The helicase domain of Dicer ensures essential accuracy of miRNA biogenesis in mice

Dicer ribonucleases, which generate small RNAs for the RNA interference (RNAi) and microRNA (miRNA) pathways, often have an N-terminal DExD helicase subdomain (also called HEL1). In the mammalian Dicer, HEL1 inhibits RNAi while it appears unnecessary for miRNA biogenesis. To determine its functional significance, we genetically eliminated HEL1 from Dicer in mice. We observed embryonic growth retardation, defects in the cardiopulmonary system, and perinatal lethality. HEL1 suppresses biogenesis of mirtrons, a non-canonical low-abundant class of miRNAs, and is required for high-fidelity cleavage and strand selection during biogenesis of canonical miRNAs. The HEL1 domain itself is essential rather than its helicase activity, because mutations of critical catalytic amino acids do not affect mouse viability or fertility and have minimal impact on miRNA biogenesis. Therefore, HEL1 is a critical structural component of the mammalian Dicer, supporting its role of a conserved structural “mold” that ensures high-fidelity processing and adaptive evolution of mammalian miRNA precursors. Overall design: Bulk small RNA-seq of 9 mouse ESC samples: - 3x WT transfected with MosIR - 3x DicerX homozygotes transfected with MosIR - 3x DicerX homozygotes & PKR null transfected with MosIR

Dicer核糖核酸酶（Dicer ribonucleases）可为RNA干扰（RNA interference, RNAi）与微小RNA（microRNA, miRNA）通路生成小RNA，其通常带有N端DExD解旋酶亚结构域（又称HEL1）。在哺乳动物Dicer核糖核酸酶中，HEL1可抑制RNAi过程，但对miRNA的生物发生似乎并非必需。为明确其功能重要性，我们在小鼠体内通过遗传学手段敲除了Dicer的HEL1结构域。实验结果显示，小鼠出现胚胎生长迟缓、心肺系统缺陷以及围产期致死现象。HEL1能够抑制内含子微小RNA（mirtrons）——一类非经典的低丰度miRNA类群——的生物发生，同时在经典miRNA的生物发生过程中，对于高保真切割与链选择是必需的。HEL1结构域本身是关键结构组分，而非其解旋酶活性：关键催化氨基酸残基的突变既不会影响小鼠的生存能力与生育能力，也对miRNA的生物发生影响极小。因此，HEL1是哺乳动物Dicer核糖核酸酶的关键结构组分，发挥保守结构“模具”的作用，确保哺乳动物miRNA前体的高保真加工与适应性进化。

整体实验设计：对9份小鼠胚胎干细胞（ESC）样本开展批量小RNA测序：
- 3份转染MosIR的野生型（WT）样本
- 3份转染MosIR的DicerX纯合子样本
- 3份转染MosIR的DicerX纯合且PKR敲除样本