Specialized regulation of non-AUG translation

非AUG翻译的专门监管

• 批准号: 9979891
• 负责人: Michael G Kearse
• 金额: $ 24.9万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9979891
• 关键词: Address; Affect; Affinity; Amino Acids; Anisomycin; Attenuated; Binding; Binding Proteins; Biochemistry; Bioinformatics; Candidate Disease Gene; Cell Survival; Cells; Cellular Stress; Code; Codon Nucleotides; Complex; Cycloheximide; Data; Elements; Environment; Event; Faculty; Foundations; Frequencies; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Human; Human Biology; Human Genome; Huntington Disease; Initiator Codon; Laboratories; Link; Luciferases; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mentors; Messenger RNA; Modification; Molecular; Mutation; Neurodegenerative Disorders; Open Reading Frames; Oxidative Stress; Phase; Plasmids; Positioning Attribute; Primer Extension; Protein Biosynthesis; Proteins; Regulation; Regulatory Pathway; Reporter; Reporter Genes; Research; Resistance; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Scanning; Stress; Terminator Codon; Testing; Time; Training; Translating; Translation Initiation; Translations; Work; attenuation; base; biological adaptation to stress; cell type; crosslink; endoplasmic reticulum stress; experimental study; genome-wide; human disease; inhibitor/antagonist; innovation; insight; novel; novel strategies; novel therapeutic intervention; recruit; response; ribosome profiling; skills; stoichiometry; therapeutic development

PROJECT SUMMARY/ABSTRACT Although it has long been thought that an AUG start codon universally marks the beginning of an open reading frame, recent advances in ribosome footprint mapping have revealed that ribosomes initiate translation at non- AUG codons (e.g., CUG or GUG) at an astonishing frequency. Canonical AUG start codons are recognized by the initiator tRNAi within the scanning pre-initiation complex, but it is still largely unclear how non-AUG start Met codons are recognized or regulated. Considering that aberrant forms of non-AUG translation have been shown to cause multiple neurodegenerative diseases and contribute to cancer malignancy, a clearer understanding of how non-AUG translation is controlled on the molecular level is greatly needed. Using plasmids that express the same reporter protein but from different start codons, I surprisingly found that a reporter expressed from a CUG or GUG start codon was not inhibited by well-characterized translation inhibitors and, in fact, increased over time. This is in stark contrast to the expected inhibition of translation I observed when the reporter initiated from a canonical AUG. These data strongly indicate that ribosomes translating proteins from non-AUG start codons are fundamentally different from ribosomes that start at AUG codons. During the mentored phase, I will gain new training in biochemistry and bioinformatics to address how translation at different start codons is uniquely regulated by both the ribosome and sequence elements within mRNAs. In Aim 1, I will affinity purify translating reporter mRNAs that initiate from an AUG or GUG start codon and determine the composition of the bound ribosomes, as well as identify other associated proteins that may confer resistance to translation inhibitors. These experiments will reveal important insights into how ribosomes translating distinct open reading frames can be different and be controlled by separate regulatory pathways. In Aim 2, I will use a candidate gene approach along with genome-wide ribosome profiling to identify sequences that regulate non- AUG translation from endogenous genes. By identifying and characterizing the key regulatory sequences, this aim should allow us to predict and validate which genes are translated by separate ribosome classes within cells. In Aim 3, I will take full advantage of this training and determine in my own laboratory how non-AUG translation is regulated when protein synthesis is naturally attenuated during cell stress. In particular, we will use ribosome profiling to identify non-AUG open reading frames that are regulated during cell stress and characterize how these events impact the stress response and cell survival. In the short term, the proposed new training (Aims 1 & 2) by my expert mentors and collaborators, along with complementary formal coursework, will provide a foundation on which I can continue to reveal in my independent laboratory how non- AUG translation is regulated and controls human biology (Aim 3). The excellent training environment in the Wilusz & Dreyfuss laboratories and at Penn will greatly facilitate not only the mentored research, but also endow me with the necessary professional skills to transition to an independent faculty position.

项目概要/摘要 尽管长期以来人们一直认为 AUG 起始密码子普遍标志着开放阅读的开始 框架，核糖体足迹图谱的最新进展表明，核糖体在非 AUG 密码子（例如 CUG 或 GUG）以惊人的频率出现。规范 AUG 起始密码子被识别 扫描预启动复合物中的启动子 tRNAi，但目前仍不清楚非 AUG 是如何启动的 蛋氨酸 密码子被识别或调节。考虑到已显示非 AUG 翻译的异常形式 引起多种神经退行性疾病并导致癌症恶性肿瘤，更清楚地了解 如何在分子水平上控制非AUG翻译是非常需要的。使用表达的质粒 相同的报告蛋白但来自不同的起始密码子，我惊讶地发现报告蛋白从 CUG 或 GUG 起始密码子不会被充分表征的翻译抑制剂抑制，事实上，增加 随着时间的推移。这与我在记者启动时观察到的预期翻译抑制形成鲜明对比 来自规范的 AUG。这些数据强烈表明核糖体翻译非 AUG 蛋白 起始密码子与以 AUG 密码子起始的核糖体根本不同。辅导期间 阶段，我将获得生物化学和生物信息学方面的新培训，以解决不同开始时的翻译问题 密码子受到核糖体和 mRNA 内序列元件的独特调节。在目标 1 中，我将 亲和纯化从 AUG 或 GUG 起始密码子起始的翻译报告基因 mRNA，并确定 结合核糖体的组成，以及识别可能赋予抗性的其他相关蛋白 翻译抑制剂。这些实验将揭示核糖体如何翻译不同的重要见解 开放阅读框可以不同，并由单独的调控途径控制。在目标 2 中，我将使用 候选基因方法与全基因组核糖体分析一起识别调节非 来自内源基因的 AUG 翻译。通过识别和表征关键调控序列， 目标应该使我们能够预测和验证哪些基因是由不同核糖体类别翻译的 细胞。在目标 3 中，我将充分利用这次培训，并在我自己的实验室中确定非 AUG 如何 当蛋白质合成在细胞应激期间自然减弱时，翻译就会受到调节。特别是，我们将 使用核糖体分析来识别在细胞应激和 描述这些事件如何影响应激反应和细胞存活。短期内，建议 由我的专家导师和合作者提供的新培训（目标 1 和 2），以及补充性的正式培训 课程作业将为我提供一个基础，在此基础上我可以继续在我的独立实验室中揭示如何非 AUG 翻译受到调控并控制人类生物学（目标 3）。优良的培训环境 Wilusz 和 Dreyfuss 实验室以及宾夕法尼亚大学不仅将极大地促进指导研究，而且 赋予我必要的专业技能，以过渡到独立的教职职位。