Identification of C-terminally and N-terminally truncated estrogen receptor alpha variants in the mouse
Identification of C-terminally and N-terminally truncated estrogen receptor alpha variants in the mouse
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小鼠中 C 端和 N 端截短的雌激素受体 α 变体的鉴定
DOI：
10.1016/j.jsbmb.2011.01.003
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发表时间：
2011
影响因子：
4.1
通讯作者：
Sakuma Y
中科院分区：
文献类型：
--
作者：
Ishii H;Shoda Y;Yomogida K;Hamada T;Sakuma Y
我们使用cDNA末端快速扩增（RACE）和RT-PCR重新检测了小鼠ERα mRNA变异体。我们的分析表明存在几种含有独特的5′或3′核苷酸序列的mRNA变体。我们绘制了小鼠基因组上的cDNA序列，并在外显子4和5之间的内含子区域中鉴定了4个新的3′-末端和5′-前导外显子。RT-PCR分析表明，C端截短的ERα产物（CTERP）的表达模式与野生型ERα相似，而N端截短的ERα产物（NTERP）的表达模式则不同。此外，我们构建了表达载体，并利用免疫细胞化学和荧光素酶报告基因分析了变异蛋白在HEK 293细胞中的亚细胞定位和转录激活能力。CTERP变体位于细胞核中并组成性激活雌激素反应元件（ERE）驱动的启动子，而NTERP变体位于核外区域，并且在存在或不存在10 nM雌二醇的情况下没有能力激活ERE启动子。我们的研究结果表明，小鼠ERα基因比以前认为的基因组结构更复杂，内含子启动子的选择性剪接和选择性使用有助于ERα mRNA和蛋白质的显着多样性。
We re-examined mouse ERα mRNA variants using rapid amplification of cDNA ends (RACE) and RT-PCR. Our analysis showed the presence of several mRNA variants containing unique 5′- or 3′-nucleotide sequences. We mapped the cDNA sequences on the mouse genome, and identified four novel 3′-terminal and 5′-leader exons in the intronic region between exons 4 and 5. RT-PCR analysis revealed that the expression patterns of the C-terminally truncated ERα products (CTERPs) were similar to that of Wild-type ERα and that the N-terminally truncated ERα products (NTERPs) appeared to have different expression profiles. Moreover, we constructed expression vectors and analyzed the subcellular localization and the transcriptional activation abilities of the variant proteins in transfected HEK293 cells using immunocytochemistry and luciferase reporter assay. The CTERP variants localized in the nuclei and constitutively activated estrogen response element (ERE)-driven promoters, while the NTERP variant was located in the extra-nuclear regions and had no ability to activate the ERE promoters in the presence or absence of 10nM estradiol. Our results indicate that the mouse ERα gene is more complex than previously thought in terms of genomic organization and that alternative splicing and alternative usage of intronic promoters contribute to the remarkable diversity of ERα mRNAs and proteins.