Cytoplasmic transport of mRNAs in the myelin sheath

描述(由申请人提供) 这是纽约西奈山的David R.Colman和阿根廷布宜诺斯艾利斯Leloir研究所(前生化研究所坎波马基金会)的Graciela L.Boccaccio的实验室之间的合作提案。这项建议将我们正在进行的髓鞘轴突连接功能组织的工作(授予#1R01 NS40560；07/01/00-06/30/05)扩展到邻近连接的神经胶质细胞胞质的细胞内成分的研究。具体地说，我们的目的是阐明中枢神经系统髓鞘细胞中蛋白质局部合成的细胞机制，这是申请人和外国合作者的研究小组长期以来都主要感兴趣的。我们将研究双链RNA结合蛋白Staufen在少突胶质细胞中mRNAs的定位和翻译中的作用。这可能与两个相关的过程有关：1.膜维持和重塑所需成分的合成；2.轴突连接处少突胶质细胞侧的信号转导。 我们将1)使用质谱学来明确地识别信使核糖核酸定位装置的新的蛋白质组分，以及2)开始评估Staufen和新的组分在信使核糖核酸定位中的作用。 这项建议的一个重要方面是它的互补性，结合了两个实验室的科学兴趣和方法优势。Boccaccio博士的团队已经开发出了少突胶质细胞的原代培养和脑亚细胞成分的生化分析，以及培养细胞中mRNA定位的功能分析。我们在西奈山的实验室已经建立了许多本项目严格要求的方法，即通过质谱学进行蛋白质分析，通过酵母双杂交和“下拉”实验分析蛋白质-蛋白质相互作用。因此，该项目将允许对外国调查员和博士生进行最先进的技术培训，这些技术目前尚未在其本国研究所建立，因此是一项宝贵的贡献。

DESCRIPTION (provided by applicant) This is a collaborative proposal between the laboratories of David R. Colman at Mount Sinai, NY, and of Graciela L. Boccaccio at the Institute Leloir (formerly Institute for Biochemical Research Campomar Foundation), Buenos Aires, Argentina. This proposal extend our ongoing work on the Functional Organization of the Myelin Axoglial Junction (grant # 1R01 NS40560; 07/01/00 - 06/30/05) to the study of intracellular elements of the glial cell cytoplasm adjacent to the junction. Specifically, we are aimed to elucidate the cell mechanisms mediating the localized synthesis of proteins in the myelinating cells of the central nervous system, that has being for long time of major interest to both, the applicant's and the foreign collaborator's research group. We will investigate the participation of the double stranded RNA binding protein Staufen in the localization and translation of mRNAs in oligodendrocytes. This is likely relevant to two related processes: 1. synthesis of components required for membrane maintenance and remodeling, and 2, signal transduction at the oligodendrocyte side of the axoglial junction. We will 1) use mass spectrometry to unambiguously identify novel protein components of the mRNA localization apparatus and 2) begin to asses the role of Staufen and novel components in mRNA localization. An important aspect of this proposal is its complementary nature combining the scientific interests and the methodological strength of two laboratories. Dr. Boccaccio's group have developed primary culture of oligodendrocytes and biochemical analysis of brain subcellular components, as well as a functional assay for mRNA localization in cultured cells. A number of methods that are strictly required for the present project have been established in our laboratory in Mount Sinai, namely, protein analysis via mass spectrometry and analysis of protein-protein interactions by yeast two-hybrid and "pull-down" experiments. Thus, this project will allow the training of the foreign investigator and Ph.D. students on state-of-the-art techniques that are currently not established at their home institute, thus representing a valuable contribution