Biochemistry zyxwvu 1984, 23, zyxwvu 1171-1 176 1171 Evidence That the Nucleic Acid Base Queuine zyxw Is Incorporated Intact into tRNA by Animal Cellst Jon R. Katze,* Ufuk Giindiiz,t David L. Smith, Chin Shu Cheng, and James A. McCloskey ABSTRACT: Queuine (the base of queuosine, Q) catalytically reduced with tritium or deuterium yields a derivative in which the proton at C-8 (purine numbering system) has been ex- changed and the cyclopentene ring has been reduced to a cyclopentane ring. Mouse fibroblast tRNA has been labeled by culturing the cells in medium supplemented with [3H]- and [ZH]dihydroqueuine. Such tRNA yields, upon hydrolysis, the nucleoside dihydroqueuosine and a saccharide derivative of dihydroqueuosine. Each product has been identified unam- T e modified nucleoside Q' occurs exclusively in the first position of the anticodon of specific tRNAs (Kasai et al., 1975a). In mammalian cells, Q is found in tRNAHiS and tRNAh", galactosyl-Q is found in tRNATy, and mannosyl-Q is found in tRNAASp (the hexose moieties are attached to C-4 of the cyclopentenediol substituent of Q) (Kasai et al., 1976). The function of Q is unknown: long-term Q-deficient, germ- free mice appear normal (Farkas, 1980; Reyniers et al., 1981), and the only phenotypic change thus far observed in an Es- cherichia coli mutant that lacks Q in its tRNA is a marked reduction in viability in the stationary phase of growth (No- guchi et al., 1982). On the other hand, in intact Xenopus oocytes, (Q-)tRNATy from Drosophila is a suppressor of the amber (UAG) stop codon of tobacco mosaic virus RNA, while the otherwise identical Drosophilia (Q+)tRNATy is not (Bienz zyxwvut & Kubli, 1981). The appearance of Q in tRNA is unique in that it results from a base exchange: in eukaryotes, guanine in the un- modified tRNA is excised and replaced with queuine, the base of Q, to form (Q+)tRNA (Katze & Farkas, 1979; Shindo- Okada et al., 1980; Crain et al., 1980). The enzyme for this base exchange, tRNA-guanine ribosyltransferase (EC 2.4.2.29), has been purified from rabbit erythrocytes (Howes & Farkas, 1978), rat liver (Shindo-Okada et al., 1980), and wheat germ (Walden & Farkas, 1981). The E. coli enzyme differs from the eukaryotic ones in that 7-(aminomethyl)-7- deazaguanine is inserted into the tRNA (queuine is not a substrate for the bacterial enzyme), with completion of the synthesis of Q occurring at the polynucleotide level (Okada et al., 1979). In mice, however, queuine is not synthesized de novo but must be obtained from the diet or gut flora (Farkas, 1980; Reyniers et al., 1981). Significant levels of free queuine occur in plant and animal products common to the diet (Katze et al., 1982). Moreover, the ability of germ-free mice to use dietary (Q+)tRNA as a source of queuine implies a mecha- nism for the salvage of free queuine from dietary (Q+)tRNA From the Department of Microbiology and Immunology, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163 (J.R.K. and U.G.), and the Departments of Medicinal Chemistry and Biochemistry, University of Utah, Salt Lake City, Utah 841 12 (D.L.S., C.S.C., and J.A.M.). Received August 30, 1983. This work was sup- ported by National Institutes of Health Grants CA20919 (J.R.K.) and GM29812 (J.A.M.). 'Present address: Department of Biological Sciences, Middle East Technical University, Ankara, Turkey. 0006-2960/84/0423- 1 17 1$01.50/0 biguously by mass spectrometry and chromatography. Both the 3H- and *H-labeled material coeluted, and no unlabeled Q nucleoside was .found. Therefore, dihydroqueuine is in- corporated intact into tRNA in mammalian cells. Further- more, fractionation of the labeled tRNA on concanavalin Aagarose, which specifically binds the mannosyl-Q-containing tRNAASp, has shown that the zyx dihydroqueuosine-containing tRNA&P is mannosylated. This is the first direct evidence that queuine is incorporated intact into mammalian tRNA in vivo. (Reyniers et al., 1980), and the salvage of Q base subsequent to the degradation of endogenous tRNA has been demon- strated in tissue-cultured cells (Giinduz & Katze, 1982) and in vitro (Gunduz & Katze, 1984). Because queuine is incorporated intact into tRNA in vitro (Katze & Farkas, 1979; Shindo-Okada et al., 1980), it has been assumed that exogenous queuine also is incorporated intact into tRNA in vivo. However, the lack of isotopically labeled queuine has prevented a direct test of queuine incor- poration into tRNA in vivo. In the present study, two labeled queuine analogues have been synthesized and used as sub- strates for the insertion reaction in vivo. The labeled compound [ZH]dihydroqueuine, rQD3, was prepared by catalytic reduc- tion and exchange of queuine (eq 1) to provide a product 2 ; q C NH (1) r ZH1 Pt02 - z 0 CH;! YH H ~ N ~ N ~ N " y y H H~NAN H N q zyxwvutsrqponmlkjihgfedcbaZYXWV H QUEUINE rQD, having deuterium labels in both the upper and lower portions of the molecule. The radioactive analogue [ 3H]dihydro- queuine, rQT3, was prepared similarly. The mass spectrum of the isolated nucleoside from tRNA can therefore be used to indicate whether incorporation of the intact base has taken place by establishing the integrity of both the upper and lower portions of the molecule. Tissue-cultured mammalian cells readily incorporate ex- ogenous queuine into their tRNAs. The mouse fibroblast cell ' Abbreviations: queuosine or Q, 5-([(( lS,4S,SR)-4,5-dihydroxy-2- cyclopenten- l-yl)amino]methyl)-7-deazaguanosine (Nishimura, 1983), queuine is the corresponding base; rQD,, queuine reduced with deuterium in the cyclopentene ring and with H replaced by 2H at C-8 in the de- azaguanine nucleus; rQT,, tritiated derivative of queuine analogous to rQD3, above; Q*, queuosine containing galactose or mannose bound to C-4 of the cyclopentenediol substituent; (Q+)tRNA, tRNA that contains Q in the first position of the anticodon; RPC-5, reversed-phase 5 chro- matography; HPLC, high-performance liquid chromatography; AMT, N-(aminomethylene)-2,2,2-trifluoroacetamide; Me&, trimethylsilyl (TMS in structures); M, molecular ion; A2@ unit, amount of material that has an absorbance of 1.0 at 260 nm when dissolved in 1 mL of water and measured with a 1-cm light path. zyxwvutsrqponmlkjihgfedcbaZYXWVU 0 1984 American Chemical Society