Binding and Cleavage Specificities of Human Argonaute2
Binding and Cleavage Specificities of Human Argonaute2
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DOI：
10.1074/jbc.m109.010835
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发表时间：
2009-09-18
影响因子：
4.8
通讯作者：
Crooke, Stanley T.
中科院分区：
文献类型：
--
作者：
Lima, Walt F.;Wu, Hongjiang;Crooke, Stanley T.
核酸内切酶 Argonaute2 (Ago2) 介导 RNA 诱导的沉默复合物中靶标 mRNA 的降解。我们确定了重组人 Ago2 的结合和切割特性。人 Ago2 无法切割预先形成的 RNA 双链体，并且与单链 RNA 相比，对 RNA 双链体表现出较弱的结合亲和力。与 Mg2+ 相比，该酶在 Mn2+ 存在下表现出更高的 RNase H 活性。人 Ago2 对具有 5' 末端羟基或脱碱基核苷酸的反义 RNA 表现出较弱的结合亲和力和降低的切割活性。结合动力学表明，5'-末端杂环碱基使酶和反义 RNA 之间的相互作用成核，而 5'-磷酸则稳定了这种相互作用。 Mn2+ 改善了 5'-末端羟基或脱碱基核苷酸对 Ago2 切割活性和结合亲和力的影响。反义RNA 3'末端的核苷酸取代对人Ago2切割活性没有影响，而2'-甲氧基乙基取代降低了结合和切割活性，12-14降低了切割活性。 RNase 保护测定表明，人 Ago2 与反义 RNA 5' 极的前 14 个核苷酸相互作用。预载反义 RNA 的人 Ago2 对较长的有义 RNA 表现出更大的结合亲和力，表明该酶与有义 RNA 中反义杂交位点之外的区域相互作用。最后，从 HeLa 细胞中瞬时表达的人 Ago2 免疫沉淀含有双链 RNA 结合蛋白人类免疫缺陷病毒 1 型、反式激活反应 RNA 结合蛋白，Ago2 的缺失突变体表明反式激活反应 RNA 结合蛋白与酶的 PIWI 结构域相互作用。
The endonuclease Argonaute2 (Ago2) mediates the degradation of the target mRNA within the RNA-induced silencing complex. We determined the binding and cleavage properties of recombinant human Ago2. Human Ago2 was unable to cleave preformed RNA duplexes and exhibited weaker binding affinity for RNA duplexes compared with the single strand RNA. The enzyme exhibited greater RNase H activity in the presence of Mn2+ compared with Mg2+. Human Ago2 exhibited weaker binding affinities and reduced cleavage activities for antisense RNAs with either a 5'-terminal hydroxyl or abasic nucleotide. Binding kinetics suggest that the 5'-terminal heterocycle base nucleates the interaction between the enzyme and the antisense RNA, and the 5'-phosphate stabilizes the interaction. Mn2+ ameliorated the effects of the 5'-terminal hydroxyl or abasic nucleotide on Ago2 cleavage activity and binding affinity. Nucleotide substitutions at the 3' terminus of the antisense RNA had no effect on human Ago2 cleavage activity, whereas 2'-methoxyethyl substitutions at position 2 reduced binding and cleavage activity and 12-14 reduced the cleavage activity. RNase protection assays indicated that human Ago2 interacts with the first 14 nucleotides at the 5'-pole of the antisense RNA. Human Ago2 preloaded with the antisense RNA exhibited greater binding affinities for longer sense RNAs suggesting that the enzyme interacts with regions in the sense RNA outside the site for antisense hybridization. Finally, transiently expressed human Ago2 immunoprecipitated from HeLa cells contained the double strand RNA-binding protein human immunodeficiency virus, type 1, trans-activating response RNA-binding protein, and deletion mutants of Ago2 showed that trans-activating response RNA-binding protein interacts with the PIWI domain of the enzyme.