ABSTRACT Mitochondria are essential organelles that serve as the cellular hub for metabolism, ATP production, and redox signaling. We identified a novel mechanism of signaling in the mitochondria mediated by a post translational modification known as AMPylation, the covalent addition of adenosine monophosphate (AMP) to protein substrates. Our previous studies revealed that Selenoprotein O catalyzes AMPylation of multiple mitochondrial proteins involved in redox homeostasis and cellular metabolism. There are several critical gaps in our current knowledge of AMPylation including the functional importance of AMPylation, as well as the enzymes that reverse AMPylation. To gain a mechanistic understanding of AMPylation, we developed an enrichment strategy for AMPylated proteins and identified RNase Z as a deAMPylase that catalyzes the removal of AMP from AMPylated substrates. RNase Z was previously shown to be a conserved endoribonuclease that cleaves the 3’ trailer of precursor tRNAs to generate mature tRNAs. Our studies establish RNase Z as a multifunctional protein with previously unrecognized functional roles beyond tRNA processing. Thus, the goal of this proposal is to determine the biochemical and molecular mechanisms of RNase Z-mediated deAMPylation. Mutations in RNase Z result in severe hypertrophic cardiomyopathy and increased prostate cancer susceptibility. We hypothesize that the deAMPylation activity, in addition to the endoribonuclease activity, contributes to the functional importance of RNase Z in the mitochondria. However, our understanding of mitochondrial AMPylation is in its infancy due to the lack of tools to study the AMPylated proteins. Thus, we developed a novel enrichment strategy for the identification and functional characterization of AMPylated proteins. We anticipate these studies will reveal previously undocumented roles for AMPylation in cellular signaling and the molecular mechanisms of RNase Z-associated diseases.