title: A spliceosome intermediate with loosely associated tri-snRNP accumulates in the absence of Prp28 ATPase activity.
abstract: The precise role of the spliceosomal DEAD-box protein Prp28 in higher eukaryotes remains unclear. We show that stable tri-snRNP association during pre-catalytic spliceosomal B complex formation is blocked by a dominant-negative hPrp28 mutant lacking ATPase activity. Complexes formed in the presence of ATPase-deficient hPrp28 represent a novel assembly intermediate, the pre-B complex, that contains U1, U2 and loosely associated tri-snRNP and is stalled before disruption of the U1/5'ss base pairing interaction, consistent with a role for hPrp28 in the latter. Pre-B and B complexes differ structurally, indicating that stable tri-snRNP integration is accompanied by substantial rearrangements in the spliceosome. Disruption of the U1/5'ss interaction alone is not sufficient to bypass the block by ATPase-deficient hPrp28, suggesting hPrp28 has an additional function at this stage of splicing. Our data provide new insights into the function of Prp28 in higher eukaryotes, and the requirements for stable tri-snRNP binding during B complex formation.
authors.affiliation: Department of Cellular Biochemistry, MPI for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Boesler
authors.affiliation: Department of Cellular Biochemistry, MPI for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
authors.forename: Norbert
authors.initials: N
authors.lastname: Rigo
authors.affiliation: Department of Cellular Biochemistry, MPI for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
authors.forename: Maria M
authors.initials: MM
authors.lastname: Anokhina
authors.affiliation: Department of Molecular Structural Biology, Institute for Microbiology and Genetics, GZMB, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany.
authors.forename: Marcel J
authors.initials: MJ
authors.lastname: Tauchert
authors.affiliation: Department of Cellular Biochemistry, MPI for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
authors.forename: Dmitry E
authors.initials: DE
authors.lastname: Agafonov
authors.affiliation: Department of Cellular Biochemistry, MPI for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
authors.forename: Berthold
authors.initials: B
authors.lastname: Kastner
authors.affiliation: Bioanalytical Mass Spectrometry Group, MPI for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.|Bioanalytics Group, Institute for Clinical Chemistry, University Medical Center Göttingen, Robert-Koch-Straße 40, D-37075 Göttingen, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.affiliation: Department of Molecular Structural Biology, Institute for Microbiology and Genetics, GZMB, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Ficner
authors.affiliation: Department of Cellular Biochemistry, MPI for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
authors.forename: Cindy L
authors.initials: CL
authors.lastname: Will
authors.affiliation: Department of Cellular Biochemistry, MPI for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lührmann
mesh_terms: D055162:Biocatalysis; D003432:Cross-Linking Reagents / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D012313:RNA / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D003432:Cross-Linking Reagents; D017411:Ribonucleoproteins, Small Nuclear; D012313:RNA; C498290:DDX23 protein, human; D053487:DEAD-box RNA Helicases