fSHAPE, fSHAPE-eCLIP, and SHAPE-eCLIP probe transcript regions that interact with specific proteins

STAR Protoc. 2021 Aug 25;2(3):100762. doi: 10.1016/j.xpro.2021.100762. eCollection 2021 Sep 17.

Abstract

Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) structure probing techniques characterize the secondary structure of RNA molecules, which influence their functions and interactions. A variation of SHAPE, footprinting SHAPE (fSHAPE), probes RNA in the presence and absence of protein to identify RNA bases that hydrogen-bond with protein. SHAPE or fSHAPE coupled with enhanced crosslinking and immunoprecipitation (SHAPE-eCLIP or fSHAPE-eCLIP) pulls down RNAs bound by any protein of interest and returns their structure or protein interaction information, respectively. Here, we describe detailed protocols for SHAPE-eCLIP and fSHAPE-eCLIP and an analysis protocol for fSHAPE. For complete details on the use and execution of these protocols, please refer to Corley et al. (2020).

Keywords: Antibody; Genomics; Molecular Biology; RNAseq.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acylation
  • Blotting, Western
  • Computational Biology / methods
  • Cross-Linking Reagents / chemistry
  • Gene Library
  • Humans
  • Hydrogen Bonding
  • Immunoprecipitation / methods
  • K562 Cells
  • Molecular Probe Techniques* / instrumentation
  • Molecular Probes / chemistry*
  • Polymerase Chain Reaction
  • Proteins / chemistry
  • Proteins / genetics*
  • Proteins / metabolism
  • RNA / chemistry*
  • RNA / isolation & purification
  • Ultraviolet Rays

Substances

  • Cross-Linking Reagents
  • Molecular Probes
  • Proteins
  • RNA primers
  • RNA