Competition between the ATPase Prp5 and branch region-U2 snRNA pairing modulates the fidelity of spliceosome assembly

Mol Cell. 2007 Dec 14;28(5):838-49. doi: 10.1016/j.molcel.2007.09.022.

Abstract

ATPase-facilitated steps during spliceosome function have been postulated to afford opportunities for kinetic proofreading. Spliceosome assembly requires the ATPase Prp5p, whose activity might thus impact fidelity during initial intron recognition. Using alanine mutations in S. cerevisiae Prp5p, we identified a suboptimal intron whose splicing could be improved by altered Prp5p activity and then, using this intron, screened for potent prp5 mutants. These prp5 alleles specifically alter branch region selectivity, with improved splicing in vivo of suboptimal substrates correlating with reduced ATPase activity in vitro for a series of mutants in ATPase motif III (SAT). Because these effects are abrogated by compensatory U2 snRNA mutations or other changes that increase branch region-U2 pairing, these results explicitly link a fidelity event with a defined physical structure, the branch region-U2 snRNA duplex, and provide strong evidence that progression of the splicing pathway requires branch region-U2 snRNA pairing prior to Prp5p-facilitated conformational change.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenosine Triphosphate / metabolism*
  • Alanine / genetics
  • Alleles
  • Base Pairing
  • DEAD-box RNA Helicases / chemistry
  • DEAD-box RNA Helicases / genetics*
  • DEAD-box RNA Helicases / metabolism
  • Mutation
  • Nucleic Acid Conformation
  • Phenotype
  • RNA Splicing*
  • RNA, Fungal / genetics
  • RNA, Fungal / metabolism
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA, Small Nuclear / genetics*
  • RNA, Small Nuclear / metabolism
  • RNA-Binding Proteins / genetics*
  • RNA-Binding Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Spliceosomes / metabolism*
  • Substrate Specificity
  • Trans-Activators / genetics*
  • Trans-Activators / metabolism

Substances

  • CUS2 protein, S cerevisiae
  • RNA, Fungal
  • RNA, Messenger
  • RNA, Small Nuclear
  • RNA-Binding Proteins
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • Trans-Activators
  • U2 small nuclear RNA
  • Adenosine Triphosphate
  • PRP5 protein, S cerevisiae
  • DEAD-box RNA Helicases
  • Alanine