RNA degradation by the plant RNA exosome involves both phosphorolytic and hydrolytic activities

The yeast and human RNA exosome is structurally related to prokaryotic phosphorylases but degrades RNA only via associated hydrolytic activities. Here the authors show that the RNA exosome of plants, and likely those of a few basal eukaryotes, combines phosphorolytic and hydrolytic activities to degrade RNA

Respective Contributions of URT1 and HESO1 to the Uridylation of 5′ Fragments Produced From RISC-Cleaved mRNAs

In plants, post-transcriptional gene silencing (PTGS) represses gene expression by translation inhibition and cleavage of target mRNAs. The slicing activity is provided by argonaute 1 (AGO1), and the cleavage site is determined by sequence complementarity between the target mRNA and the microRNA (miRNA) or short interfering RNA (siRNA) loaded onto AGO1, to form the core of the RNA induced silencing complex (RISC). Following cleavage, the resulting 5′ fragment is modified at its 3′ end by the untemplated addition of uridines. Uridylation is proposed to facilitate RISC recycling and the degradation of the RISC 5′-cleavage fragment. Here, we detail a 3′ RACE-seq method to analyze the 3′ ends of 5′ fragments produced from RISC-cleaved transcripts. The protocol is based on the ligation of a primer at the 3′ end of RNA, followed by cDNA synthesis and the subsequent targeted amplification by PCR to generate amplicon libraries suitable for Illumina sequencing. A detailed data processing pipeline is provided to analyze nibbling and tailing at high resolution. Using this method, we compared the tailing and nibbling patterns of RISC-cleaved MYB33 and SPL13 transcripts between wild-type plants and mutant plants depleted for the terminal uridylyltransferases (TUTases) HESO1 and URT1. Our data reveal the respective contributions of HESO and URT1 in the uridylation of RISC-cleaved MYB33 and SPL13 transcripts, with HESO1 being the major TUTase involved in uridylating these fragments. Because of its depth, the 3′ RACE-seq method shows at high resolution that these RISC-generated 5′ RNA fragments are nibbled by a few nucleotides close to the cleavage site in the absence of uridylation. 3′ RACE-seq is a suitable approach for a reliable comparison of uridylation and nibbling patterns between mutants, a prerequisite to the identification of all factors involved in the clearance of RISC-generated 5′ mRNA fragments

Malnutrition impact on stroke outcome: an analysis of a patient cohort 3 months after recanalization treatment

Background: Malnutrition is thought to affect 30% to 70% of hospitalized patients. Little is known about consequences of undernutrition during the acute phase of stroke. We would like to assess the impact of pre-infarction malnutrition on stroke patients treated with thrombolysis and/ or thrombectomy. Material and methods: We performed a retrospective observational study on a cohort of stroke patients who benefited from thrombolysis and/ or thrombectomy during 2015 in Saint Joseph Hospital’s Stroke Unit. The main objective of our research was to observe the clinical course of undernourished patients compared to the non-undernourished ones, using the NIHSS score at the stroke unit discharged and the 3 months modified Rankin score. Undernutrition was defined by a body mass index of (BMI) ≤ 21. Results: A total of 81 patients with thrombolysis and/or thrombectomy treatment were included. The median BMI in < 70-year-old patients was 24.5 and 25 in > 70-year-old patients. Initial severity measured by baseline NIHSS score was comparable among the undernourished and non-undernourished patients. Undernourished patients over 70 had a more severe neurological state at discharge (NIHSS 8.5 versus 3.9, NS) and were more disabled 3 months after discharge (Rankin 3.1 versus 2, NS). Conclusions: Undernutrition prior to stroke seems to have an impact on the functional prognosis of cerebral infarction, especially in patients > 70 years of age. Lack of significant results may be explained by the limited statistical means. A complementary study with a larger cohort is planned in order to assess this hypothesis

Sultr4;1 mutant seeds of Arabidopsis have an enhanced sulphate content and modified proteome suggesting metabolic adaptations to altered sulphate compartmentalization

<p>Abstract</p> <p>Background</p> <p>Sulphur is an essential macronutrient needed for the synthesis of many cellular components. Sulphur containing amino acids and stress response-related compounds, such as glutathione, are derived from reduction of root-absorbed sulphate. Sulphate distribution in cell compartments necessitates specific transport systems. The low-affinity sulphate transporters SULTR4;1 and SULTR4;2 have been localized to the vacuolar membrane, where they may facilitate sulphate efflux from the vacuole.</p> <p>Results</p> <p>In the present study, we demonstrated that the <it>Sultr4;1 </it>gene is expressed in developing Arabidopsis seeds to a level over 10-fold higher than the <it>Sultr4;2 </it>gene. A characterization of dry mature seeds from a <it>Sultr4;1 </it>T-DNA mutant revealed a higher sulphate content, implying a function for this transporter in developing seeds. A fine dissection of the <it>Sultr4;1 </it>seed proteome identified 29 spots whose abundance varied compared to wild-type. Specific metabolic features characteristic of an adaptive response were revealed, such as an up-accumulation of various proteins involved in sugar metabolism and in detoxification processes.</p> <p>Conclusions</p> <p>This study revealed a role for SULTR4;1 in determining sulphate content of mature Arabidopsis seeds. Moreover, the adaptive response of <it>sultr4;1 </it>mutant seeds as revealed by proteomics suggests a function of SULTR4;1 in redox homeostasis, a mechanism that has to be tightly controlled during development of orthodox seeds.</p

Characterization of two novel proteins involved in mitochondrial DNA anchoring in Trypanosoma brucei.

Trypanosoma brucei is a single celled eukaryotic parasite in the group of the Kinetoplastea. The parasite harbors a single mitochondrion with a singular mitochondrial genome that is known as the kinetoplast DNA (kDNA). The kDNA consists of a unique network of thousands of interlocked circular DNA molecules. To ensure proper inheritance of the kDNA to the daughter cells, the genome is physically linked to the basal body, the master organizer of the cell cycle in trypanosomes. The connection that spans, cytoplasm, mitochondrial membranes and the mitochondrial matrix is mediated by the Tripartite Attachment Complex (TAC). Using a combination of proteomics and RNAi we test the current model of hierarchical TAC assembly and identify TbmtHMG44 and TbKAP68 as novel candidates of a complex that connects the TAC to the kDNA. Depletion of TbmtHMG44 or TbKAP68 each leads to a strong kDNA loss but not missegregation phenotype as previously defined for TAC components. We demonstrate that the proteins rely on both the TAC and the kDNA for stable localization to the interface between these two structures. In vitro experiments suggest a direct interaction between TbmtHMG44 and TbKAP68 and that recombinant TbKAP68 is a DNA binding protein. We thus propose that TbmtHMG44 and TbKAP68 are part of a distinct complex connecting the kDNA to the TAC

Sulfur metabolism and transport in seeds of the model species M. truncatula and Arabidopsis : functional study of sulfate transporters

Le soufre est un macronutriment essentiel contribuant à l’élaboration du rendement et de la qualité des graines. Chez les espèces modèles M. truncatula et Arabidopsis, les gènes associés à la réduction du sulfate et à la biosynthèse des acides aminés soufrés sont exprimés dans l’embryon lors du remplissage de la graine, soulignant l’importance du transport de sulfate jusqu’à ce tissu. Chez M. truncatula, trois gènes codant des transporteurs de sulfate putatifs, MtSultr3;5, MtSultr2;2, et MtSultr4;1, sont fortement exprimés dans la graine. Le criblage des populations de mutants disponibles a permis d’identifier des mutants Tnt1 et EMS pour ces gènes. Cette étude a parallèlement été élargie à la caractérisation de mutants ADN-T d’Arabidopsis pour les cinq transporteurs de sulfate du groupe 3 (de la membrane plasmique) et pour SULTR4;1 (vacuolaire), dont les gènes s’expriment dans la graine en développement. Un rôle des transporteurs du groupe 3 dans les échanges de sulfate à l’intérieur de la graine a été mis en évidence. En particulier, l’analyse du protéome des graines de ces mutants a révélé un défaut d’accumulation des formes processées des protéines de réserve au sein de l’embryon (sultr3;5) et des modulations spécifiques de la composition protéique suggérant l’utilisation de sources alternatives de soufre (sultr3;4). Enfin, les résultats contrastés obtenus pour le mutant sultr4;1 suggèrent un rôle de l’efflux de sulfate des vacuoles pour le maintien de l’homéostasie redox lors du développement de la graine.Sulfur is an essential macronutrient contributing to crop yield and seed quality. In the model species M. truncatula and Arabidopsis, genes involved in sulfate reduction and sulfur amino acid biosynthesis are expressed in the embryo during seed filling, underlying the importance of sulfate transport until this tissue. In M. truncatula, three genes encoding putative sulfate transporters, MtSultr3;5, MtSultr2;2, et MtSultr4;1, are strongly expressed in seeds. By screening mutant collections, we identified Tnt1 and EMS mutants for these genes. In parallel, this study was extended to the characterization of Arabidopsis T-DNA mutants for the five sulfate transporters belonging to group 3 (plasmalemma-located) and SULTR4;1 (vacuolar), whose genes are expressed in developing seeds. A role of the group 3 sulfate transporters in sulfate exchange between seed tissue was revealed. In particular, seed proteome analysis for these mutants revealed a reduced accumulation of storage protein processing within the embryo (sultr3;5), and specific modulations of protein composition suggesting the utilization of alternative sulfur sources (sultr3;4). Finally, contrasted results obtained for the sultr4;1 mutant suggest a role of sulfate efflux from vacuole for maintaining redox homeostasis during seed development

Métabolisme et transport du soufre dans les graines des espèces modèles M. truncatula et Arabidopsis (étude fonctionnelle des transporteurs de sulfate)

Le soufre est un macronutriment essentiel contribuant à l élaboration du rendement et de la qualité des graines. Chez les espèces modèles M. truncatula et Arabidopsis, les gènes associés à la réduction du sulfate et à la biosynthèse des acides aminés soufrés sont exprimés dans l embryon lors du remplissage de la graine, soulignant l importance du transport de sulfate jusqu à ce tissu. Chez M. truncatula, trois gènes codant des transporteurs de sulfate putatifs, MtSultr3;5, MtSultr2;2, et MtSultr4;1, sont fortement exprimés dans la graine. Le criblage des populations de mutants disponibles a permis d identifier des mutants Tnt1 et EMS pour ces gènes. Cette étude a parallèlement été élargie à la caractérisation de mutants ADN-T d Arabidopsis pour les cinq transporteurs de sulfate du groupe 3 (de la membrane plasmique) et pour SULTR4;1 (vacuolaire), dont les gènes s expriment dans la graine en développement. Un rôle des transporteurs du groupe 3 dans les échanges de sulfate à l intérieur de la graine a été mis en évidence. En particulier, l analyse du protéome des graines de ces mutants a révélé un défaut d accumulation des formes processées des protéines de réserve au sein de l embryon (sultr3;5) et des modulations spécifiques de la composition protéique suggérant l utilisation de sources alternatives de soufre (sultr3;4). Enfin, les résultats contrastés obtenus pour le mutant sultr4;1 suggèrent un rôle de l efflux de sulfate des vacuoles pour le maintien de l homéostasie redox lors du développement de la graine.Sulfur is an essential macronutrient contributing to crop yield and seed quality. In the model species M. truncatula and Arabidopsis, genes involved in sulfate reduction and sulfur amino acid biosynthesis are expressed in the embryo during seed filling, underlying the importance of sulfate transport until this tissue. In M. truncatula, three genes encoding putative sulfate transporters, MtSultr3;5, MtSultr2;2, et MtSultr4;1, are strongly expressed in seeds. By screening mutant collections, we identified Tnt1 and EMS mutants for these genes. In parallel, this study was extended to the characterization of Arabidopsis T-DNA mutants for the five sulfate transporters belonging to group 3 (plasmalemma-located) and SULTR4;1 (vacuolar), whose genes are expressed in developing seeds. A role of the group 3 sulfate transporters in sulfate exchange between seed tissue was revealed. In particular, seed proteome analysis for these mutants revealed a reduced accumulation of storage protein processing within the embryo (sultr3;5), and specific modulations of protein composition suggesting the utilization of alternative sulfur sources (sultr3;4). Finally, contrasted results obtained for the sultr4;1 mutant suggest a role of sulfate efflux from vacuole for maintaining redox homeostasis during seed development.DIJON-BU Doc.électronique (212319901) / SudocSudocFranceF

Examination of the role of sulfate transporters expressed in seeds by using Arabidopsis T-DNA mutants

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