Objective: To examine (1) the -22C > T point mutation which introduces a premature start codon upstream of the normal open reading frame (ORF) of the GCH1 gene and (2) characterize its effect on normal GCHI translation in patients with L-dopa responsive dystonia.

Background: Within the human genome, approximately 28% of genes contain at least one translation start codon upstream of the main or canonical start codon ATG (cATG). Initiation of translation at the upstream start codon (uATG) recruits ribosomes and competes with the translation initiation at the cATG to maintain optimal levels of physiological cATG-protein. On the other hand, single nucleotide polymorphisms (SNPs) may introduce aberrant uATG(s) in genes that are normally devoid of uATGs. In such cases, translation of the cATG protein may be completely shut down or at least drastically downregulated. Such SNPs have been linked with manifestation of disorders including cancer, thrombotic predisposition, cleft lip syndrome, β-thalassemia.

Methods: We used reporter assays to analyze the direct effect of the SNP induced uATG on translation initiation at the cATG. Using immunocytochemistry, we characterized the localization of the uATG and cATG generated peptide. To analyze the mutant clearance of the uATG generated peptide from the transfected cells, we implemented a recovery assay. Western blot analysis was performed to compare and quantify levels of cATG generated peptide in the presence and absence of the uATG.

Results: Introduction of the -22T uATG downregulates physiological GCH1 protein and leads to the generation of a short, mutant peptide with a distinct amino-acid sequence and cellular localization compared to normal GCH1 enzyme. When cellular clearance is stalled, the levels of uATG generated peptide increases. We generated a stable cell line expressing the -22T (uATG) for sensitive high-throughput screening (HTS) to identify compounds that promote read-through of the uATG and allow initiation of translation at the normal cATG.

Conclusions: The presence of the uATG leads to the production of a mutant peptide and a significant reduction in the normal GCHI enzyme level suggesting competition for translational machinery between the uATG and cATG. Compounds that tilt the balance toward the cATG restore normal cATG-translation levels and thus can serve as potential therapeutic agents for DRD and other uATG-related diseases.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/functional-characterization-of-the-upstream-start-codon-in-the-gch1-gene/