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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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Chemical structure of 5-methoxycarmonylmethyl-2-thiouridene (mcm5s2U) and secondary structure of tRNAGlu. (A) Chemical structures of mcm5U and mcm5s2U. 2-thiolation of mcm5s2U utilizes cysteine and ATP as substrates. (B) Secondary structure of S. cerevisiae tRNAGlu with modified nucleosides: 5-methoxycarmonylmethyl-2-thiouridine (mcm5s2U), pseudouridine (Ψ), dihydrouridine (D), 5-methylcytidine (m5C) and 5-methyluridine (m5U). Arrows indicate the sites for RNase T1 cleavage needed to produce the anticodon-containing fragment.
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Figure 1: Chemical structure of 5-methoxycarmonylmethyl-2-thiouridene (mcm5s2U) and secondary structure of tRNAGlu. (A) Chemical structures of mcm5U and mcm5s2U. 2-thiolation of mcm5s2U utilizes cysteine and ATP as substrates. (B) Secondary structure of S. cerevisiae tRNAGlu with modified nucleosides: 5-methoxycarmonylmethyl-2-thiouridine (mcm5s2U), pseudouridine (Ψ), dihydrouridine (D), 5-methylcytidine (m5C) and 5-methyluridine (m5U). Arrows indicate the sites for RNase T1 cleavage needed to produce the anticodon-containing fragment.

Mentions: Post-transcriptional RNA modifications are characteristic structural feature of RNA molecules. RNA modifications play critical roles in various aspects of RNA molecules, including biogenesis, metabolism, structural stability and functions. To date, more than 100 species of RNA modifications have been reported (1). The majority of these modifications have been identified and characterized in tRNA molecules (2–4). In the region of the tRNA anticodon in particular, various modified nucleosides with diverse chemical structures have been found at the anticodon first (wobble) position 34, and at position 37 3′-adjacent to the anticodon. The wobble modifications play pivotal roles in deciphering genetic codes that are mediated by the precise codon–anticodon interactions at the ribosomal A-site. The wobble base of the tRNAs for Glu, Gln and Lys are universally modified to 5-methyl-2-thiouridine derivatives (xm5s2U), such as 5-methylaminomethyl-2-thiouridine (mnm5s2U) and 5-carboxymethyl-2-thiouridine (cmnm5s2U) in bacterial tRNAs, 5-taurinomethyl-2-thiouridine (τm5s2U) in mammalian mitochondrial tRNAs and 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) in cytoplasmic tRNAs in eukaryotes (4) (Figure 1). It is known that the conformation of xm5s2U is largely fixed in the C3′-endo form of its ribose puckering, since the large van der Waals’ radius of the 2-thio group causes a steric repulsion with its 2′ OH group (5). The xm5s2U modification base-pairs preferentially with purine and prevents misreading of near cognate codons ending in pyrimidine due to this conformational rigidity (5,6). It is known that the 2-thio group of mnm5s2U is required for efficient codon recognition on the ribosome (7). In addition, the 2-thio group of mnm5s2U in tRNAGlu acts as the identifying element for specific recognition by glutaminyl-tRNA synthetase (8). Lack of xm5s2U modification in the mutant mitochondrial tRNALys from myoclonus epilepsy associated with ragged-red fibers (MERRF), results in a marked defect in whole mitochondrial translation (9,10). Thus, the 2-thio modification xm5s2U plays a critical role in protein synthesis.Figure 1.


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)

Chemical structure of 5-methoxycarmonylmethyl-2-thiouridene (mcm5s2U) and secondary structure of tRNAGlu. (A) Chemical structures of mcm5U and mcm5s2U. 2-thiolation of mcm5s2U utilizes cysteine and ATP as substrates. (B) Secondary structure of S. cerevisiae tRNAGlu with modified nucleosides: 5-methoxycarmonylmethyl-2-thiouridine (mcm5s2U), pseudouridine (Ψ), dihydrouridine (D), 5-methylcytidine (m5C) and 5-methyluridine (m5U). Arrows indicate the sites for RNase T1 cleavage needed to produce the anticodon-containing fragment.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Chemical structure of 5-methoxycarmonylmethyl-2-thiouridene (mcm5s2U) and secondary structure of tRNAGlu. (A) Chemical structures of mcm5U and mcm5s2U. 2-thiolation of mcm5s2U utilizes cysteine and ATP as substrates. (B) Secondary structure of S. cerevisiae tRNAGlu with modified nucleosides: 5-methoxycarmonylmethyl-2-thiouridine (mcm5s2U), pseudouridine (Ψ), dihydrouridine (D), 5-methylcytidine (m5C) and 5-methyluridine (m5U). Arrows indicate the sites for RNase T1 cleavage needed to produce the anticodon-containing fragment.
Mentions: Post-transcriptional RNA modifications are characteristic structural feature of RNA molecules. RNA modifications play critical roles in various aspects of RNA molecules, including biogenesis, metabolism, structural stability and functions. To date, more than 100 species of RNA modifications have been reported (1). The majority of these modifications have been identified and characterized in tRNA molecules (2–4). In the region of the tRNA anticodon in particular, various modified nucleosides with diverse chemical structures have been found at the anticodon first (wobble) position 34, and at position 37 3′-adjacent to the anticodon. The wobble modifications play pivotal roles in deciphering genetic codes that are mediated by the precise codon–anticodon interactions at the ribosomal A-site. The wobble base of the tRNAs for Glu, Gln and Lys are universally modified to 5-methyl-2-thiouridine derivatives (xm5s2U), such as 5-methylaminomethyl-2-thiouridine (mnm5s2U) and 5-carboxymethyl-2-thiouridine (cmnm5s2U) in bacterial tRNAs, 5-taurinomethyl-2-thiouridine (τm5s2U) in mammalian mitochondrial tRNAs and 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) in cytoplasmic tRNAs in eukaryotes (4) (Figure 1). It is known that the conformation of xm5s2U is largely fixed in the C3′-endo form of its ribose puckering, since the large van der Waals’ radius of the 2-thio group causes a steric repulsion with its 2′ OH group (5). The xm5s2U modification base-pairs preferentially with purine and prevents misreading of near cognate codons ending in pyrimidine due to this conformational rigidity (5,6). It is known that the 2-thio group of mnm5s2U is required for efficient codon recognition on the ribosome (7). In addition, the 2-thio group of mnm5s2U in tRNAGlu acts as the identifying element for specific recognition by glutaminyl-tRNA synthetase (8). Lack of xm5s2U modification in the mutant mitochondrial tRNALys from myoclonus epilepsy associated with ragged-red fibers (MERRF), results in a marked defect in whole mitochondrial translation (9,10). Thus, the 2-thio modification xm5s2U plays a critical role in protein synthesis.Figure 1.

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