Regulatory functions of intrinsically disordered electronegative clusters (ENC) in RNA-binding proteins

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Interaction between biomolecules is essential for life. While most proteins have stable three-dimensional structures, a significant number of proteins have been shown to lack a stable three-dimensional structure and are known as intrinsically disordered proteins. It has been shown that proteins that interact with RNA contain regions that are intrinsically disordered, which are essential for the protein-RNA binding. The investigators of this project have shown that most abundant disordered regions in RNA-binding proteins encode clusters of acidic residues or phosphorylation sites that give these regions an overall negative charge. Like tails that help animals to balance and to swish away insects, the disordered protein regions help proteins to balance surface charges for stability and swish away nonspecific ligands. This project will explore how these electronegative clusters modulate protein function. Studying these proteins will provide insights into key biological processes, such as transient folding of disordered proteins, site selection in ribosomal biogenesis, and alternative splicing. In addition, understanding this phenomenon will inform development of novel protein design techniques for more robust and specific RNA-binding proteins, which will ultimately contribute to US bioeconomy. This project will provide training opportunities to high school and undergraduate students, including members of underrepresented groups, in biochemical research and bioinformatics-based coding. This project will study three model proteins: histone-mRNA Stem-Loop Binding Protein, ribosome biogenesis protein 15, and Serine-Arginine Rich Splicing Factor 1. These proteins were selected because they play essential roles in critical biological processes, such as histone pre-mRNA processing, ribosomal biogenesis, and alternative splicing, respectively. Because these proteins encode different types of electronegative clusters and have unique degrees of three-dimensional folding status, this project will systematically elucidate the function of electronegative clusters in mediating both protein folding and stability as well as RNA binding specificity and kinetics. Cutting-edge biophysical and biochemical techniques, such nuclear magnetic resonance, crystallography, stopped-flow fluorescence, and next generation sequencing will be used. Completion of this work will not only produce knowledge generally applicable to all RNA-binding proteins, but also elucidate specific and important processes where these proteins play essential roles.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.