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Formation and function of wobble uridine modifications in transfer RNA of Saccharomyces cerevisiae
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
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 [en]
Transfer RNA, Modified nucleoside, Elongator complex, Wobble uridine, Decoding
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-1433ISBN: 978-91-7264-450-2 (print)OAI: oai:DiVA.org:umu-1433DiVA: diva2:141044
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
List of papers
1. An early step in wobble uridine tRNA modification requires the Elongator complex
Open this publication in new window or tab >>An early step in wobble uridine tRNA modification requires the Elongator complex
2005 (English)In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 11, no 4, 424-436 p.Article in journal (Refereed) Published
Abstract [en]

Elongator has been reported to be a histone acetyltransferase complex involved in elongation of RNA polymerase II transcription. In Saccharomyces cerevisiae, mutations in any of the six Elongator protein subunit (ELP1–ELP6) genes or the three killer toxin insensitivity (KTI11–KTI13) genes cause similar pleiotropic phenotypes. By analyzing modified nucleosides in individual tRNA species, we show that the ELP1–ELP6 and KTI11–KTI13 genes are all required for an early step in synthesis of 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) groups present on uridines at the wobble position in tRNA. Transfer RNA immunoprecipitation experiments showed that the Elp1 and Elp3 proteins specifically coprecipitate a tRNA susceptible to formation of an mcm5 side chain, indicating a direct role of Elongator in tRNA modification. The presence of mcm5U, ncm5U, or derivatives thereof at the wobble position is required for accurate and efficient translation, suggesting that the phenotypes of elp1–elp6 and kti11–kti13 mutants could be caused by a translational defect. Accordingly, a deletion of any ELP1–ELP6 or KTI11KTI13 gene prevents an ochre suppressor tRNA that normally contains mcm5U from reading ochre stop codons.

Keyword
5-methoxycarbonylmethyl-2-thiouridine, 5-methoxycarbonylmethyluridine, 5-carbamoylmethyluridine, Elongator complex, KTI genes
Identifiers
urn:nbn:se:umu:diva-2772 (URN)10.1261/rna.7247705 (DOI)15769872 (PubMedID)
Available from: 2007-11-14 Created: 2007-11-14 Last updated: 2017-12-14Bibliographically approved
2. A genome-wide screen in Saccharomyces cerevisiae for mutants defective in the formation of wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine.
Open this publication in new window or tab >>A genome-wide screen in Saccharomyces cerevisiae for mutants defective in the formation of wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine.
(English)Manuscript (Other (popular science, discussion, etc.))
Identifiers
urn:nbn:se:umu:diva-2246 (URN)
Available from: 2007-04-23 Created: 2007-04-23 Last updated: 2010-01-14Bibliographically approved
3. 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
4. A conserved modified wobble nucleoside (mcm5s2U) in lysyl-tRNA is required for viability in yeast.
Open this publication in new window or tab >>A conserved modified wobble nucleoside (mcm5s2U) in lysyl-tRNA is required for viability in yeast.
2007 (English)In: RNA, ISSN 1355-8382, Vol. 13, no 8, 1245-55 p.Article in journal (Refereed) Published
Abstract [en]

Transfer RNAs specific for Gln, Lys, and Glu from all organisms (except Mycoplasma) and organelles have a 2-thiouridine derivative (xm(5)s(2)U) as wobble nucleoside. These tRNAs read the A- and G-ending codons in the split codon boxes His/Gln, Asn/Lys, and Asp/Glu. In eukaryotic cytoplasmic tRNAs the conserved constituent (xm(5)-) in position 5 of uridine is 5-methoxycarbonylmethyl (mcm(5)). A protein (Tuc1p) from yeast resembling the bacterial protein TtcA, which is required for the synthesis of 2-thiocytidine in position 32 of the tRNA, was shown instead to be required for the synthesis of 2-thiouridine in the wobble position (position 34). Apparently, an ancient member of the TtcA family has evolved to thiolate U34 in tRNAs of organisms from the domains Eukarya and Archaea. Deletion of the TUC1 gene together with a deletion of the ELP3 gene, which results in the lack of the mcm(5) side chain, removes all modifications from the wobble uridine derivatives of the cytoplasmic tRNAs specific for Gln, Lys, and Glu, and is lethal to the cell. Since excess of the unmodified form of these three tRNAs rescued the double mutant elp3 tuc1, the primary function of mcm(5)s(2)U34 seems to be to improve the efficiency to read the cognate codons rather than to prevent mis-sense errors. Surprisingly, overexpression of the mcm(5)s(2)U-lacking tRNA(Lys) alone was sufficient to restore viability of the double mutant.

Keyword
Amino Acid Sequence, Cytoplasm/metabolism, Molecular Sequence Data, Mutation, Nucleosides/chemistry/metabolism, RNA; Transfer; Amino Acyl/chemistry/*metabolism, RNA; Transfer; Gln/chemistry/metabolism, Saccharomyces cerevisiae/chemistry/cytology/genetics/*metabolism, Saccharomyces cerevisiae Proteins/*metabolism, Sequence Alignment
Identifiers
urn:nbn:se:umu:diva-16717 (URN)doi:10.1261/rna.558707 (DOI)17592039 (PubMedID)
Available from: 2008-01-04 Created: 2008-01-04 Last updated: 2011-01-11Bibliographically approved
5. 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

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