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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.

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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.

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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.

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This report summarizes the scientific content and activities of the second edition of the Latin American Symposium (LA-SCS), organized by the Student Council (SC) of the International Society for Computational Biology (ISCB), held in conjunction with the Fourth Latin American conference from the International Society for Computational Biology (ISCB-LA 2016) in Buenos Aires, Argentina, on November 19, 2016.

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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.

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Regulatory network plasticity is a key attribute underlying changes in bacterial gene expression and a source of phenotypic diversity to interact with the surrounding environment. Here, we sought to study the transcriptional circuit of HutC, a regulator of both metabolic and virulence genes of the facultative intracellular pathogen Brucella. Using in silico and biochemical approaches, we identified a novel functional HutC-binding site upstream of btaE, a trimeric-autotransporter adhesin involved in the attachment of Brucella to host extracellular matrix components. Moreover, we identified two additional regulators, one of which, MdrA, acts in concert with HutC to exert a combinatorial control of both btaE promoter activity and attachment of Brucella to HeLa cells. Analysis of btaE promoter sequences of different species indicated that this HutC-binding site was generated de novo by a single point mutation in a virulent Brucella strain, indicative of a transcriptional rewiring event. In addition to major domain organization differences existing between BtaE proteins within the genus Brucella, our analyses revealed that sequences upstream of btaE display high variability probably associated to intrinsic promoter structural features, which may serve as a substrate for reciprocal selection during co-evolution between this pathogen and its mammalian host.

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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.

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Light modulates plant growth and development to a great extent by regulating gene expression programs. Here, we evaluated the effect of light on alternative splicing (AS) in light-grown Arabidopsis thaliana plants using high-throughput RNA sequencing (RNA-seq). We found that an acute light pulse given in the middle of the night, a treatment that simulates photoperiod lengthening, affected AS events corresponding to 382 genes. Some of these AS events were associated with genes involved in primary metabolism and stress responses, which may help to adjust metabolic and physiological responses to seasonal changes. We also found that several core clock genes showed changes in AS in response to the light treatment, suggesting that light regulation of AS may play a role in clock entrainment. Finally, we found that many light-regulated AS events were associated with genes encoding RNA processing proteins and splicing factors, supporting the idea that light regulates this posttranscriptional regulatory layer through AS regulation of splicing factors. Interestingly, the effect of a red-light pulse on AS of a gene encoding a splicing factor was not impaired in a quintuple phytochrome mutant, providing unequivocal evidence that nonphotosensory photoreceptors control AS in light-grown plants.

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BACKGROUND: Methylation at arginine residues (R) is an important post-translational modification that regulates a myriad of essential cellular processes in eukaryotes, such as transcriptional regulation, RNA processing, signal transduction and DNA repair. Arginine methylation is catalyzed by a family of enzymes known as protein arginine methyltransferases (PRMTs). PRMTs are classified as Type I or Type II, depending on the position of the methyl group on the guanidine of the methylated arginine. Previous reports have linked symmetric R methylation to transcriptional repression, while asymmetric R methylation is generally associated with transcriptional activation. However, global studies supporting this conclusion are not available. RESULTS: Here we compared side by side the physiological and molecular roles of the best characterized plant PRMTs, the Type II PRMT5 and the Type I PRMT4, also known as CARM1 in mammals. We found that prmt5 and prmt4a;4b mutants showed similar alterations in flowering time, photomorphogenic responses and salt stress tolerance, while only prmt5 mutants exhibited alterations in circadian rhythms. An RNA-seq analysis revealed that expression and splicing of many differentially regulated genes was similarly enhanced or repressed by PRMT5 and PRMT4s. Furthermore, PRMT5 and PRMT4s co-regulated the expression and splicing of key regulatory genes associated with transcription, RNA processing, responses to light, flowering, and abiotic stress tolerance, being candidates to mediate the physiological alterations observed in the mutants. CONCLUSIONS: Our global analysis indicates that two of the most important Type I and Type II arginine methyltransferases, PRTM4 and PRMT5, have mostly overlapping as well as specific, but not opposite, roles in the global regulation of gene expression in plants.

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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.

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Mitochondrial disorders are a frequent cause of neurological disability affecting children and adults. Traditionally, molecular diagnosis of mitochondrial diseases was mostly accomplished by the use of Sanger sequencing and PCR-RFLP. However, there are particular drawbacks associated with the use of these methods. Recent multidisciplinary advances have led to new sequencing methods that may overcome these limitations. Our goal was to explore the use of a next generation sequencing platform in the molecular diagnosis of mitochondrial diseases reporting our findings in adult patients that present with a clinical-pathological diagnosis of a mitochondrial encephalomyopathy. Complete genomic sequences of mitochondrial DNA were obtained by 454 massive pyrosequencing from blood samples. The analysis of these sequences allowed us to identify two diagnostic pathogenic mutations and 74 homoplasmic polymorphisms, useful for obtaining high-resolution mitochondrial haplogroups. In summary, molecular diagnosis of mitochondrial disorders could be efficiently done from readily accessible samples, such as blood, with the use of a new sequencing platform.

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The high-altitude Andean lakes (HAAL) in the Argentinean Puna-high Andes region represent an almost unexplored ecosystem exposed to extreme conditions (high UV irradiation, hypersalinity, drastic temperature changes, desiccation, and high pH). Here we present the first genome sequence, a Sphingomonas sp., isolated from this extreme environment.

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