Diphosphate End Research Articles

Structural insights into human co-transcriptional capping

Co-transcriptional capping of the nascent pre-mRNA 5' end prevents degradation of RNA polymerase (Pol) II transcripts and suppresses the innate immune response. Here, we provide mechanistic insights into the three major steps of human co-transcriptional pre-mRNA capping based on six different cryoelectron microscopy (cryo-EM) structures. The human mRNA capping enzyme, RNGTT, first docks to the Pol II stalk to position its triphosphatase domain near the RNA exit site. The capping enzyme then moves onto the Pol II surface, and its guanylyltransferase receives the pre-mRNA 5'-diphosphate end. Addition of a GMP moiety can occur when the RNA is ∼22 nt long, sufficient to reach the active site of the guanylyltransferase. For subsequent cap(1) methylation, the methyltransferase CMTR1 binds the Pol II stalk and can receive RNA after it is grown to ∼29 nt in length. The observed rearrangements of capping factors on the Pol II surface may be triggered by the completion of catalytic reaction steps and are accommodated by domain movements in the elongation factor DRB sensitivity-inducing factor (DSIF).

Approved drugs screening against the nsP1 capping enzyme of Venezuelan equine encephalitis virus using an immuno-based assay

Alphaviruses such as the Venezuelan equine encephalitis virus (VEEV) are important human emerging pathogens transmitted by mosquitoes. They possess a unique viral mRNA capping mechanism catalyzed by the viral non-structural protein nsP1, which is essential for virus replication. The alphaviruses capping starts by the methylation of a GTP molecule by the N7-guanine methyltransferase (MTase) activity; nsP1 then forms a covalent link with m7GMP releasing pyrophosphate (GT reaction) and the m7GMP is next transferred onto the 5′-diphosphate end of the viral mRNA to form a cap-0 structure. The cap-0 structure decreases the detection of foreign viral RNAs, prevents RNA degradation by cellular exonucleases, and promotes viral RNA translation into proteins. Additionally, reverse-genetic studies have demonstrated that viruses mutated in nsP1 catalytic residues are both impaired towards replication and attenuated. The nsP1 protein is thus considered an attractive antiviral target for drug discovery. We have previously demonstrated that the guanylylation of VEEV nsP1 can be monitored by Western blot analysis using an antibody recognizing the cap structure. In this study, we developed a high throughput ELISA screening assay to monitor the GT reaction through m7GMP-nsP1 adduct quantitation. This assay was validated using known nsP1 inhibitors before screening 1220 approved compounds. 18 compounds inhibiting the nsP1 guanylylation were identified, and their IC50 determined. Compounds from two series were further characterized and shown to inhibit the nsP1 MTase activity. Conversely, these compounds barely inhibited a cellular MTase demonstrating their specificity towards nsP1. Analogues search and SAR were also initiated to identify the active pharmacophore features. Altogether the results show that this HT enzyme-based assay is a convenient way to select potent and specific hit compounds targeting the viral mRNA capping of Alphaviruses.

Virtual High-Throughput Screening Identifies Mycophenolic Acid as a Novel RNA Capping Inhibitor

The RNA guanylyltransferase (GTase) is involved in the synthesis of the m7Gppp-RNA cap structure found at the 5′ end of eukaryotic mRNAs. GTases are members of the covalent nucleotidyl transferase superfamily, which also includes DNA and RNA ligases. GTases catalyze a two-step reaction in which they initially utilize GTP as a substrate to form a covalent enzyme-GMP intermediate. The GMP moiety is then transferred to the diphosphate end of the RNA transcript in the second step of the reaction to form the Gppp-RNA structure. In the current study, we used a combination of virtual database screening, homology modeling, and biochemical assays to search for novel GTase inhibitors. Using this approach, we demonstrate that mycophenolic acid (MPA) can inhibit the GTase reaction by preventing the catalytic transfer of the GMP moiety onto an acceptor RNA. As such, MPA represents a novel type of inhibitor against RNA guanylyltransferases that inhibits the second step of the catalytic reaction. Moreover, we show that the addition of MPA to S. cerevisiae cells leads to a reduction of capped mRNAs. Finally, biochemical assays also demonstrate that MPA can inhibit DNA ligases through inhibition of the second step of the reaction. The biological implications of these findings for the MPA-mediated inhibition of members of the covalent nucleotidyl superfamily are discussed.

Mutational effects of the consensus aromatic residues in the mRNA capping domain of Bamboo mosaic virus on GTP methylation and virus accumulation

RNA viruses classified in the alphavirus-like superfamily possess a distinct capping domain, catalyzing GTP methylation and subsequent transfer of the m7GMP moiety from m7GTP to the 5′-diphosphate end of viral RNA. The H68A mutation in the capping domain of Bamboo mosaic virus enhanced GTP methylation but disabled the following transguanylation, making it possible to characterize the enzyme's methyltransferase activity separately. To explore the involvement of aromatic amino acids in substrate recognition, consensus aromatic residues in the viral domain were subjected to mutational analysis in the background of H68A. Several residues, including Y126, F144, F161, Y192, Y203, Y213, and W222, were found to be critical for GTP methylation and S-adenosylmethionine (AdoMet) binding. These mutations, except for Y213, also adversely affected the GTP binding, but less extensively. In general, the mutations decreasing the activity for GTP methylation also had correspondingly detrimental effects on virus accumulation.

The flavivirus NS5 protein is a true RNA guanylyltransferase that catalyzes a two-step reaction to form the RNA cap structure

The 5'-end of the flavivirus genome harbors a methylated (m7)GpppA(2'OMe) cap structure, which is generated by the virus-encoded RNA triphosphatase, RNA (guanine-N7) methyltransferase, nucleoside 2'-O-methyltransferase, and RNA guanylyltransferase. The presence of the flavivirus guanylyltransferase activity in NS5 has been suggested by several groups but has not been empirically proven. Here we provide evidence that the N-terminus of the flavivirus NS5 protein is a true RNA guanylyltransferase. We demonstrate that GTP can be used as a substrate by the enzyme to form a covalent GMP-enzyme intermediate via a phosphoamide bond. Mutational studies also confirm the importance of a specific lysine residue in the GTP binding site for the enzymatic activity. We show that the GMP moiety can be transferred to the diphosphate end of an RNA transcript harboring an adenosine as the initiating residue. We also demonstrate that the flavivirus RNA triphosphatase (NS3 protein) stimulates the RNA guanylyltransferase activity of the NS5 protein. Finally, we show that both enzymes are sufficient and necessary to catalyze the de novo formation of a methylated RNA cap structure in vitro using a triphosphorylated RNA transcript. Our study provides biochemical evidence that flaviviruses encode a complete RNA capping machinery.

Unconventional Mechanism of mRNA Capping by the RNA-Dependent RNA Polymerase of Vesicular Stomatitis Virus

All known eukaryotic and some viral mRNA capping enzymes (CEs) transfer a GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA via a covalent enzyme-GMP intermediate to generate the cap structure. In striking contrast, the putative CE of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand (NNS) RNA viruses including rabies, measles, and Ebola, incorporates the GDP moiety of GTP into the cap structure of transcribing mRNAs. Here, we report that the RNA-dependent RNA polymerase L protein of VSV catalyzes the capping reaction by an RNA:GDP polyribonucleotidyltransferase activity, in which a 5'-monophosphorylated viral mRNA-start sequence is transferred to GDP generated from GTP via a covalent enzyme-RNA intermediate. Thus, the L proteins of VSV and, by extension, other NNS RNA viruses represent a new class of viral CEs, which have evolved independently from known eukaryotic CEs.

A Versatile Conformational Switch Regulates Reactivity in Human Branched-Chain α-Ketoacid Dehydrogenase

The dehydrogenase/decarboxylase (E1b) component of the 4 MD human branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) is a thiamin diphosphate (ThDP)-dependent enzyme. We have determined the crystal structures of E1b with ThDP bound intermediates after decarboxylation of alpha-ketoacids. We show that a key tyrosine residue in the E1b active site functions as a conformational switch to reduce the reactivity of the ThDP cofactor through interactions with its thiazolium ring. The intermediates do not assume the often-postulated enamine state, but likely a carbanion state. The carbanion presumably facilitates the second E1b-catalyzed reaction, involving the transfer of an acyl moiety from the intermediate to a lipoic acid prosthetic group in the transacylase (E2b) component of the BCKDC. The tyrosine switch further remodels an E1b loop region to promote E1b binding to E2b. Our results illustrate the versatility of the tyrosine switch in coordinating the catalytic events in E1b by modulating the reactivity of reaction intermediates.

Crystal Structure of Baculovirus RNA Triphosphatase Complexed with Phosphate

Baculovirus RNA 5'-triphosphatase (BVP) exemplifies a family of RNA-specific cysteine phosphatases that includes the RNA triphosphatase domains of metazoan and plant mRNA capping enzymes. Here we report the crystal structure of BVP in a phosphate-bound state at 1.5 A resolution. BVP adopts the characteristic cysteine-phosphatase alpha/beta fold and binds two phosphate ions in the active site region, one of which is proposed to mimic the phosphate of the product complex after hydrolysis of the covalent phosphoenzyme intermediate. The crystal structure highlights the role of backbone amides and side chains of the P-loop motif (118)HCTHGXNRT(126) in binding the cleavable phosphate and stabilizing the transition state. Comparison of the BVP structure to the apoenzyme of mammalian RNA triphosphatase reveals a concerted movement of the Arg-125 side chain (to engage the phosphate directly) and closure of an associated surface loop over the phosphate in the active site. The structure highlights a direct catalytic role of Asn-124, which is the signature P-loop residue of the RNA triphosphatase family and a likely determinant of the specificity of BVP for hydrolysis of phosphoanhydride linkages.

Sendai Virus RNA-dependent RNA Polymerase L Protein Catalyzes Cap Methylation of Virus-specific mRNA

The Sendai virus (SeV) RNA-dependent RNA polymerase complex, which consists of L and P proteins, participates in the synthesis of viral mRNAs that possess a methylated cap structure. To identify the SeV protein(s) involved in mRNA cap methylation, we developed an in vitro assay system to detect mRNA (guanine-7-)methyltransferase (G-7-MTase) activity. Viral ribonucleoprotein complexes and purified recombinant L protein but not P protein exhibited G-7-MTase activity. On the other hand, mRNA synthesis in a reconstituted transcription system using purified N-RNA (N protein-genomic RNA) complex as a template required both the L and P proteins. The enzymatic properties of SeV G-7-MTase were different from those of cellular G-7-MTase. In particular, unlike cellular G-7-MTase, the SeV enzyme preferentially methylated capped RNA containing the viral mRNA 5'-end sequences (GpppApGpG-). The C-terminal part (amino acid residues 1,756-2,228) of the L protein catalyzed cap methylation, whereas the N-terminal half (residues 1-1,120) containing putative RNA polymerase subdomains did not. This is to our knowledge the first direct biochemical evidence that supports the idea that mononegavirus L protein catalyzes cap methylation as well as RNA synthesis.

The Broad Spectrum Antiviral Nucleoside Ribavirin as a Substrate for a Viral RNA Capping Enzyme

The broad spectrum antiviral nucleoside ribavirin displays activity against a variety of RNA and DNA viruses. A number of possible mechanisms have been proposed during the past 30 years to account for the antiviral activity of ribavirin, including the possibility that ribavirin might have a negative effect on the synthesis of the RNA cap structure of viral RNA transcripts. In the present study, we investigated the possibility that ribavirin can directly serve as a substrate for the vaccinia virus RNA capping enzyme. We demonstrate that ribavirin triphosphate can be used as a substrate by the capping enzyme and can form a covalent ribavirin monophosphate-enzyme intermediate reminiscent of the classical GMP-enzyme intermediate. Furthermore, our data indicate that ribavirin monophosphate can be transferred to the diphosphate end of an RNA transcript to form the unusual RpppN structure. Finally, we provide evidence that RNA transcripts that possess ribavirin as the blocking nucleoside are more stable than unblocked transcripts. However, in vitro translation assays indicate that RNA transcripts blocked with ribavirin are not translated efficiently. Our study provides the first biochemical evidences that ribavirin can directly interact with a viral capping enzyme. The ability of a purified RNA capping enzyme to utilize ribavirin as a substrate has not been previously documented and has implications for our understanding of the catalytic mechanisms of RNA capping enzymes. The biological implications of these findings for the proposed ribavirin-mediated inhibition of capping are discussed.

A new method for the study of calcium carbonate growth on steel surfaces

The growth of calcium carbonate crystals on a steel surface, known as scaling, has been investigated. A heat exchanger cell was built for this purpose, and the crystals formed on the surface were examined in a scanning electron microscope. Two surface active polymers with a diphosphate end group, differing in hydrophobic/hydrophilic ratio, were synthesized. The effect on crystal formation of addition of these polymers was studied. Both polymers decreased the amount of calcium carbonate deposition on the surface. When the experiment started at low pH the polymers promoted the formation of very symmetrical spheres of calcium carbonate, ranging from 1 to 10 μm in size. When the experiment was started at neutral pH only amorphous calcium carbonate was obtained on the steel surface.

A Plasmodium falciparum protein related to fungal RNA 5′-triphosphatases

Messenger RNA of eukaryotic cells and most of their viruses carry a ‘cap’ structure at their 5 end. RNA 5 -triphosphatase (RTPase) activity is required for the first step of cap biosynthesis. This activity removes the -phosphate from the 5 end of a nascent mRNA to leave a diphosphate end. mRNA guanylyltransferase (GTase) subsequently adds GMP in a 5 –5 orientation to form the structure, GpppN1-. These two activities are tightly associated and co-purify as ‘mRNA capping enzyme’ [1]. One or more methyltransferase completes the capping modifications. At least two groups of cellular RTPases have been identified. Metazoan capping enzymes consist of a single polypeptide with two separable domains; a GTase domain and a RTPase domain that resembles the protein tyrosine phosphatases (PTPs) [2–4]. This type of RTPase domain appears to use a mechanism similar to that of PTPs, as a conserved nucleophilic cysteine is required for activity [2,5–8]. The capping enzymes of the yeasts Saccharomyces cere isiae and Candida albicans define a second class of RTPases. In these organisms, GTase and RTPase are carried by distinct proteins that form a heterodimer complex. The amino acid sequence of the catalytic region of yeast RTPase subunit has no obvious similarity to PTPs [9,10]. Unlike metazoan RTPases, the yeast proteins require divalent cations for activity [11,12]. It has been suggested that fungal RTPases comprise a new family of metal-dependent nucleotide phosphohydrolase (NTPase)/RTPase. This family may also include capping RTPases encoded by some DNA viruses. Although there is no extensive sequence similarity between the viral and yeast RTPases, several short sequence elements found in all of these proteins have been shown to be essential for the activity [11–14]. To identify other possible members of the yeast RTPase family, we used the BLAST algorithm [15] to search GenBank for proteins with similarity to Cet1, the S. cere isiae capping enzyme RTPase subunit [9]. Known relatives of Cet1 include CaCet1, the C. albicans RTPase subunit [10] and Ctl1 (also known as Cth1), a second RTPase from S. cere isiae whose function is unknown but nonessential for viability [16,17]. In addition to these fungal proteins, an open reading frame from human parasite Plasmodium falciparum (PFC0985c, accession number: CAB39040) was found to have significant similarity to Cet1 (P=3e-5). No related proteins were found in the complete Drosophila, C. elegans, or human genomes. PFC0985c is a hypothetical protein with 591 amino acids whose predicted size is 69 kDa. This protein has 109 asparagine residues and 89 acidic residues that are largely clustered in the N-terminal (amino acids 100– 250) and the C-terminal (aa 405–591) regions (Fig. Abbre iations: CIP, calf intestine alkaline phopshatase; GTase, mRNA guanylytransferase; IPTG, isopropyl -D-thiogalactopyranoside; NTPase, nucleotide phosphohydrolase; PCR, polymerase chain reaction; PEI, polyethyleneimine; PTP, protein tyrosine phosphatase; RTPase, RNA 5 -triphosphatase; TLC, thin-layer chromatography. * Corresponding author. Tel.: +1-617-4320696; fax: +1-6177380516. E-mail address: steveb@hms.harvard.edu (S. Buratowski). 1 Present address: Shionogi BioResearch Corporation, 45 Hartwell Avenue, Lexington, MA 02421, USA.

Kluyveromyces lactis cytoplasmic plasmid pGKL2: heterologous expression of Orf3p and proof of guanylyltransferase and mRNA-triphosphatase activities.

The predicted ORF3 polypeptide (Orf3p) of the linear genetic element pGKL2 from Kluyveromyces lactis was expressed in Bacillus megaterium as a fusion protein with a His(6X)-tag at the C-terminus for isolation by Ni-affinity chromatography. This is the first time that a yeast cytoplasmic gene product has been expressed heterologously as a functional protein in a bacterial system. The purified protein was found to display both RNA 5'-triphosphatase and guanylyltransferase activities. When the lysine residue present at position 177 of the protein within the sequence motif (KXDG), highly conserved in capping enzymes and other nucleotidyl transferases, was substituted by alanine, the guanylyltransferase activity was lost, thereby proving an important role for the transfer of GMP from GTP to the 5'-diphosphate end of the mRNA. Our in vitro data provides the first direct evidence that the polypeptide encoded by ORF3 of the cytoplasmic yeast plasmid pGKL2 functions as a plasmid-specific capping enzyme. Since genes equivalent to ORF3 of pGKL2 have been identified in all autonomous cytoplasmic yeast DNA elements investigated so far, our findings are of general significance for these widely distributed yeast extranuclear genetic elements.

RNA triphosphatase component of the mRNA capping apparatus of Paramecium bursaria Chlorella virus 1.

Paramecium bursaria chlorella virus 1 (PBCV-1) elicits a lytic infection of its unicellular green alga host. The 330-kbp viral genome has been sequenced, yet little is known about how viral mRNAs are synthesized and processed. PBCV-1 encodes its own mRNA guanylyltransferase, which catalyzes the addition of GMP to the 5' diphosphate end of RNA to form a GpppN cap structure. Here we report that PBCV-1 encodes a separate RNA triphosphatase (RTP) that catalyzes the initial step in cap synthesis: hydrolysis of the gamma-phosphate of triphosphate-terminated RNA to generate an RNA diphosphate end. We exploit a yeast-based genetic system to show that Chlorella virus RTP can function as a cap-forming enzyme in vivo. The 193-amino-acid Chlorella virus RTP is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi and other large eukaryotic DNA viruses (poxviruses, African swine fever virus, and baculoviruses). Chlorella virus RTP is more similar in structure to the yeast RNA triphosphatases than to the enzymes of metazoan DNA viruses. Indeed, PBCV-1 is unique among DNA viruses in that the triphosphatase and guanylyltransferase steps of cap formation are catalyzed by separate viral enzymes instead of a single viral polypeptide with multiple catalytic domains.

Characterization of Schizosaccharomyces pombe RNA triphosphatase.

RNA triphosphatase catalyzes the first step in mRNA cap formation which entails the cleavage of the beta-gamma phosphoanhydride bond of triphosphate-terminated RNA to yield a diphosphate end that is then capped with GMP by RNA guanylyltransferase. Here we characterize a 303 amino acid RNA triphosphatase (Pct1p) encoded by the fission yeast SCHIZOSACCHAROMYCES: pombe. Pct1p hydrolyzes the gamma phosphate of triphosphate-terminated poly(A) in the presence of magnesium. Pct1p also hydrolyzes ATP to ADP and P(i) in the presence of manganese or cobalt (K(m) = 19 microM ATP; k(cat) = 67 s(-1)). Hydrolysis of 1 mM ATP is inhibited with increasing potency by inorganic phosphate (I(0.5) = 1 mM), pyrophosphate (I(0.5) = 0.4 mM) and tripolyphosphate (I(0.5) = 30 microM). Velocity sedimentation indicates that Pct1p is a homodimer. Pct1p is biochemically and structurally similar to the catalytic domain of Saccharomyces cerevisiae RNA triphosphatase Cet1p. Mechanistic conservation between Pct1p and Cet1p is underscored by a mutational analysis of the putative metal-binding site of Pct1p. Pct1p is functional in vivo in S.cerevisiae in lieu of Cet1p, provided that it is coexpressed with the S.pombe guanylyltransferase. Pct1p and other yeast RNA triphosphatases are completely unrelated, mechanistically and structurally, to the metazoan RNA triphosphatases, suggesting an abrupt evolutionary divergence of the capping apparatus during the transition from fungal to metazoan species.

Rubber molecular weight regulation, in vitro, in plant species that produce high and low molecular weights in vivo.

In three rubber-producing species, in vitro, the rates of initiation and polymerization and the biopolymer molecular weight produced were affected by the concentration of farnesyl diphosphate (FPP) initiator and isopentenyl diphosphate (IPP) elongation substrate (monomer). Ficus elastica, a low molecular weight-producer in vivo, synthesized rubber polymers approximately twice the molecular weight of those made by Hevea brasiliensis or Parthenium argentatum (which produce high molecular weights in vivo), possibly due to its lower IPP Km. In all species, increasing FPP concentrations increased rubber biosynthetic rate and new molecules initiated but decreased molecular weight by competition with the allylic diphosphate (APP) end of elongating rubber molecules for the APP binding site. Increasing IPP concentrations increased rubber biosynthetic rate and rubber molecular weight, but only when FPP concentrations were below the FPP Km's or where negative cooperativity operated. In conclusion, rubber transferase is not the prime regulator of rubber molecular weight in vivo.

Yeast and Viral RNA 5′ Triphosphatases Comprise a New Nucleoside Triphosphatase Family

Saccharomyces cerevisiae Cet1p catalyzes the first step of mRNA capping, the hydrolysis of the gamma phosphate of triphosphate-terminated RNA to form a 5' diphosphate end. The RNA triphosphatase activity of Cet1p is magnesium-dependent and has a turnover number of 1 s-1. Here we show that purified recombinant Cet1p possesses a robust ATPase activity (Km = 2.8 microM; Vmax = 25 s-1) in the presence of manganese. Cobalt is also an effective cofactor, but magnesium, calcium, copper, and zinc are not. Cet1p displays broad specificity in converting ribonucleoside triphosphates and deoxynucleoside triphosphates to their respective diphosphates. The manganese- and cobalt-dependent nucleoside triphosphatase of Cet1p resembles the nucleoside triphosphatase activities of the baculovirus LEF-4 and vaccinia virus D1 capping enzymes. Cet1p, LEF-4, and D1 share three collinear sequence motifs. Mutational analysis establishes that conserved glutamate and arginine side chains within these motifs are essential for the RNA triphosphatase and ATPase activities of Cet1p in vitro and for Cet1p function in vivo. These findings are in accord with the effects of single alanine mutations at analogous positions of vaccinia capping enzyme. We suggest that the metal-dependent RNA triphosphatases encoded by yeast and DNA viruses comprise a novel family of phosphohydrolase enzymes with a common active site.

Human mRNA Capping Enzyme (RNGTT) and Cap Methyltransferase (RNMT) Map to 6q16 and 18p11.22–p11.23, Respectively

Eukaryotic cellular and viral mRNAs contain a 59-terminal mGpppN cap structure, which has important consequences at multiple levels of gene expression including RNA splicing, 39 end formation, transcript stability, nuclear export, and translation initiation (6, 12, 15, 16). Recent studies have demonstrated that capping enzyme selectively binds to the phosphorylated, elongating form of RNA polymerase II (pol II) (23). This finding accounts for the specific capping of pol II transcripts. It may also provide new insights into how HIV tat protein promotes pol II phosphorylation by tat-activated kinase (TAK) to enhance polymerase processivity (5). Caps are formed on nascent pre-mRNAs by the sequential action of RNA 59-triphosphatase, which removes the g-phosphate of the initiating nucleotide, RNA guanylyltransferase, which transfers GMP from GTP to the resulting diphosphate end, and RNA (guanine-7) methyltransferase, which methylates the guanine N7 position of the newly formed GpppN termini (4, 12, 15). These three enzymatic activities are present as separate polypeptides in yeasts, and strains null for the triphosphatase (18), guanylyltransferase (14), or methyltransferase (7) are nonviable. Metazoans including humans contain a separate cap methyltransferase (RNA guanine-7-methyltransferase, RNMT), but the RNA 59-triphosphatase and guanylyltransferase activities are contained as Nand C-terminal domains in a bifunctional capping enzyme (RNGTT) encoded by a single gene (23). Despite this difference in genomic organization between uniand multicellular organisms, capping enzyme is functionally conserved, and Saccharomyces cerevisiae strains null for guanylyltransferase or RNA 59-triphosphatase are complemented for growth by the mammalian counterpart (21, 23). In light of the importance of mRNA capping for gene expression, we determined the chromosomal location of the human capping enzyme gene (RNGTT) by PCR using as template the NIGMS human/rodent somatic cell hybrid panel 2, version 3 (Coriell Institute for Medical Research Cell Repositories, Camden, NJ). This panel consists of DNA isolated from 24 human/rodent somatic cell hybrid lines that each retained one intact human chromosome. Primers 1, 59-CAGTGTGTCCATTCTGGCAGTGGTTTTG-39, and 2, 59GTGGGAGAGCATTTTCTGGTGACAGC-39, from the 39-UTR of the human capping enzyme cDNA sequence (Accession No. AF025654) were designed to give a human-specific 318-bp PCR product. PCR results demonstrated that the human capping enzyme gene RNGTT is located on chromosome 6 (data not shown). Fine-mapping was performed by PCR analysis of the GeneBridge 4 radiation hybrid panel (Research Genetics, Inc., Huntsville, AL) with 25 ng template genomic DNA and primers 1 and 2 in a 12.5-ml reaction. After an initial denaturation step at 94°C for 5 min, 35 cycles of amplification consisting of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s were performed, followed by a final extension of 5 min at 72°C. Amplification products were analyzed by agarose gel electrophoresis. Data were submitted to the Whitehead Institute/MIT Center for Genome Research STS mapping server for statistical analysis with the RHMAPPER software package (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl). The data vector for RNGTT was 00100 01101 02101 0011

Trypanosome capping enzymes display a novel two-domain structure.

The ubiquitous m7G cap of eukaryotic mRNAs and of precursors to the spliceosomal small nuclear RNAs (snRNAs) is the result of an essential RNA modification acquired during transcript elongation. In trypanosomes, the m7G cap is restricted to the spliced leader (SL) RNA and the precursors of U2, U3, and U4 snRNAs. mRNA capping in these organisms occurs posttranscriptionally by trans splicing, which transfers the capped SL sequence to the 5' ends of all mRNAs. The SL cap is the most elaborate cap structure known in nature and has been shown to consist of an m7G residue followed by four methylated nucleotides. Using Crithidia fasciculata, we have characterized and purified the guanylyltransferase (capping enzyme), which transfers GMP from GTP to the diphosphate end of RNA. The corresponding gene codes for a protein of 697 amino acids, with the carboxy-terminal half of the C. fasciculata guanylyltransferase containing the six signature motifs previously identified in yeast capping enzymes. The amino-terminal half contains a domain that displays no resemblance to any other domain associated with capping enzymes. Intriguingly, this region harbors a consensus sequence for a phosphate-binding loop which is found in ATP- and GTP-binding proteins. This two-domain structure is also present in the Trypanosoma brucei capping enzyme, which shows 44% overall identity with the C. fasciculata capping enzyme. Thus, this structure appears to be common to all trypanosomatid protozoa and defines a novel class of capping enzymes.

Expression and characterization of an RNA capping enzyme encoded by Chlorella virus PBCV-1.

We report that the A103R protein of Chlorella virus PBCV-1 is an mRNA capping enzyme that catalyzes the transfer of GMP from GTP to the 5' diphosphate end of RNA. This is a two-step reaction in which the enzyme first condenses with GTP to form a covalent enzyme-GMP intermediate and then transfers the GMP to an RNA acceptor to form a GpppN cap. Purified recombinant Al03R is a 38-kDa monomer that lacks RNA (guanine-7-) methyltransferase activity. With respect to its size, amino acid sequence, and biochemical properties, A103R is more closely related to the yeast RNA guanylyltransferases than it is to the multifunctional capping enzymes coded for by other large DNA viruses--the poxviruses and African swine fever virus. We surmise that in order to cap its transcripts, PBCV-l must either encode additional 5' processing activities or else rely on the host alga to provide these functions.