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.

Alternate approaches to repress endogenous microRNA activity inArabidopsis thaliana

“…miRNAs and target mimics in plants, the mechanistic relatedness of these two approaches for repressing miRNA function was further demonstrated by Todesco et al 3 and Ebert et al 60 respectively. Todesco et al 3 showed that by introducing mimiclike miRNA target sites in the 3' UTR of a protein-coding gene, the activity of the targeted miRNA was sequestered, target transcript levels remained constant and protein levels translated from the cis-linked mRNA were reduced.…”

“…Todesco et al 3 showed that by introducing mimiclike miRNA target sites in the 3' UTR of a protein-coding gene, the activity of the targeted miRNA was sequestered, target transcript levels remained constant and protein levels translated from the cis-linked mRNA were reduced. Furthermore, and as shown in plants harboring miRNA target mimicry vectors, Ebert et al 55 demonstrated that the levels of the targeted miRNA were reduced in mammalian cell lines expressing target mRNA sponges.…”

“…Using this approach we demonstrated that the expression of individual miRnAs or entire miRnA families, can be readily and efficiently knocked-down. Our approach is in addition to two previously reported methodologies that also allow for the targeted suppression of either individual miRnAs, or all members of a MIR gene family; these include miRnA target mimicry 2,3 and transcriptional gene silencing (TGS) of MIR gene promoters. 4 All three methodologies rely on endogenous gene regulatory machinery and in this article we provide an overview of these technologies and discuss their strengths and weaknesses in inhibiting the activity of their targeted miRnA(s).…”

The use of high-throughput sequencing methods for plant microRNA research

“…95,96 The mechanism of target mimicry enabled the researchers to introduce manually designed mimics into the plants for investigation of the biological outputs through loss-of-function of specific miRNAs. 96,97 It is an efficient way for the study on the biological functions of a specific miRNA or even a miRNA family. Similarly, in animals, manually introduced sponges were utilized for functional studies on certain miRNAs.…”

Nicotine biosynthesis is regulated by two more layers: Small and long non-protein-coding RNAs

“…Since the first eTM from a long non-protein-coding mRNA (lncRNA) gene INDUCED BY PHOSPHATE STARV-TION1 was reported by Franco-Zorrilla et al 24 in Arabidopsis to sequester miR399 and to disrupt its cleavage activity, noncoding eTMs for various miRNAs have been reported in several plant species. [25][26][27] Tobacco nta-eTMX27 is a 1,213 bp long lncRNA having a binding site for nta-miRX27 with a 3-nucleotide bulge between the ninth and 10th position. The topping experiment showed that the expression levels of nta-eTMX27 and nta-miRX27 were negatively correlated with correlation coefficiency of ¡0.956 while those of nta-eTMX27 and QPT2 were correlated positively with correlation coefficiency of 0.986 when their transcriptional levels were quantified at 3, 6, 12, 24 and 48 h after topping.…”