Giardia lamblia RNA Cap Guanine-N2 Methyltransferase (Tgs2) *

Tgs1 is the enzyme responsible for converting 7-methyl-guanosineRNAcapstothe2,2,7-trimethylguanosinecapstructures of small nuclear and small nucleolar RNAs. Whereas budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe encode a single Tgs1 protein, the primitive eukaryote Giardia lamblia encodes two paralogs, Tgs1 and Tgs2. Here we show that purified Tgs2 is a monomeric enzyme that catalyzes methyl transfer from AdoMet ( K m of 6 (cid:1) M ) to m 7 GDP ( K m of 65 (cid:1) M ; k cat of 14 min (cid:2) 1 ) to form m 2,7 GDP. Tgs2 also methylates m 7 GTP ( K m of 30 (cid:1) M ; k cat of 13 min (cid:2) 1 ) and m 7 GpppA ( K m of 7 (cid:1) M ; k cat of 14 min (cid:2) 1 ) but is unreactive with GDP, GTP, GpppA, ATP, CTP, or UTP. We find that the conserved residues Asp-68, Glu-91, and Trp-143 are essential for Tgs2 methyltransferase activity in vitro . The m 2,7 GDP product formed by Tgs2 can be converted to m 2,2,7 GDP by S. pombe Tgs1 in the presence of excess AdoMet. However, Giardia Tgs2 itself is apparently unable to add a second methyl group at guanine-N2. This result implies that 2,2,7-trimethylguanosine caps in

Many small noncoding eukaryotic RNAs contain a hypermodified 2,2,7-trimethylguanosine (TMG) 2 5Ј-cap structure (1,2). TMG caps are also found on nematode mRNAs generated via trans-splicing (3). TMG cap formation in Saccharomyces cerevisiae depends on the Tgs1 protein (4). The presence of a putative AdoMet binding motif in the Tgs1 polypeptide, the mutation of which affects TMG formation in vivo (4,5), suggested that Tgs1 might be directly involved in TMG formation. Biochemical studies of Schizosaccharomyces pombe Tgs1 showed that it is indeed a catalyst of TMG synthesis (6). Methylation of guanine-N2 by S. pombe Tgs1 in vitro is strictly dependent on the prior methylation of guanine-N7, indicating that TMG caps are formed by post-transcriptional methylation of standard m 7 G caps (6). Guanine-N2 methylation by S. pombe Tgs1 in vitro requires no RNA component and no protein cofactor (6). Although early models suggested that the TMG synthase reaction might require cis-acting RNA signals or the assembly of specific ribonucleoprotein structures (7)(8)(9)(10), the recent work on S. pombe Tgs1 instates a more conservative model in which ribonucleoprotein components might simply target Tgs1 to a particular subset of cellular RNAs that already have an m 7 G cap.
Given the ubiquity of TMG caps in eukaryotic species, it is surprising that an S. cerevisiae tgs1 deletion mutant is viable, even though the small nuclear RNAs and small nucleolar RNAs in the tgs1⌬ strain lack TMG caps (4). Genetic analysis indicates that Tgs1 is also nonessential for growth of S. pombe. 3 In contrast, TMG synthesis is essential in Drosophila, where mutations in the putative Tgs1 active site cause lethality at the early pupal stage of development that correlates with depletion of TMG-containing RNAs (11). The protozoan parasite Giardia lamblia is posited to occupy a deeply branching position in eukaryotic phylogeny. Analysis of the Giardia genome is providing important insights to the early origins of RNA processing mechanisms that are regarded as uniquely eukaryotic (12). Although there had been some debate whether Giardia mRNAs even have a 5Ј-cap structure (13,14), recent studies show that Giardia does possess the enzymatic machinery for m 7 G cap synthesis (15), caps the 5Ј ends of its mRNAs, and exploits the m 7 G cap for enhanced translation of a reporter mRNA in vivo (15)(16)(17).
The fact that Giardia encodes two homologs of the cap-binding translation initiation factor eIF4E (17) suggests that Giardia is not an exception to the general reliance on the cap structure for optimal gene expression in eukaryotes. However, characterization of the Giardia eIF4E proteins revealed that one paralog, eIF4E2, binds to m 7 G caps, whereas the other paralog, eIF4E1, binds to m 2,2,7 G caps (17). The existence of a TMG-specific cap-binding protein is consistent with an early study identifying several small RNAs in Giardia that reacted with antibody to the TMG cap (18). Because transfected TMG-capped mRNAs are not translated in Giardia (17), it is inferred that eIF4E1 and TMGcapped RNAs are involved in RNA transactions unrelated to bulk mRNA translation.
The apparent complexity of cap function in Giardia is highlighted by our finding of two Tgs-like proteins in this primitive organism. One of the Tgs paralogs, which we name Tgs1, is a 300-amino acid polypeptide that is 32% identical to S. pombe Tgs1 over a 174-amino acid segment of sequence similarity that is shown in Fig. 1. The second paralog, Tgs2, is a 258-amino acid polypeptide that is 25% identical to S. pombe Tgs1 across this segment. Giardia Tgs1 and Tgs2 are 22% identical to each other over the same region. To assess what biochemical activities, if any, are associated with the Giardia Tgs homologs, we produced the recombinant proteins in bacteria. Giardia Tgs1 was intractably insoluble despite the use of several expression and tagging strategies and was therefore not amenable to study. Giardia Tgs2 was expressed as a soluble His 10 fusion and purified for enzymatic and physical characterization. We report that Tgs2 is a monomeric m 7 G-specific N2 methyltransferase that catalyzes addition of a single methyl group to form a 2,7-dimethylguanosine cap.

EXPERIMENTAL PROCEDURES
Recombinant Giardia Tgs2-The open reading frame encoding Tgs2 (GenBank TM accession number EAA46438) was amplified from G. lam-blia genomic DNA with primers that introduced an NdeI site at the start codon and a BglII site 3Ј of the stop codon. The PCR product was digested with NdeI and BglII and inserted into pET16b. The resulting pET-His 10 Tgs2 plasmid was transformed into Escherichia coli BL21CodonPlus(DE3). A 500-ml culture amplified from a single transformant was grown at 37°C in Luria-Bertani medium containing 50 g/ml kanamycin and 50 g/ml chloramphenicol until the A 600 reached 0.6. The culture was adjusted to 2% ethanol and 0.2 mM isopropyl 1-thio-␤-D-galactopyranoside and then incubated at 17°C for 20 h with constant shaking. Cells were harvested by centrifugation, and the pellet was stored at Ϫ80°C. All subsequent procedures were performed at 4°C. Thawed bacteria were resuspended in 25 ml of buffer A (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, and 10% glycerol). Cell lysis was achieved by the addition of lysozyme to 100 g/ml. 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. The column was washed with 10 ml of the same buffer and then eluted stepwise with 2-ml aliquots of buffer A containing 50, 100, 250, and 500 mM imidazole. The polypeptide compositions of the column fractions were monitored by SDS-PAGE. The recombinant His 10 -Tgs2 polypeptide was recovered predominantly in the 250 mM imidazole fractions. The 250 mM imidazole eluate was dialyzed against a buffer containing 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2 mM DTT, 1 mM EDTA, and 10% glycerol and then stored at Ϫ80°C. The protein concentration was determined with the Bio-Rad dye reagent using bovine serum albumin as the standard. The single alanine mutations D68A, E91A, and W143A were introduced into the TGS2 gene by the PCR-based two-stage overlap extension method (32). The mutated genes were inserted into the pET16b. The inserts were sequenced completely to exclude the acquisition of unwanted mutations during amplification and cloning. The D68A, E91A, and W143A proteins were produced in E. coli and isolated from soluble bacterial extracts as described above for wild-type Tgs2.
Methyltransferase   m 7 GDP, and enzyme were incubated at 37°C. Aliquots (4 l) were spotted on PEI-cellulose TLC plates, which were developed with 0.05 M ammonium sulfate. The AdoMet-and m 2,7 GDP-containing portions of the lanes were cut out, and the radioactivity in each was quantified by liquid scintillation counting.
Glycerol Gradient Sedimentation-An aliquot (40 g) of the nickelagarose preparation of Tgs2 was mixed with catalase (45 g), bovine serum albumin (45 g), and cytochrome c (45 g). The mixture was applied to a 4.8-ml 15-30% glycerol gradient containing 50 mM Tris-HCl (pH 8.0), 0.2 M NaCl, 1 mM EDTA, and 2 mM DTT. The gradient was centrifuged for 18 h at 4°C in a Beckman SW50 rotor at 50,000 rpm. Fractions (ϳ0.19 ml) were collected from the bottom of the tube.
Materials-[methyl-3 H]AdoMet was purchased from New England Nuclear. m 7 GTP, m 7 GDP, AdoMet, AdoHcy sinefungin, and sodium periodate were purchased from Sigma. m 7 GpppA and GpppA were purchased from New England Biolabs.

RESULTS
Tgs2 Is an AdoMet and m 7 G-dependent Methyltransferase-The Giardia Tgs2 protein was produced in E. coli as a His 10 fusion and puri-fied from a soluble bacterial extract by adsorption to nickel-agarose and elution with imidazole (Fig. 2). The methyltransferase activity of Tgs2 was demonstrated by incubating increasing amounts of the protein with 50 M [methyl-3 H]AdoMet and 2.5 mM m 7 GDP at 37°C, which, at saturating enzyme, resulted in 93% label transfer from AdoMet to the m 7 GDP (Fig. 2) to form a 3 H-labeled product that was separated from the labeled AdoMet by PEI-cellulose TLC in 0.1 M ammonium sulfate (Fig. 3A). The labeled product (m 2,7 GDP; see below) migrated immediately ahead of the input m 7 GDP substrate, which was visualized by UV illumination of the chromatogram (not shown).
The extent of methylation of m 7 GDP increased with time and was proportional to input enzyme (Fig. 4A). From the initial rate, we estimated a turnover number of 14 min Ϫ1 . Methyl transfer activity was optimal at pH 7.5-9.0 in Tris-HCl buffer; activity declined to 32% of the optimum at pH 6.5 (Tris acetate), and was nil at pH Յ5.0 (Fig. 4B).
Methylation of m 7 GDP by Tgs2 displayed a hyperbolic dependence on AdoMet concentration (Fig. 4C); from a double-reciprocal plot of the data we calculated a K m value of 6 M AdoMet and k cat of 12 min Ϫ1 . Methylation of 2.5 mM m 7 GDP in the presence of 50 M [methyl-3 H]AdoMet was inhibited in a concentration-dependent fashion by the  product AdoHcy and the AdoMet analog sinefungin (Fig. 4D). The apparent IC 50 values for AdoHcy and sinefungin were 75 and 45 M, respectively. Thus, Tgs2 has similar affinity for AdoMet, AdoHcy, and sinefungin.
Sedimentation Analysis of Tgs2-The recombinant protein was subjected to zonal velocity sedimentation in a 15-30% glycerol gradient (Fig. 5). Marker proteins catalase (native size 248 kDa), bovine serum albumin (66 kDa), and cytochrome c (12 kDa) were included as internal standards. His 10 -Tgs2 (calculated to be a 32-kDa polypeptide) sedimented as a discrete peak (fraction 19) between bovine serum albumin and cytochrome c. The methyltransferase activity profile paralleled the abundance of the Tgs2 polypeptide and peaked at fraction 19. We surmise from these results that the methyltransferase activity is intrinsic to Tgs2 and that the enzyme is a monomer in solution.
Substrate Specificity and Affinity-Various nucleoside diphosphates were tested as methyl acceptors at a 2.5 mM concentration (Fig. 3). Whereas Tgs2 catalyzed near complete transfer of label from AdoMet to m 7 GDP, no new labeled product was formed in the presence of GDP, ADP, CDP, or UDP (Fig. 3A). Tgs2 also catalyzed near quantitative methyl transfer from AdoMet to m 7 GTP to form a major labeled product that migrated immediately ahead of the input m 7 GTP substrate during PEI-cellulose TLC in 0.2 M ammonium sulfate (Fig. 3C). A minor product migrating just slower than AdoMet was formed by reaction of Tgs2 with contaminating m 7 GDP nucleotide present in the commercial m 7 GTP preparation.
Tgs2 was capable of near quantitative label transfer from AdoMet to the cap dinucleotide m 7 GpppA to form a single product, presumed to be m 2,7 GpppA, that was resolved from AdoMet by PEI-cellulose TLC in 0.05 M ammonium sulfate (Fig. 3B). This product migrated immediately ahead of the input m 7 GpppA substrate, which was visualized by UV illumination of the chromatogram (not shown). No novel product was formed in the presence of the unmethylated cap dinucleotide GpppA (Fig. 3B). Simultaneous TLC analysis of the reaction product formed with m 7 GTP revealed a major species (m 2,7 GTP) that barely migrated off the origin in 0.05 M ammonium sulfate plus a minor contaminant (m 2,7 GDP) that migrated slower than m 2,7 GpppA (Fig. 3B). Tgs2 formed no new labeled product when reacted with 2.5 mM GTP, ATP, CTP, or UTP (Fig. 3B). Collectively, these results highlight the requirement for prior N7 methylation of the guanine nucleotide substrate, which can be either a cap dinucleotide, a mononucleoside triphosphate, or a mononucleoside diphosphate.
The extent of methyl transfer by Tgs2 in the presence of 50 M AdoMet displayed a hyperbolic dependence on m 7 GDP (Fig. 6A) or m 7 GTP (Fig. 6B) concentration. From double-reciprocal plots of the data, we calculated a K m for m 7 GDP of 65 M with a k cat of 14 min Ϫ1 and a K m for m 7 GTP of 30 M with a k cat of 13 min Ϫ1 . The cap dinucleotide m 7 GpppA was a more avid methyl acceptor than either m 7 GDP or m 7 GTP (Fig. 6C); we calculated a K m of 7 M m 7 GpppA and a k cat of 14 min Ϫ1 . These experiments show that, whereas the affinity of Tgs2 for the m 7 G nucleotide methyl acceptor is enhanced 2-fold by the ␥-phosphate of m 7 GTP and an additional 4-fold by the 5Ј-nucleoside of the cap analog, k cat is unaffected by either the ␥-phosphate or the 5Ј-nucleoside.
Characterization of the Reaction Product-The primary structure similarity between Tgs2 and the yeast Tgs1 trimethylguanosine synthase enzymes engenders a prediction that the exocyclic N2 atom of m 7 G is the methyl acceptor in the Tgs2-catalyzed reaction. Nonetheless, we considered the possibility that Tgs2 might transfer the methyl group to either the ribose or the base of the nucleotide substrate. If methyl transfer occurred at either the ribose O2Ј or O3Ј atoms of m 7 GDP, then the resulting 2Ј-OCH 3 or 3Ј-OCH 3 products should be resistant to oxidation by periodate, whereas methylation at guanine-N2 would preserve the vicinal ribose hydroxyls and leave them sensitive to periodate oxidation. Fig. 7 shows that treatment of the 3 H-labeled product of Tgs2-catalyzed methyl transfer from AdoMet to m 7 GDP with sodium periodate caused the labeled nucleotide to be retained at the origin during PEI-cellulose TLC. The untreated product migrated at its usual position, slower than AdoMet and immediately ahead of the m 7 GDP substrate. Retention at the origin is a consequence of oxidation of the ribose to a ring-opened 2Ј,3Ј-dialdehyde, which forms a covalent Schiff base adduct to the PEI at the site of application to the TLC plate. Control experiments showed that periodate treatment of unlabeled GTP quantitatively shifted the nucleotide to the origin, whereas periodate treatment of 3Ј-OCH 3 GTP had no effect on migration during TLC (not shown). We conclude that Tgs2 does not catalyze methylation of the ribose hydroxyls.
The labeled product formed by Tgs2 in reactions containing excess m 7 GDP methyl acceptor comigrated during TLC with the m 2,7 GDP product synthesized by S. pombe Tgs1 (data not shown). The absence of a 2,2,7-trimethylguanosine product of the Tgs2 reaction implies one of the following possibilities: (i) that Tgs2 is not able to perform the second methylation reaction at N2; or (ii) that Tgs2 does catalyze a second methylation reaction, but we are precluded from detecting it because the enzyme acts distributively, i.e. the labeled m 2,7 GDP product dissociates after a single round of catalysis and must compete with a large molar excess of unlabeled m 7 GDP for rebinding to Tgs2. Previous studies showed that S. pombe Tgs1 does catalyze sequential methylation reactions at N2 via a distributive mechanism (6). To address this issue for the Giardia enzyme, we analyzed the products of a Tgs2 methylation reaction at equal concentrations of the methyl donor and acceptor. As outlined in Fig. 8, Tgs2 was incubated with 50 M m 7 GDP and 50 M [methyl-3 H]AdoMet for 30 min, at which time most of the label had been transferred to the substrate to form a single methylated product. The reaction mixture was then split and supplemented with 1 mM cold AdoMet and either S. pombe Tgs1 or fresh Giardia Tgs2. The reactions were continued for another 60 min, and the products were analyzed by TLC. The instructive finding was that about half of the m 2,7 GDP formed during the pulse-labeling phase was subsequently converted by S. pombe Tgs1 to the slightly more rapidly migrating trimethylated product m 2,2.7 GDP during the chase in the presence of excess cold AdoMet (Fig. 8). In contrast, adding more Giardia Tgs2 resulted in no change in the radiolabeled product distribution. These results indicate that Tgs2 is a dimethylguanosine synthase capable of catalyzing only one methyl addition reaction at the exocyclic amino nitrogen of m 7 G.
Asp-68, Glu-91, and Trp-143 Are Essential for Tgs2 Methyltransferase Activity-Amino acid sequence comparisons of Tgs-like proteins and structural model building (5) led to the prediction of a canonical AdoMet binding site in S. cerevisiae Tgs1 composed of two peptide motifs highlighted in shaded boxes in Fig. 1 (corresponding to Tgs2 peptides 66 VIDGTACVGG 75 and 88 VAIE 91 ). A putative methyl acceptor site was predicted to reside within the conserved proline/glycine containing motif ( 140 DPPWGGV 146 in Tgs2; see Fig. 1). Bordonné and colleagues (4,5) have shown that alanine mutations of S. cerevisiae Tgs1 at three conserved positions in these motifs (Asp-103, Asp-126, and Trp-178, corresponding to Asp-68, Glu-91, and Trp-143 in Giardia Tgs2) cause defects in TMG cap formation in vivo. To gauge the bio-  l of water (lane marked with minus sign) and then incubated for 2 h at room temperature. The mixtures were supplemented with 5 l of 75% glycerol, and then aliquots (5 l) were spotted onto a PEI-cellulose TLC plate, which was developed with 0.1 M ammonium sulfate. The chromatogram was treated with Enhance (PerkinElmer Life Sciences), and 3 H-labeled material was visualized by autoradiography. chemical effects of such changes, we produced Giardia Tgs2 mutants D68A, E91A, and W143A in bacteria as His 10 fusions and isolated them from soluble bacterial extracts by nickel-agarose chromatography (Fig.  2). We found that the Tgs2 mutants were inert in catalysis of methyl transfer from AdoMet to m 7 GDP, at a level of sensitivity of Յ1% of the wild-type specific activity (Fig. 2). These results verify that the methyltransferase activity is intrinsic to the recombinant Tgs2 protein. Based on the crystal structure of the cap guanine-N7 methyltransferase Ecm1 bound to AdoMet and on mutational analysis of that enzyme (19,20), we suspect that essential Tgs2 residues Asp-68 and Glu-91 coordinate the methionine amine and adenosine ribose hydroxyls of AdoMet, respectively.

DISCUSSION
The experiments presented here show that Giardia Tgs2 is a monomeric m 7 G-specific methyltransferase that catalyzes addition of one methyl group to the exocyclic guanine-N2 atom. The recombinant Giardia enzyme resembles the fission yeast Tgs1 protein in its requirement for prior methylation at guanine-N7 and its ability to methylate m 7 G mononucleotides and m 7 G cap dinucleotides in the absence of an RNA polynucleotide or a separate protein cofactor. Tgs2 has a K m for AdoMet (6 M) similar to that of S. pombe Tgs1 (9 M) and, like the fission yeast enzyme, Tgs2 is inhibited by its reaction product AdoHcy. Tgs2 displays a higher affinity for m 7 GDP (K m of 65 M) than does S. pombe Tgs1 (K m of 570 M), and the turnover number of Tgs2 (14 min Ϫ1 ) is higher than that of S. pombe Tgs1 (2 min Ϫ1 ).
The most distinctive property of Giardia Tgs2 is that its activity is apparently limited to a single round of N2 methylation, resulting in the synthesis of a 2,7-dimethylguanosine product. In contrast, S. pombe Tgs1, the only other cap-specific N2 methyltransferase that has been characterized (6), is able to catalyze two sequential N2 methylations leading to TMG cap formation. tRNA-specific guanine-N2 methyltransferases also fall into two classes, depending on whether they catalyze either one methylation step to form 2-methylguanosine (21) or two sequential steps to generate 2,2-dimethylguanosine (22)(23)(24)(25). The rRNA-specific guanine-N2 methyltransferase RsmC performs only one methylation step to generate 2-methylguanosine (26). Given the proposal that that members of the Tgs-like family are structurally homologous to RsmC (5) and the present biochemical characterization of Giardia Tgs2 as a 2,7-dimethylguanosine synthase, it is appropriate to sound a note of caution in attributing TMG cap synthetic roles to Tgs1like proteins in the absence of direct evidence that they catalyze two methylation steps.
The substrate specificity of Giardia Tgs2 makes it unlikely, in our view, that it plays a role in either tRNA or rRNA modifications, because Tgs2 activity requires prior guanine-N7 methylation, and the affinity of Tgs2 for the methyl acceptor is enhanced when the m 7 G nucleoside has a 5Ј-triphosphate and a second nucleoside attached in an inverted 5Ј-5Ј orientation; these are signature features of the RNA cap. Taken at face value, our findings concerning Tgs2 suggest that any TMG caps present in Giardia small nuclear RNAs or small nucleolar RNAs are either synthesized by the paralogous protein Tgs1 alone or by the sequential action of Tgs2 and Tgs1. We cannot exclude a more elaborate scenario in which there exists in Giardia a regulatory factor that confers upon Tgs2 the ability to catalyze a second guanine-N2 methylation reaction.
The specificity of Tgs2 raises the prospect that some Giardia mRNAs might have dimethylguanosine caps. Note that the mRNAs of two eukaryotic RNA viruses (Sindbis virus and Semliki Forest virus) have been reported to contain a significant fraction of 2,7-dimethyguanosine caps (27,28). Whereas indirect assays reveal that Giardia mRNAs contained blocked 5Ј-terminal structures that are resistant to phosphatase but sensitive to pyrophosphatase (15), the structure of the blocking nucleoside is unknown. Also, whereas Giardia contains small RNAs that can be recovered using anti-TMG antibody (18), the structures of those caps have not been determined directly. The recent finding that Giardia has two eIF4Es with preferential affinity for m 7 G and m 2,2,7 G caps, respectively (17), did not examine whether the TMG-specific eIF4E2 protein might bind as well or better to a 2,7-dimethylguanosine cap. Several independent studies have shown that 2,7-dimethylguanosine (DMG)-capped reporter mRNAs are translated better than standard m 7 G-capped transcripts in vitro, whereas TMG-capped reporter mRNAs are translated with low efficiency (29 -31) or, in the case of Giardia RNA transfection experiments in vivo, not at all (17). In conclusion, the existence of cap-specific N2 methylating enzymes with TMG versus DMG synthase activities raises questions about the structural basis for the different reaction outcomes and the potential existence and function of DMG caps in eukaryotic RNA transactions.