Center for integrated protein science Munich, Laboratory of RNA Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
Nucleic Acids Res. 2011 Mar;39(6):2330-43. doi: 10.1093/nar/gkq1032. Epub 2010 Nov 10.
Argonaute (Ago) proteins are highly conserved between species and constitute a direct-binding platform for small RNAs including short-interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi interacting RNAs (piRNAs). Small RNAs function as guides whereas Ago proteins are the actual mediators of gene silencing. Although the major steps in RNA-guided gene silencing have been elucidated, not much is known about Ago-protein regulation. Here we report a comprehensive analysis of Ago2 phosphorylation in human cells. We find that the highly conserved tyrosine Y529, located in the small RNA 5'-end-binding pocket of Ago proteins can be phosphorylated. By substituting Y529 with a negatively charged glutamate (E) mimicking a phosphorylated tyrosine, we show that small RNA binding is strongly reduced. Our data suggest that a negatively charged phospho-tyrosine generates a repulsive force that prevents efficient binding of the negatively charged 5' phosphate of the small RNA.
Argonaute（AGO）蛋白在物种间高度保守，构成了小 RNA 的直接结合平台，包括短干扰 RNA（siRNA）、微 RNA（miRNA）和 Piwi 相互作用 RNA（piRNA）。小 RNA 作为向导，而 AGO 蛋白则是基因沉默的实际介导物。尽管已经阐明了 RNA 指导的基因沉默的主要步骤，但对 AGO 蛋白的调节知之甚少。在这里，我们报告了对人细胞中 Ago2 磷酸化的全面分析。我们发现，位于 AGO 蛋白小 RNA 5'-末端结合口袋中的高度保守的酪氨酸 Y529 可以被磷酸化。通过用模拟磷酸化酪氨酸的带负电荷的谷氨酸（E）取代 Y529，我们表明小 RNA 的结合大大减少。我们的数据表明，带负电荷的磷酸酪氨酸会产生排斥力，从而阻止小 RNA 的带负电荷的 5' 磷酸基团的有效结合。
Zotero 插件
