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.

微小RNA（micro RNAs，miRNAs）是一类重要的基因表达调控因子，由具有精确界定的二级茎环结构的前体RNA分子加工而来。ARGONAUTE1（AGO1）是植物miRNA通路的核心效应组分，其表达在植物中受miR168的活性调控。由MIR168a前体衍生的miR168的AGO1装载过程受到强烈限制，导致未结合蛋白的miR168在细胞质中大量积累。本研究发现，MIR168a前体的RNA二级结构不仅决定了miR168的加工过程，还能精确调控AGO1的装载效率，进而决定miR168池中具有生物活性的分子子集。研究结果显示，对MIR168a前体片段的miRNA双链结构进行修饰，或通过人工前体进行表达，会改变miR168经过精细调控的装载效率，进而引发AGO1蛋白缺失及表型异常。在dcl1-9突变体中，除miR168外绝大多数miRNA的生成均严重受损，该机制可通过增强miR168对AGO1的装载，清除未结合的AGO1蛋白。我们提出，miRNA双链的结构特征可通过精确调控其在小RNA环境竞争中结合有限量AGO1蛋白的效率，决定miRNA的生物活性。