Publications

• An RNA 5'-triphosphatase related to the protein tyrosine phosphatases.

  Takagi T., Moore C.R., Diehn F., Buratowski S.

  mRNA capping requires the sequential action of three enzymatic activities: RNA triphosphatase, guanylyl-transferase, and methyltransferase. Here we characterize a gene (CEL-1) believed to encode the C. elegans capping enzyme. CEL-1 has a C-terminal domain containing motifs found in yeast and vacci ... >> More

  mRNA capping requires the sequential action of three enzymatic activities: RNA triphosphatase, guanylyl-transferase, and methyltransferase. Here we characterize a gene (CEL-1) believed to encode the C. elegans capping enzyme. CEL-1 has a C-terminal domain containing motifs found in yeast and vaccinia virus capping enzyme guanylyltransferases. The N-terminal domain of CEL-1 has RNA triphosphatase activity. Surprisingly, this domain does not resemble the vaccinia virus capping enzyme but does have significant sequence similarity to the protein tyrosine phosphatase (PTP) enzyme family. However, CEL-1 has no detectable PTP activity. The mechanism of the RNA triphosphatase is similar to that of PTPs: the active site contains a conserved nucleophilic cysteine required for activity. These results broaden the superfamily of PTP-like phosphatases to include enzymes with RNA substrates. << Less

  Cell 89:867-873(1997) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• Biochemical analysis of the multifunctional vaccinia mRNA capping enzyme encoded by a temperature sensitive virus mutant.

  Tate J., Boldt R.L., McFadden B.D., D'Costa S.M., Lewandowski N.M., Shatzer A.N., Gollnick P., Condit R.C.

  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 sensitiv ... >> More

  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. << Less

  Virology 487:27-40(2016) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structural insights to how mammalian capping enzyme reads the CTD code.

  Ghosh A., Shuman S., Lima C.D.

  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 compone ... >> More

  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. << Less

  Mol. Cell 43:299-310(2011) [PubMed] [EuropePMC]

  This publication is cited by 6 other entries.

• mRNA capping: biological functions and applications.

  Ramanathan A., Robb G.B., Chan S.H.

  The 5' m7G cap is an evolutionarily conserved modification of eukaryotic mRNA. Decades of research have established that the m7G cap serves as a unique molecular module that recruits cellular proteins and mediates cap-related biological functions such as pre-mRNA processing, nuclear export and cap ... >> More

  The 5' m7G cap is an evolutionarily conserved modification of eukaryotic mRNA. Decades of research have established that the m7G cap serves as a unique molecular module that recruits cellular proteins and mediates cap-related biological functions such as pre-mRNA processing, nuclear export and cap-dependent protein synthesis. Only recently has the role of the cap 2'O methylation as an identifier of self RNA in the innate immune system against foreign RNA has become clear. The discovery of the cytoplasmic capping machinery suggests a novel level of control network. These new findings underscore the importance of a proper cap structure in the synthesis of functional messenger RNA. In this review, we will summarize the current knowledge of the biological roles of mRNA caps in eukaryotic cells. We will also discuss different means that viruses and their host cells use to cap their RNA and the application of these capping machineries to synthesize functional mRNA. Novel applications of RNA capping enzymes in the discovery of new RNA species and sequencing the microbiome transcriptome will also be discussed. We will end with a summary of novel findings in RNA capping and the questions these findings pose. << Less

  Nucleic Acids Res 44:7511-7526(2016) [PubMed] [EuropePMC]

  This publication is cited by 15 other entries.

• Enzymology of RNA cap synthesis.

  Ghosh A., Lima C.D.

  The 5' guanine-N7 methyl cap is unique to cellular and viral messenger RNA (mRNA) and is the first co-transcriptional modification of mRNA. The mRNA cap plays a pivotal role in mRNA biogenesis and stability, and is essential for efficient splicing, mRNA export, and translation. Capping occurs by a ... >> More

  The 5' guanine-N7 methyl cap is unique to cellular and viral messenger RNA (mRNA) and is the first co-transcriptional modification of mRNA. The mRNA cap plays a pivotal role in mRNA biogenesis and stability, and is essential for efficient splicing, mRNA export, and translation. Capping occurs by a series of three enzymatic reactions that results in formation of N7-methyl guanosine linked through a 5'-5' inverted triphosphate bridge to the first nucleotide of a nascent transcript. Capping of cellular mRNA occurs co-transcriptionally and in vivo requires that the capping apparatus be physically associated with the RNA polymerase II elongation complex. Certain capped mRNAs undergo further methylation to generate distinct cap structures. Although mRNA capping is conserved among viruses and eukaryotes, some viruses have adopted strategies for capping mRNA that are distinct from the cellular mRNA capping pathway. << Less

  Wiley Interdiscip Rev RNA 1:152-172(2010) [PubMed] [EuropePMC]

  This publication is cited by 11 other entries.

• Structure and mechanism of yeast RNA triphosphatase: an essential component of the mRNA capping apparatus.

  Lima C.D., Wang L.K., Shuman S.

  RNA triphosphatase is an essential mRNA processing enzyme that catalyzes the first step in cap formation. The 2.05 A crystal structure of yeast RNA triphosphatase Cet1p reveals a novel active site fold whereby an eight-stranded beta barrel forms a topologically closed triphosphate tunnel. Interact ... >> More

  RNA triphosphatase is an essential mRNA processing enzyme that catalyzes the first step in cap formation. The 2.05 A crystal structure of yeast RNA triphosphatase Cet1p reveals a novel active site fold whereby an eight-stranded beta barrel forms a topologically closed triphosphate tunnel. Interactions of a sulfate in the center of the tunnel with a divalent cation and basic amino acids projecting into the tunnel suggest a catalytic mechanism that is supported by mutational data. Discrete surface domains mediate Cet1p homodimerization and Cet1p binding to the guanylyltransferase component of the capping apparatus. The structure and mechanism of fungal RNA triphosphatases are completely different from those of mammalian mRNA capping enzymes. Hence, RNA triphosphatase presents an ideal target for structure-based antifungal drug discovery. << Less

  Cell 99:533-543(1999) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Structure and mechanism of the RNA triphosphatase component of mammalian mRNA capping enzyme.

  Changela A., Ho C.K., Martins A., Shuman S., Mondragon A.

  The 5' capping of mammalian pre-mRNAs is initiated by RNA triphosphatase, a member of the cysteine phosphatase superfamily. Here we report the 1.65 A crystal structure of mouse RNA triphosphatase, which reveals a deep, positively charged active site pocket that can fit a 5' triphosphate end. Struc ... >> More

  The 5' capping of mammalian pre-mRNAs is initiated by RNA triphosphatase, a member of the cysteine phosphatase superfamily. Here we report the 1.65 A crystal structure of mouse RNA triphosphatase, which reveals a deep, positively charged active site pocket that can fit a 5' triphosphate end. Structural, biochemical and mutational results show that despite sharing an HCxxxxxR(S/T) motif, a phosphoenzyme intermediate and a core alpha/beta-fold with other cysteine phosphatases, the mechanism of phosphoanhydride cleavage by mammalian capping enzyme differs from that used by protein phosphatases to hydrolyze phosphomonoesters. The most significant difference is the absence of a carboxylate general acid catalyst in RNA triphosphatase. Residues conserved uniquely among the RNA phosphatase subfamily are important for function in cap formation and are likely to play a role in substrate recognition. << Less

  EMBO J. 20:2575-2586(2001) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Chlorella virus RNA triphosphatase. Mutational analysis and mechanism of inhibition by tripolyphosphate.

  Gong C., Shuman S.

  Chlorella virus RNA triphosphatase (cvRtp1) is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, poxviruses, and baculoviruses. The primary structure of cvRtp1 is more similar to that of the yeast RNA triphosphatase Cet1 ... >> More

  Chlorella virus RNA triphosphatase (cvRtp1) is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, poxviruses, and baculoviruses. The primary structure of cvRtp1 is more similar to that of the yeast RNA triphosphatase Cet1 than it is to the RNA triphosphatases of other DNA viruses. To evaluate the higher order structural similarities between cvRtp1 and the fungal enzymes, we performed an alanine scan of individual residues of cvRtp1 that were predicted, on the basis of the crystal structure of Cet1, to be located at or near the active site. Twelve residues (Glu(24), Glu(26), Asp(64), Arg(76), Lys(90), Glu(112), Arg(127), Lys(129), Arg(131), Asp(142), Glu(163), and Glu(165)) were deemed essential for catalysis by cvRtp1, insofar as their replacement by alanine reduced phosphohydrolase activity to <5% of the wild-type value. Structure-activity relationships were elucidated by introducing conservative substitutions at the essential positions. The mutational results suggest that the active site of cvRtp1 is likely to adopt a tunnel fold like that of Cet1 and that a similar constellation of side chains within the tunnel is responsible for metal binding and reaction chemistry. Nonetheless, there are several discordant mutational effects in cvRtp1 versus Cet1, which suggest that different members of the phosphohydrolase family vary in their reliance on certain residues within the active site tunnel. We found that tripolyphosphate and pyrophosphate were potent competitive inhibitors of cvRtp1 (K(i) = 0.6 microm tripolyphosphate and 2.4 microm pyrophosphate, respectively), whereas phosphate had little effect. cvRtp1 displayed a weak intrinsic tripolyphosphatase activity (3% of its ATPase activity) but was unable to hydrolyze pyrophosphate. << Less

  J Biol Chem 277:15317-15324(2002) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.