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  Studying RNA-protein interactions of pre-mRNA complexes by mass spectrometry.

Qamar, S., Kramer, K., & Urlaub, H. (2015). Studying RNA-protein interactions of pre-mRNA complexes by mass spectrometry. In S. A. Woodson, & F. H. T. Allain (Eds.), Structures of large RNA molecules and their complexes (pp. 417-463). Amsterdam: Elsevier. doi:10.1016/bs.mie.2015.02.010.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0027-839B-B Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002A-17B8-C
Genre: Book Chapter

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2161211.pdf (Publisher version), 4MB
 
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 Creators:
Qamar, S.1, Author              
Kramer, K.1, 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: RNA–protein interactions; UV-induced cross-linking; Mass spectrometry; Protein; RNA; RNP
 Abstract: RNA–protein interactions play a crucial role in gene expression. These interactions take place in so-called ribonucleoprotein (RNP) complexes. To investigate which proteins interact with RNA in these complexes, and how they do so, UV-light-induced cross-linking has proven to be a valuable, yet straightforward technique. UV irradiation induces a covalent bond between the RNA and the proteins, whereafter cross-linked proteins can be identified by mass spectrometric (MS) approaches. Moreover, the cross-linked region of the protein, and often the actual cross-linked amino acid, can be identified by state-of-the-art MS, as can the cross-linked RNA moiety. This protocol describes in detail how to isolate peptide–RNA oligonucleotide cross-links from UV-irradiated human pre-mRNA RNPs and to perform the subsequent MS investigation of these peptide–RNA conjugates in combination with a dedicated computational analysis, in order to obtain sequence information about the cross-linked peptide and oligoribonucleotide. The described workflow can be applied to any RNP, irrespective of its origin, e.g., RNPs assembled in vitro (as described here) or RNPs isolated from UV-irradiated cells, either ex vivo or in vivo.

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Language(s): eng - English
 Dates: 2015-05-052015
 Publication Status: Published in print
 Pages: -
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 Table of Contents: -
 Rev. Method: Peer
 Identifiers: DOI: 10.1016/bs.mie.2015.02.010
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Title: Structures of large RNA molecules and their complexes
Source Genre: Book
 Creator(s):
Woodson, S. A., Editor
Allain, F. H. T., Editor
Affiliations:
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Publ. Info: Amsterdam : Elsevier
Pages: 631 Volume / Issue: - Sequence Number: - Start / End Page: 417 - 463 Identifier: ISBN: 978-0-12-801934-4

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Title: Methods in Enzymology
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Pages: - Volume / Issue: 558 Sequence Number: - Start / End Page: - Identifier: -