Hodges P E, Navaratnam N, Greeve J C, Scott J
Division of Molecular Medicine, MRC Clinical Research Centre, Harrow, Middlesex, UK.
Nucleic Acids Res. 1991 Mar 25;19(6):1197-201. doi: 10.1093/nar/19.6.1197.
Human apolipoprotein (apo) B mRNA is edited in a tissue specific reaction, to convert glutamine codon 2153 (CAA) to a stop translation codon. The RNA editing product templates and hybridises as uridine, but the chemical nature of this reaction and the physical identity of the product are unknown. After editing in vitro of [32P] labelled RNA, we are able to demonstrate the production of uridine from cytidine; [alpha 32P] cytidine triphosphate incorporated into RNA gave rise to [32P] uridine monophosphate after editing in vitro, hydrolysis with nuclease P1 and thin layer chromatography using two separation systems. By cleaving the RNA into ribonuclease T1 fragments, we show that uridine is produced only at the authentic editing site and is produced in quantities that parallel an independent primer extension assay for editing. We conclude that apo B mRNA editing specifically creates a uridine from a cytidine. These observations are inconsistent with the incorporation of a uridine nucleotide by any polymerase, which would replace the alpha-phosphate and so rule out a model of endonucleolytic excision and repair as the mechanism for the production of uridine. Although transamination and transglycosylation remain to be formally excluded as reaction mechanisms our results argue strongly in favour of the apo B mRNA editing enzyme as a site-specific cytidine deaminase.
人类载脂蛋白（apo）B信使核糖核酸（mRNA）在一种组织特异性反应中发生编辑，将谷氨酰胺密码子2153（CAA）转变为一个终止翻译密码子。RNA编辑产物以尿苷形式进行模板化和杂交，但该反应的化学性质以及产物的物理特性尚不清楚。在对[32P]标记的RNA进行体外编辑后，我们能够证明胞苷产生了尿苷；在体外编辑、用核酸酶P1水解以及使用两种分离系统进行薄层色谱分析后，掺入RNA的[α32P]胞苷三磷酸生成了[32P]尿苷一磷酸。通过将RNA切割成核糖核酸酶T1片段，我们表明尿苷仅在真实的编辑位点产生，且产生的量与用于编辑的独立引物延伸测定结果平行。我们得出结论，apo B mRNA编辑特异性地从胞苷产生了尿苷。这些观察结果与任何聚合酶掺入尿苷核苷酸的情况不一致，因为这会取代α-磷酸，从而排除了以内切核酸酶切除和修复作为尿苷产生机制的模型。尽管转氨作用和转糖基化作为反应机制仍有待正式排除，但我们的数据有力地支持apo B mRNA编辑酶是一种位点特异性胞苷脱氨酶的观点。
