Differential splicing of Pneumocystis cariniif. sp. carinii inosine 5′-monophosphate dehydrogenase pre-mRNA

Abstract

Inosine 5′-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in guanine nucleotide metabolism that has drawn attention as a drug target in several organisms. Pneumocystis cariniif. sp. carinii IMPDH mRNA (GeneBank Accession No: U42442) previously identified from cultured organisms yielded a predicted amino acid sequence about 70 amino acids shorter at the amino terminus than IMPDH from other species. Recent research has shown that the amino terminal region is important for enzyme activity, suggesting that the previous putative P. carinii IMPDH might not represent full length, functional enzyme. To test this hypothesis, RT-PCR was performed with total RNA isolated from P. carinii f. sp. carinii. Three IMPDH splicing variants were found and splicing preference was observed: P. carinii isolated from infected rat lung contained primarily splicing variant one (introns two and four deleted), but organisms from spinner flask culture contained primarily splicing variant three (all four introns deleted). Importantly, splicing variant one (GeneBank Accession No: AF196975) contained an open reading frame for 529 amino acids, a size comparable to that of other eukaryotic IMPDH forms. The other variants contained the same open reading frame (454 amino acids) previously reported. Sequence analysis and complementation studies suggest variant one represents the full length, catalytically active form of P. carinii IMPDH. The differential splicing of the enzyme may reflect a mechanism by which the organism regulates the expression of IMPDH in response to environmental stresses.