Specifying RNA-Binding Regions in Proteins by Peptide Cross-Linking and Affinity Purification

RNA-binding proteins (RBPs) allow
cells to carry out pre-RNA processing
and post-transcriptional regulation of gene expression, and aberrations
in RBP functions have been linked to many diseases, including neurological
disorders and cancer. Human cells encode thousands of RNA-binding
proteins with unique RNA-binding properties. These properties are
regulated through modularity of a large variety of RNA-binding domains,
rendering RNA–protein interactions difficult to study. Recently,
the introduction of proteomics methods has provided novel insights
into RNA-binding proteins at a systems level. However, determining
the exact protein sequence regions that interact with RNA remains
challenging and laborious, especially considering that many RBPs lack
canonical RNA-binding domains. Here we describe a streamlined proteomic
workflow called peptide cross-linking and affinity purification (pCLAP)
that allows rapid characterization of RNA-binding regions in proteins.
pCLAP is based upon the combined use of UV cross-linking and enzymatic
digestion of RNA-bound proteins followed by single-shot mass spectrometric
analysis. To benchmark our method, we identified the binding regions
for polyadenylated RNA-binding proteins in HEK293 cells, allowing
us to map the mRNA interaction regions of more than 1000 RBPs with
very high reproducibility from replicate single-shot analyses. Our
results show specific enrichment of many known RNA-binding regions
on many known RNA-binding proteins, confirming the specificity of
our approach.