Arginine methylation of SM-LIKE PROTEIN 4 antagonistically affects alternative splicing during Arabidopsis stress responses.

Arabidopsis (Arabidopsis thaliana) PROTEIN ARGININE METHYLTRANSFERASE5 (PRMT5) post-translationally modifies RNA-binding proteins by arginine (R) methylation. However, the impact of this modification on the regulation of RNA processing is largely unknown. We used the spliceosome component, SM-LIKE PROTEIN 4 (LSM4), as a paradigm to study the role of R-methylation in RNA processing. We found that LSM4 regulates alternative splicing (AS) of a suite of its in vivo targets identified here. The lsm4 and prmt5 mutants show a considerable overlap of genes with altered AS raising the possibility that splicing of those genes could be regulated by PRMT5-dependent LSM4 methylation. Indeed, LSM4 methylation impacts AS, particularly of genes linked with stress response. Wild-type LSM4 and an unmethylable version complement the lsm4-1 mutant, suggesting that methylation is not critical for growth in normal environments. However, LSM4 methylation increases with abscisic acid and is necessary for plants to grow under abiotic stress. Conversely, bacterial infection reduces LSM4 methylation, and plants that express unmethylable-LSM4 are more resistant to Pseudomonas than those expressing wild-type LSM4. This tolerance correlates with decreased intron retention of immune-response genes upon infection. Taken together, this provides direct evidence that R-methylation adjusts LSM4 function on pre-mRNA splicing in an antagonistic manner in response to biotic and abiotic stress.

MsTFL1A delays flowering and regulates shoot architecture and root development in Medicago sativa.

KEY MESSAGE: MsTFL1A is an important gene involved in flowering repression in alfalfa (Medicago sativa) which conditions not only above-ground plant shoot architecture but also root development and growth. Delayed flowering is an important trait for forage species, as it allows harvesting of high-quality forage for a longer time before nutritional values decline due to plant architecture changes related to flowering onset. Despite the relevance of delayed flowering, this trait has not yet been thoroughly exploited in alfalfa. This is mainly due to its complex genetics, sensitivity to inbreeding and to the fact that delayed flowering would be only advantageous if it allowed increased forage quality without compromising seed production. To develop new delayed-flowering varieties, we have characterized the three TERMINAL FLOWERING 1 (TFL1) family of genes in alfalfa: MsTFL1A, MsTFL1B and MsTFL1C. Constitutive expression of MsTFL1A in Arabidopsis caused late flowering and changes in inflorescence architecture, indicating that MsTFL1A is the ortholog of Arabidopsis TFL1. Overexpression of MsTFL1A in alfalfa consistently led to delayed flowering in both controlled and natural field conditions, coupled to an increase in leaf/stem ratio, a common indicator of forage quality. Additionally, overexpression of MsTFL1A reduced root development, reinforcing the role of MsTFL1A not only as a flowering repressor but also as a regulator of root development.We conclude that the precise manipulation of MsTFL1A gene expression may represent a powerful tool to improve alfalfa forage quality.

COLD REGULATED GENE 27 and 28 antagonize the transcriptional activity of the RVE8/LNK1/LNK2 circadian complex.

Many molecular and physiological processes in plants occur at a specific time of day. These daily rhythms are coordinated in part by the circadian clock, a timekeeper that uses daylength and temperature to maintain rhythms of ∼24 h in various clock-regulated phenotypes. The circadian MYB-like transcription factor REVEILLE 8 (RVE8) interacts with its transcriptional coactivators NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1 (LNK1) and LNK2 to promote the expression of evening-phased clock genes and cold tolerance factors. While genetic approaches have commonly been used to discover connections within the clock and between clock elements and other pathways, here, we used affinity purification coupled with mass spectrometry (APMS) to identify time-of-day-specific protein interactors of the RVE8-LNK1/LNK2 complex in Arabidopsis (Arabidopsis thaliana). Among the interactors of RVE8/LNK1/LNK2 were COLD-REGULATED GENE 27 (COR27) and COR28, which coprecipitated in an evening-specific manner. In addition to COR27 and COR28, we found an enrichment of temperature-related interactors that led us to establish a previously uncharacterized role for LNK1 and LNK2 in temperature entrainment of the clock. We established that RVE8, LNK1, and either COR27 or COR28 form a tripartite complex in yeast (Saccharomyces cerevisiae) and that the effect of this interaction in planta serves to antagonize transcriptional activation of RVE8 target genes, potentially through mediating RVE8 protein degradation in the evening. Together, these results illustrate how a proteomic approach can be used to identify time-of-day-specific protein interactions. Discovery of the RVE8-LNK-COR protein complex indicates a previously unknown regulatory mechanism for circadian and temperature signaling pathways.

PICLN modulates alternative splicing and light/temperature responses in plants.

Plants undergo transcriptome reprograming to adapt to daily and seasonal fluctuations in light and temperature conditions. While most efforts have focused on the role of master transcription factors, the importance of splicing factors modulating these processes is now emerging. Efficient pre-mRNA splicing depends on proper spliceosome assembly, which in plants and animals requires the methylosome complex. Ion Chloride nucleotide-sensitive protein (PICLN) is part of the methylosome complex in both humans and Arabidopsis (Arabidopsis thaliana), and we show here that the human PICLN ortholog rescues phenotypes of Arabidopsis picln mutants. Altered photomorphogenic and photoperiodic responses in Arabidopsis picln mutants are associated with changes in pre-mRNA splicing that partially overlap with those in PROTEIN ARGININE METHYL TRANSFERASE5 (prmt5) mutants. Mammalian PICLN also acts in concert with the Survival Motor Neuron (SMN) complex component GEMIN2 to modulate the late steps of UsnRNP assembly, and many alternative splicing events regulated by PICLN but not PRMT5, the main protein of the methylosome, are controlled by Arabidopsis GEMIN2. As with GEMIN2 and SM PROTEIN E1/PORCUPINE (SME1/PCP), low temperature, which increases PICLN expression, aggravates morphological and molecular defects of picln mutants. Taken together, these results establish a key role for PICLN in the regulation of pre-mRNA splicing and in mediating plant adaptation to daily and seasonal fluctuations in environmental conditions.

The 5'-3' mRNA Decay Pathway Modulates the Plant Circadian Network in Arabidopsis.

Circadian rhythms enable organisms to anticipate and adjust their physiology to periodic environmental changes. These rhythms are controlled by biological clocks that consist of a set of clock genes that regulate each other's expression. Circadian oscillations in messenger RNA (mRNA) levels require the regulation of mRNA production and degradation. While transcription factors controlling clock function have been well characterized from cyanobacteria to humans, the role of factors controlling mRNA decay is largely unknown. Here, we show that mutations in SM-LIKE PROTEIN 1 (LSM1) and exoribonucleases 4 (XRN4), components of the 5'-3' mRNA decay pathway, alter clock function in Arabidopsis. We found that lsm1 and xrn4 mutants display long-period phenotypes for clock gene expression. In xrn4, these circadian defects were associated with changes in circadian phases of expression, but not overall mRNA levels, of several core-clock genes. We then used noninvasive transcriptome-wide mRNA stability analysis to identify genes and pathways regulated by XRN4. Among genes affected in the xrn4 mutant at the transcriptional and posttranscriptional level, we found an enrichment in genes involved in auxin, ethylene and drought recovery. Large effects were not observed for canonical core-clock genes, although the mRNAs of several auxiliary clock genes that control the pace of the clock were stabilized in xrn4 mutants. Our results establish that the 5'-3' mRNA decay pathway constitutes a novel posttranscriptional regulatory layer of the circadian gene network, which probably acts through a combination of small effects on mRNA stability of several auxiliary and some core-clock genes.

Functional convergence of growth responses to shade and warmth in Arabidopsis.

Shade and warmth promote the growth of the stem, but the degree of mechanistic convergence and functional association between these responses is not clear. We analysed the quantitative impact of mutations and natural genetic variation on the hypocotyl growth responses of Arabidopsis thaliana to shade and warmth, the relationship between the abundance of PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and growth stimulation by shade or warmth, the effects of both cues on the transcriptome and the consequences of warm temperature on carbon balance. Growth responses to shade and warmth showed strong genetic linkage and similar dependence on PIF4 levels. Temperature increased growth and phototropism even within a range where damage by extreme high temperatures is unlikely to occur in nature. Both cues enhanced the expression of growth-related genes and reduced the expression of photosynthetic genes. However, only warmth enhanced the expression of genes involved in responses to heat. Warm temperatures substantially increased the amount of light required to compensate for the daily carbon dioxide balance. We propose that the main ecological function of hypocotyl growth responses to warmth is to increase the access of shaded photosynthetic organs to light, which implies functional convergence with shade avoidance.

Emergency response for evaluating SARS-CoV-2 immune status, seroprevalence and convalescent plasma in Argentina.

We report the emergency development and application of a robust serologic test to evaluate acute and convalescent antibody responses to SARS-CoV-2 in Argentina. The assays, COVIDAR IgG and IgM, which were produced and provided for free to health authorities, private and public health institutions and nursing homes, use a combination of a trimer stabilized spike protein and the receptor binding domain (RBD) in a single enzyme-linked immunosorbent assay (ELISA) plate. Over half million tests have already been distributed to detect and quantify antibodies for multiple purposes, including assessment of immune responses in hospitalized patients and large seroprevalence studies in neighborhoods, slums and health care workers, which resulted in a powerful tool for asymptomatic detection and policy making in the country. Analysis of antibody levels and longitudinal studies of symptomatic and asymptomatic SARS-CoV-2 infections in over one thousand patient samples provided insightful information about IgM and IgG seroconversion time and kinetics, and IgM waning profiles. At least 35% of patients showed seroconversion within 7 days, and 95% within 45 days of symptoms onset, with simultaneous or close sequential IgM and IgG detection. Longitudinal studies of asymptomatic cases showed a wide range of antibody responses with median levels below those observed in symptomatic patients. Regarding convalescent plasma applications, a protocol was standardized for the assessment of end point IgG antibody titers with COVIDAR with more than 500 plasma donors. The protocol showed a positive correlation with neutralizing antibody titers, and was used for clinical trials and therapies across the country. Using this protocol, about 80% of convalescent donor plasmas were potentially suitable for therapies. Here, we demonstrate the importance of providing a robust and specific serologic assay for generating new information about antibody kinetics in infected individuals and mitigation policies to cope with pandemic needs.

It's a matter of time: the role of transcriptional regulation in the circadian clock-pathogen crosstalk in plants.

Most living organisms possess an internal timekeeping mechanism known as the circadian clock, which enhances fitness by synchronizing the internal timing of biological processes with diurnal and seasonal environmental changes. In plants, the pace of these biological rhythms relies on oscillations in the expression level of hundreds of genes tightly controlled by a group of core clock regulators and co-regulators that engage in transcriptional and translational feedback loops. In the last decade, the role of several core clock genes in the control of defense responses has been addressed, and a growing amount of evidence demonstrates that circadian regulation is relevant for plant immunity. A reciprocal connection between these pathways was also established following the observation that in Arabidopsis thaliana, as well as in crop species like tomato, plant-pathogen interactions trigger a reconfiguration of the circadian transcriptional network. In this review, we summarize the current knowledge regarding the interaction between the circadian clock and biotic stress responses at the transcriptional level, and discuss the relevance of this crosstalk in the plant-pathogen evolutionary arms race. A better understanding of these processes could aid in the development of genetic tools that improve traditional breeding practices, enhancing tolerance to plant diseases that threaten crop yield and food security all around the world.

Prefoldins contribute to maintaining the levels of the spliceosome LSM2-8 complex through Hsp90 in Arabidopsis.

Although originally identified as the components of the complex aiding the cytosolic chaperonin CCT in the folding of actins and tubulins in the cytosol, prefoldins (PFDs) are emerging as novel regulators influencing gene expression in the nucleus. Work conducted mainly in yeast and animals showed that PFDs act as transcriptional regulators and participate in the nuclear proteostasis. To investigate new functions of PFDs, we performed a co-expression analysis in Arabidopsis thaliana. Results revealed co-expression between PFD and the Sm-like (LSM) genes, which encode the LSM2-8 spliceosome core complex, in this model organism. Here, we show that PFDs interact with and are required to maintain adequate levels of the LSM2-8 complex. Our data indicate that levels of the LSM8 protein, which defines and confers the functional specificity of the complex, are reduced in pfd mutants and in response to the Hsp90 inhibitor geldanamycin. We provide biochemical evidence showing that LSM8 is a client of Hsp90 and that PFD4 mediates the interaction between both proteins. Consistent with our results and with the role of the LSM2-8 complex in splicing through the stabilization of the U6 snRNA, pfd mutants showed reduced levels of this snRNA and altered pre-mRNA splicing patterns.

Global transcriptome analysis reveals circadian control of splicing events in Arabidopsis thaliana.

The circadian clock of Arabidopsis thaliana controls many physiological and molecular processes, allowing plants to anticipate daily changes in their environment. However, developing a detailed understanding of how oscillations in mRNA levels are connected to oscillations in co/post-transcriptional processes, such as splicing, has remained a challenge. Here we applied a combined approach using deep transcriptome sequencing and bioinformatics tools to identify novel circadian-regulated genes and splicing events. Using a stringent approach, we identified 300 intron retention, eight exon skipping, 79 alternative 3' splice site usage, 48 alternative 5' splice site usage, and 350 multiple (more than one event type) annotated events under circadian regulation. We also found seven and 721 novel alternative exonic and intronic events. Depletion of the circadian-regulated splicing factor AtSPF30 homologue resulted in the disruption of a subset of clock-controlled splicing events. Altogether, our global circadian RNA-seq coupled with an in silico, event-centred, splicing analysis tool offers a new approach for studying the interplay between the circadian clock and the splicing machinery at a global scale. The identification of many circadian-regulated splicing events broadens our current understanding of the level of control that the circadian clock has over this co/post-transcriptional regulatory layer.

Bacterial Infection Disrupts Clock Gene Expression to Attenuate Immune Responses.

The circadian clock modulates immune responses in plants and animals; however, it is unclear how host-pathogen interactions affect the clock. Here we analyzed clock function in Arabidopsis thaliana mutants with defective immune responses and found that enhanced disease susceptibility 4 (eds4) displays alterations in several circadian rhythms. Mapping by sequencing revealed that EDS4 encodes the ortholog of NUCLEOPORIN 205, a core component of the inner ring of the nuclear pore complex (NPC). Consistent with the idea that the NPC specifically modulates clock function, we found a strong enrichment in core clock genes, as well as an increased nuclear to total mRNA accumulation, among genes that were differentially expressed in eds4 mutants. Interestingly, infection with Pseudomonas syringae in wild-type (WT) plants downregulated the expression of several morning core clock genes as early as 1 h post-infection, including all members of the NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED (LNK) gene family, and this effect was attenuated in eds4. Furthermore, lnk mutants were more susceptible than the WT to P. syringae infection. These results indicate that bacterial infection, acting in part through the NPC, alters core clock gene expression and/or mRNA accumulation in a way that favors bacterial growth and disease susceptibility.

Improvement of alfalfa forage quality and management through the down-regulation of MsFTa1.

Alfalfa (Medicago sativa L.) is one of the most important forage crops worldwide. As a perennial, alfalfa is cut several times each year. Farmers face a dilemma: if cut earlier, forage nutritive value is much higher but regrowth is affected and the longevity of the stand is severely compromised. On the other hand, if alfalfa is cut later at full flower, stands persist longer and more biomass may be harvested, but the nutritive value diminishes. Alfalfa is a strict long-day plant. We reasoned that by manipulating the response to photoperiod, we could delay flowering to improve forage quality and widen each harvesting window, facilitating management. With this aim, we functionally characterized the FLOWERING LOCUS T family of genes, represented by five members: MsFTa1, MsFTa2, MsFTb1, MsFTb2 and MsFTc. The expression of MsFTa1 correlated with photoperiodic flowering and its down-regulation led to severe delayed flowering. Altogether, with late flowering, low expression of MsFTa1 led to changes in plant architecture resulting in increased leaf to stem biomass ratios and forage digestibility. By manipulating photoperiodic flowering, we were able to improve the quality of alfalfa forage and management, which may allow farmers to cut alfalfa of high nutritive value without compromising stand persistence.

Shade delays flowering in Medicago sativa.

Shade-intolerant plants respond to the decrease in the red (R) to far-red (FR) light ratio (R:FR) occurring under shade by elongating stems and petioles and by re-positioning leaves, in a race to outcompete neighbors for the sunlight resource. In some annual species, the shade avoidance syndrome (SAS) is accompanied by the early induction of flowering. Anticipated flowering is viewed as a strategy to set seeds before the resources become severely limiting. Little is known about the molecular mechanisms of SAS in perennial forage crops like alfalfa (Medicago sativa). To study SAS in alfalfa, we exposed alfalfa plants to simulated shade by supplementing with FR light. Low R:FR light produced a classical SAS, with increased internode and petiole lengths, but unexpectedly also with delayed flowering. To understand the molecular mechanisms involved in uncoupling SAS from early flowering, we used a transcriptomic approach. The SAS is likely to be mediated by increased expression of msPIF3 and msHB2 in low R:FR light. Constitutive expression of these genes in Arabidopsis led to SAS, including early flowering, strongly suggesting that their roles are conserved. Delayed flowering was likely to be mediated by the downregulation of msSPL3, which promotes flowering in both Arabidopsis and alfalfa. Shade-delayed flowering in alfalfa may be important to extend the vegetative phase under suboptimal light conditions, and thus assure the accumulation of reserves necessary to resume growth after the next season.

FITNESS, a CCT domain-containing protein, deregulates reactive oxygen species levels and leads to fine-tuning trade-offs between reproductive success and defence responses in Arabidopsis.

Environmental stresses are the major factors that limit productivity in plants. Here, we report on the function of an uncharacterized gene At1g07050, encoding a CCT domain-containing protein, from Arabidopsis thaliana. At1g07050 expression is highly repressed by oxidative stress. We used metabolomics, biochemical, and genomic approaches to analyse performance of transgenic lines with altered expression of At1g07050 under normal and oxidative stress conditions. At1g07050 overexpressing lines showed increased levels of reactive oxygen species (ROS), whereas knock-out mutants exhibited decreased levels of ROS and higher tolerance to oxidative stress generated in the chloroplast. Our results uncover a role for At1g07050 in cellular redox homeostasis controlling H2 O2 levels, due to changes in enzymes, metabolites, and transcripts related to ROS detoxification. Therefore, we call this gene FITNESS. Additionally, several genes such as ACD6, PCC1, and ICS1 related to salicylic acid signalling and defence were found differentially expressed among the lines. Notably, FITNESS absence significantly improved seed yield suggesting an effective fine-tuning trade-off between reproductive success and defence responses.

Rewiring of auxin signaling under persistent shade.

Light cues from neighboring vegetation rapidly initiate plant shade-avoidance responses. Despite our detailed knowledge of the early steps of this response, the molecular events under prolonged shade are largely unclear. Here we show that persistent neighbor cues reinforce growth responses in addition to promoting auxin-responsive gene expression in Arabidopsis and soybean. However, while the elevation of auxin levels is well established as an early event, in Arabidopsis, the response to prolonged shade occurs when auxin levels have declined to the prestimulation values. Remarkably, the sustained low activity of phytochrome B under prolonged shade led to (i) decreased levels of PHYTOCHROME INTERACTING FACTOR 4 (PIF4) in the cotyledons (the organs that supply auxin) along with increased levels in the vascular tissues of the stem, (ii) elevated expression of the PIF4 targets INDOLE-3-ACETIC ACID 19 (IAA19) and IAA29, which in turn reduced the expression of the growth-repressive IAA17 regulator, (iii) reduced abundance of AUXIN RESPONSE FACTOR 6, (iv) reduced expression of MIR393 and increased abundance of its targets, the auxin receptors, and (v) elevated auxin signaling as indicated by molecular markers. Mathematical and genetic analyses support the physiological role of this system-level rearrangement. We propose that prolonged shade rewires the connectivity between light and auxin signaling to sustain shade avoidance without enhanced auxin levels.

SPF45-related splicing factor for phytochrome signaling promotes photomorphogenesis by regulating pre-mRNA splicing in Arabidopsis.

Light signals regulate plant growth and development by controlling a plethora of gene expression changes. Posttranscriptional regulation, especially pre-mRNA processing, is a key modulator of gene expression; however, the molecular mechanisms linking pre-mRNA processing and light signaling are not well understood. Here we report a protein related to the human splicing factor 45 (SPF45) named splicing factor for phytochrome signaling (SFPS), which directly interacts with the photoreceptor phytochrome B (phyB). In response to light, SFPS-RFP (red fluorescent protein) colocalizes with phyB-GFP in photobodies. sfps loss-of-function plants are hyposensitive to red, far-red, and blue light, and flower precociously. SFPS colocalizes with U2 small nuclear ribonucleoprotein-associated factors including U2AF65B, U2A', and U2AF35A in nuclear speckles, suggesting SFPS might be involved in the 3' splice site determination. SFPS regulates pre-mRNA splicing of a large number of genes, of which many are involved in regulating light signaling, photosynthesis, and the circadian clock under both dark and light conditions. In vivo RNA immunoprecipitation (RIP) assays revealed that SFPS associates with EARLY FLOWERING 3 (ELF3) mRNA, a critical link between light signaling and the circadian clock. Moreover, PHYTOCHROME INTERACTING FACTORS (PIFs) transcription factor genes act downstream of SFPS, as the quadruple pif mutant pifq suppresses defects of sfps mutants. Taken together, these data strongly suggest SFPS modulates light-regulated developmental processes by controlling pre-mRNA splicing of light signaling and circadian clock genes.

Rhythmic Behavior Is Controlled by the SRm160 Splicing Factor in Drosophila melanogaster.

Circadian clocks organize the metabolism, physiology, and behavior of organisms throughout the day-night cycle by controlling daily rhythms in gene expression at the transcriptional and post-transcriptional levels. While many transcription factors underlying circadian oscillations are known, the splicing factors that modulate these rhythms remain largely unexplored. A genome-wide assessment of the alterations of gene expression in a null mutant of the alternative splicing regulator SR-related matrix protein of 160 kDa (SRm160) revealed the extent to which alternative splicing impacts on behavior-related genes. We show that SRm160 affects gene expression in pacemaker neurons of the Drosophila brain to ensure proper oscillations of the molecular clock. A reduced level of SRm160 in adult pacemaker neurons impairs circadian rhythms in locomotor behavior, and this phenotype is caused, at least in part, by a marked reduction in period (per) levels. Moreover, rhythmic accumulation of the neuropeptide PIGMENT DISPERSING FACTOR in the dorsal projections of these neurons is abolished after SRm160 depletion. The lack of rhythmicity in SRm160-downregulated flies is reversed by a fully spliced per construct, but not by an extra copy of the endogenous locus, showing that SRm160 positively regulates per levels in a splicing-dependent manner. Our findings highlight the significant effect of alternative splicing on the nervous system and particularly on brain function in an in vivo model.

Transcriptional and post-transcriptional control of the plant circadian gene regulatory network.

The circadian clock drives rhythms in multiple physiological processes allowing plants to anticipate and adjust to periodic changes in environmental conditions. These physiological rhythms are associated with robust oscillations in the expression of thousands of genes linked to the control of photosynthesis, cell elongation, biotic and abiotic stress responses, developmental processes such as flowering, and the clock itself. Given its pervasive effects on plant physiology, it is not surprising that circadian clock genes have played an important role in the domestication of crop plants and in the improvement of crop productivity. Therefore, identifying the principles governing the dynamics of the circadian gene regulatory network in plants could strongly contribute to further speed up crop improvement. Here we provide an historical as well as a current description of our knowledge of the molecular mechanisms underlying circadian rhythms in plants. This work focuses on the transcriptional and post-transcriptional regulatory layers that control the very core of the circadian clock, and some of its complex interactions with signaling pathways that help synchronize plant growth and development to daily and seasonal changes in the environment. This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer.

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.