Analysis of RNA-containing compartments by hybridization and proximity labeling in cultured human cells

TY - JOUR

T1 - Analysis of RNA-containing compartments by hybridization and proximity labeling in cultured human cells

AU - Yap, Karen

AU - Chung, Tek Hong

AU - Makeyev, Eugene V.

N1 - Funding Information: We thank Alexander Alexandrovich for the help with protein purification. This work was supported by the Biotechnology and Biological Sciences Research Council ( BB/M001199/1 , BB/M007103/1 , and BB/R001049/1 ), British Society for Antimicrobial Chemotherapy ( BSAC-COVID-24 ), and European Commission (H2020-MSCA-RISE-2016; Project ID 734791 ). Publisher Copyright: © 2022 The Author(s)

PY - 2022/3/18

Y1 - 2022/3/18

N2 - This protocol describes a hybridization-proximity labeling (HyPro) approach for identification of proteins and RNAs co-localizing with a transcript of interest in genetically unperturbed cells. It outlines steps required for purification of a recombinant HyPro enzyme, hybridization of fixed and permeabilized cells with digoxigenin-labeled probes, HyPro enzyme binding, proximity biotinylation, and downstream analyses of the biotinylated products. Although the protocol is optimized for relatively abundant noncoding transcripts, recommendations are provided for improving the signal-to-noise ratio in case of scarcer RNA “baits.” For complete details on the use and execution of this protocol, please refer to Yap et al. (2021).

AB - This protocol describes a hybridization-proximity labeling (HyPro) approach for identification of proteins and RNAs co-localizing with a transcript of interest in genetically unperturbed cells. It outlines steps required for purification of a recombinant HyPro enzyme, hybridization of fixed and permeabilized cells with digoxigenin-labeled probes, HyPro enzyme binding, proximity biotinylation, and downstream analyses of the biotinylated products. Although the protocol is optimized for relatively abundant noncoding transcripts, recommendations are provided for improving the signal-to-noise ratio in case of scarcer RNA “baits.” For complete details on the use and execution of this protocol, please refer to Yap et al. (2021).

KW - Cell Biology

KW - Gene Expression

KW - In Situ Hybridization

KW - Mass Spectrometry

KW - Microscopy

KW - Molecular Biology

KW - Molecular/Chemical Probes

KW - Protein Biochemistry

KW - Protein expression and purification

UR - http://www.scopus.com/inward/record.url?scp=85123583351&partnerID=8YFLogxK

U2 - 10.1016/j.xpro.2022.101139

DO - 10.1016/j.xpro.2022.101139

M3 - Article

AN - SCOPUS:85123583351

SN - 2666-1667

VL - 3

JO - STAR Protocols

JF - STAR Protocols

IS - 1

M1 - 101139

ER -