Transcriptional Mutagenesis Induced by 8-Oxoguanine in Mammalian Cells

Most of the somatic cells of adult metazoans, including mammals, do not undergo continuous cycles of replication. Instead, they are quiescent and devote most of their metabolic activity to gene expression. The mutagenic consequences of exposure to DNA–damaging agents are well documented, but less is known about the impact of DNA lesions on transcription. To investigate this impact, we developed a luciferase-based expression system. This system consists of two types of construct composed of a DNA template containing an 8-oxoguanine, paired either with a thymine or a cytosine, placed at defined positions along the transcribed strand of the reporter gene. Analyses of luciferase gene expression from the two types of construct showed that efficient but error-prone transcriptional bypass of 8-oxoguanine occurred in vivo, and that this lesion was not repaired by the transcription-coupled repair machinery in mammalian cells. The analysis of luciferase activity expressed from 8OG:T-containing constructs indicated that the magnitude of erroneous transcription events involving 8-oxoguanine depended on the sequence contexts surrounding the lesion. Additionally, sequencing of the transcript population expressed from these constructs showed that RNA polymerase II mostly inserted an adenine opposite to 8-oxoguanine. Analysis of luciferase expression from 8OG:C-containing constructs showed that the generated aberrant mRNAs led to the production of mutant proteins with the potential to induce a long-term phenotypical change. These findings reveal that erroneous transcription over DNA lesions may induce phenotypical changes with the potential to alter the fate of non-replicating cells

ERREURS LORS DE LA TRANSMISSION DE L'INFORMATION GENETIQUE (ETUDES DE CAS ET INFLUENCES DE L'ENVIRONNEMENT)

ON PEUT CONSIDERER LE FLUX DE L'INFORMATION GENETIQUE SOUS DEUX ASPECTS SELON QUE L'ON CONSIDERE LE COPIAGE OU L'EXPRESSION DE L'INFORMATION FAISANT INTERVENIR RESPECTIVEMENT LA REPLICATION OU LA TRANSCRIPTION ET LA TRADUCTION DONT LES TAUX D'ERREUR SONT TRES FAIBLES. DURANT MA THESE, JE ME SUIS INTERESSE AUX ERREURS PRODUITES LORS DES DIFFERENTS PROCESSUS DE TRANSMISSION DE L'INFORMATION GENETIQUE. EN CE QUI CONCERNE LA REPLICATION, J'AI VOULU ETUDIER SA FIDELITE EN FONCTION DE LA PHYSIOLOGIE DES CELLULES D'ESCHERICHIA COLI ET L'INFLUENCE DE L'ENVIRONNEMENT SUR CETTE FIDELITE. L'ANALYSE DE LA DESCENDANCE D'UN ADN DU PHAGE M13 CONTENANT UN MESAPPARIEMENT PRECIS M'A PERMIS DE VOIR QUE L'EFFICACITE DU SYSTEME DE REPARATION DES MESAPPARIEMENTS DE BASE DIMINUAIT PROGRESSIVEMENT POUR DES BACTERIES ENTRANT EN PHASE STATIONNAIRE DE CROISSANCE. LA QUANTIFICATION DE LA FREQUENCE D'APPARITION DE REVERTANTS LAC + AU SEIN DE COLONIES D'E. COLI AYANT UNE MUTATION DEFINIE DANS LE GENE LACZ M'A PERMIS D'IDENTIFIER LA VARIATION DU SPECTRE DE MUTATION EN FONCTION DE LA PRESENCE OU NON D'OXYGENE. JE ME SUIS AUSSI INTERESSE AUX MECANISMES CONTROLANT LA FIDELITE DE L'EXPRESSION D'UN ALLELE DU GENE LACZ CONTENANT UNE INSERTION D'UN DINUCLEOTIDE GA. LES ANALYSES GENETIQUES FAITES A PARTIR DE CETTE SOUCHE ONT PERMIS DE REVELER QUE LES ERREURS D'EXPRESSION DE CET ALLELE ETAIENT NOTAMMENT DUES A L'HYPOMODIFICATION DE CERTAINS ARNT. DE PLUS, UNE INCUBATION PROLONGEE DE 7 JOURS EN MILIEU STRUCTURE INDUIT UNE AUGMENTATION DES ERREURS D'EXPRESSION DE CET ALLELE. CETTE OBSERVATION N'EST D'AILLEURS PAS RESTREINTE A CET ALLELE, PUISQU'ON OBSERVE EN GENERAL QUE LES ERREURS DE DECALAGE DE PHASE LORS DE LA TRANSCRIPTION ET/OU DE LA TRADUCTION SONT AUGMENTEES APRES 7 JOURS D'INCUBATION. AINSI, IL SEMBLE QUE LES TAUX D'ERREURS DES MECANISMES ETUDIES SONT CONTROLES GENETIQUEMENT, MAIS SONT AUSSI INFLUENCES PAR LES CONDITIONS ENVIRONNEMENTALES.PARIS-BIUSJ-Thèses (751052125) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

An extended dsRBD is required for post-transcriptional modification in human tRNAs

International audienceIn tRNA, dihydrouridine is a conserved modified base generated by the post-transcriptional reduction of uridine. Formation of dihydrouridine 20, located in the D-loop, is catalyzed by dihydrouridine synthase 2 (Dus2). Human Dus2 (HsDus2) expression is upreg-ulated in lung cancers, offering a growth advantage throughout its ability to interact with components of the translation apparatus and inhibit apoptosis. Here, we report the crystal structure of the individual domains of HsDus2 and their functional characterization. HsDus2 is organized into three major modules. The N-terminal catalytic domain contains the flavin cofactor involved in the reduction of uridine. The second module is the conserved ␣-helical domain known as the tRNA binding domain in HsDus2 homologues. It is connected via a flexible linker to an unusual extended version of a dsRNA binding domain (dsRBD). Enzymatic assays and yeast comple-mentation showed that the catalytic domain binds selectively NADPH but cannot reduce uridine in the absence of the dsRBD. While in Dus enzymes from bacteria, plants and fungi, tRNA binding is essentially achieved by the ␣-helical domain, we showed that in HsDus2 this function is carried out by the dsRBD. This is the first reported case of a tRNA-modifying enzyme carrying a dsRBD used to bind tRNAs

tRNA 2′-O-methylation by a duo of TRM7/FTSJ1 proteins modulates small RNA silencing in Drosophila

International audience2'-O-Methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structure and function with crucial roles in various RNA-mediated processes ranging from RNA silencing, translation, self versus non-self recognition to viral defense mechanisms. Here, we identify two Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1. Genetic knockout studies together with MALDI-TOF mass spectrometry and RiboMethSeq mapping revealed that CG7009 is responsible for methylating the wobble position in tRNAPhe, tRNATrp and tRNALeu, while CG5220 methylates position C32 in the same tRNAs and also targets additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as lifespan reduction, small RNA pathways dysfunction and increased sensitivity to RNA virus infections. Our results provide the first detailed characterization of two TRM7 family members in Drosophila and uncover a molecular link between enzymes catalyzing Nm at specific tRNAs and small RNA-induced gene silencing pathways

Fink , a new generation of broker for the LSST community

accepted in MNRASInternational audienceFink is a broker designed to enable science with large time-domain alert streams such as the one from the upcoming Vera C. Rubin Observatory Legacy Survey of Space and Time (LSST). It exhibits traditional astronomy broker features such as automatised ingestion, annotation, selection and redistribution of promising alerts for transient science. It is also designed to go beyond traditional broker features by providing real-time transient classification which is continuously improved by using state-of-the-art Deep Learning and Adaptive Learning techniques. These evolving added values will enable more accurate scientific output from LSST photometric data for diverse science cases while also leading to a higher incidence of new discoveries which shall accompany the evolution of the survey. In this paper we introduce Fink, its science motivation, architecture and current status including first science verification cases using the Zwicky Transient Facility alert stream

Blood Gene Expression Predicts Bronchiolitis Obliterans Syndrome

Bronchiolitis obliterans syndrome (BOS), the main manifestation of chronic lung allograft dysfunction, leads to poor long-term survival after lung transplantation. Identifying predictors of BOS is essential to prevent the progression of dysfunction before irreversible damage occurs. By using a large set of 107 samples from lung recipients, we performed microarray gene expression profiling of whole blood to identify early biomarkers of BOS, including samples from 49 patients with stable function for at least 3 years, 32 samples collected at least 6 months before BOS diagnosis (prediction group), and 26 samples at or after BOS diagnosis (diagnosis group). An independent set from 25 lung recipients was used for validation by quantitative PCR (13 stables, 11 in the prediction group, and 8 in the diagnosis group). We identified 50 transcripts differentially expressed between stable and BOS recipients. Three genes, namely POU class 2 associating factor 1 (POU2AF1), T-cell leukemia/lymphoma protein 1A (TCL1A), and B cell lymphocyte kinase, were validated as predictive biomarkers of BOS more than 6 months before diagnosis, with areas under the curve of 0.83, 0.77, and 0.78 respectively. These genes allow stratification based on BOS risk (log-rank test p < 0.01) and are not associated with time posttransplantation. This is the first published large-scale gene expression analysis of blood after lung transplantation. The three-gene blood signature could provide clinicians with new tools to improve follow-up and adapt treatment of patients likely to develop BOS