Oxidative Stress Triggers Body-Wide Skipping of Multiple Exons of the Spinal Muscular Atrophy Gene

Humans carry two nearly identical copies of Survival Motor Neuron gene: SMN1 and SMN2. Loss of SMN1 leads to spinal muscular atrophy (SMA), the most frequent genetic cause of infant mortality. While SMN2 cannot compensate for the loss of SMN1 due to predominant skipping of exon 7, correction of SMN2 exon 7 splicing holds the promise of a cure for SMA. Previously, we used cell-based models coupled with a multi-exon-skipping detection assay (MESDA) to demonstrate the vulnerability of SMN2 exons to aberrant splicing under the conditions of oxidative stress (OS). Here we employ a transgenic mouse model and MESDA to examine the OS-induced splicing regulation of SMN2 exons. We induced OS using paraquat that is known to trigger production of reactive oxygen species and cause mitochondrial dysfunction. We show an overwhelming co-skipping of SMN2 exon 5 and exon 7 under OS in all tissues except testis. We also show that OS increases skipping of SMN2 exon 3 in all tissues except testis. We uncover several new SMN2 splice isoforms expressed at elevated levels under the conditions of OS. We analyze cis-elements and transacting factors to demonstrate the diversity of mechanisms for splicing misregulation under OS. Our results of proteome analysis reveal downregulation of hnRNP H as one of the potential consequences of OS in brain. Our findings suggest SMN2 as a sensor of OS with implications to SMA and other diseases impacted by low levels of SMN protein.

人类体内存在两份几乎完全一致的运动神经元生存基因（Survival Motor Neuron, SMN）拷贝：SMN1与SMN2。SMN1的缺失会引发脊髓性肌萎缩症（Spinal Muscular Atrophy, SMA），这是婴儿死亡最常见的遗传性病因。由于SMN2的7号外显子普遍发生剪接跳跃，其无法弥补SMN1缺失带来的功能缺陷，但校正SMN2 7号外显子的剪接过程，有望成为治愈SMA的潜在手段。此前，我们结合基于细胞的实验模型与多外显子跳跃检测实验（Multi-exon-skipping detection assay, MESDA），证实了氧化应激（Oxidative Stress, OS）条件下，SMN2外显子极易发生异常剪接。本研究利用转基因小鼠模型与MESDA，探究OS诱导的SMN2外显子剪接调控机制。我们采用百草枯构建OS模型——百草枯可诱导活性氧簇生成并引发线粒体功能障碍。实验结果显示，在OS条件下，除睾丸组织外，所有受试组织中SMN2的5号与7号外显子均发生显著的共同剪接跳跃。同时我们证实，OS可提升除睾丸组织外所有组织中SMN2 3号外显子的剪接跳跃率。我们还发现了数种在OS条件下表达量显著升高的新型SMN2剪接异构体。通过分析顺式作用元件与反式作用因子，我们阐明了OS条件下剪接异常调控的多种分子机制。蛋白质组学分析结果显示，大脑组织中异质核糖核蛋白H（heterogeneous nuclear ribonucleoprotein H, hnRNP H）的表达下调，是OS引发的潜在效应之一。本研究结果表明，SMN2可作为OS的感应因子，这一发现对SMA及其他因SMN蛋白水平低下所累及的疾病均具有重要研究价值。