNA included on interferon-encoding mRNAs correlates with their nuclear retention

Co-smFISH for the CDS and DoG-1 regions of IFNB1 sixteen hours post-lipofection of poly(I:C). (B)

Scatter plot of the ratio (nucleus/cytoplasm) of IFNB1-CDS foci 800 (x-axis) and nuclear IFNB1-DoG foci (y-axis) show positive correlation between DoG RNA and nuclear 801 retention. (D) Scatter plots of the quantity of nuclear IFNB1-DoG foci (y-axis) and the quantity of nuclear 802 IFNB1-CDS foci shows IFNB1 DoG RNA increase as the absolute number of IFNB1 CDS smFISH in the 803 nucleus increases. (E) Co-smFISH for the CDS and DoG-1 regions of IFNL1 sixteen hours post-lipofection 804 of poly(I:C). (F and G) Quantification of (F) nuclear IFNL1-DoG-1 foci or (G) IFNL1-CDS foci as

RNase L promotes nuclear RBP influx and DoG transcriptional read-through of IFNB1 during 809 SARS-CoV-2 infection

A) Immunofluorescence assay for PABP in WT ACE2 and RL-KO ACE2 A549 cells forty-eight hours post-811 infection with SARS-CoV-2 (MOI=5) or mock-infected WT cells. To identify infected cells

SARS-CoV-2 ORF1b mRNA was performed. (B) Scatter plot of mean intensity values for PABP staining