Abstract
Eukaryotic organisms possess a complex RNA-directed gene expression regulatory network allowing the production of unique gene expression patterns. A recent addition to the repertoire of RNA-based gene regulation is miRNA target decoys, endogenous RNA that can negatively regulate miRNA activity. miRNA decoys have been shown to be a valuable tool for understanding the function of several miRNA families in plants and invertebrates. Engineering and precise manipulation of an endogenous RNA regulatory network through modification of miRNA activity also affords a significant opportunity to achieve a desired outcome of enhanced plant development or response to environmental stresses. Here we report that expression of miRNA decoys as single or heteromeric non-cleavable microRNA (miRNA) sites embedded in either non-protein-coding or within the 3′ untranslated region of protein-coding transcripts can regulate the expression of one or more miRNA targets. By altering the sequence of the miRNA decoy sites, we were able to attenuate miRNA inactivation, which allowed for fine regulation of native miRNA targets and the production of a desirable range of plant phenotypes. Thus, our results demonstrate miRNA decoys are a flexible and robust tool, not only for studying miRNA function, but also for targeted engineering of gene expression in plants. Computational analysis of the Arabidopsis transcriptome revealed a number of potential miRNA decoys, suggesting that endogenous decoys may have an important role in natural modulation of expression in plants.
Highlights
RNA-based gene regulation has been identified as one of the major conserved mechanisms in eukaryotes [1] and represents an attractive modality for trait engineering in plants
Artificial miRNA decoys expressed from different RNA scaffolds result in miRNA inactivation Initially, two types of synthetic miRNA decoys were tested: first, miRNA decoys with three nucleotide insertions between the nucleotide positions corresponding to nucleotides 10–11 of the miRNA, similar to that described previously [8], creating a bulge upon miRNA annealing; and second, a miRNA decoy with two mismatches corresponding to positions 10–11 of the miRNA sequence (Fig. S1A)
We predicted that a target miRNA would bind to the synthetic miRNA decoy when both miRNA and miRNA decoy were expressed in plants, resulting in sequestration of the miRNA and stabilization of a reporter gene harboring a corresponding miRNA target site (Fig. S1B)
Summary
RNA-based gene regulation has been identified as one of the major conserved mechanisms in eukaryotes [1] and represents an attractive modality for trait engineering in plants. MicroRNAs (miRNAs) are small RNAs that regulate gene expression by targeting one or more sequences of high complementarity in plants [2]. Functional non-coding RNAs with natural non-cleavable miRNA sites have recently been described in Arabidopsis. These transcripts are able to bind their complementary miRNAs and, in this context, have a role in trans-acting siRNA biogenesis [7] as well as the regulation of miR399 activity in Arabidopsis by functioning as miRNA decoys [8]. While an optimized miRNA decoy approach has become a very popular tool for regulating miRNAs in various animal applications, and is a proposed natural mechanism of animal gene regulation (reviewed in [10]), there are a limited number of publications [8,11] demonstrating the utility of this approach in plants
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