Department of Pediatrics and Genetics, Stanford University, Stanford, CA 94305, USA.
Proc Natl Acad Sci U S A. 2011 May 31;108(22):9208-13. doi: 10.1073/pnas.1018023108. Epub 2011 May 16.
MicroRNAs and siRNAs interact with target sequences in mRNAs, inducing cleavage- and non-cleavage-based gene repression through the RNA-induced silencing complex (RISC) that consists of one of four mammalian Argonaute proteins, Ago1-Ago4. The process of how Dicer substrate small hairpin RNAs (shRNAs) are loaded into different mammalian Agos in vivo is not well established. Here we report that shRNAs are loaded into mammalian Agos in two stepwise processes, physical association and activation, with the latter being the rate-limiting step with noncleaving RISC. We establish that, although RNA duplexes processed from shRNAs bind to Agos in cells with similar affinity, the degree by which the complexes are activated (coupled with the removal of the passenger strand) correlates with the thermodynamic instability of RNA duplexes being loaded rather than the structure of the RNA, as was previously demonstrated in Drosophila. Interestingly, Ago loading of siRNAs is less sensitive to thermostability than that of their shRNA equivalents. These results may have important implications for the future design of RNAi-based therapeutics.
MicroRNAs 和 siRNAs 与 mRNAs 中的靶序列相互作用，通过由四种哺乳动物 Argonaute 蛋白（Ago1-Ago4）之一组成的 RNA 诱导沉默复合物 (RISC) 诱导基于切割和非切割的基因抑制。Dicer 底物小发夹 RNA (shRNA) 在体内加载到不同哺乳动物 Agos 中的过程尚未很好确立。在这里，我们报告说，shRNA 以两个逐步的过程加载到哺乳动物 Agos 中，即物理结合和激活，后者是具有非切割 RISC 的限速步骤。我们确定，尽管从 shRNA 加工的 RNA 双链在细胞中与 Agos 结合的亲和力相似，但复合物被激活的程度（伴随着过客链的去除）与被加载的 RNA 双链的热力学不稳定性相关，而不是 RNA 的结构，如以前在果蝇中所证明的那样。有趣的是，siRNA 的 Ago 加载对热稳定性的敏感性不如其 shRNA 等价物。这些结果可能对未来基于 RNAi 的治疗药物的设计具有重要意义。
