Events - Daily
|Friday, November 30|
Markus Hafner, Rockefeller U, Comprehensive identification of miRNA target sites and characterization of ribonucleoprotein complexes by PAR-CLIP
Comprehensive identification of miRNA target sites and characterization of ribonucleoprotein complexes by PAR-CLIP
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. To map interaction sites between RNA-binding proteins (RBPs) and their RNA target sites we developed Photoactivatable Ribonucleoside Enhanced Crosslinking and immunoprecipitation (PAR-CLIP), which relies on the incorporation of photoreactive nucleosides into nascent RNA followed by UV-crosslinking of RNA and interacting proteins. Sequencing of crosslinked RNA allows for the large-scale identification of target RNA binding sites and the respective RNA recognition elements (RREs). PAR-CLIP yields characteristic sequence changes upon crosslinking that facilitate the separation of signal from noise. PAR-CLIP provides precise information 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. I will illustrate in detail the application of PAR-CLIP to identify human miRNA target sites on a large-scale, a task previously requiring computational prediction followed by individual validation of target sites. 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 post-transcriptional gene regulation and thereby lead to new strategies for therapeutic treatment of genetic diseases perturbing these processes.
Lewis Thomas Lab 003 · 9:30 a.m.–10:30 a.m.