Dimerization and protein binding specificity of the U2AF homology motif of the splicing factor Puf60

J Biol Chem. 2009 Jan 2;284(1):630-9. doi: 10.1074/jbc.M805395200. Epub 2008 Oct 29.

Abstract

PUF60 is an essential splicing factor functionally related and homologous to U2AF(65). Its C-terminal domain belongs to the family of U2AF (U2 auxiliary factor) homology motifs (UHM), a subgroup of RNA recognition motifs that bind to tryptophan-containing linear peptide motifs (UHM ligand motifs, ULMs) in several nuclear proteins. Here, we show that the Puf60 UHM is mainly monomeric in physiological buffer, whereas its dimerization is induced upon the addition of SDS. The crystal structure of PUF60-UHM at 2.2 angstroms resolution, NMR data, and mutational analysis reveal that the dimer interface is mediated by electrostatic interactions involving a flexible loop. Using glutathione S-transferase pulldown experiments, isothermal titration calorimetry, and NMR titrations, we find that Puf60-UHM binds to ULM sequences in the splicing factors SF1, U2AF65, and SF3b155. Compared with U2AF65-UHM, Puf60-UHM has distinct binding preferences to ULMs in the N terminus of SF3b155. Our data suggest that the functional cooperativity between U2AF65 and Puf60 may involve simultaneous interactions of the two proteins with SF3b155.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs / physiology
  • Animals
  • Carrier Proteins / chemistry*
  • Carrier Proteins / metabolism
  • Crystallography, X-Ray
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / metabolism
  • Dimerization
  • Humans
  • Nuclear Magnetic Resonance, Biomolecular
  • Nuclear Proteins / chemistry*
  • Nuclear Proteins / metabolism
  • Phosphoproteins / chemistry*
  • Phosphoproteins / metabolism
  • Protein Binding / physiology
  • Protein Structure, Quaternary / physiology
  • Protein Structure, Tertiary / physiology
  • RNA Splicing Factors
  • RNA-Binding Proteins
  • Ribonucleoprotein, U2 Small Nuclear / chemistry*
  • Ribonucleoprotein, U2 Small Nuclear / metabolism
  • Ribonucleoproteins / chemistry*
  • Ribonucleoproteins / metabolism
  • Sequence Homology, Amino Acid
  • Sodium Dodecyl Sulfate / chemistry
  • Splicing Factor U2AF
  • Transcription Factors / chemistry*
  • Transcription Factors / metabolism

Substances

  • Carrier Proteins
  • DNA-Binding Proteins
  • Nuclear Proteins
  • Phosphoproteins
  • RNA Splicing Factors
  • RNA-Binding Proteins
  • Ribonucleoprotein, U2 Small Nuclear
  • Ribonucleoproteins
  • SF1 protein, human
  • SF3B1 protein, human
  • SIAHBP1 protein, human
  • Splicing Factor U2AF
  • Transcription Factors
  • U2AF2 protein, human
  • Sodium Dodecyl Sulfate

Associated data

  • PDB/3DXB