Transcriptional gene silencing and RNA silencing in plants

Responsible : Thierry LAGRANGE (DR1 CNRS)

Research in our team is focused on understanding the mechanisms of gene regulation in plants. In the past ten years, we put a special emphasis on the study of small RNA (siRNA)-directed DNA Methylation (RdDM), an RNA interference-mediated heterochromatin pathway that contributes to transcriptional gene silencing and heterochromatin formation in plants. Silent domain formation via the RdDM pathway is mostly targeted to transposable elements (TE) and other types of genomic repeats, as a means of defending the host genome against the potentially deleterious effects of these sequences. In 2005, our team and others showed that two plant-specific RNA polymerase II (PolII)-related enzymes, known as PolIV and PolV, were required for RdDM activity (Pontier et al., 2005, Genes & Dev.), leading us to propose a model for RdDM in which PolIV, in concert with RNA-dependent RNA polymase 2 (RDR2) and DCL3 produce 24-nt siRNAs, while PolV and siRNA/AGO4, act downstream guiding DNA methylation at target loci (see: RNA-directed DNA methylation in Arabidopsis). This model received further support from data showing that PolV interacts with siRNA/AGO4 through WG (tryptophan-glycine) AGO hook motifs present in the polV/NRPE1 C-terminal extension (Li et al., 2006, Cell; El-Shami et al., 2007, Genes & Dev.). Interestingly, a similar AGO hook platform was found in SPT5like (SPT5l), a novel RdDM component that shares similarity to the SPT5-type transcription elongation factors (Bies-Etheve et al., 2009; EMBO reports). More recently, in a long term effort to decipher RdDM mode of action, we used genetic and laser UV-assisted zero-length cross-linking to assess the dynamics of siRNA/AGO4 in RdDM, demonstrating the esssential role of NRPE1/SPT5L AGO hook platforms and the existence of functionally relevant siRNA/AGO4–DNA interaction at RdDM targets, leading us to propose an alternative model of RdDM (Lahmy et al., 2016, Genes & Dev.). Our current work is focused on the mechanisms of PolV function in RdDM, using a combination of approaches ranging from genetics, biochemistry, molecular biology.

In parallel to our previous discoveries, we have developed a bioinformatic approach to assess the genome-wide distribution of AGO hook motif in eukaryotic/prokaryotic proteomes (Karlowski et al., 2010, Nucleic Acids Research). Our genome sequence-based algorithmic approach identifies numerous candidate proteins, harboring between 2 and 40 contiguous AGO hooks, in both plant and animal proteomes but also in the proteomes of plant/animal associated pathogens (Azevedo et al., 2011, Curr. Opin. Plant Biol.). Functional analysis of the candidate proteins further demonstrates that the AGO hook motifs has been used as a versatile tool for regulating the activities of RNA silencing pathways in both positive and negative manners (see: AGO hook motif in Arabidopsis).

Finally, in addition to epigenetic regulation based on DNA methylation, we currently study how the mRNA N6-Methyl-Adenosine (m6A) modification, collectively referred to as epitranscriptomic modification, regulates gene expression in Arabidopsis. The current view is that m6A modification occurs co-transcriptionally in the nucleus driven by a conserved “writer” complex, and that it controls the fate of mRNA mostly by recruiting specific “readers”. The action of m6A modification can also be reversed by specific “erasers”. Despite the evidence that m6A players are conserved throughout the green lineage, plants lagged behind other eukaryotes in the understanding of m6A function. In line with this statement, we have recently uncovered an m6A-assisted polyadenylation/m-ASP mechanism that ensures transcriptome integrity in Arabidopsis by suppressing unterminated transcription at specific loci (see: m6A RNA modification and gene regulation in Arabidopsis). Interestingly, the m-ASP pathway is unique in that it does require a plant-specific YTH-type reader protein (CPSF30L), which corresponds to a longer isoform of conserved CLEAVAGE AND POLYADENYLATION SPECIFICITY FACTOR30/CPSF30.


Team :

AZEVEDO FAVORY Jacinthe - Chercheur CNRS

ETHEVE-BIES Natacha - Maître de conférences UPVD

KAIROUANI Alicia - Doctorante

LAGRANGE Thierry - Directeur de recherche CNRS

LAUDIE Michèle - Assistant Ingénieur CNRS

PICART Claire - Assistant ingénieur CNRS

PONTIER Dominique - Chercheur CNRS

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