• Gerovac M, Vogel J, Smirnov A (2021) The world of stable ribonucleoproteins and its mapping with Grad-seq and related approaches. Front Mol Biosci 8:661448

Currently the only review about YbeY in existence - a great piece from Zhen & Cédric! Congratulations!

• Liao Z, Schelcher C, Smirnov A (2021) YbeY, éminence grise of ribosome biogenesis. Biochem Soc Trans 49:727-45

A fantastic chapter relating the first-hand experience of using high-resolution microscopy methods to locate transcripts and proteins at a submitochondrial level - a fruit of a great collaboration with the Yves Mély's team!

• Smirnova A, Richert L, Smirnov A, Mély Y, Tarassov I (2021) Suborganellar localisation of mitochondrial proteins and transcripts in human cells. Methods Mol Biol 2277:157-73

Another great chapter about using lipophilic conjugates to deliver RNAs into cells nad toward mitochondria without transfection reagents - a result of a long-standing collaboration with the Alya Venyaminova's group!

• Dovydenko I, Meschaninova M, Heckel AM, Tarassov I, Venyaminova A, Entelis N (2021) Lipophilic conjugates for carrier-free delivery of RNA importable into human mitochondria. Methods Mol Biol 2277:49-67

Early View

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

Abstract

Objective

To demonstrate the causal role in disease of the MT-TP m.15992A>T mutation observed in

patients from 5 independent families.

Methods

Lactate measurement, muscle histology, and mitochondrial activities in patients; PCR-based

analyses of the size, amount, and sequence of muscle mitochondrial DNA (mtDNA) and

proportion of the mutation; respiration, mitochondrial activities, proteins, translation, transfer

RNA (tRNA) levels, and base modification state in skin fibroblasts and cybrids; and reactive

oxygen species production, proliferation in the absence of glucose, and plasma membrane

potential in cybrids.

Results

All patients presented with severe exercise intolerance and hyperlactatemia. They were associated

with prominent exercise-induced muscle swelling, conspicuous in masseter muscles (2

families), and/or with congenital cataract (2 families). MRI confirmed exercise-induced muscle

edema. Muscle disclosed severe combined respiratory defect. Muscle mtDNA had normal size

and amount. Its sequence was almost identical in all patients, defining the haplotype as J1c10,

and sharing 31 variants, only 1 of which, MT-TP m.15992A>T, was likely pathogenic. The

mutation was homoplasmic in all tissues and family members. Fibroblasts and cybrids with

homoplasmic mutation had defective respiration, low complex III activity, and decreased

tRNAPro amount. Their respiratory complexes amount and tRNAPro aminoacylation appeared

normal. Low proliferation in the absence of glucose demonstrated the relevance of the defects

on cybrid biology while abnormal loss of cell volume when faced to plasma membrane depolarization

provided a link to the muscle edema observed in patients.

Conclusions

The homoplasmic MT-TP m.15992A>T mutation in the J1c10 haplotype causes exerciseinduced

muscle swelling and fatigue.

Voir l'article 1

Voir l'article 2

IUBMB Life. 2018 Sep 5. doi: 10.1002/iub.1919. [Epub ahead of print]

Abstract

Mutations in mitochondrial DNA are an important source of severe and incurable human diseases. The vast majority of these mutations are heteroplasmic, meaning that mutant and wild-type genomes are present simultaneously in the same cell. Only a very high proportion of mutant mitochondrial DNA (heteroplasmy level) leads to pathological consequences. We previously demonstrated that mitochondrial targeting of small RNAs designed to anneal with mutant mtDNA can decrease the heteroplasmy level by specific inhibition of mutant mtDNA replication, thus representing a potential therapy. We have also shown that 5S ribosomal RNA, partially imported into human mitochondria, can be used as a vector to deliver anti-replicative oligoribonucleotides into human mitochondria. So far, the efficiency of cellular expression of recombinant 5S rRNA molecules bearing therapeutic insertions remained very low. In the present study, we designed new versions of anti-replicative recombinant 5S rRNA targeting a large deletion in mitochondrial DNA which causes the KSS syndrome, analyzed their specific annealing to KSS mitochondrial DNA and demonstrated their import into mitochondria of cultured human cells. To obtain an increased level of the recombinant 5S rRNA stable expression, we created transmitochondrial cybrid cell line bearing a site for Flp-recombinase and used this system for the recombinase-mediated integration of genes coding for the anti-replicative recombinant 5S rRNAs into nuclear genome. We demonstrated that stable expression of anti-replicative 5S rRNA versions in human transmitochondrial cybrid cells can induce a shift in heteroplasmy level of KSS mutation in mtDNA. This shift was directly dependent on the level of the recombinant 5S rRNA expression and the sequence of the anti-replicative insertion. Quantification of mtDNA copy number in transfected cells revealed the absence of a non-specific effect on wild type mtDNA replication, indicating that the decreased proportion between mutant and wild type mtDNA molecules is not a consequence of a random repopulation of depleted pool of mtDNA genomes. The heteroplasmy change could be also modulated by cell growth conditions, namely increased by cells culturing in a carbohydrate-free medium, thus forcing them to use oxidative phosphorylation and providing a selective advantage for cells with improved respiration capacities. We discuss the advantages and limitations of this approach and propose further development of the anti-replicative strategy based on the RNA import into human mitochondria.

BIOMATERIALS is one of the leading journals in the field of molecular therapy and targeting, IF 2014 = 8.5; IF (5y) = 9.3

Abstract

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 cannot 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.

"Mise au point d'aptamères aux capacités thérapeutiques basés sur les ARN importables dans les mitochondries humaines"

Sera soutenue le 23 septembre 2015 devant la commission d’examen :

Pr Volkmar WEISSIG Rapporteur externe

Pr Ruslan AFASIZHEV Rapporteur externe

Dr Marie SISSLER Examinateur interne

Dr Valentin VLASSOV Examinateur

Dr Dmitrii PYSHNYI Examinateur

Dr Ivan TARASSOV Examinateur

Dr Nina ENTELIS Directeur de thèse

Dr Alia VENYAMINOVA Co-directeur de thèse

La soutenance aura lieu à la Salle des Thèses (Nouveau Patio), Mercredi le 23 Septembre, à 14.30