Abstract

Human transfer RNA nucleotidyltransferase (tRNA-NT; also known as a CCA-adding enzyme and encoded by the TRNT1 gene) catalyzes the essential, template-independent addition of the invariant cytidine–cytidine–adenosine (CCA) trinucleotide to the 3′ termini of all tRNAs. This step is indispensable for aminoacylation and protein synthesis. The enzyme adopts a conserved seahorse-shaped architecture composed of head, neck, body, and tail domains. Mutations in TRNT1 cause sideroblastic anemia with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD), a rare multisystem disorder. Several of the variants studied here including K416E, T123S, and the frameshift mutations S418Kfs and S289Kfs have been reported in patients, yet the mechanistic basis of their dysfunction remains incompletely defined.
This thesis integrates structural biology, yeast genetics, enzymology, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and quantitative proteomics to characterize the effects of TRNT1 variants outside the canonical catalytic motifs. In Chapter 2, analysis of C-terminal variants demonstrates that while overall folding and stability are preserved, amino acid substitutions such as K416E altered local dynamics in the body-tail interface and reduced substrate engagement, yielding partial activity. In contrast, larger truncations (CΔ10, CΔ33) and the S418Kfs frameshift variant detected in patients abolished enzymatic function.
Chapter 3 examined variants located within the head domain of TRNT1. The conservative T123S substitution largely preserved global folding and overall catalytic capacity, but the alteration subtly perturbed local hydrogen bonding between motifs A and B. These localized structural changes appear to impair only the most sensitive steps of the reaction cycle specifically, the initial C addition and the terminal A addition, placing T123S within a hypomorphic or partial loss-of-function category. In contrast, the S289Kfs frameshift variant, also found in patients, truncates the C-terminal region that contributes essential elements for substrate positioning and catalysis. Consistent with the loss of these domains, the S289Kfs allele behaves as a severe loss-of-function or near-null variant. Together, these findings illustrate how conservative substitutions can produce graded, allele-specific defects, whereas frameshift mutations that disrupt core structural elements yield substantially more severe functional consequences.
Chapter 4 employed stable isotope labeling by amino acids in cell culture (SILAC) based proteomics to examine the E189F variant in yeast. Using isogenic strains YFT17-1 (E189F, temperature-sensitive) and YEA31-1 (wild type), we found that at 21°C the mutant maintained wild-type-like growth through compensatory proteomic adjustments despite reduced enzymatic activity. At 31.5°C, however, broad remodeling emerged, including depletion of translation factors, signatures of stress granule formation, induction of amino acid biosynthesis enzymes, and retrograde pathway activation.
Together, by integrating structural, biochemical, genetic, and proteomic approaches, this work establishes a comprehensive framework for understanding how TRNT1 mutations impair enzyme activity and disturb cellular homeostasis. The results also highlight how defects in this essential enzyme can propagate across multiple levels of cellular regulation.