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Functional aspects of wobble uridine modifications in yeast tRNA
Umeå University, Faculty of Medicine, Molecular Biology.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Transfer RNAs (tRNA) function as adaptor molecules in the translation of mRNA into protein. These adaptor molecules require modifications of a subset of their nucleosides for optimal function. The most frequently modified nucleoside in tRNA is position 34 (wobble position), and especially uridines present at this position. Modified nucleosides at the wobble position are important in the decoding process of mRNA, i.e., restriction or improvement of codon-anticodon interactions. This thesis addresses the functional aspects of the wobble uridine modifications.

The Saccharomyces cerevisiae Elongator complex consisting of the six Elp1-Elp6 proteins has been proposed to participate in three distinct cellular processes; elongation of RNA polymerase II transcription, regulation of polarized exocytosis, and formation of modified wobble nucleosides in tRNA. In Paper I, we show that the phenotypes of Elongator deficient cells linking the complex to transcription and exocytosis are counteracted by increased level of and . These tRNAs requires the Elongator complex for formation of the 5-methoxycarbonylmethyl (mcmlnGUUGsmcm25tRNALysUUUsmcm25tRNA5) group of their modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). Our results therefore indicate that the relevant function of the Elongator complex is in formation of modified nucleosides in tRNAs and the defects observed in exocytosis and transcription are indirectly caused by inefficient translation of mRNAs encoding gene products important for these processes.

The lack of defined mutants in eukaryotes has led to limited understanding about the role of the wobble uridine modifications in this domain of life. In Paper II, we utilized recently characterized mutants lacking the 2-thio (s2) or 5-carbamoylmethyl (ncm5) and mcm5 groups to address the in vivo function of eukaryotic wobble uridine modifications. We show that ncm5 and mcm5 side-chains promote reading of G-ending codons, and that presence of a mcm5 and an s2 group cooperatively improves reading of both A- and G-ending codons.

Previous studies revealed that a S. cerevisiae strain deleted for any of the six Elongator subunit genes shows resistance towards a toxin (zymocin) secreted by the dairy yeast Kluyveromyces lactis. In Paper III, we show that the cytotoxic γ subunit of zymocin is a tRNA endonuclease that target the anticodon of mcm5s2U34 containing tRNAs and that the wobble mcm5 modification is required for efficient cleavage. This explains the γ-toxin resistant phenotype of Elongator mutants which are defective in the synthesis of the mcm5 group.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet) , 2007. , 53 p.
Series
Doctoral thesis / Umeå University, Department of Molecular Biology
Keyword [en]
Molecular biology, Transfer RNA, Modified nucleosides, Elongator complex, Translation, Kluyveromyces lactis γ-toxin
Keyword [sv]
Molekylärbiologi
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-1093ISBN: 978-91-7264-302-4 (print)OAI: oai:DiVA.org:umu-1093DiVA: diva2:140167
Public defence
2007-05-16, Major Groove, 6L, Umeå University, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2007-04-23 Created: 2007-04-23 Last updated: 2010-01-08Bibliographically approved
List of papers
1. Elevated levels of two tRNA species bypass the requirement for elongator complex in transcription and exocytosis.
Open this publication in new window or tab >>Elevated levels of two tRNA species bypass the requirement for elongator complex in transcription and exocytosis.
2006 (English)In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 24, no 1, 139-148 p.Article in journal (Refereed) Published
Abstract [en]

The Saccharomyces cerevisiae Elongator complex consisting of the six Elp1-Elp6 proteins has been proposed to participate in three distinct cellular processes: transcriptional elongation, polarized exocytosis, and formation of modified wobble uridines in tRNA. Therefore it was important to clarify whether Elongator has three distinct functions or whether it regulates one key process that leads to multiple downstream effects. Here, we show that the phenotypes of Elongator-deficient cells linking the complex to transcription and exocytosis are suppressed by increased expression of two tRNA species. Elongator is required for formation of the mcm(5) group of the modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U) in these tRNAs. Hence, in cells with normal levels of these tRNAs, presence of mcm(5)s(2)U is crucial for posttranscriptional expression of gene products important in transcription and exocytosis. Our results indicate that the physiologically relevant function of the evolutionary-conserved Elongator complex is in formation of modified nucleosides in tRNAs.

Keyword
Anticodon/genetics/*metabolism, Chromatin Assembly and Disassembly, Exocytosis/genetics/*physiology, Gene Dosage, Mutation, Peptide Elongation Factors/genetics/metabolism/*physiology, Saccharomyces cerevisiae/genetics/*metabolism, Saccharomyces cerevisiae Proteins/genetics/metabolism/*physiology, Temperature, Transcription; Genetic/*physiology, Uridine/metabolism/physiology
Identifiers
urn:nbn:se:umu:diva-16739 (URN)10.1016/j.molcel.2006.07.031 (DOI)17018299 (PubMedID)
Available from: 2007-10-09 Created: 2007-10-09 Last updated: 2017-12-14Bibliographically approved
2. Eukaryotic wobble uridine modifications promote a functionally redundant decoding system.
Open this publication in new window or tab >>Eukaryotic wobble uridine modifications promote a functionally redundant decoding system.
Show others...
2008 (English)In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 28, no 10, 3301-3312 p.Article in journal (Refereed) Published
Abstract [en]

The translational decoding properties of tRNAs are modulated by naturally occurring modifications of their nucleosides. Uridines located at the wobble position (nucleoside 34 [U34]) in eukaryotic cytoplasmic tRNAs often harbor a 5-methoxycarbonylmethyl (mcm(5)) or a 5-carbamoylmethyl (ncm(5)) side chain and sometimes an additional 2-thio (s2) or 2'-O-methyl group. Although a variety of models explaining the role of these modifications have been put forth, their in vivo functions have not been defined. In this study, we utilized recently characterized modification-deficient Saccharomyces cerevisiae cells to test the wobble rules in vivo. We show that mcm5 and ncm5 side chains promote decoding of G-ending codons and that concurrent mcm5 and s2 groups improve reading of both A- and G-ending codons. Moreover, the observation that the mcm5U34- and some ncm5U34-containing tRNAs efficiently read G-ending codons challenges the notion that eukaryotes do not use U-G wobbling.

Identifiers
urn:nbn:se:umu:diva-20533 (URN)10.1128/MCB.01542-07 (DOI)18332122 (PubMedID)
Available from: 2009-03-20 Created: 2009-03-20 Last updated: 2017-12-13
3. The Kluyveromyces lactis γ-toxin targets tRNA anticodons.
Open this publication in new window or tab >>The Kluyveromyces lactis γ-toxin targets tRNA anticodons.
Show others...
2005 (English)In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 11, no 11, 1648-1654 p.Article in journal (Refereed) Published
Abstract [en]

Kluyveromyces lactis killer strains secrete a heterotrimeric toxin (zymocin), which causes an irreversible growth arrest of sensitive yeast cells. Despite many efforts, the target(s) of the cytotoxic gamma-subunit of zymocin has remained elusive. Here we show that three tRNA species tRNA(Glu)(mcm(5)s(2)UUC), tRNA(Lys)(mcm(5)s(2)UUU), and tRNA(Gln)(mcm(5)s(2)UUG) are the targets of gamma-toxin. The toxin inhibits growth by cleaving these tRNAs at the 3' side of the modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). Transfer RNA lacking a part of or the entire mcm(5) group is inefficiently cleaved by gamma-toxin, explaining the gamma-toxin resistance of the modification-deficient trm9, elp1-elp6, and kti11-kti13 mutants. The K. lactis gamma-toxin is the first eukaryotic toxin shown to target tRNA.

Keyword
Anticodon/*drug effects, Drug Resistance; Fungal/genetics, Gene Expression, Kluyveromyces/*chemistry, Mutation/genetics, Mycotoxins/genetics/*pharmacology, Nucleic Acid Conformation, RNA; Transfer/*drug effects, Saccharomyces cerevisiae/genetics, Saccharomyces cerevisiae Proteins/physiology
Identifiers
urn:nbn:se:umu:diva-16758 (URN)10.1261/rna.2172105 (DOI)16244131 (PubMedID)
Available from: 2007-10-10 Created: 2007-10-10 Last updated: 2017-12-14Bibliographically approved

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