Carbohydrate Binding Protein 35

描述(申请人提供)：本项目建议继续我们对碳水化合物结合蛋白35的研究，该蛋白已被重新命名为Galectin-3(MR-30,000)。Galectins由半乳糖/乳糖特异性糖结合蛋白家族组成，它们在多肽的碳水化合物识别结构域中共享特征氨基酸序列。我们对Galectin-3的兴趣源于观察到这种蛋白质可以以核糖核蛋白复合体的形式存在于细胞核中。利用来自HeLa细胞的核提取物(NE)，能够在无细胞的实验中进行Pre-mRNA的剪接，耗竭和重建实验证明Galectin-3是一个剪接因子。在这些研究过程中，我们发现Galectin家族的另一个成员Galectin-1(MR-14,000)也在细胞核中被检测到，并且它也是一个剪接因子。在剪接实验中，Galectin-1和Galectin-3的活性是多余的。酵母菌双杂交筛选发现Gin4是Galectin-1的相互作用配体。Gmin4是一种大分子复合体SMN(运动神经元存活)复合体的一种成分，这种复合体在被称为宝石的亚核结构中被可视化。SMN复合体的功能是在剪接体的组装中提供SnRNPs。免疫沉淀研究表明，SMN复合体同时含有Galectins-1和Galectins-3。因此，我们现在有活体证据表明Galectins与参与剪接的蛋白质复合体相互作用。在这些和其他观察的基础上，拟议的研究的具体目标包括：(1)描述和表征SMN复合体的组成和相互作用，在其上已发现GMin4和Galectins-1和-3；(2)测试Galectins-1/-3在剪接体组装的早期步骤中的作用；(3)测试Galectin-3和Gin4在体内对前mRNA剪接的影响；以及(4)继续我们的研究Galectin-3的核质穿梭特性，从输入信号和在mRNA运输中的可能作用。

DESCRIPTION (provided by applicant): This project proposes to continue our studies on Carbohydrate Binding Protein 35, which has been renamed galectin-3 (Mr-30,000). The galectins comprise a family of galactose/lactose-specific saccharide-binding proteins that share characteristic amino acid sequences in the carbohydrate recognition domain of the polypeptides. Our interest in galectin-3 stemmed from the observation that the protein could be found in the nucleus, in the form of a ribonucleoprotein complex. Using nuclear extracts (NE) derived from HeLa cells, capable of carrying out splicing of pre-mRNA in a cell-free assay, depletion and reconstitution experiments demonstrated that galectin-3 was a splicing factor. In the course of these studies, we found another member of the galectin family, galectin-1 (Mr -14,000), was also detected in the nucleus and that it, too, served as a splicing factor. The activities of galectin-1 and galectin-3 in the splicing assay were redundant. A yeast two-hybrid screen has identified Gemin4 as an interacting ligand of galectin-1. Gemin4 is one component of a macromolecular complex, the SMN (Survival of Motor Neuron) complex, which has been visualized in subnuclear structures designated as gems. The function of the SMN complex is to supply snRNPs in the assembly of spliceosomes. Immunoprecipitation studies showed that the SMN complex contains both galectins-1 and -3. Thus, we now have in vivo evidence that galectins interact with a protein complex involved in splicing. On the basis of these and other observations, the specific objectives of the proposed research include: (1) to delineate and characterize the components and interactions of the SMN complex, on which Gemin4 and galectins-1 and -3 have been found; (2) to test for a role of galectins-1/-3 in the early steps of spliceosome assembly; (3) to test for an in vivo effect of galectin-3 and Gemin4 on pre-mRNA splicing; and (4) to continue our studies on the nucleo-cytoplasmic shuttling properties of galectin-3, in terms of import signals and in terms of a possible role in mRNA transport.