Involvement of SUMO conjugation in pre-mRNA splicing

SREBROW ANABELLA

Poznan

Simposio; 4th Post-Eurasnet Symposium on RNA alternative splicing; 2016

EURANET

Pre-mRNA splicing is catalyzed by the spliceosome, a multi-megadalton ribonucleoprotein machine comprised of five small nuclear ribonucleoprotein particles (snRNPs) and numerous associated proteins. While protein modification by phosphorylation, acetylation and ubiquitylation has been reported to influence spliceosome assembly and dynamics, hardly anything is known about the regulation of proteins involved in the splicing process by SUMO conjugation. This is indeed curious considering that proteomic studies have identified the RNA- binding proteins as one of the major groups among SUMO conjugation substrates, including splicing auxiliary factors and spliceosome components, and furthermore SUMO conjugation has been shown to regulate other aspects of mRNA maturation such as pre-mRNA 3?end processing. Previous work from our laboratory revealed a novel role for the splicing factor SRSF1 as a regulator of the SUMO conjugation pathway both in vitro and in living cells. We demonstrated that SRSF1 displays certain characteristics of SUMO E3 ligases and also affects the activity of an already known member of this group, PIAS1. These results lead us to explore a connection between the SUMO pathway and the splicing machinery. We have recently found that addition of a recombinant SUMO-protease decreases pre-mRNA splicing efficiency as evaluated by an in vitro splicing system. By performing anti-SUMO immunoprecipitation of purified splicing complexes formed at different time points of the splicing reaction, followed by mass spectrometry analysis, we have identified several spliceosomal proteins as SUMO conjugation substrates. We have focused on one of them, the spliceosomal component Prp3, mapping SUMO attachment sites within this protein and generating a Prp3 SUMOylation-deficient mutant by site directed mutagenesis. When overexpressed in cultured cells, this mutant fails to increase splicing efficiency, shows reduced interaction with certain snRNP components and a diminished recruitment to active spliceosomes, compared to the wt protein. Our findings clearly indicate that SUMO conjugation plays a role throughout the splicing process and suggest an involvement of Prp3 SUMOylation during tri-snRNP formation.