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Methods. 2017 Apr 15;118-119:82-92. doi: 10.1016/j.ymeth.2016.12.008. Epub 2016 Dec 16.

RNA interactome capture in yeast.

Author information

1
European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany; IRI for Life Sciences & Institut für Biologie, Humboldt-Universität zu Berlin, Philippstrasse 13, 10115 Berlin, Germany. Electronic address: benedikt.beckmann@iri-lifesciences.de.

Abstract

RNA-binding proteins (RBPs) are key players in post-transcriptional regulation of gene expression in eukaryotic cells. To be able to unbiasedly identify RBPs in Saccharomyces cerevisiae, we developed a yeast RNA interactome capture protocol which employs RNA labeling, covalent UV crosslinking of RNA and proteins at 365nm wavelength (photoactivatable-ribonucleoside-enhanced crosslinking, PAR-CL) and finally purification of the protein-bound mRNA. The method can be easily implemented in common workflows and takes about 3days to complete. Next to a comprehensive explanation of the method, we focus on our findings about the choice of crosslinking in yeast and discuss the rationale of individual steps in the protocol.

KEYWORDS:

RBP; RNA labeling; UV crosslinking; Yeast; mRNA

PMID:
27993706
PMCID:
PMC5421583
DOI:
10.1016/j.ymeth.2016.12.008
[Indexed for MEDLINE]
Free PMC Article

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