APOBEC-1, THE CATALYTIC SUBUNIT OF THE MAMMALIAN APOLIPOPROTEIN-B MESSENGER-RNA EDITING ENZYME, IS A NOVEL RNA-BINDING PROTEIN
APOBEC-1, THE CATALYTIC SUBUNIT OF THE MAMMALIAN APOLIPOPROTEIN-B MESSENGER-RNA EDITING ENZYME, IS A NOVEL RNA-BINDING PROTEIN
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DOI：
10.1074/jbc.270.24.14762
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发表时间：
1995-06-16
影响因子：
4.8
通讯作者：
DAVIDSON, NO
中科院分区：
文献类型：
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作者：
ANANT, S;MACGINNITIE, AJ;DAVIDSON, NO
载脂蛋白B（apo B）mRNA编辑由包括催化亚基apobec-1的酶复合物介导。重组GST/APOBEC-1与大鼠apoB RNA模板高度特异性结合，如通过UV交联和电泳迁移率变动分析（ERISA）所证明的，ApoB RNA结合被poly（U）、poly（A，U）和tRNA竞争，但不被poly（A）或其他均聚核糖核苷酸竞争，GST/APOBEC-1与apoB RNA模板的UV交联不受类似于60和类似于44 kDa的蛋白质的结合的影响，这些蛋白的结合同样不受GST/APO-BEC-1同时结合的影响。此外，在RNA结合反应中加入异源S100提取物完全消除了tRNA对GST/APOBEC-1的竞争性置换。EMSA显示RNA结合在1-2分钟内开始，并且其特异性通过抗GST/APOBEC-1抗血清的超位移证实。从EMSA推断，apoB RNA结合的结构特异性似乎与apoB RNA编辑不同，因为野生型鸡apoB RNA（不可编辑）和几种在最小apoB RNA编辑盒内含有成簇突变的突变体鸡apoB RNA的结合效率与大鼠apoB RNA模板相似。总之，虽然数据表明apobec-1结合富含AU的模板，但这一观察结果在哺乳动物apoB mRNA编辑背景下的重要性仍然未知。
Apolipoprotein B (apoB) mRNA editing is mediated by an enzyme complex which includes the catalytic subunit, apobec-1. Recombinant GST/APOBEC-1 binds with high specificity to a rat apoB RNA template as demonstrated by UV cross-linking and electrophoretic mobility shift assay (ERISA), ApoB RNA binding was competed by poly(U), poly(A,U), and tRNA, but not by poly(A) or other homopolymeric ribonucleotides, UV cross-linking of GST/APOBEC-1 to an apoB RNA template was uninfluenced by the binding of proteins of similar to 60 and similar to 44 kDa, present in S100 extracts prepared from different sources, The binding of these proteins was similarly uninfluenced by the simultaneous binding of GST/APO-BEC-1. Moreover, the inclusion of heterologous S100 extracts in the RNA binding reactions completely abrogated the competitive displacement of GST/APOBEC-1 by tRNA. EMSA revealed the onset of RNA binding within 1-2 min, and its specificity was confirmed by a supershift with anti-GST/APOBEC-1 antisera. The structural specificity for apoB RNA binding, as inferred from EMSA, appears to be distinct from apoB RNA editing since wild-type chicken apoB RNA,which is not editable, and several mutant chicken apoB RNAs containing clustered mutations within the minimal apoB RNA editing cassette, bound with efficiency similar to the rat apoB RNA template. In conclusion, while the data suggest that apobec-1 binds AU-rich templates, the importance of this observation in the context of mammalian apoB mRNA editing remains unknown.