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

TY - JOUR

T1 - Specifying RNA-Binding Regions in Proteins by Peptide Cross-Linking and Affinity Purification

AU - Mullari, Meeli

AU - Lyon, David

AU - Jensen, Lars Juhl

AU - Nielsen, Michael L

PY - 2017/8/4

Y1 - 2017/8/4

N2 - 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.

AB - 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.

U2 - 10.1021/acs.jproteome.7b00042

DO - 10.1021/acs.jproteome.7b00042

M3 - Journal article

C2 - 28648085

SN - 1535-3893

VL - 16

SP - 2762

EP - 2772

JO - Journal of Proteome Research

JF - Journal of Proteome Research

IS - 8

ER -