Principal investigator:

Maizel, Alexis, Prof. Dr.,
Center for Organismal Studies (COS)

University of Heidelberg

Im Neuenheimer Feld 230

69120 Heidelberg

Phone: +49 6221 54 64 56

E-mail: alexis.maizelspam prevention@cos.uni-heidelberg.de

Summary:

In plants, small interfering RNA (siRNA) are produced by cleavage of perfectly double stranded RNA molecules generated by RNA dependent RNA polymerases (RDRs). siRNA have a role in antiviral defence but also important endogenous functions, including the control of chromatin structure and the regulation of cellular gene expression. Trans acting (ta-)siRNAs form a class of endogenous siRNAs produced by the initial ARGONAUTE (AGO)/microRNA (miR)-mediated cleavage of TAS RNAs which subsequently are processed by RDR to generate siRNAs. The ta-siRNA pathway is evolutionary ancient and has been implicated in several aspects of plant development including leaf patterning and root growth. The ta-siRNA pathway allows amplification of the effect of the miRNA and also of the propagation of its effects several cells away, as siRNA are mobile. What determines how, upon miRNA cleavage, only TAS precursors but not any other miRNA-cleaved mRNA, enter the siRNA pathway is unknown. Unravelling how specificity is generated is essential for our understanding of the RNA silencing machinery. Recent evidences point toward an essential role for the initial AGO/miRNA-mediated cleavage in routing precursors to the siRNA pathway. In Arabidopsis, four TAS precursors exist (TAS1-4). Ta-siRNA production from TAS1, TAS2 and TAS4 is initiated by AGO1-mediated cleavage but only when AGO1 is loaded by an asymmetric miRNA/miRNA* duplex. As AGO1 mediates the bulk of miRNA action, without triggering ta-siRNA production, it indicates that AGO1then adopts a specific conformation. On the contrary, ta-siRNA production from TAS3 RNA is triggered by AGO7 which exclusively binds the symmetric miR390/miR390* precursor. We postulate, that the highly specialised miR390/AGO7 pair, which exclusively process TAS3 and no other mRNA, mimics a conformational state that AGO1 only adopts when loaded by an asymmetric precursor. AGO7 provides therefore a unique opportunity to decipher the molecular mechanism leading to specificity in ta-siRNA production. We have previously shown that AGO7 associates with endomembranes and localises to cytoplasmic foci in which the key enzymes for siRNA biogenesis RDR6 also accumulate. We put forward the additional hypothesis that association of AGO7 to membranes is instrumental to siRNA biogenesis. The goal of the proposal is to identify the molecular determinants underpinning the specific ability of AGO7 to route its substrates to the siRNA pathway. Our specific aims are:

• to identify the proteins associated with AGO7 by co-immunoprecipitation coupled to mass-spectrometry;
• to identify residues in AGO7 that determine its specific ability to trigger ta-siRNAs biogenesis;
• to reveal the role of AGO7 subcellular localisation and membrane association toward ta-siRNAs biogenesis.
  

This project will reveal the molecular and cellular determinants responsible for the specific partitioning of miRNA silencing targets between degradation and entry into the siRNA pathway. More globally it offers the chance to unravel the mechanisms by which aberrant RNAs (produced by a miRNA cut) are diverted from the RNA degradation pathway, a fundamental insight to understand how RNA quality control and silencing mechanisms co-exist and inter-operate in an eukaryotic cell.