sRNA-seq of chimeric MIR168 miRNA precursor over-expressing plant tissue

RNA interference mediated via the action of micro (mi) RNAs plays pivotal role in developmental and stress response pathways. In the nucleus, plant miRNAs are generated by subsequent enzymatic cuts of the miRNA precursors (pre-miRNAs), having specific hairpin like secondary structures, to liberate the miRNA/miRNA* duplex. The mature miRNA strands are then loaded with various efficiencies mostly into the main executor ARGONAUTE 1 (AGO1) protein and trigger the downregulation of the target mRNA expression, while the miRNA* strands are eliminated. Here we revealed that structural elements, not overlapping with the miRNA/miRNA* duplex part, of an artificial pre-miRNA can enhance the AGO-loading efficiency of the produced miRNAs. Using transient and transgenic expression studies we revealed that a chimeric pre-miR168 hairpin structure containing the stem region of pre-miR171 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. The exchange of the loop parts of the various precursors did not induce significant changes in AGO-loading. The discovery of signals on remote structures of the pre-miRNA suggest that miRNA biogenesis and AGO-loading can be spatially more connected in nucleus and/or signalization events mediated by these non-duplex structural features can determine the fate of the miRNA/miRNA* duplexes in separated AGO-loading processes.