Unsworth B R, Hayman G T, Carroll A, Lelkes P I
Department of Biology, Marquette University, Milwaukee, WI 53233, USA.
Int J Dev Neurosci. 1999 Feb;17(1):45-55. doi: 10.1016/s0736-5748(98)00058-6.
The expression of phenylethanolamine N-methyl transferase (EC 2. 1.1.2.8, PNMT), the final enzyme in the cascade of catecholamine synthesis, is differentially regulated in adrenergic neurons in the brain and in adrenal chromaffin cells. Using reverse transcription-polymerase chain reaction-based techniques, we detected in the prenatal developing rat brainstem, two species of PNMT mRNA which were produced by a rare alternative splicing mechanism known as intron retention. The spliced, intronless message was downregulated postnatally, while the intron-retained mRNA species continued to be constitutively expressed through adulthood. By contrast in the adrenals, at all stages of development examined, only the intronless message was expressed. In line with previous reports on the failure of glucocorticoids to induce PNMT expression in the brain, the pattern of PNMT splicing in brainstem explants was not affected by the presence of the synthetic glucocorticoid dexamethasone. Undifferentiated sympathoadrenal PC12 pheochromocytoma cells expressed very low basal levels of both mRNA variants, accompanied by a very low basal PNMT enzymatic activity. Exposure of PC12 cells to dexamethasone resulted in the upregulation of only the spliced mRNA variant concomitant with a 3-fold increase in PNMT enzymatic activity. In contrast, treatment of PC 12 cells with nerve growth factor (NGF) enhanced the expression of both the intron-retained and the intronless mRNA species without changes in the basal enzyme activity. This latter result suggests that the translation of the intronless mRNA species may be regulated by the intron-retained mRNA species, which by itself may yield a truncated, yet enzymatically functional translational product. Our data suggest that the tissue-specific regulation of PNMT expression is based on a rare alternative splicing mechanism termed intron retention, and that in the adrenal, but not in the brain, this mechanism is sensitive to regulation by glucocorticoids. Thus, this system is uniquely suited for studying the hormonal control of tissue-specific splicing in the nervous system.
苯乙醇胺N-甲基转移酶（EC 2.1.1.2.8，PNMT）是儿茶酚胺合成级联反应中的最后一种酶，在大脑中的肾上腺素能神经元和肾上腺嗜铬细胞中受到不同的调节。利用基于逆转录-聚合酶链反应的技术，我们在产前发育的大鼠脑干中检测到两种PNMT mRNA，它们是由一种罕见的可变剪接机制即内含子保留产生的。剪接后的无内含子信息在出生后被下调，而保留内含子的mRNA种类在成年期持续组成性表达。相比之下，在肾上腺中，在所检查的所有发育阶段，只表达无内含子的信息。与之前关于糖皮质激素不能在大脑中诱导PNMT表达的报道一致，脑干外植体中PNMT的剪接模式不受合成糖皮质激素地塞米松的影响。未分化的交感肾上腺PC12嗜铬细胞瘤细胞两种mRNA变体的基础表达水平都非常低，同时基础PNMT酶活性也非常低。将PC12细胞暴露于地塞米松导致仅剪接后的mRNA变体上调，同时PNMT酶活性增加3倍。相反，用神经生长因子（NGF）处理PC12细胞增强了保留内含子和无内含子的mRNA种类的表达，而基础酶活性没有变化。后一个结果表明，无内含子的mRNA种类的翻译可能受保留内含子的mRNA种类的调节，而保留内含子的mRNA种类本身可能产生一种截短但仍具有酶功能的翻译产物。我们的数据表明，PNMT表达的组织特异性调节基于一种罕见的可变剪接机制，即内含子保留，并且在肾上腺而非大脑中，这种机制对糖皮质激素的调节敏感。因此，这个系统非常适合研究神经系统中组织特异性剪接的激素控制。
