Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: Implications for tauopathies
Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: Implications for tauopathies
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DOI：
10.1073/pnas.0503150102
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发表时间：
2005-10-25
影响因子：
11.1
通讯作者：
Gallo, JM
中科院分区：
文献类型：
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作者：
Rodriguez-Martin, T;Garcia-Blanco, MA;Gallo, JM
与17号染色体相关的额颞叶痴呆伴帕金森综合征（FTDP-17）是由编码微管相关蛋白tau的基因突变引起的。一些FTDP-17突变影响外显子10剪接。为了纠正异常外显子10剪接，同时保留内源性转录控制，我们评估了使用剪接体介导的RNA反式剪接（SMaRT）重新编程tau mRNA的可行性。我们设计了一种含有人tau外显子10至13和与tau内含子9的3'端互补的结合结构域的预反式剪接分子。使用包含tau外显子9、10和11以及最小侧翼内含子序列的小基因作为靶标。使用针对预测剪接点相对侧的引物，对用小基因和前反式剪接分子共转染的SH-SY 5 Y细胞或COS细胞进行RT-PCR分析，产生含有外显子9至13的产物。嵌合产物的测序显示已经产生了精确的外显子9-外显子10连接，从而证明tau RNA可以通过反式剪接重编程。此外，通过使用含有全长内含子序列的小基因的相同范例，我们表明外显子10的顺式剪接排除可以通过反式剪接绕过，并且可以以约34%的效率实现外显子10（-）tau RNA到外显子10（+）tau RNA的转换。我们的研究结果表明，一个选择性剪接外显子可以被反式剪接取代，并开辟了新的治疗应用的SMaRT的tau蛋白病和其他疾病的异常选择性剪接。
Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the gene encoding the microtubule-associated protein, tau. Some FTDP-17 mutations affect exon 10 splicing. To correct aberrant exon 10 splicing while retaining endogenous transcriptional control, we evaluated the feasibility of using spliceosome-mediated RNA trans-splicing (SMaRT) to reprogram tau mRNA. We designed a pre-trans-splicing molecule containing human tau exons 10 to 13 and a binding domain complementary to the 3' end of tau intron 9. A minigene comprising tau exons 9, 10, and 11 and minimal flanking intronic sequences was used as a target. RT-PCR analysis of SH-SY5Y cells or COS cells cotransfected with a minigene and a pre-trans-splicing molecule using primers to opposite sides of the predicted splice junction generated products containing exons 9 to 13. Sequencing of the chimeric products showed that an exact exon 9-exon 10 junction had been created, thus demonstrating that tau RNA can be reprogrammed by trans-splicing. Furthermore, by using the same paradigm with a minigene containing full-length intronic sequences, we show that cis-splicing exclusion of exon 10 can be by-passed by trans-splicing and that conversion of exon 10(-) tau RNA into exon 10(+) tau RNA could be achieved with approximate to 34% efficiency. Our results demonstrate that an alternatively spliced exon can be replaced by trans-splicing and open the way to novel therapeutic applications of SMaRT for tauopathies and other disorders linked to aberrant alternative splicing.