GTP cyclohydrolase I mRNA: novel splice variants in the slime mould Physarum polycephalum and in human monocytes (THP-1) indicate conservation of mRNA processing

CTP cyclohydrolase I (EC 3.5.4.16) is the first enzyme in the biosynthesis of tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H-4-biopterin] in mammals and of folic acid in bacteria. Here we have characterized the C TP cyclohydrolase I gene structure and two mRNA species from Physarum polyc ephalum, an acellular slime mould that synthesizes H-4-biopterin and metabo lites of the folic acid biosynthetic pathway. Its GTP cyclohydrolase I gene consists of seven exons, and the two GTP cyclohydrolase I cDNA species iso lated from Physarum encode for proteins with 228 (25.7 kDa) and 195 (22.1 k Da) amino acids. Furthermore, we identified two previously undescribed mRNA species in interferon-gamma -treated human myelo-monocytoma cells (THP-I) in addition to the cDNA coding for the fully functional 25D-residue (27.9 k Da) protein, which is identical with that in human phaeochromocytoma cells. One of the new splice variants codes for a 233-residue (25.7 kDa) protein, whereas the other codes for the full-length protein but is alternatively s pliced within the 3'-untranslated region. In heterologous expression, the s horter proteins of Physarum as well as of THP-1 cells identified here are d egraded by proteolysis. Accordingly, only the 27.9 kDa protein was detectab le in Western blots from THP-1 cell extracts. Quantification of GTP cycle h ydrolase I mRNA species in different human cell types with and without cyto kine treatment showed that in addition to the correct mRNA the two splice v ariants isolated here, as well as the two splice variants known from human liver, are strongly induced by cytokines in cell types with inducible GTP c yclohydrolase I (THP-1, dermal fibroblasts), but not in cell types with con stitutive GTP cyclohydrolase I expression (SK-N-SH, Hep-G2). As in human li ver, splicing of the new mRNA variant found in THP-1 cells occurs at the bo undary of exons 5 and 6. Strikingly, the 195-residue protein from Physarum is alternatively spliced at a homologous position, i.e. at the boundary of exons 6 and 7. Thus alternative splicing of GTP cyclohydrolase I at this po sition occurs in two species highly distant from each other in terms of evo lution. It remains to be seen whether variant proteins encoded by alternati vely spliced GTP cyclohydrolase I mRNA transcripts do occur in that and whe ther they participate in regulation of enzyme activity.