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  Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry.

Sharma, K., Hrle, A., Kramer, K., Sachsenberg, T., Staals, R. H. J., Randau, L., et al. (2015). Analysis of protein–RNA interactions in CRISPR proteins and effector complexes by UV-induced cross-linking and mass spectrometry. Methods, 89, 138-148. doi:10.1016/j.ymeth.2015.06.005.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-335F-0 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002A-E4D2-3
Genre: Journal Article

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 Creators:
Sharma, K.1, Author              
Hrle, A., Author
Kramer, K.1, Author              
Sachsenberg, T., Author
Staals, R. H. J., Author
Randau, L., Author
Marchfelder, A., Author
van der Oost, J., Author
Kohlbacher, O., Author
Conti, E., Author
Urlaub, H.1, Author              
Affiliations:
1Research Group of Bioanalytical Mass Spectrometry, MPI for Biophysical Chemistry, Max Planck Society, ou_578613              

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Free keywords: Protein–RNA interactions; UV cross-linking; CRISPR-Cas; Cas7; Mass spectrometry
 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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Language(s): eng - English
 Dates: 2015-06-102015-11-01
 Publication Status: Published in print
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 Rev. Method: Peer
 Identifiers: DOI: 10.1016/j.ymeth.2015.06.005
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Title: Methods
Source Genre: Journal
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Pages: - Volume / Issue: 89 Sequence Number: - Start / End Page: 138 - 148 Identifier: -