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Human Rnase L Tunes Gene Expression by Selectively Destabilizing the Microrna-Regulated Transcriptome

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Human Rnase L Tunes Gene Expression by Selectively Destabilizing the Microrna-Regulated Transcriptome Human RNase L tunes gene expression by selectively destabilizing the microRNA-regulated transcriptome Sneha Ratha, Jesse Donovana, Gena Whitneya, Alisha Chitrakara, Wei Wanga,b, and Alexei Korennykha,1 aDepartment of Molecular Biology, Princeton University, Princeton, NJ 08544; and bSequencing Core Facility, Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544 Edited by Jonathan S. Weissman, University of California, San Francisco, Howard Hughes Medical Institute, and California Institute for Quantitative Biosciences, San Francisco, CA, and approved November 11, 2015 (received for review July 2, 2015) Double-stranded RNA (dsRNA) activates the innate immune system mechanism is not documented. To understand mammalian gene of mammalian cells and triggers intracellular RNA decay by the regulation by RNase L, we mapped RNase L-dependent decay pseudokinase and endoribonuclease RNase L. RNase L protects from (RLDD) in human cells. We show that RNase L does not cleave pathogens and regulates cell growth and differentiation by desta- all cellular transcripts and identify biologically related groups of bilizing largely unknown mammalian RNA targets. We developed RLDD targets and nontargets. an approach for transcriptome-wide profiling of RNase L activity in human cells and identified hundreds of direct RNA targets and Results nontargets. We show that this RNase L-dependent decay selectively Direct Cleavage of Human Transcripts by RNase L. To observe affects transcripts regulated by microRNA (miR)-17/miR-29/miR-200 cleavage of human transcripts by RNase L and minimize off- and other miRs that function as suppressors of mammalian cell pathway effects from RNase L-independent decay and tran- adhesion and proliferation. RNase L mimics the effects of these miRs scription, we used HeLa S10 extracts. This approach removes and acts as a suppressor of proliferation and adhesion in mamma- the nucleus and the plasma membrane and permits selective lian cells. Our data suggest that RNase L-dependent decay serves to and rapid RNase L activation. We exposed the human tran- scriptome to activated RNase L using two complementary establish an antiproliferative state via destabilization of the miR- – regulated transcriptome. techniques and used either synthetic 2 5A to activate endoge- nous RNase L (Fig. 1A) or crystallization-purity recombinant human RNase L premixed with 2–5A. Both techniques activate RNase L | microRNA | miR-200 | adhesion | dsRNA RNase L and produce the characteristic pattern of 28S rRNA cleavage (Fig. 1B). + Nase L is a mammalian endoribonuclease regulated by the We analyzed the resulting RNA samples using poly-A RNA- Raction of dsRNA and IFNs α/β/λ, which induce the intra- seq, which captures the poly-A tails and reveals cleavage by the cellular synthesis of a specific RNase L activator, 2–5A (1). RNA loss of 5′-terminal reads (Fig. 1C). Incubation of untreated S10 cleavage is thought to account for all biological functions of the extracts had no considerable effects on the mRNA levels (Fig. RNase L·2–5A complex, including innate immunity during the S1A), whereas addition of 2–5A or the RNase L·2–5A complex IFN response (2, 3), and regulation of cell cycle (4), proliferation produced a broad response (Fig. S1 B and C and Dataset S1). (5), adipocyte differentiation (6), and apoptosis (7). RNase L We identified transcripts that exhibited a strong loss of reads inhibits translation by site-specific cleavage of 18S and 28S (targets) as well as transcripts that remained intact (nontargets; C – rRNA (8) and activates transcription and the inflammasome Fig. 1 ). The levels of multiple mRNAs fell by 100 1,000-fold B “ ” NLRP3 by releasing signaling RNA fragments (2, 9, 10). These when most of the 28S rRNA was still uncleaved (Fig. 1 , 2 ). mechanisms complement or operate in parallel with post- Based on this quantification, the ribosomes are not the preferred transcriptional gene control via regulated decay of some mRNAs, targets of RNase L and mRNA cleavage may be substantial even including myogenic regulatory factor MyoD (11), components of IFN signaling ISG43 and ISG15 (12), translation-inhibiting Significance kinase PKR (13), cathepsin E gastric protease (3), 3′-UTR– binding protein HuR (4), as well as ribosomal and mitochondrial The mammalian innate immune system recognizes double- protein-encoding mRNAs (14–16). Although the repression of stranded RNA (dsRNA) as a signature of infections and cell these transcripts depends on RNase L, it remains unknown damage. Cells exposed to dsRNA release interferons to activate how many of them are cleaved physically and how many are down- protective programs in surrounding tissues. One of these pro- regulated indirectly—for example, via transcription. tective programs triggers regulated decay of intracellular RNA by A recent RNA-sequencing (RNA-seq) study described some the pseudokinase/endoribonuclease RNase L. Here we map the of the direct targets of RNase L (8). Cleavages were reported pathway of this RNA decay transcriptome-wide and identify for 18S rRNA and U6 snRNA, however the experiment was groups of selectively destabilized human messenger RNAs. We designed to detect predominantly ribosomal reads and the direct show that RNA decay by RNase L has an important role in ho- impact of RNase L on mRNAs was not defined. Structural and meostasis and serves as a suppressor of cell adhesion. Our work biochemical studies found that RNase L cleaves RNA at the defines the targets of RNase L and clarifies the role of the dsRNA- consensus sequence UN^N ( N = A, U, G, or C;^is the cleavage activated messenger RNA decay in the interferon response. location) (17–19). The UN^N motifs are abundant in all mam- malian RNAs, suggesting that RNase L may degrade every Author contributions: S.R., J.D., and A.K. designed research; S.R., J.D., G.W., A.C., and W.W. mRNA it encounters, which surprisingly contrasts regulation of performed research; A.C. contributed new reagents/analytic tools; A.K. supervised work; S.R., RNase L by the highly specific stimuli dsRNA, IFNs, and 2–5A. J.D., W.W., and A.K. analyzed data; and S.R. and A.K. wrote the paper. Mammalian cells contain a transmembrane RNase L homolog, The authors declare no conflict of interest. a kinase/RNase Ire1, which drives the unfolded protein response This article is a PNAS Direct Submission. and regulated Ire1-dependent decay (RIDD) (18, 20). The Data deposition: The data reported in this paper have been deposited in the Gene Ex- cleavage consensus sequence of Ire1 (UG^C) is similarly relaxed, pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE75530). however Ire1 targets only specific mRNAs. The specificity is 1To whom correspondence should be addressed. Email: [email protected]. achieved by colocalization with cognate mRNAs at the ER mem- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. brane (21). For the cytosolic enzyme RNase L, such a specificity 1073/pnas.1513034112/-/DCSupplemental. 15916–15921 | PNAS | December 29, 2015 | vol. 112 | no. 52 www.pnas.org/cgi/doi/10.1073/pnas.1513034112 Downloaded by guest on September 28, 2021 A BC Dreads treated / reads control GENE COVERED 1 2 Belong to 2,000 most cleaved S10 cytosolic extract naїve AAAAAAAAAAAAAAAA targets // HDAC5 + 0.0.7777 0.0.2121 no AAAAAAAA reads lost ELAVL1 (HuR) + 0.0.4949 0.39 no non-targets +2-5A // STAT3 + 0.50.599 0.0.4141 no / / EIF2AK2 (PKR) + 0.80 0.59 no endogenous recombinant 5’ KLF13 3’ RNase L RNase L DDIT3 + 00.88.88 0.80 no +2-5A +2-5A + naїve ZFP36 (TTP) 11.43.43 11.05.05 no 1 hour 5 seconds ISG15 + 11.93.93 44.79.79 no CTSE - - - - 1 2 +2-5A MYOD1 - - - - AAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAA AAAAAAAAA AAAAAAAA MYOG - - - - 5’ RPL8 3’ AEBP1 - - - - USP18 (ISG43) - - - - Poly-A+ RNA sequencing EF1 2 1 2 GH 4 4 3 3 2 2 1 1 0 0 5’ 3’ 5’UTR 3’UTR Fig. 1. RNA-seq analysis of mRNA cleavage by RNase L in HeLa extracts. (A) Two RNA-seq approaches for detection of direct mRNA cleavage by RNase L. (B) Cleavage of rRNA. Samples 1 and 2 were used for RNA-seq; the numbering is as in A.(C) RNA-seq traces for a target (KLF13) and a nontarget (RPL8). (D) Cleavage properties of previously reported RNase L targets. (E) Frequency of UU+UA dinucleotides as a function of RNase L sensitivity. (F) Running average frequency of motifs as a function of RNase L sensitivity or (G) mRNA length. (H) Distribution of UAUAU sites along mRNA length in targets and nontargets of RNase L. when 28S rRNA is visibly intact. Seven previously reported RNase L Selects MicroRNA-Regulated mRNAs. By combining the data mRNA targets of RNase L (22) were abundant sufficiently for from both RNA-seq strategies, we assembled the lists of RNase L detection in our samples. Five were cleaved weakly, whereas two targets (S10+ signatures) and nontargets (S10– signatures) (Fig. (TTP and ISG15) were resistant and belonged to nontargets 2A and Dataset S1). These S10 signatures provide the reference (Fig. 1D). None belong to 2,000 most strongly cleaved transcripts for evaluation of mechanistic RNase L activity in high-throughput (Fig. 1D and Dataset S1). datasets. To this end, we used a probability-based approach that RNase L cleaves the UN^N pattern but prefers UU^N and uses signed P′ calculations (SI Methods). This method shows UA^Nsites in model substrates (17, 23). The UU/UA preference positive peaks in the regions of data that have enrichments for is recapitulated in our RNA-seq data (Fig. 1E). We extended this signature genes, negative peaks where the signature genes are analysis to other sequences and calculated running average counts depleted (Fig.
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