The mechanism and regulation of mRNA recruitment during eukaryotic translation initiation

真核翻译起始过程中mRNA招募的机制和调控

• 批准号: 10578362
• 负责人: Ruben L Gonzalez
• 金额: $ 36.34万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-19 至 2027-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10578362
• 关键词: Affinity; Antineoplastic Agents; Binding; Binding Proteins; Biochemical; Biological Assay; Biophysics; Cell Extracts; Complex; Cryoelectron Microscopy; Data; Defect; Development; Discrimination; Drug Targeting; Drug resistance; Eukaryota; Eukaryotic Initiation Factor-1; Eukaryotic Initiation Factors; Event; Exhibits; FDA approved; Fluorescence Microscopy; Future; Gene Expression; Hand; Human; In Vitro; Individual; Initiator Codon; Initiator tRNA; Label; Laboratories; Length; Letters; Link; Maintenance; Malignant Neoplasms; Measures; Mediating; Messenger RNA; Modeling; Molecular; Neoplasm Metastasis; Oncogenic; Pathogenesis; Physiological; Positioning Attribute; Preparation; Principal Investigator; Process; Proteins; Reagent; Recombinants; Regulation; Resolution; Ribosomal Proteins; Ribosomes; Role; Saccharomyces cerevisiae; Series; Signal Transduction; Site; Specificity; Structure; System; Text; Therapeutic; Time; Translating; Translation Initiation; Translations; Tumor-Derived; Yeasts; anti-cancer therapeutic; cancer therapy; data exchange; eukaryotic initiation factor-5B; fluorophore; mutant; overexpression; polyadenosine; public health relevance; reconstitution; recruit; single molecule; single-molecule FRET; time use; tumor growth; tumorigenesis

PROJECT SUMMARY The efficiency with which messenger RNAs (mRNAs) are translated into proteins by the ribosome is a fundamental determinant of gene expression. This efficiency is often determined during the mRNA recruitment step of translation by the ribosome. Consequently, this step is a crucial point of control for gene expression. In eukaryotes, mRNA recruitment is an elaborate, multi-step, and highly regulated process that depends upon the activities of ~13 eukaryotic initiation factors (eIFs). Dysregulation of eIF activity and mRNA recruitment has been causally linked to tumorigenesis, tumor growth, drug resistance, and metastasis in an increasing list of human cancers. Consequently, several eIFs and their roles in mRNA recruitment are emerging as very attractive anticancer drug targets, with an existing, FDA-approved, eIF-targeting compound already having been successfully repurposed as an anticancer therapy. In order to expand and fully exploit this therapeutic potential, however, it is necessary to understand the molecular events that underlie eIF function and mRNA recruitment. Here, we will use a highly purified, fluorophore-labeled, Saccharomyces cerevisiae in vitro translation system that we developed and that includes a full-length, site-specifically labeled eIF4G and a fully reconstituted, site-specifically labeled eIF3, reagents that have been difficult to generate. With these reagents in hand, we will use state-of-the-art, single-molecule fluorescence microscopy and cryogenic electron microscopy (cryo-EM), including a pioneering, time-resolved cryo-EM approach developed by our collaborator, Dr. Joachim Frank, to directly observe and characterize the dynamics of mRNA recruitment during eukaryotic translation initiation. In Aim 1, we will investigate the mechanism through which the multi-component eIF4F complex activates different classes of mRNAs for loading onto ribosomal 43S pre-initiation complexes (PICs), and how changes to the composition of the eIF4F complex can alter which classes of mRNAs are activated. We hypothesize that the structural dynamics of the activated mRNA complex are critical for mRNA loading and will quantify how these dynamics contribute to mRNA selection. In Aim 2, we will investigate how the multi-component eIF3 complex interacts with different classes of mRNAs and/or the 43S PIC in order to facilitate mRNA activation and/or loading onto a 43S PIC, as well as how biologically active subcomplexes of eIF3 can modulate these activities. A large- scale structural rearrangement of 43S PIC-bound eIF3 is thought to control its mRNA loading activity and we will therefore characterize how this rearrangement facilitates formation of the 48S PIC on mRNAs of different classes. In Aim 3, we will investigate the mechanism through which eIF1A and eIF5B mediate mRNA start-codon recognition within a 48S PIC. Start-codon recognition by eIF1A has recently been associated with a large-scale rearrangement of the 48S PIC in which eIF5B and initiator transfer RNA (Met-tRNAi) are repositioned in preparation for joining of the large subunit to the 48S PIC to form the elongation-competent 80S IC. We will characterize these 48S PIC dynamics and determine their role in start-codon recognition and subunit joining.

项目概要 核糖体将信使 RNA (mRNA) 翻译成蛋白质的效率是 基因表达的基本决定因素。这种效率通常是在 mRNA 招募过程中确定的 核糖体翻译的步骤。因此，这一步是控制基因表达的关键点。在 在真核生物中，mRNA 招募是一个复杂的、多步骤的、高度调控的过程，取决于 ~13 种真​​核起始因子 (eIF) 的活性。 eIF 活性和 mRNA 募集失调 与越来越多的人类肿瘤发生、肿瘤生长、耐药性和转移有因果关系 癌症。因此，几种 eIF 及其在 mRNA 招募中的作用变得非常有吸引力 抗癌药物靶点，FDA 批准的现有 eIF 靶向化合物已被 成功地重新用作抗癌疗法。为了扩大和充分利用这种治疗潜力， 然而，有必要了解 eIF 功能和 mRNA 募集背后的分子事件。 在这里，我们将使用高度纯化的、荧光团标记的酿酒酵母体外翻译 我们开发的系统，包括全长、特定位点标记的 eIF4G 和完全重构的、 位点特异性标记的 eIF3，是很难生成的试剂。有了这些试剂，我们就可以 使用最先进的单分子荧光显微镜和低温电子显微镜 (cryo-EM)， 包括由我们的合作者 Joachim Frank 博士开发的开创性的时间分辨冷冻电镜方法， 直接观察和表征真核翻译起始过程中 mRNA 招募的动态。 在目标 1 中，我们将研究多组分 eIF4F 复合物激活的机制 用于加载到核糖体 43S 预起始复合物 (PIC) 上的不同类别的 mRNA，以及如何改变 eIF4F 复合物的组成可以改变哪些类别的 mRNA 被激活。我们假设 激活的 mRNA 复合物的结构动力学对于 mRNA 加载至关重要，并将量化这些复合物如何 动力学有助于 mRNA 选择。在目标 2 中，我们将研究多组分 eIF3 复合物如何 与不同类别的 mRNA 和/或 43S PIC 相互作用，以促进 mRNA 激活和/或加载 到 43S PIC 上，以及 eIF3 的生物活性子复合物如何调节这些活性。一个大- 43S PIC 结合的 eIF3 的规模结构重排被认为控制其 mRNA 负载活性，我们将 因此，描述这种重排如何促进不同 mRNA 上 48S PIC 的形成 类。在目标 3 中，我们将研究 eIF1A 和 eIF5B 介导 mRNA 起始密码子的机制 48S PIC 内的识别。 eIF1A 的起始密码子识别最近与大规模 48S PIC 的重排，其中 eIF5B 和起始转移 RNA (Met-tRNAi) 重新定位 准备将大亚基连接到 48S PIC 以形成具有伸长能力的 80S IC。我们将 表征这些 48S PIC 动力学并确定它们在起始密​​码子识别和亚基连接中的作用。