Qi Hank H, Ongusaha Pat P, Myllyharju Johanna, Cheng Dongmei, Pakkanen Outi, Shi Yujiang, Lee Sam W, Peng Junmin, Shi Yang
Department of Pathology, Harvard Medical School, New Research Building 854, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.
Nature. 2008 Sep 18;455(7211):421-4. doi: 10.1038/nature07186. Epub 2008 Aug 6.
Human Argonaute (Ago) proteins are essential components of the RNA-induced silencing complexes (RISCs). Argonaute 2 (Ago2) has a P-element-induced wimpy testis (PIWI) domain, which folds like RNase H and is responsible for target RNA cleavage in RNA interference. Proteins such as Dicer, TRBP, MOV10, RHA, RCK/p54 and KIAA1093 associate with Ago proteins and participate in small RNA processing, RISC loading and localization of Ago proteins in the cytoplasmic messenger RNA processing bodies. However, mechanisms that regulate RNA interference remain obscure. Here we report physical interactions between Ago2 and the alpha-(P4H-alpha(I)) and beta-(P4H-beta) subunits of the type I collagen prolyl-4-hydroxylase (C-P4H(I)). Mass spectrometric analysis identified hydroxylation of the endogenous Ago2 at proline 700. In vitro, both Ago2 and Ago4 seem to be more efficiently hydroxylated than Ago1 and Ago3 by recombinant human C-P4H(I). Importantly, human cells depleted of P4H-alpha(I) or P4H-beta by short hairpin RNA and P4H-alpha(I) null mouse embryonic fibroblast cells showed reduced stability of Ago2 and impaired short interfering RNA programmed RISC activity. Furthermore, mutation of proline 700 to alanine also resulted in destabilization of Ago2, thus linking Ago2 P700 and hydroxylation at this residue to its stability regulation. These findings identify hydroxylation as a post-translational modification important for Ago2 stability and effective RNA interference.
人类AGO蛋白是RNA诱导沉默复合体（RISC）的重要组成部分。AGO2具有一个P元件诱导的弱精睾丸（PIWI）结构域，其折叠方式类似于核糖核酸酶H，负责RNA干扰中的靶RNA切割。诸如Dicer、TRBP、MOV10、RHA、RCK/p54和KIAA1093等蛋白与AGO蛋白结合，并参与小RNA加工、RISC装载以及AGO蛋白在细胞质信使RNA加工小体中的定位。然而，调节RNA干扰的机制仍不清楚。在此，我们报道了AGO2与I型胶原脯氨酰-4-羟化酶（C-P4H(I)）的α-（P4H-α(I)）和β-（P4H-β）亚基之间的物理相互作用。质谱分析确定了内源性AGO2在脯氨酸700处的羟基化。在体外，重组人C-P4H(I)对AGO2和AGO4的羟基化作用似乎比对AGO1和AGO3更有效。重要的是，通过短发夹RNA耗尽P4H-α(I)或P4H-β的人类细胞以及P4H-α(I)基因敲除的小鼠胚胎成纤维细胞显示出AGO2稳定性降低以及短干扰RNA编程的RISC活性受损。此外，将脯氨酸700突变为丙氨酸也导致AGO2不稳定，从而将AGO2的P700及其在该残基处的羟基化与其稳定性调节联系起来。这些发现确定羟基化是一种对AGO2稳定性和有效RNA干扰很重要的翻译后修饰。
