POSTTRANSCRIPTIONAL REGULATION--CATECHOLAMINE SYNTHESIS

转录后调控——儿茶酚胺合成

• 批准号: 2772811
• 负责人: Maria F Czyzyk-Krzeska
• 金额: $ 7.62万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2772811
• 关键词: POSTTRANSCRIPTIONAL; REGULATION; CATECHOLAMINE; SYNTHESIS

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.

儿茶酚胺（CA）是一种重要的神经递质， 在呼吸和心血管的神经通路中 适应急性和慢性缺氧。CA合成的调节 缺氧常发生在CA限速酶水平 合成，酪氨酸羟化酶（THE）。多巴胺能PC 12细胞系是 常用于THE调节的分子研究。在PC 12细胞中， 缺氧会增加THE蛋白的浓度（从而增加多巴胺 合成）由THE基因的转录诱导产生， THE mRNA半衰期从10小时延长至30小时。的目的 目前的建议是研究参与调控的分子机制， mRNA的稳定性。这项规定很重要，因为它导致长期- 在mRNA的长期变化，因此蛋白质，并积极 有效和经济的维持高水平的mRNA， 能量缺乏，如慢性缺氧。 缺氧期间THE mRNA稳定性的增加伴随着 低氧诱导蛋白（HIP）与27个碱基长的 在THE的3个非翻译区中的富含胞苷的序列（1551-1579） mRNA（缺氧诱导蛋白结合序列，HIPBS）。的假设 对于拟议的研究是，HIPBS及其结合蛋白是 THE mRNA组成型和O2调节半衰期的调节剂。是 假设HIPBS与核糖核酸内切酶位点相关 活性和蛋白质结合保护mRNA免受切割。因此 在缺氧过程中，蛋白质与HIPBS的结合增加， mRNA稳定性我们将确定HIPBS是否必要， 足以调节组成性半衰期和O2调节半衰期 以及这种调节是否特异于 儿茶酚胺能或O2敏感细胞。体外无细胞RNA降解 将开发分析以确定HIPBS是否是核酸酶的位点， 切割以及蛋白质与HIPBS的结合是否保护mRNA免受 降解HIPBS结合蛋白将使用poly（C）RNA纯化 亲和力，克隆，其表达和潜在的调节O2将 在不同的儿茶酚胺能组织中进行研究。最后，PC 12细胞 将开发出在一个控制下稳定表达THE mRNA的细胞系。 嵌合四环素调控和THE组织特异性启动子。等 系统是必要的，以研究mRNA的稳定性调控，而没有非 特异性转录阻断剂，并将促进未来的研究mRNA 转基因动物的稳定性。