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

Abstract

GTP cyclohydrolase I (EC 3.5.4.16) is the first enzyme in the biosynthesis of tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H4-biopterin] in mammals and of folic acid in bacteria. Here we have characterized the GTP cyclohydrolase I gene structure and two mRNA species from Physarum polycephalum, an acellular slime mould that synthesizes H4-biopterin and metabolites of the folic acid biosynthetic pathway. Its GTP cyclohydrolase I gene consists of seven exons, and the two GTP cyclohydrolase I cDNA species isolated from Physarum encode for proteins with 228 (25.7kDa) and 195 (22.1kDa) amino acids. Furthermore, we identified two previously undescribed mRNA species in interferon-γ-treated human myelomonocytoma cells (THP-1) in addition to the cDNA coding for the fully functional 250-residue (27.9kDa) protein, which is identical with that in human phaeochromocytoma cells. One of the new splice variants codes for a 233-residue (25.7kDa) protein, whereas the other codes for the full-length protein but is alternatively spliced within the 3′-untranslated region. In heterologous expression, the shorter proteins of Physarum as well as of THP-1 cells identified here are degraded by proteolysis. Accordingly, only the 27.9kDa protein was detectable in Western blots from THP-1 cell extracts. Quantification of GTP cyclohydrolase I mRNA species in different human cell types with and without cytokine treatment showed that in addition to the correct mRNA the two splice variants isolated here, as well as the two splice variants known from human liver, are strongly induced by cytokines in cell types with inducible GTP cyclohydrolase I (THP-1, dermal fibroblasts), but not in cell types with constitutive GTP cyclohydrolase I expression (SK-N-SH, Hep-G2). As in human liver, splicing of the new mRNA variant found in THP-1 cells occurs at the boundary 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 position occurs in two species highly distant from each other in terms of evolution. It remains to be seen whether variant proteins encoded by alternatively spliced GTP cyclohydrolase I mRNA transcripts do occur in vivo and whether they participate in regulation of enzyme activity.

Characterization of GTP cyclohydrolase I gene expression in the human neuroblastoma SKN‐BE(2)M17: enhanced transcription in response to cAMP is conferred by the proximal promoter

“…1995). Moreover, Golderer et al . (2001) have recently reported additional alternatively spliced forms of human GTPCH.…”

“…While we could not detect Type 2 and 3 mRNA by nuclease protection assay, these transcripts can be amplified by RT‐PCR using RNA isolated from human liver RNA (Togari et al . 1992) and the human neuroblastoma cell line SK‐N‐SH (Golderer et al . 2001) and are reported to make up as much as 20% of the GTPCH mRNA found in some human tissues (Hirano et al .…”

“…1992; Ichinose et al . 1995) or more (Golderer et al . 2001) transcripts with different coding sequences which introduces an additional level of complexity to the study of human GTPCH gene expression.…”

Discovery of common human genetic variants of GTP cyclohydrolase 1 (GCH1) governing nitric oxide, autonomic activity, and cardiovascular risk

“…Although the C+243T region lies in the 3′-UTRs of the terminal exon of GCH1 in the major mRNA isoform 1 (clone NM_00016) in humans, and mRNA splicing of GCH1 displays substantial cross-species conservation (e.g., in dog and slime mold) (10), 2 additional human splice variants (NM_001024070, NM_001024071) that exclude C+243T from the mature mRNA are known in this region, and the region may be intronic in the reported isoforms of some species (mouse), or even downstream from (i.e., 3′ to) the reported terminal exon in other species (chicken, rat, zebrafish, Xenopus, roundworm, and eremoth). Cytokine induction of GCH1 transcription may also trigger its alternative splicing (10).…”

Low tetrahydrobiopterin biosynthetic capacity of human monocytes is caused by exon skipping in 6-pyruvoyl tetrahydropterin synthase

“…Total RNA was isolated using the TRIzol R reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer's instructions. A 647 bp BamHI-EcoRI PTPS cDNA fragment containing the open reading frame (ORF) and flanking sequences, which was amplified initially from isolated human PTPS cDNA [13] using primers PTPS11/14 [14] and cloned into pUC18, as well as a 578 bp probe for human GTP cyclohydrolase I [15], were used as probes. For Northern-blot analysis, total RNA (20 µg) was resolved in 1 % (w/v) agarose/6 % (v/v) formaldehyde gels, blotted on to Duralon-UV nylon membranes (Stratagene, La Jolla, CA, U.S.A.) by means of vacuum and cross-linked by UV irradiation (Stratalinker; Stratagene).…”