REGULATION OF PNMT ACTIVITY BY ALTERNATIVE MRNA SPLICING

描述：这项研究的长期目标是识别分子 控制组织特异性表达的机制 苯乙醇胺甲基转移酶(PNMT)。PNMT与健康相关 重要性，因为它是一种将去甲肾上腺素转化为 神经递质肾上腺素。我们最近发现，在体内， 脑干，以及在体外培养的PC12细胞，两种 PNMT mRNA由另一种剪接机制产生，称为 内含子保留。我们假设这种剪接机制，很少 在哺乳动物中遇到的，形成了组织特异性调节的基础 PNMT在多种肾上腺素能组织中的表达。在脑干中， 剪接的无内含子信息的表达在转录后下调 与生俱来，而内含子重新训练的信使核糖核酸物种具有结构性 表达。这种物种反应的荷尔蒙控制是由我们的 初步数据，如体外将PC12细胞暴露于合成的 糖皮质激素(GC)、地塞米松在体内诱导剪接变异体 控制细胞，或在暴露于神经生长因子(NGF)时，只有 观察到内含子保留的变异。剪接变异体的外观 伴随着PNMT活性的诱导。在肾上腺，主要器官 对于PNMT活性和肾上腺素合成，只有辛辣变异体是 很明显。我们建议研究PNMT的生理意义 脑干、肾上腺和颈上神经节的mRNA剪接 RT-PCR法。所有这些组织都表达PNMT在发育受调控的 举止。我们将通过生理上调节胚胎PNMT的活动 给怀孕的动物和它们的后代注射 地塞米松或糖皮质激素受体(GCRs)的特异性抑制剂。 MRNA剪接变异体的相对水平与 用放射酶测定法测定功能，即酶活性， 用Western blotting表达PNMT蛋白，用免疫印迹法表达儿茶酚胺 高效液相色谱柱。在体外，GC和GCRs在这种剪接反应中的作用将是 用PC12细胞、纯化的嗜铬细胞和宫颈 培养神经节，加或不加地塞米松，或 没有GCR。PNMT mRNA剪接变异体的相对水平将是 与PNMT功能相关。理解基因的分子基础 PNMT功能的差异调节。理解分子 脑内和脑内PNMT活性的差异调节基础 肾上腺可能揭示治疗干预的可能靶点 肾上腺素分泌受扰的疾病状态。

DESCRIPTION: The long-term goal of this study is to identify molecular mechanisms controlling the tissue-specific expression of phenylethanolamine methyl transferase (PNMT). PNMT has health-related importance, as it is the enzyme that converts noradrenaline to the neurotransmitter adrenaline. We recently discovered that both in vivo in the brainstem, as well as in vitro in cultured PC12 cells, two species of PNMT mRNA are produced by an alternative splicing mechanism known as intron retention. We hypothesize that this splicing mechanism, rarely encountered in mammals, forms the basis for tissue-specific regulation of PNMT expression in various adrenergic tissues. In brainstem, the expression of a spliced intron-less message is down-regulated post- natally, while an intron-retrained mRNA species is constitutively expressed. Hormonal control of this species reaction is indicated by our preliminary data, as in vitro exposure of PC12 cells to the synthetic glucocorticoid (GC), dexamethasone, induce the spliced variant while in control cells, or upon exposure to nerve growth factor (NGF), only the intron-retained variant is observed. The appearance of the spliced variant is accompanied by induction of PNMT activity. In adrenal, the main organ for PNMT activity and epinephrine synthesis, only the spiced variant is evident. We propose to investigate the physiological importance of PNMT mRNA splicing in brainstem, adrenal and superior cervical ganglia using the RT-PCR. All these tissues express PNMT in a developmentally-regulated manner. We will physiologically modulate embryonic PNMT activity by injecting both pregnant animals, as well as their offspring, with dexamethasone or a specific inhibitor of glucocorticoid receptors (GCRs). The relative levels of mRNA spliced variants will be correlated with measurements of function, viz., enzyme activity by radio-enzyme assay, expression of PNMT protein by Western blotting, and catecholamines by HPLC. In vitro, the role of GCs and GCRs in this splicing reaction will be investigated using PC12 cells, purified chromaffin cells and cervical ganglia, cultured with or without dexamethasone and in the presence or absence of GCRs. Relative levels of PNMT mRNA spliced variants will be correlated with PNMT function. Understanding the molecular basis for the differential regulation of PNMT function. Understanding the molecular basis for the differential regulation of PNMT activity in the brain and adrenal may reveal possible targets for therapeutic intervention in diseased states with perturbed adrenaline production.