Epigenetics in the extreme - investigating heritability driven by disordered RNA binding proteins in development and cancer

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Heritable protein conformations, ‘prions’, were discovered as the cause of debilitating transmissible spongiform encephalopathies. Long considered to be a fascinating biological oddity, the recent discovery of prions in diverse organisms from fungi to sea slugs has led to the watershed realization that ‘protein-based genes’ are much more common than previously appreciated. We recently discovered that intrinsically disordered proteins can drive the formation of such protein-based molecular memories that can be epigenetically inherited over hundreds of generations without any alteration to its genome sequence. RNA binding proteins are particularly enriched in this dataset. An emergent area of study has demonstrated that disordered regions in RNA binding proteins are very common and often drives the formation of liquid-liquid de-mixing states critical for forming membrane-less organelles. My preliminary studies establish that disordered RNA binding proteins are uniquely poised to relay changes in environment to an altered cellular program. I will address the following aims during my remaining mentored training and initial independent research career: 1. To determine the biochemical and functional implication of intrinsically disordered regions of RNA binding proteins. I have developed a methodology to quantitatively investigate RNA-protein interactions across a genome. Coupling this platform with a robust system that I have developed to purify these intrinsically disordered RNA binding proteins, I intend to biochemically investigate the effect of intrinsically disordered regions on RNA binding proteins. Additionally, I will examine the effect of these disordered domains on phenotype using quantitative genetics and cell biology. Finally, I will extend the method to the human exome to interrogate human RBPs and their frequent oncogenic mutants. 2. To investigate the prion-like behavior of transient overexpression of disordered RNA binding protein ySmaug. Using high-throughput gene expression analysis, quantitative genetics and cell biology, I will examine the prion-like behavior of an evolutionarily ancient and highly disordered RNA binding protein ySmaug. 3. To establish the mechanism of assembly and transmission of prion-like RBPs. Using toolkits of biochemistry, microscopy and structural biology (NMR & EM methodologies), I will establish the mechanistic basis of assembly and transmission of these prion-like RBPs that exhibit canonical genetic hallmarks of prions but surprisingly do not form amyloids. 4. To investigate the impact of overexpression of disordered oncogenic proteins. Finally, I will harness a defining property of all known prions – protease resistance to examine the impact of three disordered nucleic acid binding proteins – p53 (one of its hotspot mutants is known to form amyloid fibers), SAMD4A (human homolog of ySmaug) and Musashi RNA binding proteins (Msi1 & Msi2) that is overexpressed in a variety of cancers and been attributed to be drivers of pancreatic adenocarcinomas. Following initial studies in yeast, I will investigate these proteins in their natural context – specifc mammalian cancer cell lines. The data obtained from this study will not only provide mechanistic insights into how higher- order heritable protein assemblies of RNA binding proteins are formed and heritably alter gene expression, but will also provide a framework for potential therapeutic intervention into a complex disease like cancer.