A yeast-like mRNA capping apparatus in Plasmodium falciparum

Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2001; 98(6):3050-5. DOI: 10.1073/pnas.061636198
Source: PubMed


Analysis of the mRNA capping apparatus of the malaria parasite Plasmodium falciparum illuminates an evolutionary connection to fungi rather than metazoans. We show that P. falciparum encodes separate RNA guanylyltransferase (Pgt1) and RNA triphosphatase (Prt1) enzymes and that the triphosphatase component is a member of the fungal/viral family of metal-dependent phosphohydrolases, which are structurally and mechanistically unrelated to the cysteine-phosphatase-type RNA triphosphatases found in metazoans and plants. These results highlight the potential for discovery of mechanism-based antimalarial drugs designed to specifically block the capping of Plasmodium mRNAs. A simple heuristic scheme of eukaryotic phylogeny is suggested based on the structure and physical linkage of the triphosphatase and guanylyltransferase enzymes that catalyze cap formation.

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Available from: C. Kiong Ho, Jan 08, 2015
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    • "Post-transcriptional regulatory mechanisms can interfere with the eIF4F complex assembly, either through eIF4E binding proteins [8], or by specific mRNA decay in which the mRNA is de-capped [9]. The human malaria parasite Plasmodium falciparum caps its mRNAs with a canonical cap structure, as it has functional mRNA capping enzymes [10] and cDNA cloning of full-length cDNAs by " oligo capping " has been demonstrated, indicating the presence of capped mRNA [11]. In contrast, few details are known of P. falciparum translation, including functional characterization of its translation factors. "
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    ABSTRACT: The mRNA 5' cap is an essential structural feature for translation of eukaryotic mRNA. Translation is initiated by recognition of the cap by the translation initiation factor eIF4E. To further our understanding of mRNA translation in the human malaria parasite Plasmodium falciparum, we have investigated the parasite eIF4E and its interaction with capped mRNA. We have purified P. falciparum eIF4E as a recombinant protein and demonstrated that it has canonical mRNA cap binding activity. We used this protein to purify P. falciparum capped mRNAs from total parasite RNA. Microarray analysis comparing total and eIF4E-purified capped mRNAs shows that 34 features were more than twofold under-represented in the purified RNA sample, including 19 features representative of nuclear transcripts. The putatively uncapped nuclear transcripts may represent a class of mRNAs targeted for storage and cap removal.
    Molecular and Biochemical Parasitology 11/2007; 155(2):146-55. DOI:10.1016/j.molbiopara.2007.07.003 · 1.79 Impact Factor
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    • "These positions are denoted by d under the aligned sequences of fungal, viral, and protozoan tunnel-family RNA triphosphatases shown in Figure 3. The alignment of Prt1 is modified compared to that reported initially (Ho and Shuman 2001a) in light of the present finding that Arg103 (formerly predicted to define the third b-strand of the tunnel and to correspond to Cet1 Arg393) is clearly not essential for catalysis. In the revised structural alignment , Arg103 is located upstream of the newly assigned equivalents of the second and third b-strands of the Cet1 triphosphate tunnel (Fig. 3). "
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    ABSTRACT: RNA triphosphatase catalyzes the first step in mRNA capping. The RNA triphosphatases of fungi and protozoa are structurally and mechanistically unrelated to the analogous mammalian enzyme, a situation that recommends RNA triphosphatase as an anti-infective target. Fungal and protozoan RNA triphosphatases belong to a family of metal-dependent phosphohydrolases exemplified by yeast Cet1. The Cet1 active site is unusually complex and located within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel). Here we probe the active site of Plasmodium falciparum RNA triphosphatase by targeted mutagenesis and thereby identify eight residues essential for catalysis. The functional data engender an improved structural alignment in which the Plasmodium counterparts of the Cet1 tunnel strands and active-site functional groups are located with confidence. We gain insight into the evolution of the Cet1-like triphosphatase family by noting that the heretofore unique tertiary structure and active site of Cet1 are recapitulated in recently deposited structures of proteins from Pyrococcus (PBD 1YEM) and Vibrio (PDB 2ACA). The latter proteins exemplify a CYTH domain found in CyaB-like adenylate cyclases and mammalian thiamine triphosphatase. We conclude that the tunnel fold first described for Cet1 is the prototype of a larger enzyme superfamily that includes the CYTH branch. This superfamily, which we name "triphosphate tunnel metalloenzyme," is distributed widely among bacterial, archaeal, and eukaryal taxa. It is now clear that Cet1-like RNA triphosphatases did not arise de novo in unicellular eukarya in tandem with the emergence of caps as the defining feature of eukaryotic mRNA. They likely evolved by incremental changes in an ancestral tunnel enzyme that conferred specificity for RNA 5'-end processing.
    RNA 09/2006; 12(8):1468-74. DOI:10.1261/rna.119806 · 4.94 Impact Factor
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    • "To address these issues, we have characterized the RNA triphosphatases of two other fungi, including the human pathogen Candida albicans and the fission yeast Schizosaccharomyces pombe[10-12]. The fungal triphosphatases, S. cerevisiae Cet1, C. albicans CaCet1 and S. pombe Pct1, belong to a new family of metal-dependent phosphohydrolases that embraces the triphosphatase components of DNA virus and protozoan mRNA capping systems [1,7,13,14]. The defining features of the metal-dependent RNA triphosphatases are two glutamate-containing motifs that are required for catalysis and comprise the metal-binding site in the crystal structure of S. cerevisiae Cet1. "
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    ABSTRACT: Background The first two steps in the capping of cellular mRNAs are catalyzed by the enzymes RNA triphosphatase and RNA guanylyltransferase. Although structural and mechanistic differences between fungal and mammalian RNA triphosphatases recommend this enzyme as a potential antifungal target, it has not been determined if RNA triphosphatase is essential for the growth of fungal species that cause human disease. Results We show by classical genetic methods that the triphosphatase (Pct1) and guanylyltransferase (Pce1) components of the capping apparatus in the fission yeast Schizosaccharomyces pombe are essential for growth. We were unable to disrupt both alleles of the Candida albicans RNA triphosphatase gene CaCET1, implying that the RNA triphosphatase enzyme is also essential for growth of C. albicans, a human fungal pathogen. Conclusions Our results provide the first genetic evidence that cap synthesis is essential for growth of an organism other than Saccharomyces cerevisiae and they validate RNA triphosphatase as a target for antifungal drug discovery.
    BMC Microbiology 11/2001; 1(1). DOI:10.1186/1471-2180-1-29 · 2.73 Impact Factor
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