Charroux B, Pellizzoni L, Perkinson R A, Yong J, Shevchenko A, Mann M, Dreyfuss G
Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6148, USA.
J Cell Biol. 2000 Mar 20;148(6):1177-86. doi: 10.1083/jcb.148.6.1177.
The survival of motor neurons (SMN) protein, the product of the neurodegenerative disease spinal muscular atrophy (SMA) gene, is localized both in the cytoplasm and in discrete nuclear bodies called gems. In both compartments SMN is part of a large complex that contains several proteins including Gemin2 (formerly SIP1) and the DEAD box protein Gemin3. In the cytoplasm, the SMN complex is associated with snRNP Sm core proteins and plays a critical role in spliceosomal snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing by serving in the regeneration of spliceosomes. These functions are likely impaired in cells of SMA patients because they have reduced levels of functional SMN. Here, we report the identification by nanoelectrospray mass spectrometry of a novel component of the SMN complex that we name Gemin4. Gemin4 is associated in vivo with the SMN complex through a direct interaction with Gemin3. The tight interaction of Gemin4 with Gemin3 suggests that it could serve as a cofactor of this DEAD box protein. Gemin4 also interacts directly with several of the Sm core proteins. Monoclonal antibodies against Gemin4 efficiently immunoprecipitate the spliceosomal U snRNAs U1 and U5 from Xenopus oocytes cytoplasm. Immunolocalization experiments show that Gemin4 is colocalized with SMN in the cytoplasm and in gems. Interestingly, Gemin4 is also detected in the nucleoli, suggesting that the SMN complex may also function in preribosomal RNA processing or ribosome assembly.
运动神经元存活（SMN）蛋白是神经退行性疾病脊髓性肌萎缩症（SMA）基因的产物，定位于细胞质和称为宝石样小体的离散核小体中。在这两个区室中，SMN都是一个大型复合物的一部分，该复合物包含几种蛋白质，包括Gemin2（以前称为SIP1）和DEAD盒蛋白Gemin3。在细胞质中，SMN复合物与snRNP Sm核心蛋白相关，并在剪接体snRNP组装中起关键作用。在细胞核中，SMN通过参与剪接体的再生而参与前体mRNA剪接。SMA患者细胞中的这些功能可能受损，因为他们功能性SMN的水平降低。在这里，我们报告通过纳米电喷雾质谱鉴定出一种新的SMN复合物成分，我们将其命名为Gemin4。Gemin4在体内通过与Gemin3的直接相互作用与SMN复合物相关联。Gemin4与Gemin3的紧密相互作用表明它可以作为这种DEAD盒蛋白的辅因子。Gemin4还直接与几种Sm核心蛋白相互作用。针对Gemin4的单克隆抗体可从非洲爪蟾卵母细胞细胞质中有效免疫沉淀剪接体U snRNAs U1和U5。免疫定位实验表明，Gemin4与SMN在细胞质和宝石样小体中共定位。有趣的是，在核仁中也检测到了Gemin4，这表明SMN复合物也可能在核糖体前体RNA加工或核糖体组装中起作用。
