Method of carrier-free delivery of therapeutic RNA importable into human mitochondria: Lipophilic conjugates with cleavable bonds:

Defects in mitochondrial DNA often cause neuromuscular pathologies, for which no efficient therapy has yet been developed. MtDNA targeting nucleic acids might therefore be promising therapeutic candidates. Nevertheless, mitochondrial gene therapy has never been achieved because DNA molecules can not penetrate inside mitochondria in vivo. In contrast, some small non-coding RNAs are imported into mitochondrial matrix, and we recently designed mitochondrial RNA vectors that can be used to address therapeutic oligoribonucleotides into human mitochondria. Here we describe an approach of carrier-free targeting of the mitochondrially importable RNA into living human cells. For this purpose, we developed the protocol of chemical synthesis of oligoribonucleotides conjugated with cholesterol residue through cleavable covalent bonds. Conjugates containing pH-triggered hydrazone bond were stable during the cell transfection procedure and rapidly cleaved in acidic endosomal cellular compartments. RNAs conjugated to cholesterol through a hydrazone bond were characterized by efficient carrier-free cellular uptake and partial co-localization with mitochondrial network. Moreover, the imported oligoribonucleotide designed to target a pathogenic point mutation in mitochondrial DNA was able to induce a decrease in the proportion of mutant mitochondrial genomes. This newly developed approach can be useful for a carrier-free delivery of therapeutic RNA into mitochondria of living human cells

Induced tRNA import into human mitochondria: implication of a host aminoacyl-tRNA-synthetase:

In human cell, a subset of small non-coding RNAs is imported into mitochondria from the cytosol. Analysis of the tRNA import pathway allowing targeting of the yeast tRNA(Lys)(CUU) into human mitochondria demonstrates a similarity between the RNA import mechanisms in yeast and human cells. We show that the cytosolic precursor of human mitochondrial lysyl-tRNA synthetase (preKARS2) interacts with the yeast tRNA(Lys)(CUU) and small artificial RNAs which contain the structural elements determining the tRNA mitochondrial import, and facilitates their internalization by isolated human mitochondria. The tRNA import efficiency increased upon addition of the glycolytic enzyme enolase, previously found to be an actor of the yeast RNA import machinery. Finally, the role of preKARS2 in the RNA mitochondrial import has been directly demonstrated in vivo, in cultured human cells transfected with the yeast tRNA and artificial importable RNA molecules, in combination with preKARS2 overexpression or downregulation by RNA interference. These findings suggest that the requirement of protein factors for the RNA mitochondrial targeting might be a conserved feature of the RNA import pathway in different organisms

Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18:

5S rRNA is an essential component of ribosomes of all living organisms, the only known exceptions being mitochondrial ribosomes of fungi, animals, and some protists. An intriguing situation distinguishes mammalian cells: Although the mitochondrial genome contains no 5S rRNA genes, abundant import of the nuclear DNA-encoded 5S rRNA into mitochondria was reported. Neither the detailed mechanism of this pathway nor its rationale was clarified to date. In this study, we describe an elegant molecular conveyor composed of a previously identified human 5S rRNA import factor, rhodanese, and mitochondrial ribosomal protein L18, thanks to which 5S rRNA molecules can be specifically withdrawn from the cytosolic pool and redirected to mitochondria, bypassing the classic nucleolar reimport pathway. Inside mitochondria, the cytosolic 5S rRNA is shown to be associated with mitochondrial ribosomes

Staphylococcus aureus Panton‐Valentine Leukocidin triggers an alternative NETosis process targeting mitochondria

Panton-Valentine Leukocidin (PVL) is a bicomponent leukotoxin produced by 3%-10% of clinical Staphylococcus aureus (SA) strains involved in the severity of hospital and community-acquired infections. Although PVL was long known as a pore-forming toxin, recent studies have challenged the formation of a pore at the plasma membrane, while its endocytosis and the exact mode of action remain to be defined. In vitro immunolabeling of human neutrophils shows that Neutrophil Extracellular Traps (NETosis) is triggered by the action of purified PVL, but not by Gamma hemolysin CB (HlgCB), a structurally similar SA leukotoxin. PVL causes the ejection of chromatin fibers (NETs) decorated with antibacterial peptides independently of the NADPH oxidase oxidative burst. Leukotoxin partially colocalizes with mitochondria and enhances the production of reactive oxygen species from these organelles, while showing an increased autophagy, which results unnecessary for NETs ejection. PVL NETosis is elicited through Ca2+-activated SK channels and Myeloperoxidase activity but is abolished by Allopurinol pretreatment of neutrophils. Moreover, massive citrullination of the histone H3 is performed by peptidyl arginine deiminases. Inhibition of this latter enzymes fails to abolish NET extrusion. Unexpectedly, PVL NETosis does not seem to involve Src kinases, which is the main kinase family activated downstream the binding of PVL F subunit to CD45 receptor, while the specific kinase pathway differs from the NADPH oxidase-dependent NETosis. PVL alone causes a different and specific form of NETosis that may rather represent a bacterial strategy conceived to disarm and disrupt the immune response, eventually allowing SA to spread

A Moonlighting Human Protein Is Involved in Mitochondrial Import of tRNA

In yeast Saccharomyces cerevisiae, ~3% of the lysine transfer RNA acceptor 1 (tRK1) pool is imported into mitochondria while the second isoacceptor, tRK2, fully remains in the cytosol. The mitochondrial function of tRK1 is suggested to boost mitochondrial translation under stress conditions. Strikingly, yeast tRK1 can also be imported into human mitochondria in vivo, and can thus be potentially used as a vector to address RNAs with therapeutic anti-replicative capacity into mitochondria of sick cells. Better understanding of the targeting mechanism in yeast and human is thus critical. Mitochondrial import of tRK1 in yeast proceeds first through a drastic conformational rearrangement of tRK1 induced by enolase 2, which carries this freight to the mitochondrial pre-lysyl-tRNA synthetase (preMSK). The latter may cross the mitochondrial membranes to reach the matrix where imported tRK1 could be used by the mitochondrial translation apparatus. This work focuses on the characterization of the complex that tRK1 forms with human enolases and their role on the interaction between tRK1 and human pre-lysyl-tRNA synthetase (preKARS2)

Valine Aminoacyl-tRNA Synthetase Promotes Therapy Resistance in Melanoma

Transfer RNA dynamics contribute to cancer development through regulation of codon-specific messenger RNA translation. Specific aminoacyl-tRNA synthetases can either promote or suppress tumourigenesis. Here we show that valine aminoacyl-tRNA synthetase (VARS) is a key player in the codon-biased translation reprogramming induced by resistance to targeted (MAPK) therapy in melanoma. The proteome rewiring in patient-derived MAPK therapy-resistant melanoma is biased towards the usage of valine and coincides with the upregulation of valine cognate tRNAs and of VARS expression and activity. Strikingly, VARS knockdown re-sensitizes MAPK-therapy-resistant patient-derived melanoma in vitro and in vivo. Mechanistically, VARS regulates the messenger RNA translation of valine-enriched transcripts, among which hydroxyacyl-CoA dehydrogenase mRNA encodes for a key enzyme in fatty acid oxidation. Resistant melanoma cultures rely on fatty acid oxidation and hydroxyacyl-CoA dehydrogenase for their survival upon MAPK treatment. Together, our data demonstrate that VARS may represent an attractive therapeutic target for the treatment of therapy-resistant melanoma

Modeling of antigenomic therapy of mitochondrial diseases by mitochondrially addressed RNA targeting a pathogenic point mutation in mitochondrial DNA

Defects in mitochondrial genome can cause a wide range of clinical disorders, mainly neuromuscular diseases. Presently, no efficient therapeutic treatment has been developed against this class of pathologies. Because most of deleterious mitochondrial mutations are heteroplasmic, meaning that wild type and mutated forms of mitochondrial DNA (mtDNA) coexist in the same cell, the shift in proportion between mutant and wild type molecules could restore mitochondrial functions. Recently, we developed mitochondrial RNA vectors that can be used to address anti-replicative oligoribonucleotides into human mitochondria and thus impact heteroplasmy level in cells bearing a large deletion in mtDNA. Here, we show that this strategy can be also applied to point mutations in mtDNA. We demonstrate that specifically designed RNA molecules containing structural determinants for mitochondrial import and 20-nucleotide sequence corresponding to the mutated region of mtDNA, are able to anneal selectively to the mutated mitochondrial genomes. After being imported into mitochondria of living human cells in culture, these RNA induced a decrease of the proportion of mtDNA molecules bearing a pathogenic point mutation in the mtDNA ND5 gene

Mitochondrial targeting of recombinant RNAs modulates the level of a heteroplasmic mutation in human mitochondrial DNA associated with Kearns Sayre Syndrome

Mitochondrial mutations, an important cause of incurable human neuromuscular diseases, are mostly heteroplasmic: mutated mitochondrial DNA is present in cells simultaneously with wild-type genomes, the pathogenic threshold being generally >70% of mutant mtDNA. We studied whether heteroplasmy level could be decreased by specifically designed oligoribonucleotides, targeted into mitochondria by the pathway delivering RNA molecules in vivo. Using mitochondrially imported RNAs as vectors, we demonstrated that oligoribonucleotides complementary to mutant mtDNA region can specifically reduce the proportion of mtDNA bearing a large deletion associated with the Kearns Sayre Syndrome in cultured transmitochondrial cybrid cells. These findings may be relevant to developing of a new tool for therapy of mtDNA associated diseases

A Deubiquitylating Complex Required for Neosynthesis of a Yeast Mitochondrial ATP Synthase Subunit

The ubiquitin system is known to be involved in maintaining the integrity of mitochondria, but little is known about the role of deubiquitylating (DUB) enzymes in such functions. Budding yeast cells deleted for UBP13 and its close homolog UBP9 displayed a high incidence of petite colonies and slow respiratory growth at 37°C. Both Ubp9 and Ubp13 interacted directly with Duf1 (DUB-associated factor 1), a WD40 motif-containing protein. Duf1 activates the DUB activity of recombinant Ubp9 and Ubp13 in vitro and deletion of DUF1 resulted in the same respiratory phenotype as the deletion of both UBP9 and UBP13. We show that the mitochondrial defects of these mutants resulted from a strong decrease at 37°C in the de novo biosynthesis of Atp9, a membrane-bound component of ATP synthase encoded by mitochondrial DNA. The defect appears at the level of ATP9 mRNA translation, while its maturation remained unchanged in the mutants. This study describes a new role of the ubiquitin system in mitochondrial biogenesis

Studying the mechanisms of tRNALys(CUU) translocation into mitochondria of Saccharomyces cerevisiae

L import d ARN dans les mitochondries est un processus ubiquitaire chez les eucaryotes. Dans Saccharomyces cerevisiae un isoaccepteur cytosolique de l ARNtLys, l ARNtLys(CUU) (tRK1), est partiellement importé dans les mitochondries. L adressage vers la surface mitochondriale a été étudié en détail mais l étape de translocation dans les mitochondries reste toujours à démontrer. L objectif principal de ce travail de thèse était l identification et la caractérisation des protéines qui participent dans ce processus. Deux protéines de la membranes externe capables de former des canaux, Tom40 et VDAC1, ont été identifiées et leur rôle dans l étape de translocation a été évalué in vitro et in vivo en utilisant des souches mutantes appropriées ou des agents capables de bloquer de manière spécifique les canaux formés par les protéines Tom40 et VDAC1. Ainsi il a été démontré que la délétion de VDAC1 ou l inhibition du canal VDAC1 a conduit à une inhibition importante, néanmoins pas complète, de l import de tRK1. Le blocage simultané des canaux Tom40 et VDAC1 par contre a causé un arrêt complet de l import de tRK1 in vitro. Vu ces résultats, nous proposons que la translocation de tRK1 à travers la membrane mitochondriale externe puisse suivre deux chemins alternatifs.RNA import into mitochondria is a ubiquitous process in eukaryotic cells. In Saccharomyces cerevisiae one cytosolic isoacceptor of tRNALys, tRNALys(CUU) (tRK1), is partially imported into mitochondria. Targeting of tRK1 to the mitochondrial surface is well described but the translocation of tRK1 into mitochondria is still poorly understood. This PhD work aimed to study this translocation step and the main objective was the identification and characterization of mitochondrial membrane proteins participating in this process. The two channel-forming proteins of the mitochondrial outer membrane, Tom40 and VDAC1, were identified and their role in tRK1 translocation further investigated with the help of in vitro and in vivo import studies using appropriate mutant strains or specific agents capable of blocking Tom40 and VDAC1. In this way, it could be demonstrated that deletion of the VDAC1 gene or inhibition of VDAC1 led to an important yet not complete inhibition of tRK1 import into mitochondria. Simultaneous blocking of the two channels formed by Tom40 and VDAC1, however, resulted in a complete inhibition of tRK1 import in vitro. Regarding these results we propose that tRK1 translocation through the mitochondrial outer membrane can use two alternative pathways.STRASBOURG-Bib.electronique 063 (674829902) / SudocSudocFranceF