Analysis of RNA conformation in endogenously assembled RNPs by icSHAPE

Abstract

Summary: The majority of the mammalian genome is transcribed into non-coding RNAs, many of which co-evolve with RNA-binding proteins (RBPs) to function as biochemically defined and tractable ribonucleoproteins (RNPs). Here, we applied icSHAPE- a robust and versatile RNA structural probing pipeline- to endogenous RNPs purified from nuclei, providing base-resolution structural rationale for RNP activity and subcellular localization. Combining with genetic and biochemical reconstitutions, structural and functional alternations can be directly attributed to a given RBP without ambiguity.For complete details on the use and execution of this protocol, please refer to Chen et al. (2018).

Keywords

• Cell separation/fractionation
• Sequencing
• RNA-seq
• Molecular Biology
• Gene Expression
• CRISPR