Recombinant Human mRNA Cap Methyltransferase Binds Capping Enzyme/RNA Polymerase IIo Complexes*

Guanine N-7 methylation is an essential step in the formation of the m7GpppN cap structure that is characteristic of eukaryotic mRNA 5′ ends. The terminal 7-methylguanosine is recognized by cap-binding proteins that facilitate key events in gene expression including mRNA processing, transport, and translation. Here we describe the cloning, primary structure, and properties of human RNA (guanine-7-)methyltransferase. Sequence alignment of the 476-amino acid human protein with the corresponding yeast ABD1 enzyme demonstrated the presence of several conserved motifs known to be required for methyltransferase activity. We also identified a Drosophila open reading frame that encodes a putative RNA (guanine-7-)methyltransferase and contains these motifs. Recombinant human methyltransferase transferred a methyl group fromS-adenosylmethionine to GpppG 5′ends, which are formed on RNA polymerase II transcripts by the sequential action of RNA 5′-triphosphatase and guanylyltransferase activities in the bifunctional mammalian capping enzyme. Binding studies demonstrated that the human cap methyltransferase associated with recombinant capping enzyme. Consistent with selective capping of RNA polymerase II transcripts, methyltransferase also formed ternary complexes with capping enzyme and the elongating form of RNA polymerase II.

Several functions of the cap structure are mediated by a family of cap-binding protein complexes that specifically recognize m 7 GpppN (19,21). For example, in the nucleus, a capbinding protein complex facilitates pre-mRNA splicing accuracy and efficiency and possibly nuclear export (19). In the cytoplasm, the heterotrimeric initiation factor eIF4F, which includes the cap-binding subunit eIF4E, promotes ribosome binding and translation initiation (21,22). Although capping stabilizes mRNAs (13,14), cap m 7 G is also recognized by the yeast decapping enzyme (23), and loss of the blocked 5Ј end leads to 5Ј 3 3Ј exonucleolytic degradation (13,24). Consistent with the multiple effects of cap on gene expression, the RNA (guanine-7-)methyltransferase, like the RNA 5Ј-triphosphatase (25) and guanylyltransferase (26), is essential for viability in yeast (27).
In an effort to gain a more complete understanding of the role of the cap in mammalian RNA metabolism, we identified human ESTs homologous to the cloned Saccharomyces cerevisiae RNA (guanine-7-)methyltransferase and used them to clone a cDNA encoding the human enzyme (hMet). hMet contains several motifs that are conserved among other RNA (guanine-7-)methyltransferases. In the presence of S-adenosylmethionine (Adomet), purified recombinant hMet converted the GpppG ends of transcripts to m 7 GpppG. In addition, hMet formed ternary complexes with human capping enzyme (HCE) and the hyperphosphorylated elongating form of human RNA polymerase II (pol IIo).

MATERIALS AND METHODS
Cloning of hMet cDNA-Yeast RNA (guanine-7-)methyltransferase (ABD1, accession number P32783) was used to search for homologous sequences in the EST division of GenBank TM using the BLAST server at the National Center for Biotechnology Information (28). Two human ESTs were identified (accession numbers C04619 and C03306) that overlapped and displayed significant homology to ABD1. Searches with these ESTs yielded three additional, overlapping ESTs (accession numbers AA566003, AA568320, and AA642424) that were assembled to predict a partial ORF coding for 389 amino acids of hMet. BLAST searches also identified an ORF from Drosophila (accession number AC002502) that encodes a putative RNA (guanine-7-)methyltransferase with homology to other methyltransferases.
The human EST sequences facilitated design of a gene-specific primer, met-GSP1: 5Ј-CTGCAGATGTCAAAACACATTTGTGGGTCAC-G-3Ј, which was used to obtain the 5Ј-untranslated region of hMet by rapid amplification of cDNA ends (RACE) with Marathon ready HeLa cDNA as template (CLONTECH). The resulting 1.2-kilobase 5Ј-RACE * This work was supported by a grant from the New Jersey Commission on Science and Technology (to A. J. S.), Grant 797-033 from the New Jersey Commission on Cancer Research (to R. P.), and a grant from the FONDECYT of Chile (to E. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  product was cloned into pCR2.1 (Invitrogen) and sequenced using an Applied Biosystems-373 automated sequencer. Several different primers designed from the EST sequences failed to amplify the complete 3Ј end of the hMet cDNA, possibly because of the presence of an internal A-rich stretch at codons 391-394. However, the 3Ј end of hMet was obtained by identifying a motif, GTLSKSEWEA, near the C terminus that was completely conserved between Drosophila and Caenorhabditis elegans (accession number Z81038). From the codon frequency usage within the hMet gene, we designed primer met-DEG1: 5Ј-GCTTCCCA-TTCAGATTTAGAAAGAGTTCC-3Ј for use in RACE to amplify the 3Ј end of the cDNA. 2 Northern Blot Analysis-A 734-bp fragment was obtained by PCR with HeLa cDNA as template and primers met-GSP1 and met-GSP2: 5Ј-GCTTCTGGGACTGGGCTTTGTGAAAAGAC-3Ј. This fragment was used to probe human multiple tissue Northern blots (CLON-TECH) in ExpressHyb solution (CLONTECH) as recommended by the manufacturer.
Full-length HCE cloned into a baculovirus expression vector, pBlueBacHis2A (Invitrogen), under the control of the polyhedrin promoter was introduced into Sf9 insect cells by transfection with the Bac-N-Blue transfection kit (Invitrogen). Recombinant virus was used to produce His 6 -tagged HCE in Sf9 cells according to the manufacturer's instructions.
RNA (Guanine-7-)methyltransferase Activity Assay-T7 polymerase run-off transcripts were synthesized and capped by incubation with vaccinia virus CE in the presence of [␣-32 P]GTP (7). Purified transcripts containing 32 P-labeled GpppG 5Јends were incubated in 20-l reaction mixtures containing 50 mM Tris, pH 8.0, 2 mM dithiothreitol, 50 M Adomet, and recombinant GST-hMet (2.5 g) at 37°C for 30 min. After phenol/chloroform extraction and ethanol precipitation, the RNA samples were dissolved in 10 l of 50 mM sodium acetate, pH 5.3, and digested with 4 g of P1 nuclease at 37°C for 30 min. Digests were spotted on polyethyleneimine TLC plates, resolved in 0.4 M ammonium sulfate, and analyzed by autoradiography (7). GpppG, m 7 GpppG, and m 7 GpppG m standards resolved alongside were detected under UV illumination.
Interaction of hMet with pol II and HCE-␣-His (monoclonal antibody against His 6 (1 g) CLONTECH) was incubated with protein G-agarose beads (Boehringer Mannheim) for 30 min at room temperature. The antibody-protein G beads were washed and equilibrated with buffer A (50 mM Tris, pH 7.85, 100 mM KCl, 0.1% Nonidet P-40) and then incubated with purified, recombinant His 6 -tagged HCE (0.25 g) for 1 h at 4°C. The immune complexes were washed extensively with buffer A and incubated for 1 h at 4°C with pol II (1.2 g of DEAE-5PW HeLa protein fraction; Ref. 29) and/or recombinant GST-hMet (1 g). The immune complexes were washed extensively with buffer A and analyzed by 4 -20% SDS-polyacrylamide gel electrophoresis, followed by Western blotting with specific antisera as indicated. Blots were developed by chemiluminescence (ECL detection system, NEN Life Science Products).

RESULTS
Cloning and Expression of a cDNA Encoding Human RNA (Guanine-7-)methyltransferase-A human cDNA containing a single ORF of 1431 bp encoding hMet was isolated based on sequence homology to the S. cerevisiae, C. elegans, and Drosophila enzymes. The predicted protein consists of 476 amino acids with a calculated molecular mass of ϳ55 kDa, which is in close agreement with that reported earlier for the purified enzyme (30). hMet shows significant homology to its counterparts from yeast (27) and C. elegans (C25A1.f gene product; Ref. 31). GenBank TM searches also revealed an ORF from Drosophila that encodes a putative RNA (guanine-7-)methyltransferase. Alignment of the predicted amino acid sequence of the human enzyme with the yeast, C. elegans, and Drosophila proteins indicates the presence of several conserved motifs (Fig. 1). Three motifs have been described as characteristic of diverse methyltransferases including motif I, VL(D/ E)XGXGXG, which has been shown to be part of the AdoMet binding pocket (32,33). hMet contains this conserved motif (residues 201-209) and the previously described motif III (residues 308 -316) but not motif II (Fig. 1). This is expected because motif II is known to be absent in RNA methyltransferases (32). hMet also shows homology to methyltransferases from vaccinia virus and Shope fibroma virus within these motifs. In addition, we identified a conserved sequence near the C 2 After we determined the sequence of full-length hMet (accession number AF067791), the same cDNA sequence from human brain was entered in the Genbank TM data base (accession number AB007858) as an unidentified gene potentially coding for a protein of unknown function. terminus, GTLSKSEWEA (referred to as motif X), that is completely conserved in human, C. elegans, and Drosophila but absent in yeast ABD1 (Fig. 1).
Full-length hMet cDNA is 6203 bp (accession number AF067791) and contains a single ORF of 1431 bp that starts with the initiator codon in the context TAAATGG. The 5Јuntranslated region consists of 196 bp, whereas the 3Ј-untranslated region is strikingly longer (4576 bp) and contains a polyadenylation signal, AATAAA 18 residues from the 3Ј end. Consistent with the length of the cloned hMet cDNA, Northern blot analyses of poly (A) ϩ RNA from normal adult human tissues revealed a specific transcript of 6.2 kilobases (Fig. 2).
RNA (Guanine-7-)methyltransferase Activity of Recombinant hMet-Purified GST-tagged hMet, either on glutathione beads or in solution, was incubated with Adomet and T7 run-off transcripts containing G 32 pppG 5Јends. Samples were then digested with P1 nuclease and analyzed for methylation. As shown in Fig. 3A (lanes 1 and 2), recombinant hMet converted GpppG ends to m 7 GpppG, whereas purified GST control (lane 3) did not show any methyltransferase activity. In contrast to hMet, vaccinia virus CE complex, which contains both RNA (guanine-7-)methyltransferase and 2Ј-O-methyltransferase activities (12) formed m 7 GpppG m caps (lane 4). His 6 -tagged hMet 1-389 and full-length proteins were also expressed in vitro, immobilized on protein A beads, and assayed for RNA (guanine-7-)methyltransferase activity. Results of this experiment demonstrated that truncation of the C-terminal 87 amino acids eliminated methyltransferase activity (Fig. 3B).
Interactions of hMet with CE and the Elongating Form of RNA pol II in Vitro-We have shown that mammalian CE binds the hyperphosphorylated, elongating form of RNA polymerase II (7). Yeast methyltransferase has also been shown to bind selectively to the hyperphosphorylated CTD of yeast RNA pol II (8). We therefore investigated whether hMet binds human RNA pol II. Immune complexes containing recombinant hMet on protein G beads did not form stable complexes with either the initiating (pol IIa) or elongating (pol IIo) form of RNA pol II but did bind recombinant HCE (data not shown). On the basis of these results, we tested for the formation of ternary complexes of hMet, HCE, and RNA pol II. Immune complexes containing HCE on protein G beads were incubated with purified human RNA pol II and/or hMet. Western blot analysis demonstrated that the elongating form of pol II was selectively immunoprecipitated with HCE (Fig. 4, lane 2) from a mixture containing approximately equal amounts of pol IIa and pol IIo (lane 1). HCE also bound hMet (Fig. 4, lane 4). When RNA pol II and hMet were incubated together with HCE, ternary complexes were formed (Fig. 4, lane 3). These results suggest that hMet may not interact with pol IIo by itself but forms complexes with HCE that bind to pol IIo. In this context it is of interest that the level of pol IIo immunoprecipitated by HCE increased in the presence of hMet (Fig. 4, compare lanes 2 and  3). DISCUSSION The cap structure that is characteristic of RNA pol II transcripts consists of m 7 G linked via a 5Ј-5Ј-triphosphate bridge to the first residue in pre-mRNAs. Capping of nascent transcripts proceeds by the sequential action of RNA 5Ј-triphosphatase, guanylyltransferase, and guanine-7-methyltransferase to form the monomethylated cap 0 structure, m 7 GpppN. Although typical of yeast mRNAs (34) important biological consequences have been described for m 7 G.
Many if not all aspects of mRNA synthesis and function depend on cap-binding protein complexes that specifically recognize m 7 GpppN. Ribosome binding during initiation of translation is facilitated by interactions of eIF4F with cap (21,22). Nuclear events such as in pre-mRNA splicing, 3Ј end formation, and nucleocytoplasmic transport are mediated by a different cap-binding protein complex (19). In addition, guanine N-7 methylation of 5Ј ends can influence transcript stability both positively and negatively. Guanine N-7 methylation blocks pyrophosphorolysis of GpppN structures that protect transcripts against 5Ј-exonucleolytic degradation (13). On the other hand, the 7-methyl group contributes to the substrate specificity of the yeast mRNA decapping enzyme, Dcp1p (23), which increases transcript susceptibility to exonuclease.
Cap methylation studies have focused on virion-associated enzymes (11,12) and on the yeast methyltransferase (27). Although cap methyltransferases have been purified from mammalian sources (30,40), the corresponding gene was not previously cloned and characterized. Here we report the identification and primary structure of a human cDNA that encodes a protein with activity and sequence motifs characteristic of RNA methyltransferases (32,33). A C-terminal truncation mutant of hMet (1-389) that retained previously identified methyltransferase motifs but not motif X was without enzymatic activity. However, motif X is not present in ABD1, and thus its possible role in RNA methylation needs to be further investigated. In this context, it is interesting to note that C-terminal deletion of as few as 55 amino acids from the 436-amino acid ABD1 protein was shown to be lethal (27). Comparison of the human and yeast sequences in this region shows several conserved residues (Fig. 1), but which if any are specifically required for methyl group transfer from Adomet to caps remains to be determined.
Selective capping of RNAs transcribed by pol II, but not pol I and III, was recently explained by the finding of specific association between capping enzymes and the hyperphosphorylated CTD of RNA polymerase II (7)(8)(9). Yeast RNA (guanine-7-)methyltransferase has also been shown by affinity chromatography to bind specifically to hyperphosphorylated GST-CTD (8). Although we did not detect binding of full-length hMet to pol II in immunoprecipitation experiments, preliminary studies of hMet 1-389 indicate that the truncation mutant can bind pol IIo. 3 Thus association of hMet with pol II may be hindered by C-terminal residues (including motif X) that are not present in ABD1. Binding of hMet to HCE and to HCE/pol IIo complexes suggests that it is recruited to the transcription machinery by association with HCE.