Isolation and Characterization of the Yeast mRNA Capping Enzyme β Subunit Gene Encoding RNA 5′-Triphosphatase, Which Is Essential for Cell Viability

The Saccharomyces cerevisiae RNA triphosphatase (Cet1) requires the presence of metal ion cofactors to catalyze its phosphohydrolase activity, the first step in the formation of the 5 -terminal cap structure of mRNAs. We have used endogenous tryptophan fluorescence studies to elucidate both the nature and the role(s) of the metal ions in the Cet1-mediated phosphohydrolase reaction. The association of Mg 2؉ , Mn 2؉ , and Co 2؉ ions with the enzyme resulted in a decrease in the intensity of the tryptophan emission spectrum. This decrease was then used to determine the apparent dissociation constants for these ions. Subsequent dual ligand titration experiments demonstrated that the metal ions bind to a common site, for which they compete. The kinetics of real-time metal ion binding to the Cet1 protein were also investigated, and the effects on RNA and nucleotide binding were evaluated. To provide additional insight into the relationship between Cet1 structure and metal ion binding, we correlated the effect of ion binding on protein structure using both circular dichroism and guanidium hydrochloride-induced denaturation as structural indicators. Our data indicate that binding of RNA, nucleotides, and metal ion cofactors does not lead to significant structural modifications of the Cet1 architecture. This suggests a model in which Cet1 possesses a preformed active site, and where major domain rearrangements are not required to form an active catalytic site. Finally, denaturation studies demonstrate that the metal ion cofactors can act by stabilizing the ground state binding of the phosphohydrolase substrate.Eukaryotic mRNAs possess a 5Ј-terminal cap structure that plays a critical role in the translation, stability, splicing, and transport of these mRNAs from the nucleus to the cytoplasm (1). The capping of mRNAs occurs immediately following the synthesis of pre-mRNAs, and involves three distinct enzymatic activities. First, the 5Ј-end of the pre-mRNA is hydrolyzed to a diphosphate by an RNA triphosphatase. This diphosphate end is then capped with GMP by an RNA guanylyltransferase, and finally methylated by an RNA (guanine-7) methyltransferase (2). Genetic experiments performed in Saccharomyces cerevisiae elegantly demonstrated that each of these activities is essential for yeast cell growth (3-8).The S. cerevisiae RNA triphosphatase (Cet1) 1 is a member of a family of metal-dependent phosphohydrolases that all possess the ability to hydrolyze the triphosphate end of mRNAs to a diphosphate in the presence of magnesium while also being able to hydrolyze NTPs to NDPs in the presence of either manganese or cobalt (9 -13). The protein has been extensively studied by a wide range of techniques, including crystallography, kinetics, and site-directed mutagenesis (14 -17). Additional studies showed that Cet1 shares many structural and mechanistic similarities with the RNA triphosphatases of various fungi, protozoan parasites, and DNA viruses (8, 11, 13, 14, 19 -23).Analysis of the Cet1 crystal structure reveals that the acti...

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confidence: 99%

Investigating the Role of Metal Ions in the Catalytic Mechanism of the Yeast RNA Triphosphatase

Bisaillon

Bougie

2003

“…Eukaryotic mRNAs are modified at the 5Ј end by addition of a m 7 GpppN "cap" (1). This characteristic structure is conserved among cellular and most viral gene transcripts, in many cases synthesized by different RNA polymerases (2,3).…”

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confidence: 99%

“…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).…”

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confidence: 99%

“…These TPases, like the protein phosphatases, form a cysteinyl-phosphate intermediate as part of their catalytic mechanism (8 -11). TPases of unicellular eukaryotes do not contain the Cys motif and use a different catalytic mechanism (7,12). GTases of metazoan and unicellular origin share several identifiable motifs characteristic of a nucleotidyltransferase superfamily (13), including a highly conserved KXDG sequence.…”

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confidence: 99%

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mRNA Capping Enzyme Requirement forCaenorhabditis elegans Viability

Srinivasan¹,

Piano²,

Shatkin³

2003

Capping of the initiated 5 ends of RNA polymerase II products is evolutionarily and functionally conserved from yeasts to humans. The m 7 GpppN cap promotes RNA stability, processing, transport, and translation. Deletion of capping enzymes in yeasts was shown to be lethal due to rapid exonucleolytic degradation of uncapped transcripts or failure of capped but unmethylated RNA to initiate protein synthesis. Using RNA interference and Caenorhabditis elegans we have found that RNA capping is also essential for metazoan viability. C. elegans bifunctional capping enzyme was cloned, and capping activity by the expressed protein as well as growth complementation of yeast deletion strains missing either RNA triphosphatase or guanylyltransferase required terminal sequences not present in the previously isolated cel-1 clone. By RNA interference analysis we show that cel-1 is required for embryogenesis. cel-1(RNAi) embryos formed cytoplasmic granules characteristic of a phenocluster of RNA processing genes and died early in development.Eukaryotic mRNAs are modified at the 5Ј end by addition of a m 7 GpppN "cap" (1). This characteristic structure is conserved among cellular and most viral gene transcripts, in many cases synthesized by different RNA polymerases (2, 3). Caps are produced co-transcriptionally on RNA polymerase II nascent transcripts via three enzymatic reactions: (i) RNA triphosphatase (TPase) 1 removes the ␥-phosphate from the initiated RNA 5Ј end, (ii) RNA guanylyltransferase (GTase) adds GMP from GTP to the resulting diphosphate terminus, and (iii) RNA (guanine-7) methyltransferase methylates the added guanosine. These early events in gene expression protect pre-mRNAs from exonucleolytic attack and, facilitated by cap-binding proteins (4), promote processing, transport of mature mRNAs to the cytoplasm, and translation initiation.Plants and metazoans contain bifunctional capping enzymes consisting of N-terminal TPase and C-terminal GTase domains, whereas the RNA methyltransferase is a separate protein (2, 3). By contrast, in yeast species the three reactions for cap synthesis are each catalyzed by a separate enzyme (5-7). 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). These TPases, like the protein phosphatases, form a cysteinyl-phosphate intermediate as part of their catalytic mechanism (8 -11). TPases of unicellular eukaryotes do not contain the Cys motif and use a different catalytic mechanism (7, 12). GTases of metazoan and unicellular origin share several identifiable motifs characteristic of a nucleotidyltransferase superfamily (13), including a highly conserved KXDG sequence. This motif, and notably the lysine, is necessary for formation of an enzyme-guanylylate GTase intermediate via phosphoamide linkage of GMP to the ⑀-amino group of the conserved lysine (2, 3).In yeast species loss of any one of the three cap-forming enzymes is lethal (5-7). Despite differences in sequenc...

“…Mammals encode a two-component capping system consisting of a bifunctional triphosphatase-guanylyltransferase polypeptide (named Mce1p in the mouse and Hce1p in humans) and a separate methyltransferase polypeptide (Hcm1p in humans) (2)(3)(4)(5)(6)(7)(8). The budding yeast Saccharomyces cerevisiae encodes a three-component system consisting of separate triphosphatase (Cet1p), guanylyltransferase (Ceg1p), and methyltransferase (Abd1p) gene products (9)(10)(11)(12). Cet1p and Ceg1p interact in trans to form a bifunctional heteromeric enzyme (13).…”

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confidence: 99%

Characterization of the mRNA Capping Apparatus of Candida albicans

Schwer¹,

Lehman²,

Saha³

et al. 2001

The mRNA capping apparatus of the pathogenic fungus Candida albicans consists of three components: a 520-amino acid RNA triphosphatase (CaCet1p), a 449-amino acid RNA guanylyltransferase (Cgt1p), and a 474-amino acid RNA (guanine-N7-)-methyltransferase (Ccm1p). The fungal guanylyltransferase and methyltransferase are structurally similar to their mammalian counterparts, whereas the fungal triphosphatase is mechanistically and structurally unrelated to the triphosphatase of mammals. Hence, the triphosphatase is an attractive antifungal target. Here we identify a biologically active C-terminal domain of CaCet1p from residues 202 to 520. We find that Binding is enhanced when the C-terminal domain is phosphorylated on both Ser-2 and Ser-5 of the YSPTSPS heptad repeat. We show that the entire threecomponent Saccharomyces cerevisiae capping apparatus can be replaced by C. albicans enzymes. Isogenic yeast cells expressing "all-Candida" versus "all-mammalian" capping components can be used to screen for cytotoxic agents that specifically target the fungal capping enzymes.