Functional Consequences of Ribosome Heterogeneity

描述（由申请人提供）： 翻译是真核生物基因表达调控的关键环节，翻译的失调与许多人类疾病有关。尽管众所周知，mRNA水平不能预测大多数基因的蛋白质水平，并且mRNA特异性翻译效率按数量级变化，但对基因表达的这种广泛和定量显著的翻译效应的分子机制知之甚少。大多数翻译控制的研究强调mRNA特征和mRNA结合因子（例如蛋白质和miRNA）之间的功能差异，并认为“核糖体”是该过程中不变的组分。这种观点忽视了一个有争议的证据，即核糖体功能的分子特化在调节基因表达中也可能起重要作用。通过核糖体特化的翻译控制是三十多年前首次提出的。尽管越来越多的蛋白质组学证据生产的生长条件和发育阶段特定的核糖体，这种核糖体的功能后果的“专业化”从来没有被严格测试。本实验室研究了单倍体S.酿酒酵母经历丝状分化，这是由长时间葡萄糖饥饿诱导的发育程序，其用作环境调节的细胞分化的良好建立的模型。我们的初步结果表明，该模型系统的适用性研究的生化和生理效应的核糖体特化基因表达的调控。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的核糖体复合物具有不同的蛋白质组成;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的影响翻译效率。拟议的工作将调查这些有趣的影响背后的机制。我们的方法利用敏感的全基因组翻译谱分析方法来确定由核糖体的特定改变所影响的翻译控制的范围。通过从全球角度出发，我们确定了最生理相关的mRNA底物。然后，我们使用这些mRNA底物进行体外生化分析，以剖析全球翻译反应的分子机制。这种强大的方法结合了体内的广度和体外的机械深度，目前在翻译领域尚未得到充分利用。真核生物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见解将为理解人类发育和疾病中的翻译控制提供范例。 公共卫生相关性： 翻译调控对人类健康和发育至关重要，但目前只有少数翻译调控机制被了解。这项工作将提供第一个详细了解的生化和生理功能的核糖体专业化，在翻译控制领域的研究课题。我们预计，我们的研究结果将有广泛的影响，真核基因表达的研究，也将阐明疾病状态的病因，包括癌症，与核糖体功能失调。

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. PUBLIC HEALTH RELEVANCE: Project Narrative Translational regulation is essential for human health and development, but only a handful of translational regulatory mechanisms are understood. The proposed work will provide the first detailed understanding of the biochemical and physiological functions of ribosome specialization, an under-studied topic in the translational control field. We anticipate that our results will have broad implications for the study of eukaryotic gene expression, and will also illuminate the etiology of disease states, including cancer, that are associated with dysregulation of ribosome function.