StaufenC facilitates utilization of the ERAD pathway to transport dsRNA through the endoplasmic reticulum to the cytosol

Jinmo Koo; Subba Reddy Palli

doi:10.1073/pnas.2322927121

StaufenC facilitates utilization of the ERAD pathway to transport dsRNA through the endoplasmic reticulum to the cytosol

Jinmo Koo
, Subba Reddy Palli

Research output: Contribution to journal › Article › peer-review

19 Scopus citations

Abstract

RNA interference (RNAi) is more efficient in coleopteran insects than other insects. StaufenC (StauC), a coleopteran-specific double-stranded RNA (dsRNA)-binding protein, is required for efficient RNAi in coleopterans. We investigated the function of StauC in the intracellular transport of dsRNA into the cytosol, where dsRNA is digested by Dicer enzymes and recruited by Argonauts to RNA-induced silencing complexes. Confocal microscopy and cellular organelle fractionation studies have shown that dsRNA is trafficked through the endoplasmic reticulum (ER) in coleopteran Colorado potato beetle (CPB) cells. StauC is localized to the ER in CPB cells, and StauC-knockdown caused the accumulation of dsRNA in the ER and a decrease in the cytosol, suggesting that StauC plays a key role in the intracellular transport of dsRNA through the ER. Using immunoprecipitation, we showed that StauC is required for dsRNA interaction with ER proteins in the ER-associated protein degradation (ERAD) pathway, and these interactions are required for RNAi in CPB cells. These results suggest that StauC works with the ERAD pathway to transport dsRNA through the ER to the cytosol. This information could be used to develop dsRNA delivery methods aimed at improving RNAi.

Original language	English
Article number	e2322927121
Journal	Proceedings of the National Academy of Sciences of the United States of America
Volume	121
Issue number	26
DOIs	https://doi.org/10.1073/pnas.2322927121
State	Published - Jun 1 2024

Bibliographical note

Publisher Copyright:
© 2024 the Author(s). Published by PNAS. This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Funding

ACKNOWLEDGMENTS. Research reported in this publication was supported by the National Institute of General Medical Sciences of the NIH under Award Number R01GM070559 and the US Department of Agriculture (under HATCH Project 2353057000). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the US Department of Agriculture. We thank Kyungbo Kim (BioMed X Institute) for helping with the plasmid construct design. We thank Dr. Nicholas Teets, Dr. Xuguo Zhou, Dr. Douglas Harrison, and Dr. Elizabeth Duncan from the University of Kentucky and anonymous reviewers for their valuable comments on the manuscript. Research reported in this publication was supported by the National Institute of General Medical Sciences of the NIH under Award Number R01GM070559 and the US Department of Agriculture (under HATCH Project 2353057000). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the US Department of Agriculture. We thank Kyungbo Kim (BioMed X Institute) for helping with the plasmid construct design. We thank Dr. Nicholas Teets, Dr. Xuguo Zhou, Dr. Douglas Harrison, and Dr. Elizabeth Duncan from the University of Kentucky and anonymous reviewers for their valuable comments on the manuscript.

Funders	Funder number
National Institute of General Medical Sciences DP2GM119177 Sophie Dumont National Institute of General Medical Sciences
University of Kentucky
National Institutes of Health (NIH)	R01GM070559
U.S. Department of Agriculture	2353057000

Keywords

C-terminal KDEL motif
Colorado potato beetle
RNA interference
intracellular trafficking
protein localization

ASJC Scopus subject areas

General

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10.1073/pnas.2322927121

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title = "StaufenC facilitates utilization of the ERAD pathway to transport dsRNA through the endoplasmic reticulum to the cytosol",

abstract = "RNA interference (RNAi) is more efficient in coleopteran insects than other insects. StaufenC (StauC), a coleopteran-specific double-stranded RNA (dsRNA)-binding protein, is required for efficient RNAi in coleopterans. We investigated the function of StauC in the intracellular transport of dsRNA into the cytosol, where dsRNA is digested by Dicer enzymes and recruited by Argonauts to RNA-induced silencing complexes. Confocal microscopy and cellular organelle fractionation studies have shown that dsRNA is trafficked through the endoplasmic reticulum (ER) in coleopteran Colorado potato beetle (CPB) cells. StauC is localized to the ER in CPB cells, and StauC-knockdown caused the accumulation of dsRNA in the ER and a decrease in the cytosol, suggesting that StauC plays a key role in the intracellular transport of dsRNA through the ER. Using immunoprecipitation, we showed that StauC is required for dsRNA interaction with ER proteins in the ER-associated protein degradation (ERAD) pathway, and these interactions are required for RNAi in CPB cells. These results suggest that StauC works with the ERAD pathway to transport dsRNA through the ER to the cytosol. This information could be used to develop dsRNA delivery methods aimed at improving RNAi.",

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author = "Jinmo Koo and Palli, \{Subba Reddy\}",

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AU - Koo, Jinmo

AU - Palli, Subba Reddy

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N2 - RNA interference (RNAi) is more efficient in coleopteran insects than other insects. StaufenC (StauC), a coleopteran-specific double-stranded RNA (dsRNA)-binding protein, is required for efficient RNAi in coleopterans. We investigated the function of StauC in the intracellular transport of dsRNA into the cytosol, where dsRNA is digested by Dicer enzymes and recruited by Argonauts to RNA-induced silencing complexes. Confocal microscopy and cellular organelle fractionation studies have shown that dsRNA is trafficked through the endoplasmic reticulum (ER) in coleopteran Colorado potato beetle (CPB) cells. StauC is localized to the ER in CPB cells, and StauC-knockdown caused the accumulation of dsRNA in the ER and a decrease in the cytosol, suggesting that StauC plays a key role in the intracellular transport of dsRNA through the ER. Using immunoprecipitation, we showed that StauC is required for dsRNA interaction with ER proteins in the ER-associated protein degradation (ERAD) pathway, and these interactions are required for RNAi in CPB cells. These results suggest that StauC works with the ERAD pathway to transport dsRNA through the ER to the cytosol. This information could be used to develop dsRNA delivery methods aimed at improving RNAi.

AB - RNA interference (RNAi) is more efficient in coleopteran insects than other insects. StaufenC (StauC), a coleopteran-specific double-stranded RNA (dsRNA)-binding protein, is required for efficient RNAi in coleopterans. We investigated the function of StauC in the intracellular transport of dsRNA into the cytosol, where dsRNA is digested by Dicer enzymes and recruited by Argonauts to RNA-induced silencing complexes. Confocal microscopy and cellular organelle fractionation studies have shown that dsRNA is trafficked through the endoplasmic reticulum (ER) in coleopteran Colorado potato beetle (CPB) cells. StauC is localized to the ER in CPB cells, and StauC-knockdown caused the accumulation of dsRNA in the ER and a decrease in the cytosol, suggesting that StauC plays a key role in the intracellular transport of dsRNA through the ER. Using immunoprecipitation, we showed that StauC is required for dsRNA interaction with ER proteins in the ER-associated protein degradation (ERAD) pathway, and these interactions are required for RNAi in CPB cells. These results suggest that StauC works with the ERAD pathway to transport dsRNA through the ER to the cytosol. This information could be used to develop dsRNA delivery methods aimed at improving RNAi.

KW - C-terminal KDEL motif

KW - Colorado potato beetle

KW - RNA interference

KW - intracellular trafficking

KW - protein localization

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StaufenC facilitates utilization of the ERAD pathway to transport dsRNA through the endoplasmic reticulum to the cytosol

Abstract

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