Tandem Mass Spectrometry for Structure Assignments of Wye Nucleosides from Transfer RNA

Zhou, Shaolian; Sitaramaiah, Devarasetty; Pomerantz, Steven C.; Crain, Pamela F.; McCloskey, James A.

doi:10.1081/ncn-120027816

Cited by 12 publications

(10 citation statements)

References 20 publications

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“…The protonated base fragment (BH 2 + ) of the each tRNA was selected as a parent mass and decomposed in collision with helium gas to obtain the spectrum of product ions (Supplementary Figure S3). Most of our data could be interpreted by referring to the CID spectra for chemically synthesized yW derivatives, part of the pioneering work by J McCloskey's group (Zhou et al , 2004a, 2004b). Interpretation of each CID spectrum and determination of chemical structure of each yW intermediate are described in the legend for Supplementary Figure S3.…”

Section: Resultsmentioning

confidence: 99%

Biosynthesis of wybutosine, a hyper-modified nucleoside in eukaryotic phenylalanine tRNA

Noma

Kirino

Ikeuchi

et al. 2006

EMBO J

190

254

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Wybutosine (yW) is a tricyclic nucleoside with a large side chain found at the 3 0 -position adjacent to the anticodon of eukaryotic phenylalanine tRNA. yW supports codon recognition by stabilizing codon-anticodon interactions during decoding on the ribosome. To identify genes responsible for yW synthesis from uncharacterized genes of Saccharomyces cerevisiae, we employed a systematic reverse genetic approach combined with mass spectrometry ('ribonucleome analysis'). Four genes YPL207w, YML005w, YGL050w and YOL141w (named TYW1, TYW2, TYW3 and TYW4, respectively) were essential for yW synthesis. Mass spectrometric analysis of each modification intermediate of yW revealed its sequential biosynthetic pathway. TYW1 is an iron-sulfur (Fe-S) cluster protein responsible for the tricyclic formation. Multistep enzymatic formation of yW from yW-187 could be reconstituted in vitro using recombinant TYW2, TYW3 and TYW4 with S-adenosylmethionine, suggesting that yW synthesis might proceed through sequential reactions in a complex formed by multiple components assembled with the precursor tRNA. This hypothesis is also supported by the fact that plant ortholog is a large fusion protein consisting of TYW2 and TYW3 with the C-terminal domain of TYW4.

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Section: Resultsmentioning

confidence: 99%

Biosynthesis of wybutosine, a hyper-modified nucleoside in eukaryotic phenylalanine tRNA

Noma

Kirino

Ikeuchi

et al. 2006

EMBO J

190

254

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“…After thin layer chromatography (Fig. 3), a single fluorescent spot (R F ϭ 0.47) 5 was detected in extracts prepared from the wild-type strain, and mass spectrometric analysis of this fluorescent material gave a mass of 377.1 Da corresponding to that of yW (57). Extracts from the ⌬YPL207w deletion strain lacked the fluorescent material.…”

Section: Identification Of Cog0731 As a Probable Wyebutosine Synthesimentioning

confidence: 99%

Discovery of a Gene Family Critical to Wyosine Base Formation in a Subset of Phenylalanine-specific Transfer RNAs

Waas

Crécy‐Lagard

Schimmel

2005

Journal of Biological Chemistry

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In all organisms, the functions of tRNAs in translation are enhanced by a series of post-transcriptional modifications (4). Over 80 modifications are known, and their presence vastly expands the structural and chemical diversity of native tRNA (2). (In a putative RNA world, base modifications may have provided a way to diversify the chemical and structural properties of RNAs.) The modification-dependent structural stability and function correlate with increased cellular fitness and viability (5, 9). The importance of these modifications is underscored by a large investment of resources for their biosynthesis, with estimates suggesting nearly 1% of some bacterial genomes being dedicated to tRNA modification genes (4).Wyebutosine (yW) 3 of yeast phenylalanine-specific tRNA (tRNA Phe )was one of the earliest tRNA modifications to be discovered (10 -14).Wyebutosine is a fluorescent, tricyclic base and a member of the wyosine family of hypermodified guanosines. All wyosine bases (Ybs) are characterized by a 1H-imidazo[1,2-␣]purine core structure and a strict occurrence in archaeal and eukaryal tRNA Phe ( Figs. 1 and 2). Wyosine bases, isolated from different organisms, show variations in ring methylation and side chain structure (15)(16)(17)(18)(19)(20). Generally, archaeal Yb structures are less differentiated then their eukaryotic counterparts.The hydrophobic nature of yW 37 promotes stacking with adjacent bases (A 36 and A 38 ) and restricts the conformational flexibility of the anticodon (21-25). Removal of yW produced local changes in anticodon conformation, as well as long range perturbations in tRNA Phe tertiary structure (26). These structural changes were accompanied by subtle differences in codon specificity 4 and a modest increase in retroviral ribosomal frameshifting (determined in cell-free extract) (27-29). Most interesting, the tRNA Phe from mouse neuroblastoma cell lacked Yb but was more efficient than the fully modified tRNA Phe in a cell-free translation system (30 -32). Although it is unclear if hypomodified tRNAs contribute to tumor-specific properties, these tRNAs support the high levels of translation required by rapidly dividing cells. Thus, despite a good understanding of its role in maintaining anticodon structure, the function of yW in translation is unclear.Although the biosynthesis of wyebutosine has been partially characterized, the genes involved are largely unknown. Several structural components of yW were identified by metabolic labeling experiments. The purine substructure was shown as being derived from the coded guanosine (33,34). NMR studies of 13 C-enriched tRNA Phe implicated the methyl group of methionine as a source for carbon-10 (refer to Fig. 1 for numbering), and for the side chain ester and N 3 -methyl moieties (35). Conflicting evidence obscures understanding the origin of the 3-amino-3-carboxypropyl side chain (36, 37). The in vivo kinetics of Yb biosynthesis of Xenopus laevis were investigated (34). By using the sitespecifically labeled [ 32 P]tRNA Phe transcript in X. ...

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“…The authors also observed facile loss of a CH 3 -radical from other protonated wye bases and explained this uncommon loss by charge stabilization and radical delocalization through the nitrogen-rich tricyclic ring [45].…”

Section: Resultsmentioning

confidence: 99%

A Nano-Chip-LC/MSⁿ Based Strategy for Characterization of Modified Nucleosides Using Reduced Porous Graphitic Carbon as a Stationary Phase

Giessing

Scott

Kirpekar

2011

J. Am. Soc. Mass Spectrom.

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LC/MS analysis of ribonucleosides is traditionally performed by reverse phase chromatography on silica based C18 type stationary phases using MS compatible buffers and methanol or acetonitrile gradients. Due to the hydrophilic and polar nature of nucleosides, down-scaling C18 analytical methods to a two-column nano-flow setup is inherently difficult. We present a nanochip LC/MS ion-trap strategy for routine characterization of RNA nucleosides in the fmol range. Nucleosides were analyzed in positive ion mode by reverse phase chromatography using a 75 μ×150 mm, 5 μ particle porous graphitic carbon (PGC) chip with an integrated 9 mm, 160 nL trapping column. Nucleosides were separated using a formic acid/acetonitrile gradient. The method was able to separate isobaric nucleosides as well as nucleosides with isotopic overlap to allow unambiguous MS n identification on a low resolution ion-trap. Synthesis of 5-hydroxycytidine (oh 5 C) was achieved from 5-hydroxyuracil in a novel three-step enzymatic process. When operated in its native state using formic acid/acetonitrile, PGC oxidized oh 5 C to its corresponding glycols and formic acid conjugates. Reduction of the PGC stationary phase was achieved by flushing the chip with 2.5 mM oxalic acid and adding 1 mM oxalic acid to the online solvents. Analyzed under reduced chromatographic conditions oh 5 C was readily identified by its MH + m/z 260 and MS n fragmentation pattern. This investigation is, to our knowledge, the first instance where oxalic acid has been used as an online reducing agent for LC/MS. The method was subsequently used for complete characterization of nucleosides found in tRNAs using both PGC and C18 chips.

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Tandem Mass Spectrometry for Structure Assignments of Wye Nucleosides from Transfer RNA

Cited by 12 publications

References 20 publications

Biosynthesis of wybutosine, a hyper-modified nucleoside in eukaryotic phenylalanine tRNA

Biosynthesis of wybutosine, a hyper-modified nucleoside in eukaryotic phenylalanine tRNA

Discovery of a Gene Family Critical to Wyosine Base Formation in a Subset of Phenylalanine-specific Transfer RNAs

A Nano-Chip-LC/MSⁿ Based Strategy for Characterization of Modified Nucleosides Using Reduced Porous Graphitic Carbon as a Stationary Phase

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Tandem Mass Spectrometry for Structure Assignments of Wye Nucleosides from Transfer RNA

Cited by 12 publications

References 20 publications

Biosynthesis of wybutosine, a hyper-modified nucleoside in eukaryotic phenylalanine tRNA

Biosynthesis of wybutosine, a hyper-modified nucleoside in eukaryotic phenylalanine tRNA

Discovery of a Gene Family Critical to Wyosine Base Formation in a Subset of Phenylalanine-specific Transfer RNAs

A Nano-Chip-LC/MSn Based Strategy for Characterization of Modified Nucleosides Using Reduced Porous Graphitic Carbon as a Stationary Phase

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A Nano-Chip-LC/MSⁿ Based Strategy for Characterization of Modified Nucleosides Using Reduced Porous Graphitic Carbon as a Stationary Phase