High-resolution profiling of protein occupancy on polyadenylated RNA transcripts

Abstract

A key prerequisite to understand how gene regulatory processes are controlled by the interplay of RNA-binding proteins and ribonucleoprotein complexes with RNAs is the generation of comprehensive high-resolution maps of protein-RNA interactions. Recent advances in next-generation sequencing technology accelerated the development of various crosslinking and immunoprecipitation (CLIP) approaches to broadly identify RNA regions contacted by RNA-binding proteins. However these methods only consider single RNA-binding proteins and their contact sites, irrespective of the overall cis-regulatory sequence space contacted by other RNA interacting factors. Here we describe the application of protein occupancy profiling, a novel approach that globally displays the RNA contact sites of the poly(A)+ RNA-bound proteome. Protein occupancy profiling enables the generation of transcriptome-wide maps of protein-RNA interactions on polyadenylated transcripts and narrows the sequence search space for transcript regions involved in cis-regulation of gene expression in response to internal or external stimuli, altered cellular programs or disease.

Keywords: CLIP; Next-generation sequencing; Non-coding RNA; PAR-CLIP; Protein occupancy profiling; Protein–RNA interaction; RNA-binding protein; lincRNA; mRNA.