RNA silencing is a conserved mechanism of regulation of gene expression mediated by small RNAs (sRNAs). Intracellular double-stranded RNAs (dsRNAs) are recognized and cleaved by RNase III-like ribonuclease enzyme of the Dicer (DCR) family into sRNAs which are incorporated into an ARGONAUTE (AGO)-containing complex to guide the sequence-specific targeting of complementary RNAs. In some organisms, RNA dependant RNA polymerases (RdRPs) synthesis dsRNAs from single-stranded RNAs to initiate silencing and/or amplify the silencing signal. Gene silencing occurs at the transcriptional level via methylation of DNA-cytosine bases by RNA-directed DNA methylation (RdDM) or post transcriptional level (PTGS) either by cleavage or translational inhibition of targeted mRNA. RNA silencing has been shown to play pivotal roles in growth and development, maintenance of genome integrity via repression of transposable elements and transgenes, defense against virus, and response to abiotic stressors (Baulcombe, Nature 2004; Sarkies & Miska, Nat. Rev. Mol. Cell Biol. 2014 ).

Diatoms are unicellular microalgae belonging to the eukaryotic supergroups of Chromalveolata playing a pivotal role in the marine environment and global carbon cycle, and presenting promising biotechnological applications. The feasibility to achieve targeted gene silencing by expression of dsRNAs the model diatom species Phaeodactylum tricornutum has been demonstrated (De Riso et al., NAR 1999). Comprehensive and combinatorial analysis of sRNAs, gene expression and DNA methylation levels in P. tricornutum suggest the presence of an endogenous RNA silencing pathway which may play a role during the acclimatory response to environmental stress conditions (Rogato et al., BMC Genomics 2014; Veluchamy et al., Nat. Comm. 2013)

To date, no diatom DCR, AGO and RdRP homologue has been cloned and direct evidences for their respective contribution and mode of action in RNA silencing as well as for the importance of RNA silencing in diatom physiology is still lacking. The project RADIO aims to characterize the function of DCR, AGO and RdRP and unveil the physiological role of RNA silencing in P. tricornutum.

WP.1: Cloning and subcellular localization of P. tricornutum DCR, AGO and RdRP

Cloning and subcellular localization of P. tricornutum DCR, AGO and RdRP

WP.2: Functional characterization of DCR, AGO and RdRPin RNAi

Assess the importance of DCR, AGO and RdRP in the initiation and maintenance of RNAi.

WP.3: Biochemical characterization of DCR activity

Characterize the apparent substrates specificityand products of recombinant DCL in vitro.

WP.4: Composition and targets of the AGO-complexes

Characterize AGO-complexesboundsRNAs and proteins

WP.5: Physiological role of RNA silencing

Characterize the importance and mechanisms of RNA silencing in regulating endogenous gene and TE expression under optimal growth condition and during the acclimatory response to changes in nitrate availability.

WP.6: Communication and Dissemination

Frédéric Verret – Project leader, IMBBC-HCMR, Greece

Emilia Grypioti – IMBB-FoRTH, Greece

Hugues Richard  – Robert Koch Institute, Germany / Sorbonne University, France

GiorgosKotoulas – IMBBC-HCMR, Greece

KritonKalantidis- Laboratory of Plant Molecular Biology, IMBB-FoRTH & UoCrete, Greece

Angela Falciatore – Institute of Physico-Chemical Biology, Sorbonne University, France

Funding Body: Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT)

Call:  « First call for HFRI Research Project for the support of Post-doctoral researchers »

Project code: 483

Budget: 200.000 EUR

Duration: 36 months (July 2018-2021)

This project has received funding from the Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT), under grant agreement No 483

IMBBC-HCMR: http://www.imbbc.hcmr.gr/

IMBB-FoRTH: https://www.imbb.forth.gr/en/

University of Crete, Biology Department: https://www.biology.uoc.gr/en

Laboratory Plant Molecular Biology, IMBB-FoRTH & UoCrete: https://www.imbb.forth.gr/imbb-people/index.php/en/kalantidis-laboratory

Laboratory of Computational and Quantitative Biology, Sorbonne University: http://www.lcqb.upmc.fr/users/falciatore

Hellenic Foundation for Research and Innovation: http://www.elidek.gr/en/homepage/

General Secretary for Research and Technology: http://www.gsrt.gr/central.aspx?sId=119I428I1089I323I488743