The tRNA-like domains of E coli and A.aeolicus transfer-messenger RNA: structural and functional studies.

International audienceTransfer-messenger RNA (tmRNA, 10Sa RNA or ssrA) acts to rescue stalled bacterial ribosomes while encoding a peptide tag added trans-translationally to the nascent peptide, targeting it for proteolysis. The understanding at molecular level of this ubiquitous quality control system in eubacteria requires structural information. Here, we describe the purification and structural analysis of a functional fragment of both Aquifex aeolicus and Escherichia coli tmRNA, recapitulating their tRNA-like domain, which were expressed in vivo from synthetic genes. Both recombinant RNA are correctly processed at both 5' and 3' ends and are produced in quantities suitable for structural analysis by NMR and/or X-ray crystallography. The sequence and solution structure of the tRNA-like domains were analysed by various methods including structural mapping with chemical and enzymatic probes and 2D NMR spectroscopy. The minimalist RNAs contain two post-transcriptional base modifications, 5-methyluridine and pseudouridine, as the full-length tmRNA. Both RNAs fold into three stems, a D-analogue, a T-loop and a GAAA tetra-loop. 2D NMR analysis of the imino proton resonances of both RNAs allowed the assignment of the three stems and of a number of tertiary interactions. It shows the existence of interactions between the TPsiC-loop and the D-analogue, exhibiting a number of similarities and also differences with the canonical tRNA fold, indicating that RNA tertiary interactions can be modulated according to the sequence and secondary structure contexts. Furthermore, the E.coli minimalist RNA is aminoacylatable with alanine with a catalytic efficiency an order of magnitude higher than that for full-length tmRNA

Identification of glucocorticoid-induced leucine zipper as a key regulator of tumor cell proliferation in epithelial ovarian cancer

<p>Abstract</p> <p>Background</p> <p>Little is known about the molecules that contribute to tumor progression of epithelial ovarian cancer (EOC), currently a leading cause of mortality from gynecological malignancies. Glucocorticoid-Induced Leucine Zipper (GILZ), an intracellular protein widely expressed in immune tissues, has been reported in epithelial tissues and controls some of key signaling pathways involved in tumorigenesis. However, there has been no report on GILZ in EOC up to now. The objectives of the current study were to examine the expression of GILZ in EOC and its effect on tumor cell proliferation.</p> <p>Results</p> <p>GILZ expression was measured by immunohistochemical staining in tissue sections from 3 normal ovaries, 7 benign EOC and 50 invasive EOC. GILZ was not detected on the surface epithelium of normal ovaries and benign tumors. In contrast, it was expressed in the cytoplasm of tumor cells in 80% EOC specimens. GILZ immunostaining scores correlated positively to the proliferation marker Ki-67 (Spearman test in univariate analysis, <it>P </it>< 0.00001, r = 0.56). They were also higher in tumor cells containing large amounts of phosphorylated protein kinase B (p-AKT) (unpaired t test, <it>P </it>< 0.0001). To assess the effect of GILZ on proliferation and AKT activation, we used the BG-1 cell line derived from ovarian tumor cells as a cellular model. GILZ expression was either enhanced by stable transfection or decreased by the use of small interfering (si) RNA targeting GILZ. We found that GILZ increased cell proliferation, phospho-AKT cellular content and AKT kinase activity. Further, GILZ upregulated cyclin D1 and phosphorylated retinoblastoma (p-Rb), downregulated cyclin-dependent kinase inhibitor p21, and promoted the entry into S phase of cell cycle.</p> <p>Conclusion</p> <p>The present study is the first to identify GILZ as a molecule produced by ovarian cancer cells that promotes cell cycle progression and proliferation. Our findings clearly indicate that GILZ activates AKT, a crucial signaling molecule in tumorigenesis. GILZ thus appears as a potential key molecule in EOC.</p

A smooth tubercle bacillus from Ethiopia phylogenetically close to the Mycobacterium tuberculosis complex

The Mycobacterium tuberculosis complex (MTBC) includes several human- and animal-adapted pathogens. It is thought to have originated in East Africa from a recombinogenic Mycobacterium canettii-like ancestral pool. Here, we describe the discovery of a clinical tuberculosis strain isolated in Ethiopia that shares archetypal phenotypic and genomic features of M. canettii strains, but represents a phylogenetic branch much closer to the MTBC clade than to the M. canettii strains. Analysis of genomic traces of horizontal gene transfer in this isolate and previously identified M. canettii strains indicates a persistent albeit decreased recombinogenic lifestyle near the emergence of the MTBC. Our findings support that the MTBC emergence from its putative free-living M. canettii-like progenitor is evolutionarily very recent, and suggest the existence of a continuum of further extant derivatives from ancestral stages, close to the root of the MTBC, along the Great Rift Valley

Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance : a retrospective cohort study

BACKGROUND : Diagnosing drug-resistance remains an obstacle to the elimination of tuberculosis. Phenotypic drugsusceptibility testing is slow and expensive, and commercial genotypic assays screen only common resistancedetermining mutations. We used whole-genome sequencing to characterise common and rare mutations predicting drug resistance, or consistency with susceptibility, for all fi rst-line and second-line drugs for tuberculosis. METHODS : Between Sept 1, 2010, and Dec 1, 2013, we sequenced a training set of 2099 Mycobacterium tuberculosis genomes. For 23 candidate genes identifi ed from the drug-resistance scientifi c literature, we algorithmically characterised genetic mutations as not conferring resistance (benign), resistance determinants, or uncharacterised. We then assessed the ability of these characterisations to predict phenotypic drug-susceptibility testing for an independent validation set of 1552 genomes. We sought mutations under similar selection pressure to those characterised as resistance determinants outside candidate genes to account for residual phenotypic resistance. FINDINGS : We characterised 120 training-set mutations as resistance determining, and 772 as benign. With these mutations, we could predict 89·2% of the validation-set phenotypes with a mean 92·3% sensitivity (95% CI 90·7–93·7) and 98·4% specifi city (98·1–98·7). 10·8% of validation-set phenotypes could not be predicted because uncharacterised mutations were present. With an in-silico comparison, characterised resistance determinants had higher sensitivity than the mutations from three line-probe assays (85·1% vs 81·6%). No additional resistance determinants were identifi ed among mutations under selection pressure in non-candidate genes. INTERPRETATION : A broad catalogue of genetic mutations enable data from whole-genome sequencing to be used clinically to predict drug resistance, drug susceptibility, or to identify drug phenotypes that cannot yet be genetically predicted. This approach could be integrated into routine diagnostic workfl ows, phasing out phenotypic drugsusceptibility testing while reporting drug resistance early.Wellcome Trust, National Institute of Health Research, Medical Research Council, and the European Union.http://www.thelancet.com/infectionhb201

Nouvelles caractérisations structurales de l'ARN transfert-messager

La "trans-traduction" est un mécanisme bactérien qui permet la libération de ribosomes bloqués en cours de synthèse protéique. Ce mécanisme fait intervenir l'ARN transfert-messager (ARNtm), comportant un domaine ARNt et un cadre ouvert de lecture, et la protéine SmpB. Chez certaines espèces, le gène codant pour l'ARNtm a subi une permutation circulaire dont la transcription aboutit à un ARN précurseur maturé sous la forme de deux ARN distincts. Nous avons caractérisé par cartographie chimique et enzymatique la structure secondaire de cet ARNtm en deux parties. D'autre part, nous avons déterminé, par résonance magnétique nucléaire, le degré de similarité structurale entre le domaine assurant la fonction ARNt de la molécule (TLD) et les ARNt standards. Nous avons montré que ce domaine interagit avec SmpB. Afin d'obtenir des informations supplémentaires sur l'initiation de la trans-traduction, nous avons entrepris de résoudre la structure du complexe TLD/SmpB par RMN.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Générer une symétrie bilatérale à partir de composants asymétriques

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Two-piece tmRNA in cyanobacteria and its structural analysis.

International audiencetmRNA acts to rescue stalled bacterial ribosomes while encoding a peptide tag added trans-translationally to the nascent peptide, targeting it for proteolysis. The permuted gene structure found in a group of cyanobacteria is shown to produce a two-piece mature tmRNA, as had been observed previously for the independently permuted gene of alpha-proteobacteria. The pieces have been mapped onto the gene sequence and aligned for the permuted cyanobacterial tmRNA sequences, including four novel sequences. Structural probing and base pair co-variations support a secondary structure model in which two pairings in the tRNA-like domain hold the two pieces together, and the coding piece bearing the tag reading frame additionally contains a single transient pseudoknot and three other stem-loops. This represents a dramatic reduction in pseudoknot number from the five present in one-piece cyanobacterial tmRNA

Polyamine derivatives as selective RNaseA mimics

International audienceSite-selective scission of ribonucleic acids (RNAs) has attracted considerable interest, since RNA is an intermediate in gene expression and the genetic material of many pathogenic viruses. Polyamine-imidazole conjugates for site-selective RNA scission, without free imidazole, were synthesized and tested on yeast phenylalanine transfer RNA. These molecules catalyze RNA hydrolysis non-randomly. Within the polyamine chain, the location of the imidazole residue, the numbers of nitrogen atoms and their relative distances have notable influence on cleavage selectivity. A norspermine derivative reduces the cleavage sites to a unique location, in the anticodon loop of the tRNA, in the absence of complementary sequence. Experimental results are consistent with a cooperative participation of an ammonium group of the polyamine moiety, in addition to it's binding to the negatively charged ribose-phosphate backbone, as proton source, and the imidazole moiety as a base. There is correlation between the location of the magnesium binding sites and the RNA cleavage sites, suggesting that the protonated nitrogens of the polycationic chain compete with some of the magnesium ions for RNA binding. Therefore, the cleavage pattern is specific of the RNA structure. These compounds cleave at physiological pH, representing novel reactive groups for antisense oligonucleotide derivatives or to enhance ribozyme activity

How Well Do Routine Molecular Diagnostics Detect Rifampin Heteroresistance in Mycobacterium tuberculosis ?

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