SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing
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 t...
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2017
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_03051048_v45_n11_p6729_Pozzi http://hdl.handle.net/20.500.12110/paper_03051048_v45_n11_p6729_Pozzi |
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paper:paper_03051048_v45_n11_p6729_Pozzi2023-06-08T15:30:32Z SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing lysine Prp3 protein small nuclear ribonucleoprotein SUMO protein U2 SF3a120 protein U5 Snu114 protein unclassified drug cysteine proteinase messenger RNA nuclear protein PRPF3 protein, human RNA precursor SENP1 protein, human small nuclear ribonucleoprotein Article binding site controlled study human human cell in vitro study mass spectrometry mutant priority journal protein binding protein protein interaction RNA splicing spliceosome sumoylation wild type chemistry HEK293 cell line HeLa cell line metabolism physiology spliceosome sumoylation Cysteine Endopeptidases HEK293 Cells HeLa Cells Humans Nuclear Proteins Ribonucleoprotein, U4-U6 Small Nuclear RNA Precursors RNA Splicing RNA, Messenger Spliceosomes Sumoylation 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/U6U5 tri-snRNP formation and/or recruitment. © The Authors 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. 2017 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_03051048_v45_n11_p6729_Pozzi http://hdl.handle.net/20.500.12110/paper_03051048_v45_n11_p6729_Pozzi |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
lysine Prp3 protein small nuclear ribonucleoprotein SUMO protein U2 SF3a120 protein U5 Snu114 protein unclassified drug cysteine proteinase messenger RNA nuclear protein PRPF3 protein, human RNA precursor SENP1 protein, human small nuclear ribonucleoprotein Article binding site controlled study human human cell in vitro study mass spectrometry mutant priority journal protein binding protein protein interaction RNA splicing spliceosome sumoylation wild type chemistry HEK293 cell line HeLa cell line metabolism physiology spliceosome sumoylation Cysteine Endopeptidases HEK293 Cells HeLa Cells Humans Nuclear Proteins Ribonucleoprotein, U4-U6 Small Nuclear RNA Precursors RNA Splicing RNA, Messenger Spliceosomes Sumoylation |
| spellingShingle |
lysine Prp3 protein small nuclear ribonucleoprotein SUMO protein U2 SF3a120 protein U5 Snu114 protein unclassified drug cysteine proteinase messenger RNA nuclear protein PRPF3 protein, human RNA precursor SENP1 protein, human small nuclear ribonucleoprotein Article binding site controlled study human human cell in vitro study mass spectrometry mutant priority journal protein binding protein protein interaction RNA splicing spliceosome sumoylation wild type chemistry HEK293 cell line HeLa cell line metabolism physiology spliceosome sumoylation Cysteine Endopeptidases HEK293 Cells HeLa Cells Humans Nuclear Proteins Ribonucleoprotein, U4-U6 Small Nuclear RNA Precursors RNA Splicing RNA, Messenger Spliceosomes Sumoylation SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing |
| topic_facet |
lysine Prp3 protein small nuclear ribonucleoprotein SUMO protein U2 SF3a120 protein U5 Snu114 protein unclassified drug cysteine proteinase messenger RNA nuclear protein PRPF3 protein, human RNA precursor SENP1 protein, human small nuclear ribonucleoprotein Article binding site controlled study human human cell in vitro study mass spectrometry mutant priority journal protein binding protein protein interaction RNA splicing spliceosome sumoylation wild type chemistry HEK293 cell line HeLa cell line metabolism physiology spliceosome sumoylation Cysteine Endopeptidases HEK293 Cells HeLa Cells Humans Nuclear Proteins Ribonucleoprotein, U4-U6 Small Nuclear RNA Precursors RNA Splicing RNA, Messenger Spliceosomes Sumoylation |
| description |
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/U6U5 tri-snRNP formation and/or recruitment. © The Authors 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. |
| title |
SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing |
| title_short |
SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing |
| title_full |
SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing |
| title_fullStr |
SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing |
| title_full_unstemmed |
SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing |
| title_sort |
sumo conjugation to spliceosomal proteins is required for efficient pre-mrna splicing |
| publishDate |
2017 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_03051048_v45_n11_p6729_Pozzi http://hdl.handle.net/20.500.12110/paper_03051048_v45_n11_p6729_Pozzi |
| _version_ |
1768542082706702336 |