Navaratnam N, Shah R, Patel D, Fay V, Scott J
Medical Research Council Molecular Medicine Group, Royal Postgraduate Medical School, London, United Kingdom.
Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):222-6. doi: 10.1073/pnas.90.1.222.
Apolipoprotein (apo) B100 mRNA undergoes editing of C-6666 to a U residue, which generates a stop-translation codon and defines the carboxyl terminus of apoB48. To aid purification of the editing enzyme we have undertaken UV crosslinking of a 32P-labeled substrate for apoB mRNA editing in vitro to proteins in an enterocyte editing extract. Proteins of 60 (p60) and 43 (p43) kDa, prominent among crosslinking bands, were competed for by unlabeled substrate, but not by nonspecific RNA, and did not crosslink to antisense RNA. Editing in vitro and UV crosslinking were inhibited by NaCl and vanadyl ribonucleoside complexes and by chemical modification of sulfhydryl, imidazolium, and guanidinium groups on the protein. The editing activity copurified predominantly with p60. To define the binding site for p60 on the substrate RNA, a series of scanning and point mutant RNAs, previously used to define nucleotides 6671-6681 as essential for editing, were used in competition studies with wild-type substrate. Results demonstrated that p60 binding is centered on nucleotides 6671-6674. We suggest that p60 contains the RNA-recognition component of the apoB mRNA-editing enzyme.
载脂蛋白（apo）B100信使核糖核酸（mRNA）的C-6666位点会被编辑为尿嘧啶（U）残基，这会产生一个终止翻译密码子，并确定apoB48的羧基末端。为了有助于纯化编辑酶，我们在体外对apoB mRNA编辑的32P标记底物与肠细胞编辑提取物中的蛋白质进行了紫外线交联。在交联条带中突出的60 kDa（p60）和43 kDa（p43）蛋白质，可被未标记的底物竞争，但不能被非特异性RNA竞争，并且不会与反义RNA交联。体外编辑和紫外线交联受到氯化钠和钒核糖核苷复合物以及蛋白质上巯基、咪唑鎓和胍基的化学修饰的抑制。编辑活性主要与p60共纯化。为了确定p60在底物RNA上的结合位点，在与野生型底物的竞争研究中使用了一系列先前用于确定6671-6681核苷酸对编辑至关重要的扫描和点突变RNA。结果表明，p60的结合集中在6671-6674核苷酸上。我们认为p60包含apoB mRNA编辑酶的RNA识别成分。
