Identification of RNA–protein interaction networks using PAR‐CLIP

Abstract

All mRNA molecules are subject to some degree of post-transcriptional gene regulation (PTGR) involving sequence-dependent modulation of splicing, cleavage and polyadenylation, editing, transport, stability, and translation. The recent introduction of deep-sequencing technologies enabled the development of new methods for broadly mapping interaction sites between RNA-binding proteins (RBPs) and their RNA target sites. In this article, we review crosslinking and immunoprecipitation (CLIP) methods adapted for large-scale identification of target RNA-binding sites and the respective RNA recognition elements. CLIP methods have the potential to detect hundreds of thousands of binding sites in single experiments although the separation of signal from noise can be challenging. As a consequence, each CLIP method has developed different strategies to distinguish true targets from background. We focus on photoactivatable ribonucleoside-enhanced CLIP, which relies on the intracellular incorporation of photoactivatable ribonucleoside analogs into nascent transcripts, and yields characteristic sequence changes upon crosslinking that facilitate the separation of signal from noise. The precise knowledge of the position and distribution of binding sites across mature and primary mRNA transcripts allows critical insights into cellular localization and regulatory function of the examined RBP. When coupled with other systems-wide approaches measuring transcript and protein abundance, the generation of high-resolution RBP-binding site maps across the transcriptome will broaden our understanding of PTGR and thereby lead to new strategies for therapeutic treatment of genetic diseases perturbing these processes.

The human RBPome: From genes and proteins to human disease

“…Hence, it becomes important to understand the structural and functional characteristics of RBPs in humans. Increasing interest in RBPs has led to the development of various experimental protocols like SELEX (systematic evolution of ligands by exponential enrichment) [9,10], CLIP [11], PAR-CLIP [12,13], iCLIP [14] and RNA compete [15], to identify the binding specificities of RBPs; thus adding context and dynamics to the regulation of gene expression at post-transcriptional level.…”

CLIP: viewing the RNA world from an RNA-protein interactome perspective

“…The PAR-CLIP protocol employs ribonucleoside analogs, such as 4-thiouridine (4SU) and 6-thioguanosine (6SG), which can be selectively photoactivated at long wavelength UV (>320 nm), to enhance cross-linking between nucleic acids and proteins [11]. 4SU is the preferred option because it has higher cross-linking efficiency [12] and uridines are the most frequent cross-linking sites [13]. The incorporation of 4SU into RNAs results in thymidine (T) to cytidine (C) transitions during the RT reaction, which is a good indicator of binding sites and can be used to subtract non-crosslinking background signals [14].…”

“…Although PAR-CLIP improves cross-linking efficiency by introducing 4SU or 6SG, its performance varies among different RBPs. Some BPS, such as HuR, have an efficiency similar to HITS-CLIP [13]. Searching high efficiency, low side effects cross-linking aids is in need for improving performance of CLIP.…”

Connecting cis-elements and trans-factors with mechanisms of developmental regulation of mRNA translation in meiotic and haploid mammalian spermatogenic cells

“…co-immunoprecipitation and microarray analysis or deep sequencing (reviewed in Kishore et al (2010), Morris et al (2010) and Ascano et al (2012)). Each approach is distinguished by tradeoffs in simplicity, cost, and reliability in distinguishing bona fide targets and false positives, and every approach is capable of producing misleading information.…”

“…It is estimated that the mammalian genome encodes about 400 miRNAs and 600 RBPs recognized by various types of RNA-binding domains (RBDs) (Bartel 2009, Ascano et al 2012. However, the number of RBPs is potentially much greater because the genome encodes an additional w1400 uncharacterized proteins with domains that can bind RNA and/or DNA (Morris et al 2010, Ascano et al 2012. In addition, polyadenylated mRNAs in somatic cells bind several hundred proteins that lack recognizable RBDs (Baltz et al 2012, Castello et al 2012.…”