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

我们已经鉴定了由选择性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NET的PC12细胞中NET mRNA表达与去甲肾上腺素（NE）转运之间的关系。我们分离了12种不同的PC12细胞系，这些细胞系显示出不同程度的NE转运和NETb mRNA水平，但它们之间没有相关性。另一方面，包括rNETa和rNETb在内的NET mRNA总水平与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，我们克隆了大鼠3′区NET基因，并将携带该克隆的表达载体转染不同细胞系。在这些细胞系中观察到的转录本分别是rNETa和rNETb。这些结果表明，NET前mrna的物种特异性剪接对NET表达的调控主要基于物种间剪接区域核苷酸序列的差异。

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.