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          Institute: MPI für Entwicklungsbiologie     Collection: Abteilung 6 - Molecular Biology (D. Weigel)     Display Documents

ID: 706651.0, MPI für Entwicklungsbiologie / Abteilung 6 - Molecular Biology (D. Weigel)
Dissection of miRNA pathways using Arabidopsis mesophyll protoplasts
Authors:Martinho, C.; Confraria, A.; Elias, C. A.; Crozet, P.; Rubio-Somoza, I.; Weigel, D.; Baena-Gonzalez, E.
Date of Publication (YYYY-MM-DD):2014-10-24
Title of Journal:Molecular Plant
Journal Abbrev.:Mol Plant
Issue / Number:2
Start Page:261
End Page:275
Sequence Number of Article:25343984
Review Status:Internal review
Audience:Experts Only
Abstract / Description: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 paper, we describe a transient expression system in Arabidopsis mesophyll protoplasts that 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, either by overexpression or by 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.
Free Keywords:Arabidopsis thaliana; miRNA; miRNA activity; miRNA sensor; protoplasts
External Publication Status:published
Document Type:Article
Affiliations:MPI für Entwicklungsbiologie/Abteilung 6 - Molekulare Biologie (Detlef Weigel)
External Affiliations:Plant Stress Signaling, Instituto Gulbenkian de Ciencia, Rua da Quinta Grande - 6, 2780-156 Oeiras, Portugal. Department of Molecular Biology, Max Planck Institute for Developmental Biology, D-72076 Tubingen, Germany. Plant Stress Signaling, Instituto Gulbenkian de Ciencia, Rua da Quinta Grande - 6, 2780-156 Oeiras, Portugal ebaena@igc.gulbenkian.pt.
Identifiers:ISSN:1752-9867 (Electronic) 1674-2052 (Linking) %R 10.1... [ID No:1]
URL:http://www.ncbi.nlm.nih.gov/pubmed/25343984 [ID No:2]
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