Estefania Mancini

LNK genes integrate light and clock signaling networks at the core of the Arabidopsis oscillator

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 spliceosome assembly factor GEMIN2 attenuates the effects of temperature on alternative splicing and circadian rhythms

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 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.

Role for LSM genes in the regulation of circadian rhythms.

Significance There is increasing evidence that previously considered core constituents of multi-subunit complexes involved in RNA processing play regulatory rather than passive roles in the control of gene expression, but specific signaling pathways in which they participate are not known. Here we show that SM-like (LSM) genes, which encode core components of the spliceosome, are regulated by the circadian clock and control clock function in plants and mammals, revealing convergent evolutionary mechanisms mediating posttranscriptional regulation of circadian networks across kingdoms. Growing evidence suggests that core spliceosomal components differentially affect RNA processing of specific genes; however, whether changes in the levels or activities of these factors control specific signaling pathways is largely unknown. Here we show that some SM-like (LSM) genes, which encode core components of the spliceosomal U6 small nuclear ribonucleoprotein complex, regulate circadian rhythms in plants and mammals. We found that the circadian clock regulates the expression of LSM5 in Arabidopsis plants and several LSM genes in mouse suprachiasmatic nucleus. Further, mutations in LSM5 or LSM4 in Arabidopsis, or down-regulation of LSM3, LSM5, or LSM7 expression in human cells, lengthens the circadian period. Although we identified changes in the expression and alternative splicing of some core clock genes in Arabidopsis lsm5 mutants, the precise molecular mechanism causing period lengthening remains to be identified. Genome-wide expression analysis of either a weak lsm5 or a strong lsm4 mutant allele in Arabidopsis revealed larger effects on alternative splicing than on constitutive splicing. Remarkably, large splicing defects were not observed in most of the introns evaluated using RNA-seq in the strong lsm4 mutant allele used in this study. These findings support the idea that some LSM genes play both regulatory and constitutive roles in RNA processing, contributing to the fine-tuning of specific signaling pathways.

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.

Genome wide comparative analysis of the effects of PRMT5 and PRMT4/CARM1 arginine methyltransferases on the Arabidopsis thaliana transcriptome.

BackgroundMethylation 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.ResultsHere 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.ConclusionsOur 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.

Acute Effects of Light on Alternative Splicing in Light-Grown Plants.

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.

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

Significance Pre-mRNA processing not only enhances the diversity encoded in the genome without the need to increase the number of genes but also provides a means to adjust cellular transcript abundance. Environmental light has a profound effect on transcript accumulation, but how this is partitioned between transcriptional and posttranscriptional processes is largely unknown. Here we describe the identification and characterization of the splicing factor for phytochrome signaling (SFPS), which directly interacts with the photoreceptor phytochrome B. sfps seedlings are hyposensitive to light and display pre-mRNA splicing defects in a large number of genes, many of which regulate light signaling and the circadian clock. Thus, light might control pre-mRNA splicing in addition to transcription of many genes through SFPS to promote photomorphogenesis. 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.

Combinatorial control of adhesion of Brucella abortus 2308 to host cells by transcriptional rewiring of the trimeric autotransporter btaE gene.

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.

Second ISCB Latin American Student Council Symposium (LA-SCS) 2016

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.

Functional annotation pipeline for high-throughput sequencing microbiomes

We have developed a novel pipeline for metagenomics and metatranscriptomics data analysis. It was testet on the first genomic datasets from rhizospheric microbial communities under intense agriculture in Argentina. We consider this pipeline an useful contribution for upcoming experiments in environmental microbiology. Functional annotation pipeline for high-throughput sequencing microbiomes Estefania Mancini*, Santiago Revale, M. Belen Carbonetto, Nicolas Rascovan, Martin Vazquez. INDEAR-CONICET, Ocampo 210 bis, predio CCT, Rosario, Argentina *estefania.mancini@indear.com Motivation Metagenomics allow the study of microbial communities from complex environments in a culture-independent manner by DNA sequence analysis. Genomic data provides metabolic information of microbial communities but cannot reveal which functions are actually active at a specific moment or condition. Metatranscriptomics involve random sequencing of microbial community RNA, allowing the study of gene expression and metabolic activity in response to environmental factors or biotic interactions. If both metagenomics and metatranscriptomics are done for the same community, the most interesting question to address is which of the identified metabolic pathways are active. When the analysis involves different environmental or experimental conditions the analysis focus on the identification of those metabolic functions that changed between treatments. A metagenomic approach will thus tell us which functions are present and metatranscriptomics which are active. Here we present a custom developed pipeline based on well known bioinformatic tools that allows filtering, functional annotation and comparative analysis of cDNA and gDNA sequence datasets obtained by high throughput sequencing. All the scripts of the pipeline were written in Perl and R.