Deciphering the molecular mechanisms of non-canonical translation orchestrating cell fate decisions

破译非规范翻译协调细胞命运决定的分子机制

• 批准号: 422676875
• 负责人: Dr. Daniel Friedrich
• 金额: --
• 依托单位: Department of Molecular and Cellular Biology
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/422676875?language=en
• 关键词: Deciphering; molecular; mechanisms; non; canonical

Non-canonical initiation of protein translation plays a central role during cellular stress, apoptosis and cell survival. Death-associated protein 5 (DAP5) acts as the major scaffolding initiation factor to promote cap-independent translation of cellular mRNAs critical in cell fate, for example p53, Bcl2, Apaf1 and XIAP. Potentially, translation of such transcripts can be initiated via internal ribosome entry sites (IRESs) in mRNAs, an alternative to canonical cap-dependent translation. The structural and molecular mechanism of how DAP5 regulates IRES-driven translation is not fully understood. To resolve the inherent molecular processes, I will investigate IRES recognition by DAP5 in target mRNAs through novel NMR techniques and biophysical tools. My research is aimed to solve the first three-dimensional structure at atomic resolution of a cellular IRES along with its functional characterization. I will first validate the functionality of putative IRESs in the mRNAs of p53, XIAP and DAP5 itself. Depending on the determined sequences, I will then solve the structure of one of these IRESs. Based on this, I will study the interaction between this IRES and DAP5 that is relevant for cell fate decisions structurally and biophysically. I will analyze not only the role of the structured MIF4G domain of DAP5, but also the so far unknown function of its intrinsically disordered regions in IRES recognition. I want to understand the influence of these regions on mRNA binding through NMR interaction studies, small angle X-ray scattering, isothermal titration calorimetry and circular dichroism spectroscopy. My project thus comprehensively aims to decipher the mRNA recognition mechanism of DAP5 that controls IRES-driven, cap-independent translation linked to cell fate decisions.

蛋白质翻译的非规范起始在细胞应激、细胞凋亡和细胞存活过程中发挥着核心作用。死亡相关蛋白 5 (DAP5) 作为主要的支架起始因子，促进对细胞命运至关重要的细胞 mRNA 的帽独立翻译，例如 p53、Bcl2、Apaf1 和 XIAP。此类转录物的翻译可能可以通过 mRNA 中的内部核糖体进入位点 (IRES) 启动，这是典型的帽子依赖性翻译的替代方案。 DAP5 如何调节 IRES 驱动的翻译的结构和分子机制尚不完全清楚。为了解决固有的分子过程，我将通过新型 NMR 技术和生物物理工具研究目标 mRNA 中 DAP5 的 IRES 识别。我的研究旨在解决细胞 IRES 的第一个原子分辨率三维结构及其功能表征。我将首先验证 p53、XIAP 和 DAP5 本身 mRNA 中假定的 IRES 的功能。根据确定的序列，我将解析这些 IRES 之一的结构。基于此，我将研究 IRES 和 DAP5 之间的相互作用，该相互作用在结构和生物物理上与细胞命运决定相关。我不仅将分析 DAP5 的结构化 MIF4G 结构域的作用，还将分析其内在无序区域在 IRES 识别中迄今为止未知的功能。我想通过 NMR 相互作用研究、小角 X 射线散射、等温滴定量热法和圆二色光谱来了解这些区域对 mRNA 结合的影响。因此，我的项目全面旨在破译 DAP5 的 mRNA 识别机制，该机制控制与细胞命运决定相关的 IRES 驱动的、与帽无关的翻译。