Équipe Tarassov-Smirnov - MITO - GMGM - Génétique moléculaire, génomique, microbiologie - Université de Strasbourg https://gmgm.unistra.fr fr Équipe Tarassov-Smirnov - MITO - GMGM - Génétique moléculaire, génomique, microbiologie - Université de Strasbourg Tue, 16 Jul 2024 08:55:03 +0200 Tue, 16 Jul 2024 08:55:03 +0200 TYPO3 EXT:news news-16657 Mon, 10 Jun 2024 14:01:50 +0200 New publication in Nature Cell Biology by the MITO team in collaboration with the University of Liège /actualites/actualite/new-publication-in-nature-cell-biology-by-the-mito-team-in-collaboration-with-the-university-of-liege Valine aminoacyl-tRNA synthetase promotes therapy resistance in melanoma.

doi.org/10.1038/s41556-024-01439-2

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.

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Actualités du GMGM Publications du GMGM Actualités de l'Équipe Tarassov-Smirnov Publications de l'Équipe Tarassov-Smirnov
news-14807 Mon, 16 Oct 2023 15:09:10 +0200 Bacteria, mitochondria, ribosomes & evolution at the Fête de la Science 2023 /actualites/actualite/bacteria-mitochondria-ribosomes-evolution-at-the-fete-de-la-science-2023 Friday, October 13, 2023 was the school day in the Village des Sciences, at the Palais Universitaire. The MITO team was there to familiarise the "youngest generation of scientists" with bases of cellular and molecular biology. Kids and adults from over 20 Alsatian schools discovered the amazing world of bacteria, mitochondria and ribosomes & their evolutionary journey. They could see (and smell!) real bacteria grown in the lab, play with structures of ribosomes to see how they changed over a billion years of evolution, and study electron microscopy photos to learn to recognise healthy and dysfunctional mitochondria.

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Actualités du GMGM Actualités de l'Équipe Tarassov-Smirnov Focus de l'équipe Tarassov-Smirnov
news-14766 Wed, 04 Oct 2023 14:00:17 +0200 GTPase Era at the heart of ribosome assembly /actualites/actualite/gtpase-era-at-the-heart-of-ribosome-assembly Our comprehensive review on the fascinating ribosome assembly GTPase Era/ERAL1 is out! Gruffaz C & Smirnov A (2023) GTPase Era at the heart of ribosome assembly. Front Mol Biosci 10:1263433

Ribosome biogenesis is a key process in all organisms. It relies on coordinated work of multiple proteins and RNAs, including an array of assembly factors. Among them, the GTPase Era stands out as an especially deeply conserved protein, critically required for the assembly of bacterial-type ribosomes from Escherichia coli to humans. In this review, we bring together and critically analyze a wealth of phylogenetic, biochemical, structural, genetic and physiological data about this extensively studied but still insufficiently understood factor. We do so using a comparative and, wherever possible, synthetic approach, by confronting observations from diverse groups of bacteria and eukaryotic organelles (mitochondria and chloroplasts). The emerging consensus posits that Era intervenes relatively early in the small subunit biogenesis and is essential for the proper shaping of the platform which, in its turn, is a prerequisite for efficient translation. The timing of Era action on the ribosome is defined by its interactions with guanosine nucleotides [GTP, GDP, (p)ppGpp], ribosomal RNA, and likely other factors that trigger or delay its GTPase activity. As a critical nexus of the small subunit biogenesis, Era is subject to sophisticated regulatory mechanisms at the transcriptional, post-transcriptional, and post-translational levels. Failure of these mechanisms or a deficiency in Era function entail dramatic generalized consequences for the protein synthesis and far-reaching, pleiotropic effects on the organism physiology, such as the Perrault syndrome in humans.

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Actualités de l'Équipe Tarassov-Smirnov Publications de l'Équipe Tarassov-Smirnov
news-14728 Thu, 21 Sep 2023 17:04:54 +0200 Our detailed CoLoC-seq protocol is published in Bio-protocol /actualites/actualite/our-detailed-coloc-seq-protocol-is-published-in-bio-protocol Anyone who wants to have a clear & reliable idea about RNA localisation inside/outside membrane-bounded organelles, extracellular vesicles, or viral particles can now use this detailed protocol. It includes all reagents & equipment necessary to perform CoLoC-seq experiments, a step-by-step protocol, and useful tips for data analysis & interpretation! Smirnova AJeandard DSmirnov A (2023) Controlled Level of Contamination Coupled to Deep Sequencing (CoLoC-seq) probes the global localisation topology of organelle transcriptomesBio-protocol 13: e4820

Information on RNA localisation is essential for understanding physiological and pathological processes, such as gene expression, cell reprogramming, host–pathogen interactions, and signalling pathways involving RNA transactions at the level of membrane-less or membrane-bounded organelles and extracellular vesicles. In many cases, it is important to assess the topology of RNA localisation, i.e., to distinguish the transcripts encapsulated within an organelle of interest from those merely attached to its surface. This allows establishing which RNAs can, in principle, engage in local molecular interactions and which are prevented from interacting by membranes or other physical barriers. The most widely used techniques interrogating RNA localisation topology are based on the treatment of isolated organelles with RNases with subsequent identification of the surviving transcripts by northern blotting, qRT-PCR, or RNA-seq. However, this approach produces incoherent results and many false positives. Here, we describe Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq), a more refined subcellular transcriptomics approach that overcomes these pitfalls. CoLoC-seq starts by the purification of organelles of interest. They are then either left intact or lysed and subjected to a gradient of RNase concentrations to produce unique RNA degradation dynamics profiles, which can be monitored by northern blotting or RNA-seq. Through straightforward mathematical modelling, CoLoC-seq distinguishes true membrane-enveloped transcripts from degradable and non-degradable contaminants of any abundance. The method has been implemented in the mitochondria of HEK293 cells, where it outperformed alternative subcellular transcriptomics approaches. It is applicable to other membrane-bounded organelles, e.g., plastids, single-membrane organelles of the vesicular system, extracellular vesicles, or viral particles.

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Actualités de l'Équipe Tarassov-Smirnov Publications de l'Équipe Tarassov-Smirnov
news-14686 Fri, 15 Sep 2023 09:31:51 +0200 New publication by MITO-team /actualites/actualite/new-publication-by-mito-team Targeting of CRISPR-Cas12a crRNAs into human mitochondria BIOCHIMIE

Mitochondrial gene editing holds great promise as a therapeutic approach for mitochondrial diseases caused by mutations in the mitochondrial DNA (mtDNA). Current strategies focus on reducing mutant mtDNA heteroplasmy levels through targeted cleavage or base editing. However, the delivery of editing components into mitochondria remains a challenge. Here we investigate the import of CRISPR-Cas12a system guide RNAs (crRNAs) into human mitochondria and study the structural requirements for this process by northern blot analysis of RNA isolated from nucleases-treated mitoplasts. To investigate whether the fusion of crRNA with known RNA import determinants (MLS) improve its mitochondrial targeting, we added MLS hairpin structures at 3’-end of crRNA and demonstrated that this did not impact crRNA ability to program specific cleavage of DNA in lysate of human cells expressing AsCas12a nuclease. Surprisingly, mitochondrial localization of the fused crRNA molecules was not improved compared to non-modified version, indicating that structured scaffold domain of crRNA can probably function as MLS, assuring crRNA mitochondrial import. Then, we designed a series of crRNAs targeting different regions of mtDNA and demonstrated their ability to program specific cleavage of mtDNA fragments in cell lysate and their partial localization in mitochondrial matrix in human cells transfected with these RNA molecules. We hypothesize that mitochondrial import of crRNAs may depend on their secondary structure/sequence. We presume that imported crRNA allow reconstituting the active crRNA/Cas12a system in human mitochondria, which can contribute to the development of effective strategies for mitochondrial gene editing and potential future treatment of mitochondrial diseases.

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Actualités du GMGM Publications du GMGM Actualités de l'Équipe Tarassov-Smirnov
news-14618 Mon, 04 Sep 2023 11:40:13 +0200 The MITO team welcomes a new PhD student, Théo Markezic /actualites/actualite/the-mito-team-welcomes-a-new-phd-student-theo-markezic Théo Markezic, funded by IMCBio, starts his PhD studies in the "RNA-binding hubs" group. Théo will work on pervasive and antisense transcription in phylogenetically distant bacteria, E. coli and S. aureus, using experimental evolution approaches. His work will take place in GMGM and IBMC under the supervision of Alexandre Smirnov and Isabelle Caldelari. Théo is funded by IMCBio, via the Inter-Labex PhD programme (MitoCross - NetRNA).

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Actualités de l'Équipe Tarassov-Smirnov
news-13132 Thu, 02 Mar 2023 10:34:51 +0100 Review on the evolutionary aspects of the FinO/ProQ family proteins /actualites/actualite/review-on-the-evolutionary-aspects-of-the-fino-proq-family-proteins Our review paper on the evolutionary significance of RNA-binding proteins of the FinO/ProQ family has been published in Bioscience Reports. FinO/ProQ-family proteins: an evolutionary perspective

Zhen Liao & Alexandre Smirnov

Biosci Rep (2023) 43 (3): BSR20220313

RNA-binding proteins are key actors of post-transcriptional networks. Almost exclusively studied in the light of their interactions with RNA ligands and the associated functional events, they are still poorly understood as evolutionary units. In this review, we discuss the FinO/ProQ family of bacterial RNA chaperones, how they evolve and spread across bacterial populations and what properties and opportunities they provide to their host cells. We reflect on major conserved and divergent themes within the family, trying to understand how the same ancestral RNA-binding fold, augmented with additional structural elements, could yield either highly specialised proteins or, on the contrary, globally acting regulatory hubs with a pervasive impact on gene expression. We also consider dominant convergent evolutionary trends that shaped their RNA chaperone activity and recurrently implicated the FinO/ProQ-like proteins in bacterial DNA metabolism, translation and virulence. Finally, we offer a new perspective in which FinO/ProQ-family regulators emerge as active evolutionary players with both negative and positive roles, significantly impacting the evolutionary modes and trajectories of their bacterial hosts.

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Actualités de l'Équipe Tarassov-Smirnov Publications de l'Équipe Tarassov-Smirnov
news-12988 Wed, 25 Jan 2023 13:58:42 +0100 The MITO team welcomes a new member, Dr Marie SISSLER (CNRS) /actualites/actualite/the-mito-team-welcomes-a-new-member-dr-marie-sissler-cnrs Dr Sissler works on diverse aspects of mitochondrial RNA biology. She is particularly interested in mitochondrial aminoacyl-tRNA synthetases in as diverses biological models as humans and trypanosomatids. Dr Sissler studies the basic principles of their orhanisation and molecular functions as well as their involvement in human pathologies. 

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Actualités de l'Équipe Tarassov-Smirnov
news-12872 Wed, 21 Dec 2022 13:43:45 +0100 Editorial for the Special issue "Research progress in RNA-binding proteins" - International Journal of Molecular Sciences /actualites/actualite/editorial-for-the-special-issue-research-progress-in-rna-binding-proteins-international-journal-of-molecular-sciences We are proud to present a great collection of papers published in the Special issue "Research progress in RNA-binding proteins" of International Journal of Molecular Sciences - editorial by Alexandre Smirnov. An editorial by Alexandre Smirnov introduces a great collection of 9 papers dedicated to various aspects of the biology of RNA-binding proteins - now published in International Journal of Molecular Sciences.

Smirnov A (2023) Research progress in RNA-binding proteins. Int J Mol Sci 24:58.

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Actualités de l'Équipe Tarassov-Smirnov Publications de l'Équipe Tarassov-Smirnov
news-12871 Tue, 20 Dec 2022 15:56:02 +0100 The MITO team created an innovative subcellular transcriptomics approach, CoLoC-seq - Nucleic Acids Research /actualites/actualite/the-mito-team-created-an-innovative-subcellular-transcriptomics-approach-coloc-seq-nucleic-acids-research A new genome-wide method that marries classical enzymatic kinetics, deep sequencing, and mathematical modelling - CoLoC-seq - has been created in the MITO team and successfully applied to human mitochondria to tell resident RNAs from contaminants. Damien Jeandard, Anna Smirnova, Akinyemi Mandela Fasemore, Léna Coudray, Nina Entelis, Konrad U Förstner, Ivan Tarassov, Alexandre Smirnov (2023) CoLoC-seq probes the global topology of organelle transcriptomesNucleic Acids Research 51:e16.

Proper RNA localisation is essential for physiological gene expression. Various kinds of genome-wide approaches permit to comprehensively profile subcellular transcriptomes. Among them, cell fractionation methods, that couple RNase treatment of isolated organelles to the sequencing of protected transcripts, remain most widely used, mainly because they do not require genetic modification of the studied system and can be easily implemented in any cells or tissues, including in non-model species. However, they suffer from numerous false-positives since incompletely digested contaminant RNAs can still be captured and erroneously identified as resident transcripts. Here we introduce Controlled Level of Contamination coupled to deep sequencing (CoLoC-seq) as a new subcellular transcriptomics approach that efficiently bypasses this caveat. CoLoC-seq leverages classical enzymatic kinetics and tracks the depletion dynamics of transcripts in a gradient of an exogenously added RNase, with or without organellar membranes. By means of straightforward mathematical modelling, CoLoC-seq infers the localisation topology of RNAs and robustly distinguishes between genuinely resident, luminal transcripts and merely abundant surface-attached contaminants. Our generic approach performed well on human mitochondria and is in principle applicable to other membrane-bounded organelles, including plastids, compartments of the vacuolar system, extracellular vesicles, and viral particles.

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Actualités du GMGM Publications du GMGM Actualités de l'Équipe Tarassov-Smirnov Publications de l'Équipe Tarassov-Smirnov
news-12893 Thu, 15 Dec 2022 12:00:00 +0100 The MITO team is looking for prospective graduate students interested in RNA biology and experimental evolution /actualites/actualite/mito-team-is-looking-for-prospective-graduate-students-interested-in-rna-biology-and-experimental-evolution The IMCBio International PhD Program 2023 call is open for applications. As a member of the Integrative Molecular and Cellular Biology (IMCBio), the MITO team, in collaboration with ARN (IBMC), participates in its annual PhD Program call and offers a PhD thesis opportunity in microbial experimental evolution and RNA biology. For more details see this page.

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Agenda de l'Équipe Tarassov-Smirnov
news-12765 Mon, 21 Nov 2022 15:40:05 +0100 MITO team : editorial in Frontiers Physiology /actualites/actualite/mito-team-editorial-in-frontiers-physiology Editorial for a special issue of Frontiers Physiology Burzio VA, Barrey E, Leucci E, Entelis N,
Hollander JM and Das S (2022)
Editorial:
Role of mitochondria-associated noncoding
RNAs inintracellular communication.
Front. Physiol. 13:980674.
doi: 10.3389/fphys.2022.980674
 

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Actualités du GMGM Publications du GMGM Actualités de l'Équipe Tarassov-Smirnov Publications de l'Équipe Tarassov-Smirnov
news-12713 Mon, 14 Nov 2022 11:05:46 +0100 New publication of the MITO team in Computational and Structural Biotechnology Journal /actualites/actualite/new-publication-of-the-mito-team-in-computational-and-structural-biotechnology-journal Theoretical and mathematical biology paper about hub-centred RNA-protein networks Smirnov A (2022) How global RNA-binding proteins coordinate the behaviour of RNA regulons: an information approach. Comput Struct Biotechnol J doi:10.1016/j.csbj.2022.11.019

RNA-binding proteins are central players in post-transcriptional regulation. Some of them, such as the well-studied bacterial RNA chaperones Hfq and ProQ or the eukaryotic RNAi factor Argonaute, interact with hundreds-to-thousands of different RNAs and thereby globally affect gene expression. As a shared yet limited resource, these and other RNA-binding hubs drive strong competition between their multiple ligands. This creates a ground for significant cross-communication between RNA targets, which enables them to share information, “synchronise” their behaviour, and produce interesting biochemical effects, sometimes propagating across the highly connected RNA-protein network. This property is likely universally present in hub-centred networks and plays a key role in global gene expression programmes. It is also an important factor in biotechnology and synthetic biology applications of RNA/protein-based circuits. However, few studies so-far focused on describing and explaining this phenomenon from first principles. Here we introduce an information theory-based framework to comprehensively and exactly describe information flow in hub-centred networks. We show that information sharing can achieve significant levels in relatively small networks, provided the hub is present in limiting concentrations. The transmitted information is sufficient to noticeably affect the binding probabilities of competing targets but drops exponentially along the network. Target overexpression can disrupt communication between other targets, while hub sequestration boosts the crosstalk. We also find that overlaps between the interactomes of two different hubs create both entropic challenges and new forms of long-range communication between RNAs and proteins.

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Actualités du GMGM Publications du GMGM Actualités de l'Équipe Tarassov-Smirnov Publications de l'Équipe Tarassov-Smirnov
news-12102 Tue, 07 Jun 2022 15:28:00 +0200 Chaires juniors dans le domaine de recherches sur les mitochondries /actualites/actualite/chaires-juniors-dans-le-domaine-de-recherches-sur-les-mitochondries Le Laboratoire d'excellence (LabEx) MitoCross annonce l'appel aux candidatures de deux chaires juniors dans le domaine de recherches sur les mitochondries. Voir les détails ICI.

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Actualités du GMGM Actualités de l'Équipe Becker Actualités de l'Équipe Tarassov-Smirnov Actualités de l'Équipe Schacherer
news-10578 Fri, 14 Jan 2022 16:48:16 +0100 We welcome XuChu Duan, a visiting researcher from the Nantong University! /actualites/actualite/we-welcome-xuchu-duan-a-visiting-researcher-from-the-nantong-university XuChu, who is a specialist in zebrafish and developmental biology, is doing his sabbatical in our team. He will work on the mechanistic roles of YBEY in coordination of the human mitoribosome assembly.

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Actualités de l'Équipe Tarassov-Smirnov
news-10570 Wed, 12 Jan 2022 11:27:09 +0100 New publication of the MITO team in Nucleic Acids Research /actualites/actualite/new-publication-of-the-mito-team-in-nucleic-acids-research-1 Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA

 

Nucleic Acids Res. 2021 Dec 24. pii: gkab1227. doi: 10.1093/nar/gkab1227. Online ahead of print.

Regina Shebanova 1Natalia Nikitchina 1 2Nikita Shebanov 1Vladimir Mekler 3Konstantin Kuznedelov 3Egor Ulashchik 4Ruslan Vasilev 5 6Olga Sharko 4Vadim Shmanai 4Ivan Tarassov 2Konstantin Severinov 1 3 7Nina Entelis 2Ilya Mazunin 1

 

Abstract

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5'-scaffold moiety and variable 3'-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.

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Actualités du GMGM Actualités de l'Équipe Tarassov-Smirnov Publications de l'Équipe Tarassov-Smirnov
news-10246 Tue, 12 Oct 2021 15:30:54 +0200 Our paper in collaboration with the Alya Venyaminova's team has been published in Molecules /actualites/actualite/ninas-paper-a-versatile-solid-phase-approach-to-the-synthesis-of-oligonucleotide-conjugates-with-biodegradable-hydrazone-linker-published-in-molecules-congratulations Meschaninova MI, Entelis NS, Chernolovskaya EL, Venyaminova AG (2021) A versatile solid-phase approach to the synthesis of oligonucleotide conjugates with biodegradable hydrazone linkerMolecules 26:2119

One of the ways to efficiently deliver various drugs, including therapeutic nucleic acids, into the cells is conjugating them with different transport ligands via labile or stable bonds. A convenient solid-phase approach for the synthesis of 5′-conjugates of oligonucleotides with biodegradable pH-sensitive hydrazone covalent bonds is proposed in this article. The approach relies on introducing a hydrazide of the ligand under aqueous/organic media to a fully protected support-bound oligonucleotide containing aldehyde function at the 5′-end. We demonstrated the proof-of-principle of this approach by synthesizing 5′-lipophilic (e.g., cholesterol and α-tocopherol) conjugates of modified siRNA and non-coding RNAs imported into mitochondria (antireplicative RNAs and guide RNAs for Mito-CRISPR/system). The developed method has the potential to be extended for the synthesis of pH-sensitive conjugates of oligonucleotides of different types (ribo-, deoxyribo-, 2′-O-methylribo-, and others) with ligands of different nature.

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Publications de l'Équipe Tarassov-Smirnov
news-10245 Tue, 12 Oct 2021 15:10:56 +0200 Congratulations on a fantastic 2021 series of review & protocol papers from the MITO team! /actualites/actualite/congratulations-on-a-fantastic-2021-series-of-review-protocol-papers-from-the-mito-team Our big review on stable RNPs and the complexomic approaches to profile them , including Grad-seq, co-authored with the Jörg Vogel's lab, is availbale in Frontiers in Molecular Biosciences:

Macromolecular complexes of proteins and RNAs are essential building blocks of cells. These stable supramolecular particles can be viewed as minimal biochemical units whose structural organization, i.e., the way the RNA and the protein interact with each other, is directly linked to their biological function. Whether those are dynamic regulatory ribonucleoproteins (RNPs) or integrated molecular machines involved in gene expression, the comprehensive knowledge of these units is critical to our understanding of key molecular mechanisms and cell physiology phenomena. Such is the goal of diverse complexomic approaches and in particular of the recently developed gradient profiling by sequencing (Grad-seq). By separating cellular protein and RNA complexes on a density gradient and quantifying their distributions genome-wide by mass spectrometry and deep sequencing, Grad-seq charts global landscapes of native macromolecular assemblies. In this review, we propose a function-based ontology of stable RNPs and discuss how Grad-seq and related approaches transformed our perspective of bacterial and eukaryotic ribonucleoproteins by guiding the discovery of new RNA-binding proteins and unusual classes of noncoding RNAs. We highlight some methodological aspects and developments that permit to further boost the power of this technique and to look for exciting new biology in understudied and challenging biological models.

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

YbeY is an ultraconserved small protein belonging to the unique heritage shared by most existing bacteria and eukaryotic organelles of bacterial origin, mitochondria and chloroplasts. Studied in more than a dozen of evolutionarily distant species, YbeY is invariably critical for cellular physiology. However, the exact mechanisms by which it exerts such penetrating influence are not completely understood. In this review, we attempt a transversal analysis of the current knowledge about YbeY, based on genetic, structural, and biochemical data from a wide variety of models. We propose that YbeY, in association with the ribosomal protein uS11 and the assembly GTPase Era, plays a critical role in the biogenesis of the small ribosomal subunit, and more specifically its platform region, in diverse genetic systems of bacterial type.

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!

Mitochondria have complex ultrastructure which includes continuous subcompartments, such as matrix, intermembrane space, and two membranes, as well as focal structures, such as nucleoids, RNA granules, and mitoribosomes. Comprehensive studies of the spatial distribution of proteins and RNAs inside the mitochondria are necessary to understand organellar gene expression processes and macromolecule targeting pathways. Here we give examples of distribution analysis of mitochondrial proteins and transcripts by conventional microscopy and the super-resolution technique 3D STORM. We provide detailed protocols and discuss limitations of immunolabeling of mitochondrial proteins and newly synthesized mitochondrial RNAs by bromouridine incorporation and single-molecule RNA FISH in hepatocarcinoma cells.

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!

Defects in human mitochondrial genome can cause a wide range of clinical disorders that still do not have efficient therapies. The natural pathway of small noncoding RNA import can be exploited to address therapeutic RNAs into the mitochondria. To create an approach of carrier-free targeting of RNA into living human cells, we designed conjugates containing a cholesterol residue and developed the protocols of chemical synthesis of oligoribonucleotides conjugated with cholesterol residue through cleavable pH-triggered hydrazone bond. The biodegradable conjugates of importable RNA with cholesterol can be internalized by cells in a carrier-free manner; RNA can then be released in the late endosomes due to a change in pH and partially targeted into mitochondria. Here we provide detailed protocols for solid-phase and "in solution" chemical synthesis of oligoribonucleotides conjugated to a cholesterol residue through a hydrazone bond. We describe the optimization of the carrier-free cell transfection with these conjugated RNA molecules and methods for evaluating the cellular and mitochondrial uptake of lipophilic conjugates.

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Publications de l'Équipe Tarassov-Smirnov
news-9594 Thu, 26 Nov 2020 09:32:00 +0100 A FASEB J. paper by the MITO team /actualites/actualite/a-faseb-j-paper-by-the-mito-team

Early View

RESEARCH ARTICLE 

Open Access 

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

 

First published: 25 November 2020

Gaëlle Zimmermann-Meisse and Anna Smirnova are participated equally to this article.

Abstract

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.

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Publications du GMGM Publications de l'Équipe Tarassov-Smirnov
news-9583 Thu, 16 Jul 2020 11:44:00 +0200 New publication by MITO team in Neurology: Genetics /actualites/actualite/new-publication-by-mito-team-in-neurology-genetics Karine Auré, Guillemette Fayet, Ivan Chicherin, Benoit Rucheton, Sandrine Filaut, Anne-Marie Heckel, Julie Eichler, Florence Caillon, Yann Péréon, Nina Entelis, Ivan Tarassov, Anne Lombès (2020) A homoplasmic mitochondrial tRNAPro mutation causing exercise-induced muscle swelling and fatigue. Neurology: Genetics (in press) Neurol Genet 2020;6:e480. doi:10.1212/NXG.0000000000000480

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.

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Publications de l'Équipe Tarassov-Smirnov
news-9580 Wed, 22 Apr 2020 16:46:00 +0200 New publication of the MITO team in Nucleic Acids Research /actualites/actualite/new-publication-of-the-mito-team-in-nucleic-acids-research

YBEY is an essential biogenesis factor for mitochondrial ribosomes

Nucleic Acids Res. 2020 Mar 17. pii: gkaa148. doi: 10.1093/nar/gkaa148. [Epub ahead of print]

Abstract

Ribosome biogenesis requires numerous trans-acting factors, some of which are deeply conserved. In Bacteria, the endoribonuclease YbeY is believed to be involved in 16S rRNA 3'-end processing and its loss was associated with ribosomal abnormalities. In Eukarya, YBEY appears to generally localize to mitochondria (or chloroplasts). Here we show that the deletion of human YBEY results in a severe respiratory deficiency and morphologically abnormal mitochondria as an apparent consequence of impaired mitochondrial translation. Reduced stability of 12S rRNA and the deficiency of several proteins of the small ribosomal subunit in YBEY knockout cells pointed towards a defect in mitochondrial ribosome biogenesis. The specific interaction of mitoribosomal protein uS11m with YBEY suggests that the latter helps to properly incorporate uS11m into the nascent small subunit in its late assembly stage. This scenario shows similarities with final stages of cytosolic ribosome biogenesis, and may represent a late checkpoint before the mitoribosome engages in translation.

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Actualités du GMGM Actualités de l'Équipe Tarassov-Smirnov Publications de l'Équipe Tarassov-Smirnov
news-9578 Thu, 12 Mar 2020 14:59:00 +0100 USIAS 2020 award for Alexandre Smirnov /actualites/actualite/usias-2020-award-for-alexandre-smirnov The Institute for Advanced Studies at the University of Strasbourg (USIAS) is committed to supporting an original type of research and potentially pioneering research, so as to promote a high level of reflection and synthesis.

Among 2020 USIAS supported projects is that of Alexandre Smirnov: "Experimental evolution of an organism with devastating impairment of regulatory networks".

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Actualités du GMGM Actualités de l'Équipe Tarassov-Smirnov
news-9557 Mon, 03 Dec 2018 09:37:00 +0100 Anne-Marie Heckel à la rencontre des Lycéens /actualites/actualite/anne-marie-heckel-a-la-rencontre-des-lyceens Anne-Marie Heckel, ingénieure de l'équipe MITO, avait présenté nos thématiques de recherche aux lycéens. La rencontre a été mise en lumière par deux publications locales (DNA).

Voir l'article 1

Voir l'article 2

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Actualités du GMGM Actualités de l'Équipe Tarassov-Smirnov
news-9554 Thu, 06 Sep 2018 13:46:00 +0200 New paper in IUBMB life by MITO-team. /actualites/actualite/new-paper-in-iubmb-life-by-mito-team

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

Can Mitochondrial DNA be CRISPRized: Pro and Contra.

Abstract

Mitochondria represent a chimera of macromolecules encoded either in the organellar genome, mtDNA, or in the nuclear one. If the pathway of protein targeting to different sub-compartments of mitochondria was relatively well studied, import of small noncoding RNAs into mammalian mitochondria still awaits mechanistic explanations and its functional issues are often not understood thus raising polemics. At the same time, RNA mitochondrial import pathway has an obvious attractiveness as it appears as a unique natural mechanism permitting to address nucleic acids into the organelles. Deciphering the function(s) of imported RNAs inside the mitochondria is extremely complicated due to their relatively low abundance, which suggests their regulatory role. We previously demonstrated that mitochondrial targeting of small noncoding RNAs able to specifically anneal with the mutant mitochondrial DNA led to a decrease of the mtDNA heteroplasmy level by inhibiting mutant mtDNA replication. We then demonstrated that increasing level of expression of such antireplicative recombinant RNAs increases significantly the antireplicative effect. In this report, we present a new data investigating the possibility to establish a CRISPR-Cas9 system targeting mtDNA exploiting of the pathway of RNA import into mitochondria. Mitochondrially addressed Cas9 versions and a set of mitochondrially targeted guide RNAs were tested in vitro and in vivo and their effect on mtDNA copy number was demonstrated. So far, the system appeared as more complicated for use than previously found for nuclear DNA, because only application of a pair of guide RNAs produced the effect of mtDNA depletion. We discuss, in a critical way, these results and put them in a broader context of polemics concerning the possibilities of manipulation of mtDNA in mammalians. The findings described here prove the potential of the RNA import pathway as a tool for studying mtDNA and for future therapy of mitochondrial disorders.

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Actualités du GMGM Publications de l'Équipe Tarassov-Smirnov
news-9552 Tue, 19 Jun 2018 08:53:00 +0200 New paper in PLoS ONE for the MITO-team /actualites/actualite/new-paper-in-plos-one-for-the-mito-team R. Loutre, AM Heckel, D Jeandard, I Tarassov, N Entelis (2018) "Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner" PLOS ONE https://doi.org/10.1371/journal.pone.0199258

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.

 

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Actualités du GMGM Publications de l'Équipe Tarassov-Smirnov
news-9545 Thu, 26 Oct 2017 16:45:00 +0200 Le projet d’École Universitaire de recherche en biologie moléculaire et cellulaire intégrative incluant le LabEx MitoCross - lauréat du Programme d’investissements d’avenir. /actualites/actualite/le-projet-decole-universitaire-de-recherche-en-biologie-moleculaire-et-cellulaire-integrative-incluant-le-labex-mitocross-laureat-du-programme-dinvestissements-davenir Basé sur un regroupement stratégique des forces strasbourgeoises en biologie moléculaire et cellulaire des trois Laboratoire d'excellence (LabEx) INRT (IGBMC), MitoCross (GMGM, IBMP et IBMC(ARN)) et NetRNA (IBMC (ARN, RIDI), IBMP), avec le concours d’outils technologiques de pointe tels que les quatre Infrastructures nationales en biologie santé (INBS) CELPHEDIA/PHENOMIN, FRISBI, France Génomique et Ingestem ainsi que l’Équipement d'excellence (EquipEx) I2MC, le projet d’Ecole universitaire de recherche (EUR) Integrative Molecular and Cellular Biology (IMCBio) a été retenu, mardi 24 octobre, dans le cadre du Programme d’investissements d’avenir (PIA3). Ce projet de formation inédit est l’une des quatre EUR lauréates portées par l’Université de Strasbourg qui se voit dotée d’un montant de 25 717 000 € pour l’ensemble de ces projets. À travers des stages en laboratoires additionnels, des modules de formations complémentaires au niveau Master, des écoles d’étés, ou encore un programme de formation de haut niveau pour le doctorat, l’EUR IMCBio souhaite attirer des étudiants ayant une forte motivation pour la recherche pour former les futurs chercheurs capables de travailler aux interfaces de la Biologie. Avec le soutien conjoint de l’Université de Strasbourg (Faculté des sciences de la Vie et Ecole doctorale Vie et Santé), du CNRS et de l’Inserm, ce projet, qui s’appuie sur le modèle de « graduate school », a pour triple ambition de lier fortement la formation à la recherche, de valoriser les points forts thématiques du site de Strasbourg mais aussi de concourir à son rayonnement international.

Le troisième PIA qui finance ce projet se veut en effet structurant pour le paysage de l’enseignement supérieur et de la recherche française et vise à offrir aux sites universitaires la possibilité de renforcer l’impact et l’attractivité internationale de leur recherche et de leur formation dans un ou plusieurs domaine(s) scientifique(s). Dans le cadre des EUR, cette structuration passe par le rassemblement des formations de master et de doctorat adossées à un ou plusieurs laboratoires de recherche de haut niveau. Sur 195 projets déposés, 29 lauréats de cette première vague du PIA3, pour un montant de 216 M€ financés sur dix ans, ont été conjointement annoncés par le ministère de l’Enseignement supérieur, de la Recherche et de l’Innovation et le Commissaire général à l’investissement, précisant avoir suivi l’avis d’un jury international.

 

Contacts :

Directeur du projet : Bertrand Séraphin, directeur de recherche, CNRS

Codirecteur du projet : Nicolas Matt, Maître de conférences, Université de Strasbourg

Représentants des LabEx: INRT (Bertrand Séraphin), MitoCross (Ivan Tarassov), NetRNA (Pascale Romby)

 

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Actualités du GMGM Actualités de l'Équipe Tarassov-Smirnov
news-9492 Mon, 02 Nov 2015 09:16:00 +0100 MITO-Team : paper accepted in BIOMATERIALS /actualites/actualite/mito-team-paper-accepted-in-biomaterals I. Dovydenko, I. Tarassov,  A. Venyaminova, N. Entelis (2015) Method of carrier-free delivery of therapeutic RNA importable into human mitochondria: lipophilic conjugates with cleavable bonds. Biomaterials (in press)

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.

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Publications de l'Équipe Tarassov-Smirnov
news-9489 Wed, 23 Sep 2015 14:30:00 +0200 Soutenance de thèse de Ilya DOVYDENKO /actualites/actualite/soutenance-de-these-de-ilya-dovydenko La Thèse de Ilya DOVYDENKO :

 

"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

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Agenda de l'Équipe Tarassov-Smirnov
news-9445 Wed, 19 Dec 2012 14:30:00 +0100 Inauguration du LabEx MitoCross /actualites/actualite/inauguration-du-labex-mitocross Le lancement officiel du laboratoire d'excellence MitoCross, qui implique deux équipes de l'unité (N. Entelis & I. Tarassov et H. Becker), a été célebré lors d'une réunion scientifique "Mito@Strass2012", au Forum de la faculté de médicine de l'Université de Strasbourg.

 

 

Le programme de l'évenement est disponible ici.

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Actualités du GMGM Agenda de l'Équipe Tarassov-Smirnov
news-9443 Fri, 02 Nov 2012 16:21:00 +0100 Le consortium MitoCross se dote d'un site web ! /actualites/actualite/le-consortium-mitocross-se-dote-dun-site-web L'objectif global du consortium MitoCross est d'approfondir les connaissances et mécanismes moléculaires fins gouvernant la biogenèse des mitochondries, leur fonctionnement et leur communication avec les autres compartiments cellulaires. En exploitant des modèles d'étude différents que sont la levure, les plantes et l'homme, les nouvelles connaissances acquises permettront de développer des outils innovants en termes biotechnologique et biomédical.

Le consortium MitoCross se dote d'un site web dédié. Sa conception a été assurée conjointement par les personnels du consortium et le département Web et diffusion scientifique de la DUN. Ce site est en outre hébergé sur les serveurs de la Direction Informatique (DI) de l'université.

Nous espérons qu'il répondra à vos attentes et vous souhaitons une excellente navigation : mitocross.unistra.fr

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Actualités du GMGM Actualités de l'Équipe Tarassov-Smirnov