Artículo

Pozzi, B.; Bragado, L.; Will, C.L.; Mammi, P.; Risso, G.; Urlaub, H.; Lührmann, R.; Srebrow, A. "SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing" (2017) Nucleic Acids Research. 45(11):6729-6745
Estamos trabajando para incorporar este artículo al repositorio
Consulte el artículo en la página del editor
Consulte la política de Acceso Abierto del editor

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/U6U5 tri-snRNP formation and/or recruitment. © The Authors 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Registro:

Documento: Artículo
Título:SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing
Autor:Pozzi, B.; Bragado, L.; Will, C.L.; Mammi, P.; Risso, G.; Urlaub, H.; Lührmann, R.; Srebrow, A.
Filiación:Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Fisiologia, Biologia Molecular y Celular, Buenos Aires, Argentina
CONICET-Universidad de Buenos Aires, Instituto de Fisiologia Biologia Molecular y Neurociencias (IFIBYNE), Buenos Aires, Argentina
Department of Cellular Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen, D-37077, Germany
Bioanalytical Mass Spectrometry Group, MPI for Biophysical Chemistry, Am Fassberg 11, Göttingen, D-37077, Germany
Bioanalytics Group, Institute for Clinical Chemistry, University Medical Center Göttingen, Robert-Koch-Strabe 40, Göttingen, D-37075, Germany
Palabras clave: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
Año:2017
Volumen:45
Número:11
Página de inicio:6729
Página de fin:6745
DOI: http://dx.doi.org/10.1093/nar/gkx213
Título revista:Nucleic Acids Research
Título revista abreviado:Nucleic Acids Res.
ISSN:03051048
CODEN:NARHA
CAS:lysine, 56-87-1, 6899-06-5, 70-54-2; cysteine proteinase, 37353-41-6; Cysteine Endopeptidases; Nuclear Proteins; PRPF3 protein, human; Ribonucleoprotein, U4-U6 Small Nuclear; RNA Precursors; RNA, Messenger; SENP1 protein, human
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_03051048_v45_n11_p6729_Pozzi

Referencias:

  • Will, C.L., Luhrmann, R., Spliceosome structure and function (2011) Cold Spring Harb. Perspect. Biol., 3, p. a003707
  • Fica, S.M., Tuttle, N., Novak, T., Li, N.S., Lu, J., Koodathingal, P., Dai, Q., Piccirilli, J.A., RNA catalyses nuclear pre-mRNA splicing (2013) Nature, 503, pp. 229-234
  • Anokhina, M., Bessonov Miao, S.Z., Westhof, E., Hartmuth, K., Luhrmann, R., RNA structure analysis of human spliceosomes reveals a compact 3D arrangement of snRNAs at the catalytic core (2013) EMBO J., 32, pp. 2804-2818
  • Galej, W.P., Oubridge, C., Newman, A.J., Nagai, K., Crystal structure of Prp8 reveals active site cavity of the spliceosome (2013) Nature, 493, pp. 638-643
  • Wahl, M.C., Will, C.L., Luhrmann, R., The spliceosome: Design principles of a dynamic RNP machine (2009) Cell, 136, pp. 701-718
  • Raghunathan, P.L., Guthrie, C., RNA unwinding in U4/U6 snRNPs requires ATP hydrolysis and the DEIH-box splicing factor Brr2 (1998) Curr. Biol., 8, pp. 847-855
  • Bell, M., Schreiner, S., Damianov, A., Reddy, R., Bindereif, A., P110, a novel human U6 snRNP protein and U4/U6 snRNP recycling factor (2002) EMBO J., 21, pp. 2724-2735
  • Raghunathan, P.L., Guthrie, C., A spliceosomal recycling factor that reanneals U4 and U6 small nuclear ribonucleoprotein particles (1998) Science, 279, pp. 857-860
  • Anthony, J.G., Weidenhammer, E.M., Woolford, J.L., Jr., The yeast Prp3 protein is a U4/U6 snRNP protein necessary for integrity of the U4/U6 snRNP and the U4/U6.U5 tri-snRNP (1997) RNA, 3, pp. 1143-1152
  • Ayadi, L., Callebaut, I., Saguez, C., Villa, T., Mornon, J.P., Banroques, J., Functional and structural characterization of the prp3 binding domain of the yeast prp4 splicing factor (1998) J. Mol. Biol., 284, pp. 673-687
  • Gonzalez-Santos, J.M., Wang, A., Jones, J., Ushida, C., Liu, J., Hu, J., Central region of the human splicing factor Hprp3p interacts with Hprp4p (2002) J. Biol. Chem., 277, pp. 23764-23772
  • Liu, S., Rauhut, R., Vornlocher, H.P., Luhrmann, R., The network of protein-protein interactions within the human U4/U6.U5 tri-snRNP (2006) RNA, 12, pp. 1418-1430
  • Liu, S., Mozaffari-Jovin, S., Wollenhaupt, J., Santos, K.F., Theuser, M., Dunin-Horkawicz, S., Fabrizio, P., Wahl, M.C., A composite double-/single-stranded RNA-binding region in protein Prp3 supports tri-snRNP stability and splicing (2015) Elife, 4, p. e07320
  • Long, J.C., Caceres, J.F., The SR protein family of splicing factors: Master regulators of gene expression (2009) Biochem. J., 417, pp. 15-27
  • Pelisch, F., Gerez, J., Druker, J., Schor, I.E., Munoz, M.J., Risso, G., Petrillo, E., Arzt, E., The serine/arginine-rich protein SF2/ASF regulates protein sumoylation (2010) Proc. Natl. Acad. Sci. U.S.A., 107, pp. 16119-16124
  • Becker, J., Barysch, S.V., Karaca, S., Dittner, C., Hsiao, H.H., Berriel Diaz, M., Herzig, S., Melchior, F., Detecting endogenous SUMO targets in mammalian cells and tissues (2013) Nat. Struct. Mol. Biol., 20, pp. 525-531
  • Gareau, J.R., Lima, C.D., The SUMO pathway: Emerging mechanisms that shape specificity, conjugation and recognition (2010) Nat. Rev. Mol. Cell Biol., 11, pp. 861-871
  • Geiss-Friedlander, R., Melchior, F., Concepts in sumoylation: A decade on (2007) Nat. Rev. Mol. Cell. Biol., 8, pp. 947-956
  • Impens, F., Radoshevich, L., Cossart, P., Ribet, D., Mapping of SUMO sites and analysis of SUMOylation changes induced by external stimuli (2014) Proc. Natl. Acad. Sci. U.S.A., 111, pp. 12432-12437
  • Liang, Y.C., Lee, C.C., Yao, Y.L., Lai, C.C., Schmitz, M.L., Yang, W.M., SUMO5, a novel poly-SUMO isoform, regulates PML nuclear bodies (2016) Sci. Rep., 6, p. 26509
  • Matic, I., Schimmel, J., Hendriks, I.A., Van Santen, M.A., Van De Rijke, F., Van Dam, H., Gnad, F., Vertegaal, A.C., Site-specific identification of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic cluster SUMOylation motif (2010) Mol. Cell, 39, pp. 641-652
  • Hickey, C.M., Wilson, N.R., Hochstrasser, M., Function and regulation of SUMO proteases (2012) Nat. Rev. Mol. Cell. Biol., 13, pp. 755-766
  • Jentsch, S., Psakhye, I., Control of nuclear activities by substrate-selective and protein-group SUMOylation (2013) Annu. Rev. Genet., 47, pp. 167-186
  • Geoffroy, M.C., Hay, R.T., An additional role for SUMO in ubiquitin-mediated proteolysis (2009) Nat. Rev. Mol. Cell. Biol., 10, pp. 564-568
  • Johnson, P.R., Hochstrasser, M., SUMO-1: Ubiquitin gains weight (1997) Trends Cell Biol., 7, pp. 408-413
  • Nacerddine, K., Lehembre, F., Bhaumik, M., Artus, J., Cohen-Tannoudji, M., Babinet, C., Pandolfi, P.P., Dejean, A., The SUMO pathway is essential for nuclear integrity and chromosome segregation in mice (2005) Dev. Cell, 9, pp. 769-779
  • Panse, V.G., Kressler, D., Pauli, A., Petfalski, E., Gnadig, M., Tollervey, D., Hurt, E., Formation and nuclear export of preribosomes are functionally linked to the small-ubiquitin-related modifier pathway (2006) Traffic, 7, pp. 1311-1321
  • Westman, B.J., Verheggen, C., Hutten, S., Lam, Y.W., Bertrand, E., Lamond, A.I., A proteomic screen for nucleolar SUMO targets shows SUMOylation modulates the function of Nop5/Nop58 (2010) Mol. Cell, 39, pp. 618-631
  • Finkbeiner, E., Haindl, M., Raman, N., Muller, S., SUMO routes ribosome maturation (2011) Nucleus, 2, pp. 527-532
  • Cubenas-Potts, C., Matunis, M.J., SUMO: A multifaceted modifier of chromatin structure and function (2013) Dev. Cell, 24, pp. 1-12
  • Ihara, M., Stein, P., Schultz, R.M., UBE2I (UBC9), a SUMO-conjugating enzyme, localizes to nuclear speckles and stimulates transcription in mouse oocytes (2008) Biol. Reprod., 79, pp. 906-913
  • Spector, D.L., Lamond, A.I., Nuclear speckles (2011) Cold Spring Harb. Perspect. Biol., 3, p. a000646
  • Schulz, S., Chachami, G., Kozaczkiewicz, L., Winter, U., Stankovic-Valentin, N., Haas, P., Hofmann, K., Wittbrodt, J., Ubiquitin-specific protease-like 1 (USPL1) is a SUMO isopeptidase with essential, non-catalytic functions (2012) EMBO Rep., 13, pp. 930-938
  • Blomster, H.A., Hietakangas Wu, V.J., Kouvonen, P., Hautaniemi, S., Sistonen, L., Novel proteomics strategy brings insight into the prevalence of SUMO-2 target sites (2009) Mol. Cell. Proteomics, 8, pp. 1382-1390
  • Vertegaal, A.C., Ogg, S.C., Jaffray, E., Rodriguez, M.S., Hay, R.T., Andersen, J.S., Mann, M., Lamond, A.I., A proteomic study of SUMO-2 target proteins (2004) J. Biol. Chem., 279, pp. 33791-33798
  • Golebiowski, F., Matic, I., Tatham, M.H., Cole, C., Yin, Y., Nakamura, A., Cox, J., Hay, R.T., System-wide changes to SUMO modifications in response to heat shock (2009) Sci. Signal., 2, p. ra24
  • Vethantham, V., Rao, N., Manley, J.L., Sumoylation modulates the assembly and activity of the pre-mRNA 3- processing complex (2007) Mol. Cell. Biol., 27, pp. 8848-8858
  • Desterro, J.M., Keegan, L.P., Jaffray, E., Hay, R.T., O'Connell, M.A., Carmo-Fonseca, M., SUMO-1 modification alters ADAR1 editing activity (2005) Mol. Biol. Cell, 16, pp. 5115-5126
  • Rappsilber, J., Ryder, U., Lamond, A.I., Mann, M., Large-scale proteomic analysis of the human spliceosome (2002) Genome Res., 12, pp. 1231-1245
  • Song, E.J., Werner, S.L., Neubauer, J., Stegmeier, F., Aspden, J., Rio, D., Harper, J.W., Rape, M., The Prp19 complex and the Usp4Sart3 deubiquitinating enzyme control reversible ubiquitination at the spliceosome (2010) Genes Dev., 24, pp. 1434-1447
  • Zillmann, M., Zapp, M.L., Berget, S.M., Gel electrophoretic isolation of splicing complexes containing U1 small nuclear ribonucleoprotein particles (1988) Mol. Cell Biol., 8, pp. 814-821
  • Dignam, J.D., Lebovitz, R.M., Roeder, R.G., Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei (1983) Nucleic Acids Res., 11, pp. 1475-1489
  • Das, R., Reed, R., Resolution of the mammalian e complex and the ATP-dependent spliceosomal complexes on native agarose mini-gels (1999) RNA, 5, pp. 1504-1508
  • Blaustein, M., Pelisch, F., Tanos, T., Munoz, M.J., Wengier, D., Quadrana, L., Sanford, J.R., Caceres, J.F., Concerted regulation of nuclear and cytoplasmic activities of SR proteins by AKT (2005) Nat. Struct. Mol. Biol., 12, pp. 1037-1044
  • Makarova, O.V., Makarov, E.M., Urlaub, H., Will, C.L., Gentzel, M., Wilm, M., Luhrmann, R., A subset of human 35S U5 proteins, including Prp19, function prior to catalytic step 1 of splicing (2004) EMBO J., 23, pp. 2381-2391
  • Will, C.L., Urlaub, H., Achsel, T., Gentzel, M., Wilm, M., Luhrmann, R., Characterization of novel SF3b and 17S U2 snRNP proteins, including a human Prp5p homologue and an SF3b DEAD-box protein (2002) EMBO J., 21, pp. 4978-4988
  • Fabrizio, P., Laggerbauer, B., Lauber, J., Lane, W.S., Luhrmann, R., An evolutionarily conserved U5 snRNP-specific protein is a GTP-binding factor closely related to the ribosomal translocase EF-2 (1997) EMBO J., 16, pp. 4092-4106
  • Tatham, M.H., Rodriguez, M.S., Xirodimas, D.P., Hay, R.T., Detection of protein SUMOylation in vivo (2009) Nat. Protoc., 4, pp. 1363-1371
  • Pillai, R.S., Will, C.L., Luhrmann, R., Schumperli, D., Muller, B., Purified U7 snRNPs lack the Sm proteins D1 and D2 but contain Lsm10, a new 14 kDa Sm D1-like protein (2001) EMBO J., 20, pp. 5470-5479
  • Flotho, A., Melchior, F., Sumoylation: A regulatory protein modification in health and disease (2013) Annu. Rev. Biochem., 82, pp. 357-385
  • Pelisch, F., Pozzi, B., Risso, G., Munoz, M.J., Srebrow, A., DNA damage-induced heterogeneous nuclear ribonucleoprotein K sumoylation regulates p53 transcriptional activation (2012) J. Biol. Chem., 287, pp. 30789-30799
  • Listerman, I., Sapra, A.K., Neugebauer, K.M., Cotranscriptional coupling of splicing factor recruitment and precursor messenger RNA splicing in mammalian cells (2006) Nat. Struct. Mol. Biol., 13, pp. 815-822
  • Dybkov, O., Will, C.L., Deckert, J., Behzadnia, N., Hartmuth, K., Luhrmann, R., U2 snRNA-protein contacts in purified human 17S U2 snRNPs and in spliceosomal A and B complexes (2006) Mol. Cell Biol., 26, pp. 2803-2816
  • Absmeier, E., Wollenhaupt, J., Mozaffari-Jovin, S., Becke, C., Lee, C.T., Preussner, M., Heyd, F., Santos, K.F., The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation (2015) Genes Dev., 29, pp. 2576-2587
  • Bartels, C., Klatt, C., Luhrmann, R., Fabrizio, P., The ribosomal translocase homologue Snu114p is involved in unwinding U4/U6 RNA during activation of the spliceosome (2002) EMBO Rep., 3, pp. 875-880
  • Wahl, M.C., Luhrmann, R., SnapShot: Spliceosome dynamics i (2015) Cell, 161, p. 1474
  • Hay, R.T., SUMO: A history of modification (2005) Mol. Cell, 18, pp. 1-12
  • Corrionero, A., Minana, B., Valcarcel, J., Reduced fidelity of branch point recognition and alternative splicing induced by the anti-tumor drug spliceostatin A (2011) Genes Dev., 25, pp. 445-459
  • Papasaikas, P., Tejedor, J.R., Vigevani, L., Valcarcel, J., Functional splicing network reveals extensive regulatory potential of the core spliceosomal machinery (2015) Mol. Cell, 57, pp. 7-22
  • Bellare, P., Small, E.C., Huang, X., Wohlschlegel, J.A., Staley, J.P., Sontheimer, E.J., A role for ubiquitin in the spliceosome assembly pathway (2008) Nat. Struct. Mol. Biol., 15, pp. 444-451
  • Gunderson, F.Q., Merkhofer, E.C., Johnson, T.L., Dynamic histone acetylation is critical for cotranscriptional spliceosome assembly and spliceosomal rearrangements (2011) Proc. Natl. Acad. Sci. U.S.A., 108, pp. 2004-2009
  • Choudhary, C., Kumar, C., Gnad, F., Nielsen, M.L., Rehman, M., Walther, T.C., Olsen, J.V., Mann, M., Lysine acetylation targets protein complexes and co-regulates major cellular functions (2009) Science, 325, pp. 834-840
  • Ward, A.J., Cooper, T.A., The pathobiology of splicing (2010) J. Pathol., 220, pp. 152-163
  • Wahl, M.C., Luhrmann, R., SnapShot: Spliceosome dynamics III (2015) Cell, 162, p. 690
  • Bonnal, S., Vigevani, L., Valcarcel, J., The spliceosome as a target of novel antitumour drugs (2012) Nat. Rev. Drug Discov., 11, pp. 847-859
  • Mozaffari-Jovin, S., Wandersleben, T., Santos, K.F., Will, C.L., Luhrmann, R., Wahl, M.C., Novel regulatory principles of the spliceosomal Brr2 RNA helicase and links to retinal disease in humans (2014) RNA Biol., 11, pp. 298-312
  • Ramsay, A.J., Rodriguez, D., Villamor, N., Kwarciak, A., Tejedor, J.R., Valcarcel, J., Lopez-Guillermo, A., Campo, E., Frequent somatic mutations in components of the RNA processing machinery in chronic lymphocytic leukemia (2013) Leukemia, 27, pp. 1600-1603
  • Yoshida, K., Sanada, M., Shiraishi, Y., Nowak, D., Nagata, Y., Yamamoto, R., Sato, Y., Nagasaki, M., Frequent pathway mutations of splicing machinery in myelodysplasia (2011) Nature, 478, pp. 64-69
  • Chakarova, C.F., Hims, M.M., Bolz, H., Abu-Safieh, L., Patel, R.J., Papaioannou, M.G., Inglehearn, C.F., Moore, A.T., Mutations in HPRP3, a third member of pre-mRNA splicing factor genes, implicated in autosomal dominant retinitis pigmentosa (2002) Hum. Mol. Genet., 11, pp. 87-92
  • Boon, K.L., Grainger, R.J., Ehsani, P., Barrass, J.D., Auchynnikava, T., Inglehearn, C.F., Beggs, J.D., Prp8 mutations that cause human retinitis pigmentosa lead to a U5 snRNP maturation defect in yeast (2007) Nat. Struct. Mol. Biol., 14, pp. 1077-1083

Citas:

---------- APA ----------
Pozzi, B., Bragado, L., Will, C.L., Mammi, P., Risso, G., Urlaub, H., Lührmann, R.,..., Srebrow, A. (2017) . SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing. Nucleic Acids Research, 45(11), 6729-6745.
http://dx.doi.org/10.1093/nar/gkx213
---------- CHICAGO ----------
Pozzi, B., Bragado, L., Will, C.L., Mammi, P., Risso, G., Urlaub, H., et al. "SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing" . Nucleic Acids Research 45, no. 11 (2017) : 6729-6745.
http://dx.doi.org/10.1093/nar/gkx213
---------- MLA ----------
Pozzi, B., Bragado, L., Will, C.L., Mammi, P., Risso, G., Urlaub, H., et al. "SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing" . Nucleic Acids Research, vol. 45, no. 11, 2017, pp. 6729-6745.
http://dx.doi.org/10.1093/nar/gkx213
---------- VANCOUVER ----------
Pozzi, B., Bragado, L., Will, C.L., Mammi, P., Risso, G., Urlaub, H., et al. SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing. Nucleic Acids Res. 2017;45(11):6729-6745.
http://dx.doi.org/10.1093/nar/gkx213