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Elevated levels of two tRNA species bypass the requirement for elongator complex in transcription and exocytosis.
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Byström)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Byströn)
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). (Byström)
2006 (English)In: Molecular Cell, ISSN 1097-2765, Vol. 24, no 1, 139-148 p.Article in journal (Refereed) Published
Description
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.

Place, publisher, year, edition, pages
2006. Vol. 24, no 1, 139-148 p.
Keyword [en]
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: urn:nbn:se:umu:diva-16739DOI: 10.1016/j.molcel.2006.07.031PubMedID: 17018299OAI: oai:DiVA.org:umu-16739DiVA: diva2:156412
Available from: 2007-10-09 Created: 2007-10-09 Last updated: 2017-05-23Bibliographically approved
In thesis
1. Functional aspects of wobble uridine modifications in yeast tRNA
Open this publication in new window or tab >>Functional aspects of wobble uridine modifications in yeast tRNA
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
Molecular biology, Transfer RNA, Modified nucleosides, Elongator complex, Translation, Kluyveromyces lactis γ-toxin, Molekylärbiologi
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-1093 (URN)978-91-7264-302-4 (ISBN)
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
2. Formation and function of wobble uridine modifications in transfer RNA of Saccharomyces cerevisiae
Open this publication in new window or tab >>Formation and function of wobble uridine modifications in transfer RNA of Saccharomyces cerevisiae
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Transfer RNAs (tRNAs) act as adaptor molecules in decoding messenger RNA into protein. Frequently found in tRNAs are different modified nucleosides, which are derivatives of the four normal nucleosides, adenosine (A), guanosine (G), cytidine (C), and uridine (U). Although modified nucleosides are present at many positions in tRNAs, two positions in the anticodon region, position 34 (wobble position) and position 37, show the largest variety of modified nucleosides. In Saccharomyces cerevisiae, the xm5U type of modified uridines found at position 34 are 5-carbamoylmethyluridine (ncm5U), 5-carbamoylmethyl-2´-O-methyluridine, (ncm5Um), 5-methoxycarbonylmethyluridine (mcm5U), and 5-methoxycarbonyl-methyl-2-thiouridine (mcm5s2U). Based on the complex structure of these nucleosides, it is likely that their formation requires several synthesis steps.

The Elongator complex consisting of proteins Elp1p - Elp6p, and the proteins Kti11p - Kti14p, Sit4p, Sap185p, and Sap190p were shown to be involved in 5-carbamoylmethyl (ncm5) and 5-methoxycarbonylmethyl (mcm5) side-chain synthesis at position 34 in eleven tRNA species. The proteins Urm1p, Uba4p, Ncs2p, Ncs6p, and Yor251cp were also identified to be required for the 2-thio (s2) group formation of the modified nucleoside mcm5s2U at wobble position.

Modified nucleosides in the anticodon region of tRNA influence the efficiency and fidelity of translation. The identification of mutants lacking ncm5-, mcm5-, or s2-group at the wobble position allowed the investigation of the in vivo role of these nucleosides in the tRNA decoding process. It was revealed that the presence of ncm5-, mcm5- or s2-group promotes reading of G-ending codons. The concurrent presence of the mcm5- and the s2-groups in the wobble nucleoside mcm5s2U improves reading of A- and G-ending codons, whereas absence of both groups is lethal to the yeast cell.

The Elongator complex was previously proposed to regulate polarized exocytosis and to participate in elongation of RNA polymerase II transcription. The pleiotropic phenotypes observed in Elongator mutants were therefore suggested to be caused by defects in exocytosis and transcription of many genes. Here it is shown that elevated levels of hypomodified tRNALys [mcm5s2UUU] and tRNAGln[mcm5s2UUG] can efficiently suppress these pleiotropic phenotypes, suggesting that the defects in transcription and exocytosis are indirectly caused by inefficient translation of mRNAs encoding proteins important in these processes.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknisk-naturvetenskaplig fakultet), 2007. 44 p.
Keyword
Transfer RNA, Modified nucleoside, Elongator complex, Wobble uridine, Decoding
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-1433 (URN)978-91-7264-450-2 (ISBN)
Public defence
2007-12-12, Major Groove, Byggnad 6L, Dept. of Molecular Biology, Umeå University, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2007-11-14 Created: 2007-11-14 Last updated: 2009-10-22Bibliographically approved

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Esberg, AndersHuang, BoByström, Anders

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