Regulation of Protein Production Dynamics:RNA Binding Proteins and the Ribosome Code

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Summary No changes from parent grant The central dogma of molecular biology assumes a linear path of gene expression from gene to protein. It is now clear that gene expression is tightly controlled at several levels - from transcription to translation to protein degradation - yet the fields of molecular, cell and developmental biology have mostly focused on transcriptional control as the primary mode of gene regulation. Yet, the mammalian genome encodes over 1,500 RNA binding proteins (RBPs), several of which are recurrently mutated in diseases, such as cancer and neurological disorders, suggesting that post-transcriptional gene expression regulation and especially mRNA translation are important in both health and human disease. Furthermore, the regulatory role of the ribosome itself has so far been under-explored. Evidence is mounting that specialized ribosomes, which vary in ribosomal protein stoichiometry and post-translational modifications, exist that may impact the translation of specific mRNAs through an as-yet-undefined ‘ribosome code’. The overarching research goal of the lab is to understand the principles and mechanisms by which translational regulation controls the dynamics of gene expression and therefore affects processes like differentiation, stress response and pathogenesis. Over the next five years, we will focus on two specific aspects of translational control in the context of mouse embryonic stem cell differentiation. First, we will systematically identify and characterize RNA binding proteins (RBPs) that regulate translational changes. Based on our previous work, we will combine high-throughput CRISPR-based screening with global measurements of RNA dynamics, and protein production and degradation. This will link RBPs to their mRNA targets, providing the foundation for future detailed functional follow-ups, allowing us to elucidate functional and causal insights of how RBPs regulate mRNA translation. Second, we are looking at the extent of ribosomal heterogeneity, testing the hypothesis that specialized ribosomes exist that selectively translate subsets of mRNAs, thereby introducing an additional level of regulation in gene expression – a ribosome code. By applying high accuracy mass spectrometry, we are focusing right now on two potential sources of ribosomal heterogeneity – differential expression in core ribosomal proteins (RPs) and changes in their post-translational modifications. Based on these measured changes in ribosome composition, we are selecting RPs and PTMs with the strongest changes for further functional characterization. The detailed follow up will provide for a selected set of RPs and PTMs the principles and mechanistic insight how ribosome specialization regulates translation. Together these two approaches will provide unprecedented insight on the dynamics of protein production in an important physiological context, potentially unravelling novel paradigms of gene expression regulation.