A Plasmodium falciparum protein related to fungal RNA 5'-triphosphatases.

Messenger RNA of eukaryotic cells and most of their viruses carry a ‘cap’ structure at their 5 end. RNA 5 -triphosphatase (RTPase) activity is required for the first step of cap biosynthesis. This activity removes the -phosphate from the 5 end of a nascent mRNA to leave a diphosphate end. mRNA guanylyltransferase (GTase) subsequently adds GMP in a 5 –5 orientation to form the structure, GpppN1-. These two activities are tightly associated and co-purify as ‘mRNA capping enzyme’ [1]. One or more methyltransferase completes the capping modifications. At least two groups of cellular RTPases have been identified. Metazoan capping enzymes consist of a single polypeptide with two separable domains; a GTase domain and a RTPase domain that resembles the protein tyrosine phosphatases (PTPs) [2–4]. This type of RTPase domain appears to use a mechanism similar to that of PTPs, as a conserved nucleophilic cysteine is required for activity [2,5–8]. The capping enzymes of the yeasts Saccharomyces cere isiae and Candida albicans define a second class of RTPases. In these organisms, GTase and RTPase are carried by distinct proteins that form a heterodimer complex. The amino acid sequence of the catalytic region of yeast RTPase subunit has no obvious similarity to PTPs [9,10]. Unlike metazoan RTPases, the yeast proteins require divalent cations for activity [11,12]. It has been suggested that fungal RTPases comprise a new family of metal-dependent nucleotide phosphohydrolase (NTPase)/RTPase. This family may also include capping RTPases encoded by some DNA viruses. Although there is no extensive sequence similarity between the viral and yeast RTPases, several short sequence elements found in all of these proteins have been shown to be essential for the activity [11–14]. To identify other possible members of the yeast RTPase family, we used the BLAST algorithm [15] to search GenBank for proteins with similarity to Cet1, the S. cere isiae capping enzyme RTPase subunit [9]. Known relatives of Cet1 include CaCet1, the C. albicans RTPase subunit [10] and Ctl1 (also known as Cth1), a second RTPase from S. cere isiae whose function is unknown but nonessential for viability [16,17]. In addition to these fungal proteins, an open reading frame from human parasite Plasmodium falciparum (PFC0985c, accession number: CAB39040) was found to have significant similarity to Cet1 (P=3e-5). No related proteins were found in the complete Drosophila, C. elegans, or human genomes. PFC0985c is a hypothetical protein with 591 amino acids whose predicted size is 69 kDa. This protein has 109 asparagine residues and 89 acidic residues that are largely clustered in the N-terminal (amino acids 100– 250) and the C-terminal (aa 405–591) regions (Fig. Abbre iations: CIP, calf intestine alkaline phopshatase; GTase, mRNA guanylytransferase; IPTG, isopropyl -D-thiogalactopyranoside; NTPase, nucleotide phosphohydrolase; PCR, polymerase chain reaction; PEI, polyethyleneimine; PTP, protein tyrosine phosphatase; RTPase, RNA 5 -triphosphatase; TLC, thin-layer chromatography. * Corresponding author. Tel.: +1-617-4320696; fax: +1-6177380516. E-mail address: steveb@hms.harvard.edu (S. Buratowski). 1 Present address: Shionogi BioResearch Corporation, 45 Hartwell Avenue, Lexington, MA 02421, USA.