Driscoll D M, Lakhe-Reddy S, Oleksa L M, Martinez D
Department of Cell Biology, Cleveland Clinic Foundation, Ohio 44195.
Mol Cell Biol. 1993 Dec;13(12):7288-94. doi: 10.1128/mcb.13.12.7288-7294.1993.
An RNA editing mechanism modifies apolipoprotein B (apo-B) mRNA in the intestine by converting cytosine at nucleotide (nt) 6666 to uracil. To define the sequence requirements for editing, mutant apo-B RNAs were analyzed for the ability to be edited in vitro by enterocyte extracts. Editing was detected by a sensitive and linear primer extension assay. An upstream region (nt 6648 to 6661) which affected the efficiency of editing was identified. RNAs with mutations in this efficiency sequence were edited at 22 to 160% of wild-type levels. Point mutations in a downstream 11-nt mooring sequence (nt 6671 to 6681) abolished editing, confirming previous studies (R. R. Shah, T. J. Knott, J. E. Legros, N. Navaratnam, J. C. Greeve, and J. Scott, J. Biol. Chem. 266:16301-16304, 1991). The optimal distance between the editing site and the mooring sequence is 5 nt, but a C positioned 8 nt upstream is edited even when nt 6666 contains U. The efficiency and mooring sequences were inserted individually and together adjacent to a heterologous C in apo-B mRNA. The mooring sequence alone induced editing of the C at nt 6597 both in vitro and in transfected rat hepatoma cells. Editing at nt 6597 was specific, was independent of editing at nt 6666, and was stimulated to wild-type levels when the efficiency sequence was also inserted. Introduction of the mooring sequence into a heterologous mRNA, luciferase mRNA, induced editing of an upstream cytidine. Although UV cross-linking studies have previously shown that proteins of 60 to 66 kDa cross-link to apo-B mRNA, these proteins did not cross-link to the luciferase translocation mutants.
一种RNA编辑机制通过将核苷酸（nt）6666处的胞嘧啶转换为尿嘧啶来修饰肠道中的载脂蛋白B（apo-B）mRNA。为了确定编辑的序列要求，分析了突变型apo-B RNA在体外被肠上皮细胞提取物编辑的能力。通过灵敏的线性引物延伸试验检测编辑情况。确定了一个影响编辑效率的上游区域（nt 6648至6661）。在该效率序列中发生突变的RNA的编辑水平为野生型水平的22%至160%。下游11个核苷酸的停泊序列（nt 6671至6681）中的点突变消除了编辑，这证实了先前的研究（R. R. Shah、T. J. Knott、J. E. Legros、N. Navaratnam、J. C. Greeve和J. Scott，《生物化学杂志》266:16301 - 16304，1991）。编辑位点与停泊序列之间的最佳距离为5个核苷酸，但即使nt 6666含有尿嘧啶，位于上游8个核苷酸处的胞嘧啶也会被编辑。将效率序列和停泊序列单独或一起插入apo-B mRNA中与异源胞嘧啶相邻的位置。单独的停泊序列在体外和转染的大鼠肝癌细胞中均诱导了nt 6597处胞嘧啶的编辑。nt 6597处的编辑具有特异性，独立于nt 6666处的编辑，并且当也插入效率序列时被刺激到野生型水平。将停泊序列引入异源mRNA（荧光素酶mRNA）中诱导了上游胞苷的编辑。尽管紫外线交联研究先前表明60至66 kDa的蛋白质与apo-B mRNA交联，但这些蛋白质未与荧光素酶易位突变体交联。
