Rational design of a synthetic mammalian riboswitch as a ligand-responsive -1 ribosomal frame-shifting stimulator

Lin, Ya-Hui; Chang, Kung-Yao

The construction of a ligand-responsive pseudoknot does not necessarily lead to ligand-responsive −1 PRF stimulation activity. Here, we present concepts and designs in using SARS-CoV −1 PRF pseudoknot stimulator as the framework to rationally building a ligand-dependent stimulator for mammalian application. Notably, we demonstrated that the extra stem of this IBV-type pseudoknot variant can be replaced by an RNA aptamer to provide a general approach for building a ligand-responsive pseudoknot and a liganddependent −1 PRF stimulator simultaneously. This engineered mammalian riboswitch possesses activity that rivals metabolite-responsive and viral −1 PRF stimulators, with potential for further improvement by adding intermolecular kissing interaction to the terminal loop of aptamer. Finally, ligand-dependency of the mammalian riboswitch engineered in this study could be swapped to other ligands by starting from replacing the extra stem of SARS-PK with other aptamer domains. In the left side of arrow, the attenuator hairpin (in red) of theoOFF2 is ON to attenuate −1 PRF without theophylline. However, the attenuator hairpin is switched off upon theophylline treatment in the right. As the ON and OFF switches of Switch-1 respond in an opposite way to theophylline treatment, the ON switch-1 and OFF theoOFF2 result in synergistic up-regulation of −1 PRF activity in the presence of theophylline. (B) 12% SDS-PAGE analysis of radioactivity-based −1 PRF activity of an upstream attenuator module (theoOFF2) in combination with different downstream stimulators in reticulocyte lysate. −1 PRF activities of p2luc reporters containing theoOFF2-SARSPK, Switch-1 and theoOFF2-Switch1 under different ligand conditions are shown with 0 and −1 frame products annotated. theoOFF2-RFC1 and theoOFF2-ZFC1 represent read-through and 0-frame product controls of theoOFF2-Switch1, respectively. (C) Relative −1 PRF activity of reporter constructs in (B) in reticulocyte lysate with the ligand-free activity being treated as 1 (in gray). −1 PRF efficiency was calculated from dual-luciferase activity calibrated by using theoOFF2-RFC and theoOFF2-RFC1 as the read-through controls of theoOFF2-SARS and theoOFF2-Switch1, respectively. Value for each bar is the mean of seven independent experiments with standard error of the mean. P-values were determined by a Student's t-test with P-value < 0.05 designated by an '*'. (D) Relative −1 PRF activity of 293T cells transfected with reporter constructs in (B) with the ligand-free activity being treated as 1 (in gray). −1 PR efficiency was calculated as those in (B). Value for each bar is the mean of five independent experiments with standard error of the mean. P-values were determined by a Student's t-test with P-value < 0.05 designated by an '*'. (E) Fluorescence microscopy images of 293T cells, transfected with a pNinsertC-Venus −1 PRF reporter harboring theoOFF2-SARSPK, theoOFF2-Switch1 or theoOFF2-RFC1, with or without 1 mM theophylline (Scale bar, 20 m). (F) Western blot results of 293T cell lysates from cells transfected with the −1 PRF vector in (E). N-Venus (corresponding to 0 frame product) and fused Venus containing full-length product (corresponding to −1 frame product) were detected by a polyclonal anti-GFP antibody. Cellular ␤-actin was treated as the internal loading control.