CONICET | Buscador de Institutos y Recursos Humanos

Post-translational modification by ubiquitin and ubiquitin-like proteins exert many non-proteasomal functions. Ubiquitin chains linked by residues other than K48 have been known to mediate important protein-protein interactions, as it is the case for the ubiquitin-like protein SUMO. In both cases, the regulation involves ubiquitin and SUMO binding entities such as ubiquitin binding domains (UBDs) and SUMO interacting motifs (SIMs). In particular for SUMO, proteomic analyses have identified multiple putative sumoylation targets in complexes functioning in almost all aspects of mRNA metabolism, including capping, splicing and polyadenylation of mRNA precursors. SUMO is also involved in regulating RNA editing and RNA binding by hnRNP proteins, and recent reports have suggested the involvement of the SUMO pathway in mRNA export. Recently, we have shown that the SR protein SRSF1 enhances SUMO conjugation to mRNA processing factors. As for ubiquitin, a general role in spliceosome assembly has been shown and it has also been reported that the U4 snRNP component hPrp3 ubiquitylation is important for its recognition by hPrp8 and consequent tri-snRNP formation.  In the present work we have confirmed that the U5 snRNP components hPrp28 and hSnu114, the U4 snRNP component hPrp3, and SmD2 are both bona fide SUMO and ubiquitin substrates. In all cases, ubiquitin conjugation is MG132-independent suggesting a proteasome-independent role for this modification. Moreover, we have found that SRSF1 enhances SUMO conjugation to hPrp28 and hSnu114, while it does not affect SUMO conjugation to hPrp3. On the other hand, preliminary results indicate that SRSF1 over-expression inhibits hPrp3 and hSnu114 ubiquitylation. We are currently mapping the SUMO and ubiquitin attachment sites on each substrate protein and trying to determine the functional role of these modifications on the spliceosome assembly/disassembly cycle.