Penultimate Nucleoside Research Articles

Synthesis of ABBV-168, a 2'-Bromouridine for the Treatment of Hepatitis C.

ABBV-168 is a dihalogenated nucleotide under investigation for the treatment of hepatitis C virus. Three synthetic routes aimed at achieving the stereoselective installation of the C2' gem-Br,F substitution and subsequent Vorbruggen glycosylation were explored to prepare the penultimate nucleoside intermediate. Development culminated in a route to ABBV-168 featuring a de novo chromatography-free furanose synthesis, protecting group-directed Vorbruggen glycosylation, and highly selective phosphoramidation to furnish the API.

Cleavage of 3\u2032-terminal adenosine by archaeal ATP-dependent RNA ligase

Methanothermobacter thermoautotrophicus RNA ligase (MthRnl) catalyzes formation of phosphodiester bonds between the 5′-phosphate and 3′-hydroxyl termini of single-stranded RNAs. It can also react with RNA with a 3′-phosphate end to generate a 2′,3′-cyclic phosphate. Here, we show that MthRnl can additionally remove adenosine from the 3′-terminus of the RNA to produce 3′-deadenylated RNA, RNA(3′-rA). This 3′-deadenylation activity is metal-dependent and requires a 2′-hydroxyl at both the terminal adenosine and the penultimate nucleoside. Residues that contact the ATP/AMP in the MthRnl crystal structures are essential for the 3′-deadenylation activity, suggesting that 3′-adenosine may occupy the ATP-binding pocket. The 3′-end of cleaved RNA(3′-rA) consists of 2′,3′-cyclic phosphate which protects RNA(3′-rA) from ligation and further deadenylation. These findings suggest that ATP-dependent RNA ligase may act on a specific set of 3′-adenylated RNAs to regulate their processing and downstream biological events.

Structures of bacterial polynucleotide kinase in a michaelis complex with nucleoside triphosphate (NTP)-Mg2+ and 5'-OH RNA and a mixed substrate-product complex with NTP-Mg2+ and a 5'-phosphorylated oligonucleotide.

Clostridium thermocellum polynucleotide kinase (CthPnk), the 5'-end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from a nucleoside triphosphate (NTP) donor to a 5'-OH polynucleotide acceptor, either DNA or RNA. Here we report the 1.5-Å crystal structure of CthPnk-D38N in a Michaelis complex with GTP-Mg(2+) and a 5'-OH RNA oligonucleotide. The RNA-binding mode of CthPnk is different from that of the metazoan RNA kinase Clp1. CthPnk makes hydrogen bonds to the ribose 2'-hydroxyls of the 5' terminal nucleoside, via Gln51, and the penultimate nucleoside, via Gln83. The 5'-terminal nucleobase is sandwiched by Gln51 and Val129. Mutating Gln51 or Val129 to alanine reduced kinase specific activity 3-fold. Ser37 and Thr80 donate functionally redundant hydrogen bonds to the terminal phosphodiester; a S37A-T80A double mutation reduced kinase activity 50-fold. Crystallization of catalytically active CthPnk with GTP-Mg(2+) and a 5'-OH DNA yielded a mixed substrate-product complex with GTP-Mg(2+) and 5'-PO4 DNA, wherein the product 5' phosphate group is displaced by the NTP γ phosphate and the local architecture of the acceptor site is perturbed.

Structure-Function Analysis of the OB and Latch Domains of Chlorella Virus DNA Ligase

Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5'-phosphate nucleotide and the 3'-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps.

Structure-guided Mutational Analysis of the OB, HhH, and BRCT Domains of Escherichia coli DNA Ligase

NAD(+)-dependent DNA ligases (LigAs) are ubiquitous in bacteria and essential for growth. LigA enzymes have a modular structure in which a central catalytic core composed of nucleotidyltransferase and oligonucleotide-binding (OB) domains is linked via a tetracysteine zinc finger to distal helix-hairpin-helix (HhH) and BRCT (BRCA1-like C-terminal) domains. The OB and HhH domains contribute prominently to the protein clamp formed by LigA around nicked duplex DNA. Here we conducted a structure-function analysis of the OB and HhH domains of Escherichia coli LigA by alanine scanning and conservative substitutions, entailing 43 mutations at 22 amino acids. We thereby identified essential functional groups in the OB domain that engage the DNA phosphodiester backbone flanking the nick (Arg(333)); penetrate the minor grove and distort the nick (Val(383) and Ile(384)); or stabilize the OB fold (Arg(379)). The essential constituents of the HhH domain include: four glycines (Gly(455), Gly(489), Gly(521), Gly(553)), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg(487), which penetrates the minor groove at the outer margin on the 3 (R)-OH side of the nick; and Arg(446), which promotes protein clamp formation via contacts to the nucleotidyltransferase domain. We find that the BRCT domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation.

Bacterial Nonhomologous End Joining Ligases Preferentially Seal Breaks with a 3′-OH Monoribonucleotide

Many bacterial species have a nonhomologous end joining system of DNA repair driven by dedicated DNA ligases (LigD and LigC). LigD is a multifunctional enzyme composed of a ligase domain fused to two other catalytic modules: a polymerase that preferentially adds ribonucleotides to double-strand break ends and a phosphoesterase that trims 3'-oligoribonucleotide tracts until only a single 3'-ribonucleotide remains. LigD and LigC have a feeble capacity to seal 3'-OH/5'-PO(4) DNA nicks. Here, we report that nick sealing by LigD and LigC enzymes is stimulated by the presence of a single ribonucleotide at the broken 3'-OH end. The ribonucleotide effect on LigD and LigC is specific for the 3'-terminal nucleotide and is either diminished or abolished when additional vicinal ribonucleotides are present. No such 3'-ribonucleotide effect is observed for bacterial LigA or Chlorella virus ligase. We found that in vitro repair of a double-strand break by Pseudomonas LigD requires the polymerase module and results in incorporation of an alkali-labile ribonucleotide at the repair junction. These results illuminate an underlying logic for the domain organization of LigD, whereby the polymerase and phosphoesterase domains can heal the broken 3'-end to generate the monoribonucleotide terminus favored by the nonhomologous end joining ligases.

Substrate Specificity and Structure-Function Analysis of the 3′-Phosphoesterase Component of the Bacterial NHEJ Protein, DNA Ligase D

DNA ligase D (LigD) performs end remodeling and end sealing reactions during nonhomologous end joining in bacteria. Pseudomonas aeruginosa LigD consists of a central ATP-dependent ligase domain fused to a C-terminal polymerase domain and an N-terminal phosphoesterase (PE) module. The PE domain catalyzes manganese-dependent phosphodiesterase and phosphomonoesterase reactions at the 3' end of the primer strand of a primer-template. The phosphodiesterase cleaves a 3'-terminal diribonucleotide to yield a primer strand with a ribonucleoside 3'-PO4 terminus. The phosphomonoesterase converts a terminal ribonucleoside 3'-PO4 or deoxyribonucleoside 3'-PO4 of a primer-template to a 3'-OH. Here we report that the phosphodiesterase and phosphomonoesterase activities are both dependent on the presence and length of the 5' single-strand tail of the primer-template substrate. Although the phosphodiesterase activity is strictly dependent on the 2'-OH of the penultimate ribose, it is indifferent to a 2'-OH versus a2'-H on the terminal nucleoside. Incision at the ribonucleotide linkage is suppressed when the 2'-OH is moved by 1 nucleotide in the 5' direction, suggesting that LigD is an exoribonuclease that cleaves the 3'-terminal phosphodiester. We report the effects of conservative amino acid substitutions at residues: (i) His42, His48, Asp50, Arg52, His84, and Tyr88, which are essential for both the ribonuclease and 3'-phosphatase activities; (ii) Arg14, Asp15, Glu21, and Glu82, which are critical for 3'-phosphatase activity but not 3'-ribonucleoside removal; and (iii) at Lys66 and Arg76, which participate selectively in the 3'-ribonuclease reaction. The results suggest roles for individual functional groups in metal binding and/or phosphoesterase chemistry.

Novel 3′-Ribonuclease and 3′-Phosphatase Activities of the Bacterial Non-homologous End-joining Protein, DNA Ligase D

Pseudomonas aeruginosa DNA ligase D (PaeLigD) exemplifies a family of bacterial DNA end-joining proteins that consist of a ligase domain fused to a polymerase domain and a putative nuclease module. The LigD polymerase preferentially adds single ribonucleotides at blunt DNA ends and, as we show here, is also capable of adding up to 4 ribonucleotides to a DNA primer-template. We report that PaeLigD has an intrinsic ability to resect the short tract of 3'-ribonucleotides of a primer-template substrate to the point at which the primer strand has a single 3'-ribonucleotide remaining. The failure to digest beyond this point reflects a requirement for a 2'-OH group on the penultimate nucleoside of the primer strand. Replacing the 2'-OH by a 2'-F, 2'-NH2, 2'-OCH3, or 2'-H abolishes the resection reaction. The ribonucleotide resection activity resides within a 187-amino acid N-terminal nuclease domain and is the result of at least two component steps: (i) the 3'-terminal nucleoside is first removed to yield a primer strand with a ribonucleoside 3'-PO4 terminus, and (ii) the 3'-PO4 is hydrolyzed to a 3'-OH. The 3'-ribonuclease and 3'-phosphatase activities are both dependent on a divalent cation, specifically manganese. PaeLigD preferentially remodels the 3'-ends of a duplex primer-template substrate rather than a single strand of identical composition, and it prefers DNA primer strands containing a short 3'-ribonucleotide tract to an all-RNA primer. The nuclease domain of PaeLigD and its bacterial homologs has no apparent structural or mechanistic similarity to previously characterized nucleases. Thus, we surmise that it exemplifies a novel phosphoesterase family, defined in part by conserved residues Asp-50, Arg-52, and His-84, which are essential for the 3'-ribonuclease and 3'-phosphatase reactions.

An approach to inhibition of viral replication: inhibition of mRNA methylation.

Several eukaryotic viral and cellular mRNAs contain at their 5′ terminus blocked methylated structures (often referred to as a “cap”) of the type m7GpppN′mpN″mp...(Fig. 1). Additional methylation of several mRNAs occurs internally between their 5′ cap and the 3′-poly(A) end, yielding 6-methyladenosine or 5-methylcytosine (Shatkin 1976). The 5′-terminal 7-methylguanosine in mRNAs is apparently required for their efficient translation in vitro (Both et al. 1975; Muthukrishnan 1978), and is also implicated in protection of mRNA against degradation by exonucleases. The enzymatic mechanisms involved in the synthesis of the cap structure have been worked out in detail by Moss and his co-workers with vaccinia virus (Moss et al. 1976) and by others (Furuichi et al. 1976). It involves (a) the transfer of a GMP residue from GTP to the 5′-diphosphate end of mRNA by GTP:mRNA guanylyltransferase to form the blocked structure GpppN..., (b) methylation of the 5′-terminal guanosine in the 7 position by mRNA (gua-nine-7-)-methyltransferase to form m7GpppN . . ., and (c) subsequent methylation of the penultimate nucleoside by mRNA(nucleoside-2′-)-methyltransferase to form m7GpppNm. All three enzymatic activities have been purified from vaccinia virus.KeywordsVaccinia VirusPolio VirusGuanine MethylatedVaccinia mRNABroad Spectrum Antiviral AgentThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Inhibition of vaccinia mRNA methylation by 2',5'-linked oligo(adenylic acid) triphosphate.

Extracts of interferon-treated cells synthesize unique 2',5'-linked oligo(adenylic acid) 5'-phosphates in the presence of ATP and double-stranded RNA. 2',5'-linked oligo(adenylic acid) 5'-triphosphate inhibits protein synthesis at nanomolar concentrations by activating RNase. We have observed that oligo(adenylic acid) 5'-monophosphate and 5'-triphosphate are potent inhibitors of vaccinia mRNA methylation in vitro. Both the methylation of mRNA methylation is not due to degradation of the mRNA. Inhibition of the 5'-terminal guanine at the 7 position and the 2'-O-ribose methylation of the penultimate nucleoside are inhibited. Such inhibition of the requisite modification of the 5' terminus of mRNA by 2',5'-linked oligo(adenylic acids) may be a mechanism of interferon action against both DNA and RNA viruses in which mRNAs derived from them are capped.

Post-transcriptional modifications of oat coleoptile ribonucleic acids. 5'-Terminal capping and methylation of internal nucleosides in poly(A)-rich RNA.

Evidence is presented for the occurrence of 5'-terminal capping structures in poly(A)-rich RNAs from oat coleoptile tissue. Similar to the cap structures in mRNA from other eukaryotic organisms, the 5' terminus of these oat coleoptile RNA molecules consists of 7-methylguanosine joined 5' to 5' with the adjacent (penultimate) nucleoside by means of three phosphate groups in two pyrophosphate linkages. The penultimate nucleoside contains primarily purine bases, but small amounts of pyrimidines (cytidine) are also detectable. Some monophosphorylated 5'-termini were also detected, however, they appear to occur as a result of RNA degradation. In addition to the 5 cap, oat RNA molecules are also post-transcriptionally modified with a low frequency of N6-methylations of internal adenosines.

Methylated 5′-terminal caps of adenovirus type 2 early mRNA: Evidence for at least six 5′-termini

Abstract The 5′-terminal caps of early adenovirus (Ad) 2 mRNA were isolated, fractionated, and compositionally analyzed. Cultured human KB cells were labeled with l -[ methyl - 3 H]methi-onine and 32 Pi from 2 to 7 hr postinfection in the presence of cycloheximide. Labeled RNA was extracted from polysomes and fractionated into poly(A) + and poly(A) − molecules. Viral mRNA was isolated by hybridization to Ad2 DNA, then digested with RNase T2, and cap 1 [m 7 G(5′)ppp(5′)N m pNp] and cap 2 [m 7 G(5′)ppp(5')N m pN m pNp] fractions were isolated by DEAE-Sephadex chromatography in 7 M urea. m 7 G was shown to be located at the 5′ termini of early Ad2 mRNA by periodate oxidation and β elimination. Cap 1 and cap 2 fractions were fractionated by electrophoresis on DEAE-cellulose paper. The cap 1 fraction separated into three major spots ( a , b , and d ), in order of increasing negative charge), and the cap 2 fraction into four major spots ( a , b , c , and d ). The methylated constituents of each spot were determined and used to deduce the number and composition of individual caps. The following results were obtained. Cap 1 structures: Spot a is m 7 GpppU m pN1; b contains m 7 GpppU m pN2 (N1 and N2 are different unmethylated nucleosides) and m 7 GpppA m pN; c is m 7 GpppN ∗m pN, m 7 Gpppm 6 A m pN, and m 7 GpppG m pN (N ∗m is m 2 6 A m or an unknown base-modified 2′- O -methyl nucleoside). Cap 2 structures: Spot a is m 7 Gppp(U m p,N ∗m p)N1; b contains at least two components, combinations of G m , U m , N ∗m , and m 6 A m ; c contains at least two components, combinations of G m , U m , A m , and m 6 A m ; d is at least two components containing combinations of C m , A m , and m 6 A m . No significant differences in methylated constituents were detected in early mRNA repared from infected cells in the presence or absence of cycloheximide. Although we detect at least 13 caps, the minimum number of distinct 5′ termini is 6, because (i) N ∗m could be m 2 6 A m (2′- 0 -methyl N 6 , N 6 -dimethyl adenosine), (ii) m 6 A m and A m could be undermethylated precursors to m 2 6 A m , and (iii) cap 1 structures could represent cap 2 structures without methylation of the penultimate nucleoside. Therefore there may be from 6 to 13 distinct Ad2 early mRNAs, which is interesting considering that only four general areas of the genome (each close to the 3′ end of each early gene block) appear to function as promoters [Berk, A. J., and Sharp, P. A. (1977). Cell 12 , 45–55; Evans, R. M., et al. (1977). Cell 12 , 733–739]. Analysis of KB cell mRNA resolved about 12 cap 1 and 10 cap 2 structures. All Ad2 mRNA cap spots resolved by electrophoresis corresponded in mobility and composition to major cap spots found in cell mRNA. Thus, the methylated caps of early mRNA are similar to those of the host cell. In contrast, most late Ad2 mRNA molecules have an identical cap sequence [Gelinas, R. E., and Robert, R. J. (1977). Cell 11 , 533–544].

Capping Structures at the 5′-Terminus of Polyadenylated Ribonucleic Acid in Avena Coleoptiles

Evidence is presented for the occurrence of 5'-terminal capping structures in the polyadenylated RNA of oat (Avena sativa) coleoptiles. These structures are composed of an inverted terminal nucleoside containing the modified base 7-methylguanine which is joined 5' to 5' with a second (penultimate) nucleoside by means of three phosphate groups in two pyrophosphate linkages. The penultimate nucleoside is joined to the remainder of the RNA molecule by a conventional 3',5' phosphodiester bond. A significant difference between the cap structures of oat coleoptile RNA and those of previously described higher eucaryotic cellular mRNAs is the lack of ribose methylations in the penultimate nucleosides of the plant RNA.

MRNA(nucleoside-2'-)-methyltransferase from vaccinia virus. Characteristics and substrate specificity

An mRNA(nucleoside-2'-)-methyltransferase purified from vaccinia virus was shown to methylate the penultimate nucleoside of RNA ending in m7G(5')pppN-. By contrast, RNAs ending in pN-, ppN-, or even G(5')pppN- are not methyl acceptors. This specificity indicates that 2'-O-methylation is the final step in the formation of the m7G(5')pppNm- cap structure. Both adenosine and guanosine are methylated, in accordance with the presence of these nucleosides in the penultimate position of vaccinia virus mRNAs. Studies with homopolyribonucleotides containing m7G(5')pppN ends indicated that poly(A) and poly(I) were the best methyl acceptors while significant but much less activity was obtained with poly(G), poly(U), and poly(C). Simple dinucleotides of the type m7G(5')pppN, however, are poor substrates and do not compete with capped RNA. Additional studies indicate that the methyltransferase has a pH optimum of 7.5, does not require divalent cations, is inhibited by S-adenosylhomocysteine, has a Km of 2.0 micrometer for S-adenosylmethionine, a Km of approximately 5 nM for brome mosaic virus RNA, and kinetics consistent with a random bireactant mechanism.

MRNA(nucleoside-2'-)-methyltransferase from vaccinia virus. Purification and physical properties

An S-adenosyl-L-methionine:mRNA(nucleoside-2'-)-methyltransferase, one of at least three activities required for the 5'-terminal modification of mRNA, has been purified from vaccinia virus particles. Employing brome mosaic virus RNA ending in m7G(5')pppG- as substrate, a simple DEAE-cellulose filter assay measuring the incorporation of methyl groups from S-adenosyl[methyl-3H]methionine to position 2' of the penultimate nucleoside was devised. Starting from disrupted vaccinia virus cores, a 350-fold enzyme purification was achieved by successive chromatography on columns of DEAE-cellulose, CM-Sephadex, and APP-agarose. Analysis of the isolated enzyme by sodium dodecyl sulfate-polyacrylamide discontinuous gel electrophoresis revealed a single polypeptide with a molecular weight of 38,000. Similar molecular weights were obtained by sucrose gradient centrifugation and gel filtration of the native methyltransferase. The isoelectric point of the purified enzyme occurs at pH 8.4.

Wheat embryo ribonucleates. XII. Formal characterization of terminal and penultimate nucleoside residues at the 5'-ends of "capped" RNA from imbibing wheat embryos.

(1) N2,N2,7-Trimethylguanosine, not previously detected as a component of plant RNA, is shown to be present in the RNA which is isotopically labelled when dry wheat embryos imbibe water in a medium that contains[methyl-3H]methionine. (2) N2,N2,7-Trimethylguanosine and 7-methylguanosine are released as part of "capped" oligonucleotides when the isotopically labelled RNA from imbibing wheat embryos is subjected to hydrolysis by RNase T2. (3) By way of contrast with the "capped" RNA of animal cells, 5'-terminal "cap" structures (m7Gppp- and m32,2,7 Gppp-) in the "capped" RNA from the higher plant organism are not bonded to pneultimate O2'-methylnucleoside constituents. (4) In an allied study, it has been found that recovery of poly (A)-rich RNA from dry wheat embryos depends on the inclusion of sodium dodecyl sulphate (SDS) in phosphate-buffered (pH 6.8) phenolic emulsions. By way of contrast, recovery of poly (A)-rich RNA from dry wheat embryos does not depend on the inclusion of SDS in Tris (hydroxymethyl) aminomethane buffered (pH 9.0) phenolic emulsions.

5′-Terminal structures of poly(A) + cytoplasmic messenger RNA and of poly(A) + and poly(A) − heterogeneous nuclear RNA of cells of the dipteran Drosophila melanogaster

Structures at the 5′ terminus of poly (A)-containing cytoplasmic RNA and heterogeneous nuclear RNA containing and lacking poly(A) have been examined in RNA extracted from both normal and heat-shocked Drosophila cells. 32P-labeled RNA was digested with ribonucleases T 2, T 1 and A and the products fractionated by a fingerprinting procedure which separates both unblocked 5′ phosphorylated termini and the blocked, methylated, “capped” termini, known to be present in the messenger RNA of most eukaryotes. Approximately 80% of the 5′-terminal structures recovered from digests of poly(A)-containing Drosophila mRNA are cap structures of the general form m 7G 5′ppp 5′X(m)pY(m)pZp. With respect to the extent of ribose methylation and the base distribution, the 5′-terminal sequences of Drosophila capped mRNA appear to be intermediate between those of unicellular eukaryotes and those of mammals. Drosophila is the first organism known in which type 0 (no ribose methylations), type 1 (one ribose methylation), and type 2 (two ribose methylations) caps are all present. In contrast to mammalian cells, the caps of Drosophila never contain the doubly methylated nucleoside N 6,2′- O-dimethyladenosine. Both purines and pyrimidines can be found as the penultimate nucleoside of Drosophila caps and there is a wide variety of X-Y base combinations. The relative frequencies of these different base combinations, and the extent of ribose methylation, vary with the duration of labeling. The large majority of poly(A)-containing cytoplasmic RNA molecules from heat-shocked Drosophila cells are also capped, but these caps are unusual in having almost exclusively purines as the penultimate X base. Greater than 75% of the 5′ termini of heterogeneous nuclear RNA (hnRNA) containing poly(A) and greater than 50% of the termini of hnRNA lacking poly (A) are also capped. Triphosphorylated nucleotides, common as the 5′ nucleotides of mammalian hnRNA, are rare in the poly(A)-containing hnRNA of Drosophila. The frequency of the various type 0 and type 1 cap sequences of cytoplasmic and nuclear poly (A)-containing RNA are almost identical. The caps of hnRNA lacking poly(A) are also quite similar to those of poly-adenylated hnRNA, but are somewhat lower in their content of penultimate pyrimidine nucleosides, suggesting that these two populations of molecules are not identical.

Methylation and capping of RNA polymerase II primary transcripts by HeLa nuclear homogenates.

HeLa nuclear homogenates incubated in vitro incorporate [beta-32P]ATP and S-[methyl-3H]-adenosylmeth-ionine ([3H]SAM) into blocked methylated 5' termini of newly synthesized RNA. Approximately 10% of the RNA chains initiated in vitro with [beta-32P]ATP are subsequently blocked by condensation of GMP to di- or triphosphate terminated RNA. The blocked termini can then be methylated by transfer of methyl groups from [3H]SAM to the 7 position of the guanosine and 2'-O position of the adenosine to form m7Gpp*pAm- capped terminus. In addition to conventional triphosphate caps, HeLa nuclear homogenates produce capping structures containing two phosphate residues in the pyrophosphate bridge. The two distinct cap forms were separated by DEAE-cellulose chromatography and analyzed. In contrast to triphosphate caps (m7GpppXm) in which X can be any one of the four nucleosides (G, A, C, or U), in diphosphate caps (m7GppXm), more than 95% of the penultimate nucleoside Xm is G. Incorporation of both [beta-32P]ATP and [3H]SAM into caps was markedly reduced by low concentrations of alpha-amanitin. However, an ammonium sulfate fraction of the nuclear homogenate can cap beta-32P-labeled RNA (pp*pA-RNA) to form m7Gpp*pA-RNA, in the presence of 0.5 microgram/mL of alpha-amanitin. Therefore, the nuclear capping enzyme is resistant to this drug. Our results indicate that RNA polymerase II primary transcripts are the substrate for the cellular capping enzyme and that the beta phosphate in the pyrophosphate bridge (m7GgammapbetapalphapXm) is derived from the 5' ends of the RNA chains.

Influenza viral mRNA contains internal N6-methyladenosine and 5'-terminal 7-methylguanosine in cap structures.

Influenza viral complementary RNA (cRNA), i.e., viral mRNA was radioactive when purified from the cytoplasmic fraction of cordycepin-treated canine kidney cells that were incubated with [methyl-3H]methionine during infection. Approximately 55 to 60% of the methyl-3H radioactivity was in internal N6-methyladenosine, a feature distinguishing this mRNA from those viral mRNA's that are known to be synthesized in the cytoplasm. The remaining methyl-3H radioactivity was in 5'-terminal cap structures that consisted of 7-methylguanosine in pyrophosphate linkage to 2'-o-methyladenosine, N6, 2'-O-dimethyladenosine, or 2'-O-methylguanosine. Methylated adenosine was the predominant penultimate nucleoside in caps, suggesting that cRNA synthesis in infected cells initiates preferentially with adenosine at the 5' end. In contrast to cRNA, influenza virion RNA segments extracted from purified virus contained mainly 5'-terminal ppA and no detectable cap structures.

Formation of the guanylylated and methylated 5'-terminus of vaccinia virus mRNA.

Abstract mRNA was synthesized in vitro by vaccinia virus particles in the presence of S-adenosyl-[methyl-3H]methionine and α- or β,γ-32P-labeled ATP or GTP. The two 5′-terminal structures m7G(5′)ppp(5′)Gm and m7G(5′)ppp(5′)Am were isolated after P1 nuclease digestion of the RNA. The distribution of radioactivity between the methylated mononucleotides and inorganic phosphate released by nucleotide pyrophosphatase treatment of m7G(5′)ppp(5′)Gm and m7G(5′)ppp(5′)Am was consistent with the formation of the latter structures by condensation of pG residues from GTP with ppG- and ppA- at the 5′-termini of vaccinia mRNAs. An alternative mechanism involving the transfer of ppG residues to pA- at the 5′-terminus of the RNA was ruled out by the 32P-labeling data as well as by the formation of m7G(5′)ppp(5′)Am- when β,γ-methylene-GTP was substituted for GTP. At limiting concentrations of S-adenosylmethionine, the predominant methylated 5′-terminal structure was m7G(5′)ppp(5′)N- in which the penultimate nucleoside was unmethylated; in the absence of S-adenosylmethionine, G(5′)ppp(5′)N- as well as unblocked ppN- termini were detected. On the basis of the structures formed under various conditions, the following sequence of reactions was considered: γβα ppG + β′α′ ppN −→ G (5′) N −+ γβ PPi G (5′) ppp (5′) N −+ AdoMet → m 7 G (5′) ppp (5′) N −+ AdoHcy m 7 G (5′) ppp (5′) N −+ AdoMet → m 7 G (5′) ppp (5′) N m + AdoHcy