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Stewart Shuman - One of the best experts on this subject based on the ideXlab platform.
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How an mRNA Capping Enzyme reads distinct RNA polymerase II and Spt5 CTD phosphorylation codes
Genes & Development, 2014Co-Authors: Selom K. Doamekpor, Stewart Shuman, Beate Schwer, Ana M. Sanchez, Christopher D. LimaAbstract:Essential eukaryal mRNA processing events are targeted to nascent transcripts made by RNA polymerase II (Pol2) via physical interactions of the processing machineries with the C-terminal domain (CTD) of Rpb1, the largest subunit of Pol2. The Pol2 CTD consists of tandemly repeated heptapeptides of consensus sequence Y1S2P3T4S5P6S7. The inherently plastic Pol2 CTD structure is sculpted by dynamic phosphorylation and dephoshorylation of the Tyr1, Ser2, Thr4, Ser5, and Ser7 residues and by cis–trans isomerization of the prolines. With up to 128n potential Pol2 CTD primary structures (where n is the number of heptads), the Pol2 CTD provides information about the state of the transcription complex (a CTD code) that is read by diverse Pol2 CTD receptor proteins that control transcription, modify chromatin structure, and catalyze or regulate mRNA Capping, splicing, and polyadenylation (Buratowski 2009; Corden 2013; Eick and Geyer 2013; Geronimo et al. 2013). The individual amino acids of the Pol2 CTD heptad and their phosphorylation marks are coding “letters” that have distinct outputs with respect to receptor recognition and impact on cellular gene expression (Schwer et al. 2014). The number of heptad repeats comprising the Pol2 CTD, the minimal number of repeats required for viability, and the prevalence of nonconsensus coding letters tend to increase with progression from unicellular to multicellular eukarya, presumably as a means to orchestrate the actions of increasing numbers of Pol2 CTD receptors. We envision a core Pol2 CTD code that governs events common to most eukarya; for example, the essential function of the Ser5-PO4–Pro6 coding “word” in recruiting mRNA Capping Enzymes to the Pol2 elongation complex (Schwer and Shuman 2011; Schwer et al. 2012), which is conserved in budding yeast, fission yeast, and mammals, notwithstanding major differences in the genetic and physical organization of the Capping apparatus in these taxa (Lima et al. 1999; Changela et al. 2001; Pei et al. 2001; Fabrega et al. 2003; Ghosh et al. 2011). The RNA Capping Enzymes are the first processing factors to act on the growing Pol2 transcript; indeed, Capping can commence after synthesis of only a 19- to 22-mer nascent RNA, the point at which the 5′ triphosphate RNA end is extruded from the RNA-binding pocket of elongating Pol2 and becomes accessible to the Capping Enzymes (Chiu et al. 2002). The m7GpppN cap is formed by three successive enzymatic reactions: (1) RNA triphosphatase (TPase) hydrolyzes the RNA 5′ triphosphate end (pppRNA) to a diphosphate (ppRNA) plus inorganic phosphate, (2) GTP:RNA guanylyltransferase (GTase) converts ppRNA to GpppRNA via a covalent GTase-(lysyl-Nζ)–GMP intermediate, and (3) AdoMet:RNA(guanine-N7)-methyltransferase (MTase) converts GpppRNA to m7GpppRNA (Gu and Lima 2005). Whereas it had long been assumed that Capping is a constitutive and efficient process for most mRNAs, this view may be too simplistic. The existence of factors that can stimulate Capping of specific transcripts and the discovery of Enzymes that mediate the decay of partially processed RNA intermediates in the cap synthetic pathway suggest that Capping efficiency is a dynamic process (Chiu et al. 2002; Jiao et al. 2010, 2013; Chang et al. 2012). One potential way to regulate Capping is by influencing the timely recruitment of the Capping Enzymes to the Pol2 elongation complex. A conserved theme among eukarya is the direct binding of the GTase component to the Ser5-PO4 form of the Pol2 CTD (Ho and Shuman 1999; Fabrega et al. 2003; Ghosh et al. 2011). In mammals and budding yeast, the TPase components are recruited passively to the Pol2 CTD by virtue of their physical association with the GTase: in cis as a covalently fused TPase–GTase Enzyme, Mce1, in mammals (Ho and Shuman 1999; Changela et al. 2001) or in trans as separately encoded subunits of a TPase•GTase complex in budding yeast (Ho et al. 1999; Gu et al. 2010). The fission yeast Schizosaccharomyces pombe has a distinctive strategy for targeting cap formation to Pol2 transcripts whereby the TPase (Pct1) and GTase (Pce1) Enzymes are not associated physically but instead bind independently to the Ser5-phosphorylated Pol2 CTD (Pei et al. 2001). Mammalian and fungal Capping Enzymes can also gain access to nascent Pol2 transcripts via physical interactions with the transcription elongation factor Spt5 (Wen and Shatkin 1999; Pei and Shuman 2002; Pei et al. 2006; Lidschreiber et al. 2013). Spt5 is a large polypeptide (∼1000–1200 amino acids) composed of multiple domain modules that associates with the Pol2 transcription complex shortly after initiation and can exert negative and positive effects on transcription elongation (Hartzog and Fu 2013). Fission yeast Spt5 has a distinctive C-terminal repeat domain (the “Spt5 CTD”) composed of 18 repeats of a nonapeptide motif (consensus: T1P2A3W4N5S6G7S8K9) that (1) binds the fission yeast RNA Capping Enzymes Pct1 and Pce1 (Pei and Shuman 2002) and (2) is targeted for threonine phosphorylation by the fission yeast Cdk9 kinase (Pei and Shuman 2002, 2003; Viladevall et al. 2009). Alignment of Spt5 CTD elements in metazoan and fungal species reveals substantive differences, although each contains a series of Thr–Pro or Ser–Pro dipeptide motifs followed by a hydrophobic side chain two residues downstream (Pei and Shuman 2002). The hydrophobic side chain is typically tryptophan in S. pombe; this position is substituted to a tyrosine or histidine in humans. Available genetic evidence points to overlapping roles of the CTDs of fission yeast Pol2 and Spt5 in recruiting the Capping Enzymes in vivo (Schneider et al. 2010). Unlike the Capping Enzyme•Pol2 CTD interactions, which stringently depend on the Ser5-PO4 mark, the binding of fission yeast Pct1 and Pce1 to the Spt5 CTD is independent of Thr1 phosphorylation (Pei et al. 2001; Pei and Shuman 2002). The structural principles underpinning Capping Enzyme•Pol2 CTD interactions were initially elucidated via cocrystallization of Candida albicans GTase (Cgt1) and mammalian GTase (Mce1) bound to Ser5-phosphorylated Pol2 CTD peptide ligands (Fabrega et al. 2003; Ghosh et al. 2011). These two cellular GTases are structurally homologous Enzymes composed of two domains: (1) an N-terminal nucleotidyltransferase (NTase) module that contains the guanylate-binding pocket and (2) a C-terminal OB-fold module. A comparison of the Candida and mammalian GTase•Pol2 CTD structures was most revealing because, although the docking sites for Pol2 CTD lie on the surface of the NTase domain in both cases, the docking sites are physically distinct and have little overlap, the atomic contacts to Pol2 CTD are different, and virtually none of the Pol2 CTD-interacting side chains of mammalian GTase are conserved in the Candida Enzyme (Ghosh et al. 2011). Thus, Capping Enzymes from different taxa have evolved different strategies to read the Pol2 CTD code. Here we extend the structural analysis to the GTase Enzyme of fission yeast and its interactions with the CTDs of Pol2 and Spt5. We report crystal structures of Pce1 bound to Pol2 CTD and Spt5 CTD ligands. Key findings are that (1) the CTDs of Pol2 and Spt5 interact with completely distinct sites on the NTase and OB domains of the fission yeast GTase, respectively, and (2) whereas the interface of GTase with the Pol2 CTD is dependent on Ser5 phosphorylation, GTase binding to Spt5 CTD is antagonized by Thr1 phosphorylation. We probed by structure-guided mutagenesis the contributions of the GTase•CTD interfaces to CTD binding in vitro and Capping Enzyme function in vivo. Our findings cohere into a model in which the Capping Enzyme recognizes and responds to distinct coding cues and phosphorylation marks in the Pol2 and Spt5 CTDs.
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Crystal structure of vaccinia virus mRNA Capping Enzyme provides insights into the mechanism and evolution of the Capping apparatus
Structure, 2014Co-Authors: O.j.p. Kyrieleis, Stewart Shuman, Jonathan Chang, Marcos De La Peña, Stephen CusackAbstract:Summary Vaccinia virus Capping Enzyme is a heterodimer of D1 (844 aa) and D12 (287 aa) polypeptides that executes all three steps in m 7 GpppRNA synthesis. The D1 subunit comprises an N-terminal RNA triphosphatase (TPase)-guanylyltransferase (GTase) module and a C-terminal guanine-N7-methyltransferase (MTase) module. The D12 subunit binds and allosterically stimulates the MTase module. Crystal structures of the complete D1⋅D12 heterodimer disclose the TPase and GTase as members of the triphosphate tunnel metalloEnzyme and covalent nucleotidyltransferase superfamilies, respectively, albeit with distinctive active site features. An extensive TPase-GTase interface clamps the GTase nucleotidyltransferase and OB-fold domains in a closed conformation around GTP. Mutagenesis confirms the importance of the TPase-GTase interface for GTase activity. The D1⋅D12 structure complements and rationalizes four decades of biochemical studies of this Enzyme, which was the first Capping Enzyme to be purified and characterized, and provides new insights into the origins of the Capping systems of other large DNA viruses.
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Structural Insights to How Mammalian Capping Enzyme Reads the CTD Code
Molecular Cell, 2011Co-Authors: Agnidipta Ghosh, Stewart Shuman, Christopher D. LimaAbstract:Summary Physical interaction between the phosphorylated RNA polymerase II carboxyl-terminal domain (CTD) and cellular Capping Enzymes is required for efficient formation of the 5′ mRNA cap, the first modification of nascent mRNA. Here, we report the crystal structure of the RNA guanylyltransferase component of mammalian Capping Enzyme (Mce) bound to a CTD phosphopeptide. The CTD adopts an extended β-like conformation that docks Tyr1 and Ser5-PO 4 onto the Mce nucleotidyltransferase domain. Structure-guided mutational analysis verified that the Mce-CTD interface is a tunable determinant of CTD binding and stimulation of guanylyltransferase activity, and of Mce function in vivo. The location and composition of the CTD binding site on mammalian Capping Enzyme is distinct from that of a yeast Capping Enzyme that recognizes the same CTD primary structure. Thus, Capping Enzymes from different taxa have evolved different strategies to read the CTD code.
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Separable functions of the fission yeast Spt5 carboxyl-terminal domain (CTD) in Capping Enzyme binding and transcription elongation overlap with those of the RNA polymerase II CTD.
Molecular and Cellular Biology, 2010Co-Authors: Susanne Schneider, Stewart Shuman, Yi Pei, Beate SchwerAbstract:An interaction network connecting mRNA Capping Enzymes, the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD), elongation factor Spt5, and the Cdk7 and Cdk9 protein kinases is thought to comprise a transcription elongation checkpoint. A crux of this network is Spt5, which regulates early transcription elongation and has an imputed role in pre-mRNA processing via its physical association with Capping Enzymes. Schizosaccharomyces pombe Spt5 has a distinctive CTD composed of tandem nonapeptide repeats of the consensus sequence 1TPAWNSGSK9. The Spt5 CTD binds the Capping Enzymes and is a substrate for threonine phosphorylation by the Cdk9 kinase. Here we report that deletion of the S. pombe Spt5 CTD results in slow growth and aberrant cell morphology. The severity of the spt5-ΔCTD phenotype is exacerbated by truncation of the Pol II CTD and ameliorated by overexpression of the Capping Enzymes RNA triphosphatase and RNA guanylyltransferase. These results suggest that the Spt5 and Pol II CTDs play functionally overlapping roles in Capping Enzyme recruitment. We probed structure-activity relations of the Spt5 CTD by alanine scanning of the consensus nonapeptide. The T1A change abolished CTD phosphorylation by Cdk9 but did not affect CTD binding to the Capping Enzymes. The T1A and P2A mutations elicited cold-sensitive (cs) and temperature-sensitive (ts) growth defects and conferred sensitivity to growth inhibition by 6-azauracil that was exacerbated by partial truncations of the Pol II CTD. The T1A phenotypes were rescued by a phosphomimetic T1E change but not by Capping Enzyme overexpression. These results imply a positive role for Spt5 CTD phosphorylation in Pol Il transcription elongation in fission yeast, distinct from its Capping Enzyme interactions. Viability of yeast cells bearing both Spt5 CTD T1A and Pol II CTD S2A mutations heralds that the Cdk9 kinase has an essential target other than Spt5 and Pol II CTD-Ser2.
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Characterization of a Trifunctional Mimivirus mRNA Capping Enzyme and Crystal Structure of the RNA Triphosphatase Domain
Structure, 2008Co-Authors: Delphine Benarroch, Paul Smith, Stewart ShumanAbstract:The RNA triphosphatase (RTPase) components of the mRNA Capping apparatus are a bellwether of eukaryal taxonomy. Fungal and protozoal RTPases belong to the triphosphate tunnel metalloEnzyme (TTM) family, exemplified by yeast Cet1. Several large DNA viruses encode metal-dependent RTPases unrelated to the cysteinyl-phosphatase RTPases of their metazoan host organisms. The origins of DNA virus RTPases are unclear because they are structurally uncharacterized. Mimivirus, a giant virus of amoeba, resembles poxviruses in having a trifunctional Capping Enzyme composed of a metal-dependent RTPase module fused to guanylyltransferase (GTase) and guanine-N7 methyltransferase domains. The crystal structure of mimivirus RTPase reveals a minimized tunnel fold and an active site strikingly similar to that of Cet1. Unlike homodimeric fungal RTPases, mimivirus RTPase is a monomer. The mimivirus TTM-type RTPase-GTase fusion resembles the Capping Enzymes of amoebae, providing evidence that the ancestral large DNA virus acquired its Capping Enzyme from a unicellular host.
Kiyohisa Mizumoto - One of the best experts on this subject based on the ideXlab platform.
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The essential role for the RNA triphosphatase Cet1p in nuclear import of the mRNA Capping Enzyme Cet1p-Ceg1p complex of Saccharomyces cerevisiae.
PLoS ONE, 2013Co-Authors: Naoki Takizawa, Toshinobu Fujiwara, Manabu Yamasaki, Ayako Saito, Akira Fukao, Akio Nomoto, Kiyohisa MizumotoAbstract:mRNA Capping is the first cotranscriptional modification of mRNA in the nucleus. In Saccharomyces cerevisiae, the first two steps of mRNA Capping are catalyzed by the RNA triphosphatase Cet1p and the RNA guanylyltransferase Ceg1p. Cet1p and Ceg1p interact to form a mRNA Capping Enzyme complex and the guanylyltransferase activity of Ceg1p is stimulated by binding with Cet1p. The Cet1p-Ceg1p complex needs to be transported into the nucleus, where mRNA Capping occurs. However, the molecular mechanism of nuclear transport of the Cet1p-Ceg1p complex is not known. Here, we show that Cet1p is responsible and that the Cet1p-Ceg1p interaction is essential for the nuclear localization of the Cet1p-Ceg1p complex. The results indicate that the Cet1p-Ceg1p interaction is important not only for the activation of Ceg1p, but also for nuclear import of the complex.
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Cloning and characterization of mRNA Capping Enzyme and mRNA (Guanine-7-)-methyltransferase cDNAs from Xenopus laevis.
Biochemical and Biophysical Research Communications, 2000Co-Authors: Jun'ichi Yokoska, Toshihiko Tsukamoto, Kin-ichiro Miura, Koichiro Shiokawa, Kiyohisa MizumotoAbstract:The mRNA cap structure, which is synthesized by a series of reactions catalyzed by Capping Enzyme, mRNA (guanine-7-)-methyltransferase, and mRNA (ribose-2'-O-)-methyltransferase, has crucial roles for RNA processing and translation. Methylation of the cap structure is also implicated in polyadenylation-mediated translational activation during Xenopus oocyte maturation. Here we isolated two Xenopus laevis cDNAs, xCAP1a and xCAP1b, for mRNA Capping Enzyme and one cDNA for mRNA (guanine-7-)-methyltransferase, xCMT1, which encode 598, 511, and 402 amino acids, respectively. The deduced amino acid sequence of xCAP1a was highly homologous to that of human Capping Enzyme hCAP1a, having all the characteristic regions including N-terminal RNA 5'-triphosphatase as well as C-terminal mRNA guanylyltransferase domains which are conserved among animal mRNA guanylyltransferases, whereas in xCAP1b the most C-terminal motif was missing. The amino acid sequence of xCMT1 was also similar to human (guanine-7-)-methyltransferase, hCMT1a, with all the conserved motifs among cellular (guanine-7-)-methyltransferases, except for its N-terminal portion. The recombinant xCAP1a and xCMT1 exhibited cap formation and mRNA (guanine-7-)-methyltransferase activities, respectively. RT-PCR analysis showed that mRNA for xCAP1a and xCMT1 exist abundantly in fertilized eggs as maternal mRNAs, but xCMT1 mRNA gradually decreased in its amount in later stages of early development.
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Cloning and characterization of two human cDNAs encoding the mRNA Capping Enzyme.
Biochemical and Biophysical Research Communications, 1998Co-Authors: Toshihiko Tsukamoto, Yoshio Shibagaki, Teruko Murakoshi, Masako Suzuki, Akiko Nakamura, Hideo Gotoh, Kiyohisa MizumotoAbstract:Abstract Previous studies demonstrated that the mammalian mRNA Capping Enzyme is a bifunctional Enzyme containing RNA 5′-triphosphatase and mRNA guanylyltransferase activities in a single polypeptide. In yeast, both the above activities are separated into two different subunits, α and β, the genes for which we have cloned recently. It is thus interesting to compare the structural and functional relationships between the mammalian and yeast Capping Enzymes. Here we isolated two human cDNAs encoding mRNA Capping Enzymes termedhCAP1aandhCAP1bwhich encode 597 and 541 amino acids, respectively. They are different only at the region coding for the C-terminal portion of the Enzyme. Comparison of the deduced amino acid sequences with other cellular and viral Capping Enzymes showed that all the regions conserved among mRNA guanylyltransferases are observed in our clones except one conserved C-terminal region which was absent in the hCAP1b protein. The purified recombinanthCAP1agene product, hCAP1a, exhibited both RNA 5′-triphosphatase and mRNA guanylyltransferase activities. Deletion mutant analysis of hCAP1a showed that the N-terminal 213 amino acid fragment containing a tyrosine specific protein phosphatase motif catalyzed the RNA 5′-triphosphatase activity and the C-terminal 369 amino acid fragment exhibited the mRNA guanylyltransferase activity. On the other hand, hCAP1b showed RNA 5′-triphosphatase activity, but neither Enzyme-GMP covalent complex formation nor cap structure formation was detected.
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isolation and characterization of the yeast mrna Capping Enzyme beta subunit gene encoding rna 5 triphosphatase which is essential for cell viability
Biochemical and Biophysical Research Communications, 1997Co-Authors: Toshihiko Tsukamoto, Yoshio Shibagaki, Shinobu Imajohohmi, Teruko Murakoshi, Masako Suzuki, Akiko Nakamura, Hideo Gotoh, Kiyohisa MizumotoAbstract:The yeast Saccharomyces cerevisiae mRNA Capping Enzyme is composed of two subunits of alpha (52 kDa, mRNA guanylyltransferase) and beta (80 kDa, RNA 5'-triphosphatase). We have isolated the alpha subunit gene (CEG1) by immunological screening. In this report, with the aid of partial amino acid sequences of purified yeast Capping Enzyme, we isolated the gene, designated CET1, encoding the S. cerevisiae Capping Enzyme beta subunit. Amino acid sequence analysis revealed that the gene encodes for 549 amino acids with a calculated M(r) of 61,800 which is unexpectedly smaller than the size estimated by SDS-PAGE. Gene disruption experiment showed that CET1 is essential for yeast cell growth. The purified recombinant CET1 gene product, Cet1, exhibited an RNA 5'-triphosphatase activity which specifically removed the gamma-phosphate from the triphosphate-terminated RNA substrate, but not from nucleoside triphosphates, confirming the identity of the gene. Interaction between the Cet1 and the Ceg1 was also studied by the West-Western procedure using recombinant Ceg1-[32P]GMP as probe.
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Isolation of the mRNA-Capping Enzyme and ferric-reductase-related genes from Candida albicans.
Microbiology, 1996Co-Authors: Toshiko Yamada-okabe, Kiyohisa Mizumoto, Rikuo Doi, Osamu Shimmi, Mikio Arisawa, Hisafumi Yamada-okabeAbstract:The mRNA-Capping Enzyme (mRNA 5′-guanylyltransferase) gene was cloned from a Candida albicans genomic DNA library by functional complementation of a Saccharomyces cerevisiae ceg1Δ null mutation. This gene, referred to as CGT1 (C. albicans guanylyltransferase 1), can encode a 52 kDa protein that is highly homologous to S. cerevisiae Ceg1p. CGT1 in a single-copy plasmid complemented the lethality of the S. cerevisiae ceg1Δ null mutation and, like S. cerevisiae Ceg1p, bacterially expressed Cgt1p was able to form a stable complex with the GMP moiety of GTP and to synthesize the cap structure in vitro, demonstrating that CGT1 is the C. albicans mRNA 5′-guanylyltransferase gene. CGT1 seemed to exist as a single copy in the C. albicans genome and was actively transcribed into mRNA. Another ORF was found in an opposite strand very close to the CGT1 locus. This gene shared significant sequence homology with S. cerevisiae FRE1, the gene encoding ferric reductase, and therefore was designated CFL1 (C. albicans ferric-reductase-like gene 1). Despite its sequence homology with S. cerevisiae FRE1, CFL1 mRNA was not induced by iron deprivation, and CFL1 did not complement the slow growth of a S. cerevisiae fre1Δ null mutant in the absence of iron, suggesting that CFL1 is functionally distinct from S. cerevisiae FRE1.
Richard C. Condit - One of the best experts on this subject based on the ideXlab platform.
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Biochemical analysis of the multifunctional vaccinia mRNA Capping Enzyme encoded by a temperature sensitive virus mutant
Virology, 2016Co-Authors: Jessica Tate, Rachel L. Boldt, Mcfadden, Susan M. D'costa, Nicholas M. Lewandowski, Amber N. Shatzer, Paul Gollnick, Richard C. ConditAbstract:Prior biochemical analysis of the heterodimeric vaccinia virus mRNA Capping Enzyme suggests roles not only in mRNA Capping but also in early viral gene transcription termination and intermediate viral gene transcription initiation. Prior phenotypic characterization of Dts36, a temperature sensitive virus mutant affecting the large subunit of the Capping Enzyme was consistent with the multifunctional roles of the Capping Enzyme in vivo. We report a biochemical analysis of the Capping Enzyme encoded by Dts36. Of the three enzymatic activities required for mRNA Capping, the guanylyltransferase and methyltransferase activities are compromised while the triphosphatase activity and the D12 subunit interaction are unaffected. The mutant Enzyme is also defective in stimulating early gene transcription termination and intermediate gene transcription initiation in vitro. These results confirm that the vaccinia virus mRNA Capping Enzyme functions not only in mRNA Capping but also early gene transcription termination and intermediate gene transcription initiation in vivo.
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Phenotypic analysis of a temperature sensitive mutant in the large subunit of the vaccinia virus mRNA Capping Enzyme
Virology, 2008Co-Authors: Amber N. Shatzer, Sayuri E.m. Kato, Richard C. ConditAbstract:The heterodimeric vaccinia virus mRNA Capping Enzyme is a multifunctional Enzyme, encoded by genes D1R and D12L. Published biochemical experiments demonstrate that, in addition to mRNA Capping, the Enzyme is involved in early viral gene transcription termination and intermediate viral gene transcription initiation. This paper presents the phenotypic characterization of Dts36, a temperature sensitive mutant in the large subunit of the mRNA Capping Enzyme (G705D), encoded by gene D1R. At the non-permissive temperature, Dts36 displays decreased steady state levels of some early RNAs, suggesting a defect in mRNA Capping. Mutant infections also show decreased steady state levels of some early proteins, while DNA replication and post-replicative gene expression are absent. Under non-permissive conditions, the mutant directs synthesis of longer-than-normal early mRNAs from some genes, demonstrating that early gene transcription termination is defective. If mutant infections are initiated at the permissive temperature and shifted to the non-permissive temperature late during infection, steady state levels of intermediate gene transcripts decrease while the levels of late gene transcripts remain constant, consistent with a defect in intermediate gene transcription initiation. In addition to its previously described role in mRNA Capping, the results presented in this study provide the first in vivo evidence that the vaccinia virus mRNA Capping Enzyme plays a role in early gene transcription termination and intermediate gene transcription.
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Analysis of a temperature-sensitive vaccinia virus mutant in the viral mRNA Capping Enzyme isolated by clustered charge-to-alanine mutagenesis and transient dominant selection.
Virology, 1997Co-Authors: Daniel E Hassett, Jackie I. Lewis, Xuekun Xing, Luke Delange, Richard C. ConditAbstract:Abstract We have previously reported the successful development of a targeted genetic method for the creation of temperature-sensitive vaccinia virus mutants [D. E. Hassett and R. C. Condit (1994) Proc. Natl. Acad. Sci. USA 91, 4554–4558]. This method has now been applied to the large subunit of the multifunctional vaccinia virus Capping Enzyme, encoded by gene D1R. Ten clustered charge-to-alanine mutations were created in a cloned copy of D1R. Four of these mutations were successfully transferred into the viral genome using transient dominant selection, and each of these four mutations yielded viruses with plaque phenotypes different from that of wild-type virus. Two of the mutant viruses, 516 and 793, were temperature sensitive in a plaque assay. Mutant 793 was also temperature sensitive in a one-step growth experiment. Phenotypic characterization of the 793 virus under both permissive and nonpermissive conditions revealed nearly normal patterns of viral protein and mRNA synthesis. Under nonpermissive conditions the 793 virus was defective in telomere resolution and blocked at an intermediate stage of viral morphogenesis. In vitro assays of various Capping Enzyme activities revealed that in permeabilized virions, Enzyme guanylylate intermediate formation was reduced and methyltransferase activity was thermolabile, while in solubilized virion extracts Enzyme guanylylate activity was reduced and both guanylyltransferase and methyltransferase activities were absent. Thus, the 793 mutation affects at least two separate enzymatic activities of the Capping Enzyme, guanylyltransferase and methyltransferase, and when incorporated into the virus genome, the mutation yields a virus that is temperature sensitive for growth, telomere resolution, and virion morphogenesis.
S Shuman - One of the best experts on this subject based on the ideXlab platform.
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Mutational analysis of the RNA triphosphatase component of vaccinia virus mRNA Capping Enzyme.
Journal of virology, 1996Co-Authors: S ShumanAbstract:Vaccinia virus mRNA Capping Enzyme is a multifunctional protein with RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7-) methyltransferase activities. The Enzyme is a heterodimer of 95- and 33-kDa subunits encoded by the vaccinia virus D1 and D12 genes, respectively. The N-terminal 60-kDa of the D1 subunit (from residues 1 to 545) is an autonomous domain which catalyzes the triphosphatase and guanylyltransferase reactions. Mutations in the D1 subunit that specifically inactivate the guanylyltransferase without affecting the triphosphatase component have been described (P. Cong and S. Shuman, Mol. Cell. Biol. 15:6222-6231, 1995). In the present study, we identified two alanine-cluster mutations of D1(1-545), R77A-K79A and E192A-E194A, that selectively inactivated the triphosphatase, but not the guanylyltransferase. Concordant mutational inactivation of RNA triphosphatase and nucleoside triphosphatase functions (to approximately 1% of wild-type specific activity) suggests that both gamma-phosphate cleavage reactions occur at a single active site. The R77A-K79A and E192A-E194A mutant Enzymes were less active than wild-type D1(1-545) in the Capping of triphosphate-terminated poly(A) but could be complemented in vitro by D1(1-545)-K260A, which is inert in nucleotidyl transfer but active in gamma-phosphate cleavage. Whereas wild-type D1(1-545) formed only the standard GpppA cap, the R77A-K79A and E192A-E194A Enzymes synthesized an additional dinucleotide, GppppA. This finding illuminates a novel property of the vaccinia virus Capping Enzyme, the use of triphosphate RNA ends as an acceptor for nucleotidyl transfer when gamma-phosphate cleavage is rate limiting.
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Covalent catalysis in nucleotidyl transfer. A KTDG motif essential for Enzyme-GMP complex formation by mRNA Capping Enzyme is conserved at the active sites of RNA and DNA ligases.
The Journal of biological chemistry, 1993Co-Authors: Peijie Cong, S ShumanAbstract:Abstract Vaccinia virus RNA Capping Enzyme, a heterodimer of 95- and 31-kDa subunits, catalyzes transfer of GMP from GTP to the 5'-diphosphate terminus of RNA via a covalent Enzyme-guanylate intermediate. The GMP residue is attached to the 95-kDa subunit through a phosphoamide bond to the epsilon-amino group of a lysine residue. The amino acid sequence of the large subunit includes a lysine-containing motif, Tyr-X-X-X-Lys260-Thr-Asp-Gly, that is conserved in the RNA guanylyltransferases encoded by Shope fibroma virus and Saccharomyces cerevisiae. The KXDG motif is also encountered at the sites of covalent adenylylation of bacteriophage T4 RNA ligase and mammalian DNA ligase I (Thogerson, H. C., Morris, H. R., Rand, K. N., and Gait, M. J. (1985) Eur. J. Biochem. 147, 325-329; Tomkinson, A. E., Totty, N. F., Ginsburg, M., and Lindahl, T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 400-404). We find that conservative amino acid substitutions at three out of four positions within the KTDG sequence of vaccinia Capping Enzyme either prevent or strongly inhibit Enzyme-guanylate formation. The conserved motif is therefore an essential component of the guanylyltransferase domain. Lys260 is implicated as the active site. Comparison of the sequences of Capping Enzymes and polynucleotide ligases from diverse sources suggests that KX(D/N)G may be a signature element for covalent catalysis in nucleotidyl transfer.
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Methyltransferase and subunit association domains of vaccinia virus mRNA Capping Enzyme.
The Journal of biological chemistry, 1992Co-Authors: P Cong, S ShumanAbstract:Abstract RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-N7-)-methyltransferase activities are associated with the vaccinia virus mRNA Capping Enzyme, a heterodimeric protein containing polypeptides of M(r) 95,000 and 31,000. Although the RNA triphosphatase and RNA guanylyltransferase domains have been localized to a M(r) 59,000 fragment of the Capping Enzyme large subunit, the location of the methyltransferase domain within the protein and the catalytic role of individual subunits in methyl group transfer remain unclear. In the present work, through the study of methyltransferase activity of truncated forms of Capping Enzyme translated in vitro in a rabbit reticulocyte lysate, we have localized the methyltransferase domain to a complex consisting of the small subunit and the carboxyl-terminal portion of the large subunit. The M(r) 31,000 subunit translated alone was not sufficient for methyltransferase activity. This requirement for both subunits may explain the tight physical association of the two polypeptides in vivo. We have recreated the association of the large and small Enzyme subunits in vitro through the translation of synthetic mRNAs encoding the two polypeptides. Study of the ability of deleted versions of the large subunit to bind the small subunit, as detected by co-immunoprecipitation, defined a 347-amino acid carboxyl-terminal region of the large subunit that was sufficient for heterodimerization. Colocalization within the large subunit of the methyltransferase and subunit association domains suggests that dimerization of the subunits may be required for methyltransferase activity.
Beate Schwer - One of the best experts on this subject based on the ideXlab platform.
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How an mRNA Capping Enzyme reads distinct RNA polymerase II and Spt5 CTD phosphorylation codes
Genes & Development, 2014Co-Authors: Selom K. Doamekpor, Stewart Shuman, Beate Schwer, Ana M. Sanchez, Christopher D. LimaAbstract:Essential eukaryal mRNA processing events are targeted to nascent transcripts made by RNA polymerase II (Pol2) via physical interactions of the processing machineries with the C-terminal domain (CTD) of Rpb1, the largest subunit of Pol2. The Pol2 CTD consists of tandemly repeated heptapeptides of consensus sequence Y1S2P3T4S5P6S7. The inherently plastic Pol2 CTD structure is sculpted by dynamic phosphorylation and dephoshorylation of the Tyr1, Ser2, Thr4, Ser5, and Ser7 residues and by cis–trans isomerization of the prolines. With up to 128n potential Pol2 CTD primary structures (where n is the number of heptads), the Pol2 CTD provides information about the state of the transcription complex (a CTD code) that is read by diverse Pol2 CTD receptor proteins that control transcription, modify chromatin structure, and catalyze or regulate mRNA Capping, splicing, and polyadenylation (Buratowski 2009; Corden 2013; Eick and Geyer 2013; Geronimo et al. 2013). The individual amino acids of the Pol2 CTD heptad and their phosphorylation marks are coding “letters” that have distinct outputs with respect to receptor recognition and impact on cellular gene expression (Schwer et al. 2014). The number of heptad repeats comprising the Pol2 CTD, the minimal number of repeats required for viability, and the prevalence of nonconsensus coding letters tend to increase with progression from unicellular to multicellular eukarya, presumably as a means to orchestrate the actions of increasing numbers of Pol2 CTD receptors. We envision a core Pol2 CTD code that governs events common to most eukarya; for example, the essential function of the Ser5-PO4–Pro6 coding “word” in recruiting mRNA Capping Enzymes to the Pol2 elongation complex (Schwer and Shuman 2011; Schwer et al. 2012), which is conserved in budding yeast, fission yeast, and mammals, notwithstanding major differences in the genetic and physical organization of the Capping apparatus in these taxa (Lima et al. 1999; Changela et al. 2001; Pei et al. 2001; Fabrega et al. 2003; Ghosh et al. 2011). The RNA Capping Enzymes are the first processing factors to act on the growing Pol2 transcript; indeed, Capping can commence after synthesis of only a 19- to 22-mer nascent RNA, the point at which the 5′ triphosphate RNA end is extruded from the RNA-binding pocket of elongating Pol2 and becomes accessible to the Capping Enzymes (Chiu et al. 2002). The m7GpppN cap is formed by three successive enzymatic reactions: (1) RNA triphosphatase (TPase) hydrolyzes the RNA 5′ triphosphate end (pppRNA) to a diphosphate (ppRNA) plus inorganic phosphate, (2) GTP:RNA guanylyltransferase (GTase) converts ppRNA to GpppRNA via a covalent GTase-(lysyl-Nζ)–GMP intermediate, and (3) AdoMet:RNA(guanine-N7)-methyltransferase (MTase) converts GpppRNA to m7GpppRNA (Gu and Lima 2005). Whereas it had long been assumed that Capping is a constitutive and efficient process for most mRNAs, this view may be too simplistic. The existence of factors that can stimulate Capping of specific transcripts and the discovery of Enzymes that mediate the decay of partially processed RNA intermediates in the cap synthetic pathway suggest that Capping efficiency is a dynamic process (Chiu et al. 2002; Jiao et al. 2010, 2013; Chang et al. 2012). One potential way to regulate Capping is by influencing the timely recruitment of the Capping Enzymes to the Pol2 elongation complex. A conserved theme among eukarya is the direct binding of the GTase component to the Ser5-PO4 form of the Pol2 CTD (Ho and Shuman 1999; Fabrega et al. 2003; Ghosh et al. 2011). In mammals and budding yeast, the TPase components are recruited passively to the Pol2 CTD by virtue of their physical association with the GTase: in cis as a covalently fused TPase–GTase Enzyme, Mce1, in mammals (Ho and Shuman 1999; Changela et al. 2001) or in trans as separately encoded subunits of a TPase•GTase complex in budding yeast (Ho et al. 1999; Gu et al. 2010). The fission yeast Schizosaccharomyces pombe has a distinctive strategy for targeting cap formation to Pol2 transcripts whereby the TPase (Pct1) and GTase (Pce1) Enzymes are not associated physically but instead bind independently to the Ser5-phosphorylated Pol2 CTD (Pei et al. 2001). Mammalian and fungal Capping Enzymes can also gain access to nascent Pol2 transcripts via physical interactions with the transcription elongation factor Spt5 (Wen and Shatkin 1999; Pei and Shuman 2002; Pei et al. 2006; Lidschreiber et al. 2013). Spt5 is a large polypeptide (∼1000–1200 amino acids) composed of multiple domain modules that associates with the Pol2 transcription complex shortly after initiation and can exert negative and positive effects on transcription elongation (Hartzog and Fu 2013). Fission yeast Spt5 has a distinctive C-terminal repeat domain (the “Spt5 CTD”) composed of 18 repeats of a nonapeptide motif (consensus: T1P2A3W4N5S6G7S8K9) that (1) binds the fission yeast RNA Capping Enzymes Pct1 and Pce1 (Pei and Shuman 2002) and (2) is targeted for threonine phosphorylation by the fission yeast Cdk9 kinase (Pei and Shuman 2002, 2003; Viladevall et al. 2009). Alignment of Spt5 CTD elements in metazoan and fungal species reveals substantive differences, although each contains a series of Thr–Pro or Ser–Pro dipeptide motifs followed by a hydrophobic side chain two residues downstream (Pei and Shuman 2002). The hydrophobic side chain is typically tryptophan in S. pombe; this position is substituted to a tyrosine or histidine in humans. Available genetic evidence points to overlapping roles of the CTDs of fission yeast Pol2 and Spt5 in recruiting the Capping Enzymes in vivo (Schneider et al. 2010). Unlike the Capping Enzyme•Pol2 CTD interactions, which stringently depend on the Ser5-PO4 mark, the binding of fission yeast Pct1 and Pce1 to the Spt5 CTD is independent of Thr1 phosphorylation (Pei et al. 2001; Pei and Shuman 2002). The structural principles underpinning Capping Enzyme•Pol2 CTD interactions were initially elucidated via cocrystallization of Candida albicans GTase (Cgt1) and mammalian GTase (Mce1) bound to Ser5-phosphorylated Pol2 CTD peptide ligands (Fabrega et al. 2003; Ghosh et al. 2011). These two cellular GTases are structurally homologous Enzymes composed of two domains: (1) an N-terminal nucleotidyltransferase (NTase) module that contains the guanylate-binding pocket and (2) a C-terminal OB-fold module. A comparison of the Candida and mammalian GTase•Pol2 CTD structures was most revealing because, although the docking sites for Pol2 CTD lie on the surface of the NTase domain in both cases, the docking sites are physically distinct and have little overlap, the atomic contacts to Pol2 CTD are different, and virtually none of the Pol2 CTD-interacting side chains of mammalian GTase are conserved in the Candida Enzyme (Ghosh et al. 2011). Thus, Capping Enzymes from different taxa have evolved different strategies to read the Pol2 CTD code. Here we extend the structural analysis to the GTase Enzyme of fission yeast and its interactions with the CTDs of Pol2 and Spt5. We report crystal structures of Pce1 bound to Pol2 CTD and Spt5 CTD ligands. Key findings are that (1) the CTDs of Pol2 and Spt5 interact with completely distinct sites on the NTase and OB domains of the fission yeast GTase, respectively, and (2) whereas the interface of GTase with the Pol2 CTD is dependent on Ser5 phosphorylation, GTase binding to Spt5 CTD is antagonized by Thr1 phosphorylation. We probed by structure-guided mutagenesis the contributions of the GTase•CTD interfaces to CTD binding in vitro and Capping Enzyme function in vivo. Our findings cohere into a model in which the Capping Enzyme recognizes and responds to distinct coding cues and phosphorylation marks in the Pol2 and Spt5 CTDs.
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Separable functions of the fission yeast Spt5 carboxyl-terminal domain (CTD) in Capping Enzyme binding and transcription elongation overlap with those of the RNA polymerase II CTD.
Molecular and Cellular Biology, 2010Co-Authors: Susanne Schneider, Stewart Shuman, Yi Pei, Beate SchwerAbstract:An interaction network connecting mRNA Capping Enzymes, the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD), elongation factor Spt5, and the Cdk7 and Cdk9 protein kinases is thought to comprise a transcription elongation checkpoint. A crux of this network is Spt5, which regulates early transcription elongation and has an imputed role in pre-mRNA processing via its physical association with Capping Enzymes. Schizosaccharomyces pombe Spt5 has a distinctive CTD composed of tandem nonapeptide repeats of the consensus sequence 1TPAWNSGSK9. The Spt5 CTD binds the Capping Enzymes and is a substrate for threonine phosphorylation by the Cdk9 kinase. Here we report that deletion of the S. pombe Spt5 CTD results in slow growth and aberrant cell morphology. The severity of the spt5-ΔCTD phenotype is exacerbated by truncation of the Pol II CTD and ameliorated by overexpression of the Capping Enzymes RNA triphosphatase and RNA guanylyltransferase. These results suggest that the Spt5 and Pol II CTDs play functionally overlapping roles in Capping Enzyme recruitment. We probed structure-activity relations of the Spt5 CTD by alanine scanning of the consensus nonapeptide. The T1A change abolished CTD phosphorylation by Cdk9 but did not affect CTD binding to the Capping Enzymes. The T1A and P2A mutations elicited cold-sensitive (cs) and temperature-sensitive (ts) growth defects and conferred sensitivity to growth inhibition by 6-azauracil that was exacerbated by partial truncations of the Pol II CTD. The T1A phenotypes were rescued by a phosphomimetic T1E change but not by Capping Enzyme overexpression. These results imply a positive role for Spt5 CTD phosphorylation in Pol Il transcription elongation in fission yeast, distinct from its Capping Enzyme interactions. Viability of yeast cells bearing both Spt5 CTD T1A and Pol II CTD S2A mutations heralds that the Cdk9 kinase has an essential target other than Spt5 and Pol II CTD-Ser2.
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Accelerated mRNA decay in conditional mutants of yeast mRNA Capping Enzyme
Nucleic Acids Research, 1998Co-Authors: Beate Schwer, Xiangdong Mao, Stewart ShumanAbstract: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.
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The guanylyltransferase domain of mammalian mRNA Capping Enzyme binds to the phosphorylated carboxyl-terminal domain of RNA polymerase II.
Journal of Biological Chemistry, 1998Co-Authors: Verl Sriskanda, Beate Schwer, Susan Mccracken, David Bentley, Stewart ShumanAbstract:Abstract We have conducted a biochemical and genetic analysis of mouse mRNA Capping Enzyme (Mce1), a bifunctional 597-amino acid protein with RNA triphosphatase and RNA guanylyltransferase activities. The principal conclusions are as follows: (i) the mammalian Capping Enzyme consists of autonomous and nonoverlapping functional domains; (ii) the guanylyltransferase domain Mce1(211–597) is catalytically active in vitro and functional in vivo in yeast in lieu of the endogenous guanylyltransferase Ceg1; (iii) the guanylyltransferase domainper se binds to the phosphorylated RNA polymerase II carboxyl-terminal domain (CTD), whereas the triphosphatase domain, Mce1(1–210), does not bind to the CTD; and (iv) a mutation of the active site cysteine of the mouse triphosphatase elicits a strong growth-suppressive phenotype in yeast, conceivably by sequestering pre-mRNA ends in a nonproductive complex or by blocking access of the endogenous yeast triphosphatase to RNA polymerase II. These findings contribute to an emerging model of mRNA biogenesis wherein RNA processing Enzymes are targeted to nascent polymerase II transcripts through contacts with the CTD. The phosphorylation-dependent interaction between guanylyltransferase and the CTD is conserved from yeast to mammals.
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Multicopy suppressors of temperature-sensitive mutations of yeast mRNA Capping Enzyme.
Gene expression, 1996Co-Authors: Beate Schwer, Stewart ShumanAbstract:We have isolated three Saccharomyces cerevisiae genes-CES1, CES2, and CES3-- that, when present in high copy, suppress the ts growth defect caused by mutations in the CEG1 gene encoding mRNA guanylyltransferase (Capping Enzyme). Molecular characterization of the Capping Enzyme suppressor genes reveals the following. CES2 is identical to ESP1, a gene required for proper nuclear division. We show by deletion analysis that the 1573-amino acid ESP1 polypeptide is composed of distinct functional domains. The C-terminal portion of ESP1 is essential for cell growth, but dispensable for CES2 activity. The N-terminal half of ESP1, which is sufficient for CES2 function, displays local sequence similarity to the small subunit of the vaccinia virus RNA Capping Enzyme. This suggests a basis for suppression by physical or functional interaction between the CES2 domain of ESP1 and the yeast guanylyltransferase. CES1 encodes a novel hydrophilic 915-amino acid protein. The amino acid sequence of CES1 is uninformative, except for its extensive similarity to another yeast gene product of unknown function. The CES1 homologue (designated CES4) is also a multicopy suppressor of Capping Enzyme ts mutations. Neither CES1 nor CES4 is essential for cell growth, and a double deletion mutant is viable. CES3 corresponds to BUD5, which encodes a putative guanine nucleotide exchange factor. We hypothesize that CES1, CES4, and BUD5 may impact on RNA transactions downstream of cap synthesis that are cap dependent in vivo.
