High-resolution profiling of protein occupancy on polyadenylated RNA transcripts

• Published in: Methods (San Diego, Calif.) Vol. 65; no. 3; pp. 302 - 309
• Main Authors: Munschauer, Mathias, Schueler, Markus, Dieterich, Christoph, Landthaler, Markus
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2014
• Subjects: Animals; Binding Sites; Cell Line, Tumor; CLIP; Embryonic Stem Cells - chemistry; Embryonic Stem Cells - metabolism; Gene Expression Profiling - methods; Gene Expression Regulation; Gene Library; Human Umbilical Vein Endothelial Cells - chemistry; Human Umbilical Vein Endothelial Cells - metabolism; Humans; lincRNA; Mice; mRNA; Next-generation sequencing; Non-coding RNA; PAR-CLIP; Protein Binding; Protein occupancy profiling; Protein–RNA interaction; Proteome - chemistry; Proteome - metabolism; RNA, Messenger - chemistry; RNA, Messenger - metabolism; RNA-binding protein; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - metabolism; Transcriptome - genetics; RNA-binding protein; Next-generation sequencing; PAR-CLIP; Protein–RNA interaction; lincRNA; mRNA; Non-coding RNA; Protein occupancy profiling; CLIP
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2013.09.017

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.