Plant secondary siRNA production determined by microRNA-duplex structure

Processing of microRNA (miRNA) precursors results in the release of a double-stranded miRNA/miRNA* duplex. The miRNA or guide strand, is loaded onto the Argonaute (AGO) effector, and the miRNA* or passenger strand is typically degraded. The loaded AGO-containing RNA-induced silencing complex specifically recognizes a target mRNA, leading to its degradation or translational inhibition. In plants, miRNA-mediated cleavage of a target triggers in some cases the production of secondary small interfering RNAs (siRNAs), which in turn can silence other genes in trans. This alternative pathway depends on the length of the miRNA and the specific AGO in the effector complex. However, 22-nt miRNAs are sufficient, but not essential for this pathway. Using a combination of computational and experimental approaches, we show that transitivity can be triggered when the small RNA that is not retained in AGO is 22-nt long. Moreover, we demonstrate that asymmetrically positioned bulged bases in the miRNA:miRNA* duplex, regardless of miRNA or miRNA* length, are sufficient for the initiation of transitivity. We propose that the RNA-induced silencing complex reprogramming occurs during the early steps of miRNA loading, before the miRNA duplex is disassembled and the guide strand is selected