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

Research output: Contribution to journalJournal articlepeer-review

Standard

Specifying RNA-Binding Regions in Proteins by Peptide Cross-Linking and Affinity Purification. / Mullari, Meeli; Lyon, David; Jensen, Lars Juhl; Nielsen, Michael L.

In: Journal of Proteome Research, Vol. 16, No. 8, 2017, p. 2762-2772.

Research output: Contribution to journalJournal articlepeer-review

Harvard

Mullari, M, Lyon, D, Jensen, LJ & Nielsen, ML 2017, 'Specifying RNA-Binding Regions in Proteins by Peptide Cross-Linking and Affinity Purification', Journal of Proteome Research, vol. 16, no. 8, pp. 2762-2772. https://doi.org/10.1021/acs.jproteome.7b00042

APA

Mullari, M., Lyon, D., Jensen, L. J., & Nielsen, M. L. (2017). Specifying RNA-Binding Regions in Proteins by Peptide Cross-Linking and Affinity Purification. Journal of Proteome Research, 16(8), 2762-2772. https://doi.org/10.1021/acs.jproteome.7b00042

Vancouver

Mullari M, Lyon D, Jensen LJ, Nielsen ML. Specifying RNA-Binding Regions in Proteins by Peptide Cross-Linking and Affinity Purification. Journal of Proteome Research. 2017;16(8):2762-2772. https://doi.org/10.1021/acs.jproteome.7b00042

Author

Mullari, Meeli ; Lyon, David ; Jensen, Lars Juhl ; Nielsen, Michael L. / Specifying RNA-Binding Regions in Proteins by Peptide Cross-Linking and Affinity Purification. In: Journal of Proteome Research. 2017 ; Vol. 16, No. 8. pp. 2762-2772.

Bibtex

@article{46b3386816fc49788e20e718bd89376e,
title = "Specifying RNA-Binding Regions in Proteins by Peptide Cross-Linking and Affinity Purification",
abstract = "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.",
author = "Meeli Mullari and David Lyon and Jensen, {Lars Juhl} and Nielsen, {Michael L}",
year = "2017",
doi = "10.1021/acs.jproteome.7b00042",
language = "English",
volume = "16",
pages = "2762--2772",
journal = "Journal of Proteome Research",
issn = "1535-3893",
publisher = "American Chemical Society",
number = "8",

}

RIS

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

Y1 - 2017

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

VL - 16

SP - 2762

EP - 2772

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 8

ER -

ID: 184320989