The Experts below are selected from a list of 147 Experts worldwide ranked by ideXlab platform
Tsutomu Suzuki - One of the best experts on this subject based on the ideXlab platform.
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Structural and functional characterization of the TYW3/Taw3 class of SAM-dependent methyltransferases
RNA (New York N.Y.), 2016Co-Authors: Mark A. Currie, Tsutomu Suzuki, Takayuki Ohira, Greg Brown, Andrew Wong, Kei Sugiyama, Alexander F. Yakunin, Zongchao JiaAbstract:S-adenosylmethionine (SAM)-dependent methyltransferases regulate a wide range of biological processes through the modification of proteins, nucleic acids, polysaccharides, as well as various metabolites. TYW3/Taw3 is a SAM-dependent methyltransferase responsible for the formation of a tRNA modification known as Wybutosine and its derivatives that are required for accurate decoding in protein synthesis. Here, we report the crystal structure of Taw3, a homolog of TYW3 from Sulfolobus solfataricus, which revealed a novel α/β fold. The sequence motif (S/T)xSSCxGR and invariant aspartate and histidine, conserved in TYW3/Taw3, cluster to form the catalytic center. These structural and sequence features indicate that TYW3/Taw3 proteins constitute a distinct class of SAM-dependent methyltransferases. Using site-directed mutagenesis along with in vivo complementation assays combined with mass spectrometry as well as ligand docking and cofactor binding assays, we have identified the active site of TYW3 and residues essential for cofactor binding and methyltransferase activity.
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Structure-Function Analysis of Human TYW2 Enzyme Required for the Biosynthesis of a Highly Modified Wybutosine (yW) Base in Phenylalanine-tRNA
PloS one, 2012Co-Authors: Virginia Rodriguez, Akiko Noma, Bradley A. Carlson, Tsutomu Suzuki, Sona Vasudevan, Jeffrey E. Green, Settara C. ChandrasekharappaAbstract:Posttranscriptional modifications are critical for structure and function of tRNAs. Wybutosine (yW) and its derivatives are hyper-modified guanosines found at the position 37 of eukaryotic and archaeal tRNAPhe. TYW2 is an enzyme that catalyzes α-amino-α-carboxypropyl transfer activity at the third step of yW biogenesis. Using complementation of a ΔTYW2 strain, we demonstrate here that human TYW2 (hTYW2) is active in yeast and can synthesize the yW of yeast tRNAPhe. Structure-guided analysis identified several conserved residues in hTYW2 that interact with S-adenosyl-methionine (AdoMet), and mutation studies revealed that K225 and E265 are critical residues for the enzymatic activity. We previously reported that the human TYW2 is overexpressed in breast cancer. However, no difference in the tRNAPhe modification status was observed in either normal mouse tissue or a mouse tumor model that overexpresses Tyw2, indicating that hTYW2 may have a role in tumorigenesis unrelated to yW biogenesis.
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Retrograde nuclear import of tRNA precursors is required for modified base biogenesis in yeast
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Takayuki Ohira, Tsutomu SuzukiAbstract:The retrograde movement of tRNAs from the cytoplasm to the nucleus occurs constitutively in eukaryotic cells but its functional significance remains unclear. We show evidence suggesting that in Saccharomyces cerevisiae, a spliced tRNA precursor must be imported into the nucleus before the biogenesis of a modified base can occur. Wybutosine (yW) is a modified base adjacent to the anticodon of tRNAPhe and is required for accurate decoding. Glucose starvation or overexpression of the nuclear tRNA binding protein Trz1p both caused nuclear retention of cytoplasmic tRNAs, impaired the yW synthesis, and induced the accumulation of its intermediate, N1-methylgunanosine (m1G), showing that the postspliced tRNAPhe is imported to the nucleus, where m1G is formed by Trm5p, after which it is reexported to the cytoplasm, where the yW synthesis is completed by cytoplasmic enzymes.
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Crystal structure of a novel JmjC-domain-containing protein, TYW5, involved in tRNA modification
Nucleic acids research, 2010Co-Authors: Megumi Kato, Akiko Noma, Tsutomu Suzuki, Ryuichiro Ishitani, Yuhei Araiso, Asuteka Nagao, Osamu NurekiAbstract:Wybutosine (yW) is a hypermodified nucleoside found in position 37 of tRNA(Phe), and is essential for correct phenylalanine codon translation. yW derivatives widely exist in eukaryotes and archaea, and their chemical structures have many species-specific variations. Among them, its hydroxylated derivative, hydroxyWybutosine (OHyW), is found in eukaryotes including human, but the modification mechanism remains unknown. Recently, we identified a novel Jumonji C (JmjC)-domain-containing protein, TYW5 (tRNA yW-synthesizing enzyme 5), which forms the OHyW nucleoside by carbon hydroxylation, using Fe(II) ion and 2-oxoglutarate (2-OG) as cofactors. In this work, we present the crystal structures of human TYW5 (hTYW5) in the free and complex forms with 2-OG and Ni(II) ion at 2.5 and 2.8 Å resolutions, respectively. The structure revealed that the catalytic domain consists of a β-jellyroll fold, a hallmark of the JmjC domains and other Fe(II)/2-OG oxygenases. hTYW5 forms a homodimer through C-terminal helix bundle formation, thereby presenting a large, positively charged patch involved in tRNA binding. A comparison with the structures of other JmjC-domain-containing proteins suggested a mechanism for substrate nucleotide recognition. Functional analyses of structure-based mutants revealed the essential Arg residues participating in tRNA recognition by TYW5. These findings extend the repertoire of the tRNA modification enzyme into the Fe(II)/2-OG oxygenase superfamily.
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structural basis of adomet dependent aminocarboxypropyl transfer reaction catalyzed by trna Wybutosine synthesizing enzyme tyw2
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Masataka Umitsu, Akiko Noma, Tsutomu Suzuki, Ryuichiro Ishitani, Hiroshi Nishimasu, Osamu NurekiAbstract:S-adenosylmethionine (AdoMet) is a methyl donor used by a wide variety of methyltransferases, and it is also used as the source of an α-amino-α-carboxypropyl (“acp”) group by several enzymes. tRNA-yW synthesizing enzyme-2 (TYW2) is involved in the biogenesis of a hypermodified nucleotide, Wybutosine (yW), and it catalyzes the transfer of the “acp” group from AdoMet to the C7 position of the imG-14 base, a yW precursor. This modified nucleoside yW is exclusively located at position 37 of eukaryotic tRNAPhe, and it ensures the anticodon-codon pairing on the ribosomal decoding site. Although this “acp” group has a significant role in preventing decoding frame shifts, the mechanism of the “acp” group transfer by TYW2 remains unresolved. Here we report the crystal structures and functional analyses of two archaeal homologs of TYW2 from Pyrococcus horikoshii and Methanococcus jannaschii. The in vitro mass spectrometric and radioisotope-labeling analyses confirmed that these archaeal TYW2 homologues have the same activity as yeast TYW2. The crystal structures verified that the archaeal TYW2 contains a canonical class-I methyltransferase (MTase) fold. However, their AdoMet-bound structures revealed distinctive AdoMet-binding modes, in which the “acp” group, instead of the methyl group, of AdoMet is directed to the substrate binding pocket. Our findings, which were confirmed by extensive mutagenesis studies, explain why TYW2 transfers the “acp” group, and not the methyl group, from AdoMet to the nucleobase.
Akiko Noma - One of the best experts on this subject based on the ideXlab platform.
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Structure-Function Analysis of Human TYW2 Enzyme Required for the Biosynthesis of a Highly Modified Wybutosine (yW) Base in Phenylalanine-tRNA
PloS one, 2012Co-Authors: Virginia Rodriguez, Akiko Noma, Bradley A. Carlson, Tsutomu Suzuki, Sona Vasudevan, Jeffrey E. Green, Settara C. ChandrasekharappaAbstract:Posttranscriptional modifications are critical for structure and function of tRNAs. Wybutosine (yW) and its derivatives are hyper-modified guanosines found at the position 37 of eukaryotic and archaeal tRNAPhe. TYW2 is an enzyme that catalyzes α-amino-α-carboxypropyl transfer activity at the third step of yW biogenesis. Using complementation of a ΔTYW2 strain, we demonstrate here that human TYW2 (hTYW2) is active in yeast and can synthesize the yW of yeast tRNAPhe. Structure-guided analysis identified several conserved residues in hTYW2 that interact with S-adenosyl-methionine (AdoMet), and mutation studies revealed that K225 and E265 are critical residues for the enzymatic activity. We previously reported that the human TYW2 is overexpressed in breast cancer. However, no difference in the tRNAPhe modification status was observed in either normal mouse tissue or a mouse tumor model that overexpresses Tyw2, indicating that hTYW2 may have a role in tumorigenesis unrelated to yW biogenesis.
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Crystal structure of a novel JmjC-domain-containing protein, TYW5, involved in tRNA modification
Nucleic acids research, 2010Co-Authors: Megumi Kato, Akiko Noma, Tsutomu Suzuki, Ryuichiro Ishitani, Yuhei Araiso, Asuteka Nagao, Osamu NurekiAbstract:Wybutosine (yW) is a hypermodified nucleoside found in position 37 of tRNA(Phe), and is essential for correct phenylalanine codon translation. yW derivatives widely exist in eukaryotes and archaea, and their chemical structures have many species-specific variations. Among them, its hydroxylated derivative, hydroxyWybutosine (OHyW), is found in eukaryotes including human, but the modification mechanism remains unknown. Recently, we identified a novel Jumonji C (JmjC)-domain-containing protein, TYW5 (tRNA yW-synthesizing enzyme 5), which forms the OHyW nucleoside by carbon hydroxylation, using Fe(II) ion and 2-oxoglutarate (2-OG) as cofactors. In this work, we present the crystal structures of human TYW5 (hTYW5) in the free and complex forms with 2-OG and Ni(II) ion at 2.5 and 2.8 Å resolutions, respectively. The structure revealed that the catalytic domain consists of a β-jellyroll fold, a hallmark of the JmjC domains and other Fe(II)/2-OG oxygenases. hTYW5 forms a homodimer through C-terminal helix bundle formation, thereby presenting a large, positively charged patch involved in tRNA binding. A comparison with the structures of other JmjC-domain-containing proteins suggested a mechanism for substrate nucleotide recognition. Functional analyses of structure-based mutants revealed the essential Arg residues participating in tRNA recognition by TYW5. These findings extend the repertoire of the tRNA modification enzyme into the Fe(II)/2-OG oxygenase superfamily.
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Biosynthesis of wyosine derivatives in tRNA: an ancient and highly diverse pathway in Archaea.
Molecular Biology and Evolution, 2010Co-Authors: Valérie De Crécy-lagard, Céline Brochier-armanet, Jaunius Urbonavicius, Bernard Fernandez, Gabriela Phillips, Benjamin Lyons, Akiko Noma, Sophie Alvarez, Louis Droogmans, Jean ArmengaudAbstract:Wyosine (imG) and its derivatives such as Wybutosine (yW) are found at position 37 of phenylalanine-specific transfer RNA (tRNA(Phe)), 3' adjacent to the anticodon in Eucarya and Archaea. In Saccharomyces cerevisiae, formation of yW requires five enzymes acting in a strictly sequential order: Trm5, Tyw1, Tyw2, Tyw3, and Tyw4. Archaea contain wyosine derivatives, but their diversity is greater than in eukaryotes and the corresponding biosynthesis pathways still unknown. To identify these pathways, we analyzed the phylogenetic distribution of homologues of the yeast Wybutosine biosynthesis proteins in 62 archaeal genomes and proposed a scenario for the origin and evolution of wyosine derivatives biosynthesis in Archaea that was partly experimentally validated. The key observations were 1) that four of the five Wybutosine biosynthetic enzymes are ancient and may have been present in the last common ancestor of Archaea and Eucarya, 2) that the variations in the distribution pattern of biosynthesis enzymes reflect the diversity of the wyosine derivatives found in different Archaea. We also identified 7-aminocarboxypropyl-demethylwyosine (yW-86) and its N4-methyl derivative (yW-72) as final products in tRNAs of several Archaea when these were previously thought to be only intermediates of the eukaryotic pathway. We confirmed that isowyosine (imG2) and 7-methylwyosine (mimG) are two archaeal-specific guanosine-37 derivatives found in tRNA of both Euryarchaeota and Crenarchaeota. Finally, we proposed that the duplication of the trm5 gene in some Archaea led to a change in function from N1 methylation of guanosine to C7 methylation of 4-demethylwyosine (imG-14).
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structural basis of adomet dependent aminocarboxypropyl transfer reaction catalyzed by trna Wybutosine synthesizing enzyme tyw2
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Masataka Umitsu, Akiko Noma, Tsutomu Suzuki, Ryuichiro Ishitani, Hiroshi Nishimasu, Osamu NurekiAbstract:S-adenosylmethionine (AdoMet) is a methyl donor used by a wide variety of methyltransferases, and it is also used as the source of an α-amino-α-carboxypropyl (“acp”) group by several enzymes. tRNA-yW synthesizing enzyme-2 (TYW2) is involved in the biogenesis of a hypermodified nucleotide, Wybutosine (yW), and it catalyzes the transfer of the “acp” group from AdoMet to the C7 position of the imG-14 base, a yW precursor. This modified nucleoside yW is exclusively located at position 37 of eukaryotic tRNAPhe, and it ensures the anticodon-codon pairing on the ribosomal decoding site. Although this “acp” group has a significant role in preventing decoding frame shifts, the mechanism of the “acp” group transfer by TYW2 remains unresolved. Here we report the crystal structures and functional analyses of two archaeal homologs of TYW2 from Pyrococcus horikoshii and Methanococcus jannaschii. The in vitro mass spectrometric and radioisotope-labeling analyses confirmed that these archaeal TYW2 homologues have the same activity as yeast TYW2. The crystal structures verified that the archaeal TYW2 contains a canonical class-I methyltransferase (MTase) fold. However, their AdoMet-bound structures revealed distinctive AdoMet-binding modes, in which the “acp” group, instead of the methyl group, of AdoMet is directed to the substrate binding pocket. Our findings, which were confirmed by extensive mutagenesis studies, explain why TYW2 transfers the “acp” group, and not the methyl group, from AdoMet to the nucleobase.
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Abstract #2486: TRMT12 overexpression in cancer cells affects the cell cycle and the nonsense mediated decay pathways by regulating the SMG-1 kinase, a member of the PIKK family of kinases
Cancer Research, 2009Co-Authors: Virginia Rodriguez, Akiko Noma, Bradley A. Carlson, Abdel G. Elkahloun, Jeffrey Green, Tsutomu Suzuki, Settara C. ChandrasekharappaAbstract:Previously, we reported the overexpression of TRMT12 (tRNA methyltransferase 12) in breast cancer. This gene is located on chromosome 8 in the 8q24 region, an area commonly amplified in many types of cancer. The human TRMT12 gene has never been studied. TRMT12 is homologous to the yeast TYW2, a tRNA methyltransferase involved in the posttranscriptional modification that converts guanosine to Wybutosine, a highly modified base in tRNA-phenylalanine. Wybutosine is present only in tRNA-phenylalanine at the 37th position adjacent to the anticodon. This modification stabilizes the codon-anticodon interaction and functions to maintain the correct reading frame. Recently, we cloned the human TRMT12 gene and determined that TRMT12 catalyzes the third step in the biosynthetic pathway of Wybutosine. This is the first time the biological function of one of the human enzymes in the Wybutosine pathway has been demonstrated. We find that Wybutosine modification in tRNA-phenylalanine is not affected in a mouse mammary tumor model that overexpresses Trmt12. To determine the effect of TRMT12 overexpression in cancer, we evaluated the expression changes in cells treated with TRMT12 siRNAs. Results indicate that the SMG-1 gene had a >2 fold increase in expression when TRMT12 was brought down, and this was validated by RT-qPCR and Western blot analysis. SMG-1 is a critical phosphatidylinositol-3-kinase-related kinase (PIKK) that is a key player in DNA damage response and in the nonsense mediated decay pathway (NMD), a surveillance system that maintains the quality and fidelity of mRNA transcripts. Overexpression of TRMT12 results in altered expression of SMG-1, a reduction in p53 (Ser 15) phosphorylation and associated disruption to the cell cycle; overexpression of TRMT12 also results in compromising the NMD pathway. Thus, overexpression of TRMT12 in cancer is likely to disrupt SMG-1 function, which in turn may allow DNA damage to go unchecked and mutated RNA transcripts to escape degradation. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2486.
Vahe Bandarian - One of the best experts on this subject based on the ideXlab platform.
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TYW1: A Radical SAM Enzyme Involved in the Biosynthesis of Wybutosine Bases.
Methods in enzymology, 2018Co-Authors: Anthony P. Young, Vahe BandarianAbstract:Transfer RNA is extensively modified by the actions of a variety of enzymes. The radical S-adenosyl-l-methionine enzyme TYW1 modifies tRNAPhe forming the characteristic tricyclic ring via the condensation of carbons 2 and 3 of pyruvate. This chapter details methods that are required for studies of TYW1.
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Radical mediated ring formation in the biosynthesis of the hypermodified tRNA base Wybutosine.
Current opinion in chemical biology, 2013Co-Authors: Anthony P. Young, Vahe BandarianAbstract:Wyosine and its derivatives are highly modified, acid labile tricyclic bases found at position 37 of tRNAPhe in archaea and eukarya. The formation of the common 4-demethylwyosine structural feature entails condensation of pyruvate and N-methylguanosine catalyzed by TYW1. This review will focus on the mechanism of this complex radical mediated transformation.
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Radical SAM Enzymes Involved in the Biosynthesis of Purine-Based Natural Products
Biochimica et biophysica acta, 2012Co-Authors: Vahe BandarianAbstract:Abstract The radical S-adenosyl- l -methionine (SAM) superfamily is a widely distributed group of iron–sulfur containing proteins that exploit the reactivity of the high energy intermediate, 5′-deoxyadenosyl radical, which is produced by the reductive cleavage of SAM, to carry-out complex radical-mediated transformations. The reactions catalyzed by radical SAM enzymes range from simple group migrations to complex reactions in protein and RNA modification. This review will highlight three radical SAM enzymes that catalyze reactions involving modified guanosines in the biosynthesis pathways of the hypermodified tRNA base Wybutosine; secondary metabolites of 7-deazapurine structure, including the hypermodified tRNA base queuosine; and the redox cofactor F420. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.
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Pyruvate is the source of the two carbons that are required for formation of the imidazoline ring of 4-demethylwyosine.
Biochemistry, 2011Co-Authors: Anthony P. Young, Vahe BandarianAbstract:TYW1 catalyzes the condensation of N-methylguanosine with two carbon atoms from an unknown second substrate to form 4-demethylwyosine, which is a common intermediate in the biosynthesis of all of the hypermodified RNA bases related to Wybutosine found in eukaryal and archaeal tRNAPhe. Of the potential substrates examined, only incubation with pyruvate resulted in formation of 4-demethylwyosine. Moreover, incubation with C1, C2, C3, or C1,2,3-13C-labeled pyruvate showed that C2 and C3 are incorporated while C1 is not. The mechanistic implications of these results are discussed in the context of the structure of TYW1.
Osamu Nureki - One of the best experts on this subject based on the ideXlab platform.
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Crystal structure of a novel JmjC-domain-containing protein, TYW5, involved in tRNA modification
Nucleic acids research, 2010Co-Authors: Megumi Kato, Akiko Noma, Tsutomu Suzuki, Ryuichiro Ishitani, Yuhei Araiso, Asuteka Nagao, Osamu NurekiAbstract:Wybutosine (yW) is a hypermodified nucleoside found in position 37 of tRNA(Phe), and is essential for correct phenylalanine codon translation. yW derivatives widely exist in eukaryotes and archaea, and their chemical structures have many species-specific variations. Among them, its hydroxylated derivative, hydroxyWybutosine (OHyW), is found in eukaryotes including human, but the modification mechanism remains unknown. Recently, we identified a novel Jumonji C (JmjC)-domain-containing protein, TYW5 (tRNA yW-synthesizing enzyme 5), which forms the OHyW nucleoside by carbon hydroxylation, using Fe(II) ion and 2-oxoglutarate (2-OG) as cofactors. In this work, we present the crystal structures of human TYW5 (hTYW5) in the free and complex forms with 2-OG and Ni(II) ion at 2.5 and 2.8 Å resolutions, respectively. The structure revealed that the catalytic domain consists of a β-jellyroll fold, a hallmark of the JmjC domains and other Fe(II)/2-OG oxygenases. hTYW5 forms a homodimer through C-terminal helix bundle formation, thereby presenting a large, positively charged patch involved in tRNA binding. A comparison with the structures of other JmjC-domain-containing proteins suggested a mechanism for substrate nucleotide recognition. Functional analyses of structure-based mutants revealed the essential Arg residues participating in tRNA recognition by TYW5. These findings extend the repertoire of the tRNA modification enzyme into the Fe(II)/2-OG oxygenase superfamily.
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structural basis of adomet dependent aminocarboxypropyl transfer reaction catalyzed by trna Wybutosine synthesizing enzyme tyw2
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Masataka Umitsu, Akiko Noma, Tsutomu Suzuki, Ryuichiro Ishitani, Hiroshi Nishimasu, Osamu NurekiAbstract:S-adenosylmethionine (AdoMet) is a methyl donor used by a wide variety of methyltransferases, and it is also used as the source of an α-amino-α-carboxypropyl (“acp”) group by several enzymes. tRNA-yW synthesizing enzyme-2 (TYW2) is involved in the biogenesis of a hypermodified nucleotide, Wybutosine (yW), and it catalyzes the transfer of the “acp” group from AdoMet to the C7 position of the imG-14 base, a yW precursor. This modified nucleoside yW is exclusively located at position 37 of eukaryotic tRNAPhe, and it ensures the anticodon-codon pairing on the ribosomal decoding site. Although this “acp” group has a significant role in preventing decoding frame shifts, the mechanism of the “acp” group transfer by TYW2 remains unresolved. Here we report the crystal structures and functional analyses of two archaeal homologs of TYW2 from Pyrococcus horikoshii and Methanococcus jannaschii. The in vitro mass spectrometric and radioisotope-labeling analyses confirmed that these archaeal TYW2 homologues have the same activity as yeast TYW2. The crystal structures verified that the archaeal TYW2 contains a canonical class-I methyltransferase (MTase) fold. However, their AdoMet-bound structures revealed distinctive AdoMet-binding modes, in which the “acp” group, instead of the methyl group, of AdoMet is directed to the substrate binding pocket. Our findings, which were confirmed by extensive mutagenesis studies, explain why TYW2 transfers the “acp” group, and not the methyl group, from AdoMet to the nucleobase.
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Structural basis of tRNA modification with CO2 fixation and methylation by Wybutosine synthesizing enzyme TYW4
Nucleic acids research, 2009Co-Authors: Yoko Suzuki, Akiko Noma, Tsutomu Suzuki, Ryuichiro Ishitani, Osamu NurekiAbstract:Wybutosine (yW), one of the most complicated modified nucleosides, is found in the anticodon loop of eukaryotic phenylalanine tRNA. This hypermodified nucleoside ensures correct codon recognition by stabilizing codon-anticodon pairings during the decoding process in the ribosome. TYW4 is an S-adenosylmethionine (SAM)-dependent enzyme that catalyzes the final step of yW biosynthesis, methylation and methoxycarbonylation. However, the structural basis for the catalytic mechanism by TYW4, and especially that for the methoxycarbonylation, have remained elusive. Here we report the apo and cofactor-bound crystal structures of yeast TYW4. The structures revealed that the C-terminal domain folds into a β-propeller structure, forming part of the binding pocket for the target nucleoside. A comparison of the apo, SAM-bound, and S-adenosylhomocysteine-bound structures of TYW4 revealed a drastic structural change upon cofactor binding, which may sequester solvent from the catalytic site during the reaction and facilitate product release after the reaction. In conjunction with the functional analysis, our results suggest that TYW4 catalyzes both methylation and methoxycarbonylation at a single catalytic site, and in the latter reaction, the methoxycarbonyl group is formed through the fixation of carbon dioxide.
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Crystal Structure of the Radical SAM Enzyme Catalyzing Tricyclic Modified Base Formation in tRNA
Journal of molecular biology, 2007Co-Authors: Yoko Suzuki, Akiko Noma, Tsutomu Suzuki, Miki Senda, Toshiya Senda, Ryuichiro Ishitani, Osamu NurekiAbstract:Wyosine and its derivatives, such as Wybutosine, found in eukaryotic and archaeal tRNAs, are tricyclic hypermodified nucleosides. In eukaryotes, Wybutosine exists exclusively in position 37, 3'-adjacent to the anticodon, of tRNA(Phe), where it ensures correct translation by stabilizing the codon-anticodon base-pairing during the ribosomal decoding process. Recent studies revealed that the wyosine biosynthetic pathway consists of multistep enzymatic reactions starting from a guanosine residue. Among these steps, TYW1 catalyzes the second step to form the tricyclic ring structure, by cyclizing N(1)-methylguanosine. In this study, we solved the crystal structure of TYW1 from Methanocaldococcus jannaschii at 2.4 A resolution. TYW1 assumes an incomplete TIM barrel with (alpha/beta)(6) topology, which closely resembles the reported structures of radical SAM enzymes. Hence, TYW1 was considered to catalyze the cyclization reaction by utilizing the radical intermediate. Comparison with other radical SAM enzymes allowed us to build a model structure complexed with S-adenosylmethionine and two [4Fe-4S] clusters. Mutational analyses in yeast supported the validity of this complex model structure, which provides a structural insight into the radical reaction involving two [4Fe-4S] clusters to create a complex tricyclic base.
Settara C. Chandrasekharappa - One of the best experts on this subject based on the ideXlab platform.
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Structure-Function Analysis of Human TYW2 Enzyme Required for the Biosynthesis of a Highly Modified Wybutosine (yW) Base in Phenylalanine-tRNA
PloS one, 2012Co-Authors: Virginia Rodriguez, Akiko Noma, Bradley A. Carlson, Tsutomu Suzuki, Sona Vasudevan, Jeffrey E. Green, Settara C. ChandrasekharappaAbstract:Posttranscriptional modifications are critical for structure and function of tRNAs. Wybutosine (yW) and its derivatives are hyper-modified guanosines found at the position 37 of eukaryotic and archaeal tRNAPhe. TYW2 is an enzyme that catalyzes α-amino-α-carboxypropyl transfer activity at the third step of yW biogenesis. Using complementation of a ΔTYW2 strain, we demonstrate here that human TYW2 (hTYW2) is active in yeast and can synthesize the yW of yeast tRNAPhe. Structure-guided analysis identified several conserved residues in hTYW2 that interact with S-adenosyl-methionine (AdoMet), and mutation studies revealed that K225 and E265 are critical residues for the enzymatic activity. We previously reported that the human TYW2 is overexpressed in breast cancer. However, no difference in the tRNAPhe modification status was observed in either normal mouse tissue or a mouse tumor model that overexpresses Tyw2, indicating that hTYW2 may have a role in tumorigenesis unrelated to yW biogenesis.
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Abstract #2486: TRMT12 overexpression in cancer cells affects the cell cycle and the nonsense mediated decay pathways by regulating the SMG-1 kinase, a member of the PIKK family of kinases
Cancer Research, 2009Co-Authors: Virginia Rodriguez, Akiko Noma, Bradley A. Carlson, Abdel G. Elkahloun, Jeffrey Green, Tsutomu Suzuki, Settara C. ChandrasekharappaAbstract:Previously, we reported the overexpression of TRMT12 (tRNA methyltransferase 12) in breast cancer. This gene is located on chromosome 8 in the 8q24 region, an area commonly amplified in many types of cancer. The human TRMT12 gene has never been studied. TRMT12 is homologous to the yeast TYW2, a tRNA methyltransferase involved in the posttranscriptional modification that converts guanosine to Wybutosine, a highly modified base in tRNA-phenylalanine. Wybutosine is present only in tRNA-phenylalanine at the 37th position adjacent to the anticodon. This modification stabilizes the codon-anticodon interaction and functions to maintain the correct reading frame. Recently, we cloned the human TRMT12 gene and determined that TRMT12 catalyzes the third step in the biosynthetic pathway of Wybutosine. This is the first time the biological function of one of the human enzymes in the Wybutosine pathway has been demonstrated. We find that Wybutosine modification in tRNA-phenylalanine is not affected in a mouse mammary tumor model that overexpresses Trmt12. To determine the effect of TRMT12 overexpression in cancer, we evaluated the expression changes in cells treated with TRMT12 siRNAs. Results indicate that the SMG-1 gene had a >2 fold increase in expression when TRMT12 was brought down, and this was validated by RT-qPCR and Western blot analysis. SMG-1 is a critical phosphatidylinositol-3-kinase-related kinase (PIKK) that is a key player in DNA damage response and in the nonsense mediated decay pathway (NMD), a surveillance system that maintains the quality and fidelity of mRNA transcripts. Overexpression of TRMT12 results in altered expression of SMG-1, a reduction in p53 (Ser 15) phosphorylation and associated disruption to the cell cycle; overexpression of TRMT12 also results in compromising the NMD pathway. Thus, overexpression of TRMT12 in cancer is likely to disrupt SMG-1 function, which in turn may allow DNA damage to go unchecked and mutated RNA transcripts to escape degradation. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2486.
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trmt12 overexpression in breast cancer cells and its effect on the Wybutosine biosynthetic pathway
Cancer Research, 2008Co-Authors: Virginia Rodriguez, Abdel G. Elkahloun, Jeffrey E. Green, Settara C. ChandrasekharappaAbstract:2716 By combining the approaches of comparative genomic hybridization (CGH) using a high- resolution BAC array and a global expression analysis with a high density oligonucleotide array, we identified the overexpression of the TRMT12 (tRNA methyltransferase 12) gene in breast cancer cell lines. This gene is located on chromosome 8 in the 8q24 region, an area commonly amplified in many types of cancer. Seven out of eight breast cancer cell lines showed amplification at the genomic level in this region that extends from 125.5 Mb to 126.5 Mb. Within this region, the TRMT12 gene showed the highest expression across all the cell lines, and the correlation between the copy number and expression changes among the eight cell lines was significant. Validation by RT-qPCR using RNA from 30 breast tumors showed that the TRMT12 gene was overexpressed >2 fold in 87% (26/30) of the tumors. We have initiated studies to explore the consequence of overexpression of TRMT12 in cancer. The TRMT12 gene is homologous to the yeast TYW2, which is a tRNA methyltransferase that catalyzes the third step in the pathway that converts guanosine to a highly modified base, Wybutosine (yW). This five-step pathway has been described for yW biosynthesis in yeast. Wybutosine is present only in phenylalanine tRNA (tRNA-Phe) at the 37th position, adjacent to the anticodon. This modification stabilizes the codon-anticodon interaction and functions to maintain the correct reading frame. It is likely that overexpression of the TRMT12 gene in cancer cells disrupts the yW biosynthetic pathway resulting in hypomodified tRNA-Phe. RT-qPCR using RNA from normal and mouse mammary tumor tissue showed that the TRMT12 gene was overexpressed (>20 fold) in the tumors. Preliminary results suggest that the major fraction of the tRNA-Phe from tumor lacks the fully modified yW base. Studies are underway to determine the exact modification status of the tRNA-Phe in mouse tumor samples. We also evaluated the expression of genes encoding enzymes catalyzing the remaining four steps of yW biosynthesis. The genes, TRM5, TYW1 and TYW2, encoding the enzymes for the first three steps are overexpressed in tumors, and those for the last two steps were unaltered. TRMT12 expression was brought down (77-95%) using three different siRNAs in a breast cancer cell line, SKBR3; global expression changes associated with this reduction in TRMT12 expression are being evaluated.
