Department of Germline Development, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan.
Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
Nat Commun. 2024 Aug 14;15(1):6993. doi: 10.1038/s41467-024-51343-4.
RNA interference (RNAi) is a gene-silencing mechanism triggered by the cytosolic entry of double-stranded RNAs (dsRNAs). Many animal cells internalize extracellular dsRNAs via endocytosis for RNAi induction. However, it is not clear how the endocytosed dsRNAs are translocated into the cytosol across the endo/lysosomal membrane. Herein, we show that in Drosophila S2 cells, endocytosed dsRNAs induce lysosomal membrane permeabilization (LMP) that allows cytosolic dsRNA translocation. LMP mediated by dsRNAs requires the lysosomal Cl/H antiporter ClC-b/DmOstm1. In clc-b or dmostm1 knockout S2 cells, extracellular dsRNAs are endocytosed and reach the lysosomes normally but fail to enter the cytosol. Pharmacological induction of LMP restores extracellular dsRNA-directed RNAi in clc-b or dmostm1-knockout cells. Furthermore, clc-b or dmostm1 mutant flies are defective in extracellular dsRNA-directed RNAi and its associated antiviral immunity. Therefore, endocytosed dsRNAs have an intrinsic ability to induce ClC-b/DmOstm1-dependent LMP that allows cytosolic dsRNA translocation for RNAi responses in Drosophila cells.
RNA 干扰 (RNAi) 是一种由双链 RNA (dsRNAs) 胞质内进入引发的基因沉默机制。许多动物细胞通过内吞作用将细胞外的 dsRNAs 内化，从而诱导 RNAi。然而，目前尚不清楚被内吞的 dsRNAs 如何穿过内体/溶酶体膜转移到细胞质中。本文中，我们发现在果蝇 S2 细胞中，内吞的 dsRNAs 诱导溶酶体膜通透性增加 (LMP)，从而允许细胞质中的 dsRNA 转运。dsRNA 介导的 LMP 需要溶酶体 Cl/H 反向转运蛋白 ClC-b/DmOstm1。在 clc-b 或 dmostm1 敲除的 S2 细胞中，细胞外 dsRNAs 被内吞并正常到达溶酶体，但无法进入细胞质。用药物诱导 LMP 可恢复 clc-b 或 dmostm1 敲除细胞中外源 dsRNA 指导的 RNAi。此外，clc-b 或 dmostm1 突变果蝇在细胞外 dsRNA 指导的 RNAi 及其相关抗病毒免疫方面存在缺陷。因此，内吞的 dsRNAs 具有内在能力诱导 ClC-b/DmOstm1 依赖性 LMP，从而允许细胞质中的 dsRNA 转运，以在果蝇细胞中引发 RNAi 反应。
