Nikcevic G, Perhonen M, Boateng S Y, Russell B
Department of Physiology and Biophysics, University of Illinois at Chicago, 60612-7342, USA.
J Muscle Res Cell Motil. 2000;21(6):599-607. doi: 10.1023/a:1026507727700.
Posttranscriptional regulation plays an important role in alpha-myosin heavy chain (alpha-MyHC) protein synthesis in cardiac muscle cells. In the present study, we test the effects of calcium and mRNA mislocalization on alpha-MyHC translation in order to determine the mechanism(s) contributing to translational block via the 3' untranslated region (3'UTR). Neonatal rat cardiac myocytes were treated for 6 h with L-isoproterenol (10 microM) to enhance beating, with 10 microM verapamil to block beating and mislocalize mRNA, or with 3 microM colchicine to enhance beating but mislocalize mRNA by depolymerization of the microtubules. In order to determine whether translation is regulated by the 3'UTR, either a control (SV40 3'UTR) or the experimental (alpha-MyHC 3'UTR) was placed after a luciferase reporter gene and transfected into the myocytes. The amount of luciferase protein only decreased significantly in verapamil arrested cells transfected with the alpha-MyHC 3'UTR construct (P < 0.01). To control for the possibility that pharmacological treatments might affect transcription or message stability, we analyzed neomycin and luciferase mRNA levels transcribed from the same transfected plasmid. No significant changes were found with an RNase protection assay. These results suggest that calcium but not mRNA localization regulates protein synthesis and further, this is mediated by the 3' untranslated region of alpha-MyHC.
转录后调控在心肌细胞α-肌球蛋白重链（α-MyHC）蛋白合成中起重要作用。在本研究中，我们测试了钙和mRNA定位错误对α-MyHC翻译的影响，以确定通过3'非翻译区（3'UTR）导致翻译受阻的机制。新生大鼠心肌细胞用L-异丙肾上腺素（10μM）处理6小时以增强搏动，用10μM维拉帕米阻断搏动并使mRNA定位错误，或用3μM秋水仙碱增强搏动但通过微管解聚使mRNA定位错误。为了确定翻译是否受3'UTR调控，将对照（SV40 3'UTR）或实验性（α-MyHC 3'UTR）置于荧光素酶报告基因之后并转染到心肌细胞中。仅在用α-MyHC 3'UTR构建体转染的维拉帕米阻滞细胞中，荧光素酶蛋白量显著降低（P <0.01）。为了控制药物处理可能影响转录或信息稳定性的可能性，我们分析了从同一转染质粒转录的新霉素和荧光素酶mRNA水平。核糖核酸酶保护试验未发现显著变化。这些结果表明，钙而非mRNA定位调节蛋白质合成，并且进一步表明，这是由α-MyHC的3'非翻译区介导的。
