Qri7/OSGEPL, the mitochondrial version of the universal Kae1/YgjD protein, is essential for mitochondrial genome maintenance

Yeast Qri7 and human OSGEPL are members of the orthologous Kae1(OSGEP)/YgjD protein family, the last class of universally conserved proteins without assigned function. Phylogenetic analyses indicate that the eukaryotic Qri7(OSGEPL) proteins originated from bacterial YgjD proteins. We have recently shown that the archaeal Kae1 protein is a DNA-binding protein that exhibits apurinic endonuclease activity in vitro. We show here that the Qri7/OSGEPL proteins localize in mitochondria and are involved in mitochondrial genome maintenance in two model eukaryotic organisms, Saccharomyces cerevisiae and Caenorhabditis elegans. Furthermore, S. cerevisiae Qri7 complements the loss of the bacterial YgjD protein in Escherichia coli, suggesting that Qri7/OSGEPL and YgjD proteins have retained similar functions in modern organisms. We suggest to name members of the Kae1(OSGEP)/YgjD family UGMP, for Universal Genome Maintenance Proteins

Gcn4 misregulation reveals a direct role for the evolutionary conserved EKC/KEOPS in the t6A modification of tRNAs

The EKC/KEOPS complex is universally conserved in Archaea and Eukarya and has been implicated in several cellular processes, including transcription, telomere homeostasis and genomic instability. However, the molecular function of the complex has remained elusive so far. We analyzed the transcriptome of EKC/KEOPS mutants and observed a specific profile that is highly enriched in targets of the Gcn4p transcriptional activator. GCN4 expression was found to be activated at the translational level in mutants via the defective recognition of the inhibitory upstream ORFs (uORFs) present in its leader. We show that EKC/KEOPS mutants are defective for the N6-threonylcarbamoyl adenosine modification at position 37 (t6A37) of tRNAs decoding ANN codons, which affects initiation at the inhibitory uORFs and provokes Gcn4 de-repression. Structural modeling reveals similarities between Kae1 and bacterial enzymes involved in carbamoylation reactions analogous to t6A37 formation, supporting a direct role for the EKC in tRNA modification. These findings are further supported by strong genetic interactions of EKC mutants with a translation initiation factor and with threonine biosynthesis genes. Overall, our data provide a novel twist to understanding the primary function of the EKC/KEOPS and its impact on several essential cellular functions like transcription and telomere homeostasis

The C-Terminal Domain of the Novel Essential Protein Gcp Is Critical for Interaction with Another Essential Protein YeaZ of Staphylococcus aureus

Previous studies have demonstrated that the novel protein Gcp is essential for the viability of various bacterial species including Staphylococcus aureus; however, the reason why it is required for bacterial growth remains unclear. In order to explore the potential mechanisms of this essentiality, we performed RT-PCR analysis and revealed that the gcp gene (sa1854) was co-transcribed with sa1855, yeaZ (sa1856) and sa1857 genes, indicating these genes are located in the same operon. Furthermore, we demonstrated that Gcp interacts with YeaZ using a yeast two-hybrid (Y2H) system and in vitro pull down assays. To characterize the Gcp-YeaZ interaction, we performed alanine scanning mutagenesis on the residues of C-terminal segment of Gcp. We found that the mutations of the C-terminal Y317-F322 region abolished the interaction of Gcp and YeaZ, and the mutations of the D324-N329 and S332-Y336 regions alleviated Gcp binding to YeaZ. More importantly, we demonstrated that these key regions of Gcp are also necessary for the bacterial survival since these mutated Gcp could not complement the depletion of endogenous Gcp. Taken together, our data suggest that the interaction of Gcp and YeaZ may contribute to the essentiality of Gcp for S. aureus survival. Our findings provide new insights into the potential mechanisms and biological functions of this novel essential protein

Mutations in KEOPS-Complex Genes Cause Nephrotic Syndrome with Primary Microcephaly

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms

Identification and characterisation of PCC complex in yeast S. cerevisiae

Pour identifier de nouveaux facteurs d'épissage chez la levure S. cerevisiae, nous avons cherché des suppresseurs multicopies du phénotype cryosensible associé à la mutation U5A dans la particule U1 snRNA (U1-5A). Nous avons isolé des plasmides contenant une région intergénique portant une petite ORF cryptique (appelée PCC1 ) qui contient un intron avec un site d'épissage 5' non canonique. Nous avons montré que PCC1 n'est pas impliqué dans l'épissage, mais que l'épissage de son intron est le principal facteur limitant pour la croissance en présence de la mutation U1-5A. Puisque PCC1 est un gène quasi essentiel, nous avons généré et étudié le mutant thermosensible pcc1-4. Notre analyse a révélé des défauts dans le cycle cellulaire et dans la réponse à la phéromone. Nos données suggèrent que PCC1 est directement impliqué dans la transcription et affecte l'expression de certains gènes, ce qui détermine les phénotypes mutants de pcc1. Le complexe PCC contient trois protéines additionnelles: Kae1p, une métalloprotéase putative, Bud32p, une kinase et Pcc2p. De multiples interactions génétiques et physiques entre ces protéines et les phénotypes des mutants suggèrent que le complexe PCC agit comme une unité dans la cellule et que sa fonction est conservée chez les métazoaires. Nous suggérons que l'activité d'endopeptidase de Kae1p et que l'activité kinase de Bud32p sont les fonctions moléculaires qui déterminent le rôle du complexe PCC dans la transcription. Nous montrons des interactions génétiques entre PCC1 et des facteurs de modification de la chromatine, ce qui suggère que le complexe PCC pourrait influencer la transcription via une fonction de modification de la chromatine.To identify new splicing factors in yeast S. cerevisiae, we searched for high-copy number suppressors of the cryosensitive phenotype associated to the U5A mutation in the U1 snRNA (U1-5A). We isolated plasmids containing an intergenic region bearing a cryptic, small ORF(that we named PCC1) containing an intron with a non-canonical 5' splice site. We showed that Pcc1p is not a new splicing factor, but that impaired splicing of its intron is the main limiting factor for growth in the presence of the U1-5A mutation. As PCC1 is a quasi-essential gene, we generated and studied the thermosensitive pcc1-4 mutant. Our analysis of this mutant revealed cell cycle progression defects and a defect in the response to pheromone. Our data suggest that Pcc1p is directly involved in transcription and affects the expression of several genes, which underlies the pcc1 mutant phenotypes. The PCC complex contains three additional proteins: Kaelp, a putative metalloprotease, Bud32p, a kinase and Pcc2p. Multiple genetic, additional physical interactions among these proteins and the phenotype of the mutants suggest that the PCC complex function as a unit in the cell. We show that the function of the complex is conserved in metazoans. The presence of a putative endopeptidase and a kinase are the most intriguing features of the PCC complex. We suggest that the endopeptidase activity of Kaelp and the kinase activity of Bud32p are the molecular functions that underlie the role of the PCC complex in transcription. We report genetic interactions between PCC1 and chromatin modifying factors, which suggest that the PCC complex might impact transcription through a chromatin modifying function.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF