Erythropoietin mRNA levels are governed by both the rate of gene transcription and posttranscriptional events.
Erythropoietin mRNA levels are governed by both the rate of gene transcription and posttranscriptional events.
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DOI：
10.1182/blood.v77.2.271.bloodjournal772271
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发表时间：
1991-01
期刊：
影响因子：
20.3
通讯作者：
MA Goldberg;CC Gaut;H. Bunn
中科院分区：
文献类型：
--
作者：
MA Goldberg;CC Gaut;H. Bunn
人肝癌细胞系 Hep3B 在缺氧和氯化钴 (CoCl2) 的作用下以受调控的方式合成大量促红细胞生成素 (Epo) mRNA 和蛋白质。为了进一步了解Epo基因表达的调控，我们研究了缺氧和CoCl2对Epo基因转录率的影响。虽然 Northern 印迹分析表明，稳态 Epo mRNA 水平在缺氧或 CoCl2 的反应下增加了 50 倍以上，但核径流实验表明，在这些刺激下，Epo 基因转录仅增加了 10 倍。在放线菌素 D (Act D) 或放线菌酮存在下，具有生物功能的 Epo mRNA 的稳定性远高于在不存在这些药物的情况下观察到的稳定性，并且远高于体内报道的稳定性。这些发现表明 Epo mRNA 的稳定性受到快速周转基因产物的转录和翻译的调节。因此，Epo mRNA 水平由基因转录速率和转录后事件决定。这些实验表明，仅根据 Act D 追踪实验估计 mRNA 半衰期存在潜在缺陷。
The human hepatoma cell line, Hep3B, synthesizes large quantities of erythropoietin (Epo) mRNA and protein in a regulated manner in response to hypoxia and cobaltous chloride (CoCl2). To further understand the regulation of Epo gene expression, we studied the effects of hypoxia and CoCl2 on the rate of Epo gene transcription. While Northern blot analyses showed that steady-state Epo mRNA levels increase more than 50-fold in response to hypoxia or CoCl2, nuclear run-off experiments demonstrated only a 10-fold increase in Epo gene transcription in response to these stimuli. In the presence of either actinomycin D (Act D) or cycloheximide, the stability of biologically functional Epo mRNA was much greater than that observed in the absence of these agents and much greater than that which has been reported in vivo. These findings suggest that the stability of Epo mRNA is modulated by the transcription and translation of rapidly turning over gene product(s). Thus, Epo mRNA levels are determined by both the rate of gene transcription and posttranscriptional events. These experiments demonstrate a potential pitfall in estimating mRNA half-lives based on Act D chase experiments alone.