biostudies-literatureTanaka TMEXT | Japan Society for the Promotion of ScienceMitsubishi FoundationMEXT | Japan Society for the Promotion of Science (JSPS)Takeda Science FoundationInter-University Research Network for High Depth Omics, IMEG, Kumamoto UniversityMinistry of Education, Culture, Sports, Science and TechnologyMinistry of Education, Culture, Sports, Science and Technology (MEXT)6993https://www.ebi.ac.uk/biostudies/studies/S-EPMC11324899biostudies-literatureUnknown15(1)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 RNNature communicationsEndocytosed dsRNAs induce lysosomal membrane permeabilization that allows cytosolic dsRNA translocation for Drosophila RNAi responses.PMC1132489917H0368623H00364JPMXP132301548622H0275921H0480321H02489Hanyu-Nakamura KMizuta KYano TBruckner KTanaka TSeo YToyama-Sorimachi NUsuki SOkaguchi MYamaguchi MNakamura AfalseEndocytosed dsRNAs induce lysosomal membrane permeabilization that allows cytosolic dsRNA translocation for Drosophila RNAi responses.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 RN2024-01-01T00:00:00Z2024 Aug2025-04-05T18:35:02.659Z2025-04-05T18:35:02.659ZS-EPMC113248993914309810.1038/s41467-024-51343-4