An unusual RNA recognition motif acts as a scaffold for multiple proteins in the pre-mRNA retention and splicing complex

J Biol Chem. 2008 Nov 21;283(47):32317-27. doi: 10.1074/jbc.M804977200. Epub 2008 Sep 22.

Abstract

The yeast pre-mRNA retention and splicing complex counteracts the escape of unspliced pre-mRNAs from the nucleus and activates splicing of a subset of Mer1p-dependent genes. A homologous complex is present in activated human spliceosomes. In many components of the spliceosome, RNA recognition motifs (RRMs) serve as versatile protein-RNA or protein-protein interaction platforms. Here, we show that in the retention and splicing complex, an atypical RRM of the Snu17p (small nuclear ribonucleoprotein-associated protein 17) subunit acts as a scaffold that organizes the other two constituents, Bud13p (bud site selection 13) and Pml1p (pre-mRNA leakage 1). GST pull-down experiments and size exclusion chromatography revealed that Snu17p constitutes the central platform of the complex, whereas Bud13p and Pml1p do not interact with each other. Fluorimetric structure probing showed the entire Bud13p and the N-terminal third of Pml1p to be natively disordered in isolation. Mutational analysis and tryptophan fluorescence confirmed that a conserved tryptophan-containing motif in the C terminus of Bud13p binds to the core RRM of Snu17p, whereas a different interaction surface encompassing a C-terminal extension of the Snu17p RRM is required to bind an N-terminal peptide of Pml1p. Isothermal titration calorimetry revealed 1:1 interaction stoichiometries, large negative binding entropies, and dissociation constants in the low nanomolar and micromolar ranges for the Snu17p-Bud13p and the Snu17p-Pml1p interactions, respectively. Our results demonstrate that the noncanonical Snu17p RRM concomitantly binds multiple ligand proteins via short, intrinsically unstructured peptide epitopes and thereby acts as a platform that displays functional modules of the ligands, such as a forkhead-associated domain of Pml1p and a conserved polylysine motif of Bud13p.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Carrier Proteins / metabolism
  • DNA Mutational Analysis
  • Humans
  • Molecular Sequence Data
  • Peptides / chemistry
  • Polylysine / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA Precursors / metabolism*
  • RNA Splicing*
  • Ribonucleoprotein, U2 Small Nuclear / metabolism
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sequence Homology, Amino Acid

Substances

  • Bud13 protein, S cerevisiae
  • Carrier Proteins
  • IST3 protein, S cerevisiae
  • Peptides
  • Pml1 protein, S cerevisiae
  • RNA Precursors
  • Ribonucleoprotein, U2 Small Nuclear
  • Saccharomyces cerevisiae Proteins
  • Polylysine