Modified 5′-nucleotides resistant to 5′-nucleotidase: isolation of 3-(3-amino-3-carboxypropyl)uridine 5′-phosphate and N2,N2-dimethylguanosine 5′-phosphate from snake venom hydrolysates of transfer RNA

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Abstract
A procedure for the quantitative measurement of the O2''-methylnucleoside constituents of RNA was recently developed by the authors. This assay method is based on the resistance of O2''-methylnucleoside 5''-phosphates (pNm) (generated by phosphodiesterase hydrolysis of RNA) to subsequent dephosphorylation by venom 5''-nucleotidase (EC 3.1.3.5). In the present investigation, 2 base-modified 5''-nucleotides, each displaying an unusual resistance to 5''-nucleotidase, were identified. These compounds were characterized by a variety of techniques as N2, N2-dimethylguanosine 5''-phosphate .**GRAPHIC**. and 3-(3-amino-3-carboxypropyl)uridine 5''-phosphate (p4abu3U). Because of their resistance to 5''-nucleotidase, .**GRAPHIC**. and p4abu3U are isolated along with the pNm in the mononucleotide fraction of venom hydrolysates of transfer RNA. Under hydrolysis conditions, the stability of p4abu3U is comparable to that of a pNm, allowing quantitative assay of the nucleotide. The proportion (mean .+-. SD) of p4aub3U in venom hydrolysates of wheat embryo and Escherichia coli tRNA was determined to be 0.35 .+-. 0.03 (n = 5) and 0.14 .+-. 0.02 (n = 4) mol%, respectively. The absence of p4abu3U in venom hydrolysates of yeast tRNA implies the absence of the corresponding nucleoside in yeast tRNA, in agreement with existing data. The variable recovery of .**GRAPHIC**. from venom hydrolysates of wheat embryo and yeast tRNA indicates that under hydrolysis conditions, this base-modified nucleotide is only partially resistant to 5''-nucleotidase. The complete absence of .**GRAPHIC**. in venom hydrolysates of E. coli tRNA is consistent with the known absence of N2,N2-dimethylguanosine in this RNA. Resistance of 5''-nucleotidase is thus a necessary but not sufficient criterion for concluding that a 5''-nucleotide is O2''-methylated. When applied to wheat embryo ribosomal RNA, the analytical methods described failed to reveal any compound having the distinctive charge properties of p4abu3U. It therefore appears that 1-methyl-3(3-amino-3-carboxypropyl)pseudouridine, recently characterized as a constituent of the 18 S rRNA of Chinese hamster cells, may not be present in wheat embryo ribosomal RNA.

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