In eukaryotic RNA silencing, RNase-III classes of enzymes in the Dicer family process double-stranded RNA of cellular or exogenous origin into small-RNA (sRNA) molecules. sRNAs are then loaded into effector proteins known as ARGONAUTEs (AGOs), which, as part of RNA-induced silencing complexes, target complementary RNA or DNA for silencing. Plants have evolved a large variety of pathways over the Dicer-AGO consortium, which most likely underpins part of their phenotypic plasticity. Dicer-like proteins produce all known classes of plant silencing sRNAs, which are invariably stabilized via 2'-O-methylation mediated by HUA ENHANCER 1 (HEN1), potentially amplified by the action of several RNA-dependent RNA polymerases, and function through a variety of AGO proteins. Here, we review the known characteristics and biochemical properties of the core silencing factors found in the model plant Arabidopsis thaliana. We also describe how interactions between these core factors and more specialized proteins allow the production of a plethora of silencing sRNAs involved in a large array of biological functions. We emphasize in particular the biogenesis and activities of silencing sRNAs of endogenous origin.

The first step in microRNA (miRNA) biogenesis usually involves cleavage at the base of its fold-back precursor. Here, we describe a non-canonical processing mechanism for miRNAs miR319 and miR159 in Arabidopsis thaliana. We found that their biogenesis begins with the cleavage of the loop, instead of the usual cut at the base of the stemloop structure. DICER-LIKE 1 (DCL1) proceeds then with three additional cuts until the mature miRNA is released. We further show that the conserved upper stem of the miR319 precursor is essential to organize its biogenesis, whereas sequences below the miRNA/miRNA* region are dispensable. In addition, the bulges present in the fold-back structure reduce the accumulation of small RNAs other than the miRNA. The biogenesis of miR319 is conserved in the moss Physcomitrella patens, showing that this processing mechanism is ancient. These results provide new insights into the plasticity of small-RNA pathways.

Unlike in metazoans, plant microRNAs (miRNAs) undergo stepwise nuclear maturation before engaging cytosolic, sequence-complementary transcripts in association with the silencing effector protein ARGONAUTE1 (AGO1). Since their discovery, how and under which form plant miRNAs translocate to the cytosol has remained unclear, as has their sub-cellular AGO1 loading site(s). Here, we show that the N termini of all plant AGO1s contain a nuclear-localization (NLS) and nuclear-export signal (NES) that, in Arabidopsis thaliana (At), enables AtAGO1 nucleo-cytosolic shuttling in a Leptomycin-B-inhibited manner, diagnostic of CRM1(EXPO1)/NES-dependent nuclear export. Nuclear-only AtAGO1 contains the same 2'O-methylated miRNA cohorts as its nucleo-cytosolic counterpart, but it preferentially interacts with the miRNA loading chaperone HSP90. Furthermore, mature miRNA translocation and miRNA-mediated silencing both require AtAGO1 nucleo-cytosolic shuttling. These findings lead us to propose a substantially revised view of the plant miRNA pathway in which miRNAs are matured, methylated, loaded into AGO1 in the nucleus, and exported to the cytosol as AGO1:miRNA complexes in a CRM1(EXPO1)/NES-dependent manner.

MicroRNAs (miRNAs) derive from longer precursors with fold-back structures. While animal miRNA precursors have homogenous structures, plant precursors comprise a collection of fold-backs with variable size and shape. Here, we design an approach to systematically analyze miRNA processing intermediates and characterize the biogenesis of most of the evolutionarily conserved miRNAs present in Arabidopsis thaliana. We found that plant miRNAs are processed by four mechanisms, depending on the sequential direction of the processing machinery and the number of cuts required to release the miRNA. Classification of the precursors according to their processing mechanism revealed specific structural determinants for each group. We found that the complexity of the miRNA processing pathways occurs in both ancient and evolutionarily young sequences and that members of the same family can be processed in different ways. We observed that different structural determinants compete for the processing machinery and that alternative miRNAs can be generated from a single precursor. The results provide an explanation for the structural diversity of miRNA precursors in plants and new insights toward the understanding of the biogenesis of small RNAs.