POSTTRANSCRIPTIONAL REGULATION--CATECHOLAMINE SYNTHESIS

转录后调控——儿茶酚胺合成

• 批准号: 2685536
• 负责人: Maria F Czyzyk-Krzeska
• 金额: $ 22.37万
• 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/2685536
• 关键词: RNA binding protein; biosynthesis; catecholamines; complementary DNA; endoribonucleases; enzyme activity; enzyme induction /repression; hypoxia; laboratory rat; messenger RNA; molecular cloning; posttranscriptional RNA processing; protein sequence; site directed mutagenesis; tissue /cell culture; tyrosine 3 monooxygenase

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) 是表达和调节的重要神经递质 参与呼吸和心血管的神经元通路 适应急、慢性缺氧。 CA合成过程中的调节 缺氧通常发生在 CA 限速酶水平 合成，酪氨酸羟化酶（THE）。多巴胺能 PC12 细胞系是 经常用于THE调节的分子研究。在 PC12 细胞中， 缺氧会增加蛋白质的浓度（因此多巴胺 合成）由THE基因的转录诱导产生，并且 THE mRNA 半衰期从 10 小时增加至 30 小时。该计划的目标 目前的建议是研究参与调节的分子机制 mRNA 稳定性。这项规定很重要，因为会导致长期 mRNA 的术语发生变化，因此蛋白质也发生变化，并且在能量上 有效且经济地维持THE mRNA的升高水平 能量匮乏，例如慢性缺氧。 缺氧期间THE mRNA稳定性的增加伴随着 增强缺氧诱导蛋白 (HIP) 与 27 个碱基长的结合 THE 的 3 个非翻译区富含胞苷序列 (1551-1579) mRNA（缺氧诱导蛋白结合序列，HIPBS）。假设 对于拟议的研究，HIPBS 及其结合蛋白是 THE mRNA 组成型和 O2 调节半衰期的调节因子。这是 假设 HIPBS 与核糖核酸内切酶位点相关 活性和蛋白质结合可保护 mRNA 免遭切割。因此 缺氧期间蛋白质与 HIPBS 的结合增加，导致增强 mRNA 稳定性。我们将确定 HIPBS 是否必要，以及 足以调节本构半衰期和 O2 调节半衰期 THE mRNA 的变化以及该调节是否特异于 儿茶酚胺能或 O2 敏感细胞。体外游离RNA衰变 将开发测定法以确定 HIPBS 是否是核酸酶位点 裂解以及蛋白质与 HIPBS 的结合是否可以保护 mRNA 降解。 HIPBS 结合蛋白将使用聚 (C) RNA 进行纯化 亲和力、克隆及其表达和 O2 的潜在调节将 在不同的儿茶酚胺能组织中进行研究。最后是PC12电池 将在a的控制下开发稳定表达mRNA的细胞系 嵌合四环素调节的组​​织特异性启动子。这样的 系统对于研究 mRNA 稳定性调节而无需非 特异性转录阻断剂，将促进未来 mRNA 的研究 转基因动物的稳定性。