Functional Mapping of Translation Initiation Kinetics to Quantify the Protein-Coding Capacity of the Human Genome

PROJECT SUMMARY Despite their central role in protein synthesis, the rates of ribosomal initiation kinetics on different mRNAs and across various tissues remain largely unknown. Understanding the cellular regulation of initiation kinetics and its dysregulation in diseases can significantly enhance our fundamental knowledge of cellular biology and improve modern therapeutics. However, the lack of methods to directly measure initiation across cell types, combined with the vast mRNA sequence space and the complex, cell-specific interactions it supports, results in an intractable complexity. This hinders our ability to answer several central questions, including: 1) How to predict transaltion initiation rates from mRNA sequence? 2) How do initiation rates of individual mRNAs vary between cell types? 3) To what extent does initiation rate determine protein levels in cells? 4) What are the initiation rates at alternative and small ORFs translation initiation sites, and what is the protein encoding capacity of the genome? This proposal outlines a five-year research plan to address these challenges and questions by developing a measurement system that will measure the kinetic parameters that control translation initiation. These paramters will be measured using a cell-free system reconstituted from cell-specific proteomes, which will be titrated with large mRNA libraries, and followed by ribosome footprinting and sequencing-based quantification of ribosomes locked on start codons. This functional genomics platform, termed MIT-seq, was recently developed by us for bacteria and this proposal will adapt to human cells. It will be benchmarked using mRNA libraries includes the entire human transcriptiome and compared to other, massively parallel reporter assays and existing data from the litrature (Aim 1). Subsequently, we will measure the initiation affinities unprecedently large (1010) mRNA libraries to explore variations in initiation kinetics between differentiated cells, such as kidney cells and neurons (Aim 2). Finally, we will examine shifting landscapes and dysregulation of intiation kinetics and their functional consequences between healthy and diseased cells using Fragile X Syndrome, a disease caused by altered levels of the translation initiation regulator FMR1 protein, as a model (Aim 3). To achieve these aims, the candidate will leverage his unique expertise in the molecular biology of translation initiation developed during his PhD studies and the assays developed during his current postdoctoral fellowship at MIT. To transition from bacterial systems to the molecular biology of human cells, the candidate has assembled an outstanding advisory team, including relevant experts who have agreed to mentor, collaborate, and provide resources for the project's success during its training phase (K99). Upon establishing Aim 1, the candidate will pursue an independent career (R00) focusing on Aims 2 and 3, while working towards the long- term goal of developing functional genomics tools and understanding the design principles, mechanisms, and evolution of mRNA translation initiation across tissues and organisms.