Enhanced Ribonucleoprotein Immunoprecipitation (RIP) Technique for the Identification of mRNA Species in Ribonucleoprotein Complexes

Saja A. Fakhraldeen, Scott M. Berry, David J. Beebe, Caroline M. Alexander

Research output: Contribution to journalArticlepeer-review

Abstract

RNA binding proteins (RBPs) are critical regulators of cellular phenotypes, and dysregulated RBP expression is implicated in various diseases including cancer. A single RBP can bind to and regulate the expression of many RNA molecules via a variety of mechanisms, including translational suppression, prevention of RNA degradation, and alteration in subcellular localization. To elucidate the role of a specific RBP within a given cellular context, it is essential to first identify the group of RNA molecules to which it binds. This has traditionally been achieved using cross-linking-based assays in which cells are first exposed to agents that cross-link RBPs to nucleic acids and then lysed to extract and purify the RBP-nucleic acid complexes. The nucleic acids within the mixture are then released and analyzed via conventional means (e.g., microarray analysis, qRT-PCR, RNA sequencing, or Northern blot). While cross-linking-based ribonucleoprotein immunoprecipitation (RIP) has proven its utility within some contexts, it is technically challenging, inefficient, and suboptimal given the amount of time and resources (e.g., cells and antibodies) required. Additionally, these types of studies often require the use of over-expressed versions of proteins, which can introduce artifacts. Here, we describe a streamlined version of RIP that utilizes exclusion-based purification technologies. This approach requires significantly less starting material and resources compared to traditional RIP approaches, takes less time, which is tantamount given the labile nature of RNA, and can be used with endogenously expressed proteins. The method described here can be used to study RNA-protein interactions in a variety of cellular contexts.

Original languageEnglish
Article numbere4526
JournalBio-protocol
Volume12
Issue number19
DOIs
StatePublished - Oct 5 2022

Bibliographical note

Publisher Copyright:
© 2022 The Authors; exclusive licensee Bio-protocol LLC.

Keywords

  • Breast cancer
  • Cancer biology
  • Post-transcriptional regulation
  • RNA binding protein
  • RNA-protein interaction
  • Ribonucleoprotein complex

ASJC Scopus subject areas

  • General Neuroscience
  • General Biochemistry, Genetics and Molecular Biology
  • General Immunology and Microbiology
  • Plant Science

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