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Mol Plant. 2015 Feb;8(2):261-75. doi: 10.1016/j.molp.2014.10.003. Epub 2014 Dec 11.

Dissection of miRNA pathways using arabidopsis mesophyll protoplasts.

Author information

1
Plant Stress Signaling, Instituto Gulbenkian de Ciência, Rua da Quinta Grande - 6, 2780-156 Oeiras, Portugal.
2
Department of Molecular Biology, Max Planck Institute for Developmental Biology, D-72076 Tübingen, Germany.
3
Plant Stress Signaling, Instituto Gulbenkian de Ciência, Rua da Quinta Grande - 6, 2780-156 Oeiras, Portugal. Electronic address: ebaena@igc.gulbenkian.pt.

Abstract

MicroRNAs (miRNAs) control gene expression mostly post-transcriptionally by guiding transcript cleavage and/or translational repression of complementary mRNA targets, thereby regulating developmental processes and stress responses. Despite the remarkable expansion of the field, the mechanisms underlying miRNA activity are not fully understood. In this article, we describe a transient expression system in Arabidopsis mesophyll protoplasts, which is highly amenable for the dissection of miRNA pathways. We show that by transiently overexpressing primary miRNAs and target mimics, we can manipulate miRNA levels and consequently impact on their targets. Furthermore, we developed a set of luciferase-based sensors for quantifying miRNA activity that respond specifically to both endogenous and overexpressed miRNAs and target mimics. We demonstrate that these miRNA sensors can be used to test the impact of putative components of the miRNA pathway on miRNA activity, as well as the impact of specific mutations, by either overexpression or the use of protoplasts from the corresponding mutants. We further show that our miRNA sensors can be used for investigating the effect of chemicals on miRNA activity. Our cell-based transient expression system is fast and easy to set up, and generates quantitative results, being a powerful tool for assaying miRNA activity in vivo.

KEYWORDS:

Arabidopsis thaliana; miRNA; miRNA activity; miRNA sensor; protoplasts

PMID:
25680775
DOI:
10.1016/j.molp.2014.10.003
[Indexed for MEDLINE]
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