Dissecting noncoding and pathogen RNA-protein interactomes

Abstract

RNA-protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA-protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA-protein recognition across the diversity of human and pathogen RNAs.

Keywords: RNA–protein interactions; genomics; noncoding RNA; virology.

FIGURE 1.

FAST-iCLIP incorporates experimental improvements and standardized experimental interface to enable iCLIP analysis. (A) Biochemical improvements to the standard iCLIP procedure reduce experimental time by half. (B) A standard interface for analysis iCLIP data increases analysis efficiency and dissects many known sources of RNA transcripts including both the repetitive and nonrepetitive human genome as well as nonhuman genomes. (C) Histogram of the types and number of genes identified by FAST-iCLIP analysis of publically available hnRNP-C iCLIP data. (D) Percentage of all hnRNP-C iCLIP reads mapping to mRNA loci subdivided by functional domain. (E) Average histogram of hnRNP-C iCLIP reads along a normalized mRNA transcript. Each gene's functional regions (5′ UTR, CDS, and 3′ UTR) are binned into 200 units plotted along the same axis. Intronic reads are not visualized in this plot. (F) Logo visualization of the HOMER motif output from all hnRNP-C iCLIP reads with the fraction of iCLIP target regions containing that motif. hnRNP-C crosslink sites and region-shuffle control are shown.

FIGURE 2.

PCBP2 is a major mRNA-binding protein that exhibits distinct binding modes. (A) Histogram of the types and number of genes identified by FAST-iCLIP bound to PCBP2. (B) Percentage of all PCBP2 iCLIP reads mapping to mRNA loci subdivided by functional domain. (C) Logo visualization of the top two HOMER motif output generated from all PCBP2 iCLIP reads. The fraction of target regions with each motif and PCBP2 or region-shuffle results are displayed. (D) Average histogram of PCBP2 iCLIP reads along a normalized mRNA transcript. Each gene's functional regions (5′ UTR, CDS, and 3′ UTR) are binned into 200 units plotted along the same axis. (E) For each functional region in D, DAVID was run to obtain enriched GO terms.

FIGURE 3.

Noncoding and repeat RNA analysis reveals novel PCBP2–RNA interactions. (A,B) Coverage histogram of PCBP2 or hnRNP-C (respectively) iCLIP reads mapping to the 42-kb human rDNA locus. Total reads mapping to the rDNA were used to calculate a fraction of total per RT stop and binding sites are reported as such. The mature sequences of the 18S, 5.8S, and 28S rRNAs are highlighted in gray. (C) PCBP2 iCLIP reads mapping to snoRNA loci were tallied and plotted as percentages mapping to the three snoRNA classes; C/D box, H/ACA box, and scaRNAs (center). Reads mapping to C/D box or H/ACA box snoRNAs were extracted and plotted across a normalized snoRNA transcript of 100 units long and read percentages were calculated as in left and right bar plots, respectively. (D) PCBP2 iCLIP reads mapping to the Y RNA 1 (left) and Y RNA 3 (right) transcripts are plotted as in (A). Secondary structure predictions of each RNA were produced with mFold and nucleotides identified as having PCBP2 iCLIP RT stops are highlighted in red.

FIGURE 4.

Systematic mapping of PCBP2 iCLIP data to the JFH-1 HCV genome. (A) Experimental design to identify in vivo PCBP2 interaction sites across the JFH-1 HCV genome. (B) Genomic structural features within both UTRs with functional annotation. (C) Scatter plot of individual RT stops mapping to the JFH-1 genome comparing the two iCLIP biological replicates. (D,E) Coverage histogram across the 5′ UTR revealing peaks at SL1, across the SL1-SLII junction, and around the start codon (D) and across the 3′ UTR revealing peaks on hairpins involved in the kissing interaction as well as the poly-U/C tract (E). Both coverage histograms include bin size of 5 bp.