Abstract

microRNAs (miRNAs), small RNA molecules of 20–24 nts, have a number of features that make them ideal tools to regulate gene expression — small size, flexible design, target predictability and action at a late stage of the gene expression pipeline. The regulatory potential of miRNAs goes beyond gene repression, as they can confer robustness to gene expression, a feature which is desirable to implement in plant synthetic gene networks. In order to harness the regulatory potential of miRNAs, a comprehensive understanding of the quantitative parameters and mode of action of miRNAs is required, which is, however, hindered by the complexity of natural systems. By using principles of synthetic biology, we have constructed a platform to characterize regulatory properties of miRNAs in the model alga Chlamydomonas reinhardtii. Using this system, we observed that the level of repression by a miRNA depends on its abundance and degree of sequence complementarity to its target mRNA. We also found that sequence complementarity between the miRNA and its target mRNA defined the mRNA’s response curve to the miRNA, and that the mode of action of a fully complementary miRNA to its target was the result of a combined action of RNA slicing and RNA destabilization. We are now using this information to design further applications of miRNAs, and studying their effect in regulatory loops. This work will be used as a proof-of-concept for the engineering of miRNA-based gene circuits in higher plants.