P1.1 The protein-only RNase P PRORP1 interacts with the nuclease MNU2 in Arabidopsis mitochondria G. Bonnard, M. Arrivé, A. Bouchoucha, A. Gobert, C. Schelcher, F. Waltz, P. Giegé Institut de biologie moléculaire des plantes, CNRS, Université de Strasbourg, Strasbourg, France The essential endonuclease activity that removes 5’ leader sequences from transfer RNA precursors is called RNase P. While ribonucleoprotein RNase P enzymes containing a ribozyme are found in all domains of life, another type of RNase P called “PRORP”, for “PROtein-only RNase P”, only composed of protein occurs in a wide variety of eukaryotes, in organelles and the nucleus. Although PRORP proteins function as single subunit enzymes in vitro, we find that PRORP1 occurs in protein complexes and is present in polysome fractions in Arabidopsis mitochondria. The analysis of immuno- precipitated protein complexes identifies proteins involved in mitochondrial gene expression processes. In particular, direct interaction is established between PRORP1 and MNU2 another mitochondrial nuclease involved in RNA 5’ processing. A specific domain of MNU2 and a conserved signature of PRORP1 are found to be directly accountable for this protein interaction. Altogether, results reveal the existence of an RNA 5’ maturation complex in Arabidopsis mitochondria and suggest that PRORP proteins might cooperate with other gene expression regulators for RNA maturation in vivo. 111 P1.2 CytoRP, a cytosolic RNase P to target TLS-RNA phytoviruses 1 1 2 1 A. Gobert , Y. Quan , I. Jupin , P. Giegé 1Institut de biologie moléculaire des plantes, CNRS, Université de Strasbourg, Strasbourg, France 1Institut Jacques Monod, CNRS, Université Paris Diderot, Paris, France In plants, PRORP enzymes are responsible for RNase P activity that involves the removal of the 5’ extremity of tRNA precursors. We showed previously that in addition to the maturation of tRNA precursors, PRORP enzymes cleave tRNA- like structures (TLS) present on mitochondrial mRNA.Many plant viruses (phytoviruses) are RNA viruses containing TLS structures. These RNA elements are involved in virus genome replication. The best characterized TLS is found in the Turnip Yellow Mosaic Virus (TYMV) genome. TLS based viruses are found in numerous clades of viruses. Their impact on crop production is major with considerable crop yield decreases worldwide. Our project is based on the modification of PRORP enzymes in order to be able to target TLS RNA viruses to obtain plant resistance to virus infections. For this, we produced a PRORP variant called “CytoRP”, localized in the cytosol, where virus replication takes place.CytoRP is able to cleave the TYMV TLS in vitro. We have produced transgenic plants expressing CytoRP and a cisgenic plant where the nuclear localization signal of PRORP2 was deleted using CrispR/Cas9 technology. We show that the nuclear localization of these mutants is lost and CytoRP accumulates in the cytosol. The ability of these CytoRP plants to hinder TYMV infection in vivo is currently investigated. 112 P1.3 Structural probing of staphylococcal-specific glyS T-box domains and modulation of transcription by protein synthesis inhibitors Nikoleta Giarimoglou1#, Vassiliki Stamatopoulou1#, Shuang Li2, Maria Maria Apostolidi3, Eleni Kaliatsi1, Jinwei Zhang2 and Constantinos Stathopoulos1 1Department of Biochemistry, School of Medicine, University of Patras, Greece2Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, MD, USA3Department of Molecular and Cellular Physiology, School of Medicine, Yale Univeristy, West Haven, CT, USA Staphylococcus aureus glyS T-box contains the species-specific intervening sequence stem Sa as part of the antiterminator/terminator stem, which possibly confers selectivity for five different tRNAGly isoacceptor ligands. In addition, stem Sa was found to accommodate binding of protein synthesis inhibitors which induce antitermination of glyS transcription. Structural probing and in vitro analysis of a series of mutants containing swaps of stem I or apical loop between O. iheyensis and S. aureus and stem Sa deletions revealed diverse effects in the presence of tigecycline and linezolid. Deletion of Stem Sa reduced the in vitro transcription independently of the presence or absence of antibiotics, suggesting that stem Sa is important for T-box-mediated transcription. Moreover, tRNAGly mutations in crucial nucleotides of the “elbow” showed that stem I becomes insensitive of discriminating the isoacceptors and this phenotype is reversed when stem Sa is deleted. Taken together, our data support our previous observations on the role of stem Sa in the equilibrium of transcription termination/antitermination conformations, when all tRNA isoacceptors compete for T-box riboswitch binding. # equal contribution 113 P1.4 Effects of Lupus antigen (La) expression in transcription and translation components in lung cancer Eleni Kaliatsi1, Athanasios-Nasir Shaukat1, Ilias Skeparnias1, Aikaterini I. Argyriou2, Georgios A. Spyroulias2 and Constantinos Stathopoulos1 1Department of Biochemistry, School of Medicine, University of Patras, Greece 2Department of Pharmacy, University of Patras, Greece La protein is the major antigen in lupus and Sjogren’s syndrome, involved in folding of tRNAs and safeguarding miRNA pathway from tRNA fragments during selection of small non-coding RNAs for silencing events. Although aberrant expression of La has been linked with malignancy, its exact role in various cancer types remains uncharacterized. In the present study, we screened the NSCLC cell line A549 and various NSCLC biopsy samples to verify upregulation of La. Expression of La in A549 cells under various stress conditions, showed constant localization in the nucleus and nucleolus, in contrast to what has been reported for HeLa cells. Interestingly, expression of La both at transcriptional and translational level is increased under glucose deprivation but decreased under oxidative stress. Expression of La leads to upregulation of genes involved in cell cycle, tRNA maturation and transport and miRNA biogenesis and upregulation of specific miRNAs and tRFs species. Finally, FACS analysis showed that La expression does not lead to cell cycle arrest. Instead, wound healing and CFSE assays showed that La expression accelerates cell motility, suggesting possible involvement in metastasis. 114 P1.5 Nuclear regulation of mitochondrial tRNA production Oliver Rackham Harry Perkins Institute of Medical Research and Centre for Medical Research, 6 Verdun Street, Nedlands, Western Australia 6009, AustraliaSchool of Molecular Sciences, The University of Western Australia, Nedlands, Western Australia 6009, Australia The size and organization of the animal mitochondrial genome has been reduced and compacted significantly since its endosymbiosis from an α- proteobacterial ancestor. This compaction has necessitated the evolution of unique mechanisms to facilitate rapid changes in gene expression in response to the changing energy demands of the cell. The mitochondrial transcriptome encodes proteins that are subunits of the respiratory chain, responsible for most of the energy production required by the cell. Consequently the coordinated regulation of the mitochondrial transcriptome by the nucleus is of particular importance for the maintenance of cell health and energy metabolism. We have been investigating the unusual features of mitochondrial tRNAs and the RNA-binding proteins that control their production, maturation, translation and stabilization to understand the regulation of mitochondrial gene expression and its contribution to health and disease. I will highlight the devastating consequences of dysregulated mitochondrial gene expression and protein synthesis in a new model of disease caused by genetic disruption of a tRNA-binding protein. Mouse models of disease have enabled us to understand the in vivo role of fundamental processes that regulate mitochondrial tRNA metabolism and the pathogenesis of diseases caused by impaired protein synthesis. This work illustrates that balanced mitochondrial tRNA production is required for the regulation of nuclear gene expression, as well as translation in multiple cellular compartments. 115 P1.6 Characterization of protein-only RNase P in complex with tRNA. C. Schelcher1, A. Gobert1, C. Sauter2, P. Giegé1 1Institut de biologie moléculaire des plantes, Strasbourg, France 2Institut de biologie moléculaire et cellulaire, Strasbourg, France RNase P activity is the essential cleavage that removes 5'-leader sequences from transfer RNA precursors. “PRORP” (PROteinaceous RNase P) is a category of protein-only RNase P. Before the characterization of PRORP, RNase P enzymes were thought to occur universally as ribonucleoproteins (RNP). The characterization of PRORP revealed an enzyme with two main domains, an N- terminal domain containing five PPR RNA binding motifs and a C- terminal NYN domain holding catalytic activity (1, 2). We used a combination of biochemical and biophysical approaches to characterize the PRORP / tRNA precursor complex. Analytical ultracentifugation, microscale thermophoresis and activity assays show that the sole PPR motifs PPR2 and PPR3 are involved in PRORP/ tRNA precursor interaction. These results were combined with small angle X ray scaterring data of PRORP alone and with tRNA to build a model of the PRORP2 pre-tRNA complex (3). In this model, PPR2 and 3 interact, in accordance with the “PPR code”, repectively with C56 and G18 spacialy localized at the corner of the tRNA.