Catecholamines (CA) are essential neurotransmitters expressed and regulated in the neuronal pathway involved in respirator and cardiovascular adaptation to acute and chronic hypoxia. Regulation of CA synthesis during hypoxia occurs often at the level of the rate limiting enzyme in CA synthesis, tyrosine hydroxylase (THE). The dopaminergic PC12 cell line is frequently used for molecular studies of THE regulation. In PC12 cells, hypoxia increases concentration of THE protein (and thus dopamine synthesis) resulting from transcriptional induction of THE gene and an increase in the THE mRNA half-life from 10 to 30 h. The objective of the present proposal is to study molecular mechanisms involved in regulation of THE mRNA stability. This regulation is important because leads to long- term changes in THE mRNA, and therefore THE protein, and is energetically effective and economic for maintaining elevated levels of THE mRNA during energetic deprivation such as chronic hypoxia. The increased stability of the THE mRNA during hypoxia is accompanied by enhanced binding of a hypoxia-inducible protein (HIP) to a 27 base long cytidine-rich sequence (1551-1579) in the 3 untranslated region of the THE mRNA (hypoxia-inducible protein binding sequence, HIPBS). The hypothesis for the proposed research is that HIPBS and its binding protein are regulators of the THE mRNA constitutive and O2-regulated half-life. It is hypothesized that HIPBS is associated with the site for endoribonuclease activity and protein binding protects THE mRNA from cleavage. Thus increased binding of protein to HIPBS during hypoxia results in augmented mRNA stability. We shall determine whether HIPBS is necessary and sufficient for regulation of both constitutive and O2-regulated half-life of the THE mRNA and whether this regulation is specific for catecholaminergic or O2-sensitive cells. The cell-free in vitro RNA decay assays will be developed to determine whether HIPBS is a site for nuclease cleavage and whether binding of protein to HIPBS protects mRNA from degradation. The HIPBS binding protein will be purified using poly(C) RNA affinity, cloned, and its expression and potential regulation by O2 will be studied in different catecholaminergic tissues. Finally, a PC12 cell line will be developed stably expressing THE mRNA under control of a chimeric tetracycline-regulated and THE tissue-specific promoter. Such system is necessary to study THE mRNA stability regulation without non- specific transcription blockers and will facilitate future studies of mRNA stability in the transgenic animals.
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