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Physiological consequences of Elongator complex inactivation in Eukaryotes
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Anders Byström)
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 [en]
Elongator complex, wobble uridine modifications, IKBKAP, IKAP, Familial dysautonomia, Untargeted Metabolic profiling.
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-125667ISBN: 978-91-7601-514-8 (print)OAI: oai:DiVA.org:umu-125667DiVA: diva2:970588
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
List of papers
1. Loss of ncm5 and mcm5 wobble uridine side chains results in an altered metabolic profile
Open this publication in new window or tab >>Loss of ncm5 and mcm5 wobble uridine side chains results in an altered metabolic profile
2016 (English)In: Metabolomics, ISSN 1573-3882, E-ISSN 1573-3890, Vol. 12, no 12, 177Article in journal (Refereed) Published
Abstract [en]

Introduction: The Elongator complex, comprising six subunits (Elp1p-Elp6p), is required for formation of 5-carbamoylmethyl (ncm(5)) and 5-methoxycarbonylmethyl (mcm(5)) side chains on wobble uridines in 11 out of 42 tRNA species in Saccharomyces cerevisiae. Loss of these side chains reduces the efficiency of tRNA decoding during translation, resulting in pleiotropic phenotypes. Overexpression of hypomodified tRNA(s2UUU)(Lys); tRNA(s2UUG)(Gln) and tRNA(s2UUC)(Glu), which in wild-type strains are modified with mcm(5)s(2)U, partially suppress phenotypes of an elp3 Delta strain. Objectives: To identify metabolic alterations in an elp3 Delta strain and elucidate whether these metabolic alterations are suppressed by overexpression of hypomodified tRNA(s2UUU)(Lys); tRNA(s2UUG)(Gln) and tRNA(s2UUC)(Glu). Method: Metabolic profiles were obtained using untargeted GC-TOF-MS of a temperature-sensitive elp3 Delta strain carrying either an empty low-copy vector, an empty high-copy vector, a low-copy vector harboring the wild-type ELP3 gene, or a high-copy vector overexpressing tRNA(s2UUU)(Lys); tRNA(s2UUG)(Gln) and tRNA(s2UUC)(Glu). The temperature sensitive elp3 Delta strain derivatives were cultivated at permissive (30 degrees C) or semi-permissive (34 degrees C) growth conditions. Results: Culturing an elp3 Delta strain at 30 or 34 degrees C resulted in altered metabolism of 36 and 46 %, respectively, of all metabolites detected when compared to an elp3D strain carrying the wild-type ELP3 gene. Overexpression of hypomodified tRNA(s2UUU)(Lys); tRNA(s2UUG)(Gln) and tRNA(s2UUC)(Glu) suppressed a subset of the metabolic alterations observed in the elp3 Delta strain. Conclusion: Our results suggest that the presence of ncm(5)- and mcm(5)-side chains on wobble uridines in tRNA are important for metabolic homeostasis.

Place, publisher, year, edition, pages
Springer, 2016
Keyword
Elongator complex, tRNA wobble uridine modifications, Translation, ELP3, Metabolomics, Metabolic profiling
National Category
Biochemistry and Molecular Biology Endocrinology and Diabetes
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-125635 (URN)10.1007/s11306-016-1120-8 (DOI)000389604300002 ()27738410 (PubMedID)
Available from: 2016-09-13 Created: 2016-09-13 Last updated: 2017-11-21Bibliographically approved
2. Familial dysautonomia (FD) patients have reduced levels of the modified wobble nucleoside mcm(5)s(2)U in tRNA
Open this publication in new window or tab >>Familial dysautonomia (FD) patients have reduced levels of the modified wobble nucleoside mcm(5)s(2)U in tRNA
2014 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 454, no 3, 441-445 p.Article in journal (Refereed) Published
Abstract [en]

Familial dysautonomia (FD) is a recessive neurodegenerative genetic disease. FD is caused by a mutation in the IKBKAP gene resulting in a splicing defect and reduced levels of full length IKAP protein. IKAP homologues can be found in all eukaryotes and are part of a conserved six subunit protein complex, Elongator complex. Inactivation of any Elongator subunit gene in multicellular organisms cause a wide range of phenotypes, suggesting that Elongator has a pivotal role in several cellular processes. In yeast, there is convincing evidence that the main role of Elongator complex is in formation of modified wobble uridine nucleosides in tRNA and that their absence will influence translational efficiency. To date, no study has explored the possibility that FD patients display defects in formation of modified wobble uridine nucleosides as a consequence of reduced IKAP levels. In this study, we show that brain tissue and fibroblast cell lines from FD patients have reduced levels of the wobble uridine nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). Our findings indicate that FD could be caused by inefficient translation due to lower levels of wobble uridine nucleosides. 

Keyword
Familial dysautonomia (FD), Elongator complex, IKBKAP, ELP1, tRNA modification, 5- thoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-98910 (URN)10.1016/j.bbrc.2014.10.116 (DOI)000346690600015 ()25450681 (PubMedID)
Available from: 2015-01-28 Created: 2015-01-28 Last updated: 2017-12-05Bibliographically approved
3. Elongator complex enhances Rnr1p levels in response to DNA damage by influencing Ixr1p expression
Open this publication in new window or tab >>Elongator complex enhances Rnr1p levels in response to DNA damage by influencing Ixr1p expression
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Genetics
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-124966 (URN)
Available from: 2016-08-31 Created: 2016-08-31 Last updated: 2016-11-30

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