Thermodynamic stability of small hairpin RNAs highly influences the loading process of different mammalian Argonautes
Thermodynamic stability of small hairpin RNAs highly influences the loading process of different mammalian Argonautes
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DOI：
10.1073/pnas.1018023108
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发表时间：
2011-05-31
影响因子：
11.1
通讯作者：
Kay, Mark A.
中科院分区：
文献类型：
--
作者：
Gu, Shuo;Jin, Lan;Kay, Mark A.
MicroRNAs和sirna与mrna中的靶序列相互作用，通过rna诱导的沉默复合体（RISC）诱导基于切割和非切割的基因抑制，该复合体由四种哺乳动物Argonaute蛋白之一Ago1-Ago4组成。Dicer底物小发夹rna （small hairpin rna, shrna）在哺乳动物体内如何被装载到不同的Agos中，这一过程尚不清楚。在这里，我们报道shrna通过两个步骤加载到哺乳动物的Agos中，物理关联和激活，后者是非切割RISC的限速步骤。我们发现，尽管shRNAs加工的RNA双链在细胞中以相似的亲和力与Agos结合，但复合物的激活程度（加上客链的去除）与装载的RNA双链的热力学不稳定性相关，而不是与RNA的结构相关，正如之前在果蝇中所证明的那样。有趣的是，Ago装载的sirna对热稳定性的敏感性低于它们的等效shRNA。这些结果可能对未来设计基于rnai的治疗方法具有重要意义。
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.