PDF(Pubmed)
Abstract：
Pseudoexons are nonfunctional intronic sequences that can be activated by deep-intronic sequence variation. Activation increases pseudoexon inclusion in mRNA and interferes with normal gene expression. The PCCA c.1285-1416A>G variation activates a pseudoexon and causes the severe metabolic disorder propionic acidemia by deficiency of the propionyl-CoA carboxylase enzyme encoded by PCCA and PCCB. We characterized this pathogenic pseudoexon activation event in detail and identified hnRNP A1 to be important for normal repression. The PCCA c.1285-1416A>G variation disrupts an hnRNP A1-binding splicing silencer and simultaneously creates a splicing enhancer. We demonstrate that blocking this region of regulation with splice-switching antisense oligonucleotides restores normal splicing and rescues enzyme activity in patient fibroblasts and in a cellular model created by CRISPR gene editing. Interestingly, the PCCA pseudoexon offers an unexploited potential to upregulate gene expression because healthy tissues show relatively high inclusion levels. By blocking inclusion of the nonactivated wild-type pseudoexon, we can increase both PCCA and PCCB protein levels, which increases the activity of the heterododecameric enzyme. Surprisingly, we can increase enzyme activity from residual levels in not only patient fibroblasts harboring PCCA missense variants but also those harboring PCCB missense variants. This is a potential treatment strategy for propionic acidemia.
摘要：
假外显子是可以被深内含子序列变异激活的非功能性内含子序列。激活增加mRNA中的假外显子包涵体并干扰正常基因表达。PCCAc.1285-1416A>G变异激活假外显子并通过缺乏PCCA和PCCB编码的丙酰基-CoA羧化酶引起严重的代谢紊乱丙酸血症。我们详细表征了这种致病性假外显子激活事件，并确定了hnRNPA1对正常抑制很重要。PCCAc.1285-1416A>G变异破坏了hnRNPA1结合剪接沉默子，同时产生了剪接增强子。我们证明，用剪接转换反义寡核苷酸阻断该调节区域可恢复正常剪接并挽救患者成纤维细胞和通过CRISPR基因编辑创建的细胞模型中的酶活性。有趣的是,PCCA假外显子具有未开发的上调基因表达的潜力,因为健康组织显示出相对较高的包涵体水平.通过阻断包含未活化的野生型假外显子，我们可以增加PCCA和PCCB蛋白水平，这增加了异十二聚体酶的活性。令人惊讶的是,我们不仅可以提高含有PCCA错义变体的患者成纤维细胞的残留水平,还可以提高含有PCCB错义变体的患者成纤维细胞的酶活性.这是丙酸血症的潜在治疗策略。
