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. 2008 Oct;14(10):2183-94.
doi: 10.1261/rna.1184108. Epub 2008 Aug 28.

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

Bo Huang  1 , Jian Lu, Anders S Byström
Affiliations

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

Bo Huang et al. RNA. 2008 Oct.

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.

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Figures

FIGURE 1.
FIGURE 1.
Identification of mutants resistant to zymocin. (A) The S. cerevisiae deletion strains to be tested were spotted on YEPD plates, and the zymocin-producing K. lactis strain 2105-1D (Gunge and Sakaguchi 1981) was inoculated on the edge of the spot. After 24 h of incubation at 30°C, the size of the clearing zone caused by zymocin was compared between the mutants and the sensitive wild-type strain. (B) The indicated strains were transformed with a plasmid containing a PMET3-driven γ-toxin gene (pABY1728). Transformants were 10-fold serially diluted and grown on YEPD or synthetic complete plates lacking uracil and containing 0.05 mM methionine for 2 d at 30°C. All strains were from the S. cerevisiae deletion collection, except the urm1Δ/ urm1Δ strain (UMY3553).
FIGURE 2.
FIGURE 2.
HPLC analysis of tRNA from zymocin-resistant strains. HPLC analysis of modified nucleosides of total tRNA from (A) wild type (BY4743) and its isogenic (B) elp1 and (C) ncs2 strain derivatives. (A,B) The 34.2–53.8-min and (C) the 33.6–53.8-min regions of the HPLC chromatograms monitored at 254 nm are shown. The arrows indicate expected retention times of mcm5U and mcm5s2U, respectively.
FIGURE 3.
FIGURE 3.
Reactivity of tRNAGlu UUC from different strains to zymocin (in vivo) and γ-toxin (in vitro). (A) Crude zymocin was added to log-phase cultures of indicated strains. Total RNA was isolated and the level of tRNAGlu UUC was analyzed by Northern blot using oligonucleotides specific for (*) tRNASer CGA and (arrow) tRNAGlu UUC. (B) Total RNA from indicated strains was mixed with purified γ-toxin-GST at a concentration of 5 nM and incubated for 15 min at 30°C. The level of tRNAGlu UUCwas analyzed by Northern blot using oligonucleotides specific for tRNAGlu UUC. (Arrowhead) The 3′ cleavage product of tRNAGlu UUC. (Arrow) The full-length tRNAGlu UUC signals were quantified, and the ratio between γ-toxin treated and untreated samples was calculated and shown in the graph. All strains were from the S. cerevisiae deletion collection.
FIGURE 4.
FIGURE 4.
Proteins involved in the formation of wobble nucleoside mcm5s2U. So far 14 and 11 gene products have been found to be involved in the formation of the mcm5and s2 group of mcm5s2U, respectively. Note that the former group of gene products is also involved in the formation of ncm5U. Trm9p is required for the last step, formation of the esterified methyl constituent in mcm5U and mcm5s2U. Physical interaction between proteins is indicated by a dot (Luke et al. 1996; Otero et al. 1999; Wittschieben et al. 1999; Furukawa et al. 2000; Krogan and Greenblatt 2001; Winkler et al. 2001; Ho et al. 2002; Gavin et al. 2006; Krogan et al. 2006). Other interactions not shown in the figure are Kti14p, Kti12p, and Kti11p, which all interact with subunits of Elongator subcomplex (Elp1p, Elp2p, Elp3p) (Fichtner et al. 2002a, 2003; Frohloff et al. 2003; Schafer et al. 2003; Petrakis et al. 2005; Gavin et al. 2006; Krogan et al. 2006). There is also interaction between the Sit4 complex and Kti14p (Ho et al. 2002). Note that Nfs1p, Isu1p, and Isu2p are located in the mitochondria. Figure modified from Lu (2007).
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References

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