A ligand-dependent hammerhead ribozyme switch for controlling mammalian gene expression

Abstract

The possibility to externally control gene expression is of fundamental importance in both basic and applied life sciences. Although there are some techniques available to regulate expression in mammalian cells, they rely on the presence of ligand-sensing transcription factors, making it necessary to generate cell lines or organisms that stably express these regulatory factors. In recent years, mechanisms relying on direct RNA-ligand interactions for controlling gene expression have been both discovered in nature and engineered artificially. Among the latter, RNA switches relying on catalytically active RNA have been described. In principle, ligand-dependent triggering of mRNA self-cleavage should be a universal mechanism in order to control gene expression in a variety of organisms. Nevertheless, no examples of such aptazymes acting as RNA-based switches have been reported so far in mammalian cells. Here we present the theophylline-induced activation of an mRNA-based hammerhead ribozyme, resulting in an off-switch of gene expression. Starting from an artificial aptazyme switch reported to function in bacteria, we identified and optimized important parameters such as artificial start codons and the communicating sequence connecting ribozyme and aptamer, resulting in an RNA switch that allows for controlling transgenic expression in mammalian cells without the need to express a corresponding ligand-sensing transcription factor.

Thermozymes

“…So far, aptamers binding several ligands were combined with HHRs functioning as expression platforms in order to control mRNAs, tRNAs, 16S rRNA as well as RNAi in organisms as diverse as bacteria and mammalian cells. 11,15,16,21,[23][24][25]41 The present work shows that a physical stimulus such as a change in temperature can also be used in order to regulate gene expression via a hammerhead-mediated mRNA cleavage reaction. Importantly, most natural RNAT and all previously engineered RNAT are composed of secondary structures that mask the SD sequence.…”

“…[18][19][20] In our opinion, ribozyme-based devices have the advantage of almost universal applicability for controlling RNA functions. Apart from regulating mRNA translation in bacteria and mRNA integrity in mammalian cells, 21,22 they can be utilized in order to control the activity of tRNAs, 23 16S rRNA, 24 as well as RNAi in mammalia. 21 In addition, they can be combined in a modular fashion in order to yield two-input Boolean logic operators.…”

“…Apart from regulating mRNA translation in bacteria and mRNA integrity in mammalian cells, 21,22 they can be utilized in order to control the activity of tRNAs, 23 16S rRNA, 24 as well as RNAi in mammalia. 21 In addition, they can be combined in a modular fashion in order to yield two-input Boolean logic operators. 25 In addition to small molecule-responsive RNA sensors, gene expression can be controlled by temperature-sensitive RNA structures, so-called RNA thermometers (RNATs).…”

Artificial riboswitches for gene expression and replication control of DNA and RNA viruses

“…The adenoviral immediate early E1A proteins play a key role in initiating virus infection as they induce transcription of further early viral genes and modify several host cell functions, both of which are required for viral DNA replication (27). P1-F5 possessed the best fold-induction rate at the time this study was initiated, which was between three-and sixfold in reporter assays (17,23). We hypothesized that insertion of this aptazyme into the E1A gene would not affect virus replication in the absence of the P1-F5-activating ligand theophylline (theo).…”

“…Their application in mammalian systems, however, has been challenging. This is mostly because aptazymes selected in vitro, in bacteria, or in yeast showed loss or strong reduction of regulation when applied in mammalian cells (20,(22)(23)(24). Previous reports on aptazyme applications in mammalian cells were reporter gene studies or studies on regulation of therapeutic gene expression (17,23,25,26).…”