Ancient origin, functional conservation and fast evolution of DNA-dependent RNA polymerase III

RNA polymerase III contains seventeen subunits in yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and in human cells. Twelve of them are akin to the core RNA polymerase I or II. The five other are RNA polymerase III-specific and form the functionally distinct groups Rpc31-Rpc34-Rpc82 and Rpc37-Rpc53. Currently sequenced eukaryotic genomes revealed significant homology to these seventeen subunits in Fungi, Animals, Plants and Amoebozoans. Except for subunit Rpc31, this also extended to the much more distantly related genomes of Alveolates and Excavates, indicating that the complex subunit organization of RNA polymerase III emerged at a very early stage of eukaryotic evolution. The Sch.pombe subunits were expressed in S.cerevisiae null mutants and tested for growth. Ten core subunits showed heterospecific complementation, but the two largest catalytic subunits (Rpc1 and Rpc2) and all five RNA polymerase III-specific subunits (Rpc82, Rpc53, Rpc37, Rpc34 and Rpc31) were non-functional. Three highly conserved RNA polymerase III-specific domains were found in the twelve-subunit core structure. They correspond to the Rpc17-Rpc25 dimer, involved in transcription initiation, to an N-terminal domain of the largest subunit Rpc1 important to anchor Rpc31, Rpc34 and Rpc82, and to a C-terminal domain of Rpc1 that presumably holds Rpc37, Rpc53 and their Rpc11 partner

Les ARN polymérases chez Saccharomyces cerevisiae (étude des sous-unités Rpb5, Rpb7 et Rpc25)

Chez les eucaryotes, comme Saccharomyces cerevisiae, la transcription de l'ADN est assurée par trois enzymes: l'ARN polymérase I, l'ARN polymérase II et l'ARN polymérase III, qui synthétisent respectivement les ARN ribosomiques, les ARN messagers et les ARN de transfert. On sait aujourd'hui qu'elles ont une structure très conservée, faite de onze sous-unités dont cinq correspondent aux sous-unités de l'enzyme bactérienne, et six sont partagées avec les archées. La douzième sous-unité est typiquement eucaryote. Dans ce travail, nous traitons de deux composantes partagées avec les archées, la sous-unité commune, Rpb5 et la sous-unité Rpc25 de l'ARN polymérase III. Nous avons montré que les mutants de Rpb5 affectent les trois ARN polymérases ce qui conforte l'idée de son rôle commun dans les trois systèmes. Par ailleurs cette fonction semble conservée au cours de l'évolution, puisque des protéines chimères composées de plus de 90 % de séquence humaine sont fonctionnelles dans la levure. De plus, nous avons observé une colétalité entre des mutants de Rpb5 et les mutants nuls rpb9-delta et rpb4-delta de l'ARN Pol II. Ainsi, Rpb9 et Rpb4 pourraient coopérer avec Rpb5 pour stabiliser l'ARN polymérase II et structurer le site actif de l'enzyme. Nous avons établi que la sous-unité Rpc25 de l'ARN Pol III se lie à la sous-unité Rpc17 et forme un hétérodimère conservé au cours de l'évolution puisque Rpc17 et Rpc25 sont, respectivement, les orthologues des sous-unités RpoF et RpoE de l'ARN polymérase archébactérienne. Ce complexe se retrouve aussi dans les ARN polymérases I et II : Rpa14/Rpa43 et Rpb4/Rpb7, respectivement. De plus, nous avons établi que des mutants de Rpc25 sont affectés, in vitro, dans l'initiation de la transcription, et notamment dans la reconnaissance du site de pré-initiation. Ainsi, dans l'ARN Pol III l'hétérodimère Rpc17/Rpc25 jouerait un rôle dans l'initiation de la transcription, comme c'est le cas pour les complexes paralogues dans les ARN Pol I et ARN Pol II.In eukaryotes, like Saccharomyces cerevisiae, RNA is transcribed by three enzymes: RNA polymerases I, II and III that synthesize respectively ribosomal RNAs, messenger RNAs and transfer RNAs. It has been established that they have a conserved structure made of eleven subunits; five of them correspond to bacterial enzyme and six are conserved with archaea. The twelfth is solely eukaryote. This work deals with two components shared with archaea: the common subunit Rpb5 and a subunit of RNA Pol III, Rpc25. We showed here that mutants of Rpb5 affect the three RNA polymerases, in vivo, supporting the idea of a common role in all three transcriptional systems. Moreover, its function seems to be conserved, since hybrid proteins with up 90 % of the human sequence are functional in yeast. We observed a synthetic lethality between mutants of Rpb5 and null mutants rpb4-delta and rpb9-delta, in RNA Pol II. Thus, Rpb4 and Rpb9 may cooperate with Rpb5 to stabilise and structure the active site of RNA Pol-II. We established that the subunit Rpc25 of RNA Pol III binds to Rpc 17 and forms an heterodimer, conserved through evolution. Indeed, Rpc17 and Rpc25 are, respectively, the orthologues of RpoF and RpoE in archaea. This complex is also found in RNA PolI and RNA Pol II: Rpa14/Rpa43 and Rpb4/Rpb7, respectively. We also demonstrated that rpc25 mutants are impaired in the initiation step of RNA Pol III transcription, particularly in the recognition of the pre-initiation complex.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Exposure to pesticides, persistent and non − persistent pollutants in French 3.5-year-old children: Findings from comprehensive hair analysis in the ELFE national birth cohort

International audienceIntroductionExposure to endocrine disruptors during early childhood poses significant health risks. This study examines the exposure levels of French 3.5-year-old children to various persistent and non-persistent pollutants and pesticides using hair analysis as part of the ELFE national birth cohort. Differences in sex and geographical location among the children were investigated as ppossible determinants of exposure.MethodsExposure biomarkers from 32 chemical families were analyzed using LCMSMS and GCMSMS in 222 hair samples from children in the ELFE cohort. Of these, 46 mother–child pairs from the same cohort provided unique insight into prenatal and postnatal exposure. Regressions, correlations and discriminate analysis were used to assess relationships between exposure and possible confounding factors.Results and DiscussionAmong the biomarkers tested in children’s hair samples, 69 had a detection frequency of ≥ 50 %, with 20 showing a 100 % detection rate. The most detected biomarkers belonged to the bisphenol, organochlorine and organophosphate families. Sex-specific differences were observed for 26 biomarkers, indicating the role of the child’s sex in exposure levels. Additionally, regional differences were noted, with Hexachlorobenzene varying significantly across the different French regions. Nicotine presented both the highest concentration (16 303 pg/mg) and highest median concentration (81 pg/mg) measured in the children’s hair. Statistically significant correlations between the levels of biomarkers found in the hair samples of the mothers and their respective children were observed for fipronil (correlation coefficient = 0.32, p = 0.03), fipronil sulfone (correlation coefficient = 0.34, p = 0.02) and azoxystrobin (correlation coefficient = 0.29, p = 0.05).ConclusionsThe study highlights the elevated exposure levels of young children to various pollutants, highlighting the influence of sex and geography. Hair analysis emerges as a crucial tool for monitoring endocrine disruptors, offering insights into exposure risks and reinforcing the need for protective measures against these harmful substances

Multiple pesticides in mothers' hair samples and children's measurements at birth: Results from the French national birth cohort (ELFE)

International audienceBackground - A growing body of studies now suggests that the general population is continuously and ubiquitously exposed to numerous pesticides. However, studies investigating the possible role of environmental exposure to pesticides on fetal growth have focused on a limited set of substances, despite the hundreds of modern pesticides currently available. Aim - To explore the relation between maternal hair concentrations of 64 pesticides and metabolites and their newborns' measurements at birth, with data from the ELFE French nationwide birth cohort. Methods - We measured 64 compounds (10-100% detection) in bundles of hair 9 cm long collected at birth from 311 women who gave birth in France in 2011. We assessed their associations with birth weight, length, and head circumference, adjusted for potential confounders, and used elastic net regularization to simultaneously select the strongest predictors of measurements at birth. Selected variables were multiply imputed for missing values, and unpenalized estimators were assessed by standard linear regression. Results - We observed statistically significant associations between maternal hair concentrations of seven pesticides or pesticide metabolites and birth measurements (weight: fipronil sulfone; length: TCPy, bitertanol, DEP, and isoproturon; head circumference: tebuconazole and prochloraz). Analyses restricted to boys identified 12 additional compounds: 8 independently associated with birth weight (3Me4NP, DCPMU, DMST, fipronil, mecoprop, propoxur, fenhexamid, and thiabendazole), 2 with birth length (dieldrin and β-endosulfan), and 6 with head circumference (β-endosulfan, β-HCH, fenuron, DCPMU, propoxur, and thiabendazole). Conclusion - Our results suggest that prenatal exposure to 19 pesticides or metabolites from various chemical families may influence measurements at birth. As with any exploratory research findings, results should be interpreted cautiously, until they are replicated or verified by further epidemiological or mechanistic studies

Cohort Profile: The French National cohort of children ELFE: birth to 5 years

ELFE is the first French national birth cohort. Its objective is to study determinants of the development, health and socialization of children from birth to adulthood through a multidisciplinary approach. A total of 18 329 children were recruited at birth in a random sample of maternity units in metropolitan France during 25 selected days of 2011 spread over the year. Follow-up in the first 5 years consisted of telephone interviews of both parents of the child at age 2 months and 1 year and 2 years, and of one parent at age 3.5 years; a home visit at age 3.5 years; questionnaires to the child’s physician at age 2 years, the child’s nursery school doctor at age 3 to 4 years, and the child’s nursery schoolteacher at age 4 years. Participation rates at the age 2-month, 1- and 2-year and 3.5-year parental interviews were 92%, 86%, 82% and 80%, espectively, of contacted participants. The main categories of data collected concern: sociodemographic characteristics; family life; parental health, behaviour and life values; child development and health; child school performance, behaviour, and socialization; day care and school; and childhood environmental exposures. The ELFE has an open-data policy after an 18-month exclusivity period following each release of new data. The data-access policy, study protocols, questionnaires and data catalogue can be found online: [https://www.ELFE-france.fr/en/

Diagnostic pitfalls in sporadic transthyretin familial amyloid polyneuropathy (TTR-FAP).

International audienceTransthyretin familial amyloid polyneuropathies (TTR-FAPs) are autosomal dominant neuropathies of fatal outcome within 10 years after inaugural symptoms. Late diagnosis in patients who present as nonfamilial cases delays adequate management and genetic counseling. Clinical data of the 90 patients who presented as nonfamilial cases of the 300 patients of our cohort of patients with TTR-FAP were reviewed. They were 21 women and 69 men with a mean age at onset of 61 (extremes: 38 to 78 years) and 17 different mutations of the TTR gene including Val30Met (38 cases), Ser77Tyr (16 cases), Ile107Val (15 cases), and Ser77Phe (5 cases). Initial manifestations included mainly limb paresthesias (49 patients) or pain (17 patients). Walking difficulty and weakness (five patients) and cardiac or gastrointestinal manifestations (five patients), were less common at onset. Mean interval to diagnosis was 4 years (range 1 to 10 years); 18 cases were mistaken for chronic inflammatory demyelinating polyneuropathy, which was the most common diagnostic error. At referral a length-dependent sensory loss affected the lower limbs in 2, all four limbs in 20, and four limbs and anterior trunk in 77 patients. All sensations were affected in 60 patients (67%), while small fiber dysfunction predominated in the others. Severe dysautonomia affected 80 patients (90%), with postural hypotension in 52, gastrointestinal dysfunction in 50, impotence in 58 of 69 men, and sphincter disturbance in 31. Twelve patients required a cardiac pacemaker. Nerve biopsy was diagnostic in 54 of 65 patients and salivary gland biopsy in 20 of 30. Decreased nerve conduction velocity, increased CSF protein, negative biopsy findings, and false immunolabeling of amyloid deposits were the main causes of diagnostic errors. We conclude that DNA testing, which is the most reliable test for TTR-FAP, should be performed in patients with a progressive length-dependent small fiber polyneuropathy of unknown origin, especially when associated with autonomic dysfunction

PBPK modeling to support risk assessment of pyrethroid exposure in French pregnant women

International audienceBackgroundPyrethroids are widely used pesticides and are suspected to affect children's neurodevelopment. The characterization of pyrethroid exposure during critical windows of development, such as fetal development and prenatal life, is essential to ensure a better understanding of pyrethroids potential effects within the concept of Developmental Origins of Health and Disease.ObjectiveThe aim of this study was to estimate maternal exposure of French pregnant women from biomonitoring data and simulate maternal and fetal internal concentrations of 3 pyrethroids (permethrin, cypermethrin and deltamethrin) using a multi-substance pregnancy-PBPK (physiologically based pharmacokinetics) model. The estimated maternal exposures were compared to newly proposed toxicological reference values (TRV) children specific also called draft child-specific reference value to assess pyrethroid exposure risk during pregnancy i.e. during the in utero exposure period.MethodsA pregnancy-PBPK model was developed based on an existing adult pyrethroids model. The maternal exposure to each parent compound of pregnant women of the Elfe (French Longitudinal Study since Childhood) cohort was estimated by reverse dosimetry based on urinary biomonitoring data. To identify permethrin and cypermethrin contribution to their common urinary biomarkers of exposure, an exposure ratio based on biomarkers in hair was tested. Finally, exposure estimates were compared to current and draft child-specific reference values derived from rodent prenatal and postnatal exposure studies.ResultsThe main contributor to maternal pyrethroid diet intake is cis-permethrin. In blood, total internal concentrations main contributor is deltamethrin. In brain, the major contributors to internal pyrethroid exposure are deltamethrin for fetuses and cis-permethrin for mothers. Risk is identified only for permethrin when referring to the draft child-specific reference value. 2.5% of the population exceeded permethrin draft child-specific reference value.ConclusionsA new reverse dosimetry approach using PBPK model combined with human biomonitoring data in urine and hair was proposed to estimate Elfe pregnant population exposure to a pyrethroids mixture with common metabolites

Multiple pesticide analysis in hair samples of pregnant French women Results from the ELFE national birth cohort

International audienceBackground - A growing body of evidence suggests that prenatal exposure to pesticides might impair fetal development. Nonetheless, knowledge about pesticide exposure of pregnant women, especially in Europe, is largely restricted to a limited panel of molecules. Aim - To characterize the concentration of 140 pesticides and metabolites in hair strands from women in the ELFE French nationwide birth cohort. Methods - Among cohort members who gave birth in northeastern and southwestern France in 2011, we selected those with a sufficient available mass of hair (n = 311). Bundles of hair 9 cm long were collected at delivery. We screened 111 pesticides and 29 metabolites, including 112 selected a priori based on their reported usage or detection in the French environment. The bundles of hair from 47 women were split into three segments to explore the intraindividual variability of the exposure. Intraclass correlation coefficients (ICCs) were computed for the chemicals with a detection frequency >70%. Results - We detected a median of 43 chemicals per woman (IQR 38-47). Overall, 122 chemicals (>20 chemical families) were detected at least once, including 28 chemicals detected in 70-100% of hair samples. The highest median concentrations were observed for permethrin (median: 37.9 pg/mg of hair), p-nitrophenol (13.2 pg/mg), and pentachlorophenol (10.0 pg/mg). The ICCs for the 28 chemicals studied ranged from 0.59 to 0.94. Conclusion - Pregnant women are exposed to multiple pesticides simultaneously from various chemical families, including chemicals suspected to be reproductive toxicants or endocrine disruptors. The ICCs suggest that the intraindividual variability of pesticide concentrations in hair is lower than its interindividual variability

An Rpb4/Rpb7-Like Complex in Yeast RNA Polymerase III Contains the Orthologue of Mammalian CGRP-RCP

The essential C17 subunit of yeast RNA polymerase (Pol) III interacts with Brf1, a component of TFIIIB, suggesting a role for C17 in the initiation step of transcription. The protein sequence of C17 (encoded by RPC17) is conserved from yeasts to humans. However, mammalian homologues of C17 (named CGRP-RCP) are known to be involved in a signal transduction pathway related to G protein-coupled receptors, not in transcription. In the present work, we first establish that human CGRP-RCP is the genuine orthologue of C17. CGRP-RCP was found to functionally replace C17 in Δrpc17 yeast cells; the purified mutant Pol III contained CGRP-RCP and had a decreased specific activity but initiated faithfully. Furthermore, CGRP-RCP was identified by mass spectrometry in a highly purified human Pol III preparation. These results suggest that CGRP-RCP has a dual function in mammals. Next, we demonstrate by genetic and biochemical approaches that C17 forms with C25 (encoded by RPC25) a heterodimer akin to Rpb4/Rpb7 in Pol II. C17 and C25 were found to interact genetically in suppression screens and physically in coimmunopurification and two-hybrid experiments. Sequence analysis and molecular modeling indicated that the C17/C25 heterodimer likely adopts a structure similar to that of the archaeal RpoE/RpoF counterpart of the Rpb4/Rpb7 complex. These RNA polymerase subunits appear to have evolved to meet the distinct requirements of the multiple forms of RNA polymerases