Does mPER2 protein oscillate without its coding mRNA cycling?: post-transcriptional regulation by cell clock
Does mPER2 protein oscillate without its coding mRNA cycling?: post-transcriptional regulation by cell clock
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DOI：
10.1111/j.1365-2443.2006.00960.x
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发表时间：
2006-05-01
期刊：
影响因子：
2.1
通讯作者：
Okamura, H
中科院分区：
文献类型：
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作者：
Fujimoto, Y;Yagita, K;Okamura, H
哺乳动物振荡蛋白 mPER2 是否在没有编码 mRNA 循环的情况下显示节律？在这里，我们通过使用 Flp-In 系统将单个拷贝的外源 mPer2 基因插入 NIH3T3 成纤维细胞系来回答这个问题。我们生成了稳定的细胞系，该细胞系在人 EF-1 α 启动子的控制下持续表达编码 N 端 FLAG 标记的全长 mPER2 (FLAG-mPER2(full)) 或其 C 端缺失形式 (FLAG-mPER2(1-1068)) 的 mRNA，该形式缺乏与 mCRY 蛋白的结合位点。尽管血清休克诱导了这些细胞系中内源时钟机制的节律，但它并没有在 mRNA 水平上启动外源插入的 FLAG-mPer2 基因的节律。相比之下，FLAG-mPER2(full) 蛋白显示节律，但没有编码 mRNA 循环。由于表达 FLAG-mPER2(1-1068) 的细胞也显示出 FLAG-mPER2(1-1068) 蛋白的节律，因此 mCRY 和 mPER2 的直接结合对于这种蛋白振荡似乎不是必需的。该系统清楚地表明细胞内内源时钟系统具有转录后修饰 mPer2 基因的能力，使 mPER2 蛋白在其编码 mRNA 不循环的情况下振荡。
Does the mammalian oscillatory protein mPER2 show the rhythm without its coding mRNA cycling? Here we answer this question by inserting a single copy of exogenous mPer2 gene to a NIH3T3 fibroblasts cell line, using Flp-In system. We generated the stable cell lines which constantly express mRNAs coding either N-terminal FLAG-tagged full length mPER2 (FLAG-mPER2(full)) or its C-terminal deleted form (FLAG-mPER2(1-1068)), which lacks the binding site to mCRY proteins, under the control of human EF-1 alpha promoter. Although serum shock induced the rhythm of endogenous clock machinery in these cell lines, it did not initiate the rhythm of exogenously inserted FLAG-mPer2 genes at the mRNA level. In contrast, FLAG-mPER2(full) proteins showed the rhythm without their coding mRNA cycling. Since cells expressing FLAG-mPER2(1-1068) also showed the rhythm of FLAG-mPER2(1-1068) proteins, the direct binding of mCRY and mPER2 seems not necessary for this protein oscillation. This system clearly demonstrates that the intracellular endogenous clock system has an ability to modify the mPer2 gene post-transcriptionally to make mPER2 proteins oscillate without its coding mRNA cycling.