Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University, Prague 2, Czech Republic.
Hum Mutat. 2010 Apr;31(4):437-44. doi: 10.1002/humu.21206.
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 mis-splicing, 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.
深内含子突变通常被忽视为人类疾病的可能原因。MTRR 基因中的深内含子突变 c.903+469T>C 是导致同型胱氨酸尿症 cblE 型的最常见突变。它与前体 mRNA 剪接错误有关，导致假外显子包含；然而，其病理机制尚不清楚。我们使用小基因证明该突变是 MTRR 假外显子包含的直接原因，并且由于 5'剪接位点不佳，假外显子通常不被识别。在假外显子中，我们鉴定出一个外显子剪接增强子（ESE），该增强子通过突变被激活。共转染和 siRNA 实验表明，假外显子包含取决于 SF2/ASF 的细胞数量，体外 RNA 结合实验表明 SF2/ASF 与突变的 MTRR ESE 的结合显著增加。突变的 MTRR ESE 序列与可选择性剪接的 MST1R 原癌基因的 ESE 相同，这表明该 ESE 可能经常参与剪接调控。我们的研究确凿地证明，单个核苷酸的内含子变化足以通过创建功能 ESE 来激活假外显子，该 ESE 结合特定的剪接因子。我们建议，这种机制可能导致遗传疾病的发生频率比以前报道的要高得多。
