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Transfer ribonucleic acids (tRNAs) are extensively modified, especially their anticodon loops. Modifications at position 34 (wobble base) and 37 in these loops affect the tRNAs’ decoding ability, while modifications outside the anticodon loops, e.g. m¹A₅₈ of tRNAMeti, may be crucial for tRNA structure or stability. A number of gene products are required for the formation of modified nucleosides, e.g. at least 26 proteins (including Elongator complex) are needed for U₃₄ modifications in yeast, and methyl transferase activity of the Trm6/61p complex is needed to form m¹A₅₈. The aim of the studies which this thesis is based upon was to investigate the functional aspects of tRNA modifications and regulation of the modifying enzymes’ activity.

First, the hypothesis that ncm⁵U₃₄, mcm⁵U₃₄, or mcm⁵s²U₃₄ modifications may be essential for reading frame maintenance was investigated. The results show that mcm⁵ and s² group of mcm⁵s²U play a vital role in reading frame maintenance. Subsequent experiments showed that the +1 frameshifting event at Lys AAA codon occurs via peptidyl-tRNA slippage due to a slow entry of the hypomodified tRNA-Lys.

Moreover, the hypothesis that Elp1p N-terminal truncation may regulate Elongator activity was investigated. Cleavage of Elp1p was found to occur between residue 203 (Lys) and 204 (Ala) and to depend on the vacuolar protease Prb1p. However, including trichloroacetic acid (TCA) during protein extraction abolished the appearance of truncated Elp1p, showing that its truncation is a preparation artifact.

Finally, in glioma cell line C6, PKCα was found to interact with TRM61. RNA silencing of TRM6/61 causes a growth defect that can be partially suppressed by tRNAMeti overexpression. PKCα overexpression reduces the nuclear level of TRM61, likely resulting in reduced level of TRM6/61 complex in the nucleus. Furthermore, lower expression of PKCα in the highly aggressive GBM (relative to its expression in less aggressive Grade II/III glioblastomas) is accompanied by increased expression of TRM6/61 mRNAs and tRNAMeti, highlighting the clinical relevance of the studies.

Elongator is a conserved six subunit protein (Elp1p-Elp6p) complex that is required for the formation of ncm5 and mcm5 side chains at wobble uridines in transfer RNAs (tRNAs). Moreover, loss-of-function mutations in any gene encoding an Elongator subunit results in translational defects and a multitude of phenotypic effects. This thesis is based on investigations of effects of wobble uridine modifications on translation.

In Saccharomyces cerevisiae, ncm⁵U₃₄-, mcm⁵U₃₄- and mcm⁵s²U₃₄- modified wobble nucleosides in tRNAs are important for proper codonanticodon interactions. My colleagues and I (hereafter we) showed that mcm⁵ and s² groups at wobble uridine in tRNAs are vital for maintaining the reading frame during translation, as absence of these modifications increases the frequency of +1 frameshifting. We also showed that +1 frameshifting events at lysine AAA codons in Elongator mutants are due to slow entry of the hypomodified tRNA ^Lys_s2UUU to the ribosomal A-site.

Ixr1p is a protein that plays a key role in increasing production of deoxynucleotides (dNTPs) in responses to DNA damage, via induction of Ribonucleotide reductase 1 (Rnr1p), in S. cerevisiae. We showed that expression of Ixr1p is reduced in elp3Δ mutants due to a post-transcriptional defect, which results in lower levels of Rnr1p in responses to DNA damage. Collectively, these results suggest that high sensitivity of Elongator mutants to DNA damaging agents might be partially due to reductions in Ixr1p expression and hence Rnr1p levels.

Elongator mutant phenotypes are linked to several cellular processes. To probe the mechanisms involved we investigated the metabolic perturbations associated with absence of a functional ELP3 gene in S. cerevisiae. We found that its absence results in widespread metabolic perturbations under both optimal (30°C) and semi-permissive (34°C) growth conditions. We also found that changes in levels of certain metabolites (but not others) were ameliorated by elevated levels of hypomodified tRNAs, suggesting that amelioration of perturbations of these metabolites might be sufficient for suppression of the Elongator mutant phenotypes.

A mutation in the IKBKAP (hELP1) gene results in lower levels of the full-length hELP1 protein, which causes a neurodegenerative disease in humans called familial dysautonomia (FD). We showed that the levels of mcm⁵s²U-modified wobble nucleoside in tRNAs are lower in both brain tissues and fibroblast cell lines derived from FD patients than in corresponding materials derived from healthy individuals. This suggests that FD may result from inefficient translation due to partial loss of mcm⁵s²U-modified nucleosides in tRNAs.