The deep intronic c.903+469T>C mutation in the MTRR gene creates an SF2/ASF binding exonic splicing enhancer, which leads to pseudoexon activation and causes the cblE type of homocystinuria.
The deep intronic c.903+469T>C mutation in the MTRR gene creates an SF2/ASF binding exonic splicing enhancer, which leads to pseudoexon activation and causes the cblE type of homocystinuria.
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DOI：
10.1002/humu.21206
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发表时间：
2010-04
期刊：
影响因子：
3.9
通讯作者：
Andresen, Brage S.
中科院分区：
文献类型：
--
作者：
Homolova, Katerina;Zavadakova, Petra;Doktor, Thomas Koed;Schroeder, Lisbeth Dahl;Kozich, Viktor;Andresen, Brage S.
深度内含子突变作为人类疾病的可能原因经常被忽视。MTRR基因中的深度内含子突变c.903+ 469 T>C是引起cblE型同型胱氨酸尿症的最常见突变。众所周知，它与前体mRNA错误剪接有关，导致假外显子包含;然而，其病理机制仍不清楚。我们使用小基因来证明这种突变是MTRR假外显子包含的直接原因，并且由于次优的5′剪接位点，假外显子通常不被识别。在假外显子中，我们发现了一个外显子剪接增强子（ESE），它被突变激活。共转染和siRNA实验表明，假外显子包含取决于细胞的SF 2/ASF的量和体外RNA结合试验表明，显着增加SF 2/ASF结合突变体MTRR ESE。突变MTRR ESE序列与选择性剪接的MST 1 R原癌基因的ESE相同，这表明该ESE可能经常参与剪接调节。我们的研究结论性地表明，一个内含子的单核苷酸的变化是足以引起假外显子激活通过创建一个功能性ESE，它结合了一个特定的剪接因子。我们认为，这种机制可能会导致遗传性疾病更频繁地比以前报道的。Mutat 31：437-444，2010.© 2010 Wiley-Liss公司。
Deep intronic mutations are often ignored as possible causes of human diseases. A deep intronic mutation in the MTRR gene, c.903+469T>C, is the most frequent mutation causing the cblE type of homocystinuria. It is well known to be associated with pre-mRNA missplicing, resulting in pseudoexon inclusion; however, the pathological mechanism remains unknown. We used minigenes to demonstrate that this mutation is the direct cause of MTRR pseudoexon inclusion, and that the pseudoexon is normally not recognized due to a suboptimal 5′ splice site. Within the pseudoexon we identified an exonic splicing enhancer (ESE), which is activated by the mutation. Cotransfection and siRNA experiments showed that pseudoexon inclusion depends on the cellular amounts of SF2/ASF and in vitro RNA-binding assays showed dramatically increased SF2/ASF binding to the mutant MTRR ESE. The mutant MTRR ESE sequence is identical to an ESE of the alternatively spliced MST1R proto-oncogene, which suggests that this ESE could be frequently involved in splicing regulation. Our study conclusively demonstrates that an intronic single nucleotide change is sufficient to cause pseudoexon activation via creation of a functional ESE, which binds a specific splicing factor. We suggest that this mechanism may cause genetic disease much more frequently than previously reported. Hum Mutat 31:437–444, 2010. © 2010 Wiley-Liss, Inc.
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