Porter D C, Keyomarsi K
Division of Molecular Medicine, Wadsworth Center, Albany, NY 12201-0509, USA.
Nucleic Acids Res. 2000 Dec 1;28(23):E101. doi: 10.1093/nar/28.23.e101.
Cyclin E, a G(1) cyclin, is overexpressed and present in low molecular weight (LMW) isoforms in breast cancer cells and tumor tissues. In this study we have examined the possibility that the shortened mRNA splice variants could give rise to tumor-specific cyclin E LMW proteins. We used the Splice Capture method to identify, enumerate and isolate known spliced mRNAs and to look for previously undetected mRNA forms of cyclin E that might be translated into the LMW proteins. We show that a new splice variant of cyclin E found in tumor cells isolated by the Splice Capture strategy, named Delta48, activates CDK2 more robustly than full-length cyclin E when assayed from transiently transfected cells with the natural substrate GST-Rb. We also found the Splice Capture method to be superior to the conventional RNase protection assay in analyzing the cyclin E mRNA present in normal and tumor cells. Splice Capture enumerated the relative abundance of known forms of cyclin E mRNA and easily discovered new splice variants in both normal and tumor cells. We conclude that the abundance of cyclin E splice variants in cells may represent a novel form of regulation of cyclin E, and if translated they show altered substrate specificity compared to the full length form of cyclin E.
细胞周期蛋白E是一种G1期细胞周期蛋白，在乳腺癌细胞和肿瘤组织中过度表达，并以低分子量（LMW）异构体形式存在。在本研究中，我们探讨了缩短的mRNA剪接变体是否可能产生肿瘤特异性细胞周期蛋白E的LMW蛋白。我们使用剪接捕获方法来识别、计数和分离已知的剪接mRNA，并寻找可能被翻译成LMW蛋白的细胞周期蛋白E的先前未检测到的mRNA形式。我们发现，通过剪接捕获策略分离的肿瘤细胞中发现的一种新的细胞周期蛋白E剪接变体，命名为Delta48，在用天然底物GST-Rb进行瞬时转染细胞检测时，比全长细胞周期蛋白E更强烈地激活CDK2。我们还发现，在分析正常细胞和肿瘤细胞中存在的细胞周期蛋白E mRNA时，剪接捕获方法优于传统的核糖核酸酶保护分析。剪接捕获确定了细胞周期蛋白E mRNA已知形式的相对丰度，并很容易在正常细胞和肿瘤细胞中发现新的剪接变体。我们得出结论，细胞中细胞周期蛋白E剪接变体的丰度可能代表细胞周期蛋白E调控的一种新形式，如果被翻译，它们与细胞周期蛋白E的全长形式相比显示出改变的底物特异性。
