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The Caenorhabditis elegansmRNA 5′-Capping Enzyme

IN VITRO AND IN VIVO CHARACTERIZATION*
Open AccessPublished:February 07, 2003DOI:https://doi.org/10.1074/jbc.M212101200
      Eukaryotic mRNA capping enzymes are bifunctional, carrying both RNA triphosphatase (RTPase) and guanylyltransferase (GTase) activities. The Caenorhabditis elegans CEL-1 capping enzyme consists of an N-terminal region with RTPase activity and a C-terminal region that resembles known GTases, However, CEL-1 has not previously been shown to have GTase activity. Cloning of the cel-1 cDNA shows that the full-length protein has 623 amino acids, including an additional 38 residues at the C termini and 12 residues at the N termini not originally predicted from the genomic sequence. Full-length CEL-1 has RTPase and GTase activities, and the cDNA can functionally replace the capping enzyme genes in Saccharomyces cerevisiae. The CEL-1 RTPase domain is related by sequence to protein-tyrosine phosphatases; therefore, mutagenesis of residues predicted to be important for RTPase activity was carried out. CEL-1 uses a mechanism similar to protein-tyrosine phosphatases, except that there was not an absolute requirement for a conserved acidic residue that acts as a proton donor. CEL-1 shows a strong preference for RNA substrates of at least three nucleotides in length. RNA-mediated interference inC. elegans embryos shows that lack of CEL-1 causes development to arrest with a phenotype similar to that seen when RNA polymerase II elongation activity is disrupted. Therefore, capping is essential for gene expression in metazoans.
      Most eukaryotic and viral mRNAs are modified at their 5′ end by a “cap” structure that consists of a 7-methylguanosine moiety attached to the 5′ terminus via a 5′-5′ linkage (
      • Furuichi Y.
      • Shatkin A.J.
      ). Three sequential enzymatic activities are required to form the “cap 0” structure, m7GpppN. First, an RNA 5′-triphosphatase (RTPase)
      The abbreviations used are: RTPase
      RNA 5′-triphosphatase
      GTase
      GTP::mRNA guanylyltransferase
      PTP
      protein-tyrosine phosphatase
      RNAi
      RNA interference
      5-FOA
      5-fluoroorotic acid
      pol II
      RNA polymerase II
      CTD
      C-terminal domain of the largest subunit of RNA polymerase II
      CTD-P
      phosphorylated CTD
      HA
      influenza virus hemagglutinin
      EST
      Expressed Sequence Tag
      ORF
      open reading frame
      P-TEFb
      positive transcription elongation factor b
      CDK
      cyclin-dependent kinase
      kb
      kilobase
      DTT
      dithiothreitol
      ds
      double-stranded
      MCE
      mouse capping enzyme
      BVP
      baculoviral PTP
      1The abbreviations used are: RTPase
      RNA 5′-triphosphatase
      GTase
      GTP::mRNA guanylyltransferase
      PTP
      protein-tyrosine phosphatase
      RNAi
      RNA interference
      5-FOA
      5-fluoroorotic acid
      pol II
      RNA polymerase II
      CTD
      C-terminal domain of the largest subunit of RNA polymerase II
      CTD-P
      phosphorylated CTD
      HA
      influenza virus hemagglutinin
      EST
      Expressed Sequence Tag
      ORF
      open reading frame
      P-TEFb
      positive transcription elongation factor b
      CDK
      cyclin-dependent kinase
      kb
      kilobase
      DTT
      dithiothreitol
      ds
      double-stranded
      MCE
      mouse capping enzyme
      BVP
      baculoviral PTP
      removes the γ-phosphate from the 5′ end of the RNA substrate to leave a diphosphate end. Next, a GTP::mRNA guanylyltransferase (GTase) catalyzes transfer of GMP from GTP, resulting in a 5′-5′ linkage, GpppNp. These two activities are typically associated and copurify as mRNA capping enzyme. A third protein, RNA (guanine-7-)-methyltransferase, adds a methyl group to the N-7 position of the guanine cap (
      • Furuichi Y.
      • Shatkin A.J.
      ,
      • Shuman S.
      ).
      Previously we characterized a putative capping enzyme gene, which we named cel-1, that emerged from the Caenorhabditis elegans genome sequencing project (
      • Takagi T.
      • Moore C.R.
      • Diehn F.
      • Buratowski S.
      ). The open reading frame (ORF) of this gene originally predicted by the Nematode Sequencing Project was 573 amino acids. The C-terminal 340 amino acids exhibit very strong similarity to yeast and viral GTases. The N-terminal region has significant sequence similarity to the protein-tyrosine phosphatase (PTP) family, including the active site consensus motif (I/V)HCXXGXXR(S/T)G (
      • Denu J.M.
      • Stuckey J.A.
      • Saper M.A.
      • Dixon J.E.
      ,
      • Fauman E.B.
      • Saper M.A.
      ,
      • Barford D.
      • Das A.K.
      • Egloff M.-P.
      ,
      • Denu J.M.
      • Dixon J.E.
      ). We proved that the isolated N-terminal region (residues 1–236) of CEL-1 has RTPase activity (
      • Takagi T.
      • Moore C.R.
      • Diehn F.
      • Buratowski S.
      ,
      • Takagi T
      • Taylor G.S.
      • Kusakabe T.
      • Charbonneau H.
      • Buratowski S.
      ). However, we were unable to demonstrate that the C-terminal region had GTase activity.
      The ORF used in the earlier study was based on predictions of exons within genomic sequence. Since then the C. elegans Expressed Sequence Tag (EST) data base has produced several cDNA sequences predicted to encode a longer form of CEL-1 that has additional residues at both the N and C termini. Protein produced from the longer ORF fully substitutes for the Saccharomyces cerevisiae GTase and RTPase in vivo. The longer CEL-1 C-terminal domain has GTase activity in vitro. We further characterized the isolated RTPase domain both in vivo and in vitro. We analyzed its catalytic properties, including the effect of RNA chain length on the activity. Mutagenesis confirms a mechanistically conserved role for key residues found in both the RTPase and PTPs. Surprisingly, the CEL-1 RTPase did not require linkage to the GTase for targeting to pre-mRNA in S. cerevisiae. Finally, we used RNA-mediated interference (RNAi) to demonstrate that CEL-1 is essentialin vivo for development of C. elegans.

      DISCUSSION

      Here we characterize the full-length C. elegans capping enzyme, CEL-1. Based on cDNAs in the EST databases, CEL-1 is a 623-amino acid protein with both RTPase and GTase activities. This matches the size of the enzyme-GMP intermediate detected in C. elegans nuclear extract (Fig. 1). Interestingly, multiple cDNAs for human and Xenopus capping enzymes have been described, possibly produced by alternative splicing of mRNA (
      • Tsukamoto T.
      • Shibagaki Y.
      • Murakoshi T.
      • Suzuki M.
      • Nakamura A.
      • Gotoh H.
      • Mizumoto K.
      ,
      • Yamada-Okabe T.
      • Doi R.
      • Shimmi O.
      • Arisawa M.
      • Yamada-Okabe H.
      ,
      • Yokoska J.
      • Tsukamoto T.
      • Miura K.
      • Shiokawa K.
      • Mizumoto K.
      ). These cDNA variants encode an intact N-terminal RTPase domain but have either internal deletions or truncations in the C-terminal GTase domain. As a result, the proteins from these variants would only have RTPase activity. PCR analyses showed that these short forms are expressed, but their physiological function is unknown. To date, no cDNAs corresponding to a shortened capping enzyme have been found in the C. elegans EST data base. CEL-1 was originally predicted to have 573 amino acids. CEL-1-(1–585) has RTPase activity but does not complement a S. cerevisiae GTase mutant ceg1Δ (Fig. 3). Therefore, CEL-1 residues 586–623 are not required for protein stability or proper localization but are essential for GTase activity.
      The CEL-1 RTPase domain (
      • Takagi T.
      • Moore C.R.
      • Diehn F.
      • Buratowski S.
      ) was the founding member of a subfamily of PTP-like RNA phosphatases. This subfamily includes the capping enzyme RTPases and RNA tri- and diphosphatases, whose functions are unknown (Fig. 2B). All members contain a nucleophilic cysteine necessary for activity (
      • Takagi T.
      • Moore C.R.
      • Diehn F.
      • Buratowski S.
      ,
      • Takagi T
      • Taylor G.S.
      • Kusakabe T.
      • Charbonneau H.
      • Buratowski S.
      ,
      • Ho C.K.
      • Sriskanda V.
      • McCracken S.
      • Bentley D.
      • Schwer B.
      • Shuman S.
      ,
      • Wen Y.
      • Yue Z.
      • Shatkin A.J.
      ,
      • Deshpande T.
      • Takagi T.
      • Hao L.
      • Buratowski S.
      • Charbonneau H.
      ,
      • Ho C.K.
      • Schwer B.
      • Shuman S.
      ,
      • Gross C.H.
      • Shuman S.
      ). A phosphocysteine intermediate was detected with MCE and BVP (
      • Changela A.
      • Ho C.K.
      • Martins A.
      • Shuman S.
      • Mondragon A.
      ,
      • Martins A.
      • Shuman S.
      ). Other PTP-like enzymes with substrates other than phosphotyrosine have been reported; these include the phosphoinositide phosphatase PTEN/MMAC1 and myotubularin (
      • Maehama T.
      • Taylor G.S.
      • Dixon J.E.
      ) and S. cerevisiae arsenite reductase Acr2 (
      • Mukhopadhyay R.
      • Rosen B.P.
      ).
      In PTPs, the formation and hydrolysis of the phosphocysteine intermediate of PTP requires transition-state stabilization by the arginine residue within the consensus motif (
      • Denu J.M.
      • Stuckey J.A.
      • Saper M.A.
      • Dixon J.E.
      ,
      • Fauman E.B.
      • Saper M.A.
      ,
      • Barford D.
      • Das A.K.
      • Egloff M.-P.
      ,
      • Denu J.M.
      • Dixon J.E.
      ). Mutagenesis of MCE (
      • Wen Y.
      • Yue Z.
      • Shatkin A.J.
      ,
      • Changela A.
      • Ho C.K.
      • Martins A.
      • Shuman S.
      • Mondragon A.
      ), BVP (
      • Martins A.
      • Shuman S.
      ), and CEL-1 (Fig. 6) shows that this residue is essential for RNA phosphatase activity, providing further evidence that the PTP and RNA phosphatases use the same enzymatic mechanism. On the other hand, mutation of a conserved aspartic acid residue in the RTPases (Asp-76 of CEL-1, Asp-66 of MCE, and Asp-60 of BVP; see Fig.2B) only slightly diminishes activity (Fig. 6; Refs.
      • Wen Y.
      • Yue Z.
      • Shatkin A.J.
      ,
      • Changela A.
      • Ho C.K.
      • Martins A.
      • Shuman S.
      • Mondragon A.
      ). The equivalent mutations in PTPs lower activity by 102–105-fold (
      • Denu J.M.
      • Stuckey J.A.
      • Saper M.A.
      • Dixon J.E.
      ,
      • Fauman E.B.
      • Saper M.A.
      ,
      • Barford D.
      • Das A.K.
      • Egloff M.-P.
      ,
      • Denu J.M.
      • Dixon J.E.
      ). X-ray crystallography of MCE-(1–210) shows that Asp-66 is positioned differently from the essential general acid aspartate loop described for PTPs (
      • Changela A.
      • Ho C.K.
      • Martins A.
      • Shuman S.
      • Mondragon A.
      ). Apparently, the RTPase mechanism does not conserve the function of this residue.
      Both CEL-1-(13–221) and MCE-(1–210) can remove the γ-phosphate from the 5′ end of a dinucleotide (Fig. 7 and data not shown). However, maximal activity is observed on substrates that are three nucleotides or longer (Fig. 8 and data not shown). The S. cerevisiaeRTPase Cet1 is unrelated to PTPs, and its reaction mechanism is different from that of metazoan RTPases (
      • Lima C.D.
      • Wang L.K.
      • Shuman S.
      ). However, Cet1 also acts on dinucleotide and trinucleotide RNAs efficiently (
      • Rodriguez C.R.
      • Takagi T.
      • Cho E.-J.
      • Buratowski S.
      ).3Diphosphate-ended oligonucleotides such as ppApG, ppGpC, and ppGpCpC are active as guanylyl acceptors for mammalian and yeast GTases (
      • Vankatesan S.
      • Moss B.
      ,
      • Wang D.
      • Furuichi Y.
      • Shatkin A.J.
      ,
      • Wang D.
      • Shatkin A.J.
      ,
      • Mizumoto K.
      • Kaziro Y.
      • Lipman F.
      ,
      • Itoh N.
      • Mizumoto K.
      • Kaziro Y.
      ). Structural studies on RNA polymerase II suggest that RNA exits polymerase in the vicinity of the CTD (
      • Cramer P.
      • Bushnell D.A.
      • Kornberg R.D.
      ), where capping enzymes will be bound. Capping occurs around the time mRNAs are about 30 nucleotides in length (
      • Jove R.
      • Manley J.L.
      ,
      • Rasmussen E.B.
      • Lis J.T.
      ). Therefore, capping enzyme probably recognizes the first few phosphodiester bonds of nascent RNA that emerge from the body of pol II and immediately caps the mRNA.
      RTPases and GTases are typically linked with each other, either on the same protein (metazoans) or in a complex (yeast). In S. cerevisiae, the interaction between the GTase (Ceg1) and RTPase (Cet1) subunits is essential for cell viability. Cet1 cannot be replaced by the RTPase domains from MCE or CEL-1, presumably because these RTPases cannot interact with Ceg1. It was originally proposed that the primary role of the linkage between GTase and RTPase on a single polypeptide was to guide RTPase to pol II transcription complex (
      • Cho E.-J.
      • Takagi T.
      • Moore C.R.
      • Buratowski S.
      ,
      • Ho C.K.
      • Schwer B.
      • Shuman S.
      ). However, both CEL-1-(13–221) and MCE-(1–210) can support viability when Ceg1 is replaced with MCE-(211–597), S. pombe pce1, or C. albicans Cgt1 (Fig. 4, Aand B). Because we did not detect any tight interaction between these RTPases and GTases (Fig.4C), we conclude that the metazoan RTPase domain can be targeted to pre-mRNA and function without any linkage to GTase. The primary function of the Cet1 interaction with Ceg1 is instead required for the activity of Ceg1 (
      • Cho E.-J.
      • Rodriguez C.R.
      • Takagi T.
      • Buratowski S.
      ,
      • Takase Y.
      • Takagi T.
      • Komarnitsky P.B.
      • Buratowski S.
      ). Other fungal and metazoan GTases do not require an interaction with RTPase for activity (
      • Pei Y.
      • Hausmann S.
      • Ho C.K.
      • Schwer B.
      • Shuman S.
      ,
      • Takagi T.
      • Cho E.-J.
      • Janoo R.T.K.
      • Polodny V.
      • Takase Y.
      • Keogh M.-C.
      • Woo S.-A.
      • Fresco-Cohen L.D.
      • Hoffman C.S.
      • Buratowski S.
      ).
      Although we found that the link between RTPase and GTase domains is not absolutely required for the capping enzymes of metazoans or fungi other than S. cerevisiae, this does not mean that the interaction is unimportant. To substitute for Cet1 in vivo, it was necessary to overexpress the isolated metazoan RTPase domain with a strong promoter and a high copy plasmid (Ref.
      • Hausmann S.
      • Ho C.K.
      • Schwer B.
      • Shuman S.
      and this study). In contrast, a low copy plasmid of the full-length enzyme was sufficient for rescuing a cet1Δ strain (
      • Changela A.
      • Ho C.K.
      • Martins A.
      • Shuman S.
      • Mondragon A.
      ). Transfection experiments showed that MCE-(1–210) is mostly cytoplasmic in mammalian cells (
      • Wen Y.
      • Yue Z.
      • Shatkin A.J.
      ). This may also be true in S. cerevisiae. Overexpression may be necessary to drive sufficient amounts of RTPase into the nucleus and into proximity with the mRNA 5′ end. Alternatively, RTPases may independently bind pol II or a pol II-associated protein. HIV-1 Tat protein binds to both full-length MCE as well as the isolated GTase and RTPase domains (
      • Chiu Y.-L.
      • Coronel E.
      • Ho C.K.
      • Shuman S.
      • Rana T.M.
      ). There could be a corresponding cellular protein(s) that mediates the association of RTPase domain with the pol II complex or RNA chain. Whatever mechanism is used, isolated RTPase domains function more efficiently in vivo when it is linked to a GTase domain.
      Finally, we examined the requirement for CEL-1 in vivo using RNA-mediated inactivation of the gene.cel-1(RNAi) embryos arrest development with a phenotype that is characteristic of a broad transcription defect. A similar phenotype is seen upon RNAi knockdown of ama-1 (pol II), ttb-1 (TFIIB), or multiple TAFs (
      • Shim E.Y.
      • Walker A.K.
      • Shi Y.
      • Blackwell T.K.
      ,
      • Shim E.Y.
      • Walker A.K.
      • Blackwell T.K.
      ,
      • Walker A.K.
      • Rothman J.H.
      • Shi Y.
      • Blackwell T.K.
      ). Onecel-1(RNAi) phenotype is strikingly different from effects seen upon depletion of basal initiation factors. In those cases, levels of CTD phosphorylation at both serine 5 and serine 2 were lowered in parallel, often reduced to undetectable levels. For example, in ttb-1(RNAi) embryos, in which basal factor TFIIB is knocked down, both serine 5 and serine 2 phosphorylation are reduced to background (
      • Shim E.Y.
      • Walker A.K.
      • Shi Y.
      • Blackwell T.K.
      ,
      • Walker A.K.
      • Rothman J.H.
      • Shi Y.
      • Blackwell T.K.
      ). In cel-1(RNAi) embryos, CTD serine 5 phosphorylation appears to be relatively unaffected, whereas serine 2 phosphorylation is dramatically reduced (Fig. 9B). The only other example of this “uncoupling” of CTD serine 5 and 2 phosphorylation occurred when we depleted either of the P-TEFb components, CDK-9, or cyclin T (
      • Shim E.Y.
      • Walker A.K.
      • Shi Y.
      • Blackwell T.K.
      ). Levels of the CDK-9 kinase appear normal in cel-1(RNAi) embryos, however. Because serine 5 phosphorylation occurs primarily near the promoter, the generally normal levels incel-1(RNAi) embryos suggest that transcription initiation may be close to normal. The markedly decreased levels of serine 2 phosphorylation, a modification linked to elongation phase, suggests that the absence of capping enzyme interrupts the progression of transcription. It will be interesting to determine whether the lack of capping enzyme decreases the efficiency with which P-TEFb or other elongation factors are recruited to transcribed genes. This would be the latest of many connections have recently emerged between transcription elongation and mRNA processing.

      Acknowledgments

      We thank Drs. Aaron J. Shatkin and Fabio Piano for communicating results before publication, Gary Ruvkun for supplying C. elegans nuclear extract, Dale Wigley for pET-A103R, Gerhard Wagner and Kylie Walter for pT7–911Q, Takahiro Kusakabe for designing the sequences of synthetic oligonucleotides for preparation of short RNA with T7 DNA primase, and Robin Buratowski for help with the plasmid and oligo tables.

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