POSTTRANSCRIPTIONAL REGULATION--CATECHOLAMINE SYNTHESIS

儿茶酚胺(CA)是表达和调节必需的神经递质 在参与呼吸机和心血管系统的神经通路中 适应急性和慢性缺氧。植物体内CA合成的调控 缺氧通常发生在CA的限速酶水平上 合成，酪氨酸羟基酶(The)。多巴胺能PC12细胞系 经常用于调节的分子研究。在PC12细胞中， 低氧会增加蛋白质的浓度(从而增加多巴胺 合成)由基因的转录诱导和一种 将信使核糖核酸的半衰期从10小时增加到30小时。 目前的建议是研究参与调控的分子机制。 信使核糖核酸稳定性。这一规定很重要，因为这会导致长期- 在信使核糖核酸，因此蛋白质，和能量的变化 有效且经济的维持较高水平的信使核糖核酸 能量缺乏，如慢性缺氧。 在低氧条件下，mRNA的稳定性增加伴随着 低氧诱导蛋白(HIP)与27个碱基长的结合增强 富胞苷序列(1551-1579) 低氧诱导蛋白结合序列(HIPBS)。假说 因为建议的研究是HIPBS及其结合蛋白是 调节该信使核糖核酸的构成和氧调节半衰期。它是 假设HIPBS与内切核酸酶的位置相关 活性和蛋白质结合保护信使核糖核酸不被切割。因此， 缺氧期间蛋白质与HIPBS结合增加导致HIPBS增加 信使核糖核酸稳定性。我们将确定HIPBS是否必要，以及 足以调节构成半衰期和氧气调节半衰期 以及这一调控是否是针对 儿茶酚胺能或O2敏感细胞。无细胞的体外RNA衰变 将开发检测以确定HIPBS是否是核酸酶的位点 裂解和蛋白质与HIPBS结合是否保护mRNA 退化。HIPBS结合蛋白将使用Poly(C)RNA进行纯化 亲和力，克隆，及其表达和O2对其潜在的调节 在不同的儿茶酚胺能组织中进行研究。最后，PC12细胞 将建立稳定表达mrna的品系。 嵌合四环素调控和组织特异性启动子。是这样的 在没有非核糖核酸的情况下，研究信使核糖核酸的稳定性调控是必要的 特定的转录阻滞剂，将有助于未来对mRNA的研究 转基因动物的稳定性。

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.