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

Abstract

Pre-mRNA splicing is catalyzed by the spliceosome, a multi-megadalton ribonucleoprotein machine. Previous work from our laboratory revealed the splicing factor SRSF1 as a regulator of the SUMO pathway, 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 reaction, 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 spliceosomal protein. We further demonstrated that a Prp3 SUMOylation-deficient mutant while still capable of interacting with U4/U6 snRNP components, is unable 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 spliceosomes, compared to the wild type protein. These findings indicate that SUMO conjugation plays a role during the splicing process and suggest the involvement of Prp3 SUMOylation in U4/U6•U5 tri-snRNP formation and/or recruitment.

Characterisation of the biflavonoid hinokiflavone as a pre-mRNA splicing modulator that inhibits SENP

“…A recent study by Pozzi et al, showed that several splicing factors were SUMO2-modified at different stages of the splicing reaction in vitro, including several components of U2 snRNP (SF3B1, SF3B3, SF3A1, SF3A2, SF3A3). It was further shown that inhibition of PRPF3 SUMOylation prevented the interaction of the U4/U6 di-snRNP with U5 to form the tri-snRNP ( Pozzi et al, 2017 ). Interestingly, we note that hinokiflavone also caused an increase in SUMO2 modification (>4 fold) on lysine 376 in PRPF3 ( Supplementary file 1 ).…”

“…The core splicing machinery, spliceosome assembly pathway and reaction mechanism is highly conserved across eukaryotes. Protein splicing factors have been shown to be targets for post translational modifications, including acetylation, phosphorylation and SUMOylation, which can affect the efficiency of spliceosome assembly and splicing ( Chen and Moore, 2014 ; Pozzi et al, 2017 ). In mammalian cells the splicing machinery typically shows a punctate, or ‘speckled’ localisation pattern in the nucleus, with snRNPs also located in bright nuclear foci, which are termed Cajal bodies ( Lamond and Spector, 2003 ).…”

Brassica Rapa SR45a Regulates Drought Tolerance via the Alternative Splicing of Target Genes

“…In response to stress, plant SR genes initially undergo alternative splicing prior to the alternative splicing of the target genes, including stress response genes [6]. The stress-dependent alternative splicing of SR proteins, which are key splice factors for protein-encoding genes, have distinct biological functions [9,10], suggesting the existence of a posttranscriptional level regulatory mechanism for stress response genes [11].…”

Regulating Divergent Transcriptomes through mRNA Splicing and Its Modulation Using Various Small Compounds

“…Consequently, hinokiflavone prevented transition of the spliceosome from its A to B complexes, resulting in global splicing modulation. It was also reported that inhibition of pre-mRNA processing factor 3 (PRPF3) SUMOylation prevented the interaction of U4/U6 di-snRNP with U5 to form tri-snRNP [175]. These reports suggested the notion that SUMOylation cycles were involved in an analog of isoginkgetin [173] induce SUMOylation of splicing factors by inhibiting SENP1 activity, and prevent transition of the spliceosome…”