Functional characterization and protein-protein interactions of trypanosome splicing factors U2AF35, U2AF65 and SF1

Mol Biochem Parasitol. 2009 Apr;164(2):137-46. doi: 10.1016/j.molbiopara.2008.12.009.

Abstract

Early in the assembly of eukaryotes the branch-point binding protein (BBP, also called SF1) recognizes the branch point sequence, whereas the heterodimer U2AF, consisting of a 65 and a 35 kDa subunit, contacts the polypyrimidine tract and the AG splice site, respectively. Herein, we identified, cloned and expressed the Trypanosoma cruzi and Trypanosoma brucei U2AF35, U2AF65 and SF1. Trypanosomatid U2AF65 strongly diverged from yeast and human homologues. On the contrary, trypanosomatid SF1 was conserved but lacked the C-terminal sequence present in the mammalian protein. Yeast two hybrid approaches were used to assess their interactions. The interaction between U2AF35 and U2AF65 was very weak or not detectable. However, as in other eukaryotes, the interaction between U2AF65 and SF1 was strong. At the cellular level, these results were confirmed by fractionation and affinity-selection experiments in which SF1 and U2AF65 were affinity-selected with TAP tagged SF1, but not with TAP tagged U2AF35. Silencing one of the three factors affected growth and trans-splicing in the first step of this reaction. Trypanosomes are the first described example of eukaryotic cells in which the interaction of two expressed U2AF factors seemed to be very weak, or not detectable.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Fractionation
  • Cloning, Molecular
  • Conserved Sequence
  • Gene Expression
  • Gene Silencing
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Interaction Mapping*
  • Protein Structure, Tertiary
  • Protozoan Proteins / genetics
  • Protozoan Proteins / metabolism*
  • RNA Splicing*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Sequence Homology, Amino Acid
  • Trypanosoma brucei brucei / genetics
  • Trypanosoma brucei brucei / physiology*
  • Trypanosoma cruzi / genetics
  • Trypanosoma cruzi / physiology*
  • Two-Hybrid System Techniques

Substances

  • Protozoan Proteins
  • RNA-Binding Proteins