Regulation of the functional expression of neurotransmitter transporters by their RNA processing

通过 RNA 加工调节神经递质转运蛋白的功能表达

• 批准号: 11680756
• 负责人: KITAYAMA Shigeo
• 金额: $ 2.37万
• 依托单位: HIROSHIMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680756/
• 关键词: norepinephrine; transporter; neurotransmitter; alternative splicing; antidipressant; 抗ラフ薬; 抗うつ薬

We have identified rat norepinephrine transporter (NET) isoforms produced by alternative RNA splicing, designated rNETa and rNETb, latter of which was dominant negative when expressed with rNETa. To explore the role of RNA processing in regulation of NET expression, we analyzed the relationship between NET mRNA expression and norepinephrine (NE) transport in PC12 cells which are known to express NET.We isolated 12 different cell lines of PC12 which reveal different degrees of NE transport and NETb mRNA levels, however, there was no correlation between these. On the other hand, there was relation between total NET mRNA levels including both rNETa and rNETb and NE transport, suggesting the importance of the transcriptional regulation in NET functional expression. Next, we cloned rat NET gene at 3'-region, and transfected different cell lines with the expression vector carrying this clone. The transcripts observed in these cell lines derived from the introduced minigene were both rNETa and rNETb. These results suggest that regulation of NET expression resulted from species-specific splicing of NET pre-mRNA is mainly based on the differences in nucleotide sequences at the regions involved in splicing between species.

我们已经鉴定了通过选择性 RNA 剪接产生的大鼠去甲肾上腺素转运蛋白 (NET) 亚型，命名为 rNETa 和 rNETb，后者在与 rNETa 一起表达时呈显性失活。为了探讨 RNA 加工在 NET 表达调节中的作用，我们分析了已知表达 NET 的 PC12 细胞中 NET mRNA 表达和去甲肾上腺素 (NE) 转运之间的关系。我们分离了 12 个不同的 PC12 细胞系，它们揭示了不同程度的 NE 转运和 NETb mRNA 水平，但这些之间没有相关性。另一方面，包括rNETa和rNETb在内的总NET mRNA水平与NE转运之间存在相关性，表明转录调控在NET功能表达中的重要性。接下来，我们在3'区克隆了大鼠NET基因，并用携带该克隆的表达载体转染不同的细胞系。在源自引入的小基因的这些细胞系中观察到的转录物都是rNETa和rNETb。这些结果表明，NET前体mRNA的物种特异性剪接引起的NET表达调节主要基于物种间参与剪接的区域的核苷酸序列的差异。

项目成果

S.Kitayama, T.Ikeda, C.Mitsuhata, T.Sato, K.Morita and T.Dohi: "Dominant negative isoform of rat norepinephrine transporter produced by alternative RNA splicing."J.Biol.Chem.. 274. 10731-10736 (1999)

S.Kitayama、T.Ikeda、C.Mitsuhata、T.Sato、K.Morita 和 T.Dohi：“通过替代 RNA 剪接产生的大鼠去甲肾上腺素转运蛋白的显性负亚型。”J.Biol.Chem.. 274. 10731-

T.Ikeda, S.Kitayama, K.Morita and T.Dohi: "Nerve growth factor down-regulates the expression of norepinephrine transporter in rat pheochromocytoma (PC12) cells."Mol.Brain Res.. 86. 90-100 (2000)

T.Ikeda、S.Kitayama、K.Morita 和 T.Dohi：“神经生长因子下调大鼠嗜铬细胞瘤 (PC12) 细胞中去甲肾上腺素转运蛋白的表达。”Mol.Brain Res.. 86. 90-100 (2000)

S.Kitoyama et al.: "Dominant negative isoform of rat norepinephrine transporter produced by alternative RNA splicing."J.Biol.Chem.. 274. 10731-10736 (1999)

S.Kitoyama 等人：“通过选择性 RNA 剪接产生的大鼠去甲肾上腺素转运蛋白的显性负亚型。”J.Biol.Chem.. 274. 10731-10736 (1999)

T.Sato et al.: "Selective inhibition of monoamine neurotransmitter transporters by synthetic local anesthetics."Naunyn-Schmiedeberg's Arch.Pharmacol.. 361. 214-220 (2000)

T.Sato 等人：“合成局部麻醉剂对单胺神经递质转运蛋白的选择性抑制。”Naunyn-Schmiedebergs Arch.Pharmacol.. 361. 214-220 (2000)

T.Sato et al.: "Changes in seizure susceptibility to local anesthetics by repeated administration of cocaine and nomifensine Dut not GBR12935"Jpn.J.Pharmacol.. 83. 265-268 (2000)

T.Sato 等人：“通过重复施用可卡因和诺米芬辛，改变癫痫对局部麻醉药的敏感性，而不是 GBR12935”Jpn.J.Pharmacol.. 83. 265-268 (2000)