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

ABSTRACT: Biosynthesis of (6 R )-5,6,7,8-tetrahydro-L-biopterin (H(4)-biopterin), an essential cofactor for aromatic amino acid hydroxylases and NO synthases, is effectively induced by cytokines in most of the cell types. However, human monocytes/macrophages form only a little H(4)-biopterin, but release neopterin/7,8-dihydroneopterin instead. Whereas 6-pyruvoyl tetrahydropterin synthase (PTPS) activity, the second enzyme of H(4)-biopterin biosynthesis, is hardly detectable in these cells, PTPS mRNA levels were comparable with those of cell types containing intact PTPS activity. By screening a THP-1 cDNA library, we identified clones encoding the entire open reading frame (642 bp) as well as clones lacking the 23 bp exon 3, which results in a premature stop codon. Quantification of the two mRNA species in different cell types (blood-derived cells, fibroblasts and endothelial cells) and cell lines showed that the amount of exon-3-containing mRNA is correlated closely to PTPS activity. The ratio of exon-3-containing to exon-3-lacking PTPS mRNA is not affected by differential mRNA stability or nonsense-mediated mRNA decay. THP-1 cells transduced with wild-type PTPS cDNA produced H(4)-biopterin levels and expressed PTPS activities and protein amounts comparable with those of fibroblasts. We therefore conclude that exon 3 skipping in transcription rather than post-transcriptional mechanisms is a major cause of the low PTPS protein expression observed in human macrophages and related cell types.

SUBMITTER: Leitner KL

PROVIDER: S-EPMC1223526 | biostudies-other | 2003 Aug

REPOSITORIES: biostudies-other

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Project description:BACKGROUND:At least 90% of human genes are alternatively spliced. Alternative splicing has an important function regulating gene expression and miss-splicing can contribute to risk for human diseases, including Alzheimer's disease (AD). METHODS:We developed a splicing decision model as a molecular mechanism to identify functional exon skipping events and genetic variation affecting alternative splicing on a genome-wide scale by integrating genomics, transcriptomics, and neuroimaging data in a systems biology approach. In this study, we analyzed RNA-Seq data of hippocampus brain tissue from Alzheimer's disease (AD; n?=?24) and cognitively normal elderly controls (CN; n?=?50) and identified three exon skipping events in two genes (RELN and NOS1) as significantly associated with AD (corrected p-value <?0.05 and fold change >?1.5). Next, we identified single-nucleotide polymorphisms (SNPs) affecting exon skipping events using the splicing decision model and then performed an association analysis of SNPs potentially affecting three exon skipping events with a global cortical measure of amyloid-? deposition measured by [18F] Florbetapir position emission tomography (PET) scan as an AD-related quantitative phenotype. A whole-brain voxel-based analysis was also performed. RESULTS:Two exons in RELN and one exon in NOS1 showed significantly lower expression levels in the AD participants compared to CN participants, suggesting that the exons tend to be skipped more in AD. We also showed the loss of the core protein structure due to the skipped exons using the protein 3D structure analysis. The targeted SNP-based association analysis identified one intronic SNP (rs362771) adjacent to the skipped exon 24 in RELN as significantly associated with cortical amyloid-? levels (corrected p-value <?0.05). This SNP is within the splicing regulatory element, i.e., intronic splicing enhancer. The minor allele of rs362771 conferred decreases in cortical amyloid-? levels in the right temporal and bilateral parietal lobes. CONCLUSIONS:Our results suggest that exon skipping events and splicing-affecting SNPs in the human hippocampus may contribute to AD pathogenesis. Integration of multiple omics and neuroimaging data provides insights into possible mechanisms underlying AD pathophysiology through exon skipping and may help identify novel therapeutic targets.

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

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