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PMID: 18755837 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

A genome-wide screen identifies genes required for formation of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine in Saccharomyces cerevisiae.

RNA (New York, N.Y.) ·Vol. 14 ·No. 10 ·2008-10-00 ·Pages 2183-94

Huang B, Lu J, Byström AS

Abstract

We recently showed that the gamma-subunit of Kluyveromyces lactis killer toxin (gamma-toxin) is a tRNA endonuclease that cleaves tRNA(mcm5s2UUC Glu), tRNA(mcm5s2UUU Lys), and tRNA(mcm5s2UUG Gln) 3' of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). The 5-methoxycarbonylmethyl (mcm(5)) side chain was important for efficient cleavage by gamma-toxin, and defects in mcm(5) side-chain synthesis correlated with resistance to gamma-toxin. Based on this correlation, a genome-wide screen was performed to identify gene products involved in the formation of the mcm(5) side chain. From a collection of 4826 homozygous diploid Saccharomyces cerevisiae strains, each with one nonessential gene deleted, 63 mutants resistant to Kluyveromyces lactis killer toxin were identified. Among these, eight were earlier identified to have a defect in formation of the mcm(5) side chain. Analysis of the remaining mutants and other known gamma-toxin resistant mutants revealed that sit4, kti14, and KTI5 mutants also have a defect in the formation of mcm(5). A mutant lacking two of the Sit4-associated proteins, Sap185 and Sap190, displays the same modification defect as a sit4-null mutant. Interestingly, several mutants were found to be defective in the synthesis of the 2-thio (s(2)) group of the mcm(5)s(2)U nucleoside. In addition to earlier described mutants, formation of the s(2) group was also abolished in urm1, uba4, and ncs2 mutants and decreased in the yor251c mutant. Like the absence of the mcm(5) side chain, the lack of the s(2) group renders tRNA(mcm5s2UUC Glu) less sensitive to gamma-toxin, reinforcing the importance of the wobble nucleoside mcm(5)s(2)U for tRNA cleavage by gamma-toxin.

MeSH Terms
Diploidy Drug Resistance, Fungal/genetics Genes, Fungal Killer Factors, Yeast Mycotoxins/pharmacology Protein Phosphatase 2/genetics RNA, Fungal/genetics,metabolism RNA, Transfer, Glu/genetics,metabolism Saccharomyces cerevisiae/drug effects,genetics,metabolism Saccharomyces cerevisiae Proteins/genetics Thiouridine/analogs & derivatives,metabolism
Chemicals
5-carbomethoxymethyl-2-thiouridine Killer Factors, Yeast Mycotoxins RNA, Fungal RNA, Transfer, Glu Saccharomyces cerevisiae Proteins zymocin Thiouridine Protein Phosphatase 2 SIT4 protein, S cerevisiae
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Huang Bo
Department of Molecular Biology, Umeå University, 901 87 Umeå, Sweden.
Lu Jian
Byström Anders S
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Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1469-9001
DOI
10.1261/rna.1184108
Published
2008-10-00
Epub
2008-00-28
Pages
2183-94
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC2553728
Subset
IM
External Links
PubMed source DOI: 10.1261/rna.1184108 PMC Full Text View journal details
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