Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome

Nat Chem Biol. 2015 Aug;11(8):592-7. doi: 10.1038/nchembio.1836. Epub 2015 Jun 15.

Abstract

Pseudouridine (Ψ) is the most abundant post-transcriptional RNA modification, yet little is known about its prevalence, mechanism and function in mRNA. Here, we performed quantitative MS analysis and show that Ψ is much more prevalent (Ψ/U ratio ∼0.2-0.6%) in mammalian mRNA than previously believed. We developed N3-CMC-enriched pseudouridine sequencing (CeU-Seq), a selective chemical labeling and pulldown method, to identify 2,084 Ψ sites within 1,929 human transcripts, of which four (in ribosomal RNA and EEF1A1 mRNA) are biochemically verified. We show that hPUS1, a known Ψ synthase, acts on human mRNA; under stress, CeU-Seq demonstrates inducible and stress-specific mRNA pseudouridylation. Applying CeU-Seq to the mouse transcriptome revealed conserved and tissue-specific pseudouridylation. Collectively, our approaches allow comprehensive analysis of transcriptome-wide pseudouridylation and provide tools for functional studies of Ψ-mediated epigenetic regulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Epigenesis, Genetic*
  • Humans
  • Hydro-Lyases / chemistry
  • Hydro-Lyases / genetics
  • Hydro-Lyases / metabolism
  • Mice
  • Organ Specificity
  • Peptide Elongation Factor 1 / chemistry
  • Peptide Elongation Factor 1 / genetics
  • Peptide Elongation Factor 1 / metabolism
  • Pseudouridine / chemistry
  • Pseudouridine / genetics
  • Pseudouridine / metabolism*
  • RNA Processing, Post-Transcriptional*
  • RNA, Messenger / chemistry
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism*
  • RNA, Ribosomal / chemistry
  • RNA, Ribosomal / genetics
  • RNA, Ribosomal / metabolism*
  • Staining and Labeling / methods
  • Stress, Physiological
  • Transcriptome*

Substances

  • EEF1A1 protein, human
  • Peptide Elongation Factor 1
  • RNA, Messenger
  • RNA, Ribosomal
  • Pseudouridine
  • Hydro-Lyases
  • pseudouridylate synthetase

Associated data

  • GEO/GSE63655
  • PubChem-Substance/251916864
  • PubChem-Substance/251916865
  • PubChem-Substance/251916866
  • PubChem-Substance/251916867
  • PubChem-Substance/251916868