Aberrant P-bodies accumulation and clearance in yeast and human cells.

摘要 核糖核酸-蛋白质(RNP)颗粒是在正常情况下形成的动态无膜细胞器 以一种可逆的方式促进生长和应对压力。一种类型的RNP颗粒是 加工体(P-体，或PBS)，其中包含mRNA和RNA加工 酵素。它们的作用包括封存故障的mRNAs以供降解。 以及在不需要时存储和沉默mRNA。PBS组件和行为是 在酵母和人类细胞之间高度保守。我们对作曲和音乐的理解 在过去的十年里，通过液相分离组装普通的PBS已经取得了进展。相比之下， 我们对PBS参与人类疾病的了解很少，关于PBS参与人类疾病的证据也是如此 异常PBS的发生和正常或异常PBS的清除。 在这个项目中，我们建议研究与PBS组件相关的变体 导致智力残疾的神经发育障碍。目前，还没有治愈的方法 这类疾病。我们假设这些变异会导致异常PBS的积累， 不像正常的PBS，是不可逆的。人类变种携带残基的错义突变 在酵母蛋白中完全相同。因此，我们首先对这些突变的影响进行建模 RNA加工与PBS在酵母中的积累。当正常的PBS组装和 根据细胞需要进行分解，我们在酵母细胞中的初步证据表明 持续性PBS堆积是这些突变的主要表型。我们建议 研究这些突变对酵母和人类组织培养细胞的影响，包括 神经细胞系。我们建议评估的表型是关于积累和 持续性PBS的动态，包括它们可能对细胞功能产生的不利影响。在……里面 此外，我们将探索清除正常和持续性PBS的可能途径，例如 宏观和微观自噬和泛素相关的降解。对于这些研究，我们将 使用分子遗传学和细胞方法的组合。学习的大多数方法 本实验室建立了RNP颗粒、酵母和人体细胞中的颗粒清除途径。 实现这一提议的目标将提供关于存在和行为的新范式 持续性PBS及其在神经发育障碍中所起的作用。此外，识别 清除正常和异常PBS的途径将为以下疾病提供新的治疗策略 一种与智力残疾有关的神经发育障碍，符合 美国国家神经疾病和中风研究所。

Abstract RNA-protein (RNP) granules are dynamic membrane-less organelles that form during normal growth and in response to stress in a reversible manner. One type of RNP granules is Processing Bodies (P-bodies, or PBs), which contain mRNA together with RNA processing enzymes. Their roles include sequestration of malfunctioning mRNA destined for degradation and storing and silencing of mRNAs when not needed. PBs components and behavior are highly conserved between yeast and human cells. Our understanding of composition and assembly by liquid-phase separation normal PBs has progressed in the last decade. In contrast, our knowledge about involvement of PBs in human disease is scarce as is evidence about the occurrence of aberrant PBs and clearance of normal or aberrant PBS. In this project, we propose to study variants in a PBs component associated with a neurodevelopmental disorder that causes intellectual disability. Currently, there is no cure for such disorders. We hypothesize that the variants cause accumulation of aberrant PBs that, unlike normal PBs, are not reversible. The human variants carry missense mutations in residues identical in the yeast protein. Therefore, we started by modeling the effects of these mutations on RNA processing and accumulation of PBs in yeast. While normal PBs assemble and disassemble according to cellular needs, our preliminary evidence in yeast cells points to accumulation of persistent PBs as the major phenotype of these mutations. We propose to study the effects of these mutations in yeast and in human tissue culture cells, including neuronal cell lines. The phenotypes we propose to assess are on the accumulation and dynamics of persistent PBs, including adverse effects they might have on cell function. In addition, we will explore possible pathways that can clear normal and persistent PBs, such as macro- and micro-autophagy and ubiquitin-associated degradation. For these studies we will use a combination of molecular genetics and cellular approaches. Most approaches for studying RNP granules, granule clearance pathways in yeast and human cells are established in our lab. Achieving the goals of this proposal would provide novel paradigms on existence and behavior of persistent PBs and the role they play in a neurodevelopmental disorder. Moreover, identifying pathways that can clear normal and aberrant PBs would provide novel therapeutic strategies for a neurodevelopmental disorder associated with intellectual disability, in line with the missions of the National Institute of Neurological Disorders and Stroke.