psiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation

Nat Commun. 2021 Mar 5;12(1):1488. doi: 10.1038/s41467-021-21745-9.

Abstract

RNA helicases remodel the spliceosome to enable pre-mRNA splicing, but their binding and mechanism of action remain poorly understood. To define helicase-RNA contacts in specific spliceosomal states, we develop purified spliceosome iCLIP (psiCLIP), which reveals dynamic helicase-RNA contacts during splicing catalysis. The helicase Prp16 binds along the entire available single-stranded RNA region between the branchpoint and 3'-splice site, while Prp22 binds diffusely downstream of the branchpoint before exon ligation, but then switches to more narrow binding in the downstream exon after exon ligation, arguing against a mechanism of processive translocation. Depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, suggesting that proofreading by Prp22 may sense the stability of the spliceosome during exon ligation. Thus, psiCLIP complements structural studies by providing key insights into the binding and proofreading activity of spliceosomal RNA helicases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / genetics
  • Adenosine Triphosphatases / metabolism
  • Autoantigens / chemistry
  • Autoantigens / metabolism
  • Cryoelectron Microscopy
  • DEAD-box RNA Helicases / chemistry
  • DEAD-box RNA Helicases / metabolism
  • Exons*
  • Models, Molecular
  • RNA Helicases / chemistry*
  • RNA Helicases / metabolism*
  • RNA Precursors / chemistry
  • RNA Precursors / metabolism*
  • RNA Splicing Factors / genetics
  • RNA Splicing Factors / metabolism
  • RNA Splicing*
  • RNA, Fungal / metabolism
  • Recombinant Proteins
  • Ribonucleoprotein, U5 Small Nuclear / chemistry
  • Ribonucleoprotein, U5 Small Nuclear / genetics
  • Ribonucleoprotein, U5 Small Nuclear / metabolism
  • Ribonucleoproteins, Small Nuclear / chemistry
  • Ribonucleoproteins, Small Nuclear / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Spliceosomes / chemistry
  • Spliceosomes / metabolism*

Substances

  • Autoantigens
  • PRP18 protein, S cerevisiae
  • RNA Precursors
  • RNA Splicing Factors
  • RNA, Fungal
  • Recombinant Proteins
  • Ribonucleoprotein, U5 Small Nuclear
  • Ribonucleoproteins, Small Nuclear
  • SMB protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Adenosine Triphosphatases
  • PRP16 protein, S cerevisiae
  • PRP22 protein, S cerevisiae
  • DEAD-box RNA Helicases
  • RNA Helicases