Structural analysis of O2'-methyl-5-carbamoylmethyluridine, a newly discovered constituent of yeast transfer RNA.

A compound tentatively identified as O2-methyl-5-carboxymethyluridine (cm5Um) was recently isolated in this laboratory from bulk yeast transfer RNA (Gray, M. W. (1975), Can, J. Biochem. 53, 735-746). Alkaline hydrolysis of yeast tRNA releases this nucleoside as part of an alkali-stable dinucleotide, cm5Um-Ap, from which sufficient cm5Um was prepared in the present investigation for a detailed examination of its properties. The ultraviolet absorption spectra and chromatographic and electrophoretic properties of cm5Um were consistent with the proposed structure, which was confirmed by characterization of the base and sugar moieties as 5-carboxymethyluracil and 2-O-methylribose, respectively. Snake venom hydrolysis of yeast tRNA releases cm5Um in the form of a carboxyl-blocked 5'-nucleotide, designated pU-2. Identification of the alkali-labile blocking group in pU-2 as an amide was based on quantitative assay for ammonia released upon acid hydrolysis of the corresponding nucleoside, U-2, and by chromatographic comparison of U-2 with the semisynthetic methyl ester and amide derivatives of cm5Um (mcm5Um and ncm5Um, respectively). Quantitative analysis has indicated that ncm5Um may be confined to a single species of yeast tRNA. In view of the unique localization (the "Wobble" position of the anticodon sequence) and coding properties (pairing with A but not with G) of other cm5U derivatives in transfer RNA, the dinucleotide cm5Um-Ap may be derived from the first two positions of the anticodon sequence of a yeast tRNA species recognizing an NUA codon. This predicts that O2-methyl-5-carbamoylmethyluridine will be found in an isoleucine, leucine, or valine isoacceptor.     

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.

A procedure for the quantitative measurement of the O2'-methylnucleoside constitutents 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 (pm2/2G) and 3-(3-amino-3-carboxypropyl)uridine 5'-phosphate (p4abu3U). Because of their resistance to 5'-nucleotidase, pm2/2G 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 pm2/2G from venom hydrolysates of wheat embryo and yeast tRNA indicates that under hydrolysis conditions, this base-modified nucleotide is only partially resistent to 5'-nucleotidase. The complete absence of pm2/2G 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.     

O2''-Methylinosine, a constituent of the ribosomal RNA of Crithidia fasciculata.

A novel nucleoside, O2'-methylinosine (Im), has been identified as a constituent of the ribosomal RNA of Crithidia fasciculata, a hemoflaggelate protozoan. The nucleoside is released as part of an alkali-stable dinucleotide, Im-Up, by alkaline hydrolysis of Crithidia rRNA, and as a 5'-nucleotide, pIm, by snake venom hydrolysis of the same RNA. The Im-containing derivatives isolated from Crithidia rRNA were characterized by comparison with marker compounds prepared by chemical deamination of the corresponding adenosine analogues. O2'-Methylinosine prepared from either natural Im-Up or natural pIm had the same ultraviolet absorption spectra and chromatographic properties as marker Im. Characterization of the base and sugar components of Im as hypoxanthine and 2-O-methylribose, respectively, provided final confimration of structure. Control experiments have eliminated the possibility that Im arises from O2'-methyladenosine (Am), a known constituent of ribosomal RNA, by chemical or enzymatic deamination during hydrolysis of Crithidia rRNA.     

RNA ligase in bacteria: formation of a 2',5' linkage by an E. coli extract

Ligase activity was detected in extracts of Escherichia coli, Clostridium tartarivorum, Rhodospirillum salexigens, Chromatium gracile, and Chlorobium limicola. Ligase was measured by joining of tRNA halves produced from yeast IVS-containing tRNA precursors by a yeast endonuclease. The structure of tRNATyr halves joined by an E. coli extract was examined. The ligated junction is resistant to nuclease P1 and RNAase T2 but sensitive to venom phosphodiesterase and alkaline hydrolysis, consistent with a 2',5' linkage. The nuclease-resistant junction dinucleotide comigrates with authentic (2',5') APA marker in thin-layer chromatography. The phosphate in the newly formed phosphodiester bond is derived from the pre-tRNA substrate. The widespread existence of a bacterial ligase raises the possibility of a novel class of RNA processing reactions.     

Presence of the methylester of 5-carboxymethyl uridine in the wobble position of the anticodon of tRNAIII Arg from brewer's yeast.

The methylester of 5-carboxymethyluridine (mcm5U), its degradation product 5-carboxymethyluridine (cm5U) and the corresponding nucleotide (cm5Up) were isolated from brewer's yeast tRNAIII Arg or from the dodecanucleotide containing the anticodon. Their chromatographic and electrophoretic properties and their UV absorbing spectra were identical to that of the corresponding synthetic compounds. The gas chromatographic behavior and the mass spectrum of mcm5U obtained from tRNAIII Arg and of a synthetic sample were also identical ; the rare occurence of a thermal reciprocal bimolecular methyl-hydrogen transfer in the mass spectrometer ion source was observed. A mild alkaline treatment of tRNAIII Arg leads to the saponification of mcm5U into cm5U (within the tRNA), which can be again esterified in the presence of a yeast homogenate and (methyl-14C) S adenosylmethionine. The radioactivity was found in the mcm5U located in the wobble position of the anticodon of tRNAIII Arg. The presence of this odd nucleotide in that position could possibly restrict the codon-anticodon interaction of tRNAIII Arg.     

The determination of 2'-O-methylnucleosides in RNA

A rapid and sensitive procedure is described for determining the 2′-O-methylnucleoside methylnucleoside composition of an RNA sample. The RNA is enzymatically hydrolyzed to nucleosides and the 2′-O-methylnucleoside fraction is isolated by DEAE-cellulose (borate) column chromatography. Boric acid is removed as its methyl ester and the 2′-O-methylnucleosides are resolved by liquid chromatography in the presence of ethylene glycol. The sensitivity of this method is sufficient to distinguish RNA samples which differ only 2–3% in 2′-O-methylnucleoside composition.     

Sequence of methylated nucleotides at the 5''-terminus of adenovirus-specific RNA.

RNA labeled with [methyl-3H] methionine and [14C]uridine was isolated from the cytoplasm of adenovirus-infected cells and purified by poly(U)-Sepharose chromatography and hybridization to filters containing immobilized adeovirus DNA. Analysis by dimethyl sulfoxide-sucrose gradient sedimentation suggested that the major mRNA species were methylated. 7-Methylguanosine was identified at the 5'-terminus of the advenovirus-specific RNA and could be removed by periodate oxidation and beta-elimination. Structures of the type m7G(5')ppp(5')Nm containing the unusual nucleoside N6, O2'-dimethyladenosine, and smaller amounts of 2'-O-methyladenosine were isolated by DEAE-cellulose chromatography after P1 nuclease digestion of the RNA. Evidence for some 5'-terminal sequences, m7G(5')ppp(5')m6AmpNm, with additional 2'-O-methylribonucleosides was also obtained. A base-methylated nucleoside, N6-methyladenosine, is located within the RNA chain and is released as a mononucleotide by alkali hydrolysis.     

Location of single-stranded and double-stranded regions in rat liver ribosomal 5S RNA and 5.8S RNA.

Rat liver 5S rRNA and 5.8S rRNA were end-labelled with 32P at 5'-end or 3'-end of the polynucleotide chain and partially digested with single-strand specific S1 nuclease and double-strand specific endonuclease from the cobra Naja naja oxiana venom. The parallel use of these two structure-specific enzymes in combination with rapid sequencing technique allowed the exact localization of single-stranded and double-stranded regions in 5S RNA and 5.8 S RNA. The most accessible regions to S1 nuclease in 5S RNA are regions 33-42, 74-78, 102-103 and in 5.8 S RNA 16-20, 26-29, 34-36, 74-80 and a region around 125-130. The cobra venom endonuclease cleaves the following areas in 5S RNA: 7-8, 17-20, 28-30, 49-51, 56-57, 60-64, 69-70, 81-82, 95-97, 106-112. In 5.8S RNA the venom endonuclease cleavage sites are 4-7, 10-13, 21-22, 33-35, 43-45, 51-55, 72-74, 85-87, 98-99, 105-106, 114-115, 132-135. According to these results the tRNA binding sequences proposed by Nishikawa and Takemura [(1974) FEBS Lett. 40, 106-109], in 5S RNA are located in partly single-stranded region, but in 5.8S RNA in double-stranded region.     

The ribosomal RNA of the trypanosomatid protozoan Crithidia fasciculata: physical characteristics and methylated sequences.

When extracted and analyzed under conditions which maintain noncovalently associated RNA-RNA complexes, the bulk cellular RNA of Crithidia fasciculata contains species of apparent molecular weights 1.3, 0.825, 0.08, 0.065, and 0.045 x 10(6) in addition to 5S rRNA and tRNA. Heat denaturation results in the disappearance of the 1.3 x 10(6) dalton RNA and the appearance of three new species having molecular weights of 0.67, 0.575, and 0.059 x 10(6). In addition, the apparent molecular weight of the 0.825 x 10(6) dalton component is reproducibly lowered to 0.81 x 10(6) after heat treatment. With the exception of tRNA, all of the RNA species are present in close to equimolar amounts in either undenatured or heat-denatured C. fasciculata bulk cellular RNA. On the basis of previous observations on the ribosomal RNA of the closely related organism, Crithidia oncopelti (Spencer, R. & Cross, G.A.M. (1976) J. Gen. Microbiol. 93, 82-88), the 1.3 and 0.825 x 10(6) dalton RNA's are considered to be components of the large and small subunits, respectively, of C. fasciculata ribosomes, but the subunit localization of the other RNA's described here has not yet been determined. O2'-Methylnucleosides account for about 1.4 mol% of the total nucleoside constituents of unfractionated C. fasciculata rRNA. Quantitative analysis suggests that the rRNA molecules in a C. fasciculata ribosome contain a total of 95-100 O2'-methyl groups, distributed in 80-85 Nm-Np sequences (including four 'hypermodified' Nm-Np, each containing a modification of a base or base-sugar linkage in addition to sugar methylation), six different Nm-Nm-Np sequences, and one Nm-Nm-Nm-Np sequence. While the specific pattern of O2'-methylation in the rRNA of C. fasciculata is distinct, both qualitatively and quantitatively, from the pattern observed in other organisms, Crithidia rRNA does contain certain 'universal' O2'-methylated sequences which appear to have been extensively conserved in evolution. The base-methylated nucleoside, N6,N6-dimethyladenosine (m26A), has been isolated from both C. fasciculata and wheat embryo rRNA in the form of the alkali-resistant dinucleotide, m26A-m26Ap. This dinucleotide and its enzymatic degradation products have been characterized by examination of their ultraviolet absorption spectra and electrophoretic and chromatographic properties.     

2'' phosphomonoester, 3''-5'' phosphodiester bond at a unique site in a circular viral RNA.

Solanum nodiflorum mottle virus (SNMV) RNA2 is a single-stranded, covalently closed circular molecule. RNase T2 or nuclease P1 digests of this RNA contain a minor nucleotide of unusual chromatographic and electrophoretic mobility. This nucleotide is resistant to further digestion by T2 or P1 ribonucleases, or by alkali, but is sensitive to venom phosphodiesterase digestion. Alkaline phosphatase digestion yields a product which is RNase T2 and P1 sensitive. The products of these various digests show that the minor nucleotide is a ribonuclease-resistant dinucleotide carrying a 2' phosphomonoester group with the core structure C2'p3'p5'A. This dinucleotide is found in a unique RNase T1 product of SNMV RNA2, thus establishing a unique location in the sequence for the 2' phosphomonoester group at residue 49. Identical results have been obtained with a second related virus. The phosphomonoester group probably results from the RNA ligation event by which the molecules were circularised.     

Replacement of RNA hairpins by in vitro selected tetranucleotides.

An in vitro selection method based on the autolytic cleavage of yeast tRNA(Phe) by Pb2+ was applied to obtain tRNA derivatives with the anticodon hairpin replaced by four single-stranded nucleotides. Based on the rates of the site-specific cleavage by Pb2+ and the presence of a specific UV-induced crosslink, certain tetranucleotide sequences allow proper folding of the rest of the tRNA molecule, whereas others do not. One such successful tetramer sequence was also used to replace the acceptor stem of yeast tRNA(Phe) and the anticodon hairpin of E.coli tRNA(Phe) without disrupting folding. These experiments suggest that certain tetramers may be able to replace structurally nonessential hairpins in any RNA.     

Hydrolysis of RNA to 5′-nucleotide by seed sprouts,particularly malt sprouts

Conditions for the efficient conversion of commercial RNA to nucleoside 5′-monophosphate by means of a phosphodiesterase in malt sprouts have been determined. A comparison of the enzyme content of the rootlets, stems, and kernels of various plant seedlings, including barley, rye, oat, wheat, rice, and beans shows maximum amounts in the rootlets, and minimum quantities in the ungerminated kernels. Of all the seedlings tested, (mung bean, soy bean, oat, wheat, rice, barley) barley gave the highest conversion of RNA to 5′-nucleotides. Commercial malt sprouts prepared from 6 different malted barleys including 2-rowed and 6-rowed samples all showed about the same amount of phosphodiesterase content. Besides phosphodiesterase, other enzymes capable of hydrolyzing RNA and 5′-nucleotides were found in sprouts. These included 3′-phosphodiesterases, 5′-nucleotidases, and nucleosidases. By carefully pretreating both extracts and the solid sprouts at elevated temperatures for a limited time and by the addition of minimum amounts of Zn⁺², the action of these undesirable enzymes was either effectively destroyed or minimized so that the production of 5′-nucleotides was maximized. It was found that suspensions of appropriately washed and treated barley malt rootlets are substantially more effective than aqueous extracts for converting RNA to 5′-nucleotides.     

An assay to determine the kinetics of RNA cleavage

To evaluate some synthetic catalysts that mimic ribonuclease, a quantitative assay has been developed that measures the number of phosphate diester bonds cleaved in a polymeric RNA substrate. This assay involves determining the number of 5'-oligonucleotide termini produced during the cleavage, using polyuridylic acid as the substrate. Samples withdrawn from the kinetic run are treated with venom exonuclease (phosphodiesterase I), and the increase in the concentration of uridine is determined by high-performance liquid chromatography. A related assay has been developed to monitor the catalyzed cleavage of the dinucleotide uridylyl(3'----5') uridine (UpU).     

Hypermodified alkali-stable dinucleotide sequences in each of the high-molecular-weight (26S and 18S) ribosomal RNA species of wheat.

Two hypermodified, alkali-stable dinucleotide sequences, each containing a base modification in addition to sugar methylation, are known to be present in wheat embryo 26S + 18S rRNA (Gray, M.W. (1974) Biochemistry 13, 5453-5463). Quantitative analysis of unfractionated 26S + 18S rRNA had suggested that each of these sequences (Cm-psi p and psi m-Ap, where Cm=O2'-methylcytidine and psi m-O2'-methylpseudouridine) was present in either the 18S or the 26S rRNA species, but not the both, at a frequency of not more than once per chain. In the study reported here, the individual 32P-labeled 18S and 26S rRNA species were isolated from viable wheat embryos germinated in the presence of [32P]orthophosphate. From analyses of phosphodiesterase and alkaline hydrolysates of the separated [32P]RNAs, we conclude that psi m-Ap is confined to wheat cytosol 18S rRNA, whereas Cm-psi p is localized in wheat cytosol 26S rRNA. The presence of psi m in the 18S rRNA of wheat stands in contrast with the situation in animal cells, where this hypermodified nucleoside is located in the 28S rRNA (Khan, M.S.N. & Maden, B.E.H. (1976) J. Mol. Biol. 101, 235-254).