School of Biological Sciences, Seoul National University, Seoul 151-742, Korea.
Nature. 2011 Jul 13;475(7355):201-5. doi: 10.1038/nature10198.
A hallmark of RNA silencing is a class of approximately 22-nucleotide RNAs that are processed from double-stranded RNA precursors by Dicer. Accurate processing by Dicer is crucial for the functionality of microRNAs (miRNAs). The current model posits that Dicer selects cleavage sites by measuring a set distance from the 3' overhang of the double-stranded RNA terminus. Here we report that human Dicer anchors not only the 3' end but also the 5' end, with the cleavage site determined mainly by the distance (∼22 nucleotides) from the 5' end (5' counting rule). This cleavage requires a 5'-terminal phosphate group. Further, we identify a novel basic motif (5' pocket) in human Dicer that recognizes the 5'-phosphorylated end. The 5' counting rule and the 5' anchoring residues are conserved in Drosophila Dicer-1, but not in Giardia Dicer. Mutations in the 5' pocket reduce processing efficiency and alter cleavage sites in vitro. Consistently, miRNA biogenesis is perturbed in vivo when Dicer-null embryonic stem cells are replenished with the 5'-pocket mutant. Thus, 5'-end recognition by Dicer is important for precise and effective biogenesis of miRNAs. Insights from this study should also afford practical benefits to the design of small hairpin RNAs.
RNA 沉默的一个标志是一类大约 22 个核苷酸的 RNA，它们是由 Dicer 从双链 RNA 前体中加工而来的。Dicer 的准确加工对于 microRNAs (miRNAs) 的功能至关重要。目前的模型假设 Dicer 通过测量双链 RNA 末端 3'突出端的一定距离来选择切割位点。在这里，我们报告人类 Dicer 不仅锚定 3'端，还锚定 5'端，切割位点主要由距离（约 22 个核苷酸）决定（5'计数规则）。这种切割需要 5'末端磷酸基团。此外，我们在人类 Dicer 中鉴定出一个新的碱性基序（5'口袋），它识别 5'磷酸化末端。5'计数规则和 5'锚定残基在果蝇 Dicer-1 中保守，但在贾第虫 Dicer 中不保守。5'口袋中的突变降低了体外加工效率并改变了切割位点。一致地，当用 5'口袋突变体补充 Dicer 缺失的胚胎干细胞时，miRNA 的生物发生在体内受到干扰。因此，Dicer 对 5'末端的识别对于 miRNAs 的精确和有效生物发生非常重要。这项研究的见解也应该为小发夹 RNA 的设计提供实际的好处。
