Vracar-Grabar Marina, Russell Brenda
Department of Physiology and Biophysics (M/C 901), University of Illinois at Chicago, 835 S. Wolcott Avenue, Chicago, IL 60612-1342, USA.
J Muscle Res Cell Motil. 2004;25(4-5):397-404. doi: 10.1007/s10974-004-1141-1.
Altered cardiac workload regulates the translation and localization of the alpha myosin heavy chain (alphaMyHC) messenger RNA through the 3' untranslated region (UTR) by protein-RNA interactions. We used the alphaMyHC 3'UTR from neonatal rat heart tissue in a gel shift analysis to find RNA binding proteins. One was identified by microsequencing as creatine kinase, brain form B (CKBB). The affinity of its binding interaction was evaluated using sense and antisense alphaMyHC 3'UTR and 3'UTR probes from myosin isoforms of 2B and 2X skeletal muscle. Removal of calcium by the chelating agent EGTA had a potentiating effect on the formation of the CKBB/alphaMyHC 3'UTR complex in vitro . Varying the concentration of ATP (0.1-1 mM) also enhanced this interaction, suggesting that autophosphorylation of CKBB is taking place. Our novel finding that CKBB, an energy transduction enzyme, binds to the RNA of the 3'UTR of the faster ATP consuming alphaMyHC suggests a possible regulatory linkage between the metabolic state of the cell and myosin isoform expression.
改变的心脏工作负荷通过蛋白质-RNA相互作用,经3'非翻译区(3'UTR)调控α肌球蛋白重链(αMyHC)信使RNA的翻译和定位。我们在凝胶迁移分析中使用新生大鼠心脏组织的αMyHC 3'UTR来寻找RNA结合蛋白。其中一种经微量测序鉴定为脑型肌酸激酶B(CKBB)。使用来自2B和2X骨骼肌肌球蛋白亚型的正义和反义αMyHC 3'UTR以及3'UTR探针评估其结合相互作用的亲和力。用螯合剂EGTA去除钙对体外CKBB/αMyHC 3'UTR复合物的形成有增强作用。改变ATP浓度(0.1 - 1 mM)也增强了这种相互作用,表明CKBB正在发生自磷酸化。我们的新发现,即能量转导酶CKBB与消耗ATP更快的αMyHC的3'UTR的RNA结合,提示细胞代谢状态与肌球蛋白亚型表达之间可能存在调控联系。