Laboratoire de Biologie Cellulaire des Membranes, Programme de Biologie Cellulaire, Institut Jacques-Monod, UMR 7592 CNRS, Université Paris Diderot, Sorbonne Paris Cité, 15 rue Hélène Brion, 75205 Paris cedex 13, France.
J Cell Sci. 2012 Jun 15;125(Pt 12):2862-74. doi: 10.1242/jcs.096255. Epub 2012 Mar 27.
The spinal muscular atrophy (SMA) gene product SMN forms with gem-associated protein 2-8 (Gemin2-8) and unrip (also known as STRAP) the ubiquitous survival motor neuron (SMN) complex, which is required for the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), their nuclear import and their localization to subnuclear domain Cajal bodies (CBs). The concentration of the SMN complex and snRNPs in CBs is reduced upon SMN deficiency in SMA cells. Subcellular localization of the SMN complex is regulated in a phosphorylation-dependent manner and the precise mechanisms remain poorly understood. Using co-immunoprecipitation in HeLa cell extracts and in vitro protein binding assays, we show here that the SMN complex and its component Gemin8 interact directly with protein phosphatase PP1γ. Overexpression of Gemin8 in cells increases the number of CBs and results in targeting of PP1γ to CBs. Moreover, depletion of PP1γ by RNA interference enhances the localization of the SMN complex and snRNPs to CBs. Consequently, the interaction between SMN and Gemin8 increases in cytoplasmic and nuclear extracts of PP1γ-depleted cells. Two-dimensional protein gel electrophoresis revealed that SMN is hyperphosphorylated in nuclear extracts of PP1γ-depleted cells and expression of PP1γ restores these isoforms. Notably, SMN deficiency in SMA leads to the aberrant subcellular localization of Gemin8 and PP1γ in the atrophic skeletal muscles, suggesting that the function of PP1γ is likely to be affected in disease. Our findings reveal a role of PP1γ in the formation of the SMN complex and the maintenance of CB integrity. Finally, we propose Gemin8 interaction with PP1γ as a target for therapeutic intervention in SMA.
脊髓性肌萎缩症（SMA）基因产物 SMN 与Gem 相关蛋白 2-8（Gemin2-8）和 unrip（也称为 STRAP）形成普遍存在的运动神经元存活（SMN）复合物，该复合物是剪接体小核核糖核蛋白（snRNP）组装、核内导入及其定位于亚核域 Cajal 体（CBs）所必需的。在 SMA 细胞中 SMN 缺乏时，CBs 中 SMN 复合物和 snRNP 的浓度降低。SMN 复合物的亚细胞定位受磷酸化依赖性调节，但其确切机制仍知之甚少。我们在此使用 HeLa 细胞提取物中的共免疫沉淀和体外蛋白结合测定，表明 SMN 复合物及其组分 Gemin8 与蛋白磷酸酶 PP1γ 直接相互作用。细胞中 Gemin8 的过表达增加了 CB 的数量，并导致 PP1γ 靶向 CB。此外，RNA 干扰耗尽 PP1γ 增强了 SMN 复合物和 snRNP 向 CB 的定位。因此，在 PP1γ 耗尽细胞的细胞质和核提取物中，SMN 与 Gemin8 的相互作用增加。二维蛋白质凝胶电泳显示，PP1γ 耗尽细胞的核提取物中 SMN 过度磷酸化，表达 PP1γ 可恢复这些同工型。值得注意的是，SMA 中的 SMN 缺乏导致萎缩骨骼肌中 Gemin8 和 PP1γ 的异常亚细胞定位，这表明 PP1γ 的功能可能在疾病中受到影响。我们的研究结果揭示了 PP1γ 在 SMN 复合物形成和 CB 完整性维持中的作用。最后，我们提出 Gemin8 与 PP1γ 的相互作用作为 SMA 治疗干预的靶点。
