RNA triphosphatase is essential in Schizosaccharomyces pombe and Candida albicans

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.

Figure 1

Genotype of the CaCET1/cacet1::UAU1 heterozygote strain of C. albicans. Illustrated in cartoon form are the configurations of the wild-type CaCET1 and the cacet1::UAU1 chromosomal loci in the Arg⁺ heterozygous diploids. The positions of pertinent restriction sites and the CaCET1 5'-specific (A) and 3'-specific (B) hybridization probes are shown. Also shown is the configuration of the triplicated cacet1::URA2 allele in the Arg⁺ Ura⁺ segregants.

Figure 2

Southern blot analysis. DNA isolated from the parental diploid strain (lane P), one of the Arg⁺ heterozygotes (isolate #19; lane H), and fourteen independent Arg⁺ Ura⁺ segregants were digested with BglII and resolved by agarose gel electrophoresis. A photograph of the ethidium bromide-stained gel is shown in panel C. The positions and sizes (kbp) of DNA size markers are indicated on the right. The DNA was transferred to a Hybond membrane, which was serially hybridized to ³²P-labeled DNA probes A (panel A) and B (panel B) derived from the 5' and 3' segments of the CaCET1 gene, respectively.