Human mRNA Capping Enzyme (RNGTT) and Cap Methyltransferase (RNMT) Map to 6q16 and 18p11.22–p11.23, Respectively

Abstract

Eukaryotic cellular and viral mRNAs contain a 59-terminal mGpppN cap structure, which has important consequences at multiple levels of gene expression including RNA splicing, 39 end formation, transcript stability, nuclear export, and translation initiation (6, 12, 15, 16). Recent studies have demonstrated that capping enzyme selectively binds to the phosphorylated, elongating form of RNA polymerase II (pol II) (23). This finding accounts for the specific capping of pol II transcripts. It may also provide new insights into how HIV tat protein promotes pol II phosphorylation by tat-activated kinase (TAK) to enhance polymerase processivity (5). Caps are formed on nascent pre-mRNAs by the sequential action of RNA 59-triphosphatase, which removes the g-phosphate of the initiating nucleotide, RNA guanylyltransferase, which transfers GMP from GTP to the resulting diphosphate end, and RNA (guanine-7) methyltransferase, which methylates the guanine N7 position of the newly formed GpppN termini (4, 12, 15). These three enzymatic activities are present as separate polypeptides in yeasts, and strains null for the triphosphatase (18), guanylyltransferase (14), or methyltransferase (7) are nonviable. Metazoans including humans contain a separate cap methyltransferase (RNA guanine-7-methyltransferase, RNMT), but the RNA 59-triphosphatase and guanylyltransferase activities are contained as Nand C-terminal domains in a bifunctional capping enzyme (RNGTT) encoded by a single gene (23). Despite this difference in genomic organization between uniand multicellular organisms, capping enzyme is functionally conserved, and Saccharomyces cerevisiae strains null for guanylyltransferase or RNA 59-triphosphatase are complemented for growth by the mammalian counterpart (21, 23). In light of the importance of mRNA capping for gene expression, we determined the chromosomal location of the human capping enzyme gene (RNGTT) by PCR using as template the NIGMS human/rodent somatic cell hybrid panel 2, version 3 (Coriell Institute for Medical Research Cell Repositories, Camden, NJ). This panel consists of DNA isolated from 24 human/rodent somatic cell hybrid lines that each retained one intact human chromosome. Primers 1, 59-CAGTGTGTCCATTCTGGCAGTGGTTTTG-39, and 2, 59GTGGGAGAGCATTTTCTGGTGACAGC-39, from the 39-UTR of the human capping enzyme cDNA sequence (Accession No. AF025654) were designed to give a human-specific 318-bp PCR product. PCR results demonstrated that the human capping enzyme gene RNGTT is located on chromosome 6 (data not shown). Fine-mapping was performed by PCR analysis of the GeneBridge 4 radiation hybrid panel (Research Genetics, Inc., Huntsville, AL) with 25 ng template genomic DNA and primers 1 and 2 in a 12.5-ml reaction. After an initial denaturation step at 94°C for 5 min, 35 cycles of amplification consisting of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s were performed, followed by a final extension of 5 min at 72°C. Amplification products were analyzed by agarose gel electrophoresis. Data were submitted to the Whitehead Institute/MIT Center for Genome Research STS mapping server for statistical analysis with the RHMAPPER software package (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl). The data vector for RNGTT was 00100 01101 02101 0011