Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria

Smirnov, Alexandre; Tarassov, Ivan; Mager-Heckel, Anne-Marie; Letzelter, Michel; Martin, Robert P.; Krasheninnikov, Igor A.; Entelis, Nina

doi:10.1261/rna.952208

Cited by 64 publications

(82 citation statements)

References 56 publications

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“…Two days after transfection, mitochondria were isolated and purified as described (Entelis et al 2001), and total and mitochondrial tRNAs were resolved in denaturing gels and analyzed by Northern hybridization with 59-32 P-labeled oligonucleotide probes against the 39-part of yeast tRNA CUU Lys (for tRK1 and small synthetic RNAs detection), human cytosolic tRNA Lys (to check possible cytosolic contamination in mtRNA preparation), and human mitochondrial tRNA Thr (Table 6). The import efficiency of RNAs was then estimated after quantification in a Typhoon-Trio scanner as a ratio between the signal obtained after hybridization with an anti-tRK1 probe and that obtained after hybridization with an oligonucleotide against the mitochondrial tRNA Thr in the mitochondrial RNA preparation, as described previously (Smirnov et al 2008). In order to compare import efficiencies of different RNAs, the total level of each RNA in transfected cells should be taken into account and normalized.…”

Section: Rna Import In Vivomentioning

confidence: 99%

Selection of RNA aptamers imported into yeast and human mitochondria

Kolesnikova¹,

Kazakova²,

Comte³

et al. 2010

RNA

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In the yeast Saccharomyces cerevisiae, nuclear DNA-encoded tRNA Lys CUU is partially imported into mitochondria. We previously found that the synthetic transcripts of yeast tRNA Lys and a number of their mutant versions could be specifically internalized by isolated yeast and human mitochondria. The mitochondrial targeting of tRNA Lys in yeast was shown to depend on the cytosolic precursor of mitochondrial lysyl-tRNA synthetase and the glycolytic enzyme enolase. Here we applied the approach of in vitro selection (SELEX) to broaden the spectrum of importable tRNA-derived molecules. We found that RNAs selected for their import into isolated yeast mitochondria have lost the potential to acquire a classical tRNA-shape. Analysis of conformational rearrangements in the importable RNAs by in-gel fluorescence resonance energy transfer (FRET) approach permitted us to suggest that protein factor binding and subsequent import require formation of an alternative structure, different from a classic L-form tRNA model. We show that in the complex with targeting protein factor, enolase 2, tRK1 adopts a particular conformation characterized by bringing together the 39-end and the TCC loop. This is a first evidence for implication of RNA secondary structure rearrangement in the mechanism of mitochondrial import selectivity. Based on these data, a set of small RNA molecules with significantly improved efficiency of import into yeast and human mitochondria was constructed, opening the possibility of creating a new mitochondrial vector system able to target therapeutic oligoribonucleotides into deficient human mitochondria.

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Section: Rna Import In Vivomentioning

confidence: 99%

Selection of RNA aptamers imported into yeast and human mitochondria

Kolesnikova¹,

Kazakova²,

Comte³

et al. 2010

RNA

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“…In our previous study, we identified structural elements of 5S rRNA serving as signals of its mitochondrial import, localized in the proximal part of helix I and in the loop D-helix IV region ( Fig. 1A; Smirnov et al 2008a). It had also been hypothesized that these regions correspond to binding sites with protein factors recruited to transport the RNA to the organelles.…”

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confidence: 94%

“…We show that human mitochondrial ribosomal protein L-18 (MRP-L18), a homolog of the Escherichia coli large ribosomal subunit protein L18, a member of a protein family L18/eL5 embracing the most conserved 5S rRNA-binding ribosomal proteins (Ban et The secondary structure of human 5S rRNA and its main functional sites (Smirnov et al 2008a(Smirnov et al ,b, 2010. ( , or containing deletion in the g-domain (as indicated below the panels).…”

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confidence: 99%

Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18

Smirnov

Entelis²,

Martin³

et al. 2011

Genes Dev.

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5S rRNA is an essential component of ribosomes of all living organisms, the only known exceptions being mitochondrial ribosomes of fungi, animals, and some protists. An intriguing situation distinguishes mammalian cells: Although the mitochondrial genome contains no 5S rRNA genes, abundant import of the nuclear DNA-encoded 5S rRNA into mitochondria was reported. Neither the detailed mechanism of this pathway nor its rationale was clarified to date. In this study, we describe an elegant molecular conveyor composed of a previously identified human 5S rRNA import factor, rhodanese, and mitochondrial ribosomal protein L18, thanks to which 5S rRNA molecules can be specifically withdrawn from the cytosolic pool and redirected to mitochondria, bypassing the classic nucleolar reimport pathway. Inside mitochondria, the cytosolic 5S rRNA is shown to be associated with mitochondrial ribosomes.

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“…In addition to in vitro evidence for a protein-only RNase P enzyme within mitochondria (9), the nucleus-encoded RNA components of RNase P and MRP enzymes are also imported into mitochondria and function in mitochondrial (mt)DNA replication and transcription (10)(11)(12). The 5S ribosomal RNA translocates into mitochondria, assembles with ribosomes (13,14), and may function in mtRNA translation, although the exact function of the 5S ribosomal RNA within mitochondria has yet to be firmly established. Finally, specific nucleus-encoded microRNAs have been isolated from mitochondria (7,15).…”

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Correcting human mitochondrial mutations with targeted RNA import

Wang¹,

Shimada²,

Zhang³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

120

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Mutations in the human mitochondrial genome are implicated in neuromuscular diseases, metabolic defects, and aging. An efficient and simple mechanism for neutralizing deleterious mitochondrial DNA (mtDNA) alterations has unfortunately remained elusive. Here, we report that a 20-ribonucleotide stem-loop sequence from the H1 RNA, the RNA component of the human RNase P enzyme, appended to a nonimported RNA directs the import of the resultant RNA fusion transcript into human mitochondria. The methodology is effective for both noncoding RNAs, such as tRNAs, and mRNAs. The RNA import component, polynucleotide phosphorylase (PNPASE), facilitates transfer of this hybrid RNA into the mitochondrial matrix. In addition, nucleus-encoded mRNAs for mitochondrial proteins, such as the mRNA of human mitochondrial ribosomal protein S12 (MRPS12), contain regulatory sequences in their 3′-untranslated region (UTR) that confers localization to the mitochondrial outer membrane, which is postulated to aid in protein translocation after translation. We show that for some mitochondrial-encoded transcripts, such as COX2, a 3′-UTR localization sequence is not required for mRNA import, whereas for corrective mitochondrial-encoded tRNAs, appending the 3′-UTR localization sequence was essential for efficient fusion-transcript translocation into mitochondria. In vivo, functional defects in mitochondrial RNA (mtRNA) translation and cell respiration were reversed in two human disease lines. Thus, this study indicates that a wide range of RNAs can be targeted to mitochondria by appending a targeting sequence that interacts with PNPASE, with or without a mitochondrial localization sequence, providing an exciting, general approach for overcoming mitochondrial genetic disorders.

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Two distinct structural elements of 5S rRNA are needed for its import into human mitochondria

Cited by 64 publications

References 56 publications

Selection of RNA aptamers imported into yeast and human mitochondria

Selection of RNA aptamers imported into yeast and human mitochondria

Biological significance of 5S rRNA import into human mitochondria: role of ribosomal protein MRP-L18

Correcting human mitochondrial mutations with targeted RNA import

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