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Influence of wobble uridine modifications on eukaryotic translation
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]

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: urn:nbn:se:umu:diva-125663ISBN: 978-91-7601-540-7OAI: oai:DiVA.org:umu-125663DiVA: diva2:970551
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
List of papers
1. The role of wobble uridine modifications in +1 translational frameshifting in eukaryotes
Open this publication in new window or tab >>The role of wobble uridine modifications in +1 translational frameshifting in eukaryotes
2015 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 19, 9489-9499 p.Article in journal (Refereed) Published
Abstract [en]

In Saccharomyces cerevisiae, 11 out of 42 tRNA species contain 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-methoxycarbonylmethyluridine (mcm5U), 5-carbamoylmethyluridine (ncm5U) or 5-carbamoylmethyl-2′-O-methyluridine (ncm5Um) nucleosides in the anticodon at the wobble position (U34). Earlier we showed that mutants unable to form the side chain at position 5 (ncm5 or mcm5) or lacking sulphur at position 2 (s2) of U34 result in pleiotropic phenotypes, which are all suppressed by overexpression of hypomodified tRNAs. This observation suggests that the observed phenotypes are due to inefficient reading of cognate codons or an increased frameshifting. The latter may be caused by a ternary complex (aminoacyl-tRNA*eEF1A*GTP) with a modification deficient tRNA inefficiently being accepted to the ribosomal A-site and thereby allowing an increased peptidyl-tRNA slippage and thus a frameshift error. In this study, we have investigated the role of wobble uridine modifications in reading frame maintenance, using either the Renilla/Firefly luciferase bicistronic reporter system or a modified Ty1 frameshifting site in a HIS4A::lacZ reporter system. We here show that the presence of mcm5 and s2 side groups at wobble uridines are important for reading frame maintenance and thus the aforementioned mutant phenotypes might partly be due to frameshift errors.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-109852 (URN)10.1093/nar/gkv832 (DOI)000366405600036 ()
Available from: 2015-10-07 Created: 2015-10-07 Last updated: 2016-09-14Bibliographically approved
2. 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: 2016-09-20
3. 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
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-125635 (URN)
Available from: 2016-09-13 Created: 2016-09-13 Last updated: 2016-09-20
4. 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: 2016-09-14Bibliographically approved

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