Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan 48824-1226, USA.
Biochemistry. 2010 Apr 13;49(14):3148-55. doi: 10.1021/bi902189s.
The recognition of small interfering RNAs (siRNAs) by the RNA-induced silencing complex (RISC) and its precursor, the RISC loading complex (RLC), is a key step in the RNA interference pathway that controls the subsequent sequence-specific mRNA degradation. In Drosophila, selection of the guide strand has been shown to be mediated by RLC protein R2D2, which senses the relative hybridization stability between the two ends of the siRNA. A protein with similar function has yet to be conclusively identified in humans. We show here that human TAR RNA binding protein (TRBP) alone can bind siRNAs in vitro and sense their asymmetry. We also show that TRBP can bind 21-nucleotide single-stranded RNAs, though with far lower affinity than for double-stranded siRNA, and that TRBP cross-links preferentially to the 3'-ends of the guide strands of siRNAs. This suggests that TRBP binding depends both on the sequences of the siRNA strands and on the relative hybridization stability of the ends of the duplex. Together, these results demonstrate the importance of the siRNA-TRBP interaction in the selection of the siRNA guide strand in RNAi.
小干扰 RNA（siRNA）被 RNA 诱导沉默复合物（RISC）及其前体 RISC 加载复合物（RLC）识别，是 RNA 干扰途径中控制随后的序列特异性 mRNA 降解的关键步骤。在果蝇中，已经证明向导链的选择是由 RLC 蛋白 R2D2 介导的，它可以感知 siRNA 两端的相对杂交稳定性。在人类中，尚未明确鉴定出具有类似功能的蛋白质。我们在这里表明，人类 TAR RNA 结合蛋白（TRBP）本身可以在体外结合 siRNA 并感知其不对称性。我们还表明，TRBP 可以结合 21 个核苷酸的单链 RNA，但与双链 siRNA 的亲和力要低得多，并且 TRBP 交联优先发生在 siRNA 向导链的 3'-末端。这表明 TRBP 结合不仅取决于 siRNA 链的序列，还取决于双链体末端的相对杂交稳定性。这些结果共同证明了 siRNA-TRBP 相互作用在 RNAi 中 siRNA 向导链选择中的重要性。
