Effects of small molecule binding to mRNA on gene expression Public Deposited

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  • March 20, 2019
Creator
  • Sullivan, Julie Marie
    • Affiliation: College of Arts and Sciences, Department of Chemistry
Abstract
  • RNA plays significant roles in many cellular processes and exhibits attractive features for use as a small molecule targeted therapeutic. This study focuses on using RNA as a drug target, specifically investigating the process by which small molecules can regulate gene expression through their interaction with secondary structural elements found in the 5' untranslated region (UTR) of two cellular messenger RNA (mRNA) targets, human ferritin and human preproinsulin. Previous work has identified compounds that specifically and selectively bind in the hairpin loop of the iron responsive element (IRE) in human ferritin mRNA using a chemical footprinting method. Ferritin is regulated at the translational level by IRP binding to the IRE, preventing assembly of the translational initiation complex. One small molecule, promazine, was identified that binds to an internal bulge in the IRE sequence where an iron regulatory protein (IRP) binds. The binding site identified during the initial screening was confirmed through an enzymatic footprinting assay. Using an electrophoretic mobility shift assay, we have shown that this compound can disrupt the binding of IRP proteins to the IRE sequence. In an in vitro translation assay, binding of promazine leads to up-regulation of ferritin protein synthesis by greater than 60% while showing no effect on control mRNA. The 5'UTR of the human preproinsulin mRNA exists in two forms, the native form, and a splice variant which is up-regulated in response to chronic glucose stimulation. Small molecules were screened for their ability to bind to, or change the secondary structure of, the 5'UTR of the preproinsulin mRNAs using RNase cleavage, and the transition metal cleavage agents Ru(tpy)(bpy)O2+ (RuO2+, bpy = 2,2'-bipyridine, tpy = 2,2',2''-terpyridine), and Ru(bpy)3 2+. Two small molecules, neomycin B and kanamycin B, have been identified as binding to the mRNAs using these screening methods. Binding of neomycin B, and kanamycin B decreased the amount of preproinsulin protein synthesized for both the native and splice variant forms in an in vitro translation assay. A decrease in the translation of a control RNA was also seen, indicating that the binding of these aminoglycosides are not specific to secondary structures found in preproinsulin mRNA.
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  • Thorp, H. Holden
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