PhD Oral Exam - Nathalie Reid, Biochemistry

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

The enzyme tRNA nucleotidyltransferase (tRNA-NT) is required in all eukaryotes to add a specific cytidine-cytidine-adenosine (CCA) sequence at the 3′-terminus of all tRNAs. This sequence is added post-transcriptionally during tRNA maturation or repair to prepare a tRNA for aminoacylation prior to protein synthesis. Moreover, tRNA-NTs perform additional roles in tRNA quality control and transport such that they must be present in mitochondria, the nucleus, and cytosol to perform their functions. As the majority of eukaryotes, characterized to date, have a single nuclear gene encoding tRNA-NTs that must be targeted to different cellular destinations, this enzyme has served as a model for research into how the products of a single gene can be targeted to multiple subcellular localizations.

In this study, we showed through in vivo complementation assays and in vitro activity assays that rather than having a single gene encoding a CCA-adding tRNA-NT, Schizosaccharomyces pombe was the first eukaryote to have two genes, cca1 and cca2, that added first CC and then A, respectively. To understand the differences between the eukaryotic two-enzyme system (separate CC- and A-adding activities) and the single enzyme CCA-adding system, we explored aspects of the activity and localization of these never-before-seen eukaryotic CC- and A-adding enzymes.

After defining the CC- and A-adding activities of Cca1 and Cca2, we used site-directed mutagenesis to show that, as in CCA-adding enzymes, changes in conserved motifs C and A, generated and suppressed, respectively, a temperature-sensitive phenotype. This suggests that although for CC- or A-adding enzymes the reorganization of the active site need not be as dramatic as is seen in CCA-adding enzymes which must change conformation from pyrimidine binding (CC) to purine binding (A), there are still conformational changes important for enzyme activity in the CC- and A-adding proteins. Although changes in the tail domain of Cca2, the A-adding enzyme, had little impact on A-addition, similar changes in the same region of Cca1, the CC-adding enzyme, resulted in reduced growth. As the tail domain is thought to play a role in tRNA binding this suggests that in CC-addition, substrate selection or rearrangement of the tRNA is partially controlled by this region. Conversely, the C-terminus is less important for A-addition. Moreover, in vivo the tRNA released from the CC-adding enzyme must move to the A-adding enzyme to complete CCA addition. Our co-immunoprecipitation experiments suggest that Cca1 and Cca2 can be found together when a tRNA is present suggesting direct transfer of the substrate from one enzyme to the next.

Eukaryotic tRNA-NTs must perform their functions in multiple cellular compartments and therefore must contain features not required in bacteria to regulate their subcellular localizations. We found through growth studies using heterologous expression that the absence of the native promoters of cca1 and cca2 causes a 50% increase in doubling times suggesting that some aspects of regulation occur in their promoter regions. Moreover, we showed that both Cca1 and Cca2 possess amino-terminal mitochondrial targeting signals that can restore localization to a truncated S. cerevisiae CCA-adding enzyme. Finally, through proximity-labelling, we identified 20 potential interactions of Cca1 and/or Cca2 with other proteins in S. pombe. of which a nucleoporin usually positioned at the nuclear basket is a potential interaction partner for the CC-adding but not A-adding enzyme.

This exploration of the S. pombe tRNA-NTs has highlighted differences among organisms in the activities and localization strategies for genes with multiple localizations.