Eulalio Ana, Huntzinger Eric, Izaurralde Elisa
Max Planck Institute for Developmental Biology, Spemannstrasse 35, D-72076 Tübingen, Germany.
Nat Struct Mol Biol. 2008 Apr;15(4):346-53. doi: 10.1038/nsmb.1405. Epub 2008 Mar 16.
MicroRNAs (miRNAs) silence gene expression by binding 3' untranslated regions of target mRNAs. Recent studies suggested silencing is achieved through either recruitment of eIF6, which prevents ribosome assembly, or displacement of eIF4E from the mRNA 5' cap structure. Using Drosophila melanogaster cells, we show that eIF6 is not required for silencing. In contrast, silencing is abolished by mutating Argonaute 1 (AGO1) at two conserved phenylalanine residues predicted to mediate binding to the cap structure. Notably, we found these mutations also prevented AGO1 from interacting with GW182 and miRNAs, indicating that the essential role of these residues is unrelated to cap binding. Consistently, depleting GW182 or overexpressing its AGO1 binding domain relieved silencing of all reporters tested, including those lacking a poly(A) tail. Together, our findings show that miRNA function is effected by AGO1-GW182 complexes and the role of GW182 in silencing goes beyond promoting deadenylation.
微小RNA（miRNA）通过与靶标mRNA的3'非翻译区结合来沉默基因表达。最近的研究表明，沉默是通过招募阻止核糖体组装的真核起始因子6（eIF6），或者从mRNA 5'帽结构上置换真核起始因子4E（eIF4E）来实现的。利用黑腹果蝇细胞，我们发现沉默并不需要eIF6。相反，在预测介导与帽结构结合的两个保守苯丙氨酸残基处突变AGO1会消除沉默。值得注意的是，我们发现这些突变也阻止了AGO1与GW182和miRNA相互作用，表明这些残基的重要作用与帽结合无关。一致地，敲低GW182或过表达其AGO1结合结构域可解除对所有测试报告基因的沉默，包括那些缺乏聚腺苷酸尾的报告基因。总之，我们的研究结果表明，miRNA功能受AGO1 - GW182复合物影响，并且GW182在沉默中的作用超出了促进去腺苷酸化。
