Functional contacts with a range of splicing proteins suggest a central role for Brr2p in the dynamic control of the order of events in spliceosomes of Saccharomyces cerevisiae

Genetics. 2001 Apr;157(4):1451-67. doi: 10.1093/genetics/157.4.1451.

Abstract

Mapping of functional protein interactions will help in understanding conformational rearrangements that occur within large complexes like spliceosomes. Because the U5 snRNP plays a central role in pre-mRNA splicing, we undertook exhaustive two-hybrid screening with Brr2p, Prp8p, and other U5 snRNP-associated proteins. DExH-box protein Brr2p interacted specifically with five splicing factors: Prp8p, DEAH-box protein Prp16p, U1 snRNP protein Snp1p, second-step factor Slu7p, and U4/U6.U5 tri-snRNP protein Snu66p, which is required for splicing at low temperatures. Co-immunoprecipitation experiments confirmed direct or indirect interactions of Prp16p, Prp8p, Snu66p, and Snp1p with Brr2p and led us to propose that Brr2p mediates the recruitment of Prp16p to the spliceosome. We provide evidence that the prp8-1 allele disrupts an interaction with Brr2p, and we propose that Prp8p modulates U4/U6 snRNA duplex unwinding through another interaction with Brr2p. The interactions of Brr2p with a wide range of proteins suggest a particular function for the C-terminal half, bringing forward the hypothesis that, apart from U4/U6 duplex unwinding, Brr2p promotes other RNA rearrangements, acting synergistically with other spliceosomal proteins, including the structurally related Prp2p and Prp16p. Overall, these protein interaction studies shed light on how splicing factors regulate the order of events in the large spliceosome complex.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / genetics
  • Adenosine Triphosphatases / metabolism
  • Animals
  • Cell Nucleus / metabolism
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Fungal Proteins / physiology*
  • Humans
  • Open Reading Frames
  • Precipitin Tests
  • RNA Helicases / genetics
  • RNA Helicases / metabolism
  • RNA Helicases / physiology*
  • RNA Splicing Factors
  • RNA Splicing*
  • RNA, Small Nuclear / genetics
  • RNA, Small Nuclear / metabolism
  • Repressor Proteins / genetics
  • Repressor Proteins / metabolism
  • Repressor Proteins / physiology*
  • Ribonucleoprotein, U4-U6 Small Nuclear / genetics
  • Ribonucleoprotein, U4-U6 Small Nuclear / metabolism
  • Ribonucleoprotein, U5 Small Nuclear
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae / physiology
  • Saccharomyces cerevisiae Proteins*
  • Spliceosomes / metabolism*
  • Two-Hybrid System Techniques

Substances

  • Fungal Proteins
  • PRP8 protein, S cerevisiae
  • RNA Splicing Factors
  • RNA, Small Nuclear
  • Repressor Proteins
  • Ribonucleoprotein, U4-U6 Small Nuclear
  • Ribonucleoprotein, U5 Small Nuclear
  • Saccharomyces cerevisiae Proteins
  • U5 small nuclear RNA
  • Adenosine Triphosphatases
  • BRR2 protein, S cerevisiae
  • PRP16 protein, S cerevisiae
  • RNA Helicases