Ribonucleoside analysis by reversed-phase high-performance liquid chromatography

Gehrke, Charles W.; Kuo, Kenneth C.

doi:10.1016/s0021-9673(00)94152-9

Cited by 213 publications

(210 citation statements)

References 30 publications

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“…As shown in Figure 2B-D, each of the three individual tRNAs known to have a 29-Omethylated residue at position 4 has the expected 29-Omethylated nucleoside (Cm for tRNA Pro and tRNA Gly and Am for tRNA His ), as measured by appearance of a peak in the expected location (Gehrke and Kuo 1989) and by the UV absorption spectrum of the corresponding peak. By contrast, each of the corresponding tRNAs from the trm13-D strain lack the 29-O-methylated residues (Fig.…”

Section: Resultsmentioning

confidence: 92%

“…To analyze the individual nucleoside content of each tRNA species, purified tRNA Gly(GCC) , tRNA Pro , or tRNA His isolated from yeast (1 mg) or in vitro transcribed tRNA Gly(GCC) (50 mg) incubated with Trm13 protein were treated with 5-10 mg of P1 nuclease at 37°C for 16 h, and then with 8 U of calf intestinal alkaline phosphatase at 37°C for 3 h. Nucleosides were resolved by HPLC (Waters Alliance Model 2690, equipped with Waters 996 photodiode array detector) on a reverse phase C18 column as described (Gehrke and Kuo 1989), and modified nucleosides were quantified as described Xing et al 2004), using either known extinction coefficients for the modified nucleosides where available (C, m 5 C, m 1 G, DHU, and T) or using the corresponding extinction coefficients for the unmodified major nucleosides for the species for which this information is not available (Cm, Am).…”

Section: Hplc Analysis Of Nucleosidesmentioning

confidence: 99%

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The 2′-O-methyltransferase responsible for modification of yeast tRNA at position 4

Wilkinson¹,

Crary²,

Jackman³

et al. 2007

RNA

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The methylation of the ribose 29-OH of RNA occurs widely in nature and in all stable RNAs and occurs at five positions in yeast tRNA. 29-O-methylation of tRNA at position 4 is interesting because it occurs in the acceptor stem (which is normally undermodified), it is the only 29-O-methylation that occurs in the middle of a duplex region in tRNA, the modification is conserved in eukaryotes, and the features of the tRNA necessary for substrate recognition are poorly defined. We show here that Saccharomyces cerevisiae ORF YOL125w (TRM13) is necessary and sufficient for 29-O-methylation at position 4 of yeast tRNA. Biochemical analysis of the S. cerevisiae proteome shows that Trm13 copurifies with 29-O-methylation activity, using tRNA Gly(GCC) as a substrate, and extracts made from a trm13-D strain have undetectable levels of this activity. Trm13 is necessary for activity in vivo because tRNAs isolated from a trm13-D strain lack the corresponding 29-O-methylated residue for each of the three known tRNAs with this modification. Trm13 is sufficient for 29-O-methylation at position 4 in vitro since yeast Trm13 protein purified after expression in Escherichia coli has the same activity as that produced in yeast. Trm13 protein binds substrates tRNA His and tRNA Gly(GCC) with K D values of 85 6 8 and 100 6 14 nM, respectively, and has a K M for tRNA His of 10 nM, but binds nonsubstrate tRNAs very poorly (K D > 1 mM). Trm13 is conserved in eukaryotes, but there is no sequence similarity between Trm13 and other known methyltransferases.

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Section: Resultsmentioning

confidence: 92%

Section: Hplc Analysis Of Nucleosidesmentioning

confidence: 99%

The 2′-O-methyltransferase responsible for modification of yeast tRNA at position 4

Wilkinson¹,

Crary²,

Jackman³

et al. 2007

RNA

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“…Analysis of nucleosides by reverse-phase HPLC was performed as described [11]. The nucleosides were identified according to their UV spectra, relative retention times, and by comparison with appropriate controls, including synthetic markers [7,9,26]. …”

Section: Methodsmentioning

confidence: 99%

Bacillus subtilis exhibits MnmC-like tRNA modification activities

et al. 2018

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The MnmE-MnmG complex of Escherichia coli uses either ammonium or glycine as a substrate to incorporate the 5-aminomethyl or 5-carboxymethylaminomethyl group into the wobble uridine of certain tRNAs. Both modifications can be converted into a 5-methylaminomethyl group by the independent oxidoreductase and methyltransferase activities of MnmC, which respectively reside in the MnmC(o) and MnmC(m) domains of this bifunctional enzyme. MnmE and MnmG, but not MnmC, are evolutionarily conserved. Bacillus subtilis lacks genes encoding MnmC(o) and/or MnmC(m) homologs. The glycine pathway has been considered predominant in this typical gram-positive species because only the 5-carboxymethylaminomethyl group has been detected in tRNALysUUU and bulk tRNA to date. Here, we show that the 5-methylaminomethyl modification is prevalent in B. subtilis tRNAGlnUUG and tRNAGluUUC. Our data indicate that B. subtilis has evolved MnmC(o)- and MnmC(m)-like activities that reside in non MnmC homologous protein(s), which suggests that both activities provide some sort of biological advantage.

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“…Methods like high-performance liquid chromatography (HPLC) with UV detection [16,17] and capillary electrophoresis (CE) [18] have been applied for the analysis of ribonucleosides. Recently, the coupling of HPLC with mass spectrometric detection via electrospray ionization ion trap mass spectrometry (ESI-IT MS) [15,19], ESI tandem MS [20,21], and fast atom bombardment (FAB) [22,23] has been established.…”

mentioning

confidence: 99%

Identification of urinary modified nucleosides and ribosylated metabolites in humans via combined ESI-FTICR MS and ESI-IT MS analysis

Bullinger

Fux

Nicholson

et al. 2008

J. Am. Soc. Mass Spectrom.

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The physiological response of the human body to several diseases can be reflected by the metabolite pattern in biological fluids. Cancer, like other diseases accompanied by metabolic disorders, causes characteristic effects on cell turnover rate, activity of modifying enzymes, and RNA/DNA modifications. This results in an altered excretion of modified nucleosides and biochemically related compounds. In the course of our metabolic profiling project, we screened 24-h urine of patients suffering from lung, rectal, or head and neck cancer for previously unknown ribosylated metabolites. Therefore, we developed a sample preparation procedure based on boronate affinity chromatography followed by additional prepurification with preparative TLC. The isolated metabolites were analyzed by ion trap mass spectrometry (IT MS) and Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). IT MS was applied for LC-auto MS 3 screening runs and Msn(n = 4-6) syringe pump infusion experiments, yielding characteristic fragmentation patterns. FTICR MS measurements enabled the calculation of corresponding molecular formulae based on accurate mass determination (mass accuracy: 1-5 ppm for external and sub-ppm values for internal calibration).We were able to identify 22 metabolites deriving from cellular RNA metabolism and related metabolic pathways like histidine metabolism, purine biosynthesis, methionine/polyamine cycle, and nicotinate/nicotinamide metabolism. The compounds l-ribosyl-3-hydroxypyridinium, 1-ribosyl-pyridinium, and 3-ribosyl-1-methyl-Lhistidinium as well as a series of ribosylated histamines, conjugated to carboxylic acids at the N'

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Ribonucleoside analysis by reversed-phase high-performance liquid chromatography

Cited by 213 publications

References 30 publications

The 2′-O-methyltransferase responsible for modification of yeast tRNA at position 4

The 2′-O-methyltransferase responsible for modification of yeast tRNA at position 4

Bacillus subtilis exhibits MnmC-like tRNA modification activities

Identification of urinary modified nucleosides and ribosylated metabolites in humans via combined ESI-FTICR MS and ESI-IT MS analysis

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