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Argonaute-associated factors required for translational repression in plant RNA silencing (ASTRiR)
Date du début: 1 mars 2012, Date de fin: 31 mars 2014 PROJET  TERMINÉ 

MicroRNAs (miRNAs) are key post-transcriptional regulators of eukaryotic gene expression. In plants, most miRNAs associate with AGO1 as part of RNA-induced-silencing-complexes (RISC) to regulate target mRNAs via perfectly or near-perfecly miRNA-complementarity sites. These sites differ from those found in metazoan miRNA target transcripts, where extensive mis-pairing prevents endonucleolytic cleavage, or ‘slicing’, and favors translational repression as an alternative mode of AGO action. Recently, two classes of miRNA action deficient (mad) mutants have been isolated in Arabidopsis thaliana: class I mutants carry lesions in genes required for slicing, whereas class II mutants are still able to slice but yet, fail to silence miRNA target transcripts at the protein level. Studying many examples of endogenous miRNA/target mRNA interactions in the context of class-II mad mutants, the host laboratory showed that most plant miRNAs may, in fact, concurrently slice and translationally inhibit any given pool of target mRNAs, raising the fundamental question of how slicing is avoided during translational repression? One hypothesis holds that translational repressor proteins specifically associate with AtAGO1 on polysomes and block its slicing activity. The recent purification and preliminary characterization, in the host laboratory, of several possible core components of the AtAGO1 RISC support this idea. Combining biochemical and genetic approaches, I will address (i) how these proteins interact with each other, (ii) how their inactivation/over-expression impacts the mode of action of AtAGO1 and its known functions in the various Arabidopsis silencing pathways, and (iii) how these factors may be exploited to uncover the full suite of small RNAs operating preferentially via translational repression. Parallel investigations in mammalian cell systems will further uncover the extent to which the underlying mechanisms are conserved in metazoans.