Carbohydrate Binding Protein 35

描述（由申请人提供）：本项目建议继续我们对碳水化合物结合蛋白35（已更名为半乳糖凝集素-3（Mr-30，000））的研究。半乳糖凝集素包含半乳糖/乳糖特异性糖结合蛋白家族，其在多肽的碳水化合物识别结构域中共享特征性氨基酸序列。我们对半乳糖凝集素-3的兴趣源于观察到该蛋白质可以以核糖核蛋白复合物的形式存在于细胞核中。使用来自HeLa细胞的核提取物（NE），能够在无细胞测定中进行前mRNA的剪接，消耗和重建实验证明半乳糖凝集素-3是剪接因子。在这些研究的过程中，我们发现半乳糖凝集素家族的另一个成员，半乳糖凝集素-1（Mr-14，000），也在细胞核中检测到，它也是一种剪接因子。半乳糖凝集素-1和半乳糖凝集素-3在剪接测定中的活性是多余的。酵母双杂交筛选已确定Gemin 4作为半乳糖凝集素-1的相互作用配体。Gemin 4是一种大分子复合物SMN（运动神经元存活）复合物的一种成分，该复合物已在指定为宝石的亚核结构中可视化。SMN复合物的功能是在剪接体的组装中提供snRNP。免疫沉淀研究表明，SMN复合物含有半乳糖凝集素-1和-3。因此，我们现在有体内证据表明半乳糖凝集素与参与剪接的蛋白质复合物相互作用。基于这些和其他观察，所提出的研究的具体目标包括：（1）描绘和表征SMN复合体的组分和相互作用，在SMN复合体上已经发现Gemin 4和半乳糖凝集素-1和-3;（2）测试半乳糖凝集素-1/-3在剪接体组装的早期步骤中的作用;（3）测试半乳糖凝集素-3和Gemin 4对前体mRNA剪接的体内作用;和（4）继续我们对半乳糖凝集素-3的核质穿梭性质的研究，包括输入信号和在mRNA转运中的可能作用。

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.