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| Status |
Public on Jul 07, 2024 |
| Title |
Endocytosed dsRNAs induce lysosomal membrane permeabilization that allows cytosolic dsRNA translocation for Drosophila RNAi response |
| Organism |
Drosophila melanogaster |
| Experiment type |
Non-coding RNA profiling by high throughput sequencing
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| Summary |
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.
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| Overall design |
Ago2 complex was immunopurified and copurified RNAs are deep-sequenced. Samples 1-4 were control saline injected w (1), and DCV injected w (2), clc-b_KO (3), and ostm1_KO (4) flies, and whole fly lysates were used for immunoprecipitation. Samples 5-10 were control S2 (5, 6), clc-b_KO (7, 8), and dmostm1_KO (9, 10) cell lines clutured in without (5, 7, 9) or with (6, 8, 10) dsRNA for firefly luciferase CDS in the medium.
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| Contributor(s) |
Tanaka T, Yano T, Usuki S, Hanyu-Nakamura K, Nakamura A |
| Citation(s) |
39143098 |
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| Submission date |
Oct 03, 2023 |
| Last update date |
Sep 11, 2024 |
| Contact name |
Shingo Usuki |
| E-mail(s) |
[email protected]
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| Phone |
+81-96-373-5786
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| Organization name |
Kumamoto University
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| Department |
Institute of Molecular Embryology and Genetics (IMEG)
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| Lab |
Liaison Laboratory Research Promotion Center
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| Street address |
2-2-1 Honjo, Chuo-ku
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| City |
Kumamoto |
| ZIP/Postal code |
860-0811 |
| Country |
Japan |
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| Platforms (1) |
| GPL19132 |
Illumina NextSeq 500 (Drosophila melanogaster) |
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| Samples (10)
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| GSM7818619 |
Sample 5_S2 cells withour dsRNA in the medium |
| GSM7818620 |
Sample 6_S2 cells with dsRNA in the medium |
| GSM7818621 |
Sample 7_clc-b_KO S2 cells without dsRNA in the medium |
| GSM7818622 |
Sample 8_clc-b_KO S2 cells with dsRNA in the medium |
| GSM7818623 |
Sample 9_dmostem-KO S2 cells without dsRNA in the medium |
| GSM7818624 |
Sample 10_dmostem-KO S2 cells with dsRNA in the medium |
| GSM8305921 |
control saline injected w flies |
| GSM8305922 |
DCV injected w flies |
| GSM8305923 |
DCV injected clc-b_KO flies |
| GSM8305924 |
DCV injected dmostm1_KO flies |
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| Relations |
| BioProject |
PRJNA1023479 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE244523_Count.txt.gz |
269.3 Kb |
(ftp)(http) |
TXT |
| GSE244523_Count_new.txt.gz |
325.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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