PhD Oral Exam - Hamed Tahmasbi, Chemistry

When studying for a doctoral degree (PhD), candidates submit a thesis that provides a critical review of the current state of knowledge of the thesis subject as well as the student’s own contributions to the subject. The distinguishing criterion of doctoral graduate research is a significant and original contribution to knowledge.

Once accepted, the candidate presents the thesis orally. This oral exam is open to the public.

Abstract

Transfer RNA nucleotidyltransferase (tRNA-NT; also known as CCA-adding enzyme, encoded by TRNT1) 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 A[8] 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 demonstrated that while overall folding and stability were preserved, mutations 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 A[8] frameshift variant detected in patients abolished enzymatic function.

Chapter 3 focused on mutations in the head domain. The conservative T123S substitution retained near-native folding and enzymatic activity but subtly perturbed local hydrogen bonding between motifs A and B, producing selective defects during the first C and final A addition steps. By contrast, the S289Kfs frameshift variant, also identified in patients, eliminated critical C-terminal regions and rendered the enzyme non-functional. These results highlight how conservative substitutions yield nuanced effects, while frameshift mutations are catastrophic.

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.