Association of galectin-1 and galectin-3 with Gemin4 in complexes containing the SMN protein.
Association of galectin-1 and galectin-3 with Gemin4 in complexes containing the SMN protein.
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DOI：
10.1093/nar/29.17.3595
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发表时间：
2001-09
影响因子：
14.9
通讯作者：
Jung Woo Park;P. G. Voss;Sharon Grabski;John L. Wang;R. Patterson
中科院分区：
文献类型：
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作者：
Jung Woo Park;P. G. Voss;Sharon Grabski;John L. Wang;R. Patterson
在之前的研究中，我们发现半乳糖凝集素-1和半乳糖凝集素-3是前mrna剪接所需的因子，在无细胞系统中进行了检测。利用酵母双杂交筛选，以半乳糖凝集素-1为诱饵，鉴定出Gemin4为推测的相互作用蛋白。Gemin4是一个大分子复合物的一个组成部分，包含大约15个多肽，包括运动神经元存活蛋白。兔抗半乳糖凝集素-1与半乳糖凝集素-1共从HeLa细胞核提取物中免疫沉淀，鉴定出该复合物中的多肽：SMN， Gemin2和snRNPs的Sm多肽。在谷胱甘肽s -转移酶（GST）下拉试验中证实了Gemin4和半乳糖凝集素-1之间的直接相互作用。我们还发现半乳糖凝集素-3与Gemin4相互作用，并构成与半乳糖凝集素-1共免疫沉淀复合物的一个组成部分。事实上，当加入无细胞剪接实验时，Gemin4或半乳糖凝集素-3的片段表现出显性的负作用。例如，在外源添加半乳糖凝集素-3多肽的n端结构域存在时，观察到剪接的剂量依赖性抑制。相比之下，平行添加完整的半乳糖凝集素-3多肽或c端结构域都不能产生相同的效果。使用天然凝胶电泳检测由剪接提取物形成的复合物，我们发现随着n端结构域的加入，放射性标记的pre-mRNA的主要部分被捕获在与h复合物对应的位置。由于含有smn的复合物参与了snRNPs向h复合物的传递，这些结果提供了强有力的证据，证明半乳糖凝集素-1和半乳糖凝集素-3通过与Gemin4相互作用，在体内剪接体组装中发挥作用。
In previous studies we showed that galectin-1 and galectin-3 are factors required for the splicing of pre-mRNA, as assayed in a cell-free system. Using a yeast two-hybrid screen with galectin-1 as bait, Gemin4 was identified as a putative interacting protein. Gemin4 is one component of a macromolecular complex containing approximately 15 polypeptides, including SMN (survival of motor neuron) protein. Rabbit anti-galectin-1 co-immunoprecipitated from HeLa cell nuclear extracts, along with galectin-1, polypeptides identified to be in this complex: SMN, Gemin2 and the Sm polypeptides of snRNPs. Direct interaction between Gemin4 and galectin-1 was demonstrated in glutathione S-transferase (GST) pull-down assays. We also found that galectin-3 interacted with Gemin4 and that it constituted one component of the complex co-immunoprecipitated with galectin-1. Indeed, fragments of either Gemin4 or galectin-3 exhibited a dominant negative effect when added to a cell-free splicing assay. For example, a dose-dependent inhibition of splicing was observed in the presence of exogenously added N-terminal domain of galectin-3 polypeptide. In contrast, parallel addition of either the intact galectin-3 polypeptide or the C-terminal domain failed to yield the same effect. Using native gel electrophoresis to detect complexes formed by the splicing extract, we found that with addition of the N-terminal domain the predominant portion of the radiolabeled pre-mRNA was arrested at a position corresponding to the H-complex. Inasmuch as SMN-containing complexes have been implicated in the delivery of snRNPs to the H-complex, these results provide strong evidence that galectin-1 and galectin-3, by interacting with Gemin4, play a role in spliceosome assembly in vivo.