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

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

Cdc48-associated complex bound to 60S particles is required for the clearance of aberrant translation products

International audienceRibosome stalling on eukaryotic mRNAs triggers cotranslational RNA and protein degradation through conserved mechanisms. For example, mRNAs lacking a stop codon are degraded by the exosome in association with its cofactor, the SKI complex, whereas the corresponding aberrant nascent polypeptides are ubiquitinated by the E3 ligases Ltn1 and Not4 and become proteasome substrates. How translation arrest is linked with polypeptide degradation is still unclear. Genetic screens with SKI and LTN1 mutants allowed us to identify translation-associated element 2 (Tae2) and ribosome quality control 1 (Rqc1), two factors that we found associated, together with Ltn1 and the AAA-ATPase Cdc48, to 60S ribosomal subunits. Translation-associated element 2 (Tae2), Rqc1, and Cdc48 were all required for degradation of polypeptides synthesized from Non-Stop mRNAs (Non-Stop protein decay; NSPD). Both Ltn1 and Rqc1 were essential for the recruitment of Cdc48 to 60S particles. Polysome gradient analyses of mutant strains revealed unique intermediates of this pathway, showing that the polyubiquitination of Non-Stop peptides is a progressive process. We propose that ubiquitination of the nascent peptide starts on the 80S and continues on the 60S, on which Cdc48 is recruited to escort the substrate for proteasomal degradation