Serine/arginine-rich (SR) proteins are important regulators of mRNA splicing. Several postsplicing activities have been described for a subset of shuttling SR proteins, including regulation of mRNA export and translation. Using the fibronectin gene to study the links between signal-transduction pathways and SR protein activity, we show that growth factors not only modify the alternative splicing pattern of the fibronectin gene but also alter translation of reporter messenger RNAs in an SR protein–dependent fashion, providing two coregulated levels of isoform-specific amplification. These effects are inhibited by specific small interfering RNAs against SR proteins and are mediated by the AKT kinase, which elicits opposite effects to those evoked by overexpressing SR protein kinases Clk and SRPK. These results show how SR protein activity is modified in response to extracellular stimulation, leading to a concerted regulation of splicing and translation.

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. Mapping host-pathogen interactions has proven fundamental for understanding how viruses manipulate host machinery and how cellular processes are regulated during infection. Dengue virus poses a major threat to public health: two-thirds of the world’s population is now at risk from infection by this mosquito-borne virus. In this work, using a global proteomic approach in the context of viral infections with tagged dengue viruses, we constructed a comprehensive protein-protein interaction map of the multifunctional NS5 viral protein. NS5 is central for viral RNA replication and for immune evasion. Our studies revealed the interaction of NS5 with core components of the splicing machinery, specifically with proteins of the U5 small nuclear ribonucleoprotein particle, and that viral infection reduces splicing efficiency. Mechanistic studies analyzing endogenous splicing events and in vitro splicing assays indicated that NS5 binds active spliceosomes and reduces the efficiency of pre-mRNA processing. Our results provide a new function of the dengue virus NS5 protein and support a model in which manipulation of specific splicing components favors viral infection.

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.

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.

Heterogeneous nuclear ribonucleoprotein (hnRNP) K is a nucleocytoplasmic shuttling protein that is a key player in the p53-triggered DNA damage response, acting as a cofactor for p53 in response to DNA damage. hnRNP K is a substrate of the ubiquitin E3 ligase MDM2 and, upon DNA damage, is de-ubiquitylated. In sharp contrast with the role and consequences of the other post-translational modifications, nothing is known about the role of SUMO conjugation to hnRNP K in p53 transcriptional co-activation. In the present work, we show that hnRNP K is modified by SUMO in lysine 422 within its KH3 domain, and sumoylation is regulated by the E3 ligase Pc2/CBX4. Most interestingly, DNA damage stimulates hnRNP K sumoylation through Pc2 E3 activity, and this modification is required for p53 transcriptional activation. Abrogation of hnRNP K sumoylation leads to an aberrant regulation of the p53 target gene p21. Our findings link the DNA damage-induced Pc2 activation to the p53 transcriptional co-activation through hnRNP K sumoylation.