SUMO conjugation regulates the activity of the Integrator complex.

RNA polymerase II (RNAPII) transcribes small nuclear RNA (snRNA) genes in close proximity to Cajal bodies, subnuclear compartments that depend on the SUMO isopeptidase USPL1 for their assembly. We show here that overexpression of USPL1 as well as of another nuclear SUMO isopeptidase, SENP6, alters snRNA 3'-end cleavage, a process carried out by the Integrator complex. Beyond its role in snRNA biogenesis, this complex is responsible for regulating the expression of different RNAPII transcripts. While several subunits of the complex are SUMO conjugation substrates, we found that the SUMOylation of the INTS11 subunit is regulated by USPL1 and SENP6. We defined Lys381, Lys462 and Lys475 as bona fide SUMO attachment sites on INTS11 and observed that SUMOylation of this protein modulates its subcellular localization and is required for Integrator activity. Moreover, while an INTS11 SUMOylation-deficient mutant is still capable of interacting with INTS4 and INTS9, its interaction with other subunits of the complex is affected. These findings point to a regulatory role for SUMO conjugation on Integrator activity and suggest the involvement of INTS11 SUMOylation in the assembly of the complex. Furthermore, this work adds Integrator-dependent RNA processing to the growing list of cellular processes regulated by SUMO conjugation.

Dengue virus targets RBM10 deregulating host cell splicing and innate immune response.

RNA-seq experiments previously performed by our laboratories showed enrichment in intronic sequences and alterations in alternative splicing in dengue-infected human cells. The transcript of the SAT1 gene, of well-known antiviral action, displayed higher inclusion of exon 4 in infected cells, leading to an mRNA isoform that is degraded by non-sense mediated decay. SAT1 is a spermidine/spermine acetyl-transferase enzyme that decreases the reservoir of cellular polyamines, limiting viral replication. Delving into the molecular mechanism underlying SAT1 pre-mRNA splicing changes upon viral infection, we observed lower protein levels of RBM10, a splicing factor responsible for SAT1 exon 4 skipping. We found that the dengue polymerase NS5 interacts with RBM10 and its sole expression triggers RBM10 proteasome-mediated degradation. RBM10 over-expression in infected cells prevents SAT1 splicing changes and limits viral replication, while its knock-down enhances the splicing switch and also benefits viral replication, revealing an anti-viral role for RBM10. Consistently, RBM10 depletion attenuates expression of interferon and pro-inflammatory cytokines. In particular, we found that RBM10 interacts with viral RNA and RIG-I, and even promotes the ubiquitination of the latter, a crucial step for its activation. We propose RBM10 fulfills diverse pro-inflammatory, anti-viral tasks, besides its well-documented role in splicing regulation of apoptotic genes.

When SUMO met splicing.

Spliceosomal proteins have been revealed as SUMO conjugation targets. Moreover, we have reported that many of these are in a SUMO-conjugated form when bound to a pre-mRNA substrate during a splicing reaction. We demonstrated that SUMOylation of Prp3 (PRPF3), a component of the U4/U6 di-snRNP, is required for U4/U6•U5 tri-snRNP formation and/or recruitment to active spliceosomes. Expanding upon our previous results, we have shown that the splicing factor SRSF1 stimulates SUMO conjugation to several spliceosomal proteins. Given the relevance of the splicing process, as well as the complex and dynamic nature of its governing machinery, the spliceosome, the molecular mechanisms that modulate its function represent an attractive topic of research. We posit that SUMO conjugation could represent a way of modulating spliceosome assembly and thus, splicing efficiency. How cycles of SUMOylation/de-SUMOylation of spliceosomal proteins become integrated throughout the highly choreographed spliceosomal cycle awaits further investigation.

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

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.

The Dengue Virus NS5 Protein Intrudes in the Cellular Spliceosome and Modulates Splicing.

Dengue virus NS5 protein plays multiple functions in the cytoplasm of infected cells, enabling viral RNA replication and counteracting host antiviral responses. Here, we demonstrate a novel function of NS5 in the nucleus where it interferes with cellular splicing. Using global proteomic analysis of infected cells together with functional studies, we found that NS5 binds spliceosome complexes and modulates endogenous splicing as well as minigene-derived alternative splicing patterns. In particular, we show that NS5 alone, or in the context of viral infection, interacts with core components of the U5 snRNP particle, CD2BP2 and DDX23, alters the inclusion/exclusion ratio of alternative splicing events, and changes mRNA isoform abundance of known antiviral factors. Interestingly, a genome wide transcriptome analysis, using recently developed bioinformatics tools, revealed an increase of intron retention upon dengue virus infection, and viral replication was improved by silencing specific U5 components. Different mechanistic studies indicate that binding of NS5 to the spliceosome reduces the efficiency of pre-mRNA processing, independently of NS5 enzymatic activities. We propose that NS5 binding to U5 snRNP proteins hijacks the splicing machinery resulting in a less restrictive environment for viral replication.

Akt/PKB: one kinase, many modifications.

Akt/PKB, a serine/threonine kinase member of the AGC family of proteins, is involved in the regulation of a plethora of cellular processes triggered by a wide diversity of extracellular signals and is thus considered a key signalling molecule in higher eukaryotes. Deregulation of Akt signalling is associated with a variety of human diseases, revealing Akt-dependent pathways as an attractive target for therapeutic intervention. Since its discovery in the early 1990s, a large body of work has focused on Akt phosphorylation of two residues, Thr308 and Ser473, and modification of these two sites has been established as being equivalent to Akt activation. More recently, Akt has been identified as a substrate for many different post-translational modifications, including not only phosphorylation of other residues, but also acetylation, glycosylation, oxidation, ubiquitination and SUMOylation. These modifications could provide additional regulatory steps for fine-tuning Akt function, Akt trafficking within the cell and/or for determining the substrate specificity of this signalling molecule. In the present review, we provide an overview of these different post-translational modifications identified for Akt, focusing on their consequences for this kinase activity.

Modification of Akt by SUMO conjugation regulates alternative splicing and cell cycle.

Akt/PKB is a key signaling molecule in higher eukaryotes and a crucial protein kinase in human health and disease. Phosphorylation, acetylation, and ubiquitylation have been reported as important regulatory post-translational modifications of this kinase. We describe here that Akt is modified by SUMO conjugation, and show that lysine residues 276 and 301 are the major SUMO attachment sites within this protein. We found that phosphorylation and SUMOylation of Akt appear as independent events. However, decreasing Akt SUMOylation levels severely affects the role of this kinase as a regulator of fibronectin and Bcl-x alternative splicing. Moreover, we observed that the Akt mutant (Akt E17K) found in several human tumors displays increased levels of SUMOylation and also an enhanced capacity to regulate fibronectin splicing patterns. This splicing regulatory activity is completely abolished by decreasing Akt E17K SUMO conjugation levels. Additionally, we found that SUMOylation controls Akt regulatory function at G1/S transition during cell cycle progression. These findings reveal SUMO conjugation as a novel level of regulation for Akt activity, opening new areas of exploration related to the molecular mechanisms involved in the diverse cellular functions of this kinase.