Molecular pathomechanisms of congenital defects of neuromuscular transmission

人乙酰胆碱受体（AChR）α亚基携带75-nt的额外外显子P3 A，其位于外显子3和4之间。外显子P3 A的包含导致产生非功能性α亚基，而外显子P3 A的跳跃产生功能性AChR α亚基。在人类骨骼肌中，每种剪接产物以一比一的比例产生。外显子P3 A不存在于啮齿动物或任何其他物种中。外显子P3 A的功能意义和剪接机制尚不清楚。在先天性肌无力综合征患者中，我们发现了一个G-到-A取代的位置-8内含子3紧接外显子P3 A上游。在患者的骨骼肌中，仅观察到非功能性外显子P3 A保留转录本。我们还概括了异常外显子P3 A剪接在一个小基因在HEK细胞。为了确定潜在的机制，我们在患者突变处及其周围引入了人工突变，并发现该突变可能破坏了尚未鉴定的内含子剪接沉默子。靶向外显子P3 A的siRNA有效地敲低了外显子P3 A保留的转录本，但即使siRNA可以应用于患者，功能性外显子P3 A跳过的转录本的比例仍然太低，无法改善患者终板处的AChR缺陷。为了鉴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UV交联测定、针对候选分子的过表达的免疫沉淀和候选分子的siRNA敲低，并鉴定了两种分子负责正常骨骼肌中的选择性剪接和患者中的异常剪接。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。我们还鉴定了该化合物通过增强上述剪接反式元件之一的表达来发挥其作用。

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.