Efficient tools to target DNA to Podospora anserina

Here we report the construction of two plasmids designed to target DNA sequences to two specific loci of Podospora anserina

Ribosomal protein S1 influences trans-translation in vitro and in vivo

When the bacterial ribosome stalls on a truncated mRNA, transfer–messenger RNA (tmRNA) acts initially as a transfer RNA (tRNA) and then as a messenger RNA (mRNA) to rescue the ribosome and add a peptide tag to the nascent polypeptide that targets it for degradation. Ribosomal protein S1 binds tmRNA but its functional role in this process has remained elusive. In this report, we demonstrate that, in vitro, S1 is dispensable for the tRNA-like role of tmRNA but is essential for its mRNA function. Increasing or decreasing the amount of protein S1 in vivo reduces the overall amount of trans-translated proteins. Also, a truncated S1 protein impaired for ribosome binding can still trigger protein tagging, suggesting that S1 interacts with tmRNA outside the ribosome to keep it in an active state. Overall, these results demonstrate that S1 has a role in tmRNA-mediated tagging that is distinct from its role during canonical translation

Mitochondria of the Yeasts Saccharomyces cerevisiae and Kluyveromyces lactis Contain Nuclear rDNA-Encoded Proteins

In eukaryotes, the nuclear ribosomal DNA (rDNA) is the source of the structural 18S, 5.8S and 25S rRNAs. In hemiascomycetous yeasts, the 25S rDNA sequence was described to lodge an antisense open reading frame (ORF) named TAR1 for Transcript Antisense to Ribosomal RNA. Here, we present the first immuno-detection and sub-cellular localization of the authentic product of this atypical yeast gene. Using specific antibodies against the predicted amino-acid sequence of the Saccharomyces cerevisiae TAR1 product, we detected the endogenous Tar1p polypeptides in S. cerevisiae (Sc) and Kluyveromyces lactis (Kl) species and found that both proteins localize to mitochondria. Protease and carbonate treatments of purified mitochondria further revealed that endogenous Sc Tar1p protein sub-localizes in the inner membrane in a Nin-Cout topology. Plasmid-versions of 5′ end or 3′ end truncated TAR1 ORF were used to demonstrate that neither the N-terminus nor the C-terminus of Sc Tar1p were required for its localization. Also, Tar1p is a presequence-less protein. Endogenous Sc Tar1p was found to be a low abundant protein, which is expressed in fermentable and non-fermentable growth conditions. Endogenous Sc TAR1 transcripts were also found low abundant and consistently 5′ flanking regions of TAR1 ORF exhibit modest promoter activity when assayed in a luciferase-reporter system. Using rapid amplification of cDNA ends (RACE) PCR, we also determined that endogenous Sc TAR1 transcripts possess heterogeneous 5′ and 3′ ends probably reflecting the complex expression of a gene embedded in actively transcribed rDNA sequence. Altogether, our results definitively ascertain that the antisense yeast gene TAR1 constitutes a functional transcription unit within the nuclear rDNA repeats

Biological Roles of the Podospora anserina Mitochondrial Lon Protease and the Importance of Its N-Domain

Mitochondria have their own ATP-dependent proteases that maintain the functional state of the organelle. All multicellular eukaryotes, including filamentous fungi, possess the same set of mitochondrial proteases, unlike in unicellular yeasts, where ClpXP, one of the two matricial proteases, is absent. Despite the presence of ClpXP in the filamentous fungus Podospora anserina, deletion of the gene encoding the other matricial protease, PaLon1, leads to lethality at high and low temperatures, indicating that PaLON1 plays a main role in protein quality control. Under normal physiological conditions, the PaLon1 deletion is viable but decreases life span. PaLon1 deletion also leads to defects in two steps during development, ascospore germination and sexual reproduction, which suggests that PaLON1 ensures important regulatory functions during fungal development. Mitochondrial Lon proteases are composed of a central ATPase domain flanked by a large non-catalytic N-domain and a C-terminal protease domain. We found that three mutations in the N-domain of PaLON1 affected fungal life cycle, PaLON1 protein expression and mitochondrial proteolytic activity, which reveals the functional importance of the N-domain of the mitochondrial Lon protease. All PaLon1 mutations affected the C-terminal part of the N-domain. Considering that the C-terminal part is predicted to have an α helical arrangement in which the number, length and position of the helices are conserved with the solved structure of its bacterial homologs, we propose that this all-helical structure participates in Lon substrate interaction

A Viable Hypomorphic Allele of the Essential IMP3 Gene Reveals Novel Protein Functions in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the essential IMP3 gene encodes a component of the SSU processome, a large ribonucleoprotein complex required for processing of small ribosomal subunit RNA precursors. Mutation of the IMP3 termination codon to a sense codon resulted in a viable mutant allele producing a C-terminal elongated form of the Imp3 protein. A strain expressing the mutant allele displayed ribosome biogenesis defects equivalent to IMP3 depletion. This hypomorphic allele represented a unique opportunity to investigate and better understand the Imp3p functions. We demonstrated that the +1 frameshifting was increased in the mutant strain. Further characterizations revealed involvement of the Imp3 protein in DNA repair and telomere length control, pointing to a functional relationship between both pathways and ribosome biogenesis

TAR1 (un gène hébergé sur le brin antisens de l'ADN ribosomique de Saccharomyces cerevisiae, code une protéine mitochondriale)

Mon projet de thèse a porté sur l'étude fonctionnelle du gène TAR1. Ce gène est localisé sur le brin antisens de l'ADNr 25S de Saccharomyces cerevisiae et code un polypeptide (Tar1p), de fonction inconnue, localisé dans les mitochondries. Je me suis intéressée au transcrit TAR1 afin d'en déterminer les extrémités 5' et 3'. Grâce à un système rapporteur que j'ai construit, j'ai étudié l'expression de TAR1 dans différentes conditions de culture. J'ai mis en évidence que Tar1p est localisée dans la membrane interne des mitochondries avec son extrémité amino-terminale localisée du coté matriciel et son extrémité carboxy-terminale dans l'espace intermembranaire. Une séquence clivable, non essentielle pour la localisation de la protéine dans les mitochondries, semble être présente dans les 32 derniers acides aminés. J'ai également détecté Tar1p dans les mitochondries de la levure Kluyveromyces lactis mais pas dans celles de Schizosaccharomyces pombe. Je me suis ensuite intéressée à l'inactivation du gène TAR1. Etant donné que le gène TAR1 est répété un grand nombre de fois dans le génome, une délétion classique par inactivation de l'ORF n'est pas réalisable. J'ai donc entrepris d'inactiver l'expression de la protéine Tar1p dans une souche sauvage de levure par deux techniques différentes. L'une est basée sur l'utilisation de snoARNs à boîtes C/D. L'idée étant de méthyler spécifiquement le transcrit TAR1 dans l'optique d'altérer la traduction de ce gène et donc la production de la protéine dans la cellule. L'autre est réalisée par introduction de codons stop dans la phase ouverte de lecture TAR1 dans une souche de levure contenant une seule unité d'ADNr portée par un plasmide.My PhD project focused on functional study of the TAR1 gene. TAR1 is localized on the antisense strand of the 25S rDNA of yeast Saccharomyces cerevisiae and encodes a polypeptide (Tar1p) of unknown function, which localized into mitochondria. First, I was interested to the TAR1 transcript to identify its 5' and 3' ends. Using a reporter system I set up, I studied the expression of TAR1 in various conditions. Then I have demonstrated that Tar1p is localized in the inner membrane of mitochondria with its amino-terminal end in the matrix and its carboxy-terminal end in the intermembrane space. The last 32 amino acids appear to be cleavable but this sequence is not essential for the protein import into mitochondria. I detected the endogenous Tar1p polypeptide in mitochondria of the yeast Kluyveromyces lactis but not of Schizosaccharomyces pombe. Secondly, I tried to inactivate TAR1. However, due to the high copy number of this gene in the genome, it was not possible to use classical deletion approach. I developed two approaches to inactivate TAR1. The first one is based on the use of C/D snoRNAs. SnoRNAs were constructed to target the methylation of one nucleotide within the TAR1 mRNA. One may expect that this methylation will interfere with mRNA translation. The second one is to introduce stop codons within the open reading frame of TAR1 in a yeast strain containing a single plasmidic unit of rDNA. These stop codons are expected to stop translation, preventing the synthesis of Tar1p.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Initiation de la traduction et autorégulation du gène rpsA chez Escherichia coli

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

ETUDE DU ROLE PARTICULIER JOUE PAR LE GENE RRP5 DANS LA MATURATION DES ARN RIBOSOMAUX CHEZ LA LEVURE SACCHAROMYCES CEREVISIAE

ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Contribution of ERMES subunits to mature peroxisome abundance

International audienceEukaryotic organelles share different components and establish physical contacts to communicate throughout the cell. One of the best-recognized examples of such interplay is the metabolic cooperation and crosstalk between mitochondria and peroxisomes, both organelles being functionally and physically connected and linked to the endoplasmic reticulum (ER). In Saccharomyces cerevisiae, mitochondria are linked to the ER by the ERMES complex that facilitates inter-organelle calcium and phospholipid exchanges. Recently, peroxisome-mitochondria contact sites (PerMit) have been reported and among Permit tethers, one component of the ERMES complex (Mdm34) was shown to interact with the peroxin Pex11, suggesting that the ERMES complex or part of it may be involved in two membrane contact sites (ER-mitochondria and peroxisome- mitochondria). This opens the possibility of exchanges between these three membrane compartments. Here, we investigated in details the role of each ERMES subunit on peroxisome abundance. First, we confirmed previous studies from other groups showing that absence of Mdm10 or Mdm12 leads to an increased number of mature peroxisomes. Secondly, we showed that this is not simply due to respiratory function defect, mitochondrial DNA (mtDNA) loss or mitochondrial network alteration. Finally, we present evidence that the contribution of ERMES subunits Mdm10 and Mdm12 to peroxisome number involves two different mechanisms