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Characterization of non-coding RNAs and regulatory networks involved in the pathogenicity of yeasts of the Candida genus.

Research unit

UPR 9002 - Architecture et réactivité de l'ARN (IBMC)
15, rue Rene Descartes 67084 - Strasbourg Cedex

Group

Name: Evolution des ARN non-codants chez les levures.

Group leader: JOSSINET Fabrice - f.jossinet@ibmc-cnrs.unistra.fr

Group leader's phone: 03 88 41 70 53

Website: Visit website

Group organization:
- Chercheurs: 3
- ITA: 1
- Doctorants: 0
- Post-Docs: 0
- Autres: 0

Publications of the team linked to the topic (3 last years):
1) Lehmann J, Jossinet F & Gautheret D. A universal RNA structural motif docking the elbow of tRNA in the ribosome, RNAse P and T-box leaders. Nucleic Acids Res 41: 5494–5502 (2013).
2) Hashem Y, Georges des A, Fu J, Buss SN, Jossinet F, Jobe A, Zhang Q, Liao HY, Grassucci RA, Bajaj C, Westhof E, Madison-Antenucci S & Franck J. High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome. Nature 494: 385–389 (2013).
3) Jossinet F, Ludwig TE & Westhof E. Assemble: an interactive graphical tool to analyze and build RNA architectures at the 2D and 3D levels. Bioinformatics 26, 2057-2059 (2010).

About PhD

PhD Director: JOSSINET Fabrice - f.jossinet@ibmc-cnrs.unistra.fr

Phone: 03 88 41 70 53

Junior advisor: LEH Veronique

Co-tutely: non

Co-Director: non

About PhD topic :

Title: Characterization of non-coding RNAs and regulatory networks involved in the pathogenicity of yeasts of the Candida genus.

Project: Yeasts of the Candida genus are commensal species with human and animals. Some species are opportunistic ones and are an important cause of mortality for immunocompromised people (those that have AIDS, organ transplantation or undergoing chemotherapy or radiation therapy). As for all the eukaryotic organisms studied so far with high-thoughput technologies, almost all the genome of yeast is transcribed into non- protein coding transcripts that do not overlap existing gene annotations («non-coding» RNAs or ncRNAs). Several studies have confirmed the role of such ncRNAs as key factors in the regulation of gene expression.
Consequently, this research project will concentrate on the role of ncRNAs in the pathogenic mechanisms of yeasts of the Candida genus, and more specifically of the species Candida glabrata, the second most common cause of candidiasis worldwide. This project will be hosted in a new team started in 2013, and will be divided into three main parts:
- the identification of ncRNAs candidates (estimated to 6 months/man): this will be done using high-throughput sequencing (RNA-seq). The task of the candidate will be to purify all the transcripts expressed in yeasts using pathogenic (insect infection model) and non- pathogenic (cell culture) conditions. The experimental conditions of infection and of RNA isolation will be available to the candidate at the beginning of the project. He/she will also construct and select mutant strains lacking the ability to degrade RNA (exosome mutants). These strains will allow to isolate and to sequence transient ncRNAs participating to the phenotypic switch during pathogenicity. The experimental conditions allowing an efficient homologous recombination in Candida glabrata are under investigation in the laboratory. Once sequenced and aligned to the genome, the combination of several approaches (differential expression analysis, comparative genomics of putative loci,...) will allow the candidate to suggest a list of ncRNAs potentially involved in the pathogenic mechanisms of Candida glabrata.
-the experimental validation of ncRNAs (estimated to 12 months/man): using the homologous recombination developed in the first part of the project, the candidate will have to construct and select a mutant strain of Candida glabrata for each ncRNA identified. Then he/she will study its pathogenic fitness in the insect infection model. The exhibition of a strong mutant phenotype for virulence could lead to the need to weaken more the underlying regulatory networks. Consequently, the candidate will evaluate the efficiency of a genome-wide approach based on a random insertional mutagenesis using the Tn7 transposon («signature tagged mutagenesis» or STM).
- the experimental characterization of the regulatory networks (estimated to 12 months/ man): the candidate will link each validated ncRNA to aptamer sequences, naturally recognised by proteins like the MS2 coat protein or streptavidin. Once the constructions inserted in the genome of Candida glabrata, they will allow the isolation of the ribonucleoprotein complexes (RNPs) involving the validated ncRNAs. The protein partners will be sequenced using mass spectrometry and they will be used for the production of antibodies. Using these antibodies, a new round of isolation of RNPs will be realized with the goal of identifying new coding and non-coding RNA partners («CLIP-Seq» approach). After several iterations, the RNA and protein components isolated will allow the candidate to infer their underlying regulatory networks.

Wished skills: - theoretical and practical knowledge of yeast genetics and molecular biology
- practical experience of microbiological methods

- without to be a strong prerequisites, an ability to use bioinformatics tools will be appreciated

Expertises which will be acquired during the training: - Basic domains : structural and functional evolution of ncRNAs, regulatory networks and
pathogenic mechanisms

-Methodological domains : Yeast biology and genetic engineering, isolation of ribonucleoprotein complexes, bioinformatics analysis of biological sequences and high-throughput data