Molecular Cell
Volume 46, Issue 5, 8 June 2012, Pages 674-690
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The mRNA-Bound Proteome and Its Global Occupancy Profile on Protein-Coding Transcripts

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Summary

Protein-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.

Highlights

► 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

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These authors contributed equally to this work