MOLECULAR STUDIES OF FMRP FUNCTION IN YEAST

这个项目的长期目标是阐明自然功能 FMRP和相关基因产品的研究，重点是探索 这些蛋白质作为促进因素的拟议作用 真核细胞基因表达的调控。特别是，我们 计划研究候选基因Scp160p的表达和功能 FMRP的同源物，在遗传和生化适应性酵母中 酿酒酵母。此外，我们将使用酵母作为模型 表达和研究野生型和突变型的系统 脊椎动物FMRP。 来自哺乳动物体外和体内研究的最新数据 细胞表明，FMRP和FMRP相关的FXR蛋白都是 核糖核蛋白，与核糖体结合并表面上介导 特定的代谢、运输、稳定性和/或翻译 单元格中的消息。然而，目前尚不清楚的是， FMRP功能对这些生物通路的影响，其机制是 这种影响发生的原因，以及基因的身份和关系 受FMRP中介监管的产品。我们建议解决以下问题 关于酵母Scp160p和脊椎动物FMRP的这些问题 实现以下具体目标“(1)表征 Scp160p在酵母中的亚细胞定位 在显微镜下，结合缺失和突变方案， 启动该蛋白的结构/功能研究；(2)研究 酵母中Scp160p缺失和过表达的表型 用生化方法鉴定Scp160p的蛋白质和RNA物种 在活体内相关，以及在遗传上识别其他基因座 其产品在物理或功能上与之相互作用的酵母 Scp160p；(4)在酵母中表达脊椎动物FMRP蛋白，研究 这些蛋白质的定位和大分子相互作用 与内源酵母蛋白和/或FNAs结合，并探讨其对 这些脊椎动物的FMRP序列来补充缺失的 相应的酵母内源序列。总而言之，这些研究 应该提供不仅在以下方面有意义的信息 FMRP-like序列表达和功能的基础科学 酵母，还因为从这项工作中获得的见解应该提供一种 用于解释可能复杂得多的数据的有用框架 源于对哺乳动物细胞和组织中FMRP功能的研究。

The long-term goal of this project is to elucidate the natural function of FMRP and related gene products, with emphasis on exploring the proposed roles of these proteins as factors contributing to the regulation of gene expression of eukaryotic cells. In particular, we plan to study both expression and function of Scp160p, a candidate homologue of FMRP, in the genetically and biochemically amenable yeast Saccharomyces cerevisiae. In addition, we will use yeast as a model system in which to express and study wild-type and mutant forms of vertebrate FMRP. Current data derived both in vitro and in vivo from studies of mammalian cells indicate that both FMRP and the FMRP-related FXR proteins are ribonucleoproteins that associate with ribosomes and ostensibly mediate the metabolism, transport, stability, and/or translation of specific messages in cells. What remains unknown, however, is the specific impact of FMRP function on these biological pathways, the mechanism by which this impact occurs, and the identities and relationships of gene products subject to FMRP-mediated regulation. We propose to address these questions with regard to both yeast Scp160p and vertebrate FMRP by accomplishing the following specific objectives" (1) to characterize Scp160p subcellular localization in yeast both biochemically and microscopically and, coupled with deletion and mutagenesis protocols, to initiate structure/function studies of this protein; (2) to study the phenotypes of both loss and overexpression of Scp160p in yeast; 93) to identify biochemically both protein and RNA species with which Scp160p associated in vivo, as well as to identify genetically other loci in yeast whose products interact, either physically or functionally, with Scp160p, and (4) to express vertebrate FMRP proteins in yeast, to study both the localization and macromolecular interactions of these proteins with endogenous yeast proteins and/or FNAs, and to probe the capacity of these vertebrate FMRP sequences to complement deletion of the corresponding yeast endogenous sequences. Combined, these studies should provide information that is meaningful not only in terms of the basic science of expression and function of FMRP-like sequences in yeast, but also because insights gained from this work should provide a useful framework for interpreting the potentially much more complex data derived from studies of FMRP function in mammalian cells and tissues.