Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP

Abstract

Summary RNA transcripts are subject to post-transcriptional gene regulation involving hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) expressed in a cell-type dependent fashion. We developed a cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs. The crosslinked sites are revealed by thymidine to cytidine transitions in the cDNAs prepared from immunopurified RNPs of 4-thiouridine-treated cells. We determined the binding sites and regulatory consequences for several intensely studied RBPs and miRNPs, including PUM2, QKI, IGF2BP1-3, AGO/EIF2C1-4 and TNRC6A-C. Our study revealed that these factors bind thousands of sites containing defined sequence motifs and have distinct preferences for exonic versus intronic or coding versus untranslated transcript regions. The precise mapping of binding sites across the transcriptome will be critical to the interpretation of the rapidly emerging data on genetic variation between individuals and how these variations contribute to complex genetic diseases.

Pdcd4 directly binds the coding region of c-myb mRNA and suppresses its translation

“…Our data indicate that Pdcd4 targets a 'Pdcd4-response element' that maps to the coding region of c-myb mRNA. At first glance, it appears unusual that the coding region of an mRNA is the target of a translational regulatory mechanism; however, genome-wide analyses of interactions of regulatory RNA-binding proteins with mRNAs have revealed surprisingly large numbers of interactions that take place in the coding regions of the cognate mRNAs (Hogan et al, 2008;Hafner et al, 2010). Previous studies have also identified specific regulatory proteins that bind to the coding regions of certain mRNAs, such as the thymidylate synthase and dihydrofolate reductase, both of which bind to the coding regions of their own mRNAs (Ercikan-Abali et al, 1997;Lin et al, 2000;Zhang et al, 2010) and the RNA-binding protein HuR, which interacts with the coding region of CD83 RNA (Prechtel et al, 2006).…”

MicroRNAs: An adaptive mechanism in the pancreatic β-cell…and beyond?

“…The issue of combinatorial targeting of genes by multiple miRNAs has not been well studied in vivo however the advent of new methodologies including CRISPR/Cas9 site-directed mutagenesis and crosslinkingimmunoprecipitation (CLIP) protocols can now expedite the identification of biologically relevant cis and trans sequences [15] [16]. In addition to improving our understanding miRNA target recognition, these protocols have the potential of establishing the functional importance of low expressed sequences either by loss of function studies or by identifying robust miRNA : target gene interactions.…”

“…Since these early model studies, the TargetScan algorithm has continued to refine its target predictions and remains among the most recognized in the field [22]. Recent reports have implemented biochemical approaches (crosslinking and immunoprecipitation, CLIP) to identify specific miRNA target sites by isolating and sequencing the 3'UTR region bound by Ago proteins [16]. While these protocols are currently dependent upon large quantities of source tissue material in cell culture, this limitation excludes the possibility of studying human islets however the development of the stem cell differentiation protocols may make the CLIP protocol viable for human β-cells in the near future.…”

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.…”