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.