运动神经元存活基因1（SMN1）突变通过影响SMN蛋白及其复合物的结构和/或功能而引发常染色体隐性遗传病-脊髓性肌萎缩症（SMA）。目前研究证实SMN蛋白磷酸化会调控其自身蛋白稳定性、SMN复合物形成和定位等。本课题组前期证实10余种SMN1基因突变，其中位于已证实磷酸化位点的突变（Y109C）和潜在磷酸化位点的突变（Y277C）均未影响全长SMN1基因的转录和全长SMN蛋白的表达，其深入的致病机制并未明确。本研究以SMA点突变患者样本为基础，利用体外实验结合体内研究方法共同探讨2种新突变对SMN蛋白磷酸化程度的改变，明确其对SMN蛋白自身稳定性、SMN复合物形成及其在亚细胞定位的影响。我们期望从SMN蛋白磷酸化修饰的角度探索SMN1基因新突变的致病机制，为理解新突变在SMA疾病发生中的作用提供实验数据和理论依据，并将有助于从磷酸化调控SMN复合物形成的新角度探索SMA治疗的研究方向。

Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder characterized by the degeneration of spinal motor neurons and muscle atrophy. As the primary genetic cause of infant mortality and the second most common autosomal recessive genetic disorder, SMA affects one in every six thousand live births with a carrier frequency of one in forty people. SMA is caused by the survival motor neuron gene 1 (SMN1) mutations,including homozygous deletion or subtle mutations. Full length transcripts are almost exclusively produced by SMN1 gene, wherase the predominant form encoded by SMN2 lacks exon 7.The truncated transcripts lacking exon 7 encodes a shorter unstable protein lacking C terminus. The pathogenic mechanism of SMN1 subtle mutation needs to be further elucidated especially when it does not affect the production of full length transcripts and protein..The human SMN complex consisting of nine core units (SMN protein, Germins 2-8, and the unr interacting protein (unrip))fulfils essential functions in the assembly of small nuclear ribonuclear proteins(snRNPs), which are key components in the splicing of pre-mRNAs. The SMN complex also localizes to the nucleus, where it accumulates in Cajal Bodies(CBs). The phosphorylation in the residues of serine, threonine and tyrosine of SMN protein would regulate its function in snRNPs assembly, affect its stability and play important role in the formation and/or subcelluar localization of SMN complex. Recent study has revealed that three tyrosine phosphorylations, which lie within the TUDOR domain of human SMN (Y109,Y127 and Y130), would regulate the SMN localization..Our team have confirmed more than ten SMN1 gene mutations in Chinese SMA patients. Two of them （p.Tyr109Cys and p. Tyr277Cys）were novel and located in the identified or potential phosphorylation sites. Our previous study revealed that the two mutations did not affect the production of full length transcripts and SMN protein. However,the detailed pathogenic mechanism of the missense mutations are unknown. .This project aims to study the pathogenic mechanism of the above mutations by in vivo and vitro tests. The effect of them on the SMN protein stability would be analyzed by incubation in the presence of cycloheximide. An co-immunoprecipitation assay would be done to determine whether the mutations affect the formation of SMN complex by disturbing the binding of SMN to Germin2-8/Sm/unrip. And immunofluoresence analysis is performed to evaluate the subcelluar distribution of the caja bodies formed by SMN. This research would provide the basic effect of the SMN1 gene novel mutations on SMN protein by influencing its phosphorylation,which might be helpful to reveal the pathogenic mechanism of the two SMN1 missense mutations at the post-translational level(phosphoregulation).