The dysregulation of RNAs has global effects on all cellular pathways. The regulation of RNA metabolism is thus tightly controlled. Terminal RNA nucleotidyltransferases (TENTs) regulate RNA stability and activity through the addition of non-templated nucleotides to the 3′-end. TENT-catalyzed adenylation and uridylation have opposing effects; adenylation stabilizes while uridylation silences or degrades RNA. All TENT homologs were initially characterized as adenylyltransferases; the identification of caffeine-induced death suppressor protein 1 (Cid1) in Schizosaccharomyces pombe as an uridylyltransferase led to the reclassification of many TENTs as uridylyltransferases. Cid1 uridylates mRNAs that are subsequently degraded by the exonuclease Dis-like 3′-5′ exonuclease 2 (Dis3L2), while the human homolog germline-development 2 (Gld2) has been associated with adenylation of mRNAs and miRNAs and uridylation of Group II pre-miRNAs. Mechanisms regulating these enzymes and the extent of TENT activity on cellular RNA homeostasis remain largely unknown. In this thesis, the regulation of human Gld2 and the role of the yeast Cid1/Dis3L2-mediated RNA decay pathway were investigated. An enzyme kinetic study revealed that Gld2 is a true adenylyltransferase with only weak activity for UTP. A detailed phylogenetic analysis revealed that uridylyltransferases arose multiple times during evolution through a single histidine insertion in the active site of adenylyltransferases. Insertion of the critical histidine into Gld2 changed its nucleotide preference from ATP to UTP. Next, the regulation of Gld2 through site-specific phosphorylation in the predicted disordered N-terminal domain was investigated using phosphomimetic substitutions at specific serine (S) residues. Two sites (S62, S110) increased Gld2 activity while one site (S116) drastically reduced 3′-adenylation activity. Mass spectrometry and in vitro activity assays identified protein kinases A (PKA) and B (Akt1) as kinases that specifically phosphorylate Gld2 at S116 to obliterate nucleotide addition activity similarly to the S116E phosphomimetic mutant. Finally, RNA deep sequencing of cid1 and dis3L2 S. pombe deletion strains revealed that the role of Cid1 is redundant in uridylation-dependent mRNA decay while Dis3L2 is the bottleneck to RNA decay. Deletion of either gene increases the accumulation of misfolded proteins but only the dis3L2 deletion up-regulates stress response proteins. Overall, this thesis demonstrates how terminal nucleotidyltransferases regulate RNA stability
