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. 2005 Apr;11(4):424-36.
doi: 10.1261/rna.7247705.

An early step in wobble uridine tRNA modification requires the Elongator complex

Bo Huang  1 , Marcus J O Johansson, Anders S Byström
Affiliations

An early step in wobble uridine tRNA modification requires the Elongator complex

Bo Huang et al. RNA. 2005 Apr.

Abstract

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 KTI11-KTI13 gene prevents an ochre suppressor tRNA that normally contains mcm5U from reading ochre stop codons.

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Figures

FIGURE 1.
FIGURE 1.
Structure of 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), 5-carbamoyl-methyluridine (ncm5U), and 5-carboxymethyluridine (cm5U). R represents ribose.
FIGURE 2.
FIGURE 2.
Strains with elp and kti mutations confer a loss-of-suppression phenotype. Approximately 104 cells of wild type (W303-1B), SUP4 (UMY2893), SUP4 elp1-elp6 (UMY2916, UMY3039, UMY3040, UMY2912, UMY2914, UMY2918, UMY2920, UMY2922), and SUP4 kti11-kti13 (UMY2936, UMY2938, UMY2940) strains were spotted on SC, SC-Ade, or SC-Arg+Can plates and incubated for 3 d at 30°C.
FIGURE 3.
FIGURE 3.
An elp3-null mutant is lacking mcm5 and ncm5 side chains at wobble uridines. HPLC analysis of modified tRNA nucleosides from SUP4 (UMY2893, left panels) and SUP4 elp3::KanMX4 (UMY2916, right panels) strains. (A) Analysis of modified nucleosides in tRNAGlumcm5s2UUC. The part of the chromatogram between retention times 33 and 54 min is shown. Arrows indicate the expected retention time of s2U (left panel) and mcm5s2U (right panel). (B) Analysis of modified nucleosides in tRNAArgmcm5UCU. The part of the chromatogram between retention times 33 and 45.5 min is shown. The arrow indicates the expected retention time of mcm5U (right panel). (C) Analysis of modified nucleosides in tRNAPro ncm5UGG. The part of the chromatogram between retention times 10 and 20 min is shown. The arrow indicates the expected retention time of ncm5U (right panel). The small peak at this position represents an unrelated compound with a spectrum different from that of ncm5U.
FIGURE 4.
FIGURE 4.
Elp1p and Elp3p coimmunoprecipitate tRNAGluUUC. (A) T7-transcribed 32P-labeled tRNAGlu UUC was mixed with extracts from wild-type, ELP1-(HA)3, ELP3-(HA)3, or ELP5-(HA)3 strains. After UV-cross-linking, immunoprecipitation using agarose conjugated anti-HA antibodies was performed. The immunoprecipitated material was digested with proteinase K and analyzed on a denaturing polyacrylamide gel. The signals were quantified and normalized to the background signal in the control, which was set to 1. (B) T7-transcribed 32P-labeled tRNAGluUUC or tRNAMeti was mixed with extracts from TIF4632-(HA)3, ELP1-(HA)3, ELP3-(HA)3, or ELP5-(HA)3 strains. The experiment and quantifications were performed as described in A.
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