RESUMO
Mitochondrial dysfunction underlies a large number of acute or progressive diseases, as well as aging. However, proposed therapies for mitochondrial mutations suffer from poor transformation of mitochondria with exogenous DNA, or lack of functionality of the transferred nucleic acid within the organelle. We show that a transfer RNA import complex (RIC) from the parasitic protozoon Leishmania tropica rapidly and efficiently delivered signal-tagged antisense (STAS) RNA or DNA to mitochondria of cultured human cells. STAS-induced specific degradation of the targeted mitochondrial mRNA, with downstream effects on respiration. These results reveal the existence of a novel small RNA-mediated mRNA degradation pathway in mammalian mitochondria, and suggest that RIC-mediated delivery could be used to target therapeutic RNAs to the organelle within intact cells.
Assuntos
Marcação de Genes , Mitocôndrias/genética , Estabilidade de RNA , RNA Antissenso/genética , RNA de Transferência de Tirosina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Tumoral , Respiração Celular , Humanos , Leishmania tropica/genética , Mitocôndrias/fisiologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Transporte de RNA , RNA Antissenso/química , RNA Antissenso/fisiologia , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , RNA de Protozoário/fisiologia , RNA de Transferência de Tirosina/isolamento & purificação , RNA de Transferência de Tirosina/fisiologiaRESUMO
The mitochondrial genome of trypanosomes, unlike that of most other eukaryotes, does not appear to encode any tRNAs. Therefore, mitochondrial tRNAs must be either imported into the organelle or created through a novel mitochondrial process, such as RNA editing. Trypanosomal tRNA(Tyr), whose gene contains an 11-nucleotide intron, is present in both the cytosol and the mitochondrion and is encoded by a single-copy nuclear gene. By site-directed mutagenesis, point mutations were introduced into this tRNA gene, and the mutated gene was reintroduced into the trypanosomal nuclear genome by DNA transfection. Expression of the mutant tRNA led to the accumulation of unspliced tRNA(Tyr) (A. Schneider, K. P. McNally, and N. Agabian, J. Biol. Chem. 268:21868-21874, 1993). Cell fractionation revealed that a significant portion of the unspliced mutant tRNA(Tyr) was recovered in the mitochondrial fraction and was resistant to micrococcal nuclease treatment in the intact organelle. Expression of the nuclear integrated, mutated tRNA gene and recovery of its gene product in the mitochondrial fraction directly demonstrated import. In vitro experiments showed that the unspliced mutant tRNA(Tyr), in contrast to the spliced wild-type form, was no longer a substrate for the cognate aminoacyl synthetase. The presence of uncharged tRNA in the mitochondria demonstrated that aminoacylation was not coupled to import.
Assuntos
Núcleo Celular/metabolismo , Mitocôndrias/metabolismo , Splicing de RNA , RNA de Protozoário/biossíntese , RNA de Transferência de Tirosina/biossíntese , Trypanosoma brucei brucei/metabolismo , Animais , Sequência de Bases , Northern Blotting , Linhagem Celular Transformada , Citosol/metabolismo , Íntrons , Dados de Sequência Molecular , Peso Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Mutação Puntual , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , RNA de Transferência de Tirosina/genética , RNA de Transferência de Tirosina/isolamento & purificação , Transfecção , Trypanosoma brucei brucei/genéticaRESUMO
During isolation of total ribonucleic acids from white lupin (Lupinus albus) and their subsequent separation by 10% polyacrylamide gel electrophoresis, a fast migrating RNA band is very well separated. The nucleotide sequence analysis of 76 nucleotide long sequence with many modified nucleosides was found to be identical with that of tyrosine specific tRNA of yellow lupin seeds (Lupinus luteus) and wheat germ (Triticum aestivum). Also this tRNA(Tyr) is identical with plant amber suppressor tRNA. The presented approach offers a very rapid method of purification of plant tRNA with UAG suppressor activity.
