Advisory Committee Chair
Jun Zhang
Advisory Committee Members
Champion Deivanayagam
Hui-Ting Lee
Aaron Lucius
William Placzek
Document Type
Dissertation
Date of Award
2023
Degree Name by School
Doctor of Philosophy (PhD) College of Arts and Sciences
Abstract
RNA-binding proteins (RBPs) are crucial to RNA processing and regulation. RBPs have elevated levels of disordered regions that are enriched with repetitive motifs. The longest and most common of these repetitive motifs are electronegative clusters (ENCs) that are made up of consecutive acidic amino acids and/or phosphorylatable residues. In this dissertation, we aim to characterize the function of ENCs in RBPs for the first time. We investigate the effect that ENCs have on both structured and unstructured RNA binding domains (RBDs). Using NMR and fluorescence spectroscopy we illustrate that a native ENC in ribosome biogenesis protein 15 (Nop15) increases the stability of its RNA recognition motif (RRM) and increases its specificity towards its target RNA. We also show that a grafted ENC can increase the stability of the RRM of TAR DNA binding protein 43 (TDP-43). ENCs have a different function when bordering unstructured RBDs. Using NMR spectroscopy, we show that phosphorylation of the stem loop binding protein’s (SLBP) ENC increases the population of a conformation that resembles its folded RNA-bound state. Moreover, we show that mutating an ENC adjacent to the first double stranded RNA binding motif (DRBM1) of endoribonuclease homolog 1 (DCL1) also increases the population of the folded state. Finally, we show that we can control folding and RNA binding by adding electropositive residues to the main body of DRBM1. In conclusion, this dissertation has found that ENCs can be used to control iv stability, folding, and RNA-binding affinity and specificity of RBDs. This provides novel insight into how RNA-binding is regulated in both structured and unstructured proteins and both expands our understanding of biological processes and serves as a potential framework for the development of protein therapeutics.
Recommended Citation
Zaharias, Steven Mathew, "Electronegative Clusters Modulate the Stability, folding and Binding Affinity of RNA-Binding Proteins" (2023). All ETDs from UAB. 370.
https://digitalcommons.library.uab.edu/etd-collection/370