Photo-cross-linking and high-resolution mass spectrometry for assignment of RNA-binding sites in RNA-binding proteins.
Nat Methods
; 11(10): 1064-70, 2014 Oct.
Article
em En
| MEDLINE
| ID: mdl-25173706
ABSTRACT
RNA-protein complexes play pivotal roles in many central biological processes. Although methods based on high-throughput sequencing have advanced our ability to identify the specific RNAs bound by a particular protein, there is a need for precise and systematic ways to identify RNA interaction sites on proteins. We have developed an experimental and computational workflow combining photo-induced cross-linking, high-resolution mass spectrometry and automated analysis of the resulting mass spectra for the identification of cross-linked peptides, cross-linking sites and the cross-linked RNA oligonucleotide moieties of such RNA-binding proteins. The workflow can be applied to any RNA-protein complex of interest or to whole proteomes. We applied the approach to human and yeast mRNA-protein complexes in vitro and in vivo, demonstrating its powerful utility by identifying 257 cross-linking sites on 124 distinct RNA-binding proteins. The open-source software pipeline developed for this purpose, RNP(xl), is available as part of the OpenMS project.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Espectrometria de Massas
/
RNA
/
Proteínas de Ligação a RNA
Limite:
Humans
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article