Identification of new miRNA biogenesis components by a fast-forward genetic approach

MANAVELLA, PA; HAGMANN, J AND WEIGEL, D

Encuentro; HFSP Awardees Meeting 2011; 2011

Abstract text: MicroRNAs (miRNAs) are post-transcriptional regulators of ~21 nt in length that distinguish target messenger RNA transcripts (mRNAs) based on complementary recognition usually resulting in gene silencing. The production of mature miRNAs requires multiple and coordinated steps starting on the transcription of a primary miRNA up to a target mRNA cleavage or the inhibition of its translation. Although mutant screens have identified a variety of plant proteins involved in miRNA processing and function, their mutant phenotypes are not always the same, going from strong embryonic lethal to weak morphological effects. 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. Up to now most of the screens made to identify new miRNA components are based on scoring alteration on the normal development of the plant or using reporters that affect the plant morphology. The main weakness of these approaches laid on its incapacity to detect mutants without morphological changes such as those produce by weak alleles or redundant proteins. Herein we report the development of a high-throughput screen for miRNA-mediated gene regulation in whole Arabidopsis seedlings and the identification of a group of new mutants that have an impaired miRNA production or action. Our strategy used transgenic lines carrying the luciferase gene as a reporter of the activity of an artificial miRNA (amiRNA) that targets it. Using this reporter system we were able to quickly screen thousands of mutants seeds and identify miRNA deficient plants even those showing no drastic changes on its morphology. The use of next generation sequencing of the whole mutant genomes follow by SHORE mapping allow us to speed up the mapping process and to certain identify the causal mutation within a time frame of a few months. In order to confirm the participation of the mutated genes on the miRNA pathway we perform measurement of endogenous miRNAs on these mutants and T-DNA insertion lines. Additionally, the expression level of miRNA precursors and its targets genes were analyzed on the same plants. To better understand the mechanism of action of we test the effect of the mutant backgrounds on small RNA related pathways such as siRNA and tasiRNA. Further molecular characterization on the expression, interaction, localization and other features of the identified proteins has also been done.