Poster abstracts

Poster number 108 submitted by Robert Patton

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

Robert D. Patton (Department of Physics, The Ohio State University), Manu Sanjeev (Department of Molecular Genetics, The Ohio State University), Lauren A. Woodward (Department of Molecular Genetics, The Ohio State University), Justin W. Mabin (Department of Molecular Genetics, The Ohio State University), Ralf Bundschuh (Department of Physics, The Ohio State University), Guramrit Singh (Department of Molecular Genetics, The Ohio State University)

Abstract:
In eukaryotic cells, RNA binding proteins (RBPs) regulate RNA activity to control cellular function. To fully illuminate the basis of RNA function, it is essential to identify RBPs, their mode of action on RNA, and their preferred RNA targets and binding sites. By analyzing existing human RBP catalogs that were defined based on ultraviolet light (UV)-dependent and independent approaches, we classify RBPs into direct RBPs (efficiently UV crosslinkable) and indirect RBPs (poorly UV crosslinkable). As the UV cross-linking and immunoprecipitation followed by sequencing (CLIP-Seq) approach will be ill-suited to identify binding sites of indirect RBPs, we show that formaldehyde crosslinking stabilizes indirect RBPs within ribonucleoproteins to allow for their purification under stringent conditions. Using a direct RBP (CASC3) and an indirect RBP (RNPS1) within the exon junction complex (EJC) as examples, we show that formaldehyde crosslinking of cells combined with RNA immunoprecipitation in tandem followed by sequencing (xRIPiT-Seq) far exceeds CLIP-Seq to identify binding sites of RNPS1. However, both xRIPiT-Seq and CLIP-Seq are comparable for enrichment of CASC3 binding sites. Overall, our work highlights the existence of a significant number of indirect RBPs in the human proteome, and shows that xRIPiT-Seq is more suitable to study RNA cargoes of these proteins.

References:
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large-scale protein-protein interactomes. Mol Cell. 64(2): 282-293.
2. Trendel, J., Schwarzl, T., Horos, R., Prakash, A., Bateman, A., Hentze, and M., Krijgsveld, J. (2019). The Human RNA-Binding Proteome and Its Dynamics during Translational Arrest. Cell. 176:391-403.

Keywords: CLIP-Seq, RIPiT-Seq, RBP