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

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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.