RNA:RNA interaction in ternary complexes resolved by chemical probing.

RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We use RNA:RNA binding by SHAPE (RABS) to investigate microRNA-34a (miR-34a) binding its mRNA target, the silent information regulator 1 (mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the naked miR-34a is able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.

RNA调控可由第二种靶向RNA分子来执行，比如在微小RNA（microRNA）调控机制中。通过引物延伸分析的选择性2′ - 羟基酰化（SHAPE）可探测RNA分子的结构，并能够解析RNA - 蛋白质相互作用，但该技术尚未用于解决RNA - RNA相互作用。在此，我们应用SHAPE来研究RNA - RNA以及RNA - RNA - RBP复合物中RNA介导的结合过程。我们利用基于SHAPE的RNA - RNA结合（RABS）来研究微小RNA - 34a（miR - 34a）与其mRNA靶点——沉默信息调节因子1（mSIRT1）的结合情况，包括有和没有Argonaute蛋白（构成RNA诱导沉默复合物，即RISC）的情况。我们表明，mRNA靶点的种子序列必须与加载到RISC中的微小RNA结合，才能使RISC进一步结合补偿区域，而单独的miR - 34a能够在没有种子序列相互作用的情况下结合补偿区域。这里介绍的方法为通常基于荧光素酶测定评估微小RNA在mRNA靶点上结合位点的效率和特异性提供了补充性的结构证据，因此可与之结合使用。该方法可应用于任何核酸介导的RNA或RBP结合过程，如剪接、反义RNA结合或由RISC进行的调控，为靶向RNA结构提供重要的见解。