Learning the language of post-transcriptional gene regulation

Abstract

A large-scale RNA in vitro selection study systematically identified RNA recognition elements for 205 RNA-binding proteins belonging to families conserved in most eukaryotes.

Figure 1

Overview of in vitro and in vivo methods for RRE determination. (a) SELEX and RNAcompete start with the preparation of a diverse DNA sequence pool, which is in vitro transcribed into RNA. The protein of interest is incubated with the random sequence RNA pool, followed by RBP pulldown and recovery of the bound RNA. In SELEX, high-affinity ligands are enriched by several rounds of reverse transcription, (mutagenic) PCR and selection, before sequencing of the RNA ligands. In RNAcompete, the recovered RNA is directly quantified on a microarray, rather than sequenced, and enrichment for each individual sequence over the initial pool is calculated. Enrichment scores, which directly correlate with the binding affinity of the RNA sequence, are used to derive the RRE, which serve as input for computational prediction of in vivo RNA targets. (b) CLIP-based methods use in vivo crosslinking to covalently link RBPs to their RNA targets by UV light. After cell lysis, limited RNase treatment and immunoprecipitation of the RBP, the crosslinked RNA segments are recovered, converted into cDNA libraries and deep sequenced. CLIP methods directly identify in vivo RNA targets and binding sites, and motif finding algorithms are used to deduce the RRE from the crosslinked RNA sequences. dsDNA, double-stranded DNA; HITS-CLIP, high-throughput sequencing of RNA isolated by CLIP; iCLIP, individual-nucleotide resolution CLIP; PAR-CLIP, photoactivatable-ribonucleoside-enhanced CLIP; ssDNA, single-stranded DNA; XL-RBP, crosslinked RBP.

Figure 2

Summary of human mRBPs characterized by RNAcompete. The left panel shows, categorized by selected domain types, the number of RBPs for which RREs were characterized by RNAcompete (red) or remain to be studied (dark gray). RBD categories are RRM, RRM + other (combination of RRM with KH or zinc finger), KH and other RBDs selected in Ray et al. (zinc finger, PUF, CSD, YTH, S1 and SAM). The right panel shows the fraction of human mRBPs studied by RNAcompete. Of all known/putative mRBPs (n = 717; reviewed by Ascano et al. [1]), 20% were studied by RNAcompete (red, n = 146). Another 18% of mRBPs contain at least one or more of the selected RBDs but remain to be studied (dark gray, n = 127). Sixty-two percent of mRBPs contained RBDs (for example, DSRM, La, PWI, and so on) not studied by RNAcompete (light gray, n = 444).