Limits...
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.

Show MeSH

Related in: MedlinePlus

APM Northern analyses of thiolation-status of tRNAs in mutant strains and transformants. (A) APM northern analyses of tRNAGlu in the s2U-deficient strains by introducing a series of mutant plasmids. Total RNA from each strain was resolved by denaturing polyacrylamide-gel electrophoresis in the presence (upper panels) or absence (lower panels) of APM. 2-thiolated tRNAGlu in each strain was detected as a shifted band only in the presence of APM, by northern blotting. Lanes 1 to 12 correspond to wild-tye (1), ΔURM1 (2), ΔURM1 harboring pURM1 (3), ΔURM1 harboring pURM1ΔGG (4), ΔUBA4 (5), ΔUBA4 harboring pUBA4 (6), ΔUBA4 harboring pUBA4 C225S (7), ΔUBA4 harboring pUBA4 C225A (8), ΔUBA4 harboring pUBA4 C397S (9),ΔTUM1 (10), ΔTUM1 harboring pTUM1 (11), ΔTUM1 harboring pTUM1 C259S (12), ΔTUM1 harboring pTUM1 C259A (13), ΔNCS2 (14) and ΔNCS2 harboring pNCS2 (15), respectively. (B) Quantification of the thiolation-status of tRNAGlu by APM/northern analyses. The fraction of the retarded band in the total intensity of the bands was calculated. Data are shown as values ± SD and reflect the average of four independent experiments. (C) APM/northern analyses of tRNAsLys from wild-type, ΔTRM9, ΔELP4, ΔTUM1 and ΔURM1. The right panel represents a mass chromatogram shown by triply charged ions of the anticodon-containing fragments of tRNAGlu isolated from ΔELP4. The unmodified fragment (m/z 1414.18, black line) and the s2U-containing fragment (m/z 1422.16, gray line) are specifically detected.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2651780&req=5

Figure 4: APM Northern analyses of thiolation-status of tRNAs in mutant strains and transformants. (A) APM northern analyses of tRNAGlu in the s2U-deficient strains by introducing a series of mutant plasmids. Total RNA from each strain was resolved by denaturing polyacrylamide-gel electrophoresis in the presence (upper panels) or absence (lower panels) of APM. 2-thiolated tRNAGlu in each strain was detected as a shifted band only in the presence of APM, by northern blotting. Lanes 1 to 12 correspond to wild-tye (1), ΔURM1 (2), ΔURM1 harboring pURM1 (3), ΔURM1 harboring pURM1ΔGG (4), ΔUBA4 (5), ΔUBA4 harboring pUBA4 (6), ΔUBA4 harboring pUBA4 C225S (7), ΔUBA4 harboring pUBA4 C225A (8), ΔUBA4 harboring pUBA4 C397S (9),ΔTUM1 (10), ΔTUM1 harboring pTUM1 (11), ΔTUM1 harboring pTUM1 C259S (12), ΔTUM1 harboring pTUM1 C259A (13), ΔNCS2 (14) and ΔNCS2 harboring pNCS2 (15), respectively. (B) Quantification of the thiolation-status of tRNAGlu by APM/northern analyses. The fraction of the retarded band in the total intensity of the bands was calculated. Data are shown as values ± SD and reflect the average of four independent experiments. (C) APM/northern analyses of tRNAsLys from wild-type, ΔTRM9, ΔELP4, ΔTUM1 and ΔURM1. The right panel represents a mass chromatogram shown by triply charged ions of the anticodon-containing fragments of tRNAGlu isolated from ΔELP4. The unmodified fragment (m/z 1414.18, black line) and the s2U-containing fragment (m/z 1422.16, gray line) are specifically detected.

Mentions: Next, we also employed APM–PAGE/northern blotting to detect thiolated tRNAGlu (Figure 4A and B) from each of the knockout strains. In the wild-type cells, a large fraction of the tRNAGlu migrates slowly on the PAGE in the presence of APM due to a specific interaction between the thiocarbonyl group of mcm5s2U and the APM in the gel. No retarded band for tRNAGlu from ΔURM1, ΔUBA4 and ΔNCS2 was observed, showing that no thiouridine formation occurred in these strains. The thiouridine in these deletion strains was partially or completely restored by the introduction of a plasmid encoding URM1, UBA4 or NCS2. With respect to NCS6, we failed to restore 2-thiouridine formation in the ΔNCS6 strain, by introduction of a plasmid encoding NCS6, for unknown reasons (data not shown). In the case of ΔTUM1, the large fraction of tRNAGlu lacked 2-thiouridine, but the small fraction of tRNAGlu remained 2-thiolated. This result is consistent with the LC/MS analysis (Figure 2B). Thus, although TUM1 is not essential for thiolation, it is a major component involved in this pathway.


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)

APM Northern analyses of thiolation-status of tRNAs in mutant strains and transformants. (A) APM northern analyses of tRNAGlu in the s2U-deficient strains by introducing a series of mutant plasmids. Total RNA from each strain was resolved by denaturing polyacrylamide-gel electrophoresis in the presence (upper panels) or absence (lower panels) of APM. 2-thiolated tRNAGlu in each strain was detected as a shifted band only in the presence of APM, by northern blotting. Lanes 1 to 12 correspond to wild-tye (1), ΔURM1 (2), ΔURM1 harboring pURM1 (3), ΔURM1 harboring pURM1ΔGG (4), ΔUBA4 (5), ΔUBA4 harboring pUBA4 (6), ΔUBA4 harboring pUBA4 C225S (7), ΔUBA4 harboring pUBA4 C225A (8), ΔUBA4 harboring pUBA4 C397S (9),ΔTUM1 (10), ΔTUM1 harboring pTUM1 (11), ΔTUM1 harboring pTUM1 C259S (12), ΔTUM1 harboring pTUM1 C259A (13), ΔNCS2 (14) and ΔNCS2 harboring pNCS2 (15), respectively. (B) Quantification of the thiolation-status of tRNAGlu by APM/northern analyses. The fraction of the retarded band in the total intensity of the bands was calculated. Data are shown as values ± SD and reflect the average of four independent experiments. (C) APM/northern analyses of tRNAsLys from wild-type, ΔTRM9, ΔELP4, ΔTUM1 and ΔURM1. The right panel represents a mass chromatogram shown by triply charged ions of the anticodon-containing fragments of tRNAGlu isolated from ΔELP4. The unmodified fragment (m/z 1414.18, black line) and the s2U-containing fragment (m/z 1422.16, gray line) are specifically detected.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651780&req=5

Figure 4: APM Northern analyses of thiolation-status of tRNAs in mutant strains and transformants. (A) APM northern analyses of tRNAGlu in the s2U-deficient strains by introducing a series of mutant plasmids. Total RNA from each strain was resolved by denaturing polyacrylamide-gel electrophoresis in the presence (upper panels) or absence (lower panels) of APM. 2-thiolated tRNAGlu in each strain was detected as a shifted band only in the presence of APM, by northern blotting. Lanes 1 to 12 correspond to wild-tye (1), ΔURM1 (2), ΔURM1 harboring pURM1 (3), ΔURM1 harboring pURM1ΔGG (4), ΔUBA4 (5), ΔUBA4 harboring pUBA4 (6), ΔUBA4 harboring pUBA4 C225S (7), ΔUBA4 harboring pUBA4 C225A (8), ΔUBA4 harboring pUBA4 C397S (9),ΔTUM1 (10), ΔTUM1 harboring pTUM1 (11), ΔTUM1 harboring pTUM1 C259S (12), ΔTUM1 harboring pTUM1 C259A (13), ΔNCS2 (14) and ΔNCS2 harboring pNCS2 (15), respectively. (B) Quantification of the thiolation-status of tRNAGlu by APM/northern analyses. The fraction of the retarded band in the total intensity of the bands was calculated. Data are shown as values ± SD and reflect the average of four independent experiments. (C) APM/northern analyses of tRNAsLys from wild-type, ΔTRM9, ΔELP4, ΔTUM1 and ΔURM1. The right panel represents a mass chromatogram shown by triply charged ions of the anticodon-containing fragments of tRNAGlu isolated from ΔELP4. The unmodified fragment (m/z 1414.18, black line) and the s2U-containing fragment (m/z 1422.16, gray line) are specifically detected.
Mentions: Next, we also employed APM–PAGE/northern blotting to detect thiolated tRNAGlu (Figure 4A and B) from each of the knockout strains. In the wild-type cells, a large fraction of the tRNAGlu migrates slowly on the PAGE in the presence of APM due to a specific interaction between the thiocarbonyl group of mcm5s2U and the APM in the gel. No retarded band for tRNAGlu from ΔURM1, ΔUBA4 and ΔNCS2 was observed, showing that no thiouridine formation occurred in these strains. The thiouridine in these deletion strains was partially or completely restored by the introduction of a plasmid encoding URM1, UBA4 or NCS2. With respect to NCS6, we failed to restore 2-thiouridine formation in the ΔNCS6 strain, by introduction of a plasmid encoding NCS6, for unknown reasons (data not shown). In the case of ΔTUM1, the large fraction of tRNAGlu lacked 2-thiouridine, but the small fraction of tRNAGlu remained 2-thiolated. This result is consistent with the LC/MS analysis (Figure 2B). Thus, although TUM1 is not essential for thiolation, it is a major component involved in this pathway.

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