Identifying Protein Interactomes of Target RNAs Using HyPR-MS

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2021 Oct 25

PMID 34694612

Citations 4

Authors

Katherine B Henke

Rachel M Miller

Rachel A Knoener

Mark Scalf

Michele Spiniello

Lloyd M Smith

Affiliations

Soon will be listed here.

Abstract

RNA-protein interactions are integral to maintaining proper cellular function and homeostasis, and the disruption of key RNA-protein interactions is central to many disease states. HyPR-MS (hybridization purification of RNA-protein complexes followed by mass spectrometry) is a highly versatile and efficient technology which enables multiplexed discovery of specific RNA-protein interactomes. This chapter provides extensive guidance for successful application of HyPR-MS to the system and target RNA(s) of interest, as well as a detailed description of the fundamental HyPR-MS procedure, including: (1) experimental design of controls, capture oligonucleotides, and qPCR assays; (2) formaldehyde cross-linking of cell culture; (3) cell lysis and RNA solubilization; (4) isolation of target RNA(s); (5) RNA purification and RT-qPCR analysis; (6) protein preparation and mass spectrometric analysis; and (7) mass spectrometric data analysis.

Citing Articles

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PMID: 39078123 PMC: 11693245. DOI: 10.1021/acs.jproteome.4c00505.

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Towards an Ideal Hybridization-Based Strategy to Discover Protein Interactomes of Selected RNA Molecules.

Spiniello M, Scalf M, Casamassimi A, Abbondanza C, Smith L Int J Mol Sci. 2022; 23(2).

PMID: 35055128 PMC: 8779001. DOI: 10.3390/ijms23020942.

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