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.