Abstract
RNA interference (RNAi) has been thought to be a gene-silencing pathway present in most eukaryotic cells to safeguard the genome against retrotransposition. Small interfering RNAs (siRNAs) have also become a powerful tool for studying gene functions. Given the endosymbiotic hypothesis that mitochondria originated from prokaryotes, mitochondria have been generally assumed to lack active RNAi; however, certain bacteria have Argonaute homologs and various reports suggest the presence of specific microRNAs and nuclear genome (nDNA)-encoded Ago2 in the mitochondria. Here we report that transfected siRNAs are not only able to enter the matrix of mitochondria, but also function there to specifically silence targeted mitochondrial transcripts. The mitoRNAi effect is readily detectable at the mRNA level, but only recordable on relatively unstable proteins, such as the mtDNA-encoded complex IV subunits. We also apply mitoRNAi to directly determine the postulated crosstalk between individual respiratory chain complexes, and our result suggests that the controversial observations previously made in patient-derived cells might result from differential adaptation in different cell lines. Our findings bring a new tool to study mitochondrial biology.
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Acknowledgements
This work was supported by grants from the Ministry of Science and Technology of China (2017YFA0504600, 2019YFA0508700, and 2017YFA0504400), the National Natural Science Foundation of China (31670825 and 91440102), and Chinese Academy of Science foundation grant (22KJZD-EW-L12) to X. Zhang. Work in the Murphy Lab was supported by the Medical Research Council UK (MC_U105663142) and by a Wellcome Trust Investigator award (110159/Z/15/Z). Work of K.H. is funded by the FWO (2018, 1.5.193.18N) and EU Horizon 2020 research and innovation program (665501). We also acknowledge the contribution of Drs. Yi Zhang and Yuanchao Xue in the early phase of this project, Dr. Heping Cheng for providing the Seahorse apparatus, and Hongjie Zhang for isotope experiment assistance. We are grateful to Drs. Immo Scheffler, Jing Hu, and Peter Rehling for critical comments on the manuscript.
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X. Zhang and X.-D.F. designed the experiments; K.G., M.C., and X. Zhang performed most experiments; X. Zuo helped with data analysis; J.L. contributed to experimental design and data interpretation; M.P.M. and K.H. designed the ClickIn experiment and provided the MitoOct reagents; X. Zhang, K.G., and X.-D.F. wrote the manuscript; and M.P.M. and K.H. revised the manuscript.
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Gao, K., Cheng, M., Zuo, X. et al. Active RNA interference in mitochondria. Cell Res 31, 219–228 (2021). https://doi.org/10.1038/s41422-020-00394-5
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DOI: https://doi.org/10.1038/s41422-020-00394-5
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