Molecular pathomechanisms of congenital defects of neuromuscular transmission

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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.