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

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

NUCLEIC ACIDS RESEARCH

OXFORD UNIV PRESS

Lugar: Oxford; Año: 2017 p. 6729 - 6745

0305-1048

Pre-mRNA splicing is catalyzed by the spliceosome,a multi-megadalton ribonucleoprotein machine. Previouswork from our laboratory revealed the splicingfactor SRSF1 as a regulator of the SUMO pathway,leading us to explore a connection betweenthis pathway and the splicing machinery. We showhere that addition of a recombinant SUMO-proteasedecreases the efficiency of pre-mRNA splicing invitro. By mass spectrometry analysis of anti-SUMOimmunoprecipitated proteins obtained from purifiedsplicing complexes formed along the splicing reaction,we identified spliceosome-associated SUMOsubstrates. After corroborating SUMOylation of Prp3in cultured cells, we defined Lys 289 and Lys 559 asbona fide SUMO attachment sites within this spliceosomalprotein. We further demonstrated that a Prp3SUMOylation-deficient mutant while still capable ofinteracting with U4/U6 snRNP components, is unableto co-precipitate U2 and U5 snRNA and thespliceosomal proteins U2-SF3a120 and U5-Snu114.This SUMOylation-deficient mutant fails to restorethe splicing of different pre-mRNAs to the levelsachieved by the wild type protein, when transfectedinto Prp3-depleted cultured cells. This mutant alsoshows a diminished recruitment to active spliceosomes,compared to the wild type protein. Thesefindings indicate that SUMO conjugation plays a roleduring the splicing process and suggest the involvementof Prp3 SUMOylation in U4/U6?U5 tri-snRNPformation and/or recruitment.