中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2022, Vol. 17 ›› Issue (11): 2530-2536.doi: 10.4103/1673-5374.339005

• 原著:退行性病与再生 • 上一篇    下一篇

神经-肿瘤腹侧抗原1改善脊髓性肌萎缩的机制

  

  • 出版日期:2022-11-15 发布日期:2022-04-23
  • 基金资助:
    国家自然科学基金项目（32000841）；江苏省南通市科技计划项目（JC2018090）；江苏省研究生科研创新项目（KYCX18-2415）

NOVA1 promotes SMN2 exon 7 splicing by binding the UCAC motif and increases SMN protein expression

Li-Li Du1, 2, 3, #, Jun-Jie Sun2, #, Zhi-Heng Chen1, 2, #, Yi-Xiang Shao1, 2, *, Liu-Cheng Wu1, 4, *   

  1. 1Institute of Comparative Medicine, Nantong University, Nantong, Jiangsu Province, China; 2Key Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Co-Innovation Center of Neuroregeneration, Nantong University, Nantong, Jiangsu Province, China; 3Laboratory Animal Center, Nanjing University of Chinese Medicine, Nanjing, Jiangsu Province, China; 4Institute of Neuroscience, Soochow University, Suzhou, Jiangsu Province, China
  • Online:2022-11-15 Published:2022-04-23
  • Contact: Yi-Xiang Shao, shaoyx@ntu.edu.cn; Liu-Cheng Wu, hnwulc@ntu.edu.cn.
  • Supported by:
    This work was supported by the National Natural Science Foundation of China, No. 32000841 (to JJS); a grant from Science and Technology Project of Nantong of Jiangsu Province, No. JC2018090 (to LCW); a grant from Graduate Research and Innovation Project of Jiangsu Province, No. KYCX18-2415 (to LLD).

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摘要：

脊髓性肌萎缩是一种罕见的遗传性神经肌肉疾病，其致死率很高。据报道，同源基因运动神经元存活基因1和运动神经元存活基因2是脊髓性肌萎缩的致病因素之一。研究表明，运动神经元存活基因2外显子7列入会增加运动神经元存活基因表达，从而改善脊髓性肌萎缩的严重程度。神经肿瘤腹侧抗原是一种能在神经元中特异性表达的剪接因子，其也在神经系统和神经系统疾病的发展中发挥着关键的作用，但这种剪接因子是否也能影响脊髓性肌萎缩目前尚不清楚。（1）因此实验首先对脊髓性肌萎缩小鼠（基因型 smn-/-SMN22tg/0）和正常对照小鼠（基因型smn+/-SMN22tg/0）的不同组织中运动神经元存活基因2外显子7的表达情况进行分析，发现其大脑和脊髓组织中运动神经元存活基因2外显子7列入率较高，同时剪接因子神经-肿瘤腹侧抗原1也在神经组织中高表达，且两者在中枢神经系统中的表达变化同步。（2）因此进一步研究了这种剪接因子神经-肿瘤腹侧抗原1对疾病的影响，发现SMA小鼠在出生后1-7d时，脊髓前角中神经元数量减少，同时运动神经元中的神经-肿瘤腹侧抗原1随着SMA的发展而逐渐减少。（3）进而在体外过表达神经-肿瘤腹侧抗原1，这可增强U87MG细胞中运动神经元存活基因2外显子7的列入以及运动神经元存活基因蛋白的表达；而敲除神经-肿瘤腹侧抗原1时，上述变化相反。（4）最后，通过定点突变和RNA下拉实验表明，运动神经元存活基因2外显子7中UCAC结合基序对于神经-肿瘤腹侧抗原1 结合和促进外显子7的列入至关重要，且其中的核心碱基序列CA对运动神经元存活基因2外显子7列入的影响最大。上述结果说明神经-肿瘤腹侧抗原1可与运动神经元存活基因2外显子7中UCAC结合基序相互作用，增强运动神经元存活基因2外显子7的列入，从而增加运动神经元存活基因蛋白的表达。

https://orcid.org/0000-0002-3774-8069 (Li-Li Du); https://orcid.org/0000-0001-8710-7318 (Jun-Jie Sun); https://orcid.org/0000-0001-8029-3322 (Zhi-Heng Chen); https://orcid.org/0000-0002-8787-9454 (Yi-Xiang Shao); https://orcid.org/0000-0002-8053-1340 (Liu-Cheng Wu)

关键词: 脊髓性肌萎缩, 脊髓, 运动神经元, 神经-肿瘤腹侧抗原1, UCAC结合基序, 剪接因子, 运动神经元存活基因2剪接, 外显子7列入

Abstract: Spinal muscular atrophy (SMA) is a rare hereditary neuromuscular disease with a high lethality rate in infants. Variants in the homologous genes survival of motor neuron (SMN)1 and SMN2 have been reported to be SMA pathogenic factors. Previous studies showed that a high inclusion rate of SMN2 exon 7 increased SMN expression, which in turn reduced the severity of SMA. The inclusion rate of SMN2 exon 7 was higher in neural tissues than in non-neural tissues. Neuro-oncological ventral antigen (NOVA) is a splicing factor that is specifically and highly expressed in neurons. It plays a key role in nervous system development and in the induction of nervous system diseases. However, it remains unclear whether this splicing factor affects SMA. In this study, we analyzed the inclusion of SMN2 exon 7 in different tissues in a mouse model of SMA (genotype smn–/–SMN22tg/0) and littermate controls (genotype smn+/–SMN22tg/0). We found that inclusion level of SMN2 exon 7 was high in the brain and spinal cord tissue, and that NOVA1 was also highly expressed in nervous system tissues. In addition, SMN2 exon 7 and NOVA1 were expressed synchronously in the central nervous system. We further investigated the effects of NOVA1 on disease and found that the number of neurons in the anterior horn of spinal cord decreased in the mouse model of SMA during postnatal days 1–7, and that NOVA1 expression levels in motor neurons decreased simultaneously as spinal muscular atrophy developed. We also found that in vitro expression of NOVA1 increased the inclusion of SMN2 exon 7 and expression of the SMN2 protein in the U87MG cell line, whereas the opposite was observed when NOVA1 was knocked down. Finally, point mutation and RNA pull-down showed that the UCAC motif in SMN2 exon 7 plays a critical role in NOVA1 binding and promoting the inclusion of exon 7. Moreover, CA was more essential for the inclusion of exon 7 than the order of Y residues in the motif. Collectively, these findings indicate that NOVA1 interacts with the UCAC motif in exon 7 of SMN2, thereby enhancing inclusion of exon 7 in SMN2, which in turn increases expression of the SMN protein.

Key words: exon 7 inclusion, motor neuron, neuro-oncological ventral antigen 1, SMN2 splicing, spinal cord, spinal muscular atrophy, splicing factors, UCAC motif