Translational Control by 5'-untranslated regions

项目摘要 翻译起始是真核生物基因表达过程中一个高度调控的步骤， 它的失调与遗传性人类疾病和癌症有关。翻译调控 依赖于mRNA蛋白质输出的细胞条件依赖性差异，但 区分有效翻译的mRNA的关键mRNA特征在很大程度上是未知的。一 需要对这些mRNA特征的预测性理解来利用广泛的 靶向翻译因子的药物的治疗潜力，开发新的基于 疗法，并设计治疗性mRNA。 方法：我们的工作旨在全面鉴定5′-UTR序列， 抑制翻译并阐明其潜在机制。我们假设5′-UTR 具有无法解释的高或低翻译活性的结合蛋白， 翻译激活剂或抑制剂。我们设想两大类翻译 增强子：与核心起始因子结合的5′-UTR序列或RNA结构 优先，和5′-UTR元件，其结合在核糖体募集中起作用的新因子 仍有待阐明。为了支持第一种模式，我们最近展示了强大的 酵母eIF 4G 1的序列偏好，并发现其优选的结合基序，寡尿苷， 天然存在于数百个基因的5′-UTR中，并增强其翻译。目标1将 利用我们实验室开发的一种新方法， 候选顺式调控元件，以确定特定的5′-UTR特征对 细胞裂解物中的核糖体募集活性。我们使用S。酿酒酵母（芽殖酵母），因为5′- UTR同种型在该生物体中实验性地被很好地定义，并且我们可以利用大量的UTR同种型。 遗传、生化和结构数据。目标2将揭示观察到的方差 在核糖体募集中，核心起始因子的亲和力差异直接解释了这一点。 目的3将利用免疫荧光技术鉴定结合5′-UTR增强子和沉默子元件的新因子。 交联和质谱分析;研究它们改变核糖体的机制 体外招募;并探讨其对体内调节翻译的影响。在一起，这项工作 将揭示mRNA一级序列的差异如何导致大的和受调控的差异 在翻译中。因为真核生物的翻译机制和调控模式 高度保守，这里获得的分子见解很可能是广泛的启发， 理解翻译控制，包括人类的病理生理过程。

PROJECT SUMMARY Significance: Translation initiation is a highly regulated step in eukaryotic gene expression and its dysregulation is linked to heritable human diseases and cancer. Translational regulation depends on cellular condition-dependent differences in the protein output of mRNAs, but the key mRNA features that distinguish efficiently translated mRNAs are largely unknown. A predictive understanding of these mRNA characteristics is needed to harness the broad therapeutic potential of drugs that target translation factors, develop new translation-based therapies, and engineer therapeutic mRNAs. Approach: Our work aims to comprehensively identify 5′-UTR sequences that enhance or repress translation and illuminate their underlying mechanisms. We hypothesize that 5′-UTRs with unexplained high or low translation activity bind proteins that function as mRNA-specific translational activators or repressors. We envision two broad categories of translational enhancers: 5′-UTR sequences or RNA structures that bind to core initiation factors preferentially, and 5′-UTR elements that bind novel factors whose roles in ribosome recruitment remain to be elucidated. In support of the first model, we have recently demonstrated strong sequence preferences for yeast eIF4G1 and found that its preferred binding motif, oligo-uridine, occurs naturally in the 5′-UTRs of hundreds of genes and enhances their translation. Aim 1 will leverage a new method developed in our laboratory that allows highly parallel dissection of candidate cis regulatory elements to define the contribution of specific 5′-UTR features to ribosome recruitment activity in cell lysates. We use S. cerevisiae (budding yeast) because 5′- UTR isoforms are experimentally well defined in this organism and we can exploit a wealth of genetic, biochemical and structural data. Aim 2 will reveal how much of the observed variance in ribosome recruitment is directly explained by differences in affinities for core initiation factors. Aim 3 will identify novel factors that bind 5′-UTR enhancer and silencer elements using crosslinking and mass spectrometry; investigate their mechanisms that alter ribosome recruitment in vitro; and explore their impact on regulated translation in vivo. Together, this work will reveal how differences in mRNA primary sequence lead to large and regulated differences in translation. Because the eukaryotic translational machinery and regulatory paradigms are highly conserved, the molecular insights gained here are likely to be broadly illuminating for understanding translational control, including in pathophysiological processes in humans.