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

Abstract

A procedure for the quantitative measurement of the O2′-methylnucleoside constituents of RNA has recently been developed in this laboratory (Gray, M. W. Can. J. Biochem. 53,735–746 (1975)). 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, two base-modified 5′-nucleotides, each displaying an unusual resistance to 5′-nucleotidase, have been identified. These compounds have been characterized by a variety of techniques as N2,N2-dimethylguanosine 5′-phosphate [Formula: see text] and 3-(3-amino-3-carboxypropyl)uridine 5′-phosphate (p4abu3U). Because of their resistance to 5′-nucleotidase, [Formula: see text] 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 p4abu3U in venom hydrolysates of wheat embryo and Escherichia coli tRNA has been 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 [Formula: see text] 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[Formula: see text] in venom hydrolysates of E. coli tRNA is consistent with the known absence of N2,N2-dimethylguanosine in this RNA. These observations demonstrate that resistance to 5′-nucleotidase is 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 in this report 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 (Saponara, A. G. &Enger, M. D. Biochim. Biophys. Acta 349,61–77 (1974)), may not be present in wheat embryo ribosomal RNA.