Coordinating different steps in mRNA localization

协调 mRNA 定位的不同步骤

• 批准号: 9367001
• 负责人: Paul M. Macdonald
• 金额: $ 31.3万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-07 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9367001
• 关键词: Address; Affinity; Affinity Chromatography; Anterior; Binding; Binding Sites; Biochemical; Biological Assay; Cells; Complex; Conflict (Psychology); Defect; Destinations; Drosophila genus; Dynein ATPase; Factor Analysis; Genetic; Genetic Translation; Goals; Hand; Individual; Link; Mammals; Messenger RNA; Microtubules; Modeling; Molecular Motors; Motor; Nurses; Oocytes; Pattern; Phase; Phenotype; Play; Post-Transcriptional Regulation; Process; Proteins; Publishing; RNA; RNA Transport; RNA-Binding Protein FUS; RNA-Binding Proteins; Role; Signal Transduction; Site; System; Testing; Translating; Translations; Work; base; cell type; fly; insight; mutant; novel; prevent; protein expression; stem

Expression of proteins in spatially restricted patterns frequently relies on prior localization of the encoding mRNAs. One conserved RNA transport mechanism, used in multiple cell types, involves a stem-loop transport signal within the mRNA, and proteins (Egalitarian and Bicaudal-D) to bind the signal and link the RNA cargo with the dynein motor for transit along microtubules. This transport system also anchors the mRNAs at their destination, although anchoring is poorly understood. The initial step in localization of Drosophila oskar mRNA - transport from the nurse cells to the oocyte - relies on this conserved transport system. However, oskar mRNA differs from simple examples of this system, in that the mRNA must then be transferred to a different localization machinery for a later step in localization. Notably, the cis-acting localization signals in oskar mRNA that direct the initial transport step are individually weak, even though transport is robust. Replacing the multiple weak signals with a single strong signal disrupts the later step in localization. This suggests that weak association with the machinery for the initial transport step allows the mRNA to be handed off to the other type of machinery. A model is proposed to explain what makes the oskar transport signals weak, and how the conflicting requirements for weak signals yet highly efficient transport can both be met. In part, this model relies on Staufen protein to inhibit association of the oocyte transport machinery with the oskar oocyte transport signals. Staufen is more conventionally thought to act in the later step of oskar mRNA localization, although the staufen mutant phenotype is equally consistent with both models. One Aim is to confirm, using an affinity purification approach, that oskar relies on the conserved transport system for its initial step of localization and to reveal candidates for other contributing factors. The second Aim is to test two predictions of what makes the individual transport signals weak: modest affinity for the recognition factor, Egalitarian; and displacement of Egalitarian by Staufen after the mRNA arrives in the oocyte. The third Aim is to ask if the contribution of Staufen to localization of oskar mRNA is limited to the novel role postulated here, or if Staufen also has the conventional role, or both. The novel role for Staufen could explain how it contributes to a wide variety of forms of post-transcriptional regulation, a possibility that will be tested. A final Aim is to exploit unique features of this system in a sensitized genetic assay to evaluate candidate transport/anchoring factors, and to screen for such factors. Further analysis of these factors should provide substantial insights into many aspects of mRNA localization and anchoring.

蛋白质以空间受限模式的表达通常依赖于编码蛋白质的先前定位。 mRNA。一个保守的RNA转运机制，用于多种细胞类型，涉及茎环 mRNA内的转运信号和蛋白质（平等主义者和双尾-D）结合信号并将 RNA货物与动力蛋白马达沿着微管运输。这一运输系统还将 mRNA在其目的地，尽管锚定知之甚少。本地化的第一步 果蝇oskar mRNA -从滋养细胞到卵母细胞的运输-依赖于这种保守的运输 系统然而，oskar mRNA与该系统的简单示例不同，因为mRNA必须被 转移到不同的本地化机器以用于本地化中的后续步骤。值得注意的是， 在奥斯卡mRNA定位信号，指导最初的运输步骤是个别弱，即使 运输是强大的。用单个强信号替换多个弱信号会破坏后续步骤， 本地化这表明，在最初的运输步骤中，与机器的弱关联允许 mRNA被移交给另一种机器。提出了一个模型来解释是什么使 奥斯卡运输信号弱，以及如何对弱信号的冲突要求，但高效 运输都可以满足。在某种程度上，该模型依赖于Staufen蛋白来抑制卵母细胞的结合， 运输机械与奥斯卡卵母细胞运输信号。施陶芬通常被认为是 Oskar mRNA定位的后期步骤，尽管Staufen突变体表型与 两个模型。一个目的是使用亲和纯化方法证实oskar依赖于 保守的运输系统，其初步步骤的本地化，并揭示候选人的其他贡献 因素第二个目标是测试两个预测是什么使得单个传输信号变弱： 对承认因素的适度亲和力，平等主义者;以及施陶芬在 mRNA到达卵母细胞。第三个目的是问施陶芬对奥斯卡本土化的贡献 mRNA仅限于这里假设的新作用，或者如果Staufen也有传统的作用，或者两者兼而有之。的 Staufen的一个新作用可以解释它如何有助于多种形式的转录后 监管，这是一种可能性，将受到考验。最后一个目标是利用这个系统的独特功能， 敏化遗传测定以评估候选转运/锚定因子，并筛选这些因子。 对这些因素的进一步分析将为mRNA定位的许多方面提供实质性的见解 和锚定。