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

我们已经鉴定了大鼠去甲肾上腺素转运体(Net)亚型，命名为rNETa和rNETb，后者在rNETa表达时为显性阴性。为了探讨RNA加工在Net表达调控中的作用，我们分析了已知表达NETs的PC12细胞Net mRNA表达与去甲肾上腺素(NE)转运的关系。我们分离了12个不同的PC12细胞株，它们表现出不同程度的NE转运和NETb mRNA水平，但两者之间没有相关性。另一方面，包括rNETa和rNETb在内的总净mRNA水平与NE转运有关，提示转录调控在净功能表达中的重要性。接下来，我们克隆了大鼠Net基因的3‘-区，并用携带该克隆的表达载体转染了不同的细胞系。从导入的微型基因衍生的这些细胞系中观察到的转录本都是rNETa和rNETb。这些结果表明，物种特异性剪接Net Pre-mRNA对Net表达的调控主要是基于物种间剪接所涉及区域的核苷酸序列的差异。

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.