Active RNA interference in mitochondria.
Animals
Argonaute Proteins
/ genetics
DNA, Mitochondrial
/ genetics
Fibroblasts
/ metabolism
HEK293 Cells
HeLa Cells
Humans
Membrane Potential, Mitochondrial
/ genetics
Mice
Mitochondria
/ genetics
Myocytes, Cardiac
/ metabolism
Oxygen
/ metabolism
RNA Interference
RNA, Messenger
/ genetics
RNA, Small Interfering
/ genetics
Transfection
Journal
Cell research
ISSN: 1748-7838
Titre abrégé: Cell Res
Pays: England
ID NLM: 9425763
Informations de publication
Date de publication:
02 2021
02 2021
Historique:
received:
13
03
2020
accepted:
24
07
2020
pubmed:
19
8
2020
medline:
21
12
2021
entrez:
19
8
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 32807841
doi: 10.1038/s41422-020-00394-5
pii: 10.1038/s41422-020-00394-5
pmc: PMC8027830
doi:
Substances chimiques
Ago2 protein, mouse
0
Argonaute Proteins
0
DNA, Mitochondrial
0
RNA, Messenger
0
RNA, Small Interfering
0
Oxygen
S88TT14065
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
219-228Subventions
Organisme : Wellcome Trust
Pays : United Kingdom
Organisme : Wellcome Trust
ID : 110158/Z/15/Z
Pays : United Kingdom
Organisme : Medical Research Council
ID : MC_U105663142
Pays : United Kingdom
Références
Drinnenberg I. A. et al. RNAi in budding yeast. Science 326, 544–550 (2009).
pubmed: 19745116
pmcid: 3786161
doi: 10.1126/science.1176945
Fire A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).
pubmed: 9486653
doi: 10.1038/35888
Hamilton, A. J. & Baulcombe, D. C. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286, 950–952 (1999).
pubmed: 10542148
doi: 10.1126/science.286.5441.950
Imig J. et al. miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction. Nat. Chem. Biol. 11, 107–114 (2015).
pubmed: 25531890
doi: 10.1038/nchembio.1713
Elbashir S. M. et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498 (2001).
pubmed: 11373684
doi: 10.1038/35078107
Shi Y. Mammalian RNAi for the masses. Trends Genet. 19, 9–12 (2003).
pubmed: 12493242
doi: 10.1016/S0168-9525(02)00005-7
Roger, A. J., Munoz-Gomez, S. A. & Kamikawa, R. The origin and diversification of mitochondria. Curr. Biol. 27, R1177–R1192 (2017).
pubmed: 29112874
doi: 10.1016/j.cub.2017.09.015
Liu J. et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 305, 1437–1441 (2004).
pubmed: 15284456
doi: 10.1126/science.1102513
Zhang X. R. et al. MicroRNA directly enhances mitochondrial translation during muscle differentiation. Cell 158, 607–619 (2014).
pubmed: 25083871
pmcid: 4119298
doi: 10.1016/j.cell.2014.05.047
Kren B. T. et al. MicroRNAs identified in highly purified liver-derived mitochondria may play a role in apoptosis. RNA Biol. 6, 65–72 (2009).
pubmed: 19106625
doi: 10.4161/rna.6.1.7534
Koopman, W. J. H., Distelmaier, F., Smeitink, J. A. M. & Willems, P. H. G. M. OXPHOS mutations and neurodegeneration. EMBO J. 32, 9–29 (2013).
pubmed: 23149385
doi: 10.1038/emboj.2012.300
Ojala, D., Montoya, J. & Attardi, G. tRNA punctuation model of RNA processing in human mitochondria. Nature 290, 470–474 (1981).
pubmed: 7219536
doi: 10.1038/290470a0
Moulin, C., Caumont-Sarcos, A. & Ieva, R. Mitochondrial presequence import: Multiple regulatory knobs fine-tune mitochondrial biogenesis and homeostasis. Biochim. Biophys. Acta Mol. Cell Res. 1866, 930–944 (2019).
pubmed: 30802482
doi: 10.1016/j.bbamcr.2019.02.012
Jeandard D. et al. Import of non-coding RNAs into human mitochondria: a critical review and emerging approaches. Cells 8, 286 (2019).
pmcid: 6468882
doi: 10.3390/cells8030286
Isaac, R. S., McShane, E. & Churchman, L. S. The multiple levels of mitonuclear coregulation. Annu. Rev. Genet. 52, 511–533 (2018).
pubmed: 30230928
doi: 10.1146/annurev-genet-120417-031709
Schneider A. Unique aspects of mitochondrial biogenesis in trypanosomatids. Int. J. Parasitol. 31, 1403–1415 (2001).
pubmed: 11595226
doi: 10.1016/S0020-7519(01)00296-X
Schneider A. Mitochondrial tRNA import and its consequences for mitochondrial translation. Annu. Rev. Biochem. 80, 1033–1053 (2011).
pubmed: 21417719
doi: 10.1146/annurev-biochem-060109-092838
Holzmann J. et al. RNase P without RNA: identification and functional reconstitution of the human mitochondrial tRNA processing enzyme. Cell 135, 462–474 (2008).
pubmed: 18984158
doi: 10.1016/j.cell.2008.09.013
Brown A. et al. Structure of the large ribosomal subunit from human mitochondria. Science 346, 718–722 (2014).
pubmed: 25278503
pmcid: 4246062
doi: 10.1126/science.1258026
Kiss, T. & Filipowicz, W. Evidence against a mitochondrial location of the 7-2/MRP RNA in mammalian cells. Cell 70, 11–16 (1992).
pubmed: 1377982
doi: 10.1016/0092-8674(92)90528-K
Das S. et al. Nuclear miRNA regulates the mitochondrial genome in the heart. Circ. Res. 110, 1596–1603 (2012).
pubmed: 22518031
pmcid: 3390752
doi: 10.1161/CIRCRESAHA.112.267732
Mahapatra, S., Ghosh, T. & Adhya, S. Import of small RNAs into Leishmania mitochondria in vitro. Nucleic Acids Res. 22, 3381–3386 (1994).
pubmed: 8078774
pmcid: 523732
doi: 10.1093/nar/22.16.3381
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).
pubmed: 18587046
doi: 10.1073/pnas.0804283105
pmcid: 2453747
Hoogewijs K. et al. ClickIn: a flexible protocol for quantifying mitochondrial uptake of nucleobase derivatives. Interface Focus 7, 20160117 (2017).
pubmed: 28382203
pmcid: 5311907
doi: 10.1098/rsfs.2016.0117
Logan A. et al. Assessing the mitochondrial membrane potential in cells and in vivo using targeted click chemistry and mass spectrometry. Cell Metab. 23, 379–385 (2016).
pubmed: 26712463
pmcid: 4752821
doi: 10.1016/j.cmet.2015.11.014
Wang Y. et al. Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex. Nature 456, 921–926 (2008).
pubmed: 19092929
pmcid: 2765400
doi: 10.1038/nature07666
McKee, E. E., Ferguson, M., Bentley, A. T. & Marks, T. A. Inhibition of mammalian mitochondrial protein synthesis by oxazolidinones. Antimicrobial Agents Chemother. 50, 2042–2049 (2006).
doi: 10.1128/AAC.01411-05
Stroud D. A. et al. Accessory subunits are integral for assembly and function of human mitochondrial complex I. Nature 538, 123–126 (2016).
pubmed: 27626371
doi: 10.1038/nature19754
Kehrein K. et al. Organization of mitochondrial gene expression in two distinct ribosome-containing assemblies. Cell Rep. 10, 843–853 (2015).
pubmed: 25683707
doi: 10.1016/j.celrep.2015.01.012
Bogenhagen, D. F., Martin, D. W. & Koller, A. Initial steps in RNA processing and ribosome assembly occur at mitochondrial DNA nucleoids. Cell Metab. 19, 618–629 (2014).
pubmed: 24703694
doi: 10.1016/j.cmet.2014.03.013
Rackham O. et al. Hierarchical RNA processing is required for mitochondrial ribosome assembly. Cell Rep. 16, 1874–1890 (2016).
pubmed: 27498866
doi: 10.1016/j.celrep.2016.07.031
Taylor, R. W. & Turnbull, D. M. Mitochondrial DNA mutations in human disease. Nat. Rev. Genet. 6, 389–402 (2005).
pubmed: 15861210
pmcid: 1762815
doi: 10.1038/nrg1606
Gu J. et al. The architecture of the mammalian respirasome. Nature 537, 639–643 (2016).
pubmed: 27654917
doi: 10.1038/nature19359
Wu, M., Gu, J., Guo, R., Huang, Y. & Yang, M. Structure of mammalian respiratory supercomplex I1III2IV1. Cell 167, 1598–1609 (2016).
pubmed: 27912063
doi: 10.1016/j.cell.2016.11.012
Guo, R., Zong, S., Wu, M., Gu, J. & Yang, M. Architecture of human mitochondrial respiratory megacomplex I
pubmed: 28844695
doi: 10.1016/j.cell.2017.07.050
Tiranti V. et al. A novel frameshift mutation of the mtDNA COIII gene leads to impaired assembly of cytochrome c oxidase in a patient affected by Leigh-like syndrome. Hum. Mol. Genet. 9, 2733–2742 (2000).
pubmed: 11063732
doi: 10.1093/hmg/9.18.2733
Li Y. et al. An assembled complex IV maintains the stability and activity of complex I in mammalian mitochondria. J. Biol. Chem. 282, 17557–17562 (2007).
pubmed: 17452320
doi: 10.1074/jbc.M701056200
Rahman S. et al. A missense mutation of cytochrome oxidase subunit II causes defective assembly and myopathy. Am. J. Hum. Genet. 65, 1030–1039 (1999).
pubmed: 10486321
pmcid: 1288235
doi: 10.1086/302590
Hornig-Do H. T. et al. Nonsense mutations in the COX1 subunit impair the stability of respiratory chain complexes rather than their assembly. EMBO J. 31, 1293–1307 (2012).
pubmed: 22252130
pmcid: 3297988
doi: 10.1038/emboj.2011.477
Wang G. et al. PNPASE regulates RNA import into mitochondria. Cell 142, 456–467 (2010).
pubmed: 20691904
pmcid: 2921675
doi: 10.1016/j.cell.2010.06.035
Noh J. H. et al. HuR and GRSF1 modulate the nuclear export and mitochondrial localization of the lncRNA RMRP. Genes Dev. 30, 1224–1239 (2016).
pubmed: 27198227
pmcid: 4888842
doi: 10.1101/gad.276022.115
Wiedemann, N. & Pfanner, N. Mitochondrial machineries for protein import and assembly. Annu. Rev. Biochem. 86, 685–714 (2017).
pubmed: 28301740
doi: 10.1146/annurev-biochem-060815-014352
Schinzel A. C. et al. Cyclophilin D is a component of mitochondrial permeability transition and mediates neuronal cell death after focal cerebral ischemia. Proc. Natl. Acad. Sci. USA. 102, 12005–12010 (2005).
pubmed: 16103352
doi: 10.1073/pnas.0505294102
pmcid: 1189333
Halestrap A. P. What is the mitochondrial permeability transition pore? J. Mol. Cell Cardiol. 46, 821–831 (2009).
pubmed: 19265700
doi: 10.1016/j.yjmcc.2009.02.021
Dhir A. et al. Mitochondrial double-stranded RNA triggers antiviral signalling in humans. Nature 560, 238–242 (2018).
pubmed: 30046113
pmcid: 6570621
doi: 10.1038/s41586-018-0363-0
Li H. et al. MicroRNA-21 lowers blood pressure in spontaneous hypertensive rats by upregulating mitochondrial translation. Circulation 134, 734–751 (2016).
pubmed: 27542393
pmcid: 5515592
doi: 10.1161/CIRCULATIONAHA.116.023926
Frezza, C., Cipolat, S. & Scorrano, L. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat. Protoc. 2, 287–295 (2007).
pubmed: 17406588
doi: 10.1038/nprot.2006.478
Sasarman, F. & Shoubridge, E. A. Radioactive labeling of mitochondrial translation products in cultured cells. Methods Mol. Biol. 837, 207–217 (2012).
pubmed: 22215550
doi: 10.1007/978-1-61779-504-6_14
Leary S. C. Blue native polyacrylamide gel electrophoresis: a powerful diagnostic tool for the detection of assembly defects in the enzyme complexes of oxidative phosphorylation. Methods Mol. Biol. 837, 195–206 (2012).
pubmed: 22215549
doi: 10.1007/978-1-61779-504-6_13
Khvorostov, I., Zhang, J. & Teitell, M. Probing for mitochondrial complex activity in human embryonic stem cells. J. Vis. Exp. 17, 724 (2008).
Gao Y. et al. Mammalian elongation factor 4 regulates mitochondrial translation essential for spermatogenesis. Nat. Struct. Mol. Biol. 23, 441–449 (2016).
pubmed: 27065197
doi: 10.1038/nsmb.3206
Khacho M. et al. Mitochondrial dynamics impacts stem cell identity and fate decisions by regulating a nuclear transcriptional program. Cell Stem Cell 19, 232–247 (2016).
pubmed: 27237737
doi: 10.1016/j.stem.2016.04.015