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Mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at tRNA wobble positions.

Noma A, Sakaguchi Y, Suzuki T - Nucleic Acids Res. (2009)

Bottom Line: We could successfully reconstitute the 2-thiouridine formation in vitro using recombinant proteins.This study revealed that 2-thiouridine formation shares a pathway and chemical reactions with protein urmylation.The sulfur-flow of eukaryotic 2-thiouridine formation is distinct mechanism from the bacterial sulfur-relay system which is based on the persulfide chemistry.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Bldg. 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

ABSTRACT
The wobble modification in tRNAs, 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U), is required for the proper decoding of NNR codons in eukaryotes. The 2-thio group confers conformational rigidity of mcm(5)s(2)U by largely fixing the C3'-endo ribose puckering, ensuring stable and accurate codon-anticodon pairing. We have identified five genes in Saccharomyces cerevisiae, YIL008w (URM1), YHR111w (UBA4), YOR251c (TUM1), YNL119w (NCS2) and YGL211w (NCS6), that are required for 2-thiolation of mcm(5)s(2)U. An in vitro sulfur transfer experiment revealed that Tum1p stimulated the cysteine desulfurase of Nfs1p, and accepted persulfide sulfurs from Nfs1p. URM1 is a ubiquitin-related modifier, and UBA4 is an E1-like enzyme involved in protein urmylation. The carboxy-terminus of Urm1p was activated as an acyl-adenylate (-COAMP), then thiocarboxylated (-COSH) by Uba4p. The activated thiocarboxylate can be utilized in the subsequent reactions for 2-thiouridine formation, mediated by Ncs2p/Ncs6p. We could successfully reconstitute the 2-thiouridine formation in vitro using recombinant proteins. This study revealed that 2-thiouridine formation shares a pathway and chemical reactions with protein urmylation. The sulfur-flow of eukaryotic 2-thiouridine formation is distinct mechanism from the bacterial sulfur-relay system which is based on the persulfide chemistry.

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Mass spectrometric analyses of total nucleosides and purified tRNAsGlu from S. cerevisiae wild-type and mutant cells. (A) LC/MS analyses of total nucleosides from strains of wild-type (WT), ΔYOR251c (TUM1), ΔYHR111w (UBA4), ΔYIL008w (URM1), ΔYNL119w (NCS2) and ΔYGL211w (NCS6). The upper panels show the merged mass chromatograms detecting MH+ (m/z 333) and BH2+ (m/z 201) of mcm5s2U. The lower panels show the mass chromatograms detecting MH+ (m/z 317) and BH2+ (m/z 185) of mcm5U. Arrows in the upper panels indicate the retention time for mcm5s2U. (B) LC/MS fragment analyses of RNase T1-digested tRNAsGlu obtained from wild-type strains: ΔTUM1, ΔUBA4, ΔURM1, ΔNCS2, ΔNCS6 and ΔTRM9. A graph on the top-right represents the mass spectrum for the anticodon-containing fragment (CUmcm5s2UUCACCGp) (Figure 1B) from the wild-type strain. Charge states of multiply charged ions are indicated in parentheses. Other graphs describe the mass chromatograms shown by triply charged ions of anticodon-containing fragments bearing mcm5s2U (m/z 1458.17, red line), mcm5U (m/z 1450.18, black line), ncm5s2U (m/z 1450.67, green line) and ncm5U (m/z 1443.18, blue line). The RNA sequence including the wobble modification is indicated on each graph.
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Figure 2: Mass spectrometric analyses of total nucleosides and purified tRNAsGlu from S. cerevisiae wild-type and mutant cells. (A) LC/MS analyses of total nucleosides from strains of wild-type (WT), ΔYOR251c (TUM1), ΔYHR111w (UBA4), ΔYIL008w (URM1), ΔYNL119w (NCS2) and ΔYGL211w (NCS6). The upper panels show the merged mass chromatograms detecting MH+ (m/z 333) and BH2+ (m/z 201) of mcm5s2U. The lower panels show the mass chromatograms detecting MH+ (m/z 317) and BH2+ (m/z 185) of mcm5U. Arrows in the upper panels indicate the retention time for mcm5s2U. (B) LC/MS fragment analyses of RNase T1-digested tRNAsGlu obtained from wild-type strains: ΔTUM1, ΔUBA4, ΔURM1, ΔNCS2, ΔNCS6 and ΔTRM9. A graph on the top-right represents the mass spectrum for the anticodon-containing fragment (CUmcm5s2UUCACCGp) (Figure 1B) from the wild-type strain. Charge states of multiply charged ions are indicated in parentheses. Other graphs describe the mass chromatograms shown by triply charged ions of anticodon-containing fragments bearing mcm5s2U (m/z 1458.17, red line), mcm5U (m/z 1450.18, black line), ncm5s2U (m/z 1450.67, green line) and ncm5U (m/z 1443.18, blue line). The RNA sequence including the wobble modification is indicated on each graph.

Mentions: To identify the genes responsible for 2-thiouridine formation of mcm5s2U, we performed ribonucleome analysis, a genome-wide reverse genetic approach combined with mass spectrometry (26). Since 2-thiouridine is a non-essential RNA modification, the complete set of S. cerevisiae deletion strains (4829) serves as a parent population for this analysis. To reduce the size of the starting population, we selected 3482 genes that have orthologs in Schizosaccharomyces pombe, because S. pombe tRNAs possess mcm5s2U. Next, we chose 767 genes which are described as proteins of unknown function, and proteins with weak similarity to known functions in S. cerevisiae (CYGD: http://mips.gsf.de/genre/proj/yeast) (34). We then started the ribonucleome analysis using this population. In the mass chromatogram (Figure 2A), mcm5s2U was detected as a proton adduct form (MH+; m/z 333) with its base fragment (BH2+; m/z 201) in wild-type cells. mcm5U (MH+; m/z 317) with its base fragment (BH2+; m/z 185), which is a precursor form of mcm5s2U, could also be detected in wild-type cells (Figure 2A). Among the 767 selected deletion mutants, we identified four deletion strains, YHR111w(UBA4), YNL119w(NCS2), YGL211w(NCS6) and YOR251c, in which mcm5s2U was absent (Figure 2A, upper panels), while levels of its precursor mcm5U were increased (Figure 2A, lower panels), demonstrating that these genes are specifically involved in 2-thiolation of mcm5s2U. We have renamed YOR251c as tRNA-thiouridine modification protein 1 (TUM1). NCS2 is an orthologous protein of Ctu2 in Caenorhabditis elegans and fission yeast (24). Although it was reported that NCS6/TUC1 is responsible for 2-thiouridine formation by comparative genomic approach in 2007 (23), we have independently identified the same gene in this analysis. It was reported that UBA4, NCS2 and NCS6 are essential in the ΔCLA4 strain (35), and that their deletion affects the protein urmylation, which is a ubiquitin-like conjugation pathway (36,37). UBA4 encodes a paralog of a ubiquitin-activating enzyme (E1) (38). In fact, UBA4 was shown to function as an E1-like enzyme for URM1, which is an ubiquitin-related modifier involved in protein urmylation. We therefore analyzed the total nucleosides of ΔYIL008w(URM1) and found that URM1 is also responsible for 2-thiolation of mcm5s2U (Figure 2A).Figure 2.


Mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at tRNA wobble positions.

Noma A, Sakaguchi Y, Suzuki T - Nucleic Acids Res. (2009)

Mass spectrometric analyses of total nucleosides and purified tRNAsGlu from S. cerevisiae wild-type and mutant cells. (A) LC/MS analyses of total nucleosides from strains of wild-type (WT), ΔYOR251c (TUM1), ΔYHR111w (UBA4), ΔYIL008w (URM1), ΔYNL119w (NCS2) and ΔYGL211w (NCS6). The upper panels show the merged mass chromatograms detecting MH+ (m/z 333) and BH2+ (m/z 201) of mcm5s2U. The lower panels show the mass chromatograms detecting MH+ (m/z 317) and BH2+ (m/z 185) of mcm5U. Arrows in the upper panels indicate the retention time for mcm5s2U. (B) LC/MS fragment analyses of RNase T1-digested tRNAsGlu obtained from wild-type strains: ΔTUM1, ΔUBA4, ΔURM1, ΔNCS2, ΔNCS6 and ΔTRM9. A graph on the top-right represents the mass spectrum for the anticodon-containing fragment (CUmcm5s2UUCACCGp) (Figure 1B) from the wild-type strain. Charge states of multiply charged ions are indicated in parentheses. Other graphs describe the mass chromatograms shown by triply charged ions of anticodon-containing fragments bearing mcm5s2U (m/z 1458.17, red line), mcm5U (m/z 1450.18, black line), ncm5s2U (m/z 1450.67, green line) and ncm5U (m/z 1443.18, blue line). The RNA sequence including the wobble modification is indicated on each graph.
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Related In: Results  -  Collection

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Show All Figures
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Figure 2: Mass spectrometric analyses of total nucleosides and purified tRNAsGlu from S. cerevisiae wild-type and mutant cells. (A) LC/MS analyses of total nucleosides from strains of wild-type (WT), ΔYOR251c (TUM1), ΔYHR111w (UBA4), ΔYIL008w (URM1), ΔYNL119w (NCS2) and ΔYGL211w (NCS6). The upper panels show the merged mass chromatograms detecting MH+ (m/z 333) and BH2+ (m/z 201) of mcm5s2U. The lower panels show the mass chromatograms detecting MH+ (m/z 317) and BH2+ (m/z 185) of mcm5U. Arrows in the upper panels indicate the retention time for mcm5s2U. (B) LC/MS fragment analyses of RNase T1-digested tRNAsGlu obtained from wild-type strains: ΔTUM1, ΔUBA4, ΔURM1, ΔNCS2, ΔNCS6 and ΔTRM9. A graph on the top-right represents the mass spectrum for the anticodon-containing fragment (CUmcm5s2UUCACCGp) (Figure 1B) from the wild-type strain. Charge states of multiply charged ions are indicated in parentheses. Other graphs describe the mass chromatograms shown by triply charged ions of anticodon-containing fragments bearing mcm5s2U (m/z 1458.17, red line), mcm5U (m/z 1450.18, black line), ncm5s2U (m/z 1450.67, green line) and ncm5U (m/z 1443.18, blue line). The RNA sequence including the wobble modification is indicated on each graph.
Mentions: To identify the genes responsible for 2-thiouridine formation of mcm5s2U, we performed ribonucleome analysis, a genome-wide reverse genetic approach combined with mass spectrometry (26). Since 2-thiouridine is a non-essential RNA modification, the complete set of S. cerevisiae deletion strains (4829) serves as a parent population for this analysis. To reduce the size of the starting population, we selected 3482 genes that have orthologs in Schizosaccharomyces pombe, because S. pombe tRNAs possess mcm5s2U. Next, we chose 767 genes which are described as proteins of unknown function, and proteins with weak similarity to known functions in S. cerevisiae (CYGD: http://mips.gsf.de/genre/proj/yeast) (34). We then started the ribonucleome analysis using this population. In the mass chromatogram (Figure 2A), mcm5s2U was detected as a proton adduct form (MH+; m/z 333) with its base fragment (BH2+; m/z 201) in wild-type cells. mcm5U (MH+; m/z 317) with its base fragment (BH2+; m/z 185), which is a precursor form of mcm5s2U, could also be detected in wild-type cells (Figure 2A). Among the 767 selected deletion mutants, we identified four deletion strains, YHR111w(UBA4), YNL119w(NCS2), YGL211w(NCS6) and YOR251c, in which mcm5s2U was absent (Figure 2A, upper panels), while levels of its precursor mcm5U were increased (Figure 2A, lower panels), demonstrating that these genes are specifically involved in 2-thiolation of mcm5s2U. We have renamed YOR251c as tRNA-thiouridine modification protein 1 (TUM1). NCS2 is an orthologous protein of Ctu2 in Caenorhabditis elegans and fission yeast (24). Although it was reported that NCS6/TUC1 is responsible for 2-thiouridine formation by comparative genomic approach in 2007 (23), we have independently identified the same gene in this analysis. It was reported that UBA4, NCS2 and NCS6 are essential in the ΔCLA4 strain (35), and that their deletion affects the protein urmylation, which is a ubiquitin-like conjugation pathway (36,37). UBA4 encodes a paralog of a ubiquitin-activating enzyme (E1) (38). In fact, UBA4 was shown to function as an E1-like enzyme for URM1, which is an ubiquitin-related modifier involved in protein urmylation. We therefore analyzed the total nucleosides of ΔYIL008w(URM1) and found that URM1 is also responsible for 2-thiolation of mcm5s2U (Figure 2A).Figure 2.

Bottom Line: We could successfully reconstitute the 2-thiouridine formation in vitro using recombinant proteins.This study revealed that 2-thiouridine formation shares a pathway and chemical reactions with protein urmylation.The sulfur-flow of eukaryotic 2-thiouridine formation is distinct mechanism from the bacterial sulfur-relay system which is based on the persulfide chemistry.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Bldg. 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

ABSTRACT
The wobble modification in tRNAs, 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U), is required for the proper decoding of NNR codons in eukaryotes. The 2-thio group confers conformational rigidity of mcm(5)s(2)U by largely fixing the C3'-endo ribose puckering, ensuring stable and accurate codon-anticodon pairing. We have identified five genes in Saccharomyces cerevisiae, YIL008w (URM1), YHR111w (UBA4), YOR251c (TUM1), YNL119w (NCS2) and YGL211w (NCS6), that are required for 2-thiolation of mcm(5)s(2)U. An in vitro sulfur transfer experiment revealed that Tum1p stimulated the cysteine desulfurase of Nfs1p, and accepted persulfide sulfurs from Nfs1p. URM1 is a ubiquitin-related modifier, and UBA4 is an E1-like enzyme involved in protein urmylation. The carboxy-terminus of Urm1p was activated as an acyl-adenylate (-COAMP), then thiocarboxylated (-COSH) by Uba4p. The activated thiocarboxylate can be utilized in the subsequent reactions for 2-thiouridine formation, mediated by Ncs2p/Ncs6p. We could successfully reconstitute the 2-thiouridine formation in vitro using recombinant proteins. This study revealed that 2-thiouridine formation shares a pathway and chemical reactions with protein urmylation. The sulfur-flow of eukaryotic 2-thiouridine formation is distinct mechanism from the bacterial sulfur-relay system which is based on the persulfide chemistry.

Show MeSH
Related in: MedlinePlus