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Methods. 2017 Aug 15;126:44-53. doi: 10.1016/j.ymeth.2017.07.013. Epub 2017 Jul 19.

Analysis of RNA-protein interactions in vertebrate embryos using UV crosslinking approaches.

Author information

1
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
2
Institute of Molecular Biology, Mainz, Germany.
3
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA. Electronic address: karla.neugebauer@yale.edu.

Abstract

A decade ago, we believed that at least 300 RNA binding proteins (RBPs) were encoded in our genomes based on annotations of known or predicted RNA binding domains. Deciphering the roles of those RBPs in regulated gene expression was a vast frontier awaiting exploration. Since then, the field has developed a number of key tools that navigate the landscape of cellular RNA. These rely principally on UV crosslinking to create covalent bonds between RBPs and target RNAs in vivo, revealing not only target identities but also local binding sites upon RNA-Seq. More recently, a reverse protocol - mRNA interactome capture - has enabled the identification of the proteins that interact with mRNA. Astonishingly, the number of RBPs has grown to more than 1000, and we must now understand what they do. Here, we discuss the application of these methods to model organisms, focusing on the zebrafish Danio rerio, which provide unique biological contexts for the analysis of RBPs and their functions.

KEYWORDS:

RNA binding proteins; UV crosslinking; Zebrafish embryo; iCLIP; mRNA interactome capture

PMID:
28734934
DOI:
10.1016/j.ymeth.2017.07.013
[Indexed for MEDLINE]

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