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Zusammenfassung:
The production of mature miRNAs requires multiple and coordinated steps that lead to a target mRNA cleavage or the inhibition of its translation. Although a variety of plant proteins involved in miRNA processing and function has been identified by mutant screens, their mutant phenotypes are not always the same. How much of this reflects redundancy between closely related proteins, differential requirements in several RNA silencing pathways, or differential activity during development is still unknown. An alternative to traditional forward genetic screens is chemical genetics, a powerful approach for studying signaling mechanisms in a variety of organisms. this approach utilizes small molecules to perturb a signaling pathway, permitting the identification of relevant factors at any stage of development without a continuous perturbation in a gene product, which may be essential for organismal or cellular survial. Additionally the identification of specific inhibitors of a pathway, such as miRNA biogenesis, could provide new powerful tools in further studies. Herein we report the development of a high-throughput assay for mirnA-mediated gene regulation in whole Arabidopsis seedlings and the identification of several miRNA inhibitors. Our strategy used transgenic lines carrying the luciferase gene as a reporter of the activity of an artificial miRNA (amiRNA) that targets it. We screened a commercial library of 10,000 structurally diverse small molecules, and identified a group of compounds with different levels and apparent modes of inhibition. measurement of endogenous mirnAs together with the expression level of its precursors and targets genes confirmed the inhibitory effect of these compounds. Additionally, the compounds have been tested for their ability to affect other related pathways such as sirnA and tasirnA. most affect the tasirnA production, only a few affect all the tree pathways.
