A Collection of Target Mimics for Comprehensive Analysis of MicroRNA Function in Arabidopsis thaliana

Abstract

Many targets of plant microRNAs (miRNAs) are thought to play important roles in plant physiology and development. However, because plant miRNAs are typically encoded by medium-size gene families, it has often been difficult to assess their precise function. We report the generation of a large-scale collection of knockdowns for Arabidopsis thaliana miRNA families; this has been achieved using artificial miRNA target mimics, a recently developed technique fashioned on an endogenous mechanism of miRNA regulation. Morphological defects in the aerial part were observed for ∼20% of analyzed families, all of which are deeply conserved in land plants. In addition, we find that non-cleavable mimic sites can confer translational regulation in cis. Phenotypes of plants expressing target mimics directed against miRNAs involved in development were in several cases consistent with previous reports on plants expressing miRNA–resistant forms of individual target genes, indicating that a limited number of targets mediates most effects of these miRNAs. That less conserved miRNAs rarely had obvious effects on plant morphology suggests that most of them do not affect fundamental aspects of development. In addition to insight into modes of miRNA action, this study provides an important resource for the study of miRNA function in plants.

Author Summary

MiRNAs are small RNA molecules that play an important role in regulating gene function, both in animals and in plants. In plants, miRNA target mimicry is an endogenous mechanism used to negatively regulate the activity of a specific miRNA family, through the production of a false target transcript that cannot be cleaved. This mechanism can be engineered to target different miRNA families. Using this technique, we have generated artificial target mimics predicted to reduce the activity of most of the miRNA families in Arabidopsis thaliana and have observed their effects on plant development. We found that deeply conserved miRNAs tend to have a strong impact on plant growth, while more recently evolved ones had generally less obvious effects, suggesting either that they primarily affect processes other than development, or else that they have more subtle or conditional functions or are even dispensable. In several cases, the effects on plant development that we observed closely resembled those seen in plants expressing miRNA–resistant versions of the major predicted targets, indicating that a limited number of targets mediates most effects of these miRNAs. Analyses of mimic expressing plants also support that plant miRNAs affect both transcript stability and protein accumulation. The artificial target mimic collection will be a useful resource to further investigate the function of individual miRNA families.