Translational Control by 5'-untranslated regions

项目总结 意义：翻译启动是真核基因表达和表达过程中高度调控的步骤 它的失调与可遗传的人类疾病和癌症有关。翻译调控 取决于依赖于细胞条件的mRNAs蛋白输出的差异，但 区分高效翻译的mRNAs的关键信使核糖核酸特征在很大程度上是未知的。一个 需要对这些信使核糖核酸特性的预测性理解来驾驭广泛的 靶向翻译因子的药物的治疗潜力，开发新的基于翻译的 治疗，并设计治疗性的mRNA。 方法：我们的工作旨在全面识别能够增强或 抑制翻译并阐明其潜在的机制。我们假设5‘-UTRs 具有无法解释的高或低翻译活性的结合蛋白的功能是mRNA特异性的 翻译激活剂或抑制物。我们设想了两大类翻译 增强剂：结合核心起始因子的5‘-非编码区序列或RNA结构 优先结合在核糖体募集中起作用的新因子的5‘-UTR元件 仍有待阐明。为了支持第一种模式，我们最近表现出了强劲的 对酵母eIF4G1的序列偏好，发现其首选的结合基序，寡尿苷， 自然地发生在数百个基因的5‘-UTRs中，并增强它们的翻译。目标1将 利用我们实验室开发的一种新方法，可以高度平行地剖析 候选顺式调控元素，以定义特定5‘-UTR特征对 核糖体在细胞裂解物中的募集活性。我们使用酿酒酵母(萌芽酵母)是因为5‘- Utr亚型在实验中被很好地定义在这种生物体中，我们可以利用丰富的 遗传、生化和结构数据。目标2将揭示观测到的差异有多大 在核糖体中，核心启动因子亲和力的差异直接解释了核糖体募集。 目标3将识别结合5‘-UTR增强子和消音器元件的新因子，使用 研究它们改变核糖体的机制 体外招募；并探讨它们对体内调控翻译的影响。共同努力，这项工作 将揭示信使核糖核酸一级序列的差异如何导致大的和受调控的差异 翻译过来。因为真核生物的翻译机制和调控范式是 高度保守，这里获得的分子洞察力可能会对 理解翻译控制，包括在人类的病理生理过程中。

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.