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

MPS-Authors
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Qamar,  S.
Research Group of Bioanalytical Mass Spectrometry, MPI for biophysical chemistry, Max Planck Society;

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Kramer,  K.
Research Group of Bioanalytical Mass Spectrometry, MPI for biophysical chemistry, Max Planck Society;

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Urlaub,  H.
Research Group of Bioanalytical Mass Spectrometry, MPI for biophysical chemistry, Max Planck Society;

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Citation

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.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0027-839B-B
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.