title: Multiple protein-protein interactions converging on the Prp38 protein during activation of the human spliceosome.
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.
authors.affiliation: Freie Universität Berlin, Laboratory of Structural Biochemistry, D-14195 Berlin, Germany.
authors.forename: Tonio
authors.initials: T
authors.lastname: Schütze
authors.affiliation: Freie Universität Berlin, Laboratory of Structural Biochemistry, D-14195 Berlin, Germany.
authors.forename: Alexander K C
authors.initials: AK
authors.lastname: Ulrich
authors.affiliation: Max-Planck Institute for Molecular Genetics, Otto-Warburg Laboratory, D-14195 Berlin, Germany.
authors.forename: Luise
authors.initials: L
authors.lastname: Apelt
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of Cellular Biochemistry, D-37077 Göttingen, Germany.
authors.forename: Cindy L
authors.initials: CL
authors.lastname: Will
authors.affiliation: Freie Universität Berlin, Laboratory of Structural Biochemistry, D-14195 Berlin, Germany.
authors.forename: Natascha
authors.initials: N
authors.lastname: Bartlick
authors.affiliation: Freie Universität Berlin, Laboratory of Structural Biochemistry, D-14195 Berlin, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Seeger
authors.affiliation: Freie Universität Berlin, Laboratory of Structural Biochemistry, D-14195 Berlin, Germany.
authors.forename: Gert
authors.initials: G
authors.lastname: Weber
authors.affiliation: Max Planck Institute for Biophysical Chemistry, Department of Cellular Biochemistry, D-37077 Göttingen, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lührmann
authors.affiliation: Max-Planck Institute for Molecular Genetics, Otto-Warburg Laboratory, D-14195 Berlin, Germany University of Graz, Institute of Pharmaceutical Sciences (IPW), Pharmaceutical Chemistry, A-8010 Graz, Austria.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Stelzl
authors.affiliation: Freie Universität Berlin, Laboratory of Structural Biochemistry, D-14195 Berlin, Germany.
authors.forename: Markus C
authors.initials: MC
authors.lastname: Wahl
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D000072260:RNA Splicing Factors; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D017381:Spliceosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C076234:PRP38 protein, S cerevisiae; D021122:Protein Subunits; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins
keywords: pre-mRNA processing factor 38; pre-mRNA splicing; protein–protein interactions; spliceosomal B complex; spliceosome; yeast two-hybrid analysis