WP2: Allelic variations that govern alternative splicing of the CFTR ion channel

Scientific team: Ayelet Rahat, Mira Korner, Malka Nissim-Rafinia, Liat Shushi, Michal Shuker, Efrat Ozeri, Asaf Bester, Batsheva Kerem.

Objectives: Use the Cystic fibrosis transmembrane conductance regulator (CFTR) ion channel and its splicing mutations to test the impact of alternative splicing on human disease; Create cell and animal models where CFTR splicing can be manipulated and use these to search for small molecules retrieving normal CFTR electro-physiology. Our specific aims are:

1. Exploring differences in spliceosome-component expression levels in cell lines derived from unrelated cystic fibrosis (CF) patients carrying the 3849+10kb C->T splicing mutation, and to determine how these differences affect the modulation of CFTR splicing.

2. Generating a knock-in mouse model carrying the mutation on different strain backgrounds.

3. Characterizing the histopathology, electrophysiology, and CFTR splicing pattern and spliceosome components in the different mouse strains.

4. Testing the effect of compounds that restored the CFTR chloride (Cl^-) efflux in cells, on the CFTR splicing pattern, the histopathology and electro-physiology of the knock-in mice.

Description of the work: CF is a common severe autosomal recessive disease, caused by mutations in the CFTR gene, encoding a cAMP-stimulated Clˉ channel . 13% of CFTR mutations affect the pre-mRNA splicing of the CFTR gene, by disrupting or generating intronic and exonic splicing motifs, leading to both aberrantly and correctly spliced transcripts. Transcript levels of the same splicing allele vary between different patients. This variability is inversely correlated with the level of correctly spliced transcripts, suggesting that splicing regulation is a genetic modifier of disease expression in patients carrying splicing mutations . We showed that over-expression of splicing factors, among them htra2-beta1 (WP3), increases the amount of correctly spliced CFTR RNA, in minigenes carrying the 3849+10kb C->T mutation . Similar modulation is seen in epithelial cell lines derived from unrelated CF patients, carrying this mutation. In these cells, the CFTR splicing modulation led to activation of the CFTR Clˉ channel and restored its function. Restoration of the CFTR function was also obtained by administration of sodium butyrate, a histone deacetylase inhibitor, previously shown to up-regulate the expression of splicing factors, such as htra2-beta1 . The pathophysiological mechanisms will be analyzed as follows:
Aim 1: Differences in expression levels of spliceosome components that might modify the splicing pattern will be analyzed using the chip from WP6 and candidate genes will be verified using RT-PCR. Significant differences in the expression pattern between cell lines will further be studied to identify natural sequence variations, which modify the splicing pattern of the CFTR gene.
Aim 2: To generate a knock-in mouse model carrying the 3849+10kb C->T mutation, a targeting vector will be constructed based on our 3849+10kb C->T minigene, inserted by homologous recombination in embryonic stem cells, and used to produce chimeric mice from which animals carrying the mutation will be bred.
Aim 3: The effect of spliceosome components on the phenotype will be assayed by transfer of the 3849+10kb C->T mutation into different mouse strains through mating. Crossing these mice with cholinergic signaling manipulated mice (WP6) will show the signaling dependency of CFTR functioning. The expression level of murine spliceosome components will be analyzed using micro arrays (WP6) and the histopathology will be compared with humans (Aim1).
Aim 4: To explore the effect of different compounds on the murine CFTR splicing pattern, homozygous and pregnant heterozygous knock-in mice will be treated with NaBu and with other small molecules shown to modify splicing, or to interact with signal transduction pathways from WP3. These small molecules will be added to the mice in their drinking water. The mice will be analyzed for all parameters described above.
 

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Nissim-Rafinia, M., et al., Cellular and viral splicing factors can modify the splicing pattern of CFTR transcripts carrying splicing mutations. Hum. Mol. Genet., 2000. 2000: p. 1771-1778.

Chiba-Falek, O., et al., The molecular basis of disease variability among cystic fibrosis patients carrying the 3849+10 kb C→T mutation. Genomics, 1998. 53(3): p. 276-283.

Augarten, A., et al., Mild cystic fibrosis and normal or borderline sweat test in patients with the 3849 + 10 kb C→T mutation. Lancet, 1993. 342(8862): p. 25-26.

Kerem, B., et al., Identification of the cystic fibrosis gene: genetic analysis. Science, 1989. 245(4922): p. 1073-1080.