SUMO conjugation to spliceosomal proteins is required for efficient pre-mRNA splicing

POZZI, BERTA; BRAGADO, LAUREANO; WILL, CINDY L.; MAMMI, PABLO; RISSO, GUILLERMO; URLAUB, HENNING; LÜHRMANN, REINHARD; SREBROW, ANABELLA

NUCLEIC ACIDS RESEARCH

OXFORD UNIV PRESS

Año: 2017 vol. 45 p. 6729 - 6745

0305-1048

Pre-mRNA splicing is catalyzed by the spliceosome, a multi-megadalton ribonucleoprotein machine. Pre- vious work from our laboratory revealed the splic- ing factor SRSF1 as a regulator of the SUMO path- way, 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 re- action, 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 spliceo- somal protein. We further demonstrated that a Prp3 SUMOylation-deficient mutant while still capable of interacting with U4/U6 snRNP components, is un- able 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 spliceo- somes, compared to the wild type protein. These findings indicate that SUMO conjugation plays a role during the splicing process and suggest the involve- ment of Prp3 SUMOylation in U4/U6?U5 tri-snRNP formation and/or recruitment.