Abstract
Bidirectional transcription of mammalian mitochondrial DNA generates overlapping transcripts that are capable of forming double-stranded RNA (dsRNA) structures. Release of mitochondrial dsRNA into the cytosol activates the dsRNA-sensing immune signaling, which is a defense mechanism against microbial and viral attack and possibly cancer, but could cause autoimmune diseases when unchecked. A better understanding of the process is vital in therapeutic application of this defense mechanism and treatment of cognate human diseases. In addition to exporting dsRNAs, mitochondria also export and import a variety of non-coding RNAs. However, little is known about how these RNAs are transported across mitochondrial membranes. Here we provide direct evidence showing that adenine nucleotide translocase-2 (ANT2) functions as a mammalian RNA translocon in the mitochondrial inner membrane, independent of its ADP/ATP translocase activity. We also show that mitochondrial dsRNA efflux through ANT2 triggers innate immunity. Inhibiting this process alleviates inflammation in vivo, providing a potential therapeutic approach for treating autoimmune diseases.
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References
Anderson, S. et al. Sequence and organization of the human mitochondrial genome. Nature 290, 457–465 (1981).
Calvo, S. E., Clauser, K. R. & Mootha, V. K. MitoCarta2.0: an updated inventory of mammalian mitochondrial proteins. Nucleic Acids Res. 44, D1251–D1257 (2016).
Couvillion, M. T., Soto, I. C., Shipkovenska, G. & Churchman, L. S. Synchronized mitochondrial and cytosolic translation programs. Nature 533, 499–503 (2016).
Brown, A. et al. Structure of the large ribosomal subunit from human mitochondria. Science 346, 718–722 (2014).
Greber, B. J. et al. Ribosome. The complete structure of the 55S mammalian mitochondrial ribosome. Science 348, 303–308 (2015).
Friedman, J. R. & Nunnari, J. Mitochondrial form and function. Nature 505, 335–343 (2014).
Tamura, Y., Kawano, S. & Endo, T. Lipid homeostasis in mitochondria. Biol. Chem. 401, 821–833 (2020).
Kim, J. et al. VDAC oligomers form mitochondrial pores to release mtDNA fragments and promote lupus-like disease. Science 366, 1531–1536 (2019).
Wang, L. Q. et al. Perfluoroalkyl substance pollutants activate the innate immune system through the AIM2 inflammasome. Nat. Commun. 12, 2915 (2021).
Aloni, Y. & Attardi, G. Symmetrical in vivo transcription of mitochondrial DNA in HeLa cells. Proc. Natl. Acad. Sci. USA 68, 1757–1761 (1971).
Dhir, A. et al. Mitochondrial double-stranded RNA triggers antiviral signalling in humans. Nature 560, 238–242 (2018).
Young, P. G. & Attardi, G. Characterization of double-stranded RNA from HeLa cell mitochondria. Biochem. Biophys. Res. Commun. 65, 1201–1207 (1975).
Chen, Y. G. & Hur, S. Cellular origins of dsRNA, their recognition and consequences. Nat. Rev. Mol. Cell Biol. 23, 286–301 (2022).
Hooftman, A. et al. Macrophage fumarate hydratase restrains mtRNA-mediated interferon production. Nature 615, 490–498 (2023).
Kim, S. et al. Mitochondrial double-stranded RNAs govern the stress response in chondrocytes to promote osteoarthritis development. Cell Rep. 40, 111178 (2022).
Yoon, J. et al. Mitochondrial double-stranded RNAs as a pivotal mediator in the pathogenesis of Sjögren’s syndrome. Mol. Ther. Nucleic Acids 30, 257–269 (2022).
Liu, S., Feng, M. & Guan, W. Mitochondrial DNA sensing by STING signaling participates in inflammation, cancer and beyond. Int. J. Cancer 139, 736–741 (2016).
Hwang, B. J. et al. Sensitizing immune unresponsive colorectal cancers to immune checkpoint inhibitors through MAVS overexpression. J. Immunother. Cancer 10, e003721 (2022).
Zecchini, V. et al. Fumarate induces vesicular release of mtDNA to drive innate immunity. Nature 615, 499–506 (2023).
McArthur, K. et al. BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis. Science 359, eaao6047 (2018).
Riley, J. S. et al. Mitochondrial inner membrane permeabilisation enables mtDNA release during apoptosis. EMBO J. 37, e99238 (2018).
Rongvaux, A. et al. Apoptotic caspases prevent the induction of type I interferons by mitochondrial DNA. Cell 159, 1563–1577 (2014).
White, M. J. et al. Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production. Cell 159, 1549–1562 (2014).
Zhang, W. et al. Cytosolic escape of mitochondrial DNA triggers cGAS-STING-NLRP3 axis-dependent nucleus pulposus cell pyroptosis. Exp. Mol. Med. 54, 129–142 (2022).
Liu, H. et al. Prohibitin 1 regulates mtDNA release and downstream inflammatory responses. EMBO J. 41, e111173 (2022).
Xian, H. et al. Oxidized DNA fragments exit mitochondria via mPTP- and VDAC-dependent channels to activate NLRP3 inflammasome and interferon signaling. Immunity 55, 1370–1385.e8 (2022).
Yu, C. H. et al. TDP-43 triggers mitochondrial DNA release via mPTP to activate cGAS/STING in ALS. Cell 183, 636–649.e18 (2020).
Huang, J., Liu, P. & Wang, G. Regulation of mitochondrion-associated cytosolic ribosomes by mammalian mitochondrial ribonuclease T2 (RNASET2). J. Biol. Chem. 293, 19633–19644 (2018).
Mercer, T. R. et al. The human mitochondrial transcriptome. Cell 146, 645–658 (2011).
Jeandard, D. et al. Import of non-coding RNAs into human mitochondria: a critical review and emerging approaches. Cells 8, 286 (2019).
Wang, G. et al. PNPASE regulates RNA import into mitochondria. Cell 142, 456–467 (2010).
Zhang, X. et al. MicroRNA directly enhances mitochondrial translation during muscle differentiation. Cell 158, 607–619 (2014).
Gao, K. et al. Active RNA interference in mitochondria. Cell Res. 31, 219–228 (2021).
Liu, X. et al. Identification of mecciRNAs and their roles in the mitochondrial entry of proteins. Sci. China Life Sci. 63, 1429–1449 (2020).
Cheng, Y. et al. Mitochondrial trafficking and processing of telomerase RNA TERC. Cell Rep. 24, 2589–2595 (2018).
Zheng, Q. et al. Mitochondrion-processed TERC regulates senescence without affecting telomerase activities. Protein Cell 10, 631–648 (2019).
Slomovic, S. & Schuster, G. Stable PNPase RNAi silencing: its effect on the processing and adenylation of human mitochondrial RNA. RNA 14, 310–323 (2008).
Borowski, L. S., Dziembowski, A., Hejnowicz, M. S., Stepien, P. P. & Szczesny, R. J. Human mitochondrial RNA decay mediated by PNPase-hSuv3 complex takes place in distinct foci. Nucleic Acids Res. 41, 1223–1240 (2013).
Bruni, F., Gramegna, P., Oliveira, J. M., Lightowlers, R. N. & Chrzanowska-Lightowlers, Z. M. REXO2 is an oligoribonuclease active in human mitochondria. PLoS ONE 8, e64670 (2013).
Chang, D. D. & Clayton, D. A. A mammalian mitochondrial RNA processing activity contains nucleus-encoded RNA. Science 235, 1178–1184 (1987).
Liu, P. et al. Mammalian mitochondrial RNAs are degraded in the mitochondrial intermembrane space by RNASET2. Protein Cell 8, 735–749 (2017).
Zhou, Q. et al. Mitochondrial endonuclease G mediates breakdown of paternal mitochondria upon fertilization. Science 353, 394–399 (2016).
Kunji, E. R. et al. The transport mechanism of the mitochondrial ADP/ATP carrier. Biochim. Biophys. Acta 1863, 2379–2393 (2016).
Klingenberg, M. The ADP and ATP transport in mitochondria and its carrier. Biochim. Biophys. Acta 1778, 1978–2021 (2008).
Sang, L. et al. Mitochondrial long non-coding RNA GAS5 tunes TCA metabolism in response to nutrient stress. Nat. Metab. 3, 90–106 (2021).
Luevano-Martinez, L. A. & Duncan, A. L. Origin and diversification of the cardiolipin biosynthetic pathway in the Eukarya domain. Biochem. Soc. Trans. 48, 1035–1046 (2020).
Rubio, M. A. et al. Mammalian mitochondria have the innate ability to import tRNAs by a mechanism distinct from protein import. Proc. Natl. Acad. Sci. USA 105, 9186–9191 (2008).
Dolce, V., Scarcia, P., Iacopetta, D. & Palmieri, F. A fourth ADP/ATP carrier isoform in man: identification, bacterial expression, functional characterization and tissue distribution. FEBS Lett. 579, 633–637 (2005).
Lim, C. H., Brower, J. V., Resnick, J. L., Oh, S. P. & Terada, N. Adenine nucleotide translocase 4 is expressed within embryonic ovaries and dispensable during oogenesis. Reprod. Sci. 22, 250–257 (2015).
Rodic, N. et al. DNA methylation is required for silencing of ant4, an adenine nucleotide translocase selectively expressed in mouse embryonic stem cells and germ cells. Stem Cells 23, 1314–1323 (2005).
Doerner, A. et al. Tissue-specific transcription pattern of the adenine nucleotide translocase isoforms in humans. FEBS Lett. 414, 258–262 (1997).
Stepien, G., Torroni, A., Chung, A. B., Hodge, J. A. & Wallace, D. C. Differential expression of adenine nucleotide translocator isoforms in mammalian tissues and during muscle cell differentiation. J. Biol. Chem. 267, 14592–14597 (1992).
Lu, Y. W. et al. Human adenine nucleotide translocases physically and functionally interact with respirasomes. Mol. Biol. Cell 28, 1489–1506 (2017).
Mempin, R. et al. Release of extracellular ATP by bacteria during growth. BMC Microbiol. 13, 301 (2013).
Tigano, M., Vargas, D. C., Tremblay-Belzile, S., Fu, Y. & Sfeir, A. Nuclear sensing of breaks in mitochondrial DNA enhances immune surveillance. Nature 591, 477–481 (2021).
Killarney, S. T. et al. Executioner caspases restrict mitochondrial RNA-driven Type I IFN induction during chemotherapy-induced apoptosis. Nat. Commun. 14, 1399 (2023).
Huang, Y. H. et al. Heat shock protein 60 restricts release of mitochondrial dsRNA to suppress hepatic inflammation and ameliorate non-alcoholic fatty liver disease in mice. Int. J. Mol. Sci. 23, 577 (2022).
Weber-Lotfi, F. et al. Nucleic acid import into mitochondria: New insights into the translocation pathways. Biochim. Biophys. Acta 1853, 3165–3181 (2015).
Huang, J., Wu, S., Wang, P. & Wang, G. Non-coding RNA regulated cross-talk between mitochondria and other cellular compartments. Front. Cell Dev. Biol. 9, 688523 (2021).
Leucci, E. et al. Melanoma addiction to the long non-coding RNA SAMMSON. Nature 531, 518–522 (2016).
Vendramin, R. et al. SAMMSON fosters cancer cell fitness by concertedly enhancing mitochondrial and cytosolic translation. Nat. Struct. Mol. Biol. 25, 1035–1046 (2018).
von Ameln, S. et al. A mutation in PNPT1, encoding mitochondrial-RNA-import protein PNPase, causes hereditary hearing loss. Am. J. Hum. Genet. 91, 919–927 (2012).
Chen, X. et al. An RNA degradation machine sculpted by Ro autoantigen and noncoding RNA. Cell 153, 166–177 (2013).
Smirnov, A. et al. Mitochondrial enzyme rhodanese is essential for 5 S ribosomal RNA import into human mitochondria. J. Biol. Chem. 285, 30792–30803 (2010).
Smirnov, A., Entelis, N., Martin, R. P. & Tarassov, I. Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18. Genes Dev. 25, 1289–1305 (2011).
Rinehart, J., Krett, B., Rubio, M. A., Alfonzo, J. D. & Soll, D. Saccharomyces cerevisiae imports the cytosolic pathway for Gln-tRNA synthesis into the mitochondrion. Genes Dev. 19, 583–592 (2005).
Rubio, M. A., Liu, X., Yuzawa, H., Alfonzo, J. D. & Simpson, L. Selective importation of RNA into isolated mitochondria from Leishmania tarentolae. RNA 6, 988–1003 (2000).
Oka, T. et al. Mitochondrial DNA that escapes from autophagy causes inflammation and heart failure. Nature 485, 251–255 (2012).
Salinas, T. et al. The voltage-dependent anion channel, a major component of the tRNA import machinery in plant mitochondria. Proc. Natl. Acad. Sci. USA 103, 18362–18367 (2006).
Salinas, T., Duchene, A. M. & Marechal-Drouard, L. Recent advances in tRNA mitochondrial import. Trends Biochem. Sci. 33, 320–329 (2008).
Mannella, C. A. & Kinnally, K. W. Reflections on VDAC as a voltage-gated channel and a mitochondrial regulator. J. Bioenerg. Biomembr. 40, 149–155 (2008).
Chen, H. W. et al. Mammalian polynucleotide phosphorylase is an intermembrane space RNase that maintains mitochondrial homeostasis. Mol. Cell. Biol. 26, 8475–8487 (2006).
Seo, M., Lei, L. & Egli, M. Label-free electrophoretic mobility shift assay (EMSA) for measuring dissociation constants of protein-rna complexes. Curr. Protoc. Nucleic Acid Chem. 76, e70 (2019).
Dieterich, D. C. et al. Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging. Nat. Protoc. 2, 532–540 (2007).
Sheng, W. et al. LSD1 ablation stimulates anti-tumor immunity and enables checkpoint blockade. Cell 174, 549–563.e19 (2018).
Acknowledgements
This work was supported by the Priority Research Program of the Ministry of Science and Technology of China (2017YFA0504600), and the National Natural Science Foundation of China (91649103 and 32091159).
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Conceptualization, G.W., S.W. and P.W.; Methodology, G.W., P.W. and L.Z.; Investigation, P.W., L.Z., S.C., S.W., R.L., P.L., X. Li, H.L., Y.H., Z. Zhang, Y.C., X. Liu, J.H., G.Z., Z.S., S.D., J.S., Z. Zhou, R.Y and L.L.; Writing-original draft, G.W. and P.W.; Writing-review & editing, G.W. and P.W.; Funding acquisition, G.W.; Resources, G.W.; Supervision, G.W.
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Wang, P., Zhang, L., Chen, S. et al. ANT2 functions as a translocon for mitochondrial cross-membrane translocation of RNAs. Cell Res 34, 504–521 (2024). https://doi.org/10.1038/s41422-024-00978-5
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DOI: https://doi.org/10.1038/s41422-024-00978-5
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