The mRNA-Bound Proteome and Its Global Occupancy Profile on Protein-Coding Transcripts

SummaryProtein-RNA interactions are fundamental to core biological processes, such as mRNA splicing, localization, degradation, and translation. We developed a photoreactive nucleotide-enhanced UV crosslinking and oligo(dT) purification approach to identify the mRNA-bound proteome using quantitative proteomics and to display the protein occupancy on mRNA transcripts by next-generation sequencing. Application to a human embryonic kidney cell line identified close to 800 proteins. To our knowledge, nearly one-third were not previously annotated as RNA binding, and about 15% were not predictable by computational methods to interact with RNA. Protein occupancy profiling provides a transcriptome-wide catalog of potential cis-regulatory regions on mammalian mRNAs and showed that large stretches in 3′ UTRs can be contacted by the mRNA-bound proteome, with numerous putative binding sites in regions harboring disease-associated nucleotide polymorphisms. Our observations indicate the presence of a large number of mRNA binders with diverse molecular functions participating in combinatorial posttranscriptional gene-expression networks.

Graphical AbstractFigure optionsDownload high-quality image (224 K)Download as PowerPoint slideHighlights
► System-wide characterization of protein-mRNA interactome
► Identification of 797 proteins interacting with mRNA
► Characterization of binding sites and targets of five mRNA binders by PAR-CLIP
► Protein occupancy profiling globally mapped potential cis-regulatory mRNA regions