A theophylline-responsive riboswitch regulates expression of nuclear-encoded genes in Arabidopsis

Abstract

ABSTRACTLigand-responsive synthetic riboswitches are versatile and innovative tools for external gene regulation in pro- and eukaryotes. Riboswitches are smallcis-regulatory RNA elements that regulate gene expression by conformational changes in response to ligand binding. In plants, synthetic riboswitches were used to regulate gene expression in plastids, but the application of synthetic riboswitches for the regulation of nuclear-encoded genesin plantahas not been reported so far. Here we characterize the properties of a theophylline-responsive synthetic aptazyme for control of nuclear-encoded transgenes in Arabidopsis (Arabidopsis thaliana). Activation of the aptazyme, inserted in the 3-UTR of the target gene, resulted in rapid self-cleavage and subsequent decay of the mRNA. This riboswitch allowed reversible, theophylline-dependent downregulation of theGreen Fluorescent Protein(GFP) reporter gene in a dose- and time- dependent manner. Insertion of the riboswitch into theOne Helix Protein 1(OHP1) gene allowed complementation ofohp1mutants and induction of the mutant phenotype by theophylline.GFPorOHP1transcript levels were downregulated by maximally 90%, and GFP protein levels by 95%. These results establish artificial riboswitches as tools for externally controlled gene expression in synthetic biology in plants or functional crop design.One sentence summaryArtificial, ligand-responsive RNA aptazymes are an efficient tool for dose- and time-dependent external control of nuclear gene expression in plants.