A Photocrosslinking-Based RNA Chemical Proteomics Approach to Profile m 6 A-Regulated Protein-RNA Interactions

A. Emilia Arguello, Tharan Srikumar, Ralph E. Kleiner

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

Post-transcriptional modifications play an important role in RNA biology. In particular, the addition of small chemical groups to the nucleobases of mRNA can affect how modified transcripts are processed in the cell, thereby impacting gene expression programs. In order to study the molecular mechanisms underlying these modifications, it is necessary to characterize their ‘readers’, that is, proteins that directly bind to these modifications to mediate their functional consequences; this is a major challenge because we lack approaches to precisely manipulate RNA chemistry in the cell and because protein–modified RNA interactions can be low affinity. In this unit, we describe in detail a photocrosslinking-based RNA chemical proteomics approach to profile the protein–modified RNA interactome modulated by N 6 -methyladenosine (m 6 A), the most abundant internal modification in eukaryotic mRNA. First, we present protocols for the synthesis and characterization of short, diazirine-containing synthetic RNA probes, followed by a description of their use in mass spectrometry–based proteomics with HeLa cell lysate and a short commentary on data analysis and result interpretation.

Original languageEnglish (US)
Article numbere69
JournalCurrent Protocols in Nucleic Acid Chemistry
Volume75
Issue number1
DOIs
StatePublished - Dec 2018

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Organic Chemistry

Keywords

  • epitranscriptome
  • m6A
  • mRNA modifications
  • photocrosslinking
  • proteomics

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