Mitochondrial import of a yeast cytoplasmic tRNA<sup>Lys</sup>: possible roles of aminoacylation and modified nucleosides in subcellular partitioning

Entelis, Nina; Krasheninnikov, Igor A.; Martin, Robert P.; Tarassov, Ivan

doi:10.1016/0014-5793(96)00259-1

Cited by 33 publications

(15 citation statements)

References 23 publications

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“…Although these data support some version of the synthetase model for this tRNA, it should be noted that this tRNA is the only imported tRNA in yeast. Furthermore, it does not appear to function in mitochondrial translation: It is not a substrate for the mitochondrial lysyltRNA synthetase; the yeast mitochondrial genome encodes a lysine tRNA that can decode a similar set of lysine codons; and the acylated form of this molecule is stable in isolated yeast mitochondria (Martin et al 1977(Martin et al , 1979Tarassov et al 1995;Entelis et al 1996). Therefore, the conclusions drawn from these data regarding the role of synthetases in tRNA import may not be generalizable to the import of multiple tRNAs for use in mitochondrial translation.…”

Section: The Potential Role Of Aminoacyl Trna Synthetases In Trna Importmentioning

confidence: 97%

The anticodon is the signal sequence for mitochondrial import of glutamine tRNA in Tetrahymena.

Rusconi¹,

Cech²

1996

Genes Dev.

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The import of nuclear-encoded RNAs into mitochondria is required for proper mitochondrial function in most organisms. However, the mechanisms used to achieve RNA import are largely unknown. In particular, the RNA elements that direct import have not been identified in any organism. In Tetrahymena, only one of three nuclear-encoded glutamine accepting tRNAs is imported into mitochondria. We transform Tetrahymena with marked glutamine tRNAs and quantitate their level of accumulation in mitochondria. Of several isostructural nucleotide substitutions tested, alteration of the anticodon sequence uniquely abolishes import. Furthermore, substitution of a single anticodon nucleotide (UUA-,UUG) confers import on a normally nonimported glutamine tRNA. Thus, the anticodon functions as a mitochondrial localization signal and is both necessary and sufficient for tRNA import. Given the prior evidence that neither the cytoplasmic nor the mitochondrial glutaminyl-tRNA synthetase distinguishes between the imported and nonimported glutamine tRNAs with respect to aminoacylation, we propose that some mitochondrial import factor distinct from a synthetase recognizes the anticodon of the imported glutamine tRNA.

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Section: The Potential Role Of Aminoacyl Trna Synthetases In Trna Importmentioning

confidence: 97%

The anticodon is the signal sequence for mitochondrial import of glutamine tRNA in Tetrahymena.

Rusconi¹,

Cech²

1996

Genes Dev.

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“…The molecular mechanism of discrimination between imported tRK1 and all the other cytosolic tRNAs was still unknown. Using mutagenesis followed by in vitro and in vivo import assays, we have previously found that not only is tRK1 imported into yeast mitochondria, but also several of its mutant versions, mutants of the second cytosolic lysine isoacceptor tRNA, normally nonimported tRNA Lys UÃUU (tRK2), and even a synthetic transcript of the mitochondrially localized tRNA Lys UÃUU (tRK3) (Entelis et al 1996(Entelis et al , 1998Kolesnikova et al 2002). Systematic analysis of the sequences of imported versions permitted us to discern the determinants (the first base pair G1-C72 and U73 in the acceptor stem and C34 in the anticodon) and an anti-determinant (modified U34) that controls mitochondrial import specificity (Fig.…”

Section: Introductionmentioning

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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“…The import assay was carried out at 30°C for 20 min, which corresponds to the logarithmic phase of RNA uptake (42). After treatment with a mixture of nucleases (20 units/ml micrococcal nuclease, 50 g/ml RNase A, and 25 units/ml phosphodiesterase), mitoplasts were generated by treatment with digitonin, at 100 g/1 mg of mitoprotein.…”

Section: Methodsmentioning

confidence: 99%

“…For Northern hybridization (23,42), the following 5Ј end-32 P-labeled probes were used: human U3 snRNA, CGCTACCTCTCTTCCTCGTG-GTTTTCGGTGCTCTACA; human cytoplasmic tRNA Met , TGGTAGCA-GAGGATGGTTTCG; human mitochondrial tRNA Cys , AAGCCCCG-GCAGGTTTGAAG; human 5 S rRNA, CCCGACGTTGCTTAACTTC.…”

Section: Methodsmentioning

confidence: 99%

“…HmIDPs supplemented with pre-MSK were found to direct internalization of tRK1 (24). In these previous experiments, HmIDPs were isolated by the same procedure as ScIDPs, directing tRK1 import into isolated yeast mitochondria (42,60). We tested here if we could direct tRK1 into isolated human mitochondria without addition of pre-MSK by changing the conditions of HmIDPs isolation (Fig.…”

Section: Human Proteins Can Direct Import Of a Yeast Trna Lys Into Humentioning

confidence: 99%

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5 S rRNA and tRNA Import into Human Mitochondria

Entelis¹,

Kolesnikova²,

Doğan³

et al. 2001

Journal of Biological Chemistry

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102

101

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In vivo, human mitochondria import 5 S rRNA and do not import tRNAs from the cytoplasm. We demonstrated previously that isolated human mitochondria are able to internalize a yeast tRNA Lys in the presence of yeast soluble factors. Here, we describe an assay for specific uptake of 5 S rRNA by isolated human mitochondria and compare its requirements with the artificial tRNA import. The efficiency of 5 S rRNA uptake by isolated mitochondria was comparable with that found in vivo. The import was shown to depend on ATP and the transmembrane electrochemical potential and was directed by soluble proteins. Blocking the pre-protein import channel inhibited internalization of both 5 S rRNA and tRNA, which suggests this apparatus be involved in RNA uptake by the mitochondria. We show that human mitochondria can also selectively internalize several in vitro synthesized versions of yeast tRNA Lys as well as a transcript of the human mitochondrial tRNA Lys . Either yeast or human soluble proteins can direct this import, suggesting that human cells possess all factors needed for such an artificial translocation. On the other hand, the efficiency of import directed by yeast or human protein factors varies significantly, depending on the tRNA version. Similarly to the yeast system, tRNA Lys import into human mitochondria depended on aminoacylation and on the precursor of the mitochondrial lysyl-tRNA synthetase. 5 S rRNA import was also dependent upon soluble protein(s), which were distinct from the factors providing tRNA internalization.Mitochondria, although containing their own genome, import the vast majority of their macromolecular components from the cytoplasm. If the mechanisms of pre-protein import are well understood, the import of nuclear-coded RNAs into mitochondria was investigated to a much lesser extent. Targeting of RNA into mitochondria though not universal is widely spread among organisms (1-5). Mitochondrial import of transfer RNAs was found in plants, protists, some lower animals, and fungi. The number of imported tRNA species varies from one (in yeast) to the totality (in trypanosomatids), and tRNA import mechanisms seem to differ from one organism to another. We have shown previously that, in the yeast Saccharomyces cerevisiae, import of a single tRNA CUU Lys (further referred to as tRK1) 1 occurs via formation of a complex with the precursor form of the mitochondrial lysyl-tRNA synthetase (pre-MSK) and requires the intactness of pre-protein import apparatus (6 -8).In mammalians, no tRNA import has been reported, but several other RNAs are thought to be targeted into mitochondria. One of these is the RNA component of RNase MRP, a site-specific endoribonuclease supposed to be involved in primer RNA cleavage during replication of mitochondrial DNA and to be present in the organelle in a very low amount (9). It was hypothesized that the process of mitochondrial DNA replication requires a very low number of MRP RNA molecules per mitochondrial genome (10 -12). The presence of MRP RNA in the mitochondria was recent...

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Mitochondrial import of a yeast cytoplasmic tRNA^Lys: possible roles of aminoacylation and modified nucleosides in subcellular partitioning

Cited by 33 publications

References 23 publications

The anticodon is the signal sequence for mitochondrial import of glutamine tRNA in Tetrahymena.

The anticodon is the signal sequence for mitochondrial import of glutamine tRNA in Tetrahymena.

Selection of RNA aptamers imported into yeast and human mitochondria

5 S rRNA and tRNA Import into Human Mitochondria

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Mitochondrial import of a yeast cytoplasmic tRNALys: possible roles of aminoacylation and modified nucleosides in subcellular partitioning

Cited by 33 publications

References 23 publications

The anticodon is the signal sequence for mitochondrial import of glutamine tRNA in Tetrahymena.

The anticodon is the signal sequence for mitochondrial import of glutamine tRNA in Tetrahymena.

Selection of RNA aptamers imported into yeast and human mitochondria

5 S rRNA and tRNA Import into Human Mitochondria

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Product

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Mitochondrial import of a yeast cytoplasmic tRNA^Lys: possible roles of aminoacylation and modified nucleosides in subcellular partitioning