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.

项目摘要 核糖体将信使RNA（mRNA）转化为蛋白质的效率是 基因表达的基本决定因素。这种效率通常是在mRNA募集过程中确定的 核糖体翻译步骤。因此，此步骤是基因表达的关键控制点。在 真核生物，mRNA募集是一个精心制作的，多步骤的，高度调节的过程，取决于 〜13个真核引发因子（EIF）的活动。 EIF活性和mRNA募集的失调一直是 与肿瘤发生，肿瘤生长，耐药性和转移的因果关系越来越多。 癌症。因此，几个EIF及其在mRNA招募中的作用非常吸引人 抗癌药物靶标具有现有的，FDA批准的，靶向的靶向化合​​物 成功地作为抗癌治疗。为了扩大和充分利用这种治疗潜力， 但是，有必要了解基于EIF功能和mRNA募集的分子事件。 在这里，我们将使用高度纯化的荧光团标签，酿酒酵母在体外翻译 我们开发的系统，其中包括一个全长的，特定于网站的EIF4G和一个完全重构的， 特定于位点标记的EIF3，很难生成的试剂。将这些试剂手里拿到 使用最先进的单分子荧光显微镜和低温电子显微镜（Cryo-EM）， 包括我们的合作者Joachim Frank博士开发的开创性，时间分辨的冷冻EM方法 直接观察并表征真核翻译启动过程中mRNA募集的动力学。 在AIM 1中，我们将研究多组分EIF4F复合物激活的机制 不同类别的mRNA，用于加载到核糖体43S前发射复合物（图片）以及如何变化为核糖体43 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载荷至关重要，将量化这些动力学 动力学有助于mRNA选择。在AIM 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 在43S PIC上，以及EIF3的生物活性亚复合物如何调节这些活动。大 - 据认为，43S PIC结合的EIF3的缩放结构重排可以控制其mRNA载荷活性，我们将 因此，表征了这种重排的促进在不同的mRNA上的48S图片的形成 课程。在AIM 3中，我们将研究EIF1A和EIF5B介导mRNA start-codon的机制 在48S图片中识别。 EIF1A的启动二号识别最近与大规模相关联 EIF5B和引发剂转移RNA（Met-tRNAi）重新安排的48S PIC重新排列。 将大型亚基连接到48S图片的准备工作形成了能力伸长的80S IC。我们将 表征这48S PIC动力学，并确定它们在启动二元识别和亚基加入中的作用。