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

• Published in: STAR protocols 2021-09, Vol.2 (3), p.100762, Article 100762
• Main Authors: Corley, Meredith, Flynn, Ryan A., Blue, Steven M., Yee, Brian A., Chang, Howard Y., Yeo, Gene W.
• Format: Article
• Language: English
• Subjects: Acylation; Antibody; Blotting, Western; Computational Biology - methods; Cross-Linking Reagents - chemistry; Gene Library; Genomics; Humans; Hydrogen Bonding; Immunoprecipitation - methods; K562 Cells; Molecular Biology; Molecular Probe Techniques - instrumentation; Molecular Probes - chemistry; Polymerase Chain Reaction; Proteins - chemistry; Proteins - genetics; Proteins - metabolism; Protocol; RNA - chemistry; RNA - isolation & purification; RNAseq; Ultraviolet Rays
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2021.100762

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). [Display omitted] •Protocols for probing protein-RNA hydrogen bonds (fSHAPE) and RNA structure (SHAPE)•fSHAPE-eCLIP and SHAPE-eCLIP probe RNA regions bound by immunoprecipitated protein•Samples probed in parallel under different conditions is the basis of each protocol•Probed regions yield fSHAPE or SHAPE reactivities at nucleotide resolution 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.