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An artificial riboswitch for controlling pre-mRNA splicing.
Dong-Suk Kim1, Veronica Gusti, Sailesh G Pillai
1Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
RNA (New York, N.Y.)
|October 26, 2005
Summary
Scientists engineered a riboswitch to control pre-messenger RNA (mRNA) splicing. This RNA aptamer, when inserted into a specific location, allows theophylline to repress splicing, demonstrating novel RNA-based genetic regulation.
Area of Science:
- Molecular Biology
- RNA Biology
- Gene Regulation
Background:
- Riboswitches are natural RNA aptamers regulating gene expression in bacteria.
- RNA genetic elements, including riboswitches, are found near plant and fungal introns.
- This suggests a potential role for riboswitches in regulating messenger RNA (mRNA) splicing.
Purpose of the Study:
- To demonstrate for the first time that a riboswitch can be engineered to regulate pre-mRNA splicing in vitro.
- To investigate theophylline-dependent control of pre-mRNA splicing using an engineered riboswitch.
- To assess the specificity and functional relevance of theophylline-mediated splicing control.
Main Methods:
- Insertion of a high-affinity theophylline-binding aptamer into the 3' splice site (3' ss) region of a model pre-mRNA (AdML-Theo29AG).
- In vitro splicing assays to evaluate the effect of theophylline on pre-mRNA splicing.
- Specificity tests using a structurally similar small molecule and a pre-mRNA lacking the aptamer binding site.
- Analysis of the specific step of the splicing reaction affected by theophylline.
Main Results:
- Engineered riboswitch successfully repressed pre-mRNA splicing in the presence of theophylline.
- The location of the 3' ss AG within the aptamer was crucial for theophylline-dependent control.
- Theophylline-mediated splicing control was highly specific, with no effect from a similar molecule or on pre-mRNA without the aptamer.
- Theophylline specifically inhibited step II of the splicing reaction.
- Evidence suggests functional relevance of this theophylline-dependent control mechanism.
Conclusions:
- Riboswitches can be engineered to control pre-mRNA splicing in vitro.
- Theophylline-dependent regulation of splicing is achievable and specific.
- This study establishes a novel RNA-based system for controlling mRNA splicing, with potential applications in synthetic biology and gene therapy.