Yeast mRNA Cap Methyltransferase Is a 50-Kilodalton Protein Encoded by an Essential Gene

Abstract

RNA (guanine-7-)methyltransferase, the enzyme responsible for methylating the 5 cap structure of eukaryotic mRNA, was isolated from extracts of Saccharomyces cerevisiae. The yeast enzyme catalyzed methyl group transfer from S-adenosyl-L-methionine to the guanosine base of capped, unmethylated poly(A). Cap methylation was stimulated by low concentrations of salt and was inhibited by S-adenosyl-L-homocysteine, a presumptive product of the reaction, but not by S-adenosyl-D-homocysteine. The methyltransferase sedimented in a glycerol gradient as a single discrete component of 3.2S. A likely candidate for the gene encoding yeast cap methyltransferase was singled out on phylogenetic grounds. The ABD1 gene, located on yeast chromosome II, encodes a 436-amino-acid (50-kDa) polypeptide that displays regional similarity to the catalytic domain of the vaccinia virus cap methyltransferase. That the ABD1 gene product is indeed RNA (guanine-7-)methyltransferase was established by expressing the ABD1 protein in bacteria, purifying the protein to homogeneity, and characterizing the cap methyltransferase activity intrinsic to recombinant ABD1. The physical and biochemical properties of recombinant ABD1 methyltransferase were indistinguishable from those of the cap methyltransferase isolated and partially purified from whole-cell yeast extracts. Our finding that the ABD1 gene is required for yeast growth provides the first genetic evidence that a cap methyltransferase (and, by inference, the cap methyl group) plays an essential role in cellular function in vivo.Eukaryotic mRNAs contain a 5Ј-terminal cap structure consisting of 7-methylguanosine linked to the 5Ј end of the transcript via a 5Ј-5Ј triphosphate bridge (3). Capping occurs by a series of three enzymatic reactions in which the 5Ј triphosphate terminus of a primary transcript is first cleaved to a diphosphate-terminated RNA by RNA triphosphatase, then capped with GMP by RNA guanylyltransferase, and finally methylated at the N-7 position of guanine by RNA (guanine-7-)methyltransferase: pppN(pN) n 3 ppN(pN) n ϩ P i ppN(pN) n ϩ pppG 7 G(5Ј)pppN(pN) n ϩ PP i G(5Ј)pppN(pN) n ϩ AdoMet 3 m7

Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA

“…Both Trm8 and Trm82 are widely conserved proteins that have not previously been ascribed a function, as determined by a search of the Saccharomyces Genome Database (Cherry et al+, 2002), SwissProt (Gasteiger et al+, 2001), TrEMBL (Bairoch & Apweiler, 2000), and CDD (Marchler-Bauer et al+, 2002)+ A BLAST search (Altschul et al+, 1997) (Stultz et al+, 1997), which are often implicated in macromolecular interaction and regulatory functions (Neer et al+, 1994)+ Neither Trm8 nor Trm82 is significantly related to KgmB from S. tenebrarius, a rRNA m 7 G methyltransferase associated with aminoglycoside resistance (Beauclerk & Cundliffe, 1987;Holmes & Cundliffe, 1991) or to Abd1, which catalyzes m 7 G formation in mRNA cap structures (Mao et al+, 1995), other than the methyltransferase domain shared by Trm8, Abd1, and numerous other methyltransferases (Niewmierzycka & Clarke, 1999)+ Diploid strains carrying homozygous deletions of either TRM8 or TRM82 are viable (Winzeler et al+, 1999), and closer examination of growth reveals no measurable growth rate differences, compared to wild-type control strains, in rich media containing glucose at 30 8C+ Strains lacking either Trm8 or Trm82 yield extracts with no detectable tRNA m 7 G methyltransferase activity, and have severely reduced m 7 G-modified tRNA in vivo…”

“…Virtually every tRNA in every organism carries multiple modifications of its nucleotides (Nishimura, 1979b), and the average yeast tRNA contains about 11 modifications+ More than 80 different modifications have been documented so far (Limbach et al+, 1994;Bjork, 1995), of which 22 have been found in yeast (Hopper & Martin, 1992)+ The most widely distributed and prevalent tRNA modification is methylation, which occurs in yeast tRNAs on the 29-OH of specific nucleotide residues, and at multiple base positions, including the 1 position of adenine, the 5 position of uracil, the 3 and 5 positions of cytosine, and the 1, 2, and 7 positions of guanine (Nishimura, 1979b;Sprinzl et al+, 1998)+ 7-methylguanosine (m 7 G) modification of tRNA is of particular interest for several reasons+ First, like a number of other modifications, m 7 G-modified tRNA is widely found in prokaryotes and eukaryotes (Sprinzl et al+, 1998), as well as in some archaea (Edmonds et al+, 1991), underscoring its likely importance+ In yeast, m 7 G has been reported in at least 11 tRNA species, including tRNA Cys , tRNA Trp , tRNA Pro , tRNA Met , tRNA Met-i , and two each of tRNA Val , tRNA Phe , and tRNA Lys (Sprinzl et al+, 1998)+ Second, in almost every tRNA in which m 7 G is found, it occurs at position 46 in the extra loop (Fig+ 1B), a site that is known to form tertiary interactions with the bases C-13 and G-22 in the crystal structure of yeast tRNA Phe (Kim et al+, 1974;Robertus et al+, 1974)+ Two rare exceptions include the finding of m 7 G at position 36 in some chloroplast species of tRNA Leu (Sprinzl et al+, 1998) and at position 34 in tRNA GCU Ser of starfish mito-chondria (Matsuyama et al+, 1998)+ Third, m 7 G, like m 1 A (Agris et al+, 1986), is one of the few tRNA modifications that produces a positively charged base under physiological conditions (see Fig+ 1A)+ Fourth, the same m 7 G modification occurs in every eukaryote as part of the mRNA cap; in yeast, this reaction is catalyzed by the essential Abd1 protein (Mao et al+, 1995)+ To begin to understand the role of m 7 G modification of tRNA, we sought to identify the corresponding structural gene from yeast+ Previously, a gene encoding a ribosomal RNA m 7 G methyltransferase associated with aminoglycoside resistance was cloned from Streptomyces tenebrarius (Beauclerk & Cundliffe, 1987;Holmes & Cundliffe, 1991), but this gene bears little resemblance to any yeast gene+ Purification of tRNA m 7 G me...…”

Mutational Analysis of Encephalitozoon cuniculi mRNA Cap (Guanine-N7) Methyltransferase, Structure of the Enzyme Bound to Sinefungin, and Evidence That Cap Methyltransferase Is the Target of Sinefungin's Antifungal Activity

“…Inhibition of cap methylation, in particular, has been touted as an anti-infective strategy based on two lines of evidence: (i) raising the cellular levels of AdoHcy by genetic or pharmacological inhibition of AdoHcy hydrolase blocks replication of many viruses (3, 4), and (ii) the AdoMet analog sinefungin (an inhibitor of cap methylation in vitro) inhibits the growth of diverse viruses, fungi, and protozoan parasites (5-11). Indeed, it was shown recently that sinefungin displays selectivity in inhibiting yeast cap methyltransferases versus the human enzyme in vivo (12).The Saccharomyces cerevisiae cap methyltransferase Abd1 has been extensively characterized genetically, but biochemical and structural analyses of the yeast enzyme are not as far advanced (13)(14)(15)(16). Cellular cap methyltransferases from humans, Xenopus laevis, Candida albicans, Schizosaccharomyces pombe, and Trypanosoma brucei have also been characterized (17)(18)(19)(20)(21)(22).…”

mRNA Capping Enzyme Requirement forCaenorhabditis elegans Viability

“…By contrast, in yeast species the three reactions for cap synthesis are each catalyzed by a separate enzyme (5)(6)(7). The TPase domain of metazoan capping enzymes includes a conserved active site motif, (I/V)HCXXGXXR(S/T)G, which is also characteristic of protein tyrosine phosphatases (8,9).…”