Department of Hygiene, School of Medicine, Yokohama City University, 3-9 Fukuura, Kanazawa, Yokohama 236-0004, Japan1
Department of Mycology, Nippon Roche Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan2
Author for correspondence: Hisafumi Yamada-Okabe. Tel: +81 467 45 4382. Fax: +81 467 46 5320. e-mail: hisafumi.okabe{at}roche.com
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ABSTRACT |
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Keywords: mRNA 5'-capping, mRNA cap methyltransferase, human, cDNA, Candida albicans
Abbreviations: cap MTase, RNA (guanine-N7-)-methyltransferase; 5-FOA, 5-fluoroorotic acid; GST, glutathione S-transferase; mRNA GTase, mRNA guanylyltransferase; mRNA TPase, mRNA triphosphatase; SAM, S-adenosyl-L-methionine
The GenBank accession numbers for the nucleotide sequence of CaABD1 and hMet are AB020965 and AB020966, respectively.
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INTRODUCTION |
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In Saccharomyces cerevisiae, the CET1, CEG1 (referred to as CGT1 in Candida albicans), and ABD1 genes are responsible for mRNA TPase, mRNA GTase and cap MTase, respectively (Shibagaki et al., 1992 ; Mao et al., 1995
; Tsukamoto et al., 1997
). Because deletion of any one of these genes is lethal, every step of mRNA capping is essential for viability (Shibagaki et al., 1992
; Mao et al., 1995
; Tsukamoto et al., 1997
). Furthermore, mRNA TPase and mRNA GTase are physically associated, forming a subunit structure: the association of these two enzymes is essential for the functionality of the enzyme in vivo (Ho et al., 1998
).
In higher eukaryotes, both mRNA GTase and mRNA TPase activities are intrinsic to a single polypeptide called Hce1p (McCracken et al., 1997 ; Takagi et al., 1997
; Yue et al., 1997
; Tsukamoto et al., 1998a
; Yamada-Okabe et al., 1998a
). Hce1p binds to the hyperphosphorylated C-terminal domain of the largest subunit of RNA polymerase II, which accounts for the selective capping of RNA polymerase II transcripts (McCracken et al., 1997
; Yue et al., 1997
). The N-terminal mRNA TPase domain contains an amino acid sequence motif that is characteristic of the active site of protein-tyrosine-phosphatase families (Fauman & Shaper, 1996
), suggesting that the catalytic mechanism of the higher eukaryotic mRNA TPase is similar to that of tyrosine phosphatases (Takagi et al., 1997
; Wen et al., 1998
). While the mRNA TPase domains of the higher eukaryotic capping enzyme show no sequence homology to the yeast TPases (McCracken et al., 1997
; Takagi et al., 1997
; Tsukamoto et al., 1997
, 1998a
; Yue et al., 1997
; Yamada-Okabe et al., 1998a
, b
), the C-terminal mRNA GTase domains are rather conserved (Shibagaki et al., 1992
; Shuman et al., 1994
; McCracken et al., 1997
; Yamada-Okabe et al., 1996
, 1998a
; Takagi et al., 1997
; Yue et al., 1997
; Tsukamoto et al., 1998a
).
Recently, the capping enzyme cDNAs of Crithidia fasciculata and Trypanosoma brucei were isolated (Silva et al., 1998 ). The protozoan capping enzymes also have a domain structure similar to that of the mammalian homologues; although the TPase activity of the enzyme remains to be confirmed, the enzyme consists of a single polypeptide bearing both putative mRNA TPase and mRNA GTase domains. The amino acid sequences of the putative mRNA TPase domains of the protozoan enzymes are unrelated to the yeast and mammalian mRNA TPases (McCracken et al., 1997
; Takagi et al., 1997
; Tsukamoto et al., 1997
, 1998a
; Yue et al., 1997
; Silva et al., 1998
; Yamada-Okabe et al., 1998a
, b
), however, strongly suggesting a different phylogeny of mRNA TPase in yeast, protozoa and mammals.
A cap structure is important for the binding of mRNA to the ribosomes, although some mRNAs form a certain secondary structure that mimics the function of the cap structure (Pelletier et al., 1988 ). Methylation of the terminal guanosine of mRNAs (called cap methylation in this paper) is not an absolute requirement for mRNA translation in vitro. A certain level of translation occurs even from mRNAs with an unmethylated cap structure (Held et al., 1977
); however, cap methylation significantly facilitates mRNA translation both in vitro and in vivo (Held et al., 1977
). In fact, ABD1-deficient S. cerevisiae can not survive (Mao et al., 1995
), and cap MTase activity is increased during Xenopus oocyte maturation, which stimulates translation of exogenous mRNAs bearing an unmethylated cap structure (Gillian-Daniel et al., 1998
). The S. cerevisiae ABD1 (ScABD1) gene on chromosome II and the human hMet (also called CMT1) cDNA both code for cap MTase (Pillutla et al., 1998
; Tsukamoto et al., 1998b
). By deletion and mutation analyses, the N-terminal 130 amino acids and C-terminal 10 amino acids of ScAbd1p were shown to be dispensable for the activity, and several residues important for the function have been identified (Mao et al., 1996
; Wang & Shuman, 1997
). In humans, the hMet/Cmt1p forms a ternary complex with Hce1p and the elongating form of the human RNA polymerase II (Pillutla et al., 1998
).
In this study, we isolated the cap MTase gene from the pathogenic fungus C. albicans. This gene, called CaABD1, encodes a 55 kDa protein, which exhibited cap MTase activity in vitro and functionally complemented an S. cerevisiae abd1 null mutant. Unlike the human cap MTase, the yeast Abd1 protein did not directly bind to mRNA TPase and mRNA GTase. By further exploring the highly conserved amino acid sequence motif that is characteristic of various cap MTases, additional residues important for catalysis were identified.
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METHODS |
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Expression and purification of the recombinant cap MTase.
The coding regions of CaABD1, ScABD1 and the hMet/CMT1 cDNA were cloned into the SmaI site of pGEX2T (Smith & Johnson, 1988 ). The resulting plasmids were transfected into Escherichia coli JM109, and they were induced by IPTG to express CaAbd1p, ScAbd1p and hMet/Cmt1p as fusion proteins with glutathione S-tranferase (GST) (Smith & Johnson, 1988
). Four hours after the addition of IPTG, the bacterial cells were harvested, suspended in a buffer containing 20 mM Tris/HCl (pH 7·5), 0·5 mM EDTA, 50 mM NaCl, 10 mM ß-mercaptoethanol, 10% (v/v) glycerol, 0·05% NP-40 and 1 mM PMSF, and lysed by sonication. After the cell debris was removed by centrifugation at 15000 g at 4 °C for 30 min, the recombinant Abd1 and hMet/Cmt1 proteins in the supernatant were purified by glutathioneSepharose CL-4B column chromatography as described previously (Yamada-Okabe et al., 1996
).
Assays for cap MTase.
The assays for cap MTase were carried out in a buffer containing 50 mM Tris/HCl (pH 7·5), 5 mM DTT, 50 µM S-adenosyl-L-methionine (SAM), G32pppA-terminated RNA and various amounts of the purified fusion proteins at 37 °C for 5 min, followed by incubation at 95 °C for 3 min. After adding sodium acetate (pH 5·5) to a final concentration of 50 mM and incubating with 10 U P1 nuclease (Seikagaku-kogyo) at 37 °C for 1 h, the reaction mixture was analysed by TLC using polyethylenimine cellulose plates (Mao et al., 1995 ), and the spots were visualized by an image analyser (Fuji BAS 2000). The G32pppA-terminated RNA was prepared by incubating 0·5 mg polyadenylic acid ml-1 in a buffer containing 20 mM Tris/HCl (pH 7·5), 3 mM MgCl2, 10 mM DTT, 1 µM [
-32P]GTP (10005000 c.p.m. pmol-1), 0·1mg purified GSTCaCet1p ml-1 (Yamada-Okabe et al., 1998b
) and 0·1mg purified GSTCaCgt1p ml-1 (Yamada-Okabe et al., 1996
) at 37 °C for 30 min. The produced G32pppA-terminated RNA was extracted with phenol and chloroform, and separated from unincorporated [
-32P]GTP by Sephadex G-25 column chromatography.
Generation of the S. cerevisiae abd1 null mutant strain.
To generate the S. cerevisiae abd1 null mutant strain, ScABD1 including its promoter and terminator was amplified by PCR from the S. cerevisiae genomic DNA extracted from strain A451 (MAT
can1 aro7 can1 leu2 trp1 ura3) as a template, and cloned into the HincII site of pUC19 and BamHI site of YEp24, generating pUC-ScABD1 and YEp-ScABD1, respectively. Primers used for amplifying ScABD1 were 5'-GGATCCGGATCCATCACTGAAGTCGCCGGATATTTT-3' and 5'-GGATCCGGATCCAATACTTTGCCGAGGACGAGAGTC-3'. For the disruption of ScABD1, the 1·2 kb HincIIBglII region of ScABD1 in pUC-ScABD1 was replaced with LEU2. Then, haploid S. cerevisiae YPH499 (MATa ade2 ura3 leu2 his3 trp1) was transformed with the ScABD1::LEU2 chimeric gene together with YEp-ScABD1 (Ito et al., 1983
) and several Leu+ Ura+ transformants were selected. The correct integration of LEU2 at the original ScABD1 locus was confirmed by PCR and by Southern blotting. Thus, the resulting abd1
null mutant (MATa ade2 ura3 leu2 his3 trp1 abd1
::LEU2 ABD1::URA3) grew in the absence of 5-fluoroorotic acid (5-FOA) but died in the presence of 5-FOA. To test the ability of CaABD1 and hMet/CMT1 to complement an S. cerevisiae abd1
null mutation, the coding regions of CaABD1, ScABD1 and hMet/CMT1 were cloned between the HindIII and PstI sites (between the ADH1 promoter and terminator) of pGBT9 (Clontech) that carried TRP1, and the resulting plasmids were transfected into the above S. cerevisiae abd1
null mutant strain, in which the endogenous ScABD1 gene was disrupted by LEU2, but where episomal copies of ScABD1 cloned in YEp24 were maintained (Ito et al., 1983
). To detect the proteins, the triple c-Myc sequence tag, which was excised from pMPY-3xMYC (Schneider et al., 1995
), was introduced at the 3' ends of the ScABD1, CaABD1 and hMet/CMT1 ORFs. The transformants were transferred to an agar plate containing 5-FOA and cultured at 30 °C for 3 d.
Yeast two-hybrid analysis.
The entire ORFs of the indicated yeast and human proteins were cloned into the SalI site of pGBT9 and pGAD424 (Clontech) to express these products as fusion proteins with the DNA-binding domain of Gal4p or with the transactivation domain of Gal4p. Then, the resulting plasmids were transfected into S. cerevisiae strain HF7c (MATa ura3-52 his3-200 lys2-801 ade2-101 trp1-901 leu2-3 112 gal4-542 gal80-538 LYS2::GAL1-HIS3 URA3::(GAL4 17-MERS)3CYC1-LACZ) where the HIS3 gene expression was driven by the DNA-binding and transactivation domains of Gal4p (Feilotter et al., 1994 ). After the transformation of HF7c (Ito et al., 1983
), the Leu+ Trp+ transformants were collected and tested for the ability to grow in the absence of histidine.
Site-directed mutagenesis.
A series of the CaABD1 mutants were obtained by the oligonucleotide-directed dual amber method (Hashimoto-Gotoh et al., 1995 ). The entire CaABD1 ORF was cloned into the SmaI site of pKF18k using a SmaI linker, and hybridized with oligonucleotides containing the indicated mutations. The mutant CaABD1 was excised from the vector, ligated into the SmaI site of pGEX-2T and also between the HindIII and PstI (between the ADH1 promoter and terminator) sites of pGBT9. All mutations were confirmed by DNA sequencing as described by Sambrook et al. (1989)
.
Western blotting.
The indicated amounts of proteins of the yeast cell extracts were separated on a 10% SDS-polyacrylamide gel, transferred electrophoretically to a PVDF membrane (Sambrook et al., 1989 ) and reacted with the anti-c-Myc monoclonal antibody (clone 9E10; Santa Cruz Biotechnology) and then with horseradish-peroxidase-conjugated protein A (Amersham). The Abd1 proteins were detected using an ECL protein-detection kit (Amersham).
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RESULTS |
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No direct interaction of CaAbd1p with mRNA GTase and mRNA TPase
Both ScAbd1p and hMet/Cmt1p are associated with the elongating form of RNA polymerase II (McCracken et al., 1997 ; Pillutla et al., 1998
), but their ways of associating with RNA polymerase II seem to be different. ScAbd1p is able to bind directly to the hyperphosphorylated C-terminal domain of RNA polymerase II (McCracken et al., 1997
), whereas hMet/Cmt1p is not; it is recruited to a RNA polymerase II complex through the interaction with Hce1p (Pillutla et al., 1998
). Although the yeast cap MTase activity was separated from those of mRNA GTase and mRNA TPase at an early stage of the purification (Mizumoto & Lipmann, 1979
), the above fact prompted us to examine whether the yeast cap MTase also interacts with mRNA GTase and mRNA TPase of yeast. Yeast two-hybrid analysis demonstrated that the co-transfection into the yeast HF7c cells of pGAD424-CaCET1 and pGBT9-CaCGT1 suppressed histidine auxotrophy, whereas those of pGAD424-CaABD1 and pGBT9-CaCGT1, and pGAD424-CaABD1 and pGBT9-CaCET1 did not (Fig. 4a
). Furthermore, pGAD424-hMet/CMT1 also did not support the growth of HF7c in the absence of histidine even when co-transfected with pGBT9-CaCGT1 or pGBT9-CaCET1 (Fig. 4a
). The same results were obtained with the S. cerevisiae homologues (Fig. 4b
). These results demonstrated that, unlike hMet/Cmt1p, the yeast Abd1 proteins are unable to directly interact with mRNA GTases and mRNA TPases.
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DISCUSSION |
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Although ScAbd1p directly interacts with the hyperphosphorylated C-terminal domain of RNA polymerase II (McCracken et al., 1997 ), hMet/Cmt1p forms a complex with the elongating form of RNA polymerase II not by directly binding to RNA polymerase II but by interacting with Hce1p (Pillutla et al., 1998
). By yeast two-hybrid analysis, we found that neither mRNA GTase nor mRNA TPase physically associates with the Abd1 proteins. Thus, yeast and humans apparently utilize a different mechanism to recruit cap MTase to an RNA polymerase II complex. Although cap MTases seem to be evolutionarily conserved proteins, their N-terminal regions are rather divergent. This may imply that the N-terminal regions of cap MTases are involved in proteinprotein interaction and determine the specificity of the interaction.
In ScAbd1p, the catalytic site should reside within the region between the amino acid positions 130 and 426, because the short ScAbd1p fragment encompassing the above region was active as cap MTase in vitro (Wang & Shuman, 1997 ). By comparing the amino acids of CaAbd1p and other cap MTases, we were able to define the amino acid sequence motif for cap MTase as Phe/Val-Leu-Asp/Glu-Leu/Met-Xaa-Cys-Gly-Lys-Gly-Gly-Asp-Leu-Xaa-Lys. In this motif, mutations of uncharacterized amino acids to alanine identified the additional residues essential for the activity. By combining the previous results of Mao et al. (1996)
and Wang & Shuman (1997)
, we concluded that the important amino acids in the motif are leucine at the second, aspartic acid or glutamic acid at the third, glycine at the seventh, glutamic acid at the eleventh and leucine at the twelfth positions. Moreover, Abd1p requires a hydrophobic amino acid with a certain length of side chain at the position 214 (corresponding to the leucine at the twelfth position within the motif) for its activity, because Leu214 could be substituted by isoleucine but not by valine. In contrast, Leu204 (corresponding to the leucine at the second position within the motif) was replaced either by isoleucine or valine without loss of activity and functionality. Thus, it seems that just the presence of a hydrophobic amino acid at this position is sufficient for activity. Hydrophobic amino acids are also conserved at the fourth position in the motif (see Fig. 5
). However, because the mutation of Leu206 of CaAbd1p to Ala did not affect the activity and functionality, a hydrophobic moiety at the fourth position in the motif may not be essential for the catalytic reactions by methyltransferases. Replacement of Lys210 or Gly212 with Ala reduced the enzyme activity, and the growth of S. cerevisiae abd1
null mutants, which was conferred by K210A or G212A, was retarded compared with that of the wild-type. This result coincides with the previous reports by Mao et al. (1996)
and Wang & Shuman (1997)
that the viability of yeast cells is contingent on a threshold level of MTase activity. Tyrosine at position 182 of ScAbd1p, which is adjacent to the conserved sequence motif of cap MTase, is converted to cysteine in CaAbd1p but is conserved as tryptophan in other organisms (Fig. 5
); however, alanine substitution for Tyr182 of ScAbd1p did not affect the activity and functionality of the enzyme (Wang & Shuman, 1997
).
Pillutla et al. (1998) also pointed out two other conserved amino acid sequence motifs, Leu-Ser/Lys-Pro/Ile-Gly-Gly-Xaa-Phe-Ile/Phe-Gly/Ala-Thr and Gly-Thr-Leu-Ser-Lys-Ser-Glu-Trp-Glu-Ala, which are called motif III and motif X, respectively (Pillutla et al., 1998
). Motif III is preserved in a wide variety of methyltransferases, including CaAbd1p, but motif X is missing in the fungal cap MTases (Pillutla et al., 1998
). An amino acid sequence that fits motif III is present and located at amino acid position 323 to 332 of CaAbd1p. In ScAbd1p, glycines at the second and the third positions and the last threonine in this motif were shown to be replaced by alanine without loss of the functionality (Wang & Shuman, 1997
). Neverthless, the physiological importance of other highly conserved amino acids in motif III and the role of motif III in catalysis awaits future study.
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ACKNOWLEDGEMENTS |
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Received 25 February 1999;
revised 8 June 1999;
accepted 28 June 1999.