title: SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing.
abstract: Pre-mRNA splicing is catalyzed by the spliceosome, a multi-megadalton ribonucleoprotein machine. Previous work from our laboratory revealed the splicing factor SRSF1 as a regulator of the SUMO pathway, leading us to explore a connection between this pathway and the splicing machinery. We show here that addition of a recombinant SUMO-protease decreases the efficiency of pre-mRNA splicing in vitro. By mass spectrometry analysis of anti-SUMO immunoprecipitated proteins obtained from purified splicing complexes formed along the splicing reaction, we identified spliceosome-associated SUMO substrates. After corroborating SUMOylation of Prp3 in cultured cells, we defined Lys 289 and Lys 559 as bona fide SUMO attachment sites within this spliceosomal protein. We further demonstrated that a Prp3 SUMOylation-deficient mutant while still capable of interacting with U4/U6 snRNP components, is unable to co-precipitate U2 and U5 snRNA and the spliceosomal proteins U2-SF3a120 and U5-Snu114. This SUMOylation-deficient mutant fails to restore the splicing of different pre-mRNAs to the levels achieved by the wild type protein, when transfected into Prp3-depleted cultured cells. This mutant also shows a diminished recruitment to active spliceosomes, compared to the wild type protein. These findings indicate that SUMO conjugation plays a role during the splicing process and suggest the involvement of Prp3 SUMOylation in U4/U6•U5 tri-snRNP formation and/or recruitment.
authors.affiliation: Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Fisiología, Biología Molecular y Celular, Buenos Aires, Argentina.|CONICET-Universidad de Buenos Aires, Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE), Buenos Aires, Argentina.
authors.forename: Berta
authors.initials: B
authors.lastname: Pozzi
authors.affiliation: Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Fisiología, Biología Molecular y Celular, Buenos Aires, Argentina.|CONICET-Universidad de Buenos Aires, Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE), Buenos Aires, Argentina.
authors.forename: Laureano
authors.initials: L
authors.lastname: Bragado
authors.affiliation: Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
authors.forename: Cindy L
authors.initials: CL
authors.lastname: Will
authors.affiliation: Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Fisiología, Biología Molecular y Celular, Buenos Aires, Argentina.|CONICET-Universidad de Buenos Aires, Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE), Buenos Aires, Argentina.
authors.forename: Pablo
authors.initials: P
authors.lastname: Mammi
authors.affiliation: Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Fisiología, Biología Molecular y Celular, Buenos Aires, Argentina.|CONICET-Universidad de Buenos Aires, Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE), Buenos Aires, Argentina.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Risso
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 Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Lührmann
authors.affiliation: Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Fisiología, Biología Molecular y Celular, Buenos Aires, Argentina.|CONICET-Universidad de Buenos Aires, Instituto de Fisiología, Biología Molecular y Neurociencias (IFIBYNE), Buenos Aires, Argentina.
authors.forename: Anabella
authors.initials: A
authors.lastname: Srebrow
mesh_terms: D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000502:physiology; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000737:chemistry / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*; D058207:Sumoylation
chemical_list: D009687:Nuclear Proteins; C108507:PRPF3 protein, human; D012322:RNA Precursors; D012333:RNA, Messenger; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; C404512:SENP1 protein, human; D003546:Cysteine Endopeptidases