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J. Biol. Chem., Vol. 276, Issue 49, 46182-46186, December 7, 2001

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Trypanosoma brucei RNA Triphosphatase

ANTIPROTOZOAL DRUG TARGET AND GUIDE TO EUKARYOTIC PHYLOGENY^*

C. Kiong Ho and Stewart Shuman

From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021

Received for publication, September 10, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The mRNA capping apparatus of the protozoan parasite Trypanosoma brucei consists of separately encoded RNA triphosphatase and RNA guanylyltransferase enzymes. The triphosphatase TbCet1 is a member of a new family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi and the malaria parasite Plasmodium falciparum. The protozoal/fungal enzymes are structurally and mechanistically unrelated to the RNA triphosphatases of metazoans and plants. These results highlight the potential for discovery of broad spectrum antiprotozoal and antifungal drugs that selectively block the capping of pathogen-encoded mRNAs. We propose a scheme of eukaryotic phylogeny based on the structure of RNA triphosphatase and its physical linkage to the guanylyltransferase component of the capping apparatus.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Kinetoplastid protozoan parasites of the genus Trypanosoma are major zoonotic pathogens of humans. Trypanosoma cruzi is the cause of Chagas disease, endemic in South America and affecting some 18 million people (1, 2). Trypanosoma brucei causes sleeping sickness in Africa, which has been resurgent in recent years and is estimated to affect 500,000 people (3, 4). The drugs currently in use to treat trypanosomiasis are old, ineffective, and toxic. There is a need for new therapeutic approaches, and it is anticipated that candidate drug targets will be uncovered by sequencing Trypanosoma genomes. The most promising targets are gene products or metabolic pathways that are essential for all stages of the parasite life cycle but are either absent or fundamentally different in the human host. Such targets can be identified either by whole-genome comparisons or by directed analyses of specific cellular transactions.

Here we analyze the mRNA capping apparatus of T. brucei. The m⁷GpppN cap structure (cap 0) is a defining feature of eukaryotic mRNA and is required for mRNA stability and efficient translation. The cap is formed by three enzymatic reactions: the 5' triphosphate end of the nascent pre-mRNA is hydrolyzed to a diphosphate by RNA triphosphatase; the diphosphate end is capped with GMP by RNA guanylyltransferase; and the GpppN cap is methylated by RNA (guanine-N7-)-methyltransferase (5). Each of the mRNA capping enzymes is essential for cell growth in budding yeast.

Although the three capping reactions are universal in eukaryotes, there is a surprising diversity in the genetic organization of the cap-forming enzymes in different taxa as well as a complete divergence in the structure and catalytic mechanism of the RNA triphosphatase component as one moves from lower to higher eukaryotic species (5). Metazoans and plants have a two-component capping system consisting of a bifunctional triphosphatase-guanylyltransferase polypeptide and a separate methyltransferase polypeptide, whereas fungi and the microsporidian parasite Encephalitozoon cuniculi have a three-component system consisting of separate triphosphatase, guanylyltransferase, and methyltransferase gene products (5).¹ The primary structures and biochemical mechanisms of the fungal and mammalian guanylyltransferases and cap methyltransferases are conserved. However, the atomic structures and catalytic mechanisms of the fungal and mammalian RNA triphosphatases are completely different (7, 8). Thus, it has been suggested that RNA triphosphatase is a promising target for antifungal drug discovery (7).

Relatively little is known about the organization of the mRNA capping apparatus in the many other branches of the eukaryotic phylogenetic tree, especially the protozoa. We recently identified the guanylyltransferase and triphosphatase enzymes from the malaria parasite Plasmodium falciparum, and we showed that the Plasmodium triphosphatase is structurally and mechanistically similar to the metal-dependent fungal enzymes (9). An evolutionary connection between plasmodia and fungi had not been appreciated in previous schemes of molecular taxonomy.

Trypanosome mRNAs contain a unique hypermodified "cap 4" structure, which is derived from the standard m⁷GpppN cap by cotranscriptional methylation of seven sites within the first four nucleosides of the spliced leader RNA (10, 11). Although cap formation is (in principle) an attractive target for drug treatment of trypanosomiasis, the pathway of cap synthesis has not been fully determined. An RNA guanylyltransferase has been characterized in Trypanosoma and Crithidia (12), but the triphosphatase and several methyltransferase components have not been identified. The T. brucei guanylyltransferase is mechanistically and structurally related to the guanylyltransferases from all other eukaryal species (and thus is not an especially attractive drug target). However, the 586-amino acid T. brucei guanylyltransferase contains a 250-amino acid N-terminal extension not found in fungal or metazoan guanylyltransferases; it has been speculated that this extra domain (also present in Crithidia guanylyltransferase) might contribute the triphosphatase activity during cap synthesis (12).

Here we report the identification of the triphosphatase component of the T. brucei capping apparatus as the product of a separate gene, which we named TbCET1. The TbCet1 enzyme is structurally and mechanistically related to the fungal and Plasmodium RNA triphosphatases. Indeed, TbCet1 is active in cap formation in vivo when expressed in budding yeast. We discuss the pharmacological and evolutionary implications of these results.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Yeast Expression Vector for T. brucei RNA Triphosphatase-- A DNA fragment containing the TbCET1 open reading frame was amplified by polymerase chain reaction from T. brucei genomic DNA (a gift of Vivian Bellofatto, University of Medicine and Dentistry of New Jersey) using oligonucleotide primers designed to introduce an NcoI restriction site at the predicted translation start codon and a BamHI site 3' of the predicted stop codon. The polymerase chain reaction product was digested with NcoI and BamHI and cloned into the yeast vector pYX132 (CEN TRP1). TbCET1 expression is under the transcriptional control of the yeast TPI1 promoter. The nucleotide sequence of the T. brucei DNA insert was determined and was found to be identical to the genomic sequence (GenBank^TM accession number AC091330).

Expression and Purification of Recombinant TbCet1-- The TbCET1 open reading frame was excised from pYX-TbCET1 with NcoI and BamHI, and the 5' overhangs were filled in with T4 DNA polymerase. The blunt DNA fragment was inserted into the filled-in BamHI site of pET28-His/Smt3 (a gift of Chris Lima, Cornell Medical College) to fuse the open reading frame in-frame to N-terminal His₆/Smt3. pET-His/Smt3-TbCet1 was transformed into Escherichia coli BL21-CodonPlus(DE3). A 200-ml culture amplified from a single transformant was grown at 37 °C in Luria-Bertani medium containing 60 µg/ml kanamycin and 100 µg/ml chloramphenicol until the A₆₀₀ reached 0.5. The culture was adjusted to 2% ethanol and 0.4 mM isopropyl-1-thio--D-galactopyranoside and then incubated at 17 °C for 18 h. Cells were harvested by centrifugation, and the pellet was stored at 80 °C. All subsequent procedures were performed at 4 °C. Thawed bacteria were resuspended in 10 ml of buffer A (50 mM Tris-HCl (pH 7.5), 0.25 M NaCl, 10% sucrose). Cell lysis was achieved by addition of lysozyme and Triton X-100 to final concentrations of 100 µg/ml and 0.1%, respectively. The lysate was sonicated to reduce viscosity, and insoluble material was removed by centrifugation. The soluble extract was applied to a 1-ml column of nickel-nitrilotriacetic acid-agarose resin (Qiagen) that had been equilibrated with buffer A containing 0.1% Triton X-100. The column was washed with 5 ml of the same buffer and then eluted stepwise with 2-ml aliquots of buffer B (50 mM Tris-HCl (pH 8.0), 0.25 M NaCl, 10% glycerol, 0.05% Triton X-100) containing 5, 50, 100, 200, and 500 mM imidazole. The polypeptide compositions of the column fractions were monitored by SDS-polyacrylamide gel electrophoresis. The 45-kDa recombinant His/Smt3-TbCet1 polypeptide was recovered in the 50 and 100 mM imidazole fractions. The 100 mM imidazole eluate fraction (containing 3 mg of protein) was used to characterize the triphosphatase activity of TbCet1.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of a Candidate T. brucei RNA Triphosphatase, TbCet1-- Metazoan and plant RNA triphosphatases belong to the cysteine phosphate enzyme superfamily, which is defined by the conserved phosphate-binding loop motif HCXXXXXR(S/T). Mammalian RNA triphosphatase catalyzes a two-step ping-pong phosphoryl transfer reaction in which the conserved cysteine of the signature motif attacks the  phosphorus of triphosphate-terminated RNA to form a covalent protein-cysteinyl-S-phosphate intermediate and expel the diphosphate RNA product (8). The covalent phosphoenzyme intermediate is hydrolyzed to liberate inorganic phosphate. The reaction does not require a divalent cation cofactor.

In contrast, the RNA triphosphatases of fungal species such as Saccharomyces cerevisiae, Candida albicans, and Schizosaccharomyces pombe are strictly dependent on a divalent cation. The fungal enzymes belong to a new family of metal-dependent phosphohydrolases that embraces the triphosphatase components of the poxvirus, baculovirus, Chlorella virus, and P. falciparum mRNA capping systems (13-20). The signature biochemical property of this enzyme family is the ability to hydrolyze nucleoside triphosphates to nucleoside diphosphates and inorganic phosphate in the presence of either manganese or cobalt. The defining structural features of the metal-dependent RNA triphosphatases are two glutamate-containing motifs (1 and 11 in Fig. 1) that are required for catalysis by every family member and that comprise the metal-binding site. The crystal structure of the S. cerevisiae RNA triphosphatase Cet1 revealed a novel tertiary structure in which the active site is situated within a topologically closed hydrophilic tunnel composed of eight antiparallel  strands (7). The  strands comprising the tunnel walls are displayed over the Cet1 protein sequence in Fig. 1. Each of the eight strands contributes at least one functional constituent of the active site (13, 16, 21). The 15 individual side chains within the tunnel that are important for Cet1 function in vitro and in vivo are denoted by dots in Fig. 1.

View larger version (67K): [in this window] [in a new window]   Fig. 1.   Structural conservation among fungal, viral, microsporidian, and protozoan RNA triphosphatases. The amino acid sequence of the catalytic domain of S. cerevisiae RNA triphosphatase Cet1 is aligned to the sequences of C. albicans CaCet1, S. cerevisiae Cth1, S. pombe Pct1, P. falciparum Prt1, E. cuniculi Cet1, Chlorella virus cvRtp1, and T. brucei TbCet1. Gaps in the alignment are indicated by dashes. Polyasparagine inserts in Prt1 are omitted from the alignment and are denoted by . The  strands that form the triphosphate tunnel of ScCet1 are denoted above the sequence. Peptide segments with the highest degree of conservation in all eight proteins are highlighted by the shaded boxes. Hydrophilic amino acids that comprise the active site within the ScCet1 tunnel are denoted by dots.

We searched for a candidate RNA triphosphatase in T. brucei by querying the Microbial Genomes Database (www.ncbi.nlm.nih.gov/Microb_blast) for proteins related to the S. pombe RNA triphosphatase Pct1. We thereby identified a putative RNA triphosphatase gene on T. brucei chromosome III (DNA sequence in GenBank^TM accession number AC091330) that encodes a 252-amino acid polypeptide (shown in Fig. 2A) with primary structural similarity to the catalytic domains of fungal, viral, and Plasmodium RNA triphosphatases (Fig. 1). We named this T. brucei gene product TbCet1 (capping enzyme triphosphatase). TbCet1 is slightly larger than the "minimal" RNA triphosphatases of Chlorella virus PBCV-1 (cvRtp1; 193 amino acids) and E. cuniculi (EcCet1; 221 amino acids) but is smaller than the RNA triphosphatases of P. falciparum (Prt1; 596 amino acids), S. cerevisiae (Cet1; 549 amino acids), C. albicans (CaCat1; 520 amino acids), and S. pombe (Pct1; 303 amino acids). Cet1 and CaCet1 contain large nonessential N-terminal extensions (22, 23) that are missing from the T. brucei protein.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Triphosphatase activity of recombinant TbCet1. A, sequence of the TbCet1 polypeptide. B, purification of TbCet1. Aliquots (10 µl) of the soluble bacterial lysate (L), the nickel-agarose flow-through (FT), wash (W), and the indicated imidazole eluates were analyzed by SDS-polyacrylamide gel electrophoresis. The fixed gel was stained with Coomassie Brilliant Blue dye. C, triphosphatase activity. Reaction mixtures (10 µl) containing 50 mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, 2 mM MnCl2, 0.2 mM [-32P]ATP, and TbCet1 as specified were incubated for 15 min at 30 °C. The reactions were quenched by adding 2.5 µl of 5 M formic acid. Aliquots of the mixtures were applied to a polyethyleneimine-cellulose thin layer chromatography plate, which was developed with 1 M formic acid, 0.5 M LiCl. 32Pi release was quantitated by scanning the chromatogram with a Fujix phosphorimaging system and then plotted as a function of input protein.

TbCet1 includes the two metal binding motifs characteristic of the fungal, viral, and Plasmodium triphosphatases plus putative homologs of all of the other  strands that comprise the active site tunnel of Cet1 (Fig. 2). Indeed, all of the 15 hydrophilic amino acids that are essential for catalysis by Cet1 are conserved in the T. brucei polypeptide, which suggested strongly that TbCet1 is the RNA triphosphatase component of the trypanosome mRNA capping apparatus.

Metal-dependent Triphosphatase Activity of TbCet1-- The TbCET1 gene was cloned into a T7 RNA polymerase-based pET vector to fuse the protein in-frame with an N-terminal His₆-Smt3 tag (24). The recombinant TbCet1 protein was purified from a soluble bacterial extract by adsorption to nickel-agarose and elution with 50 and 100 mM imidazole (Fig. 1B). We found that recombinant TbCet1 catalyzed the release of ³²P_i from [-³²P]ATP in the presence of manganese and that the extent of ATP hydrolysis was proportional to enzyme concentration; the reaction proceeded to completion at saturating enzyme (Fig. 1C). We calculated a specific activity of 1.3 nmol of ³²P_i formed/ng of TbCet1 during a 15-min reaction, which corresponded to a turnover number of 65 s¹. There was no detectable ATPase activity in the absence of a divalent cation (Fig. 3A). Hydrolysis of 0.2 mM ATP was optimal at 2 mM MnCl₂ and declined slightly at 5-10 mM MnCl₂ (Fig. 3A). Activity with magnesium (10 mM) was 6% of that observed at 2 mM manganese (Fig. 3A). Specificity for NTP hydrolysis in the presence of manganese is characteristic of the fungal-type RNA triphosphatase family.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Characterization of T. brucei triphosphatase. A, divalent cation dependence. Reaction mixtures (10 µl) containing 50 mM Tris-HCl (pH 7.5), 0.2 mM [-32P]ATP, 2 ng of TbCet1, and either MnCl2 or MgCl2 as specified were incubated for 15 min at 30 °C. 32Pi release is plotted as a function of divalent cation concentration. B, pH dependence. Reaction mixtures (10 µl) containing 50 mM Tris buffer (either Tris acetate, pH 5.0, 5.5, 6.0, and 6.5 or Tris-HCl, pH 7.0, 7.5, 8.0, 8.5, 9.0, and 9.5), 5 mM dithiothreitol, 2 mM MnCl2, 0.2 mM [-32P]ATP, and 2 ng of TbCet1 were incubated for 15 min at 30 °C. 32Pi release is plotted as a function of pH. C, kinetics. Reaction mixtures (100 µl) containing 50 mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, 2 mM MnCl2, 0.2 mM [-32P]ATP or [-32P]ATP, and 100 ng of TbCet1 were incubated at 30 °C. Aliquots (10 µl) were withdrawn at the times indicated and quenched immediately with formic acid. The reaction products were analyzed by thin layer chromatography. The extent of 32Pi or [-32P]ADP formation (from 2 nmol of input ATP/sample) is plotted as a function of time.

Triphosphatase activity was optimal at pH 7.5 in Tris buffer and declined sharply as the pH was lowered to 5.0 or increased to 9.0 (Fig. 3B). The rate of release of ³²P_i from [-³²P]ATP was nearly identical to the rate of conversion of [-³²P]ATP to [-³²P]ADP in a parallel reaction mixture containing the same concentration of TbCet1 (Fig. 3C). We detected no formation of [-³²P]AMP during the reaction. Hence, we conclude that TbCet1 catalyzes the hydrolysis of ATP to ADP and P_i. TbCet1 also converts [-³²P]GTP to [-³²P]GDP (not shown).

RNA Triphosphatase Activity of TbCet1 in Vivo-- We cloned the TbCET1 gene into a yeast CEN TRP1 plasmid under the transcriptional control of the constitutive yeast TPI1 promoter. The function of the TbCET1 gene was first tested by plasmid shuffle in yeast cet1 cells that contain CET1 on a CEN URA3 plasmid. The cet1 strain is unable to form colonies on medium containing 5-fluoroorotic acid (5-FOA),² a drug that selects against the URA3 plasmid unless it is transformed with a second plasmid bearing CET1 or a functional homolog from another source. We found that TbCET1 was unable to complement growth of cet1 on 5-FOA (Fig. 4A). This negative result was not surprising given that TbCet1 lacks an essential domain of Cet1 that mediates its binding to the yeast guanylyltransferase Ceg1 (25). Ceg1 is exquisitely labile and stabilized against inactivation by binding to Cet1 (26). The requirement for the guanylyltransferase binding and stabilization functions of Cet1 can be circumvented in vivo by replacing the endogenous S. cerevisiae guanylyltransferase with an inherently stable guanylyltransferase, e.g. Mce1-(211-597), the guanylyltransferase domain of mammalian capping enzyme (26, 27).

View larger version (56K): [in this window] [in a new window]   Fig. 4.   TbCet1 functions as an RNA triphosphatase in vivo. A, yeast strain YBS20 (cet1) was transformed with CEN TRP1 plasmids bearing the MCE1 or TbCET1 genes or with the CEN TRP1 vector alone. Individual Trp+ isolates were selected and streaked on agar medium containing 0.75 mg/ml 5-FOA. B, yeast strain YBS50 (cet1 ceg1) was cotransformed with a CEN ADE2 plasmid bearing MCE1-(211-597) under the control of the TPI1 promoter and a CEN TRP1 plasmid bearing either MCE1 or TbCET1 or no insert (vector). Individual Trp+ Ade+ isolates were selected and streaked on agar medium containing 0.75 mg/ml 5-FOA. The plates were photographed after incubation for 3 days at 30 °C.

Thus, the function of the TbCET1 gene was tested again by plasmid shuffle in yeast cet1 ceg1 cells that contain CET1 on a CEN URA3 plasmid and MCE1-(211-597) on a CEN ADE2 plasmid. We found that TbCET1 now supported growth on 5-FOA, whereas cells transformed with the TRP1 vector alone did not (Fig. 4B). The cells expressing TbCET1 plus MCE1-(211-597) grew as well as MCE1 cells on rich medium (YPD agar) at 25, 30, and 37 °C (not shown). These results show that TbCET1 encodes a biologically active RNA triphosphatase.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Kinetoplast mRNAs acquire their 5' caps via trans-splicing of an RNA leader sequence containing the hypermethylated cap 4 structure (10, 11). The presence of a distinctive N-terminal domain in the RNA guanylyltransferases of T. brucei and C. fasciculata, which contains a putative nucleotide binding motif, raised the possibility that the N-terminal segment comprises the triphosphatase component of the capping apparatus (12), but there is as yet no biochemical evidence for a triphosphatase activity associated with the T. brucei guanylyltransferase. Here we demonstrated that T. brucei encodes a separate RNA triphosphatase, TbCet1, which is structurally and mechanistically akin to the metal-dependent RNA triphosphatases of fungi and the malaria parasite P. falciparum. The finding that TbCet1 complements the cet1 mutation in budding yeast confirms that the T. brucei protein can function as a cap-forming enzyme in vivo.

TbCet1 is an attractive therapeutic target for trypanosomiasis because (i) the active site structure and catalytic mechanism of TbCet1 is completely different from the RNA triphosphatase domain of the metazoan RNA capping enzyme, and (ii) metazoans encode no identifiable homologs of the fungal or protozoal RNA triphosphatases. Thus, a mechanism-based inhibitor of TbCet1 should be highly selective for the kinetoplastid parasite and have minimal effect on the human host. Given the central role of the mRNA cap in eukaryotic gene expression, a drug that targets TbCet1 would presumably be effective at all stages of the life cycle of the parasite. Also, the structural similarity between T. brucei, P. falciparum, and fungal RNA triphosphatases raises the exciting possibility of achieving antitrypanosomal, antimalarial, and antifungal activity with a single class of mechanism-based inhibitors.

We have suggested that capping enzymes are a good focal point for considering eukaryotic evolution because (i) the mRNA cap structure is ubiquitous in eukarya but absent from the bacterial and archaeal domains, and (ii) differences in the capping apparatus between taxa will reflect events that postdate the emergence of ancestral nucleated cells (9). We proposed a heuristic scheme of eukaryotic phylogeny based on two features of the mRNA capping apparatus: the structure and mechanism of the triphosphatase component (metal-dependent "fungal" type versus metal-independent cysteine phosphatase type) and whether the triphosphatase is physically linked in cis to the guanylyltransferase component. By these simple criteria, relying on fundamental differences in the same metabolic pathway, one arrives at different relationships among taxa than those suggested by comparisons of sequence variations among proteins that are themselves highly conserved in all eukaryotes (28).

For example, the capping-based phylogeny would place metazoans in a common lineage with Viridiplantae (exemplified by the metaphyta Arabidopsis and the unicellular alga Chlamydomonas reinhardtii) because all of these organisms have a cysteine phosphatase-type RNA triphosphatase fused to their guanylyltransferase (Fig. 5). Fungi, microsporidia, plasmodia (which are classified as Apicomplexa along with other pathogenic parasites Toxoplasma and Cryptosporidia), and now Trypanosoma (which are classified as Euglenozoa along with the human parasite Leishmania) fall into a different lineage distinguished by a "Cet1-like" RNA triphosphatase that is physically separate from RNA guanylyltransferase. In contrast, the protein sequence variation-based scheme proposed by Baldauf et al. (28) places fungi in the same supergroup as metazoa and puts the Apicomplexa nearer to plants.

View larger version (49K): [in this window] [in a new window]   Fig. 5.   Capping enzyme-based scheme of eukaryotic phylogeny. The ancestral capping system consists of a separately encoded metal-dependent RNA triphosphatase (TPase, blue) and guanylyltransferase (GTase, yellow) enzymes. The metazoan and plant capping systems, consisting of a cysteine phosphatase-type RNA triphosphatase (TPase, red) fused to a guanylyltransferase (GTase, yellow) may have evolved via a series of intermediate steps highlighted in the shaded boxes.

Assuming that multicellular animals evolved from unicellular ancestors, we envision that the three-component capping system with a metal-dependent triphosphatase is the ancestral state from which other eukarya (and their viruses) evolved (Fig. 5). We see evidence of evolution in two directions. Certain viruses (poxviruses, baculoviruses, and African swine fever virus) and fungal cytoplasmic episomes have acquired bifunctional capping enzymes by fusion of the ancestral triphosphatase and guanylyltransferase genes (6, 14, 15, 18, 19). Metazoans and plants have experienced a different gene rearrangement event that transferred a cysteine phosphatase domain into the same transcription unit as the guanylyltransferase, leading to creation of the triphosphatase-guanylyltransferase fusion protein that we see today. A plausible pathway of evolution could entail the appearance of a new cysteine phosphatase enzyme (e.g. via duplication and mutation of one of the protein phosphatase genes present in lower eukarya) that gained the capacity to hydrolyze an RNA 5' phosphate instead of, or in addition to, a phosphoprotein (Fig. 5). The model implies a "transition state" wherein an organism contained both a metal-dependent and a cysteine phosphatase-type triphosphatase. The fusion of the cysteine phosphatase to the guanylyltransferase presumably allowed for the loss of the Cet1-like enzyme from the genome of the common metazoan/plant ancestor or else the divergence of the protein to a point that it is no longer discernable as Cet1-like. The alternative explanation would be that plants and metazoans independently experienced this gene fusion in distant branches of the phylogenetic tree, a prospect that seems less appealing to us.

The scheme is useful in that it raises some interesting questions about missing links and the order of events in the progression from fungal- and protozoal-type to metazoan- and plant-type capping systems. It is surely oversimplified because it is based on knowledge of only a fraction of eukaryal taxa. As more genomes are sequenced, we may encounter species that have a Cet1-like triphosphatase fused to a guanylyltransferase, others with a cysteine phosphatase-type RNA triphosphatase that participates in cap formation but is physically separate from the guanylyltransferase, and yet others that encode a novel class of RNA triphosphatase enzyme. Of particular interest will be the characterization of the mRNA capping apparatus in the most primitive protozoan and metazoan organisms.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 212-639-7145; Fax: 212-717-3623; E-mail: s-shuman@ski.mskcc.org.

Published, JBC Papers in Press, September 11, 2001, DOI 10.1074/jbc.M108706200

¹ Hausmann, S., Vivares, C., and Shuman, S. (2001) J. Biol. Chem., in press.

	ABBREVIATIONS

The abbreviations used are: 5-FOA, 5-fluoroorotic acid; Ade, adenine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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