miRNA mobility is a highly regulated process in plants

Small RNAs regulate many important developmental processes by targeting key regulators, such as transcription factor families, for posttranscriptional repression. Although originally assumed to function cell-autonomously, several instances of small RNAs serving as short-range mobile signals have now been reported. The recognition that small RNAs can move from cell-to-cell has broadened our thinking on how these molecules are utilized during development, serving as positional, morphogen-like, signals. However, major questions regarding the properties and function of mobile small RNAs during plant development remain. For example, is miRNA movement a developmentally regulated process? What factors are important for miRNA mobility? In order to address questions regarding the properties of miRNAs mobility, our group has developed a reporter system to assay non-cell-autonomous miRNA activity. In this system an artificial miRNA (MIR-GFP) is expressed from tissue-specific promoters and assayed for its capacity to silence a ubiquitously-expressed, cell-autonomous, nuclear-localized GFP reporter (p35S:3xNLS-GFP). Here we report that miRNA movement is a regulated process, and that competence to move is developmentally determined. In select tissue contexts, miRNAs show differential or directional movement, indicating miRNA mobility to be regulated independently from other signalling molecules. Examination of artificial miRNA (miRGFP) levels in libraries generated from whole seedlings of pCLV3:miRGFP, pSUC2:miRGFP and pRbcS:miRGFP lines and from shoots and roots of pRbcS:miRGFP and the p35S:3xNLS-GFP (no miRGFP) control line. Moreover, in this study we examined the levels of another artificial miRNA (miRGUS), as well as GUS-derived secondary siRNA production, in libraries generated from whole seedlings of p35S:miRGUS line. All lines used to generated small RNA libraries in this study, were made in an rdr6 null mutant background impaired for siRNA production.