Molecular pathomechanisms of congenital defects of neuromuscular transmission

先天性神经肌肉传递缺陷的分子病理机制

• 批准号: 17390252
• 负责人: OHNO Kinji
• 金额: $ 9.79万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17390252/
• 关键词: congenital myasthenic syndromes; mRNA splicing; 遺伝子; 神経科学; 蛋白質; 脳・神経; 脳神経疾患

The human acetylcholine receptor (AChR) a subunit carries a 75-nt extra exon P3A, which is placed between exons 3 and 4. Inclusion of exon P3A results in production of a nonfunctional a subunit, whereas skipping of exon P3A yields a functional AChR a subunit. In human skeletal muscle, each splicing product is generated at a one-to-one ratio. The exon P3A is not present in rodents or in any other species. Functional significance and splicing mechanism of exon P3A remain elusive. In a patient with congenital myasthenic syndromes, we identified a G-to-A substitution at position-8 of intron 3 immediately upstream of exon P3A. In patient's skeletal muscle, only nonfunctional exon P3A-retained transcript was observed. We also recapitulated aberrant exon P3A splicing in a minigene in HEK cells. In an effort to identify the underlying mechanisms, we introduced artificial mutations at and around the patient mutation, and found that the mutation likely disrupts a yet unidentified intronic splicing silencer. siRNA targeted against exon P3A efficiently knocked down the exon P3A-retained transcript, but the ratio of the functional exon P3A-skipped transcript was still to low to ameliorate defective AChR at the patient endplate, even if siRNA can be applied to the patient. In an effort to identify a splicing trans element that interacts with the intronic splicing silencer, we performed UV-crosslinking assay, immunoprecipitation against candidate molecules over expression and siRNA knock-down of the candidate molecules, and identified that two molecules are responsible for alternative splicing in normal skeletal muscle and also for aberrant splicing in the patient. We also screened 960 FDA pre-approved drugs that ameliorate aberrant splicing in the patient and identified a single effective compound. We also identified that the compound exerts its effect by enhancing expression of one of the splicing trans-elements identified above.

人乙酰胆碱受体（AChR）一个亚基携带一个75 nt的额外外显子P3A，位于外显子3和4之间。外显子P3A的包含导致产生无功能的a亚基，而外显子P3A的跳过产生功能的AChR a亚基。在人体骨骼肌中，每个拼接产物都以1:1的比例产生。外显子P3A不存在于啮齿类动物或任何其他物种中。外显子P3A的功能意义和剪接机制尚不清楚。在一例先天性肌无力综合征患者中，我们在P3A外显子上游的内含子3的第8位发现了G-to-A的替换。在患者骨骼肌中，仅观察到保留无功能外显子p3a的转录本。我们也在HEK细胞的一个小基因中重现了异常的外显子P3A剪接。为了确定潜在的机制，我们在患者突变处和周围引入了人工突变，并发现该突变可能破坏了一个尚未识别的内含子剪接沉默子。针对外显子P3A的siRNA有效地敲除了保留P3A的外显子转录本，但即使siRNA可以应用于患者，功能性外显子P3A跳过转录本的比例仍然很低，无法改善患者终板上有缺陷的AChR。为了确定与内含子剪接沉默子相互作用的剪接反式元件，我们进行了紫外交联试验，免疫沉淀对候选分子的过表达和候选分子的siRNA敲除，并确定了两个分子负责正常骨骼肌的选择性剪接和患者的异常剪接。我们还筛选了960种FDA预先批准的改善患者异常剪接的药物，并确定了一种有效的化合物。我们还发现，该化合物通过增强上述一种剪接反式元件的表达来发挥作用。