Structure and mechanism of the RNA triphosphatase comonent of mammalian mRNA capping enzyme

Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2153 Sheridan Road, Evanston, IL 60208-3500, USA.
The EMBO Journal (Impact Factor: 10.43). 06/2001; 20(10):2575-86. DOI: 10.1093/emboj/20.10.2575
Source: PubMed


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.


Available from: Alfonso Mondragón
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    • "Within the PTP family, we built two sub-trees including ten subfamilies of rice and Arabidopsis, using the same naming convention for the subfamily except for atypical DSP and ''Other'' (Kerk et al. 2008). We substituted mRNA capping enzyme for atypical DSP because members of this subfamily represent mRNA capping enzyme—a holo-enzyme responsible for the formation of mRNA capping—and its N-terminus has a DSP-like domain that catalyzes the removal of c-phosphate of pre-mRNA (Changela et al. 2001). The ''Other'' subfamily was further split into four new subfamilies, MKP-like, KIS, SIW14- like, and PLIP; the former three are defined according to the literature as shown in supplemental Table 2, whereas PLIP was determined based on a homolog search against the PANTHER database (Thomas et al. 2003). "
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    ABSTRACT: Protein phosphatases play essential roles in many cellular processes through the reversible protein phosphorylation that dictates many signal transduction pathways among organisms. Based on an in silico analysis, we classified 163 and 164 non-redundant protein phosphatases in rice and Arabidopsis, respectively. Protein serine/threonine phosphatases make up 67% of the total in both plants, in contrast to those of human, where this fraction is about 27%. Based on domain organization and intron composition analyses, we found that protein phosphatases in the two plants are highly conserved in structure. Evolutionary analysis suggests that segmental duplications occurring 40–70millionyears ago, contributed to the limited expansion of protein phosphatases. Gene expression analysis suggests that most phosphatases have broad expression spectra, with high abundance in four surveyed tissues (root, leaf, inflorescence, and seedling); only 46 and 12 phosphatases expressed in a single tissue of rice and Arabidopsis, respectively, regardless of their expression levels. Promoter analysis among different phosphatase subfamilies demonstrates a variable distribution of the w-box (a cis-element involved in disease resistance) between rice and Arabidopsis. KeywordsProtein phosphatases-Duplication-Expression-Promoter
    Plant Systematics and Evolution 11/2010; 289(3):111-126. DOI:10.1007/s00606-010-0336-8 · 1.42 Impact Factor
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    • "Primary structures of mammalian RNA triphosphatases considerably differ from their fungal counterparts. In addition, the catalytic mechanism of RNA triphosphatases in fungi and mammals are completely different (Ho et al., 1998; Lima et al., 1999; Pei et al., 1999; Changela et al., 2001). In fact, while mammalian RNA triphosphatases belong to the metal-independent family, fungi contain RNA triphosphatases that belong to the divalent-cationdependent group. "
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    ABSTRACT: The identification of essential genes represents a critical step in the discovery of novel therapeutic targets in Aspergillus fumigatus. Structural analyses of the Saccharomyces cerevisiae RNA triphosphatase pointed out this enzyme as an attractive therapeutic target for fungal infections. In addition, demonstration of the essentiality of the S. cerevisiae RNA triphosphatase encoding gene enhanced the value of this potential therapeutic target. Nevertheless, consideration of a fungal RNA triphosphatase as an ideal therapeutic target needs confirmation of the essentiality of the respective gene in a fungal pathogen. In this work, we analyzed the essentiality of the A. fumigatus triA gene, encoding RNA triphosphatase, by conditional gene expression and heterokaryon deletion. Using the conditional gene expression driven by the alcA promoter (alcA(P)), we found that TriA depletion causes morphological abnormalities that result in a very strong growth inhibition. Nevertheless, since a strict terminal phenotype was not observed, the essentiality of the triA gene could not be ensured. Accordingly, the essentiality of this gene was analyzed by the heterokaryon rescue technique. Results obtained unequivocally demonstrated the essentiality of the A. fumigatus triA gene, indicating the suitability of the RNA triphosphatase as an ideal therapeutic target to treat A. fumigatus infections. Besides, a second conditional gene expression system, based on the niiA promoter (niiA(P)), was utilized in this work. Although the niiA(P)-mediated repression of triA was less severe than that driven by the alcA(P), a strong growth inhibition was also found in niiA(P)-triA strains. Finally, E-tests performed to determine whether triA down-regulated cells became more sensitive to antifungals suggest a synergic effect between amphotericin B and another antifungal inhibiting the A. fumigatus RNA triphosphatase activity.
    Fungal Genetics and Biology 10/2009; 47(1):66-79. DOI:10.1016/j.fgb.2009.10.010 · 2.59 Impact Factor
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    • "A mock-purified glycerol gradient preparation from non-recombinant baculovirus-infected cells did not show RTPase activity, ruling out the presence of baculovirus triphosphatases in the L–P complex fraction (Supplementary Fig. S3, lanes 10–12). Further evidence for L-mediated RTPase activity was obtained by the metal-dependency of the reaction, whereas the cellular RTPase reaction is metal-independent (Changela et al., 2001). "
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    ABSTRACT: Rinderpest virus (RPV) large (L) protein is an integral part of the ribonucleoprotein (RNP) complex of the virus that is responsible for transcription and replication of the genome. Previously, we have shown that recombinant L protein coexpressed along with P protein (as the L-P complex) catalyses the synthesis of all viral mRNAs in vitro and the abundance of mRNAs follows a gradient of polarity, similar to the occurrence in vivo. In the present work, we demonstrate that the viral mRNAs synthesized in vitro by the recombinant L or purified RNP are capped and methylated at the N7 guanine position. RNP from the purified virions, as well as recombinant L protein, shows RNA triphosphatase (RTPase) and guanylyl transferase (GT) activities. L protein present in the RNP complex catalyses the removal of gamma-phosphate from triphosphate-ended 25 nt RNA generated in vitro representing the viral N-terminal mRNA 5' sequence. The L protein forms a covalent enzyme-guanylate intermediate with the GMP moiety of GTP, whose formation is inhibited by the addition of pyrophosphate; thus, it exhibits characteristics of cellular GTs. The covalent bond between the enzyme and nucleotide is acid labile and alkali stable, indicating the presence of phosphoamide linkage. The C-terminal region (aa 1717-2183) of RPV L protein alone exhibits the first step of GT activity needed to form a covalent complex with GMP, though it lacks the ability to transfer GMP to substrate RNA. Here, we describe the biochemical characterization of the newly found RTPase/GT activity of L protein.
    Journal of General Virology 04/2009; 90(Pt 7):1748-56. DOI:10.1099/vir.0.010975-0 · 3.18 Impact Factor
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