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

Current models of mRNA decay in yeast posit that 3' deadenylation precedes enzymatic removal of the 5' cap, which then exposes the naked end to 5' exonuclease action. Here, we analyzed gene expression in Saccharomyces cerevisiae cells bearing conditional mutations of Ceg1 (capping enzyme), a 52 kDa protein that transfers GMP from GTP to the 5' end of mRNA to form the GpppN cap structure. Shift of ceg1 mutants to restrictive temperature elicited a rapid decline in the rate of protein synthesis, which correlated with a sharp reduction in the steady-state levels of multiple individual mRNAs. ceg1 mutations prevented the accumulation of SSA1 and SSA4 mRNAs that were newly synthesized at the restrictive temperature. Uncapped poly(A)+ SSA4 mRNA accumulated in cells lacking the 5' exoribonuclease Xrn1. These findings provide genetic evidence for the long-held idea that the cap guanylate is critical for mRNA stability. The deadenylation-decapping-degradation pathway appears to be short-circuited when Ceg1 is inactivated.

RNA (guanine-7-)-methyltransferase is the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA. The Saccharomyces cerevisiae enzyme is a 436-amino-acid protein encoded by the essential ABD1 gene. In this study, deletion and point mutations in ABD1 were tested for the ability to support growth of an abd1 null strain. Elimination of 109 amino acids from the N terminus had no effect on cell viability, whereas a more extensive N-terminal deletion of 155 residues was lethal, as was a C-terminal deletion of 55 amino acids. Alanine substitution mutations were introduced at eight conserved residues within a 206-amino-acid region of similarity between ABD1 and the methyltransferase domain of the vaccinia virus capping enzyme. ABD1 alleles H253A (encoding a substitution of alanine for histidine at position 253), T282A, E287A, E361A, and Y362A were viable, whereas G174A, D178A, and Y254A were either lethal or severely defective for growth. Alanine-substituted and amino-truncated ABD1 proteins were expressed in bacteria, purified, and tested for cap methyltransferase activity in vitro. Mutations that were viable in yeast cells had either no effect or only a moderate effect on the specific methyltransferase activity of the mutated ABD1 protein, whereas mutations that were deleterious in vivo yielded proteins that were catalytically defective in vitro. These findings substantiate for the first time the long-held presumption that cap methylation is an essential function in eukaryotic cells.

The (guanine-7-)methyltransferase domain of the vaccinia virus mRNA capping enzyme is composed of the C-terminal portion of the D1 subunit, D1(498-844), heterodimerized with the D12 protein. In order to identify protein structural elements involved in cap methylation, we introduced eight alanine substitution mutations within two sequence motifs of D1(498-844)-(594)VLAIDFGNG(602) and (681)IHYSF(685)--that are conserved in the cap methyltransferase from yeast. The D1(498-844)-Ala proteins were coexpressed in bacteria with the D12 subunit, and the recombinant D1(498-844)/D12 heterodimers were purified. Alanine substitutions at five positions--Asp-598, Gly-602, Ile-681, Ser-684, and Phe-685--had little or no effect on methyltransferase activity. Mutations at three conserved residues were deleterious. Alanine substitution at Gly-600 reduced the specific activity to 4% of that of the wild-type protein. Substitutions at His-682 and Tyr-683 reduced activity to 4% and 0.05%, respectively. By further mutating Tyr-683 to Phe and Ser, we established that the aromatic group was essential for cap methylation, whereas the hydroxyl moiety was dispensable. Specific binding of the methyltransferase to the RNA cap was demonstrated by UV cross-linking to [32P]GMP-labeled capped poly(A). Label transfer occurred exclusively to the D1(498-844) subunit and was competed by the cap analogs GpppA and m7GpppA. Cap-specific cross-linking to m7GpppA(pA)n was stimulated by AdoHcy, whereas cross-linking to GpppA(pA)n was unaffected by AdoHcy, but stimulated by AdoMet. We suggest that occupancy of the methyl donor site either enhances the affinity for the cap guanosine or alters the protein interface so that a photoreactive moiety is brought closer to the cap structure. The catalytically defective H682A, Y683A, and Y683S mutant methyltransferases were unable to cross-link to the cap in the presence of AdoHcy. The catalytically defective G600A mutant did cross-link to the cap in the presence of AdoHcy, suggesting that this mutation affects the chemical step of transmethylation.

Alleles specifically defective in telomeric silencing were generated by in vitro mutagenesis of the yeast RAP1 gene. The most severe phenotypes occur with three mutations in the C-terminal 28 amino acids. Two of the alleles are nonsense mutations resulting in truncated repressor/activator protein 1 (RAP1) species lacking the C-terminal 25-28 amino acids; the third allele is a missense mutation within this region. These alleles define a novel 28-amino acid region, termed the C-terminal tail domain, that is essential for telomeric and HML silencing. Using site-directed mutagenesis, an 8-amino acid region (amino acids 818-825) that is essential for telomeric silencing has been localized within this domain. Further characterization of these alleles has indicated that the C-terminal tail domain also plays a role in telomere size control. The function of the C-terminal tail in telomere maintenance is not mediated through the RAP1 interacting factor RIF1: rap1 alleles defective in both the C-terminal tail and RIF1 interaction domains have additive effects on telomere length. Overproduction of SIR3, a dose-dependent enhancer of telomeric silencing, suppresses the telomeric silencing, but not length, phenotypes of a subset of C-terminal tail alleles. In contrast, an allele that truncates the terminal 28 amino acids of RAP1 is refractory to SIR3 overproduction. These results indicate that the C-terminal tail domain is required for SIR3-dependent enhancement of telomeric silencing. These data also suggest a distinct set of C-terminal requirements for telomere size control and telomeric silencing.