Targeting RNA Structures that Control Protein Synthesis and RNA Stability

Abstract

RNA structures play a pivotal role in many biological processes and the progression of human disease, making them an unexploited target for therapeutic development. The first chapter of this thesis reviews features related to targeting RNA structures and to the subsequent topics of this thesis: disease-associated RNA classes, advantages and disadvantages of different RNA-binding chemistries (e.g. small molecules, peptides, engineered proteins), NMR-based methods for RNA screening and structure determination, and a future perspective on the field. Chapter 2 describes the results of my research on specialized translation initiation, whereby protein synthesis is controlled by eukaryotic initiation factor 3 (eIF3) recognizing RNA structures within the 5’-untranslated regions of certain genes to regulate translation rates of specific mRNAs. I examined this mechanism by establishing the structural basis for eIF3 recognition of the c-JUN 5’-UTR cis-regulatory element (SL1). SAXS modeling and NMR structure determination identified similarities to the way eIF3 recognizes RNA motifs within internal ribosomal entry sites (IRES) in the Hepatitis C Virus (HCV) RNA, suggesting mechanistic similarities. This work establishes RNA structural features involved in c-JUN specialized translation initiation and provides a foundation to search for small molecules inhibitors of aberrant expression of the proto-oncogenic c-JUN protein. Chapter 3 reviews strategies for designing cyclic β-hairpin peptidomimetics targeting pharmaceutically relevant structured RNAs such as HIV-TAR and pre-microRNA-21 (pre-miR-21). This rational is built into several pre-microRNA targeting projects discussed in chapter 4, where I describe the development of a pre-miR-21 processing assay to characterize inhibitors ranging from cyclic peptides to engineered RNA-binding motifs (RRM*). My results demonstrate that many cyclic peptides disrupt efficient pre-miR-21 processing, as demonstrated by the formation of novel reaction intermediates only produced in the presence of cyclic peptide inhibitors.