Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing.
BACKGROUND
Despite tremendous progress in understanding the mechanisms of constitutive and alternative splicing, an important and widespread step along the gene expression pathway, our ability to deliberately regulate gene expression at this step remains rudimentary. The present study was performed to investigate whether a theophylline-dependent "splice switch" that sequesters the branchpoint sequence (BPS) within RNA-theophylline complex can regulate alternative splicing.


RESULTS
We constructed a series of pre-mRNAs in which the BPS was inserted within theophylline aptamer. We show that theophylline-induced sequestering of BPS inhibits pre-mRNA splicing both in vitro and in vivo in a dose-dependent manner. Several lines of evidence suggest that theophylline-dependent inhibition of splicing is highly specific, and thermodynamic stability of RNA-theophylline complex as well as the location of BPS within this complex affects the efficiency of splicing inhibition. Finally, we have constructed an alternative splicing model pre-mRNA substrate in which theophylline caused exon skipping both in vitro and in vivo, suggesting that a small molecule-RNA interaction can modulate alternative splicing.


CONCLUSIONS
These findings provide the ability to control splicing pattern at will and should have important implications for basic, biotechnological, and biomedical research.
Kim
Dong-Suk
DS
Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA. dskim11@gmail.com
Gusti
Veronica
V
Dery
Kenneth J
KJ
Gaur
Rajesh K
RK
D017398:Alternative Splicing; D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D006367:HeLa Cells; D006801:Humans; D008024:Ligands; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000502:physiology*; D012326:RNA Splicing / Q000502:physiology*; D013806:Theophylline / Q000378:metabolism / Q000494:pharmacology*
D052157:Aptamers, Nucleotide; D008024:Ligands; D012322:RNA Precursors; D013806:Theophylline