SUMO CONJUGATION TO SPLICEOSOMAL PROTEINS

POZZI, BERTA; RISSO, GUILLERMO; MAMMI, PABLO; LÜHRMANN, REINHARD; SREBROW, ANABELLA

Buenos Aires

Conferencia; EMBO Conference on Ubiquitin & ubiquitin-like proteins: At the crossroads from chromatin to protein.; 2014

Most eukaryotic genes transcribed by RNA polymerase II give rise to precursor messenger RNAs (pre-mRNAs) containing exons and introns. Among several steps of mRNA maturation, splicing is the process by which introns are removed from the pre-mRNA and consecutive exons are joined. This process is carried out by the ?spliceosome?, a complex of small nuclear ribonucleoprotein particles (snRNPs) and associated factors that assemble on pre-mRNA in a precise and stepwise manner, recognizing sequence-specific splice sites primarily located at the intron- exon boundaries.Proteomic studies have identified the RNA-binding proteins as one of the major groups among small ubiquitin-related modifier (SUMO) conjugation substrates, including splicing-related proteins. In fact, ubiquitylation of snRNP components modulates spliceosome assembly. Furthermore, SUMO conjugation regulates pre-mRNA 3? end processing and ARN editing. However, a possible role for SUMO conjugation in splicing regulation hasn?t yet been explored.Previous work from our laboratory has demonstrated that the splicing factor SRSF1 is a regulator of SUMO conjugation, providing a provocative link between the splicing and SUMO machineries.We propose to analyze the modification of snRNP components by SUMO conjugation and to study the role of SUMO conjugation on spliceosome assembly and, consequently, on splicing regulation. So far, we have found that knock-down of the E2 conjugation enzyme of the SUMO pathway, Ubc9, increases splicing efficiency in culture cells. In addition, Mass Spec analysis of anti-SUMO immunoprecipitated proteins obtained from pre-mRNA-bound complexes at different steps of the splicing reaction allowed us to identify several SUMO substrates. Focusing on the spliceosomal protein components, we validated SUMO conjugation to Prp3 (component of the U4/U6.U5 tri-snRNP), Prp28 and Snu-114 (components of U5 snRNP), in cultured cells. Remarkably, we have found that SRSF1 is involved in heat shock enhanced-SUMO conjugation to Prp3 and Snu114. Furthermore, preliminary results indicate that SRSF1 over-expression enhances SUMO conjugation to Prp28 and inhibits Prp3 and Snu114 ubiquitylation. Since the analysis of the primary sequences of these distinct members of snRNPs proteins allows identifying putative SUMO conjugation sites, we intend to generate mutant versions of these proteins unable to conjugate to SUMO in an attempt to evaluate the impact of SUMOylation in spliceosome assembly and catalytic activity.