Publications by authors named "Lauriane Kuhn"

72 Publications

The TUTase URT1 connects decapping activators and prevents the accumulation of excessively deadenylated mRNAs to avoid siRNA biogenesis.

Nat Commun 2021 02 26;12(1):1298. Epub 2021 Feb 26.

Institut de biologie moléculaire des plantes, CNRS, Université de Strasbourg, Strasbourg, France.

Uridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators. URT1 directly interacts with DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6, and this interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. Nanopore direct RNA sequencing reveals a global role of URT1 in shaping poly(A) tail length, notably by preventing the accumulation of excessively deadenylated mRNAs. Based on in vitro and in planta data, we propose a model that explains how URT1 could reduce the accumulation of oligo(A)-tailed mRNAs both by favoring their degradation and because 3' terminal uridines intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb Arabidopsis growth and development.
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http://dx.doi.org/10.1038/s41467-021-21382-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910438PMC
February 2021

Arabidopsis mTERF9 protein promotes chloroplast ribosomal assembly and translation by establishing ribonucleoprotein interactions in vivo.

Nucleic Acids Res 2021 01;49(2):1114-1132

Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg, France.

The mitochondrial transcription termination factor proteins are nuclear-encoded nucleic acid binders defined by degenerate tandem helical-repeats of ∼30 amino acids. They are found in metazoans and plants where they localize in organelles. In higher plants, the mTERF family comprises ∼30 members and several of these have been linked to plant development and response to abiotic stress. However, knowledge of the molecular basis underlying these physiological effects is scarce. We show that the Arabidopsis mTERF9 protein promotes the accumulation of the 16S and 23S rRNAs in chloroplasts, and interacts predominantly with the 16S rRNA in vivo and in vitro. Furthermore, mTERF9 is found in large complexes containing ribosomes and polysomes in chloroplasts. The comprehensive analysis of mTERF9 in vivo protein interactome identified many subunits of the 70S ribosome whose assembly is compromised in the null mterf9 mutant, putative ribosome biogenesis factors and CPN60 chaperonins. Protein interaction assays in yeast revealed that mTERF9 directly interact with these proteins. Our data demonstrate that mTERF9 integrates protein-protein and protein-RNA interactions to promote chloroplast ribosomal assembly and translation. Besides extending our knowledge of mTERF functional repertoire in plants, these findings provide an important insight into the chloroplast ribosome biogenesis.
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http://dx.doi.org/10.1093/nar/gkaa1244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826268PMC
January 2021

Structural Differences in Translation Initiation between Pathogenic Trypanosomatids and Their Mammalian Hosts.

Cell Rep 2020 Dec;33(12):108534

INSERM U1212 (ARNA), Institut Européen de Chimie et Biologie, Université de Bordeaux, Pessac 33607, France. Electronic address:

Canonical mRNA translation in eukaryotes begins with the formation of the 43S pre-initiation complex (PIC). Its assembly requires binding of initiator Met-tRNA and several eukaryotic initiation factors (eIFs) to the small ribosomal subunit (40S). Compared to their mammalian hosts, trypanosomatids present significant structural differences in their 40S, suggesting substantial variability in translation initiation. Here, we determine the structure of the 43S PIC from Trypanosoma cruzi, the parasite causing Chagas disease. Our structure shows numerous specific features, such as the variant eIF3 structure and its unique interactions with the large rRNA expansion segments (ESs) 9, 7, and 6, and the association of a kinetoplastid-specific DDX60-like helicase. It also reveals the 40S-binding site of the eIF5 C-terminal domain and structures of key terminal tails of several conserved eIFs underlying their activities within the PIC. Our results are corroborated by glutathione S-transferase (GST) pull-down assays in both human and T. cruzi and mass spectrometry data.
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http://dx.doi.org/10.1016/j.celrep.2020.108534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773551PMC
December 2020

Opportunistic use of catecholamine neurotransmitters as siderophores to access iron by Pseudomonas aeruginosa.

Environ Microbiol 2020 Dec 21. Epub 2020 Dec 21.

Université de Strasbourg, InnoVec, UMR7242, ESBS, Bld Sébastien Brant, F-67413 Illkirch, Strasbourg, France.

Iron is an essential nutrient for bacterial growth and the cause of a fierce battle between the pathogen and host during infection. Bacteria have developed several strategies to access iron from the host, the most common being the production of siderophores, small iron-chelating molecules secreted into the bacterial environment. The opportunist pathogen Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, and is also able to use a wide panoply of xenosiderophores, siderophores produced by other microorganisms. Here, we demonstrate that catecholamine neurotransmitters (dopamine, l-DOPA, epinephrine and norepinephrine) are able to chelate iron and efficiently bring iron into P. aeruginosa cells via TonB-dependent transporters (TBDTs). Bacterial growth assays under strong iron-restricted conditions and with numerous mutants showed that the TBDTs involved are PiuA and PirA. PiuA exhibited more pronounced specificity for dopamine uptake than for norepinephrine, epinephrine and l-DOPA, whereas PirA specificity appeared to be higher for l-DOPA and norepinephrine. Proteomic and qRT-PCR approaches showed pirA transcription and expression to be induced in the presence of all four catecholamines. Finally, the oxidative properties of catecholamines enable them to reduce iron, and we observed ferrous iron uptake via the FeoABC system in the presence of l-DOPA.
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http://dx.doi.org/10.1111/1462-2920.15372DOI Listing
December 2020

The viral protein NSP1 acts as a ribosome gatekeeper for shutting down host translation and fostering SARS-CoV-2 translation.

RNA 2020 Dec 2. Epub 2020 Dec 2.

Institut de Biologie Moleculaire et Cellulaire, Architecture et Reactivite de l ARN CNRS UPR9002, Universite de Strasbourg, 2, allee Konrad Roentgen, F-67084 Strasbourg (France);

SARS-CoV-2 coronavirus is responsible for Covid-19 pandemic. In the early phase of infection, the single-strand positive RNA genome is translated into non-structural proteins (NSP). One of the first proteins produced during viral infection, NSP1, binds to the host ribosome and blocks the mRNA entry channel. This triggers translation inhibition of cellular translation. In spite of the presence of NSP1 on the ribosome, viral translation proceeds however. The molecular mechanism of the so-called viral evasion to NSP1 inhibition remains elusive. Here, we confirm that viral translation is maintained in the presence of NSP1. The evasion to NSP1-inhibition is mediated by the cis-acting RNA hairpin SL1 in the 5'UTR of SARS-CoV-2. NSP1-evasion can be transferred on a reporter transcript by SL1 transplantation. The apical part of SL1 is only required for viral translation. We show that NSP1 remains bound on the ribosome during viral translation. We suggest that the interaction between NSP1 and SL1 frees the mRNA accommodation channel while maintaining NSP1 bound to the ribosome. Thus, NSP1 acts as a ribosome gatekeeper, shutting down host translation or fostering SARS-CoV-2 translation depending on the presence of the SL1 5'UTR hairpin. SL1 is also present and necessary for translation of sub-genomic RNAs in the late phase of the infectious program. Consequently, therapeutic strategies targeting SL1 should affect viral translation at early and late stages of infection. Therefore, SL1 might be seen as a genuine 'Achille heel' of the virus.
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http://dx.doi.org/10.1261/rna.078121.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901841PMC
December 2020

Structure of the mature kinetoplastids mitoribosome and insights into its large subunit biogenesis.

Proc Natl Acad Sci U S A 2020 11 9;117(47):29851-29861. Epub 2020 Nov 9.

Institut Européen de Chimie et Biologie, U1212 INSERM, UMR5320 CNRS, Université de Bordeaux, F-33600 Pessac, France;

Kinetoplastids are unicellular eukaryotic parasites responsible for such human pathologies as Chagas disease, sleeping sickness, and leishmaniasis. They have a single large mitochondrion, essential for the parasite survival. In kinetoplastid mitochondria, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Structural studies performed in already highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit. However, several important aspects mainly related to the large subunit (LSU) remain elusive, such as the structure and maturation of its ribosomal RNA. Here we present a cryo-electron microscopy study of the protozoans and mitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the LSU, as well as previously unidentified ribosomal proteins. In addition, we introduce the structure of an LSU assembly intermediate in the presence of 16 identified maturation factors. These maturation factors act on both the intersubunit and the solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.
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http://dx.doi.org/10.1073/pnas.2011301117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7703582PMC
November 2020

Specific features and assembly of the plant mitochondrial complex I revealed by cryo-EM.

Nat Commun 2020 10 15;11(1):5195. Epub 2020 Oct 15.

Institut Européen de Chimie et Biologie, U1212 Inserm, Université de Bordeaux, 2 rue R. Escarpit, F-33600, Pessac, France.

Mitochondria are the powerhouses of eukaryotic cells and the site of essential metabolic reactions. Complex I or NADH:ubiquinone oxidoreductase is the main entry site for electrons into the mitochondrial respiratory chain and constitutes the largest of the respiratory complexes. Its structure and composition vary across eukaryote species. However, high resolution structures are available only for one group of eukaryotes, opisthokonts. In plants, only biochemical studies were carried out, already hinting at the peculiar composition of complex I in the green lineage. Here, we report several cryo-electron microscopy structures of the plant mitochondrial complex I. We describe the structure and composition of the plant respiratory complex I, including the ancestral mitochondrial domain composed of the carbonic anhydrase. We show that the carbonic anhydrase is a heterotrimeric complex with only one conserved active site. This domain is crucial for the overall stability of complex I as well as a peculiar lipid complex composed of cardiolipin and phosphatidylinositols. Moreover, we also describe the structure of one of the plant-specific complex I assembly intermediates, lacking the whole P module, in presence of the maturation factor GLDH. GLDH prevents the binding of the plant specific P1 protein, responsible for the linkage of the P to the P module.
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http://dx.doi.org/10.1038/s41467-020-18814-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567890PMC
October 2020

Characterization of a DCL2-Insensitive Isolate Infecting .

Viruses 2020 10 2;12(10). Epub 2020 Oct 2.

Institut de Biologie de Moléculaire des Plantes, CNRS, Université de Strasbourg, 67000 Strasbourg, France.

(TBSV), the type member of the genus in the family is one of the best studied plant viruses. The TBSV natural and experimental host range covers a wide spectrum of plants including agricultural crops, ornamentals, vegetables and . However, , the well-established model organism in plant biology, genetics and plant-microbe interactions is absent from the list of known TBSV host plant species. Most of our recent knowledge of the virus life cycle has emanated from studies in , a surrogate host for TBSV that lacks crucial plant antiviral mechanisms such as RNA interference (RNAi). Here, we identified and characterized a TBSV isolate able to infect with high efficiency. We demonstrated by confocal and 3D electron microscopy that in TBSV-BS3Ng replicates in association with clustered peroxisomes in which numerous spherules are induced. A dsRNA-centered immunoprecipitation analysis allowed the identification of TBSV-associated host components including DRB2 and DRB4, which perfectly localized to replication sites, and NFD2 that accumulated in larger viral factories in which peroxisomes cluster. By challenging knock-out mutants for key RNAi factors, we showed that TBSV-BS3Ng undergoes a non-canonical RNAi defensive reaction. In fact, unlike other RNA viruses described, no 22nt TBSV-derived small RNA are detected in the absence of DCL4, indicating that this virus is DCL2-insensitive. The new TBSV-BS3Ng pathosystem should provide a valuable new model for dissecting plant-virus interactions in complement to .
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http://dx.doi.org/10.3390/v12101121DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7650723PMC
October 2020

Nocardamine-Dependent Iron Uptake in : Exclusive Involvement of the FoxA Outer Membrane Transporter.

ACS Chem Biol 2020 10 24;15(10):2741-2751. Epub 2020 Sep 24.

CNRS, UMR7242, ESBS, Université de Strasbourg, Bld Sébastien Brant, F-67412 Illkirch, France.

Iron is a key nutrient for almost all living organisms. Paradoxically, it is poorly soluble and consequently poorly bioavailable. Bacteria have thus developed multiple strategies to access this metal. One of the most common consists of the use of siderophores, small compounds that chelate ferric iron with very high affinity. Many bacteria are able to produce their own siderophores or use those produced by other microorganisms (exosiderophores) in a piracy strategy. produces two siderophores, pyoverdine and pyochelin, and is also able to use a large panel of exosiderophores. We investigated the ability of to use nocardamine (NOCA) and ferrioxamine B (DFOB) as exosiderophores under iron-limited planktonic growth conditions. Proteomic and RT-qPCR approaches showed induction of the transcription and expression of the outer membrane transporter FoxA in the presence of NOCA or DFOB in the bacterial environment. Expression of the proteins of the heme- or pyoverdine- and pyochelin-dependent iron uptake pathways was not affected by the presence of these two tris-hydroxamate siderophores. Fe uptake assays using mutants showed ferri-NOCA to be exclusively transported by FoxA, whereas ferri-DFOB was transported by FoxA and at least one other unidentified transporter. The crystal structure of FoxA complexed with NOCA-Fe revealed very similar siderophore binding sites between NOCA-Fe and DFOB-Fe. We discuss iron uptake by hydroxamate exosiderophores in cells in light of these results.
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http://dx.doi.org/10.1021/acschembio.0c00535DOI Listing
October 2020

Selenoprotein N is an endoplasmic reticulum calcium sensor that links luminal calcium levels to a redox activity.

Proc Natl Acad Sci U S A 2020 09 17;117(35):21288-21298. Epub 2020 Aug 17.

Istituto di Ricerche Farmacologiche Mario Negri IRCCS, 20156 Milan, Italy;

The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain. In vitro and in vivo experiments show that via this domain, SEPN1 responds to diminished luminal calcium levels, dynamically changing its oligomeric state and enhancing its redox-dependent interaction with cellular partners, including the ER calcium pump sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Importantly, single amino acid substitutions in the EF-hand domain of SEPN1 identified as clinical variations are shown to impair its calcium-binding and calcium-dependent structural changes, suggesting a key role of the EF-hand domain in SEPN1 function. In conclusion, SEPN1 is a ER calcium sensor that responds to luminal calcium depletion, changing its oligomeric state and acting as a reductase to refill ER calcium stores.
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http://dx.doi.org/10.1073/pnas.2003847117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474598PMC
September 2020

Proteasome subunit PSMC3 variants cause neurosensory syndrome combining deafness and cataract due to proteotoxic stress.

EMBO Mol Med 2020 07 5;12(7):e11861. Epub 2020 Jun 5.

Laboratoire de Génétique Médicale, INSERM, UMRS_1112, Institut de Génétique Médicale d'Alsace (IGMA), Université de Strasbourg, Faculté de médecine de Strasbourg, Strasbourg, France.

The ubiquitin-proteasome system degrades ubiquitin-modified proteins to maintain protein homeostasis and to control signalling. Whole-genome sequencing of patients with severe deafness and early-onset cataracts as part of a neurological, sensorial and cutaneous novel syndrome identified a unique deep intronic homozygous variant in the PSMC3 gene, encoding the proteasome ATPase subunit Rpt5, which lead to the transcription of a cryptic exon. The proteasome content and activity in patient's fibroblasts was however unaffected. Nevertheless, patient's cells exhibited impaired protein homeostasis characterized by accumulation of ubiquitinated proteins suggesting severe proteotoxic stress. Indeed, the TCF11/Nrf1 transcriptional pathway allowing proteasome recovery after proteasome inhibition is permanently activated in the patient's fibroblasts. Upon chemical proteasome inhibition, this pathway was however impaired in patient's cells, which were unable to compensate for proteotoxic stress although a higher proteasome content and activity. Zebrafish modelling for knockout in PSMC3 remarkably reproduced the human phenotype with inner ear development anomalies as well as cataracts, suggesting that Rpt5 plays a major role in inner ear, lens and central nervous system development.
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http://dx.doi.org/10.15252/emmm.201911861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7338805PMC
July 2020

SERRATE interacts with the nuclear exosome targeting (NEXT) complex to degrade primary miRNA precursors in Arabidopsis.

Nucleic Acids Res 2020 07;48(12):6839-6854

Department of Gene Expression, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Uniwersytetu Poznanskiego 6, 61-614 Poznan, Poland.

SERRATE/ARS2 is a conserved RNA effector protein involved in transcription, processing and export of different types of RNAs. In Arabidopsis, the best-studied function of SERRATE (SE) is to promote miRNA processing. Here, we report that SE interacts with the nuclear exosome targeting (NEXT) complex, comprising the RNA helicase HEN2, the RNA binding protein RBM7 and one of the two zinc-knuckle proteins ZCCHC8A/ZCCHC8B. The identification of common targets of SE and HEN2 by RNA-seq supports the idea that SE cooperates with NEXT for RNA surveillance by the nuclear exosome. Among the RNA targets accumulating in absence of SE or NEXT are miRNA precursors. Loss of NEXT components results in the accumulation of pri-miRNAs without affecting levels of miRNAs, indicating that NEXT is, unlike SE, not required for miRNA processing. As compared to se-2, se-2 hen2-2 double mutants showed increased accumulation of pri-miRNAs, but partially restored levels of mature miRNAs and attenuated developmental defects. We propose that the slow degradation of pri-miRNAs caused by loss of HEN2 compensates for the poor miRNA processing efficiency in se-2 mutants, and that SE regulates miRNA biogenesis through its double contribution in promoting miRNA processing but also pri-miRNA degradation through the recruitment of the NEXT complex.
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http://dx.doi.org/10.1093/nar/gkaa373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7337926PMC
July 2020

Structural Insights into the Mammalian Late-Stage Initiation Complexes.

Cell Rep 2020 04;31(1):107497

INSERM U1212 Acides nucléiques: Régulations Naturelle et Artificielle (ARNA), Institut Européen de Chimie et Biologie, Université de Bordeaux, Pessac 33607, France. Electronic address:

In higher eukaryotes, the mRNA sequence in the direct vicinity of the start codon, called the Kozak sequence (CRCCaugG, where R is a purine), is known to influence the rate of the initiation process. However, the molecular basis underlying its role remains poorly understood. Here, we present the cryoelectron microscopy (cryo-EM) structures of mammalian late-stage 48S initiation complexes (LS48S ICs) in the presence of two different native mRNA sequences, β-globin and histone 4, at overall resolution of 3 and 3.5 Å, respectively. Our high-resolution structures unravel key interactions from the mRNA to eukaryotic initiation factors (eIFs): 1A, 2, 3, 18S rRNA, and several 40S ribosomal proteins. In addition, we are able to study the structural role of ABCE1 in the formation of native 48S ICs. Our results reveal a comprehensive map of ribosome/eIF-mRNA and ribosome/eIF-tRNA interactions and suggest the impact of mRNA sequence on the structure of the LS48S IC.
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http://dx.doi.org/10.1016/j.celrep.2020.03.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7166083PMC
April 2020

YBEY is an essential biogenesis factor for mitochondrial ribosomes.

Nucleic Acids Res 2020 09;48(17):9762-9786

UMR7156 - Molecular Genetics, Genomics, Microbiology, University of Strasbourg, CNRS, Strasbourg F-67000, France.

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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http://dx.doi.org/10.1093/nar/gkaa148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515705PMC
September 2020

Phenotypic Adaption of by Hacking Siderophores Produced by Other Microorganisms.

Mol Cell Proteomics 2020 04 5;19(4):589-607. Epub 2020 Feb 5.

Université de Strasbourg, UMR7242, ESBS, Bld Sébastien Brant, F-67413 Illkirch, Strasbourg, France; CNRS, UMR7242, ESBS, Bld Sébastien Brant, F-67413 Illkirch, Strasbourg, France. Electronic address:

Bacteria secrete siderophores to access iron, a key nutrient poorly bioavailable and the source of strong competition between microorganisms in most biotopes. Many bacteria also use siderophores produced by other microorganisms (exosiderophores) in a piracy strategy. , an opportunistic pathogen, produces two siderophores, pyoverdine and pyochelin, and is also able to use a panel of exosiderophores. We first investigated expression of the various iron-uptake pathways of in three different growth media using proteomic and RT-qPCR approaches and observed three different phenotypic patterns, indicating complex phenotypic plasticity in the expression of the various iron-uptake pathways. We then investigated the phenotypic plasticity of iron-uptake pathway expression in the presence of various exosiderophores (present individually or as a mixture) under planktonic growth conditions, as well as in an epithelial cell infection assay. In all growth conditions tested, catechol-type exosiderophores were clearly more efficient in inducing the expression of their corresponding transporters than the others, showing that bacteria opt for the use of catechol siderophores to access iron when they are present in the environment. In parallel, expression of the proteins of the pyochelin pathway was significantly repressed under most conditions tested, as well as that of proteins of the pyoverdine pathway, but to a lesser extent. There was no effect on the expression of the heme and ferrous uptake pathways. Overall, these data provide precise insights on how adjusts the expression of its various iron-uptake pathways (phenotypic plasticity and switching) to match varying levels of iron and competition.
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http://dx.doi.org/10.1074/mcp.RA119.001829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7124469PMC
April 2020

Grad-cryo-EM: Tool to Isolate Translation Initiation Complexes from Rabbit Reticulocyte Lysate Suitable for Structural Studies.

Methods Mol Biol 2020 ;2113:329-339

Architecture et Réactivité de l'ARN - CNRS UPR 9002, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Strasbourg, France.

Since its development, single-particle cryogenic electron microscopy (cryo-EM) has played a central role in the study at medium resolution of both bacterial and eukaryotic ribosomal complexes. With the advent of the direct electron detectors and new processing software which allow obtaining structures at atomic resolution, formerly obtained only by X-ray crystallography, cryo-EM has become the method of choice for the structural analysis of the translation machinery. In most of the cases, the ribosomal complexes at different stages of the translation process are assembled in vitro from purified components, which limit the analysis to previously well-characterized complexes with known factors composition. The initiation phase of the protein synthesis is a very dynamic process during which several proteins interact with the translation apparatus leading to the formation of a chronological series of initiation complexes (ICs). Here we describe a method to isolate ICs assembled on natural in vitro transcribed mRNA directly from rabbit reticulocyte lysate (RRL) by sucrose density gradient centrifugation . The Grad-cryo-EM approach allows investigating structures and composition of intermediate ribosomal complexes prepared in near-native condition by cryo-EM and mass spectrometry analyses. This is a powerful approach, which could be used to study translation initiation of any mRNAs, including IRES containing ones, and which could be adapted to different cell extracts.
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http://dx.doi.org/10.1007/978-1-0716-0278-2_21DOI Listing
January 2021

Proteomic Analysis of DNA Synthesis on a Structured DNA Template in Human Cellular Extracts: Interplay Between NHEJ and Replication-Associated Proteins.

Proteomics 2020 02 13;20(3-4):e1900184. Epub 2020 Feb 13.

Biotechnologie et Signalisation Cellulaire, Université de Strasbourg, UMR7242, CNRS, Illkirch, 67412, France.

It is established that short inverted repeats trigger base substitution mutagenesis in human cells. However, how the replication machinery deals with structured DNA is unknown. It has been previously reported that in human cell-free extracts, DNA primer extension using a structured single-stranded template is transiently blocked at DNA hairpins. Here, the proteomic analysis of proteins bound to the DNA template is reported and evidence that the DNA-PK complex (DNA-PKcs and the Ku heterodimer) recognizes, and is activated by, structured single-stranded DNA is provided. Hijacking the DNA-PK complex by double-stranded oligonucleotides results in a large removal of the pausing sites and an elevated DNA extension efficiency. Conversely, DNA-PKcs inhibition results in its stabilization on the template, along with other proteins acting downstream in the Non-Homologous End-Joining (NHEJ) pathway, especially the XRCC4-DNA ligase 4 complex and the cofactor PAXX. Retention of NHEJ factors to the DNA in the absence of DNA-PKcs activity correlates with additional halts of primer extension, suggesting that these proteins hinder the progression of the DNA synthesis at these sites. Overall these results raise the possibility that, upon binding to hairpins formed onto ssDNA during fork progression, the DNA-PK complex interferes with replication fork dynamics in vivo.
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http://dx.doi.org/10.1002/pmic.201900184DOI Listing
February 2020

RST1 and RIPR connect the cytosolic RNA exosome to the Ski complex in Arabidopsis.

Nat Commun 2019 08 27;10(1):3871. Epub 2019 Aug 27.

Institut de biologie moléculaire des plantes, CNRS, Université de Strasbourg, Strasbourg, France.

The RNA exosome is a key 3'-5' exoribonuclease with an evolutionarily conserved structure and function. Its cytosolic functions require the co-factors SKI7 and the Ski complex. Here we demonstrate by co-purification experiments that the ARM-repeat protein RESURRECTION1 (RST1) and RST1 INTERACTING PROTEIN (RIPR) connect the cytosolic Arabidopsis RNA exosome to the Ski complex. rst1 and ripr mutants accumulate RNA quality control siRNAs (rqc-siRNAs) produced by the post-transcriptional gene silencing (PTGS) machinery when mRNA degradation is compromised. The small RNA populations observed in rst1 and ripr mutants are also detected in mutants lacking the RRP45B/CER7 core exosome subunit. Thus, molecular and genetic evidence supports a physical and functional link between RST1, RIPR and the RNA exosome. Our data reveal the existence of additional cytosolic exosome co-factors besides the known Ski subunits. RST1 is not restricted to plants, as homologues with a similar domain architecture but unknown function exist in animals, including humans.
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http://dx.doi.org/10.1038/s41467-019-11807-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6711988PMC
August 2019

Determination of protein-only RNase P interactome in Arabidopsis mitochondria and chloroplasts identifies a complex between PRORP1 and another NYN domain nuclease.

Plant J 2019 11 21;100(3):549-561. Epub 2019 Aug 21.

Institut de biologie moléculaire des plantes, CNRS, Université de Strasbourg, Strasbourg, France.

The essential type of endonuclease 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', is composed of protein that occurs only in a wide variety of eukaryotes, in organelles and in the nucleus. Here, to find how PRORP functions integrate with other cell processes, we explored the protein interaction network of PRORP1 in Arabidopsis mitochondria and chloroplasts. Although PRORP proteins function as single subunit enzymes in vitro, we found that PRORP1 occurs in protein complexes and is present in high-molecular-weight fractions that contain mitochondrial ribosomes. The analysis of immunoprecipitated protein complexes identified proteins involved in organellar gene expression processes. In particular, direct interaction was established between PRORP1 and MNU2 a mitochondrial nuclease. A specific domain of MNU2 and a conserved signature of PRORP1 were found to be directly accountable for this protein interaction. Altogether, results revealed the existence of an RNA maturation complex in Arabidopsis mitochondria and suggested that PRORP proteins cooperated with other gene expression factors for RNA maturation in vivo.
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http://dx.doi.org/10.1111/tpj.14458DOI Listing
November 2019

tRNA Maturation Defects Lead to Inhibition of rRNA Processing via Synthesis of pppGpp.

Mol Cell 2019 06 16;74(6):1227-1238.e3. Epub 2019 Apr 16.

UMR8261 (CNRS-Université Paris Diderot), Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France. Electronic address:

rRNAs and tRNAs universally require processing from longer primary transcripts to become functional for translation. Here, we describe an unsuspected link between tRNA maturation and the 3' processing of 16S rRNA, a key step in preparing the small ribosomal subunit for interaction with the Shine-Dalgarno sequence in prokaryotic translation initiation. We show that an accumulation of either 5' or 3' immature tRNAs triggers RelA-dependent production of the stringent response alarmone (p)ppGpp in the Gram-positive model organism Bacillus subtilis. The accumulation of (p)ppGpp and accompanying decrease in GTP levels specifically inhibit 16S rRNA 3' maturation. We suggest that cells can exploit this mechanism to sense potential slowdowns in tRNA maturation and adjust rRNA processing accordingly to maintain the appropriate functional balance between these two major components of the translation apparatus.
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http://dx.doi.org/10.1016/j.molcel.2019.03.030DOI Listing
June 2019

Small is big in Arabidopsis mitochondrial ribosome.

Nat Plants 2019 01 9;5(1):106-117. Epub 2019 Jan 9.

Institut de biologie de moléculaire des plantes UPR2357 du CNRS, Université de Strasbourg, Strasbourg, France.

Mitochondria are responsible for energy production through aerobic respiration, and represent the powerhouse of eukaryotic cells. Their metabolism and gene expression processes combine bacterial-like features and traits that evolved in eukaryotes. Among mitochondrial gene expression processes, translation remains the most elusive. In plants, while numerous pentatricopeptide repeat (PPR) proteins are involved in all steps of gene expression, their function in mitochondrial translation remains unclear. Here we present the biochemical characterization of Arabidopsis mitochondrial ribosomes and identify their protein subunit composition. Complementary biochemical approaches identified 19 plant-specific mitoribosome proteins, of which ten are PPR proteins. The knockout mutations of ribosomal PPR (rPPR) genes result in distinct macroscopic phenotypes, including lethality and severe growth delay. The molecular analysis of rppr1 mutants using ribosome profiling, as well as the analysis of mitochondrial protein levels, demonstrate rPPR1 to be a generic translation factor that is a novel function for PPR proteins. Finally, single-particle cryo-electron microscopy (cryo-EM) reveals the unique structural architecture of Arabidopsis mitoribosomes, characterized by a very large small ribosomal subunit, larger than the large subunit, bearing an additional RNA domain grafted onto the head. Overall, our results show that Arabidopsis mitoribosomes are substantially divergent from bacterial and other eukaryote mitoribosomes, in terms of both structure and protein content.
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http://dx.doi.org/10.1038/s41477-018-0339-yDOI Listing
January 2019

Blocking nuclear export of HSPA8 after heat shock stress severely alters cell survival.

Sci Rep 2018 11 14;8(1):16820. Epub 2018 Nov 14.

CNRS-University of Strasbourg, Biotechnology and cell signaling, Illkirch, France/Laboratory of excellence Medalis, Strasbourg, France.

The nuclear translocation of endogenous heat shock cognate protein HSPA8 is a requisite for cell survival during oxidative and heat shock stress. Upon these events, cytoplasmic HSPA8 is thought to concentrate within the nucleus and nucleolus. When the situation returns to normal, HSPA8 is released from its nuclear/nucleolar anchors and redistributes into the cytoplasm. By using different stress conditions and a 21-mer phosphopeptide tool called P140, which binds HSPA8 and hampers its chaperone properties, we deciphered the cellular and molecular effects arising during this vital cytoplasmic-nuclear-cytoplasmic shuttling process. Using the non-metastatic fibroblastoid cell line MRL/N-1 derived from a MRL/MpTn-gld/gld lupus-prone mouse, we discovered that P140 treatment neutralized the egress of HSPA8 from nucleus to cytoplasm in the cell recovery phase. This lack of relocation of HSPA8 into the cytoplasm of heat-shocked MRL/N-1 cells altered the ability of these cells to survive when a second mild oxidative stress mimicking inflammatory conditions was applied. Crosslinking experiments followed by proteomics studies showed that P140 binds regions close to nuclear import and export signal sequences encompassed within the HSPA8 structure. These data are consistent with HSPA8 having a crucial cell protective role against reactive oxygen species (ROS) production by mitochondria during inflammatory conditions.
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http://dx.doi.org/10.1038/s41598-018-34887-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235846PMC
November 2018

Control of dengue virus in the midgut of Aedes aegypti by ectopic expression of the dsRNA-binding protein Loqs2.

Nat Microbiol 2018 12 29;3(12):1385-1393. Epub 2018 Oct 29.

Department of Biochemistry and Immunology, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte/MG, Brazil.

Dengue virus (DENV) is an arbovirus transmitted to humans by Aedes mosquitoes. In the insect vector, the small interfering RNA (siRNA) pathway is an important antiviral mechanism against DENV. However, it remains unclear when and where the siRNA pathway acts during the virus cycle. Here, we show that the siRNA pathway fails to efficiently silence DENV in the midgut of Aedes aegypti although it is essential to restrict systemic replication. Accumulation of DENV-derived siRNAs in the midgut reveals that impaired silencing results from a defect downstream of small RNA biogenesis. Notably, silencing triggered by endogenous and exogenous dsRNAs remained effective in the midgut where known components of the siRNA pathway, including the double-stranded RNA (dsRNA)-binding proteins Loquacious and r2d2, had normal expression levels. We identified an Aedes-specific paralogue of loquacious and r2d2, hereafter named loqs2, which is not expressed in the midgut. Loqs2 interacts with Loquacious and r2d2 and is required to control systemic replication of DENV and also Zika virus. Furthermore, ectopic expression of Loqs2 in the midgut of transgenic mosquitoes is sufficient to restrict DENV replication and dissemination. Together, our data reveal a mechanism of tissue-specific regulation of the mosquito siRNA pathway controlled by Loqs2.
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http://dx.doi.org/10.1038/s41564-018-0268-6DOI Listing
December 2018

ATG5 is required for B cell polarization and presentation of particulate antigens.

Autophagy 2019 02 22;15(2):280-294. Epub 2018 Sep 22.

a CNRS, Immunology, Immunopathology and Therapeutic Chemistry , Institut de Biologie Moléculaire et Cellulaire/University of Strasbourg , Strasbourg , France.

The involvement of macroautophagy/autophagy proteins in B-cell receptor (BCR) trafficking, although suspected, is not well understood. We show that ATG5 (autophagy related 5) contributes to BCR polarization after stimulation and internalization into LAMP1 (lysosomal-associated membrane protein 1) and major histocompatibility complex class II (MHC-II) compartments. BCR polarization is crucial in the context of immobilized antigen processing. Moreover, antigen presentation to cognate T cells is decreased in the absence of ATG5 when the model antigen OVAL/ovalbumin is provided in an immobilized form in contrast to the normal presentation of soluble OVAL. We further show that ATG5 is required for centrosome polarization and actin nucleation in the immune synapse area. This event is accompanied by an increased interaction between ATG16L1 (autophagy related 16-like 1 []) and the microtubule-organizing center-associated protein PCM1 (pericentriolar material 1). In the human B cell line BJAB, PCM1 is required for BCR polarization after stimulation. We thus propose that the ATG12 (autophagy related 12)-ATG5-ATG16L1 complex under BCR stimulation allows its interaction with PCM1 and consequently facilitates centrosome relocalization to the immune synapse, optimizing the presentation of particulate antigens. ACTB: actin beta; ACTR2/3: ARP2/3 actin-related protein 2/3; APC: antigen-presenting cells; ATG: autophagy-related; BCR: B cell receptor; BECN1/Beclin 1: beclin 1, autophagy related; CDC42: cell division cycle 42; Cr2: complement receptor 2; CSFE: carboxyfluorescein succinimidyl ester; DAPI: 4',6-diamidino-2-phenylindole dihydrochloride; EEA1: early endosome antigen 1; ELISA: enzyme-linked immunosorbent assay; FITC: fluorescein isothyocyanate; GC: germinal center; GJA1/CX3: gap junction protein, alpha 1; Ig: immunoglobulin; LAMP1: lysosomal-associated membrane protein 1; LAP: LC3-associated phagocytosis; LM: littermate; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/ERK: mitogen activated protein kinase; MHC-II: major histocompatibility complex class II; MIIC: MHC class II compartment; OVAL: ovalbumin; PBS: phosphate-buffered saline; PCM1: pericentriolar material 1; PtdIns3K: phosphatidylinositol 3-kinase; PTPRC/CD45RB/B220; Protein tyrosine phosphatase, receptor type, C; SYK: spleen tyrosine kinase; TBS: Tris-buffered saline; TCR: T cell receptor; ULK1: unc-51 like kinase 1.
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http://dx.doi.org/10.1080/15548627.2018.1516327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333460PMC
February 2019

The Kinase IKKβ Regulates a STING- and NF-κB-Dependent Antiviral Response Pathway in Drosophila.

Immunity 2018 08 14;49(2):225-234.e4. Epub 2018 Aug 14.

Université de Strasbourg, CNRS, Insect Models of Innate Immunity (M3I; UPR9022), 67084 Strasbourg, France; Sino-French Hoffmann Institute, School of Basic Medical Science, Guangzhou Medical University, Guangzhou 511436, China. Electronic address:

Antiviral immunity in Drosophila involves RNA interference and poorly characterized inducible responses. Here, we showed that two components of the IMD pathway, the kinase dIKKβ and the transcription factor Relish, were required to control infection by two picorna-like viruses. We identified a set of genes induced by viral infection and regulated by dIKKβ and Relish, which included an ortholog of STING. We showed that dSTING participated in the control of infection by picorna-like viruses, acting upstream of dIKKβ to regulate expression of Nazo, an antiviral factor. Our data reveal an antiviral function for STING in an animal model devoid of interferons and suggest an evolutionarily ancient role for this molecule in antiviral immunity.
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http://dx.doi.org/10.1016/j.immuni.2018.07.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6267954PMC
August 2018

Toxicological effects of CdSe nanocrystals on the marine diatom Phaeodactylum tricornutum: The first mass spectrometry-based proteomic approach.

Ecotoxicol Environ Saf 2018 May 4;152:78-90. Epub 2018 Feb 4.

Institut National des Sciences et Techniques de la Mer, Conservatoire National des Arts et Métiers, 50103 Cherbourg en Cotentin Cedex, France; Laboratoire Universitaire des Sciences Appliquées de Cherbourg, EA4253, Normandie Université, UNICAEN, 50130 Cherbourg en Cotentin, France. Electronic address:

In the marine environment, benthic diatoms from estuarine and coastal sediments are among the first targets of nanoparticle pollution whose potential toxicity on marine organisms is still largely unknown. It is therefore relevant to improve our knowledge of interactions between these new pollutants and microalgae, the key players in the control of marine resources. In this study, the response of P. tricornutum to CdSe nanocrystals (CdSe NPs) of 5 nm (NP5) and 12 nm (NP12) in diameter was evaluated through microscopic, physiological, biochemical and proteomic approaches. NP5 and NP12 affected cell growth but oxygen production was only slightly decreased by NP5 after 1-d incubation time. In our experimental conditions, a high CdSe NP dissolution was observed during the first day of culture, leading to Cd bioaccumulation and oxidative stress, particularly with NP12. However, after a 7-day incubation time, proteomic analysis highlighted that P. tricornutum responded to CdSe NP toxicity by regulating numerous proteins involved in protection against oxidative stress, cellular redox homeostasis, Ca regulation and signalling, S-nitrosylation and S-glutathionylation processes and cell damage repair. These proteome changes allowed algae cells to regulate their intracellular ROS level in contaminated cultures. P. tricornutum was also capable to control its intracellular Cd concentration at a sufficiently low level to preserve its growth. To our knowledge, this is the first work allowing the identification of proteins differentially expressed by P. tricornutum subjected to NPs and thus the understanding of some molecular pathways involved in its cellular response to nanoparticles.

Significance: The microalgae play a key role in the control of marine resources. Moreover, they produce 50% of the atmospheric oxygen. CdSe NPs are extensively used in the industry of renewable energies and it is regrettably expected that these pollutants will sometime soon appear in the marine environment through surface runoff, urban effluents and rivers. Since estuarine and coastal sediments concentrate pollutants, benthic microalgae which live in superficial sediments will be among the first targets of nanoparticle pollution. Thus, it is relevant to improve our knowledge of interactions between diatoms and nanoparticles. Proteomics is a powerful tool for understanding the molecular mechanisms triggered by nanoparticle exposure, and our study is the first one to use this tool to identify proteins differentially expressed by P. tricornutum subjected to CdSe nanocrystals. This work is fundamental to improve our knowledge about the defence mechanisms developed by algae cells to counteract damage caused by CdSe NPs.
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http://dx.doi.org/10.1016/j.ecoenv.2018.01.043DOI Listing
May 2018

The IRES5'UTR of the dicistrovirus cricket paralysis virus is a type III IRES containing an essential pseudoknot structure.

Nucleic Acids Res 2017 Sep;45(15):8993-9004

Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR 9002, F-67000 Strasbourg, France.

Cricket paralysis virus (CrPV) is a dicistrovirus. Its positive-sense single-stranded RNA genome contains two internal ribosomal entry sites (IRESs). The 5' untranslated region (5'UTR) IRES5'UTR mediates translation of non-structural proteins encoded by ORF1 whereas the well-known intergenic region (IGR) IRESIGR is required for translation of structural proteins from open reading frame 2 in the late phase of infection. Concerted action of both IRES is essential for host translation shut-off and viral translation. IRESIGR has been extensively studied, in contrast the IRES5'UTR remains largely unexplored. Here, we define the minimal IRES element required for efficient translation initiation in drosophila S2 cell-free extracts. We show that IRES5'UTR promotes direct recruitment of the ribosome on the cognate viral AUG start codon without any scanning step, using a Hepatitis-C virus-related translation initiation mechanism. Mass spectrometry analysis revealed that IRES5'UTR recruits eukaryotic initiation factor 3, confirming that it belongs to type III class of IRES elements. Using Selective 2'-hydroxyl acylation analyzed by primer extension and DMS probing, we established a secondary structure model of 5'UTR and of the minimal IRES5'UTR. The IRES5'UTR contains a pseudoknot structure that is essential for proper folding and ribosome recruitment. Overall, our results pave the way for studies addressing the synergy and interplay between the two IRES from CrPV.
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http://dx.doi.org/10.1093/nar/gkx622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5587806PMC
September 2017

Definition of a RACK1 Interaction Network in Using SWATH-MS.

G3 (Bethesda) 2017 07 5;7(7):2249-2258. Epub 2017 Jul 5.

RIDI UPR 9022, Université de Strasbourg, Centre National de la Recherche Scientifique, F-67000, France

Receptor for Activated protein C kinase 1 (RACK1) is a scaffold protein that has been found in association with several signaling complexes, and with the 40S subunit of the ribosome. Using the model organism , we recently showed that RACK1 is required at the ribosome for internal ribosome entry site (IRES)-mediated translation of viruses. Here, we report a proteomic characterization of the interactome of RACK1 in S2 cells. We carried out Label-Free quantitation using both Data-Dependent and Data-Independent Acquisition (DDA and DIA, respectively) and observed a significant advantage for the Sequential Window Acquisition of all THeoretical fragment-ion spectra (SWATH) method, both in terms of identification of interactants and quantification of low abundance proteins. These data represent the first SWATH spectral library available for and will be a useful resource for the community. A total of 52 interacting proteins were identified, including several molecules involved in translation such as structural components of the ribosome, factors regulating translation initiation or elongation, and RNA binding proteins. Among these 52 proteins, 15 were identified as partners by the SWATH strategy only. Interestingly, these 15 proteins are significantly enriched for the functions translation and nucleic acid binding. This enrichment reflects the engagement of RACK1 at the ribosome and highlights the added value of SWATH analysis. A functional screen did not reveal any protein sharing the interesting properties of RACK1, which is required for IRES-dependent translation and not essential for cell viability. Intriguingly however, 10 of the RACK1 partners identified restrict replication of Cricket paralysis virus (CrPV), an IRES-containing virus.
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http://dx.doi.org/10.1534/g3.117.042564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5499132PMC
July 2017

Two proteomic methodologies for defining N-termini of mature human mitochondrial aminoacyl-tRNA synthetases.

Methods 2017 01 26;113:111-119. Epub 2016 Oct 26.

Université de Strasbourg, CNRS, Architecture et Réactivité de l'ARN, UPR 9002, F-67000 Strasbourg, France. Electronic address:

Human mitochondrial aminoacyl-tRNA synthetases (mt-aaRSs) are encoded in the nucleus, synthesized in the cytosol and targeted for importation into mitochondria by a N-terminal mitochondrial targeting sequence. This targeting sequence is presumably cleaved upon entry into the mitochondria, following a process still not fully deciphered in human, despite essential roles for the mitochondrial biogenesis. Maturation processes are indeed essential both for the release of a functional enzyme and to route correctly the protein within mitochondria. The absence of consensus sequences for cleavage sites and the discovery of possible multiple proteolytic steps render predictions of N-termini difficult. Further, the knowledge of the cleavages is key for the design of protein constructions compatible with efficient production in bacterial strains. Finally, full comprehension becomes essential because a growing number of mutations are found in genes coding for mt-aaRS. In the present study, we take advantage of proteomic methodological developments and identified, in mitochondria, three N-termini for the human mitochondrial aspartyl-tRNA synthetase. This first description of the co-existence of different forms opens new perspectives in the biological understanding of this enzyme. Those methods are extended to the whole set of human mt-aaRSs and methodological advice are provided for further investigations.
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http://dx.doi.org/10.1016/j.ymeth.2016.10.012DOI Listing
January 2017