Translational Control by 5'-untranslated regions

5-非翻译区域的翻译控制

基本信息

批准号：
10019570
负责人：
Wendy Victoria Gilbert
金额：
$ 33.5万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-17 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10019570
关键词：
5&apos Untranslated Regions Affinity Antineoplastic Agents Binding Binding Proteins Biochemical Biological Assay Biology Categories Cells Characteristics Data Development Disease Dissection Drug Targeting Elements Engineering Enhancers Eukaryota Eukaryotic Initiation Factors Gene Expression Genes Genetic Genetic Enhancer Element Genetic Translation Goals Heritability High-Throughput Nucleotide Sequencing Human In Vitro Initiator Codon Investigation Laboratories Lead Link Malignant Neoplasms Mass Spectrum Analysis Mediating Messenger RNA Methods Modeling Molecular Mutagenesis Mutate Oligonucleotides Organism Output Peptide Initiation Factors Phylogenetic Analysis Process Production Property Protein Isoforms Proteins Proteomics RNA RNA Sequences RNA Splicing RNA-Binding Proteins Recruitment Activity Regulation Regulatory Element Research Rest Ribosomes Role Saccharomyces cerevisiae Saccharomycetales Site Structure System Testing Therapeutic Transcriptional Silencer Elements Translating Translation Initiation Translational Regulation Translations Untranslated Regions Uridine Variant Virus Work Yeasts base crosslink defined contribution genome-wide human disease improved in vivo insight mRNA Precursor novel predictive modeling preference protein function recruit response structured data transcriptome translation assay translation factor

项目摘要

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.

项目概要意义：翻译起始是真核基因表达中高度调控的步骤，它的失调与遗传性人类疾病和癌症有关。取决于 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。需要对这些 mRNA 特征进行预测性理解，以利用广泛的针对翻译因子的药物的治疗潜力，开发新的基于翻译的药物疗法，并设计治疗性 mRNA。方法：我们的工作旨在全面鉴定可增强或我们勇敢地面对 5'-UTRs 的翻译并阐明其潜在机制。具有无法解释的高或低翻译活性，结合具有 mRNA 特异性功能的蛋白质我们设想了两大类翻译激活因子或抑制因子。增强子：与核心起始因子结合的 5′-UTR 序列或 RNA 结构优先，以及结合在核糖体招募中起作用的新因子的 5'-UTR 元件在支持第一个模型方面，我们最近表现出了强大的实力。酵母 eIF4G1 的序列偏好，发现其首选结合基序，寡聚尿苷，天然存在于数百个基因的 5'-UTR 中，并增强其翻译。利用我们实验室开发的一种新方法，可以高度并行地解剖候选顺式调控元件，用于定义特定 5'-UTR 特征对我们使用酿酒酵母（芽殖酵母），因为 5'- UTR亚型在该生物体中已通过实验得到明确定义，我们可以利用大量的目标 2 将揭示观察到的方差有多少。核糖体招募的直接原因是与核心起始因子的亲和力差异。目标 3 将使用以下方法识别结合 5'-UTR 增强子和沉默子元件的新因子交联和质谱分析；研究其改变核糖体的机制；体外招募；并探讨它们对体内调节翻译的影响。将揭示 mRNA 一级序列的差异如何导致巨大且受调节的差异因为真核翻译机制和调控范式是高度保守，这里获得的分子见解可能具有广泛的启发性了解翻译控制，包括人类的病理生理过程。