Structure-function analysis and genetic interactions of the yeast branchpoint binding protein Msl5

Nucleic Acids Res. 2012 May;40(10):4539-52. doi: 10.1093/nar/gks049. Epub 2012 Jan 28.

Abstract

Saccharomyces cerevisiae Msl5 (branchpoint binding protein) orchestrates spliceosome assembly by binding the branchpoint sequence 5'-UACUAAC and establishing cross intron-bridging interactions with other components of the splicing machinery. Reciprocal tandem affinity purifications verify that Msl5 exists in vivo as a heterodimer with Mud2 and that the Msl5-Mud2 complex is associated with the U1 snRNP. By gauging the ability of mutants of Msl5 to complement msl5Δ, we find that the Mud2-binding (amino acids 35-54) and putative Prp40-binding (PPxY(100)) elements of the Msl5 N-terminal domain are inessential, as are the C-terminal proline-rich domain (amino acids 382-476) and two zinc-binding CxxCxxxxHxxxxC motifs (amino acids 273-286 and 299-312). A subset of conserved branchpoint RNA-binding amino acids in the central KH-QUA2 domain (amino acids 146-269) are essential pairwise (Ile198-Arg190; Leu256-Leu259) or in trios (Leu169-Arg172-Leu176), whereas other pairs of RNA-binding residues are dispensable. We used our collection of viable Msl5 mutants to interrogate synthetic genetic interactions, in cis between the inessential structural elements of the Msl5 polypeptide and in trans between Msl5 and yeast splicing factors (Mud2, Nam8 and Tgs1) that are optional for vegetative growth. The results suggest a network of important but functionally buffered protein-protein and protein-RNA interactions between the Mud2-Msl5 complex at the branchpoint and the U1 snRNP at the 5' splice site.

Publication types

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

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Guanosine / analogs & derivatives
  • Guanosine / chemistry
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Protein Structure, Tertiary
  • RNA Splicing Factors
  • RNA-Binding Proteins / chemistry*
  • RNA-Binding Proteins / genetics*
  • RNA-Binding Proteins / metabolism
  • Ribonucleoprotein, U1 Small Nuclear / metabolism
  • Ribonucleoproteins / genetics
  • Ribonucleoproteins / isolation & purification
  • Ribonucleoproteins / metabolism
  • Ribonucleoproteins, Small Nuclear / genetics
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / isolation & purification
  • Saccharomyces cerevisiae Proteins / metabolism
  • Splicing Factor U2AF
  • Structure-Activity Relationship

Substances

  • MSL5 protein, S cerevisiae
  • MUD2 protein, S cerevisiae
  • RNA Splicing Factors
  • RNA-Binding Proteins
  • Ribonucleoprotein, U1 Small Nuclear
  • Ribonucleoproteins
  • Ribonucleoproteins, Small Nuclear
  • Saccharomyces cerevisiae Proteins
  • Splicing Factor U2AF
  • Guanosine
  • NAM8 protein, S cerevisiae
  • N(2),N(2),7-trimethylguanosine