Abstract

Spinal muscular atrophy (SMA) is caused by mutation or deletion of survival motor neuron 1 (SMN1) and retention of SMN2 leading to SMN protein deficiency. We developed an immortalized mouse embryonic fibroblast (iMEF) line in which full-length wild-type Smn (flwt-Smn) can be conditionally deleted using Cre recombinase. iMEFs lacking flwt-Smn are not viable. We tested the SMA patient SMN1 missense mutation alleles A2G, D44V, A111G, E134K and T274I in these cells to determine which human SMN (huSMN) mutant alleles can function in the absence of flwt-Smn. All missense mutant alleles failed to rescue survival in the conditionally deleted iMEFs. Thus, the function lost by these mutations is essential to cell survival. However, co-expression of two different huSMN missense mutants can rescue iMEF survival and small nuclear ribonucleoprotein (snRNP) assembly, demonstrating intragenic complementation of SMN alleles. In addition, we show that a Smn protein lacking exon 2B can rescue iMEF survival and snRNP assembly in the absence of flwt-Smn, indicating exon 2B is not required for the essential function of Smn. For the first time, using this novel cell line, we can assay the function of SMN alleles in the complete absence of flwt-Smn.

中文翻译：

---

在细胞培养中条件性删除 SMN 可识别功能性 SMN 等位基因

摘要

脊髓性肌萎缩症 (SMA) 是由存活运动神经元 1 ( SMN1 )的突变或缺失和SMN2 的保留导致 SMN 蛋白缺乏引起的。我们开发了一种永生化小鼠胚胎成纤维细胞 (iMEF) 系，其中可以使用 Cre 重组酶有条件地删除全长野生型 Smn (flwt-Smn)。缺少 flwt-Smn 的 iMEF 不可行。我们测试了 SMA 患者SMN1这些细胞中的错义突变等位基因 A2G、D44V、A111G、E134K 和 T274I，以确定哪些人类 SMN (huSMN) 突变等位基因可以在不存在 flwt-Smn 的情况下发挥作用。所有错义突变等位基因都未能挽救有条件删除的 iMEF 中的存活。因此，这些突变丧失的功能对细胞存活至关重要。然而，两种不同的 huSMN 错义突变体的共表达可以挽救 iMEF 存活和小核核糖核蛋白 (snRNP) 组装，证明 SMN 等位基因的基因内互补。此外，我们表明，在没有 flwt-Smn 的情况下，缺乏外显子 2B 的 Smn 蛋白可以挽救 iMEF 存活和 snRNP 组装，表明外显子 2B 不是 Smn 的基本功能所必需的。首次使用这种新型细胞系，我们可以在完全没有 flwt-Smn 的情况下测定 SMN 等位基因的功能。