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.

Light signaling pathways and the circadian clock interact to help organisms synchronize physiological and developmental processes with periodic environmental cycles. The plant photoreceptors responsible for clock resetting have been characterized, but signaling components that link the photoreceptors to the clock remain to be identified. Here we describe a family of night light–inducible and clock-regulated genes ( LNK ) that play a key role linking light regulation of gene expression to the control of daily and seasonal rhythms in Arabidopsis thaliana . A genomewide transcriptome analysis revealed that most light-induced genes respond more strongly to light during the subjective day, which is consistent with the diurnal nature of most physiological processes in plants. However, a handful of genes, including the homologous genes LNK1 and LNK2, are more strongly induced by light in the middle of the night, when the clock is most responsive to this signal. Further analysis revealed that the morning phased LNK1 and LNK2 genes control circadian rhythms, photomorphogenic responses, and photoperiodic dependent flowering, most likely by regulating a subset of clock and flowering time genes in the afternoon. LNK1 and LNK2 themselves are directly repressed by members of the TIMING OF CAB1 EXPRESSION / PSEUDO RESPONSE REGULATOR family of core-clock genes in the afternoon and early night. Thus, LNK1 and LNK2 integrate early light signals with temporal information provided by core oscillator components to control the expression of afternoon genes, allowing plants to keep track of seasonal changes in day length.

The mechanisms by which poikilothermic organisms ensure that biological processes are robust to temperature changes are largely unknown. Temperature compensation, the ability of circadian rhythms to maintain a relatively constant period over the broad range of temperatures resulting from seasonal fluctuations in environmental conditions, is a defining property of circadian networks. Temperature affects the alternative splicing (AS) of several clock genes in fungi, plants, and flies, but the splicing factors that modulate these effects to ensure clock accuracy throughout the year remain to be identified. Here we show that GEMIN2, a spliceosomal small nuclear ribonucleoprotein assembly factor conserved from yeast to humans, modulates low temperature effects on a large subset of pre-mRNA splicing events. In particular, GEMIN2 controls the AS of several clock genes and attenuates the effects of temperature on the circadian period in Arabidopsis thaliana. We conclude that GEMIN2 is a key component of a posttranscriptional regulatory mechanism that ensures the appropriate acclimation of plants to daily and seasonal changes in temperature conditions. Significance RNA processing, an important step in the regulation of gene expression, is mediated by proteins and RNA molecules that are highly sensitive to variations in temperature conditions. Most organisms do not control their own body temperature. Therefore, molecular mechanisms must have evolved that ensure that biological processes are robust to temperature changes. Here we identify a protein that buffers the effect of temperature on biological timing by enhancing the assembly of the spliceosome, a large ribonucleoprotein complex involved in RNA processing in organisms ranging from yeast to humans, and thereby controlling the alternative splicing of clock genes.

The spliceosome is the complex molecular machinery that sequentially assembles on eukaryotic messenger RNA precursors to remove introns (pre-mRNA splicing), a physiologically regulated process altered in numerous pathologies. We report transcriptome-wide analyses upon systematic knock down of 305 spliceosome components and regulators in human cancer cells and the reconstruction of functional splicing factor networks that govern different classes of alternative splicing decisions. The results disentangle intricate circuits of splicing factor cross-regulation, reveal that the precise architecture of late-assembling U4/U6.U5 tri–small nuclear ribonucleoprotein (snRNP) complexes regulates splice site pairing, and discover an unprecedented division of labor among protein components of U1 snRNP for regulating exon definition and alternative 5′ splice site selection. Thus, we provide a resource to explore physiological and pathological mechanisms of splicing regulation.