umu.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
The role of wobble uridine modifications in +1 translational frameshifting in eukaryotes
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
Umeå University, Faculty of Medicine, Department of Odontology.
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
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.

Place, publisher, year, edition, pages
2015. Vol. 43, no 19, 9489-9499 p.
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-109852DOI: 10.1093/nar/gkv832ISI: 000366405600036OAI: oai:DiVA.org:umu-109852DiVA: diva2:859491
Available from: 2015-10-07 Created: 2015-10-07 Last updated: 2017-12-01Bibliographically approved
In thesis
1. Functional aspects of modified nucleosides in tRNA
Open this publication in new window or tab >>Functional aspects of modified nucleosides in tRNA
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2015. 32 p.
Keyword
tRNA modification, frameshifting, Elongator complex, Elp1p, Prb1p, proteolysis, glioma, TRM6/61, PKCα
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-109491 (URN)978-91-7601-355-7 (ISBN)
Public defence
2015-10-29, Hörsal D Unod T 9, Umeå universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2015-10-08 Created: 2015-09-28 Last updated: 2015-10-08Bibliographically approved
2. 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

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Tükenmez, HasanXu, HaoEsberg, AndersByström, Anders S.
By organisation
Department of Molecular Biology (Faculty of Medicine)Department of Molecular Biology (Faculty of Science and Technology)Department of Odontology
In the same journal
Nucleic Acids Research
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar

doi
urn-nbn

Altmetric score

doi
urn-nbn
Total: 275 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf