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

蛋白质翻译的非经典起始在细胞应激、凋亡和细胞存活过程中起着核心作用。死亡相关蛋白5（DAP 5）作为主要的支架起始因子，促进细胞命运中关键的细胞mRNA（例如p53、Bcl 2、Apaf 1和XIAP）的帽非依赖性翻译。潜在地，这种转录物的翻译可以通过mRNA中的内部核糖体进入位点（IRES）启动，这是典型的帽依赖性翻译的替代。DAP 5如何调节IRES驱动的翻译的结构和分子机制尚未完全了解。为了解决固有的分子过程，我将通过新的NMR技术和生物物理工具研究目标mRNA中DAP 5的IRES识别。我的研究旨在解决第一个三维结构的原子分辨率的细胞IRES沿着其功能特性。我将首先验证p53，XIAP和DAP 5本身的mRNA中推定的IRES的功能。根据确定的序列，我将解决这些IRES之一的结构。基于此，我将研究IRES和DAP 5之间的相互作用，这与细胞命运决定的结构和生物学相关。我将不仅分析DAP 5的结构化MIF4G结构域的作用，还分析其内在无序区域在IRES识别中迄今未知的功能。我想通过核磁共振相互作用研究，小角X射线散射，等温滴定量热法和圆二色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DAP 5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，不依赖帽的翻译。

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.