A forward chemical genetic screen reveals putative inhibitors of the miRNA biogenesis pathway

MANAVELLA PA, TOTH R, COUPLAND G, WEIGEL D

Simposio; Keystone Symposia - RNA silcencing mechanisms in plants; 2010

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. For example, dcl1 and se null mutants are embryonic lethal, while hyl1 and hen1 null mutants are viable. How much of this reflects redundancy between closely related proteins, differential requirements in several RNA silencing pathways, or differential activity during different tissues or stages of development is not known. It has been shown that an alternative to the traditional genetic screening is the use of forward chemical genetics screenings as a powerful approach for studying signaling mechanisms in a variety of organisms. These kinds of screenings, utilizes small molecules to perturb a signaling pathway, permitting the identification of relevant gene products at any stage of development without a continuous perturbation in a gene product. Additionally the identification of specific inhibitors of a pathway, such as the miRNA biogenesis, could generate new powerful tools in further studies. Herein we report the development of a high-throughput screen for miRNA-mediated gene regulation in whole Arabidopsis seedlings and the identification of several putative miRNA inhibitors. Our strategy used transgenic lines carrying the Luciferase gene as a reporter of the activity of an artificial miRNA (amiRNA) able to target it. In our screen we used a commercial library of 10,000 structurally diverse small molecules and identified several compounds with different level of inhibition over the amiRNA function. Additionally we report the identification and characterization of a DNA/RNA binding molecule as a non-specific inhibitor of the pathway. In this sense, plants treated shortly with this compound show a strong reduction in the miRNA activity correlated with a marked reduction in the mature miRNAs concentration. No effects on the transcription of the miRNA genes are detected after short treatments with this chemical compound. Even though, longer treatments show no specific effects in the reporter activity indicating that the chemical could also act in other general processes.