Modulation of aberrant NF1 pre-mRNA splicing by kinetin treatment

Abstract

Neurofibromatosis type 1 is one of the most common neurocutaneous autosomal dominant disorders. It is caused by mutations in the neurofibromatosis type 1 (NF1) gene and approximately 30-40% of them affect the correct splicing of NF1 pre-mRNA. In this report, we evaluate the effect of five different drugs, previously found to modify splicing in several genetic disorders, on the splicing of mutated NF1 alleles. For this purpose, cell lines derived from patients bearing 19 different NF1-splicing defects were used. Our results showed that kinetin partially corrects the splicing defect in four of the studied mutations (c.910C>T, c.3113G>A, c.6724C>T and c.6791dupA). Our study is a valuable contribution to the field because it identifies new exon-skipping events that can be reversed by kinetin treatment and provides new information about kinetin splicing modulation. However, owing to the nature of mutations in our patients, kinetin treatment could not be used as a therapeutic agent in these cases.

Figure 1

Summary of the different treatments carried out in EBV-transformed lymphocyte cell lines from 22 patients harboring 19 different splicing mutations. Results are given as a ratio of the percentage of aberrantly spliced transcripts in treated cells vs the percentage of aberrant transcripts in untreated cells. X-axis: patients (numbered 1–22, according to Table 1). Y-axis: levels of aberrantly spliced transcripts in treated cells normalized to untreated cells. Slight variations were observed between different patients and different drugs. A clear correction of aberrant splicing was only observed in mutations c.910C>T (patients 2 and 3), c.3113G>C (patient 11), c.6724C>T (patient 16) and c.6791dupA (patient 17) after kinetin treatment. The thick black horizontal line indicates a 30% relative decrease in aberrantly spliced transcripts.

Figure 2

Effect of kinetin treatment on NF1-transcript levels, as well as on aberrantly spliced and full-length transcripts, in EBV-transformed lymphocyte cell lines from patients 3 (c.910C>T), 11 (c.3113G>C), 16 (c.6724C>T) and 17 (c.6791dupA). The effect of different drugs was evaluated before (a) and after (b) puromycin treatment; (c) dose–response after 24 h of treatment using three different concentrations of kinetin; (d) time course at 100 μ of kinetin treatment. C, untreated cells; K, kinetin (100 μ); VPA, valproic acid (50 μ); SB, sodium butyrate (5 μg/μl); EGCG, (−)-epigallocatechin gallate (25 μg/ml); A, aclarubicin (10 n); DMSO, control of aclarubicin treatment.