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The aim of the Committee for Open Science's Electronic Laboratory Notebook Working Group is to put forward an analysis methodology for scientific teams who need to choose an electronic laboratory notebook (ELN). An ELN is the dematerialised digital version of the conventional lab notebook which is basically a logbook used to detail the day-to-day activities of research projects. Researchers use it to list descriptions of the experiments and protocols they use along with details of each individual's contribution. The working group makes recommendations on the need for the different tools scientists use to be interoperable and sets out criteria to help researchers choose the right tool. It does not provide absolute elements of comparison between different tools and does not recommend a single tool for use by the whole higher education and research community. The report also includes a comparative list of existing tools along with a method for implementing an ELN.; Le groupe de travail « Cahier de laboratoire électronique », du Comité pour la science ouverte, a pour objectif de proposer une méthodologie d’analyse aux équipes scientifiques ayant à faire le choix d’un cahier de laboratoire électronique (désigné par l’acronyme ELN [Electronic Lab Notebook]). L’ELN est la version numérique dématérialisée du cahier de laboratoire. Celui-ci est un journal de bord employé pour détailler au quotidien les activités liées aux projets de recherche. Il assure le suivi du descriptif des expériences et des protocoles utilisés ainsi que les contributions de chacune et chacun. Le groupe de travail énonce des recommandations relatives à la nécessaire interopérabilité entre différents outils utilisés par les scientifiques et des critères d’aide à la décision pour le choix d’un outil. Il ne fournit pas des éléments absolus de comparaison entre différents outils, ni de préconisations pour l’adoption d’un outil unique pour l’ensemble de la communauté de l’enseignement supérieur et de la recherche. Le rapport comprend une liste comparative d’outils existants ainsi qu’une méthode pour mener à bien l’implémentation d’un tel outil.

The report of the "Electronic Laboratory Notebook" (ELN) working group provides a common vision on the definition, framing, uses and functional scope of the ELN, which must be able to integrate into existing IT and institutional environments. It makes recommendations on the requirements for the choice of a tool and includes a comparative list of existing tools.

International audience; The Set1 family of histone H3 lysine 4 (H3K4) methyltransferases is highly conserved from yeast to human. Here we show that the Set1 complex (Set1C) directly binds RNA in vitro through the regions that comprise the double RNA recognition motifs (dRRM) and N-SET domain within Set1 and its subunit Spp1. To investigate the functional relevance of RNA binding, we performed UV RNA crosslinking (CRAC) for Set1 and RNA polymerase II in parallel with ChIP-seq experiments. Set1 binds nascent transcripts through its dRRM. RNA binding is important to define the appropriate topology of Set1C distribution along transcription units and correlates with the efficient deposition of the H3K4me3 mark. In addition, we uncovered that Set1 binds to different classes of RNAs to levels that largely exceed the levels of binding to the general population of transcripts, suggesting the Set1 persists on these RNAs after transcription. This class includes RNAs derived from SET1, Ty1 retrotransposons, specific transcription factors genes and snRNAs (small nuclear RNAs). We propose that Set1 modulates adaptive responses, as exemplified by the post-transcriptional inhibition of Ty1 retrotransposition.

International audience; The yeast KEOPS protein complex comprising Kae1, Bud32, Cgi121, Pcc1 and Gon7 is responsible for the essential tRNA threonylcarbamoyladenosine (t(6)A) modification. Deletion of genes coding for the KEOPS subunits also affects telomere elongation and transcriptional regulation. In the present work, the crystal structure of Bud32/Cgi121 in complex with ADP revealed that ADP is bound in the catalytic site of Bud32 in a canonical manner characteristic of Protein Kinase A (PKA) family proteins. We found that Gon7 forms a stable heterodimer with Pcc1 and report the crystal structure of the Pcc1-Gon7 heterodimer. Gon7 interacts with the same Pcc1 region engaged in the archaeal Pcc1 homodimer. We further show that yeast KEOPS, unlike its archaeal counterpart, exists as a heteropentamer in which Gon7, Pcc1, Kae1, Bud32 and Cgi121 also adopt a linear arrangement. We constructed a model of yeast KEOPS that provides structural insight into the role of Gon7. The model also revealed the presence of a highly positively charged crater surrounding the entrance of Kae1 that likely binds tRNA.

Idiopatic Central Hypogonadism (ICH) is a rare pathology with a strong genetic component, in which hypothalamic and pituitary dysfunctions involving development and/or functionality of GnRH neurons, cause a reduced or absent gonads functionality. This disease can occur in association with anosmia or hyposmia, (Kallmann Syndrome, KS) o with a normal sense of smell (normosmic idiopathic hypogonadotropic hypogonadism, nIHH) and it shows an extreme phenotypic variability. Despite the identification of 14 genes implicated in the pathogenesis of the disease, approximatively 70% of ICH cases remains idiopathic. Among the causative genes, a role of particular importance is covered by the the Prokineticin pathway, in particular the Prokineticin-2 (PROK2) and its receptor (PROKR2). In fact, in approximately 10% of cases of ICH is possible to identify a genetic variant in one of these two genes such as pathogenetic event of the disease. The receptor PROKR2 belongs to the family of G-protein coupled receptor (GPCR). Its activation, through the binding with PROK2, determines the activation of protein Gq, Gs and Gi, a consequent production of IP3, cAMP and subsequently the mobilization of intracellular calcium. To date in literature have been described 27 PROKR2 mutations and the functional studies performed on a minority of them have only evaluated the effects on the Gq-IP3 signal transduction pathway. Nevertheless a growing number of works in the field of GPCRs demonstrates the importance of the functional studies of all the possible pathways related to a single receptor in order to interpret the functional consequences of genetic variants identified. In the present work we have developed two main lines of research starting from the wider availability of Italian cohort of ICH patients. In the first part of this work we have carried out studies of genetic screening of a cohort of 217 patients, considering the main causative known genes for that pathology, including PROKR2. Genetic variants identified in PROKR2 were then characterized by a functional point of view to test their potential pathogenic role. This screening allowed the identification of seven PROKR2 missense variants (V158I, L173R,T260M, R268C, V274D, V331M and V334M) of which 3 have not yet been described in the literature; in addition to 2 variants nonsense (15fsX45, 20fsX43). The variants identified have been inserted by site-directed mutagenesis into vectorsSPRT-PROKR2-pcDNA3, characterized by the presence of a Rhodopsin tag at the N-terminus of the receptor. This allows the display of the cellular localization of the mutants by binding with an anti-rhodopsin antibody. The constructs thus generated were transfected into HEK 293 cells and CHO for the following functional studies. The FACS analysis revealed that all variants have a reduced membrane expression (reduction of 11-55%), with the exception of the mutation V334M, which shows an expression slightly exceeding that of the wild-type receptor. Functional assays were then performed with the generation of concentration-effectcurves for both IP1 that for cAMP. The results obtained show how the mutation T260M, R268C, V274D, V331M and V334M cause a strong reduction of the signal mediated by the Gq protein , while the signal of cAMP mediated by the Gs protein is significantly reduced in the mutant L173R andV334M. The V334M and V274D mutations are characterized by a marked inactivation of both pathways. Finally analyzing the homology model of PROKR2 it appears evident that the variant V331M is localized at the level of a highly conserved domain (the motif NPXXY), involved in signal transduction. These are the first experiments that analyze both transduction pathways activated by PROKR2 receptor and showing how the different variants associated with ICH can affect signal transduction pathways in a very variable manner. In particular, some variants causing inability to stimulate the two pathways, suggesting that the integrity of both is necessary for normal development and function of GnRH-secreting neurons. The second part of this thesis, it was instead intended to further clarify the genetic mechanisms (and eventually epigenetic) about the ethiopathogenesis underlying ICH. To conduct these studies we used the techniques of SNPs and CNVs genotyping , on a selected series of familial cases of ICH. For each patient were analyzed 660,000 SNPs and CNVs 100,000, then comparing them with a large database of apparently healthy controls in our possession, in a case / control analysis. The analyzes focused on the identification of SNPs and on the presence of CNVs significantly correlated with ICH and on an family type analysis to detect extended regions of homozygosity in patients (LOH = Loss of Heterozigosity regions). A first macroscopic analysis of the data obtained shows that the number and extent of the deletions in single copy is significantly higher in cases of ICH, compared to controls. The analysis of SNPs and CNVs showed, among the 30 identified loci four genes (CNTNAP2, GPC, RAB39B and PPFIA2) that for expression and molecular function seem to be good candidates for direct sequencing screening in ICH patients. Furthermore we have identified three clusters of microRNA (mir4275, mir507/508/509,mir320D2), suggesting for the first time a potential involvement of these molecules in ICH pathogenesis. Analyzing instead the genes that reside in LOH areas , it is possible to observe an enrichment for certain pathways or protein families, such as: the FGFR pathway; cadherins and cell adhesion molecules (CAM); receptors and ligands involved in the differentiation of the central nervous system (CNS), hypothalamus and pituitary; genes associated with midline defects or with Prader-Willi and Angelmann syndrome; plexins and RAB proteins.

Recent studies on yeast transcriptome have revealed the presence of a large set of RNA polymerase II transcripts mapping to intergenic and antisense regions or overlapping canonical genes. Most of these ncRNAs (ncRNAs) are subject to termination by the Nrd1-dependent pathway and rapid degradation by the nuclear exosome and have been dubbed cryptic unstable transcripts (CUTs). CUTs are often considered as by-products of transcriptional noise, but in an increasing number of cases they play a central role in the control of gene expression. Regulatory mechanisms involving expression of a CUT are diverse and include attenuation, transcriptional interference, and alternative transcription start site choice. This review focuses on the impact of cryptic transcription on gene expression, describes the role of the Nrd1-complex as the main actor in preventing nonfunctional and potentially harmful transcription, and details a few systems where expression of a CUT has an essential regulatory function. We also summarize the most recent studies concerning other types of ncRNAs and their possible role in regulation.

findes ikke Udgivelsesdato: 5. april The nuclear exosome is involved in numerous RNA metabolic processes. Exosome degradation of rRNA, snoRNA, snRNA and tRNA in Saccharomyces cerevisiae is activated by TRAMP complexes, containing either the Trf4p or Trf5p poly(A) polymerase. These enzymes are presumed to facilitate exosome access by appending oligo(A)-tails onto structured substrates. Another role of the nuclear exosome is that of mRNA surveillance. In strains harboring a mutated THO/Sub2p system, involved in messenger ribonucleoprotein particle biogenesis and nuclear export, the exosome-associated 3' --> 5' exonuclease Rrp6p is required for both retention and degradation of nuclear restricted mRNAs. We show here that Trf4p, in the context of TRAMP, is an mRNA surveillance factor. However, unlike Rrp6p, Trf4p only partakes in RNA degradation and not in transcript retention. Surprisingly, a polyadenylation-defective Trf4p protein is fully active, suggesting polyadenylation-independent mRNA degradation. Transcription pulse-chase experiments show that HSP104 molecules undergoing quality control in THO/sub2 mutant strains fall into two distinct populations: One that is quickly degraded after transcription induction and another that escapes rapid decay and accumulates in foci associated with the HSP104 transcription site.

INTRODUCTION: Defects of prokineticin pathway affect the neuroendocrine control of reproduction, but their role in the pathogenesis of central hypogonadism remains undefined, and the functional impact of the missense PROKR2 variants has been incompletely characterized. MATERIAL AND METHODS: In a series of 246 idiopathic central hypogonadism patients, we found three novel (p.V158I, p.V334M, and p.N15TfsX30) and six already known (p.L173R, p.T260M, p.R268C, p.V274D, p.V331M, and p.H20MfsX23) germline variants in the PROKR2 gene. We evaluated the effects of seven missense alterations on two different prokineticin receptor 2 (PROKR2)-dependent pathways: inositol phosphate-Ca(2+) (Gq coupling) and cAMP (Gs coupling). RESULTS: PROKR2 variants were found in 16 patients (6.5%). Expression levels of variants p.V158I and p.V331M were moderately reduced, whereas they were markedly impaired in the remaining cases, except p.V334M, which was significantly overexpressed. The variants p.T260M, p.R268C, and p.V331M showed no remarkable changes in cAMP response (EC50) whereas the IP signaling appeared more profoundly affected. In contrast, cAMP accumulation cannot be stimulated through the p.L173R and p.V274D, but IP EC50 was similar to wt inp.L173R and increased by 10-fold in p.V274D. The variant p.V334M led to a 3-fold increase of EC50 for both cAMP and IP. CONCLUSION: Our study shows that single PROKR2 missense allelic variants can either affect both signaling pathways differently or selectively. Thus, the integrity of both PROKR2-dependent cAMP and IP signals should be evaluated for a complete functional testing of novel identified allelic variants.

La biogenèse des ribosomes est un processus hautement complexe impliquant plusieurs centaines de facteurs de synthèse dont la finalité est la production de toutes les protéines de la cellule. Chaque étapes de la ribogenèse est une source potentielle d’erreur et est vérifiée par des mécanismes de contrôle de qualité redondants et rigoureux. Dans la première partie de ma thèse, j’ai collaboré à une meilleure compréhension d’une des voies de la surveillance nucléolaire, celle qui dégrade les pré-ribosomes défectueux recrutée à partir de l’extrémité 3’ des ARN ribosomiques (ARNr). Suite à une erreur d’assemblage, les pré-ARNr sont polyadénylés par le complexe nucléaire TRAMP, ce dernier est recruté cotranscriptionnellement. Les ARNr polyadénylés deviennent alors des substrats pour l’exosome et sont dégradés. On ignore comment la synthèse des ARNr, leur maturation et la surveillance nucléolaire sont intégrées mais on suspecte l’existence d’une interface physico-fonctionnelle à l’ADNr. Dans une deuxième partie de ce travail, nous avons testé si des cofacteurs de l’exosome, les protéines Nrd1/Nab3 étaient impliquées dans la surveillance des pré-ARNr. Nous rapportons que, chez la levure S. cerevisiae, le facteur d’élongation Spt5 interagit avec l’ARN polymérase (Pol) I et avec Nrd1. L’interaction entre Spt5 et ces deux protéines requiert la présence d’un domaine particulier situé à l’extrémité C-terminal ressemblant au « Carboxy-terminal domain » (CTD) de la Pol II appelé « Carboxy terminal repeat » (CTR). Spt4/Spt5 et Nrd1/Nab3 interagissent fonctionnellement avec Rrp6, sous-unité catalytique de l’exosome. Ces complexes colocalisent à l’ADNr, comme déterminé par ChIP. Des mutations dans le domaine de liaison à l’ARN (RRM – « RNA recognition motif ») de Nrd1 mais pas dans son domaine de liaison au CTD (CID – « carboxy-terminal interacting domain ») de la Pol II et dans le RRM de Nab3 mènent à l’accumulation de transcrits ribosomiques aberrants polyadénylés. Ceci indique que Nrd1/Nab3 contribue au recrutement de la surveillance nucléolaire à la Pol en cours d’élongation pour « scruter » les transcrits ribosomiques naissants. Nous proposons un modèle dans lequel Nrd1/Nab3 sont recrutées à la machinerie d’élongation de la transcription via leur interaction avec Spt5 afin de surveiller la synthèse des transcrits ribosomiques. Si un problème dans la fabrication des transcrits naissants a lieu, des sites de liaison pour Nrd1/Nab3 sur les pré-ARNr normalement recouverts de facteurs de synthèse seraient dénudés. Ceci marquerait les transcrits ribosomiques aberrants et entrainerait leur dégradation par les machineries de dégradation TRAMP et exosome., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

La biogenèse des ribosomes est un processus hautement complexe impliquant plusieurs centaines de facteurs de synthèse dont la finalité est la production de toutes les protéines de la cellule. Chaque étapes de la ribogenèse est une source potentielle d’erreur et est vérifiée par des mécanismes de contrôle de qualité redondants et rigoureux. Dans la première partie de ma thèse, j’ai collaboré à une meilleure compréhension d’une des voies de la surveillance nucléolaire, celle qui dégrade les pré-ribosomes défectueux recrutée à partir de l’extrémité 3’ des ARN ribosomiques (ARNr). Suite à une erreur d’assemblage, les pré-ARNr sont polyadénylés par le complexe nucléaire TRAMP, ce dernier est recruté cotranscriptionnellement. Les ARNr polyadénylés deviennent alors des substrats pour l’exosome et sont dégradés. On ignore comment la synthèse des ARNr, leur maturation et la surveillance nucléolaire sont intégrées mais on suspecte l’existence d’une interface physico-fonctionnelle à l’ADNr. Dans une deuxième partie de ce travail, nous avons testé si des cofacteurs de l’exosome, les protéines Nrd1/Nab3 étaient impliquées dans la surveillance des pré-ARNr. Nous rapportons que, chez la levure S. cerevisiae, le facteur d’élongation Spt5 interagit avec l’ARN polymérase (Pol) I et avec Nrd1. L’interaction entre Spt5 et ces deux protéines requiert la présence d’un domaine particulier situé à l’extrémité C-terminal ressemblant au « Carboxy-terminal domain » (CTD) de la Pol II appelé « Carboxy terminal repeat » (CTR). Spt4/Spt5 et Nrd1/Nab3 interagissent fonctionnellement avec Rrp6, sous-unité catalytique de l’exosome. Ces complexes colocalisent à l’ADNr, comme déterminé par ChIP. Des mutations dans le domaine de liaison à l’ARN (RRM – « RNA recognition motif ») de Nrd1 mais pas dans son domaine de liaison au CTD (CID – « carboxy-terminal interacting domain ») de la Pol II et dans le RRM de Nab3 mènent à l’accumulation de transcrits ribosomiques aberrants polyadénylés. Ceci indique que Nrd1/Nab3 contribue au recrutement de la surveillance nucléolaire à la Pol en cours d’élongation pour « scruter » les transcrits ribosomiques naissants. Nous proposons un modèle dans lequel Nrd1/Nab3 sont recrutées à la machinerie d’élongation de la transcription via leur interaction avec Spt5 afin de surveiller la synthèse des transcrits ribosomiques. Si un problème dans la fabrication des transcrits naissants a lieu, des sites de liaison pour Nrd1/Nab3 sur les pré-ARNr normalement recouverts de facteurs de synthèse seraient dénudés. Ceci marquerait les transcrits ribosomiques aberrants et entrainerait leur dégradation par les machineries de dégradation TRAMP et exosome., Doctorat en Sciences, info:eu-repo/semantics/published