Human RNase L tunes gene expression by selectively destabilizing the microRNA-regulated transcriptome

Abstract

Double-stranded RNA (dsRNA) activates the innate immune system of mammalian cells and triggers intracellular RNA decay by the pseudokinase and endoribonuclease RNase L. RNase L protects from pathogens and regulates cell growth and differentiation by destabilizing largely unknown mammalian RNA targets. We developed an approach for transcriptome-wide profiling of RNase L activity in human cells and identified hundreds of direct RNA targets and nontargets. We show that this RNase L-dependent decay selectively affects transcripts regulated by microRNA (miR)-17/miR-29/miR-200 and other miRs that function as suppressors of mammalian cell adhesion and proliferation. RNase L mimics the effects of these miRs and acts as a suppressor of proliferation and adhesion in mammalian cells. Our data suggest that RNase L-dependent decay serves to establish an antiproliferative state via destabilization of the miR-regulated transcriptome.

Keywords: RNase L; adhesion; dsRNA; miR-200; microRNA.

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.

Fig. S1.

RNA-seq analysis of HeLa extracts. (A) Abundance of mRNAs measured by RNA-seq of S10 extract incubated for 1 h under the reaction conditions (Methods) versus matching naїve extract. (B) Abundance of mRNAs measured by RNA-seq of S10 extract cleaved by endogenous RNase L versus matching control extract incubated without 2–5A. (C) Abundance of mRNAs measured by RNA-seq of S10 extract treated with recombinant RNase L for 5 s versus naїve extract. (D) Correlation of AUUUA and UAUAU abundance in the transcripts covered by the RNA-seq analysis. (E) Plain count of UAUAU sites in mRNAs from HeLa S10 samples as a function of read loss upon cleavage by RNase L. Note the log10 y axis scale.

Fig. 2.

Gene set enrichment analysis of RLDD. (A) Overlay of mRNA cleavage profiles for endogenous (1) and recombinant (2) RNase L. Numbering is as in Fig. 1A. Common targets down-regulated by ≥threefold are designated as S10+ signatures; the 1,016 most resistant transcripts from sample 1 and transcripts down-regulated by no more than 10% in sample 2 are designated as S10– signatures. (B) Signed P′ profiles for RNA-seq data from samples 1 and 2. (C) GSEA profiling of the RNA-seq data. The 25% (GSEA-recommended) and the 5% (classic) limits are marked with dashed lines. Observed FDR values for each signature are shown in the right column. x axis shows the GSEA normalized enrichment score (NES).

Fig. S2.

Signed P′ and TargetScan analyses of HeLa extracts. (A) Signed P′ profile for data in Fig. 2B, after randomization of the input data. The |log10(P′)| values for a randomized dataset do not exceed the magnitude of ∼1 and lack the characteristic uniform change from left to right observed with nonrandom data (Fig. 2B). (B) Signed P′ analysis of select signatures indentified by GSEA. Nontargets of RNase L are colored blue; targets are colored red. The P′ analysis shows enrichment of the nontargets in the resistant mRNAs (left half of the x axis) and enrichment of the targets in the cleaved mRNAs (right half of the x axis), as expected. (C) MicroRNA enrichment analysis separately for transcripts with short and long 3′UTRs. The analysis describes two subsets derived from the data in Fig. 1A (experiment 1), based on the 3′UTR length. The first subset contains only mRNAs with short 3′UTRs of ≤1,000 nt (5,330 transcripts, ∼50% of the entire dataset). The second subset has the mRNAs with short 3′UTRs selectively removed and contains 5,250 transcripts (also ∼50% of the entire dataset). GSEA shows robust enrichments for microRNA targets in both subsets, indicating that RNase L preferentially targets microRNA-regulated transcripts within pools of mRNAs with comparable 3′UTR sizes.

Fig. S3.

Profiling mRNA targets using RNase L overexpression in live HeLa cells. (A) Western blot analysis of overexpressed WT and H672N RNase L. (B) Cleavage of 28S rRNA in cells overexpressing WT RNase L. The control point mutant H672N shows no rRNA cleavage. Total RNA analysis was conducted on BioAnalyzer with an RNA Nano-Chip. RNase L cleavage products are marked with an asterisk. (C) Signed P′ analysis of S10+ and S10– signatures in RNA-seq samples from HeLa cells overexpressing WT and H672N RNase L. Bottom graph shows running average of log₂[(reads, WT)/(reads, H672N)]. Data were obtained using two biological replicates for each, WT and H672N overexpression. (D) GSEA preranked enrichment analysis conducted as in Fig. 2C.

Fig. S4.

Profiling mRNA targets of RNase L in live HeLa cells using RNAi. (A) Expression of RNase L in HeLa cells transfected with RNAi for RNase L and with scrambled RNA control (scrRNA), monitored by qPCR. (B) BioAnalyzer analysis of 28S rRNA cleavage in cells with RNase L KD. Specific cleavage bands of RNase L are marked with an asterisk. RNase L was activated by transfecting poly-I:C (Methods). (C) Signed P′ analysis of the RNA-seq data. Two biological replicates (lines 5 and 7; lines 6 and 8; Table S1) were used for the analysis. (D) GSEA preranked enrichment analysis of the RNA-seq data, conducted as in Fig. 2C.

Fig. 3.

Profiling RLDD by in situ semipermeabilization in T47D cells. (A) Semipermeabilization procedure. (B) BioAnalyzer analysis of rRNA cleavage by RNase L. Cleavage products are marked with an asterisk. (C) Signed P′ analysis of the RNA-seq data. The ratios of reads in the +2–5A samples versus the corresponding mocks were used for analysis. (D) GSEA profiles for GSEA and RK signature sets. (E) GSEA profiles for TargetScan microRNA signature set. Enrichments are observed for the same groups of microRNAs as in HeLa extracts and in live HeLa cells.

Fig. S5.

RNA-seq traces for select targets (A) and nontargets (B) of RNase L. Data obtained from T47D cells treated with 2–5A for 3 and 9 min were visualized in IGV (hg19) (50). Reads near the 3′ end are unchanged, whereas reads near the 5′ end are lost due to cleavage by RNase L.

Fig. 4.

RNA-seq analysis of WT and RNase L-KO human cells. (A) The 2–5A activates cleavage of 28S rRNA in WT but not RNase L-KO HAP1 cells. (B) Signed P′ profiles for poly-A⁺ and Ribo-Zero RNA-seq data. (C) Running average motif frequency in Ribo-Zero data. (D) GSEA analysis of poly-A⁺ and Ribo-Zero samples. Gene sets that exhibit inverse enrichment compared with poly-A⁺ samples are marked with an ampersand (&). (E) Select well-characterized TFs up-regulated and down-regulated in WT versus KO HAP1 cells in poly-A⁺ data. Poly-A⁺ RNA-seq data represent two biological replicates and Ribo-Zero data represent a single replicate.

Fig. 5.

RNase L is a suppressor of proliferation and adhesion. (A) Scratch wound healing assay for WT and RNase L-KO HAP1 cells. (B) Quantitation of scratch wound healing in HAP1 and MEF cells. (C) Targeted qPCR of select down-regulated and up-regulated transcripts in WT and RNase L-KO HAP1 cells. (D) Time-dependent adhesion of HAP1 and MEF cells. (E) A schematic representation of transcriptional and posttranscriptional gene regulation by RLDD. Error bars show SE from three biological replicates. For wound healing assay, two different edge sections were used for each replicate quantification.

Fig. S6.

Inhibition of proliferation by RNase L overexpression. (A) HeLa cells were transfected with plasmids encoding WT and H672N mutant RNase L for 16 h. The 2–5A was transfected and cells were counted at several time points (Methods). (B) Quantification of the three replicates in A using ImageJ. The Left panel shows the time courses after 2–5A transfection. The Right panel includes the first 16 h of overexpressing WT or H672N RNase L. Error bars show SEs from three biological replicates. (C) Inhibition of MEF cell proliferation by overexpression of human RNase L, WT versus H672N. Experiments and quantifications were conducted as in A and B.

Fig. S7.

Inhibition of adhesion in HeLa and MEF RNase L-KO cells by RNase L overexpression. HeLa and MEF cells were transfected with plasmids encoding WT and H672N mutant RNase L. After 24 h of adhesion, kinetics was measured using three biological replicates (Methods). Error bars show SEs.