Abstract 1208 Investigating Cox7B as a Possible Human Riboswitch

Riboswitches are RNA structures that regulate gene expression by binding directly to a small-molecule ligand. They are often found in the 3' or 5' untranslated region (UTR) of an mRNA strand. Using varied mechanisms, riboswitches can control gene expression at the transcription or translation level. While riboswitches are abundant bacteria, only one has been found in fungi and plants in Eukarya. Currently, there is no evidence of riboswitches in the human transcriptome. Jeff Morgan, Ph.D. at the Rutter lab, asked if humans could also have riboswitches. Utilizing dimethyl sulfate (DMS) structure probing and mass spectrometry integrated with equilibrium dialysis (MIDAS), Dr. Morgan screened the 5' UTRs of HEK293 mRNA and found evidence of unique metabolite-RNA interactions. One of the hits from this preliminary data was the 5' UTR of Cytochrome C Oxidase Subunit 7B (COX7B) binding to cyclic adenosine monophosphate (cAMP). COX7B is a subunit protein of Complex IV in the mitochondria's electron transport chain (ETC). To date, little is known about the protein and its functions of the ETC. The goal of this project was to study whether modulating cAMP concentrations can induce changes in COX7B expression, a key trait of riboswitches. First, we utilized an in-vitro translation assay and showed that increased cAMP levels result in higher COX7B protein production. Second, using an in vivo luciferase reporter assay, we affirmed that increased intracellular cAMP levels result in upregulated translation of our luciferase reporter gene. Finally, we show that endogenous COX7B protein levels increase upon stimulation with forskolin and IBMX, two commonly used drugs that specifically and acutely raise intracellular cAMP concentrations. These data suggest that cAMP can bind to COX7B and regulate translation by increasing COX7B protein production, providing some of the first evidence for riboswitches in humans. We are continuing to investigate the COX7B riboswitch, including characterizing the binding affinity in vitro. However, why this aptamer exists is another major question to be answered. cAMP is known to impact respiration in mitochondria, although the exact mechanisms remain undefined. Given that COX7B increases upon cAMP stimulation, that may suggest it is able to impact respiration in ways currently unknown to the field. Therefore, we plan to investigate the effects of COX7B expression on respiration and the ETC as part of our future directions for this project. This project was funded in part by the Rural Underserved Utah Training Experience through the University of Utah.