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Elongator complex enhances Rnr1p levels in response to DNA damage by influencing Ixr1p expression
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Anders Byström)ORCID iD: 0000-0002-2259-6883
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Anders Byström)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
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(English)Manuscript (preprint) (Other academic)
National Category
Genetics
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-124966OAI: oai:DiVA.org:umu-124966DiVA: diva2:956999
Available from: 2016-08-31 Created: 2016-08-31 Last updated: 2016-09-20
In thesis
1. Influence of wobble uridine modifications on eukaryotic translation
Open this publication in new window or tab >>Influence of wobble uridine modifications on eukaryotic translation
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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 Lyss2UUU 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.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2016. 53 p.
National Category
Genetics
Identifiers
urn:nbn:se:umu:diva-125663 (URN)978-91-7601-540-7 (ISBN)
Public defence
2016-10-13, N200, Naturvetarhuset, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2016-09-22 Created: 2016-09-14 Last updated: 2016-09-20Bibliographically approved
2. Physiological consequences of Elongator complex inactivation in Eukaryotes
Open this publication in new window or tab >>Physiological consequences of Elongator complex inactivation in Eukaryotes
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2016. 62 p.
Keyword
Elongator complex, wobble uridine modifications, IKBKAP, IKAP, Familial dysautonomia, Untargeted Metabolic profiling.
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-125667 (URN)978-91-7601-514-8 (ISBN)
Public defence
2016-10-27, N220, Umeå Universitet, Naturvetarhuset, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2016-09-22 Created: 2016-09-14 Last updated: 2016-09-21Bibliographically approved

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