Rescue of a human mRNA splicing defect by the plant cytokinin kinetin

Abstract

The defective splicing of pre-mRNA is a major cause of human disease. Exon skipping is a common result of splice mutations and has been reported in a wide variety of genetic disorders, yet the underlying mechanism is poorly understood. Often, such mutations are incompletely penetrant, and low levels of normal transcript and protein are maintained. Familial dysautonomia (FD) is caused by mutations in IKBKAP, and all cases described to date involve an intron 20 mutation that results in a unique pattern of tissue-specific exon skipping. Accurate splicing of the mutant IKBKAP allele is particularly inefficient in the nervous system. Here we show that treatment with the plant cytokinin kinetin alters splicing of IKBKAP. Kinetin significantly increases inclusion of exon 20 from the endogenous gene, as well as from an IKBKAP minigene. By contrast the drug does not enhance inclusion of alternatively spliced exon 31 in MYO5A. Benzyladenine, the most closely related cytokinin, showed a similar but less dramatic effect. Our findings reveal a remarkable impact on splicing fidelity by these small molecules, which therefore provide new tools for the dissection of mechanisms controlling tissue-specific pre-mRNA splicing. Further, kinetin should be explored as a treatment for increasing the level of normal IKAP in FD, and for other splicing disorders that may share a similar mechanism.

Plasma Catechols in Familial Dysautonomia: A Long-term Follow-up Study

“…With recent decriptions of in vitro agents that can rescue the mRNA IKBKAP splicing defect and enhance levels of functional ELP-1 in FD cell lines [20], pathophysiology-based treatment of this disease may evolve out of future clinical trials. The present findings suggest that plama catechol profiling-in particular the DOPA:DHPG ratio-can provide biomarkers with which to track progression of the disease and effects of treatment.…”

Modulation of aberrant NF1 pre-mRNA splicing by kinetin treatment

“…13 In this regard, several drugs have been found to modify splicing in other genetic conditions. [3][4][5][6][7] In a previous exploratory study, we demonstrated that kinetin improved exon 36 inclusion in a minigene model for NF1 mutation c.6724C4T. 9 Here, we evaluated the effect of five different drugs, including kinetin, on 19 different mutations and found that kinetin was able to partially correct aberrant splicing caused by four NF1 splicing mutations (c.910C4T, c.3113G4C, c.6724C4T and c.6791dupA).…”

“…First, experimental conditions were set up in NF1 EBV-transformed cell lines to optimize drug treatment in relation to cellular toxicity and drug concentration, on the basis of previous reports [3][4][5][6][7] (Supplementary Figure 1). Cellular toxicity was only observed for EGCG treatment at 50 mg/ml.…”

“…The other studied drugs did not show any effect in our set of mutations, which confirms the existence of multiple, distinct mechanisms that contribute to exon selection during pre-mRNA processing. 7 Kinetin is a plant cytokinin used in topical applications for its antiaging properties. Its effect on splicing modulation has been demonstrated in IKBKAP, ABI2, BMP2K and NF1 genes.…”

“…1,2 Recent studies have demonstrated that several drugs can modify transcriptional patterns from alleles carrying splicing mutations in different disorders. [3][4][5][6][7][8] This modification has mainly been shown in splicing mutations giving a percentage of wildtype transcripts from the mutated allele, which is also described as having a 'leaky' effect. In a previous collaborative study in which kinetin corrected aberrant splicing for the most common splicing mutation in familial dysautonomia (FD), we also demonstrated that kinetin was able to restore normal splicing for one NF1 mutation (c.6724C4T, exon 36) in a minigene splicing model, 9 which encouraged us to extend our research to other mutations.…”