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.

项目摘要意义：翻译计划是真核基因表达和它的失调与可遗传的人类疾病和癌症有关。翻译法规取决于mRNA蛋白质输出的细胞状况依赖性差异，但是有效地翻译的mRNA的关键mRNA特征在很大程度上未知。一个需要对这些mRNA特征的预测理解来利用广泛的针对翻译因素的药物的治疗潜力，发展基于翻译的新疗法和工程师治疗mRNA。方法：我们的工作旨在全面识别增强或压制翻译并照亮其潜在机制。我们假设5'-UTRS 意外的高转化活性或低平移活性结合起作用的蛋白质，该蛋白起作用翻译激活剂或复制品。我们设想两大类的翻译增强剂：与核心起始因子结合的5'-UTR序列或RNA结构优先，和5'-UTR元素结合了新的因素，其在核糖体募集中的作用仍然有待阐明。为了支持第一个模型，我们最近证明了强大的酵母EIF4G1的序列偏好，发现其首选结合基序寡尿尿碱，自然发生在数百个基因的5'-Utrs中，并增强其翻译。目标1意志利用我们实验室开发的一种新方法，该方法允许高度平行解剖候选CIS调节元素，以定义特定5'-UTR特征的贡献细胞裂解物中的核糖体募集活性。我们使用S. cerevisiae（芽酵母），因为5'-- UTR同工型在实验中在这种生物体中定义得很好，我们可以探索很多遗传，生化和结构数据。 AIM 2将揭示观察到的差异的数量在核糖体中，募集的直接解释了核心起始因子的亲和力差异。 AIM 3将确定使用5'-UTR增强子和消音器元素结合的新因素交联和质谱；研究他们的机制改变核糖体体外招募；并探索它们对体内受调节翻译的影响。在一起，这项工作将揭示mRNA初级序列的差异如何导致较大和受调节的差异在翻译中。因为真核翻译机制和监管范例是高度保守的，这里获得的分子见解可能会广泛阐明了解翻译控制，包括人类的病理生理过程。