Källman Annika M, Sahlin Margareta, Ohman Marie
Department of Molecular Biology and Functional Genomics, Stockholm University, S-106 91 Stockholm, Sweden.
Nucleic Acids Res. 2003 Aug 15;31(16):4874-81. doi: 10.1093/nar/gkg681.
ADAR enzymes, adenosine deaminases that act on RNA, form a family of RNA editing enzymes that convert adenosine to inosine within RNA that is completely or largely double-stranded. Site-selective A-->I editing has been detected at specific sites within a few structured pre-mRNAs of metazoans. We have analyzed the editing selectivity of ADAR enzymes and have chosen to study the naturally edited R/G site in the pre-mRNA of the glutamate receptor subunit B (GluR-B). A comparison of editing by ADAR1 and ADAR2 revealed differences in the specificity of editing. Our results show that ADAR2 selectively edits the R/G site, while ADAR1 edits more promiscuously at several other adenosines in the double-stranded stem. To further understand the mechanism of selective ADAR2 editing we have investigated the importance of internal loops in the RNA substrate. We have found that the immediate structure surrounding the editing site is important. A purine opposite to the editing site has a negative effect on both selectivity and efficiency of editing. More distant internal loops in the substrate were found to have minor effects on site selectivity, while efficiency of editing was found to be influenced. Finally, changes in the RNA structure that affected editing did not alter the binding abilities of ADAR2. Overall these findings suggest that binding and catalysis are independent events.
作用于RNA的腺苷脱氨酶（ADAR）酶构成了一个RNA编辑酶家族，该家族可将完全或大部分为双链的RNA中的腺苷转化为肌苷。在后生动物的一些结构化前体mRNA的特定位点已检测到位点选择性A→I编辑。我们分析了ADAR酶的编辑选择性，并选择研究谷氨酸受体亚基B（GluR-B）前体mRNA中天然编辑的R/G位点。对ADAR1和ADAR2编辑的比较揭示了编辑特异性的差异。我们的结果表明，ADAR2选择性编辑R/G位点，而ADAR1在双链茎中的其他几个腺苷处的编辑更为随意。为了进一步了解ADAR2选择性编辑的机制，我们研究了RNA底物中内环的重要性。我们发现编辑位点周围的直接结构很重要。与编辑位点相对的嘌呤对编辑的选择性和效率都有负面影响。发现底物中更远距离的内环对位点选择性影响较小，而编辑效率则受到影响。最后，影响编辑的RNA结构变化并未改变ADAR2的结合能力。总体而言，这些发现表明结合和催化是独立的事件。
