Neural Regeneration Research ›› 2022, Vol. 17 ›› Issue (11): 2530-2536.doi: 10.4103/1673-5374.339005

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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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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