Applying UV crosslinking to study RNA-protein interactions in multicomponent ribonucleoprotein complexes

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Citation (Scopus)

Abstract

Ribonucleoprotein complexes (RNPs) play crucial roles in a wide range of biological processes. Here, we describe experimental approaches to the UV crosslinking-based identification of protein-binding sites on RNA, using multicomponent Saccharomyces cerevisiae RNPs of the RNase P/MRP family as an example. To identify the binding sites of a protein component of interest, a hexahistidine affinity tag was fused to that protein. Then RNase P/MRP RNPs were purified from yeast cells that had expressed the protein component of interest with the fused tag, subjected to UV crosslinking, and disassembled to separate the non-covalently-bound components. The protein component of interest was isolated under denaturing conditions using the hexahistidine tag as a purification handle. Provided that the isolated protein formed UV-induced crosslinks with the RNA component of the studied RNP, the isolation of the protein resulted in the co-isolation of the covalently bound RNP RNA. The isolated protein was enzymatically degraded, and the UV crosslinked RNA was purified. The locations of the crosslinks formed between the protein component of interest and the RNP RNA were identified by primer extension with a reverse transcriptase followed by gel electrophoresis; this procedure was repeated for all of the protein components of RNases P/MRP.

Original languageEnglish (US)
Title of host publicationRNA Folding
Subtitle of host publicationMethods and Protocols
PublisherHumana Press Inc.
Pages193-207
Number of pages15
ISBN (Print)9781627036665
DOIs
StatePublished - Jan 1 2014

Publication series

NameMethods in Molecular Biology
Volume1086
ISSN (Print)1064-3745

Fingerprint

Ribonucleoproteins
RNA
His-His-His-His-His-His
Ribonuclease P
Proteins
Binding Sites
Biological Phenomena
RNA-Directed DNA Polymerase
Protein Binding
Saccharomyces cerevisiae
Electrophoresis
Yeasts
Gels

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

Cite this

Krasilnikov, A. S. (2014). Applying UV crosslinking to study RNA-protein interactions in multicomponent ribonucleoprotein complexes. In RNA Folding: Methods and Protocols (pp. 193-207). (Methods in Molecular Biology; Vol. 1086). Humana Press Inc.. https://doi.org/10.1007/978-1-62703-667-2_11
Krasilnikov, Andrey S. / Applying UV crosslinking to study RNA-protein interactions in multicomponent ribonucleoprotein complexes. RNA Folding: Methods and Protocols. Humana Press Inc., 2014. pp. 193-207 (Methods in Molecular Biology).
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Krasilnikov, AS 2014, Applying UV crosslinking to study RNA-protein interactions in multicomponent ribonucleoprotein complexes. in RNA Folding: Methods and Protocols. Methods in Molecular Biology, vol. 1086, Humana Press Inc., pp. 193-207. https://doi.org/10.1007/978-1-62703-667-2_11

Applying UV crosslinking to study RNA-protein interactions in multicomponent ribonucleoprotein complexes. / Krasilnikov, Andrey S.

RNA Folding: Methods and Protocols. Humana Press Inc., 2014. p. 193-207 (Methods in Molecular Biology; Vol. 1086).

Research output: Chapter in Book/Report/Conference proceedingChapter

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Krasilnikov AS. Applying UV crosslinking to study RNA-protein interactions in multicomponent ribonucleoprotein complexes. In RNA Folding: Methods and Protocols. Humana Press Inc. 2014. p. 193-207. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-62703-667-2_11