Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, MA 02142, USA; Howard Hughes Medical Institute, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Mol Cell. 2024 Aug 8;84(15):2918-2934.e11. doi: 10.1016/j.molcel.2024.06.026. Epub 2024 Jul 17.
The RNA-induced silencing complex (RISC), which powers RNA interference (RNAi), consists of a guide RNA and an Argonaute protein that slices target RNAs complementary to the guide. We find that, for different guide-RNA sequences, slicing rates of perfectly complementary bound targets can be surprisingly different (>250-fold range), and that faster slicing confers better knockdown in cells. Nucleotide sequence identities at guide-RNA positions 7, 10, and 17 underlie much of this variation in slicing rates. Analysis of one of these determinants implicates a structural distortion at guide nucleotides 6-7 in promoting slicing. Moreover, slicing directed by different guide sequences has an unanticipated, 600-fold range in 3'-mismatch tolerance, attributable to guides with weak (AU-rich) central pairing requiring extensive 3' complementarity (pairing beyond position 16) to more fully populate the slicing-competent conformation. Together, our analyses identify sequence determinants of RISC activity and provide biochemical and conformational rationale for their action.
RNA 诱导沉默复合物（RISC）是 RNA 干扰（RNAi）的动力源，由向导 RNA 和 Argonaute 蛋白组成，可将与向导互补的靶 RNA 切割。我们发现，对于不同的向导 RNA 序列，完美互补结合的靶标切割率可能差异惊人（>250 倍），而且更快的切割能在细胞中实现更好的敲低效果。在向导 RNA 位置 7、10 和 17 的核苷酸序列同一性是导致这种切割率差异的主要原因。对其中一个决定因素的分析表明，向导核苷酸 6-7 的结构扭曲有助于促进切割。此外，由不同向导序列指导的切割具有出人意料的 600 倍的 3'-错配容忍度范围，这归因于具有较弱（富含 AU）中心配对的向导需要广泛的 3'互补性（配对超过位置 16），以更充分地填充切割能力构象。总的来说，我们的分析确定了 RISC 活性的序列决定因素，并为它们的作用提供了生化和构象依据。
