Mutations found in genes encoding human Elongator complex subunits have been linked to neurodevelopmental disorders such as familial dysautonomia (FD), rolandic epilepsy and amyotrophic lateral sclerosis. In addition, loss-of-function mutations in genes encoding Elongator complex subunits cause defects in neurodevelopment and reduced neuronal function in both mice and nematodes. The Elongator complex is a conserved protein complex comprising six subunits (Elp1p-Elp6p) found in eukaryotes. The primary function of this complex in yeast is formation of the 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) side chains found on wobble uridines (U34) in tRNAs. The aim of this thesis is to investigate the physiological consequences of Elongator complex inactivation in humans and in the yeast Saccharomyces cerevisiae. Inactivation of the Elongator complex causes widespread defects in a multitude of different cellular processes in S. cerevisiae. Thus, we investigated metabolic alterations resulting from Elongator complex inactivation. We show that deletion of the S. cerevisiae ELP3 gene leads to widespread metabolic alterations. Moreover, all global metabolic alterations observed in the elp3Δ strain are not restored in the presence of elevated levels of hypomodified tRNAs that normally have the modified nucleoside mcm5s2U. Collectively, we show that modified wobble nucleosides in tRNAs are required for metabolic homeostasis. Elongator mutants display sensitivity to DNA damage agents, but the underlying mechanism explaining this sensitivity remains elusive. We demonstrate that deletion of the S. cerevisiae ELP3 gene results in post-transcriptional reduction of Ixr1p levels. Further, we show that the reduced Ixr1p levels prevent adequate Rnr1p levels upon treatment with DNA damage agents. These findings suggest that reduced Ixr1p levels could in part explain why Elongator mutants are sensitive to DNA damage agents. Depletion of Elongator complex subunits results in loss of wobble uridine modifications in plants, nematodes, mice and yeast. Therefore, we investigated whether patients with the neurodegenerative disease familial dysautonomia (FD), who have lower levels of the ELP1 protein, display reduced amounts of modified wobble uridine nucleosides. We show that tRNA isolated from brain tissue and fibroblast cell lines derived from FD patients have 64–71% of the mcm5s2U nucleoside levels observed in total tRNA from non-FD brain tissue and non-FD fibroblasts. Overall, these results suggest that the cause for the neurodegenerative nature of FD could be translation impairment caused by reduced levels of modified wobble uridine nucleosides in tRNAs. Thus, our results give new insight on the importance of modified wobble uridine nucleosides for neurodevelopment.

Transfer RNAs (tRNAs) act as adaptor molecules in decoding messenger RNA into protein. Frequently found in tRNAs are different modified nucleosides, which are derivatives of the four normal nucleosides, adenosine (A), guanosine (G), cytidine (C), and uridine (U). Although modified nucleosides are present at many positions in tRNAs, two positions in the anticodon region, position 34 (wobble position) and position 37, show the largest variety of modified nucleosides. In Saccharomyces cerevisiae, the xm5U type of modified uridines found at position 34 are 5-carbamoylmethyluridine (ncm5U), 5-carbamoylmethyl-2´-O-methyluridine, (ncm5Um), 5-methoxycarbonylmethyluridine (mcm5U), and 5-methoxycarbonyl-methyl-2-thiouridine (mcm5s2U). Based on the complex structure of these nucleosides, it is likely that their formation requires several synthesis steps. The Elongator complex consisting of proteins Elp1p - Elp6p, and the proteins Kti11p - Kti14p, Sit4p, Sap185p, and Sap190p were shown to be involved in 5-carbamoylmethyl (ncm5) and 5-methoxycarbonylmethyl (mcm5) side-chain synthesis at position 34 in eleven tRNA species. The proteins Urm1p, Uba4p, Ncs2p, Ncs6p, and Yor251cp were also identified to be required for the 2-thio (s2) group formation of the modified nucleoside mcm5s2U at wobble position. Modified nucleosides in the anticodon region of tRNA influence the efficiency and fidelity of translation. The identification of mutants lacking ncm5-, mcm5-, or s2-group at the wobble position allowed the investigation of the in vivo role of these nucleosides in the tRNA decoding process. It was revealed that the presence of ncm5-, mcm5- or s2-group promotes reading of G-ending codons. The concurrent presence of the mcm5- and the s2-groups in the wobble nucleoside mcm5s2U improves reading of A- and G-ending codons, whereas absence of both groups is lethal to the yeast cell. The Elongator complex was previously proposed to regulate polarized exocytosis and to participate in elongation of RNA polymerase II transcription. The pleiotropic phenotypes observed in Elongator mutants were therefore suggested to be caused by defects in exocytosis and transcription of many genes. Here it is shown that elevated levels of hypomodified tRNALys [mcm5s2UUU] and tRNAGln[mcm5s2UUG] can efficiently suppress these pleiotropic phenotypes, suggesting that the defects in transcription and exocytosis are indirectly caused by inefficient translation of mRNAs encoding proteins important in these processes.