Import of desired nucleic acid sequences using addressing motif of mitochondrial ribosomal 5S-rRNA for fluorescent in vivo hybridization of mitochondrial DNA and RNA
- PMID: 24562889
- DOI: 10.1007/s10863-014-9543-2
Import of desired nucleic acid sequences using addressing motif of mitochondrial ribosomal 5S-rRNA for fluorescent in vivo hybridization of mitochondrial DNA and RNA
Abstract
Based on the matrix-addressing sequence of mitochondrial ribosomal 5S-rRNA (termed MAM), which is naturally imported into mitochondria, we have constructed an import system for in vivo targeting of mitochondrial DNA (mtDNA) or mt-mRNA, in order to provide fluorescence hybridization of the desired sequences. Thus DNA oligonucleotides were constructed, containing the 5'-flanked T7 RNA polymerase promoter. After in vitro transcription and fluorescent labeling with Alexa Fluor(®) 488 or 647 dye, we obtained the fluorescent "L-ND5 probe" containing MAM and exemplar cargo, i.e., annealing sequence to a short portion of ND5 mRNA and to the light-strand mtDNA complementary to the heavy strand nd5 mt gene (5'-end 21 base pair sequence). For mitochondrial in vivo fluorescent hybridization, HepG2 cells were treated with dequalinium micelles, containing the fluorescent probes, bringing the probes proximally to the mitochondrial outer membrane and to the natural import system. A verification of import into the mitochondrial matrix of cultured HepG2 cells was provided by confocal microscopy colocalizations. Transfections using lipofectamine or probes without 5S-rRNA addressing MAM sequence or with MAM only were ineffective. Alternatively, the same DNA oligonucleotides with 5'-CACC overhang (substituting T7 promoter) were transcribed from the tetracycline-inducible pENTRH1/TO vector in human embryonic kidney T-REx®-293 cells, while mitochondrial matrix localization after import of the resulting unlabeled RNA was detected by PCR. The MAM-containing probe was then enriched by three-order of magnitude over the natural ND5 mRNA in the mitochondrial matrix. In conclusion, we present a proof-of-principle for mitochondrial in vivo hybridization and mitochondrial nucleic acid import.
Similar articles
-
Import of Fluorescent RNA into Mitochondria of Living Cells.Methods Mol Biol. 2016;1351:175-81. doi: 10.1007/978-1-4939-3040-1_13. Methods Mol Biol. 2016. PMID: 26530682
-
Identification of Neurospora mitochondrial promoters and analysis of synthesis of the mitochondrial small rRNA in wild-type and the promoter mutant [poky].J Biol Chem. 1990 Mar 15;265(8):4515-26. J Biol Chem. 1990. PMID: 1689728
-
Fluorescent in situ hybridization of mitochondrial DNA and RNA.Acta Biochim Pol. 2010;57(4):403-8. Epub 2010 Nov 29. Acta Biochim Pol. 2010. PMID: 21125028
-
ECHO probes: a concept of fluorescence control for practical nucleic acid sensing.Chem Soc Rev. 2011 Dec;40(12):5815-28. doi: 10.1039/c1cs15025a. Epub 2011 Jun 10. Chem Soc Rev. 2011. PMID: 21660343 Review.
-
Mitochondrion-targeted RNA therapies as a potential treatment strategy for mitochondrial diseases.Mol Ther Nucleic Acids. 2022 Oct 27;30:359-377. doi: 10.1016/j.omtn.2022.10.012. eCollection 2022 Dec 13. Mol Ther Nucleic Acids. 2022. PMID: 36420220 Free PMC article. Review.
Cited by
-
CoLoC-seq probes the global topology of organelle transcriptomes.Nucleic Acids Res. 2023 Feb 22;51(3):e16. doi: 10.1093/nar/gkac1183. Nucleic Acids Res. 2023. PMID: 36537202 Free PMC article.
-
Mitochondria Encoded Non-coding RNAs in Cell Physiology.Front Cell Dev Biol. 2021 Jul 30;9:713729. doi: 10.3389/fcell.2021.713729. eCollection 2021. Front Cell Dev Biol. 2021. PMID: 34395442 Free PMC article. Review.
-
Fluorescence In Vivo Hybridization (FIVH) for Detection of Helicobacter pylori Infection in a C57BL/6 Mouse Model.PLoS One. 2016 Feb 5;11(2):e0148353. doi: 10.1371/journal.pone.0148353. eCollection 2016. PLoS One. 2016. PMID: 26848853 Free PMC article.
-
Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner.PLoS One. 2018 Jun 18;13(6):e0199258. doi: 10.1371/journal.pone.0199258. eCollection 2018. PLoS One. 2018. PMID: 29912984 Free PMC article.
-
Import of Non-Coding RNAs into Human Mitochondria: A Critical Review and Emerging Approaches.Cells. 2019 Mar 26;8(3):286. doi: 10.3390/cells8030286. Cells. 2019. PMID: 30917553 Free PMC article. Review.
References
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources