Controlled RISC loading efficiency of miR168 defined by miRNA duplex structure adjusts ARGONAUTE1 homeostasis

Micro RNAs (miRNAs) are important regulators of gene expression processed from precursor RNA molecules with precisely defined secondary stem-loop structures. ARGONAUTE1 (AGO1) is the main executor component of miRNA pathway and its expression is controlled via the activity of miR168 in plants. AGO1 loading of MIR168a precursor derived miR168 is strongly restricted leading to abundant cytoplasmic accumulation of protein-unbound miR168. Here we report, that RNA secondary structure of MIR168a precursor not only defines the processing of miR168, but also precisely adjusts AGO1 loading efficiency determining the biologically active subset of miR168 pool. Our results show, that modification of miRNA duplex structure of MIR168a precursor fragment or expression from artificial precursors can alter the finely adjusted loading efficiency of miR168 bringing about AGO1 deficiency and phenotypical abnormalities. In dcl1-9 mutant where, except for miR168, production of most miRNAs is severely reduced this mechanism ensures the elimination of unloaded AGO1 proteins via enhanced AGO1 loading of miR168. We propose that structural features of miRNA duplex can determine the biological activity of miRNAs by precisely defining their loading efficiencies into the limiting AGO1 proteins in competition with the small RNA environment.