Takarada Toru, Ar Rochmah Mawaddah, Harahap Nur Imma Fatimah, Shinohara Masakazu, Saito Toshio, Saito Kayoko, Lai Poh San, Bouike Yoshihiro, Takeshima Yasuhiro, Awano Hiroyuki, Morioka Ichiro, Iijima Kazumoto, Nishio Hisahide, Takeuchi Atsuko
Analytical Laboratory, Kobe Pharmaceutical University, Kobe, Japan.
Department of Community Medicine and Social Healthcare Science, Kobe University Graduate School of Medicine, Kobe, Japan.
Brain Dev. 2017 Aug;39(7):606-612. doi: 10.1016/j.braindev.2017.03.002. Epub 2017 Mar 31.
Most spinal muscular atrophy (SMA) patients are homozygous for survival of motor neuron 1 gene (SMN1) deletion. However, some SMA patients carry an intragenic SMN1 mutation. Such patients provide a clue to understanding the function of the SMN protein and the role of each domain of the protein. We previously identified mutations in the Tudor domain and C-terminal region of the SMN protein in three Japanese SMA patients. To clarify the effect of these mutations on protein stability, we conducted expression assays of SMN with mutated domains.
Patients A and B carried a mutation in SMN1 exon 3, which encodes a Tudor domain, c.275G>C (p.Trp92Ser). Patient C carried a mutation in SMN1 exon 6, which encodes a YG-box; c.819_820insT (p.Thr274Tyrfs). We constructed plasmid expression vectors containing wild-type and mutant SMN1 cDNAs. After transfection of HeLa cells with the expression plasmids, RNA and protein were isolated and analyzed by reverse-transcription PCR and western blot analysis.
The abundance of wild-type and mutant SMN1 transcripts in HeLa cells was almost the same. However, western blot analysis showed lower levels of mutant SMN proteins compared with wild-type SMN. In mutant SMN proteins, it is noteworthy that the level of the p.Thr274Tyrfs mutant was much reduced compared with that of the p.Trp92Ser mutant.
SMN mutations may affect the stability and levels of the protein.
大多数脊髓性肌萎缩症（SMA）患者的运动神经元存活基因1（SMN1）缺失为纯合子。然而，一些SMA患者携带基因内SMN1突变。这类患者为理解SMN蛋白的功能及其各结构域的作用提供了线索。我们之前在三名日本SMA患者中鉴定出了SMN蛋白的Tudor结构域和C末端区域的突变。为阐明这些突变对蛋白质稳定性的影响，我们对具有突变结构域的SMN进行了表达分析。
患者A和B在编码Tudor结构域的SMN1外显子3中携带一个突变，c.275G>C（p.Trp92Ser）。患者C在编码YG盒的SMN1外显子6中携带一个突变，c.819_820insT（p.Thr274Tyrfs）。我们构建了包含野生型和突变型SMN1 cDNA的质粒表达载体。用表达质粒转染HeLa细胞后，分离RNA和蛋白质，并通过逆转录PCR和蛋白质印迹分析进行检测。
HeLa细胞中野生型和突变型SMN1转录本的丰度几乎相同。然而，蛋白质印迹分析显示，与野生型SMN相比，突变型SMN蛋白水平较低。在突变型SMN蛋白中，值得注意的是，与p.Trp92Ser突变体相比，p.Thr274Tyrfs突变体的水平显著降低。
SMN突变可能影响蛋白质的稳定性和水平。
