In vitro modification of Tissue-Specific alternative splicing of the mouse TPM3 gene with antisense oligonucleotides
Johnson, C., Wilton, S. and Fletcher, S. (2005) In vitro modification of Tissue-Specific alternative splicing of the mouse TPM3 gene with antisense oligonucleotides. In: 4th Australasian Gene Therapy Society Meeting, 27 - 29 April 2005, Rydges Hotel, Carlton, VIC.
Alternative splicing is one of the principle mechanisms to increase protein diversity in higher eukaryotes. From a single pre-mRNA, multiple mRNAs can be generated by selective exon inclusion, exclusion or, in some instances, exon extension or shortening. This mechanism dramatically increases the coding capacity of a single gene. Numerous genes undergo tissue-specific and/or developmentallyregulated splicing, an example of which is the tropomyosin 3 (TPM3) gene. Antisense oligonucleotides (AOs) were applied to cultured cells to investigate modifying tissue-specific splicing of the TPM3 gene transcript. It was found that AOs targeting mutually exclusive exons 6a or 6b were able to partially redirect splicing from one tissue-specific exon to the other. AOs annealing to exonic sequences showed the highest efficacy which suggests that these sequences contain exonic splicing enhancer (ESEs) elements controlling the splicing process. With both single and multiple transfections (blocking both exons 6a and 6b) exclusion of both exons from the same transcript could not be induced, suggesting some mechanism to ensure inclusion of either mutually exclusive exon. Conversely, a single AO blocking constitutively spliced exon 7 was able to induce exclusion of this exon. These results are contrary to the general model of exon selection, where constitutive spliced exons are strongly selected for by the splicing machinery, whereas alternatively spliced exons are supposedly weakly selected. AO-induced modification of tissue-specific splicing patterns indicates that this approach could potentially to be used therapeutically to alter isoform expression in a variety of conditions.
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