Mechanism of stringent translation initiation: a probe for its biological relevance

严格翻译起始机制：对其生物学相关性的探索

• 批准号: 10660217
• 负责人: KATSURA ASANO
• 金额: $ 29.74万
• 依托单位: KANSAS STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-17 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10660217
• 关键词: Adenosine; Affect; Anticodon; Automobile Driving; Base Pairing; Binding; Biological; Biological Models; Cancer Model; Cell Survival; Codon Nucleotides; Complex; Cues; Data; Development; Disease; Elements; Enzymes; Eukaryota; Eukaryotic Cell; Eukaryotic Initiation Factors; Exclusion; Gene Expression; Genomic approach; Grant; Heat-Shock Response; High temperature of physical object; Human; Human Genome; Human Papillomavirus; Immune; Initiator Codon; Knowledge; Learning; Life; Malignant Neoplasms; Messenger RNA; Methods; Mitotic Cell Cycle; Modeling; Mutation; Neurodegenerative Disorders; Neurons; Normal Cell; Oncogenes; Oncogenic; Outcome; Patient-Focused Outcomes; Peptides; Periodicity; Phase; Process; Production; Protein Biosynthesis; Proteins; Public Health; RNA Binding; Regulation; Repression; Research; Ribosomes; Role; Saccharomyces cerevisiae; Scanning; Side; Site; Stimulus; Testing; Translation Initiation; Translational Regulation; Translations; Yeasts; biological adaptation to stress; c-myc Genes; cancer cell; cancer type; carcinogenesis; differential expression; disorder prevention; flexibility; genetic regulatory protein; genome-wide; insight; mRNA Translation; molecular dynamics; mutant; neurotoxic; novel; patient prognosis; recruit; response; stem

Eukaryotic translation initiation is a complex process involving the ribosome, mRNA, Met-tRNAiMet and numerous eukaryotic initiation factors (eIFs). Decades of studies driven by those using the model eukaryote yeast Saccharomyces cerevisiae revealed that the key process is the formation of codon-anticodon base pairing in the small ribosome P-site. Stringent initiation is enabled by formation of the 48S ribosomal pre-initiation complex (PIC) strictly at the AUG start codon while excluding initiation at other sites. Intriguingly, however, many non-canonical start sites are utilized in some biological contexts and diseases such as cancer and neurodegenerative disorders. The list of non-AUG start sites within the human genome is far from being complete and how the use of these sites and hence protein production from these sites are regulated remains an open question. Thus, Aim 1 of this grant is to make such a list of non-AUG start sites through genome-wide translation profiling of well characterized cancer model systems, verify some of these sites and determine the mechanism driving the observed non-AUG translational regulation in cancer. The Aim 2 is to study the mechanistic role of 5MP and Met-tRNAiMet adenosine N6- threonylcarbamoylation (t6A) in controlling non-AUG translation. It will be tested if 5MP mutations found in many types of cancer can alter initiation accuracy, thereby affecting patients' prognosis. Our preliminary studies suggested that t6A located 3' of Met-tRNAiMet anticodon can discriminate specifically against GUG and UUG start codons, in contrast to eIF1 being more universal non-AUG discriminator. Combining molecular dynamics simulation methods, we will test if the recently discovered cyclic t6A serves the discriminating role and determine how the cooperation or competition between t6A and eIF1 promotes stringent initiation and leaky scanning crucial for translational regulation. The Aim 3 is to study yet a distinct mechanism of start codon selection that is exploited during the heat shock response at the translational level. This mechanism was discovered through translational profiling of yeast eIF3i mutant defective in its interaction with RNA-binding eIF3g subunit at a high temperature. The working hypothesis assumes that, at a high temperature, stable PIC formation at the AUG codon requires additional mRNA elements anchoring the initiating ribosome at its entry site. These hypothetical mRNA elements are located downstream of the start codon and include a novel eIF3g-binding motif 5'-GUCG-3' and a downstream stem- loop that potentially binds the entry site-side of the 40S, thereby stabilizing the PIC formation. This model will tested using yeast as a model system.

真核翻译开始是一个复杂的过程，涉及核糖体，mRNA，met-trnairet 以及许多真核引发因子（EIF）。数十年来的研究是由使用的研究 酿酒酵母的真核生酵母菌酵母糖果实表明，关键过程是形成 在小核糖体P位中的密码子 - 抗病基碱基配对。严格的启动由 严格在Aug Start密码子上严格地形成48S核糖体预生效复合物（PIC） 在其他站点不包括启动。然而，有趣的是，使用了许多非典型的起始站点 在某些生物学环境和疾病中，例如癌症和神经退行性疾病。这 人类基因组中的非aug起始站点列表远非完整，以及如何使用 在这些地点中，从这些地点进行蛋白质的产生，仍然是开放的 问题。 因此，本赠款的目标1是通过全基因组列出这样的非aug开始站点 良好特征癌症模型系统的翻译分析，验证其中一些站点 并确定驱动观察到的癌症中观察到的非aug翻译调节的机制。 目的2是研究5MP和Met-trnaimet腺苷N6-的机械作用 在控制非aug翻译方面，三元carbamoylation（T6A）。如果5MP，它将进行测试 在许多类型的癌症中发现的突变可以改变起始准确性，从而影响患者。 预后。我们的初步研究表明，T6A位于Met-trnaimet反dantodon的3'可以 与EIF1相反，特别歧视Gug和Uug启动密码子 通用的非杰出歧视者。结合分子动力学模拟方法，我们将测试 如果最近发现的环状T6A发挥了歧视作用，并确定 T6A和EIF1之间的合作或竞争促进了严格的启动和漏水扫描 对于翻译调节至关重要。 目的3是研究被利用的独特的起始密码子选择机制 在翻译水平的热量冲击响应中。发现了这种机制 通过酵母EIF3I突变体与RNA结合的相互作用的翻译分析 在高温下EIF3G亚基。工作假设假设在高温下 Aug密码子处的稳定图片形成需要锚定的其他mRNA元素 在其入门网站上启动核糖体。这些假设的mRNA元素位于下游 起始密码子中的包含一个新颖的EIF3G结合图案5'-Gucg-3'和一个下游茎 - 循环可能结合40S的入口站点侧，从而稳定PIC的形成。 该模型将使用酵母作为模型系统进行测试。