Covalent guanylyl intermediate formed by HeLa cell mRNA capping enzyme.

Wang, D; Furuichi, Yasuhiro; Shatkin, Aaron J.

doi:10.1128/mcb.2.8.993

Cited by 31 publications

(11 citation statements)

References 23 publications

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“…1B. The mouse and human predicted proteins are 95% identical and consist of 597 amino acids with calculated molecular masses that are in close agreement with the 68 kDa reported previously for mammalian capping enzymes (10,28). mRNA capping proceeds by removal of the ␥-phosphate from 5Ј-triphosphate termini followed by GMP transfer from guanylylated capping enzyme intermediate to the resulting 5Ј-diphosphate ends.…”

Section: Nucleotidyl Transferase Superfamily Sequence Motifs Are Conssupporting

confidence: 81%

Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II

Yue

Maldonado

Pillutla

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

217

279

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ABSTRACT5-Capping is an early mRNA modification that has important consequences for downstream events in gene expression. We have isolated mammalian cDNAs encoding capping enzyme. They contain the sequence motifs characteristic of the nucleotidyl transferase superfamily. The predicted mouse and human enzymes consist of 597 amino acids and are 95% identical. Mouse cDNA directed synthesis of a guanylylated 68-kDa polypeptide that also contained RNA 5-triphosphatase activity and catalyzed formation of RNA 5-terminal GpppG. A haploid strain of Saccharomyces cerevisiae lacking mRNA guanylyltransferase was complemented for growth by the mouse cDNA. Conversion of Lys-294 in the KXDG-conserved motif eliminated both guanylylation and complementation, identifying it as the active site. The K294A mutant retained RNA 5-triphosphatase activity, which was eliminated by N-terminal truncation. Full-length capping enzyme and an active C-terminal fragment bound to the elongating form and not to the initiating form of polymerase. The results document functional conservation of eukaryotic mRNA guanylyltransferases from yeast to mammals and indicate that the phosphorylated C-terminal domain of RNA polymerase II couples capping to transcription elongation. These results also explain the selective capping of RNA polymerase II transcripts.Addition of a 5Ј-terminal cap is an important, early event in mRNA formation (1). This structural hallmark of most eukaryotic mRNAs enhances splicing (2-4), transport (5), translation (6), and stability (7,8) and is essential for viability (9).Caps are formed on nascent nuclear pre-mRNAs by conversion of 5Ј-tri-diphosphate to 5Ј-diphosphate ends, followed by addition of GMP and methylation (1, 10). The guanylyltransfer reaction characterized in various systems involves formation of an active enzyme intermediate containing GMP covalently attached to lysine (11). In yeast, mRNA capping enzyme consists of separate subunits for RNA 5Ј-triphosphatase and guanylyltransferase activities (9, 12). cDNA clones coding for mRNA guanylyltransferase in Saccharomyces cerevisiae (9), Schizosaccharomyces pombe (13), and Candida albicans (14) have been sequenced. Each contains the active site lysine in KXDG (13, 15), one of several highly conserved motifs characteristic of a superfamily of nucleotidyl transferases (16). A number of viral capping enzymes also contain these diagnostic sequence motifs, and the recently solved structure of capping enzyme from Chlorella virus PBCV-1 suggests that specific residues in these motifs are important for binding GTP (17). Despite this detail of sequence and structure information, no metazoan capping enzyme previously has been cloned and characterized.To explore the molecular interactions that result in selective capping of RNA polymerase II (pol II) transcripts in mammalian cells, we have isolated and characterized cDNA clones that code for the human and mouse capping enzymes. Functional studies demonstrated that the mammalian enzyme complements the lethality of a S. cerevisiae mu...

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Section: Nucleotidyl Transferase Superfamily Sequence Motifs Are Conssupporting

confidence: 81%

Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II

Yue

Maldonado

Pillutla

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

217

279

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“…Z75525). This is in good agreement with the sizes of the other higher eukaryotic guanylyltransferases [human (68 kDa), rat liver (69 kDa), calf thymus (65 kDa), brine shrimp (73 kDa), and wheat germ (77 kDa)], which were determined by SDS͞ PAGE analysis of the covalent enzyme-GMP catalytic intermediate (23)(24)(25)(26)(27)(28).…”

Section: Biochemistry: Wang Et Al Proc Natl Acad Sci Usa 94 (1997)supporting

confidence: 62%

Phylogeny of mRNA capping enzymes

Wang

Deng

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

108

145

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The m 7 GpppN cap structure of eukaryotic mRNA is formed cotranscriptionally by the sequential action of three enzymes: RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7)-methyltransferase. A multifunctional polypeptide containing all three active sites is encoded by vaccinia virus. In contrast, fungi and Chlorella virus encode monofunctional guanylyltransferase polypeptides that lack triphosphatase and methyltransferase activities. Transguanylylation is a two-stage reaction involving a covalent enzyme-GMP intermediate. The active site is composed of six protein motifs that are conserved in order and spacing among yeast and DNA virus capping enzymes. We performed a structurefunction analysis of the six motifs by targeted mutagenesis of Ceg1, the Saccharomyces cerevisiae guanylyltransferase. Essential acidic, basic, and aromatic functional groups were identified. The structural basis for covalent catalysis was illuminated by comparing the mutational results with the crystal structure of the Chlorella virus capping enzyme. The results also allowed us to identify the capping enzyme of Caenorhabditis elegans. The 573-amino acid nematode protein consists of a C-terminal guanylyltransferase domain, which is homologous to Ceg1 and is strictly conserved with respect to all 16 amino acids that are essential for Ceg1 function, and an N-terminal phosphatase domain that bears no resemblance to the vaccinia triphosphatase domain but, instead, has strong similarity to the superfamily of protein phosphatases that act via a covalent phosphocysteine intermediate.mRNA 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 by RNA triphosphatase, then capped with GMP by RNA guanylyltransferase, and methylated at the N7 position of guanine by RNA (guanine-7)-methyltransferase (1). To date, only the guanylyltransferase reaction mechanism has been dissected in detail. Transfer of GMP from GTP to the 5Ј-diphosphate terminus of RNA occurs in a two-stage reaction involving a covalent enzyme-GMP intermediate (2). The GMP is linked to the enzyme through a phosphoamide (P-N) bond to the -amino group of a lysine residue. This structure is analogous to the enzyme (-Lys)-AMP intermediate formed when DNA ligase reacts with ATP.

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“…To assess whether 5'-terminal guanylylation is necessary for runoff transcript formation, globin and adenovirus DNAs were tested as templates in reaction mixtures containing AMPP(NH)P in place of ATP. Capping of HeLa cell pre-mRNA involves conversion of the nascent 5'-terminal triphosphate to a diphosphorylated end which is the guanylylate acceptor (27,28). Consequently, the presence of the non-hydrolyzable ,-y-imido analog prevents capping of transcripts that normally begin with ATP, including globin 1.4-kb transcripts (15) and adenovirus-specific products.…”

Section: Resultsmentioning

confidence: 99%

Initiation by RNA polymerase II and formation of runoff transcripts containing unblocked and unmethylated 5' termini.

Ernst¹,

Filipowicz²,

Shatkin³

1983

Mol. Cell. Biol.

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Transcription of cloned adenovirus, P-globin, and retrovirus long terminal repeat DNAs in HeLa whole-cell lysate was inhibited by S-adenosylhomocysteine. However, full-length 1.7-kilobase transcripts made on adenovirus 2 late promoter DNA contained 5'-terminal GpppA, consistent with specific initiation and runoff synthesis in the absence of product methylation. Formation of runoff transcripts including retrovirus RNAs that normally contain 5'-m7GpppGpmC was not decreased by replacing GTP with non-hydrolyzable analogs, and Rousassociated virus-2 runoff products made in the presence of GTP-'y-S contained 5'-terminal y-S-pppGpC. The results indicate that capping and specific transcript synthesis by RNA polymerase II are not obligatorily linked in HeLa whole-cell lysate. Accurate initiation is dependent on ATP hydrolysis, and in contrast to GTP, replacement of ATP by 5'-adenylyl-imidodiphosphate blocked specific initiation of transcripts that start with either GTP (Rous-associated virus-2, Rousassociated virus-0) or ATP (P-globin, adenovirus).Eucaryotic cellular mRNAs contain a characteristic 5'-terminal "cap," m7GpppN, that is added to RNA polymerase II nascent products in the nucleus at an early stage of mRNA formation (1,21,24). The minimum transcript size necessary for capping in vivo has not been determined. However, results obtained for several viral mRNAs suggest that capping may be coupled to initiation in some transcription systems. For example, 5'-terminal oligonucleotides initiated in vitro by reovirion-associated transcriptase contain a cap at the dinucleotide level (6,30), and capping appears to be a pretranscriptional event in the formation of mRNA by the double-stranded RNA-dependent RNA polymerase in insect cytoplasmic polyhedrosis virus (5). In addition, it was reported recently that Sadenosylhomocysteine, a competitive inhibitor of methylation, markedly diminishes initiation by RNA polymerase II in HeLa whole-cell lysate (13).Other studies suggest that capping and initiation are not obligatorily linked. RNA polymerase II and mRNA guanylyltransferase can be readily separated during fractionation of HeLa whole-cell lysate (22) cloned adenovirus 2 late promoter DNA in HeLa nuclear extract are uncapped until they are elongated further. In addition, full-length reovirus transcripts containing 5'-terminal ppGpC or p(NH)ppGpC can be synthesized in incubation mixtures that contain S-adenosylhomocysteine (7) or the non-hydrolyzable I,-yimido analog in place of GTP (8).These diverse observations encouraged us to test the effect of blocking methylation and 5' guanylylation on specific initiation and runoff transcript formation by RNA polymerase II in HeLa whole-cell lysate. Although we confirmed that 0.5 mM S-adenosylhomocysteine inhibits runoff transcript synthesis, >95% of the fulllength adenovirus 2 products made in the presence of the inhibitor contained 5'-terminal GpppA, demonstrating that accurate initiation and generation of runoff transcripts can proceed in the absence of methylation. Guanylylation ...

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Covalent guanylyl intermediate formed by HeLa cell mRNA capping enzyme.

Cited by 31 publications

References 23 publications

Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II

Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II

Phylogeny of mRNA capping enzymes

Initiation by RNA polymerase II and formation of runoff transcripts containing unblocked and unmethylated 5' termini.

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