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Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Heinemann, Ilka U.

Abstract

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.

Summary for Lay Audience

Ribonucleic acids (RNAs) play important roles in protein production and regulating cellular processes such as cell proliferation. Dysregulation of RNA expression, maturation, and/or degradation is associated with multiple human diseases such as cancer and cardiovascular disease. RNAs can be regulated through the addition of adenine or uridine nucleotides to its 3’-end. The proteins that perform these additions are known as terminal RNA nucleotidyltransferases (TENTs). All TENTs were initially thought to add adenine residues (adenylyltranferases), but more extensive studies revealed that some TENTs preferred to add uridine residues (uridylyltransferases). The addition of adenine is associated with stability while uridine addition is associated with silencing/degradation. Thus, the simple addition of different nucleotides can change the fate of an RNA molecule. The yeast TENT uridylates RNAs which are recognized and degraded by the exonuclease Dis-like 3′-5′ exonuclease 2 (Dis3L2). On the other hand, its human counterpart, germline-development 2 (Gld2), has been associated with RNA adenylation and uridylation. Mechanisms regulating these proteins 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. First, Gld2 was shown to be a true adenylyltransferase. The simple insertion or deletion of the amino acid histidine in the active site was shown to change the nucleotide preference of TENTs. Secondly, Gld2 was shown to be regulated through phosphorylation of specific serine residues (S). Two sites (S62, S110) increased Gld2 activity while one site (S116) drastically reduced activity. Two cancer-related kinases, protein kinases A (PKA) and B (Akt1), were identified to phosphorylate Gld2 at S116 to obliterate nucleotide addition activity. This discovery provided the first link between cancer-related kinases and RNA regulation. Finally, deletion of either the Cid1 or Dis3L2 genes in yeast revealed that Cid1 is redundant in uridylation-dependent mRNA decay while Dis3L2 is the bottleneck to RNA decay. Deletion of the Dis3L2 gene elicited a larger change in the RNA population. Overall, this thesis demonstrates how terminal nucleotidyltransferases regulate RNA stability.

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