Functional Consequences of Ribosome Heterogeneity

描述(由申请人提供)： 翻译是真核基因表达调控的关键环节，翻译失调与多种人类疾病密切相关。虽然众所周知，信使核糖核酸水平不能预测大多数基因的蛋白质水平，并且信使核糖核酸特异的翻译效率在数量级上有所不同，但对基因表达产生如此广泛和数量上显著的翻译效应的分子机制却知之甚少。大多数关于翻译控制的研究强调了mRNA特征和mRNA结合因子(如蛋白质和miRNAs)之间的功能差异，并认为核糖体在这一过程中是一个不变的组成部分。这一观点忽视了具有挑衅性的证据，即核糖体功能的分子特化也可能在调节基因表达方面发挥重要作用。通过核糖体特化进行翻译调控是在30多年前首次提出的。尽管越来越多的蛋白质组学证据表明生长条件和发育阶段特定的核糖体的产生，但这种核糖体“特化”的功能后果从未得到严格的测试。我们的实验室研究了单倍体酿酒酵母丝状分化过程中的翻译调控，这是一种由长期葡萄糖饥饿诱导的发育程序，是环境调控细胞分化的成熟模型。我们的初步结果表明，该模型系统适用于研究核糖体特化对基因表达调控的生化和生理效应。我们已经获得的初步数据表明，葡萄糖饥饿的酵母细胞产生不同蛋白质组成的核糖体复合体；葡萄糖饥饿的核糖体具有不同的功能特性；饥饿诱导的核糖体蛋白质组成的变化导致基因特异性地影响翻译效率。这项拟议的工作将调查这些有趣效应背后的机制。我们的方法利用敏感的全基因组翻译图谱方法来确定核糖体的特定改变所影响的翻译控制的范围。从全球的角度出发，我们确定了最具生理学意义的信使核糖核酸底物。然后，我们使用这些信使核糖核酸底物进行体外生化分析，以剖析全球翻译反应背后的分子机制。这一强大的方法结合了体内的广度和体外的机械深度，目前在翻译领域没有得到充分利用。真核细胞翻译机制和调控过程的高度保守性表明，我们从酵母中获得的分子洞察力将为理解人类发育和疾病中的翻译控制提供范例。

DESCRIPTION (provided by applicant): Project Summary/Abstract Translation is a crucial point of regulation of eukaryotic gene expression, and dysregulation of translation is implicated in many human diseases. Although it is well known that mRNA levels fail to predict protein levels for most genes, and that mRNA-specific translational efficiencies vary by orders of magnitude, the molecular mechanisms responsible for such widespread and quantitatively significant translational effects on gene expression are poorly understood. Most studies of translational control emphasize functional differences between mRNA features and mRNA-binding factors (e.g. proteins and miRNAs), and consider 'the ribosome' as an unvarying component in the process. This view overlooks provocative evidence suggesting that molecular specialization of ribosome function may also play an important role in regulating gene expression. Translational control through ribosome specialization was first proposed more than thirty years ago. Despite increasing proteomic evidence for the production of growth condition and developmental stage specific ribosomes, the functional consequences of such ribosome 'specializations' have never been rigorously tested. Our lab studies translational regulation in haploid S. cerevisiae undergoing filamentous differentiation, a developmental program induced by prolonged glucose starvation, which serves as a well-established model for environmentally regulated cellular differentiation. Our Preliminary Results demonstrate the suitability of this model system for investigating the biochemical and physiological effects of ribosome specialization on regulation of gene expression. We have obtained preliminary data showing that glucose starved yeast cells produce ribosomal complexes with different protein compositions; that the glucose starved ribosomes have different functional properties; and that starvation-induced changes in ribosome protein composition lead to gene-specific effects on translational efficiency. The proposed work will investigate the mechanisms underlying these interesting effects. Our approach exploits sensitive genome-wide translational profiling methods to determine the scope of translational control effected by specific alterations of ribosomes. By starting with a global perspective, we identify the most physiologically relevant mRNA substrates. We then use these mRNA substrates for in vitro biochemical assays to dissect the molecular mechanisms underlying the global translational responses. This powerful approach, which combines breadth in vivo with mechanistic depth in vitro, is currently underutilized in the translation field. The high degree of conservation of eukaryotic translation mechanisms and regulatory processes argues that the molecular insights we gain from yeast will provide paradigms for understanding translational control in human development and disease.