PROJECT SUMMARY Riboswitches are regulatory RNA structures that control gene expression by binding to a small molecule and changing conformation, allowing organisms to rapidly respond to changes in their metabolic environments. They are prevalent in bacteria, but in over 20 years of study, only one eukaryotic riboswitch has been found. It has long been postulated that there are more, but the lack of high-throughput screening assays and in silico detection algorithms has limited discovery. To address these challenges, the Rutter lab has developed a high-throughput approach combining dimethyl sulfate (DMS) structure probing and MIDAS (mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically) to screen for novel eukaryotic riboswitches. A preliminary screen of HEK293 mRNA resulted in eight novel interactions in the 5’UTR regions of various transcripts. The overarching objective of this proposal is to investigate the regulatory abilities of these interactions and their downstream metabolic consequences in vivo. One of these, COX7B, is a subunit of complex IV (CIV) in the electron transport chain (ETC) and bound to cyclic AMP (cAMP) as a putative ligand. cAMP is known to stimulate respiration, but an obvious regulatory connection between cAMP and COX7B is unknown. Other subunits of CIV are known to regulate respiration through phosphorylation and substrate sensing, but COX7B’s function is yet to be defined. However, recent literature places it at the interface of complex I (CI) and CIV in supercomplex formation. We propose that COX7B’s 5’UTR is a novel riboswitch that impacts respiration through regulating supercomplex formation. Aim 1 will investigate the binding event using isothermal titration calorimetry, DMS structure probing, and reporter assays. Aim 2 will characterize COX7B’s role in CIV, testing for supercomplex formation, respiration, and CI and CIII activity via blue-native PAGE (BN-PAGE), seahorse assays, and enzymatic assays in wild-type, COX7B knock out (KO) and overexpression (OE) cell lines. Aim 3 will then define the metabolic impact of COX7B mRNA interactions with cAMP in vivo. Mutations in the 5’UTR of COX7B will be introduced and supercomplex formation, respiration, and complex activity in response to elevated cAMP levels will be assessed as described. These data will highlight novel regulatory pathways for respiration and introduce a new field of gene regulation to be explored. The Rutter Lab is the first to develop a high-throughput platform for studying novel RNA-metabolite interactions and has a dedicated team devoted to studying novel riboswitches. In addition, the University of Utah hosts a community of renowned scientists studying metabolism and RNA biochemistry such as my sponsor, Dr. Jared Rutter, and co-sponsor, Dr. Ming Hammond. Finally, this project contains a detailed training plan for developing multidisciplinary research and professional skills that will enable me to becom...