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Abstract

RNA-protein interactions critically regulate almost every facet of cellular biological processes. Beyond the canonical RNA binding proteins (RBPs) regulating the lifecycle of RNA, systematic analyses of RNA bound proteomes have revealed several unconventional RBPs. Among them, enzymes of intermediary metabolism draw attention in their potential to connect cellular energy metabolism with RNA regulation. Some metabolic enzymes function similarly to canonical RBPs, altering the expression of their target RNAs. Recent evidence also points to RNA as a regulatory molecule, controlling the metabolic function of these enzymes (e.g., glycolytic enzyme ENO1). RNA-metabolic enzyme interactions can thus reciprocally link changes in gene expression with metabolic changes. In this thesis, I explore the RNA binding function of two mitochondrial metabolic enzymes- malate dehydrogenase 2 (MDH2) and ATP synthase subunit alpha (ATP5A1). I primarily focused on characterizing MDH2 as an RNA binding protein, first verifying its RNA binding activity biochemically with orthogonal approaches. MDH2 binds mainly cytosolic RNAs- particularly tRNAs. MDH2 binds RNA predominantly outside the mitochondria, its compartment of function. Based on indications from in-vitro RNA-binding assays, the concentration of MDH2’s substrates and cofactors (particularly malate and NAD+) can direct this preference. Furthermore, MDH2’s RNA binding alters with cellular metabolic state. MDH2 binds less RNA in hepatocyte-like cells with increased mitochondrial respiration compared to a proliferative cancer state. Thus, we find that MDH2’s RNA binding function is an active, compartment-specific process that may have regulatory consequences on protein and/or RNA. ATP5A1-RNA associations were characterized in-vitro, focusing on ATP5A1’s propensity to bind TOP-mRNAs. Towards understanding regulatory consequences of these interactions, I assayed the effects of ATP5A1 depletion on nascent protein synthesis. A fraction of ATP5A1-bound targets is affected in protein synthesis, the mechanisms and implications of which need to be further explored. Evaluation of ATP5A1’s protein interactors revealed a network of cytosolic translation factors and components of RNP complexes in addition to established mitochondrial interactions. These proteins can localise close to the mitochondrial outer membrane, the site where ATP5A1 begins its mitochondrial import and where ATP5A1 interacts with RNA. Taken together, I discuss how ATP5A1’s RNA and protein interactions could be coordinated spatially with resulting functional effects on ATP5A1-bound RNAs as well as ATP5A1’s import into the mitochondria. Together, this study explores the RNA binding functions of two mitochondrial metabolic proteins providing insights into the identity of RNAs associated with them, the location of their association and potential regulation of their RNA binding function.