Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 498597
Title Analysis of protein-RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry
Author(s) Sharma, Kundan; Hrle, Ajla; Kramer, Katharina; Sachsenberg, Timo; Staals, Raymond H.J.; Randau, Lennart; Marchfelder, Anita; Oost, John van der; Kohlbacher, Oliver; Conti, Elena; Urlaub, Henning
Source Methods : a companion to Methods in enzymology 89 (2015). - ISSN 1046-2023 - p. 138 - 148.
DOI http://dx.doi.org/10.1016/j.ymeth.2015.06.005
Department(s) Microbiological Laboratory
VLAG
Publication type Refereed Article in a scientific journal
Publication year 2015
Keyword(s) Cas7 - CRISPR-Cas - Mass spectrometry - Protein-RNA interactions - UV cross-linking
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.

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