Multiple protein-protein interactions converging on the Prp38 protein during activation of the human spliceosome

RNA. 2016 Feb;22(2):265-77. doi: 10.1261/rna.054296.115. Epub 2015 Dec 16.

Abstract

Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNA-binding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein-protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38. By quantitative, alanine surface-scanning two-hybrid screens and biochemical analyses we delineated four distinct interfaces on the Prp38 amino-terminal domain. In vitro interaction assays using recombinant proteins showed that Prp38 can bind at least two proteins simultaneously via two different interfaces. Addition of excess Prp38 amino-terminal domain to in vitro splicing assays, but not of an interaction-deficient mutant, stalled splicing at a precatalytic stage. Our results show that human Prp38 is an unusual SR protein, whose amino-terminal domain is a multi-interface protein-protein interaction platform that might organize the relative positioning of other proteins during splicing.

Keywords: pre-mRNA processing factor 38; pre-mRNA splicing; protein–protein interactions; spliceosomal B complex; spliceosome; yeast two-hybrid analysis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Arabidopsis / genetics
  • Arabidopsis / metabolism
  • Binding Sites
  • Crystallography, X-Ray
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Protein Subunits / chemistry*
  • Protein Subunits / genetics
  • Protein Subunits / metabolism
  • RNA Precursors / chemistry*
  • RNA Precursors / genetics
  • RNA Precursors / metabolism
  • RNA Splicing Factors
  • RNA Splicing*
  • RNA, Messenger / chemistry*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Spliceosomes / chemistry*
  • Spliceosomes / genetics
  • Spliceosomes / metabolism

Substances

  • PRP38 protein, S cerevisiae
  • Protein Subunits
  • RNA Precursors
  • RNA Splicing Factors
  • RNA, Messenger
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins