Remote precursor elements can modulate RNA induced silencing complex-loading efficiency of miR168 in Arabidopsis

RNA interference mediated via the action of micro RNAs (miRNAs) plays a pivotal role in developmental and stress response pathways. In the nucleus, plant miRNAs are generated by subsequent enzymatic cuts of the MIRNA precursors, having specific hairpin-like secondary structures, to liberate the miRNA/miRNA* duplex. The mature miRNA strands are then loaded mostly into the ARGONAUTE 1-containing RNA induced silencing complex (RISC) with various efficiencies and trigger the down-regulation of the expression of the target mRNAs, while the miRNA* strands are eliminated. Here we revealed that MIRNA precursor structural elements, not overlapping with the miRNA/miRNA* duplex part, can have an influence on the AGO-loading efficiency of the produced miRNAs. Using transient and transgenic expression studies, we revealed that a chimeric MIR168 precursor hairpin structure containing the stem region of a hvu-MIR171 precursor can induce the enhancement of AGO-loading efficiency of the produced miR168, resulting in increased target down-regulation and in developmental defects of the transgenic plants. This effect was the most pronounced when the orientation of the wild-type miR168/miR168* duplex was inverted in the chimeric precursor, implying the cooperative action of the structural elements. In transient studies, we also showed that precursor elements of MIR168a can reduce AGO-loading efficiency of miR171. The discovery of signals on remote structures of the miRNA precursor suggests that miRNA biogenesis and AGO-loading can be spatially more connected in the nucleus and/or signalization events mediated by these non-duplex structural features during miRNA biogenesis can determine the fate of the miRNA/miRNA* duplexes in separated AGO-loading processes.