An Extended dsRBD with a Novel Zinc-Binding Motif Mediates Nuclear Retention of Fission Yeast Dicer. Schizosaccharomyces pombe

Dicer proteins function in RNA interference (RNAi) pathways by generating small RNAs (sRNAs). Here we report the solution structure of the C-terminal domain of Schizosaccharomyces pombe Dicer (Dcr1). The structure reveals an unusual double-stranded RNA binding domain (dsRBD) fold embedding a novel zinc-binding motif that is conserved among dicers in yeast. Although the C-terminal domain of Dcr1 still binds nucleic acids, this property is dispensable for proper functioning of Dcr1. In contrast, disruption of zinc coordination renders Dcr1 mainly cytoplasmic and leads to remarkable changes in gene expression and loss of heterochromatin assembly. In summary, our results reveal novel insights into the mechanism of nuclear retention of Dcr1 and raise the possibility that this new class of dsRBDs might generally function in nucleo-cytoplasmic trafficking and not substrate binding. The C-terminal domain of Dcr1 constitutes a novel regulatory module that might represent a potential target for therapeutic intervention with fungal diseases. Overall design: Small RNA libraries from total RNA isolations of wild-type, dcr1Delta, dcr1SHSS, and dcr1Deltaloop2 cells and subjected to high-throughput sequencing.

Dicer蛋白通过生成小RNA（sRNAs）参与RNA干扰（RNAi）通路。本研究解析了粟酒裂殖酵母（Schizosaccharomyces pombe）Dcr1的C端结构域的溶液结构。该结构揭示出一种罕见的双链RNA结合结构域（double-stranded RNA binding domain, dsRBD）折叠，其内部嵌入了一个在酵母Dicer蛋白中保守的新型锌结合基序。尽管Dcr1的C端结构域仍可结合核酸，但该特性对于Dcr1的正常功能并非必需。与之相反，破坏锌配位会使Dcr1主要定位于细胞质，并引发基因表达的显著改变以及异染色质组装的缺失。综上，本研究结果为Dcr1的核滞留机制提供了全新的见解，并提出这类新型dsRBD可能普遍参与核质穿梭而非底物结合的可能性。Dcr1的C端结构域构成了一个新型调控模块，有望成为真菌疾病治疗干预的潜在靶点。
实验整体设计：从野生型、dcr1Δ缺失突变体、dcr1SHSS突变体与dcr1Δloop2突变体细胞的总RNA提取物中构建小RNA文库，随后进行高通量测序。