Le T T, Coovert D D, Monani U R, Morris G E, Burghes A H
Department of Medical Biochemistry, Ohio State University, Columbus 43210, USA.
Neurogenetics. 2000 Sep;3(1):7-16. doi: 10.1007/s100480000090.
Spinal muscular atrophy (SMA) is caused by mutations in the telomeric copy of the survival motor neuron gene (SMN1) but not mutations in the centromeric copy (SMN2). The critical difference between the two genes is a nucleotide difference in exon 7 that affects splicing and causes this exon to be spliced out of most SMN2 transcripts. A majority of the SMN1 gene transcripts contain exon 7. To investigate the effect of exon loss or mutations in SMN on protein localization, 15 SMN constructs were prepared and transfected into COS-7 cells and fibroblasts derived from a type I SMA patient. Loss of exon 5 (Iso5-SMN), a putative nuclear localization signal in exon 2, and the G279V point mutation had little effect on SMN localization. Loss of both exons 5 and 7 (Iso57-SMN) resulted in low gem numbers and the localization of the majority of the SMN protein to the cytoplasm. Cells expressing constructs lacking only exon 7 (Iso7-SMN) did not produce large numbers of gems in general, although there were a few cells that had a staining pattern similar to cells transfected with a full-length (Full-SMN) construct. HeLa cells stably transfected with full-length SMN or Iso7-SMN did not overexpress SMN, and both constructs produced a similar localization of the protein, although Iso7-SMN formed gems less efficiently. Removal of the amino-terminus, deletion of the conserved domain in exon 2A, and the mutation Y272C all caused accumulation of SMN in the nucleus, sometimes in large aggregates. These findings suggest that the amino-terminal domain of SMN is essential for the correct cellular distribution of SMN, whereas Iso7-SMN is capable of forming gems, albeit at a reduced efficiency.
脊髓性肌萎缩症（SMA）是由生存运动神经元基因（SMN1）端粒拷贝中的突变引起的，而非着丝粒拷贝（SMN2）中的突变。这两个基因的关键差异在于外显子7中的一个核苷酸差异，该差异影响剪接并导致该外显子从大多数SMN2转录本中被剪接掉。大多数SMN1基因转录本包含外显子7。为了研究SMN中外显子缺失或突变对蛋白质定位的影响，制备了15种SMN构建体并将其转染到COS-7细胞和来自一名I型SMA患者的成纤维细胞中。外显子5（Iso5-SMN）缺失、外显子2中一个假定的核定位信号缺失以及G279V点突变对SMN定位影响很小。外显子5和7都缺失（Iso57-SMN）导致宝石样结构数量减少，且大多数SMN蛋白定位于细胞质。一般来说，表达仅缺失外显子7的构建体（Iso7-SMN）的细胞不会产生大量宝石样结构，尽管有少数细胞的染色模式与转染全长（Full-SMN）构建体的细胞相似。稳定转染全长SMN或Iso7-SMN的HeLa细胞并未过度表达SMN，且两种构建体产生的蛋白质定位相似，尽管Iso7-SMN形成宝石样结构的效率较低。去除氨基末端区域、缺失外显子2A中的保守结构域以及Y272C突变均导致SMN在细胞核中积累，有时形成大的聚集体。这些发现表明，SMN氨基末端区域对于SMN在细胞中的正确分布至关重要，而Iso7-SMN能够形成宝石样结构，尽管效率较低。
