摘要
In eukaryotes, the RNase-Ⅲ Dicer often produces length/sequence microRNA （miRNA） variants, called ＂isomiRs＂, owing to intrinsic structural/sequence determinants of the miRNA precursors （pre-miRNAs）. In this study, we combined biophysics, genetics and biochemistry approaches to study Arabidopsis miR168, the key feedback regulator of central plant silencing effector protein ARGONAUTE1 （AGO1）. We identified a motif conserved among plant premiR168 orthologs, which enables flexible internal basepairing underlying at least three metastable structural configurations. These configurations promote alternative, accurate Dicer cleavage events generating length and structural isomiR168 variants with distinctive AGO sorting properties and modes of action. Among these isomiR168s, a duplex with a 22-nt guide strand exhibits strikingly preferential affinity for AGO10, the closest AGO1 paralog. The 22-nt miR168-AGO10 complex antagonizes AGO1 accumulation in part via ＂transitive RNAi＂, a silencing-amplification process, to maintain appropriate AGO1 cellular homeostasis. Furthermore, we found that the tombusviral P19 silencing-suppressor protein displays markedly weaker affinity for the 22-nt form among its isomiR168 cargoes, thereby promoting AGO10-directed suppression of AGOl-mediated antiviral silencing. Taken together, these findings indicate that structural flexibility, a previously overlooked property of premiRNAs, considerably increases the versatility and regulatory potential of individual MIRNA genes, and that some pathogens might have evolved the capacity or mechanisms to usurp this property.
In eukaryotes, the RNase-Ⅲ Dicer often produces length/sequence microRNA （miRNA） variants, called ＂isomiRs＂, owing to intrinsic structural/sequence determinants of the miRNA precursors （pre-miRNAs）. In this study, we combined biophysics, genetics and biochemistry approaches to study Arabidopsis miR168, the key feedback regulator of central plant silencing effector protein ARGONAUTE1 （AGO1）. We identified a motif conserved among plant premiR168 orthologs, which enables flexible internal basepairing underlying at least three metastable structural configurations. These configurations promote alternative, accurate Dicer cleavage events generating length and structural isomiR168 variants with distinctive AGO sorting properties and modes of action. Among these isomiR168s, a duplex with a 22-nt guide strand exhibits strikingly preferential affinity for AGO10, the closest AGO1 paralog. The 22-nt miR168-AGO10 complex antagonizes AGO1 accumulation in part via ＂transitive RNAi＂, a silencing-amplification process, to maintain appropriate AGO1 cellular homeostasis. Furthermore, we found that the tombusviral P19 silencing-suppressor protein displays markedly weaker affinity for the 22-nt form among its isomiR168 cargoes, thereby promoting AGO10-directed suppression of AGOl-mediated antiviral silencing. Taken together, these findings indicate that structural flexibility, a previously overlooked property of premiRNAs, considerably increases the versatility and regulatory potential of individual MIRNA genes, and that some pathogens might have evolved the capacity or mechanisms to usurp this property.
基金
The NCCR RNA and disease funded by the Swiss National Science Foundation （SNF
51NF40_141735） for access to the Biomolecular NMR spectrometry platform at ETH ZUrich. This work was supported by an IIF Marie Curie fellowship to TI （no. 329029） and a European Research Council （ERC） advanced grant ＂Frontiers of RNAi-Ⅱ＂ to OV （no. 323071）.
