Analysis of protein-RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry.
Methods
; 89: 138-48, 2015 Nov 01.
Article
em En
| MEDLINE
| ID: mdl-26071038
ABSTRACT
Ribonucleoprotein (RNP) complexes play important roles in the cell by mediating basic cellular processes, including gene expression and its regulation. Understanding the molecular details of these processes requires the identification and characterization of protein-RNA interactions. Over the years various approaches have been used to investigate these interactions, including computational analyses to look for RNA binding domains, gel-shift mobility assays on recombinant and mutant proteins as well as co-crystallization and NMR studies for structure elucidation. Here we report a more specialized and direct approach using UV-induced cross-linking coupled with mass spectrometry. This approach permits the identification of cross-linked peptides and RNA moieties and can also pin-point exact RNA contact sites within the protein. The power of this method is illustrated by the application to different single- and multi-subunit RNP complexes belonging to the prokaryotic adaptive immune system, CRISPR-Cas (CRISPR clustered regularly interspaced short palindromic repeats; Cas CRISPR associated). In particular, we identified the RNA-binding sites within three Cas7 protein homologs and mapped the cross-linking results to reveal structurally conserved Cas7 - RNA binding interfaces. These results demonstrate the strong potential of UV-induced cross-linking coupled with mass spectrometry analysis to identify RNA interaction sites on the RNA binding proteins.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Raios Ultravioleta
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Proteínas de Ligação a RNA
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Espectrometria de Massas por Ionização por Electrospray
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Espectrometria de Massas em Tandem
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Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
Idioma:
En
Revista:
Methods
Assunto da revista:
BIOQUIMICA
Ano de publicação:
2015
Tipo de documento:
Article
País de afiliação:
Alemanha