Abstract
Mutations that affect the splicing of pre-mRNA are a major cause of human disease. Familial dysautonomia (FD) is a recessive neurodegenerative disease caused by a T to C transition at base pair 6 of IKBKAP intron 20. This mutation results in variable tissue-specific skipping of exon 20. Previously, we reported that the plant cytokinin kinetin dramatically increases exon 20 inclusion in RNA isolated from cultured FD cells. The goal of the current study was to investigate the nature of the FD splicing defect and the mechanism by which kinetin improves exon inclusion, as such knowledge will facilitate the development of future therapeutics aimed at regulating mRNA splicing. In this study, we demonstrate that treatment of FD lymphoblast cell lines with kinetin increases IKBKAP mRNA and IKAP protein to normal levels. Using a series of minigene constructs, we show that deletion of a region at the end of IKBKAP exon 20 disrupts the ability of kinetin to improve exon inclusion, pinpointing a kinetin responsive sequence element. We next performed a screen of endogenously expressed genes with multiple isoforms resulting from exon skipping events and show that kinetin’s ability to improve exon inclusion is not limited to IKBKAP. Lastly, we highlight the potential of kinetin for the treatment of other human splicing disorders by showing correction of a splicing defect in neurofibromatosis.
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Acknowledgements
We wish to thank all FD patients and their families for their continued participation in our studies. We thank Dr. Felicia Axelrod and her staff at the Dysautonomia Treatment and Evaluation Center at New York University Medical School for her long-standing contributions to this work. We also thank Dr. Chris Burge for generously providing us with the SXN minigene construct. This work was supported by grants from the Dysautonomia Foundation Inc, the National Institute for Neurological Disorders and Stroke, and the Massachusetts General Hospital-Tosteson Fund for Medical discovery. C. Lazaro was supported by Generalitat de Catalunya (2003BEAI4000094, 2004BE00368, 2005SGR00018) and by the Spanish Government (SAF2005-00833, SAF2006-05399, ISCIII C03/07).
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Hims, M.M., Ibrahim, E.C., Leyne, M. et al. Therapeutic potential and mechanism of kinetin as a treatment for the human splicing disease familial dysautonomia. J Mol Med 85, 149–161 (2007). https://doi.org/10.1007/s00109-006-0137-2
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DOI: https://doi.org/10.1007/s00109-006-0137-2