Chemical crosslinking enhances RNA immunoprecipitation for efficient identification of binding sites of proteins that photo-crosslink poorly with RNA

In eukaryotic cells, proteins that associate with RNA regulate its activity to control cellular function. To fully illuminate the basis of RNA function, it is essential to identify such RNA associated proteins, their mode of action on RNA, and their preferred RNA targets and binding sites. By analyz...

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Bibliographic Details
Published in:RNA (Cambridge) 2020-09, Vol.26 (9), p.1216-1233
Main Authors: Patton, Robert D, Sanjeev, Manu, Woodward, Lauren A, Mabin, Justin W, Bundschuh, Ralf, Singh, Guramrit
Format: Article
Language:English
Subjects:
Binding sites
Exons
Formaldehyde
Immunoprecipitation
Proteins
Ribonucleic acid
Ribonucleoproteins
RNA
RNA-binding protein
Ultraviolet radiation
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Summary:In eukaryotic cells, proteins that associate with RNA regulate its activity to control cellular function. To fully illuminate the basis of RNA function, it is essential to identify such RNA associated proteins, their mode of action on RNA, and their preferred RNA targets and binding sites. By analyzing catalogs of human RNA associated proteins defined by ultraviolet light (UV)-dependent and independent approaches, we classify these proteins into two major groups: (1) the widely-recognized RNA binding proteins (RBPs), which bind RNA directly and UV crosslink efficiently to RNA, and (2) a new group of RBP-associated factors (RAFs), which bind RNA indirectly via RBPs and UV crosslink poorly to RNA. As the UV cross-linking and immunoprecipitation followed by sequencing (CLIP-Seq) approach will be ill-suited to identify binding sites of RAFs, we show that formaldehyde crosslinking stabilizes RAFs within ribonucleoproteins to allow for their immunoprecipitation under stringent conditions. Using an RBP (CASC3) and an RAF (RNPS1) within the exon junction complex (EJC) as examples, we show that formaldehyde crosslinking combined with RNA immunoprecipitation in tandem followed by sequencing (xRIPiT-Seq) far exceeds CLIP-Seq to identify binding sites of RNPS1. xRIPiT-Seq reveals that RNPS1 occupancy is increased on exons immediately upstream of strong recursively spliced exons, which depend on EJC for their inclusion.
ISSN:1355-8382
1469-9001
DOI:10.1261/RNA.074856.120