In the yeast
Saccharomyces cerevisiae
, one of the two cytoplasmic lysine tRNAs, tRNA
CUU
Lys
, is partially associated with the mitochondrial matrix. Mitochondrial import of this tRNA requires ...binding to the precursor of the mitochondrial lysyl-tRNA synthetase, pre-MSK, and aminoacylation by the cytoplasmic lysyl-tRNA synthetase, KRS, appears to be a prerequisite for this binding. The second lysine isoacceptor tRNA
mnm
Lys
5
s
2
UUU
{where 5-(methylamino)-methyl-2-thiouridine is mnm
5
s
2
U} is exclusively localized in the cytoplasm. To study import determinants within the tRNA
CUU
Lys
molecule, we introduced a panel of replacements in the original sequences of the imported and nonimported lysine tRNAs that correspond to domains or individual residues that differ between these two isoacceptors. The mutant transcripts were tested for import, aminoacylation, and binding to pre-MSK. Import and aminoacylation efficiencies correlate well for the majority of mutant transcripts. However, some poorly aminoacylated transcripts were rather efficiently imported. Surprisingly, these transcripts retained binding capacity to pre-MSK. In fact, all imported transcripts retained pre-MSK binding capacity but nonimported versions did not, suggesting that this binding, rather than aminoacylation, is essential for import. Substitution of the anticodon arm of tRNA
CUU
Lys
with that of tRNA
mnm
Lys
5
s
2
UUU
abolished import without affecting aminoacylation. A version of tRNA
mnm
Lys
5
s
2
UUU
with an anticodon CUU was efficiently imported
in vitro
and was also found to be imported
in vivo
. This implies that the anticodon arm, especially position 34, is important for recognition by the import machinery. A nicked tRNA
CUU
Lys
transcript is still imported but its import requires reannealing of the two tRNA moieties, which implies that tRNA
CUU
Lys
is imported as a folded molecule.
In the yeast Saccharomyces cerevisiae, one of the two cytoplasmic lysine tRNAs, tRNACUU
Lys, is partially associated with the mitochondrial matrix. Mitochondrial import of this tRNA requires binding ...to the precursor of the mitochondrial lysyl-tRNA synthetase, pre-MSK, and amino-acylation by the cytoplasmic lysyl-tRNA synthetase, KRS, appears to be a prerequisite for this binding. The second lysine isoacceptor tRNAmnm5s2UUU
Lys{where 5-(methylamino)-methyl-2-thiouridine is mnm5s2U} is exclusively localized in the cytoplasm. To study import determinants within the tRNACUU
Lysmolecule, we introduced a panel of replacements in the original sequences of the imported and nonimported lysine tRNAs that correspond to domains or individual residues that differ between these two isoacceptors. The mutant transcripts were tested for import, aminoacylation, and binding to pre-MSK. Import and aminoacylation efficiencies correlate well for the majority of mutant transcripts. However, some poorly aminoacylated transcripts were rather efficiently imported. Surprisingly, these transcripts retained binding capacity to pre-MSK. In fact, all imported transcripts retained pre-MSK binding capacity but nonimported versions did not, suggesting that this binding, rather than aminoacylation, is essential for import. Substitution of the anticodon arm of tRNACUU
Lyswith that of tRNAmnm5s2UUU
Lysabolished import without affecting aminoacylation. A version of tRNAmnm5s2UUU
Lyswith an anticodon CUU was efficiently imported in vitro and was also found to be imported in vivo. This implies that the anticodon arm, especially position 34, is important for recognition by the import machinery. A nicked tRNACUU
Lystranscript is still imported but its import requires reannealing of the two tRNA moieties, which implies that tRNACUU
Lysis imported as a folded molecule.
Abstract Defects in mitochondrial DNA often cause neuromuscular pathologies, for which no efficient therapy has yet been developed. MtDNA targeting nucleic acids might therefore be promising ...therapeutic candidates. Nevertheless, mitochondrial gene therapy has never been achieved because DNA molecules can not penetrate inside mitochondria in vivo . In contrast, some small non-coding RNAs are imported into mitochondrial matrix, and we recently designed mitochondrial RNA vectors that can be used to address therapeutic oligoribonucleotides into human mitochondria. Here we describe an approach of carrier-free targeting of the mitochondrially importable RNA into living human cells. For this purpose, we developed the protocol of chemical synthesis of oligoribonucleotides conjugated with cholesterol residue through cleavable covalent bonds. Conjugates containing pH-triggered hydrazone bond were stable during the cell transfection procedure and rapidly cleaved in acidic endosomal cellular compartments. RNAs conjugated to cholesterol through a hydrazone bond were characterized by efficient carrier-free cellular uptake and partial co-localization with mitochondrial network. Moreover, the imported oligoribonucleotide designed to target a pathogenic point mutation in mitochondrial DNA was able to induce a decrease in the proportion of mutant mitochondrial genomes. This newly developed approach can be useful for a carrier-free delivery of therapeutic RNA into mitochondria of living human cells.
In human cell, a subset of small non-coding RNAs is imported into mitochondria from the cytosol. Analysis of the tRNA import pathway allowing targeting of the yeast tRNA(Lys)(CUU) into human ...mitochondria demonstrates a similarity between the RNA import mechanisms in yeast and human cells. We show that the cytosolic precursor of human mitochondrial lysyl-tRNA synthetase (preKARS2) interacts with the yeast tRNA(Lys)(CUU) and small artificial RNAs which contain the structural elements determining the tRNA mitochondrial import, and facilitates their internalization by isolated human mitochondria. The tRNA import efficiency increased upon addition of the glycolytic enzyme enolase, previously found to be an actor of the yeast RNA import machinery. Finally, the role of preKARS2 in the RNA mitochondrial import has been directly demonstrated in vivo, in cultured human cells transfected with the yeast tRNA and artificial importable RNA molecules, in combination with preKARS2 overexpression or downregulation by RNA interference. These findings suggest that the requirement of protein factors for the RNA mitochondrial targeting might be a conserved feature of the RNA import pathway in different organisms.
Can Mitochondrial DNA be CRISPRized: Pro and Contra Loutre, Romuald; Heckel, Anne‐Marie; Smirnova, Anna ...
IUBMB life,
December 2018, 2018-12-00, 20181201, 2018-12, Letnik:
70, Številka:
12
Journal Article
Abstract
CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5′-scaffold moiety and variable 3′-spacer moieties. Here, we demonstrate ...that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.
Editorial on the Research Topic Role of mitochondria-associated non-coding RNAs in intracellular communication Once considered solely as the powerhouse of eukaryotic cells, the mitochondrion is now ...known to be a hub for several other important cellular processes such as apoptosis, calcium homeostasis, regulation of innate immunity, amino acid metabolism, stem cell regulation and the cellular regulome, among others (Murphy and Steenbergen, 2011). Some of these functions are accounted for by the minimal circular 16,569-bp genome of human mitochondria (mtDNA), which contains the genes for 2 rRNAs, 22 tRNAs and 13 polypeptides (Chen et al., 2009). However, most mitochondrial proteins are nuclearencoded and imported from the cytosol (Neupert and Herrmann, 2007). Lately, there is increasing evidence of bidirectional communication between mitochondria and the nucleus, coordinating multiple cellular functions (Soledad et al., 2019). This connection, nonetheless, is not limited to mitochondrial import of nuclear-encoded proteins and export of metabolites, but has also been shown to involve noncoding RNAs (ncRNAs) connecting both cellular compartments (
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.
The yeast tRNACUU
Lys is transcribed from a nuclear gene and then unequally redistributed between the cytosol (97–98%) and mitochondria (2–3%). We have optimized the conditions for its specific ...import into isolated mitochondria. However, only a minor fraction (about 0.5%) of the added tRNA was translocated into the organelles. An in vitro transcript, once aminoacylated, appeared to be a better import substrate than the natural tRNA which carries modified nucleosides. The tRNA is translocated across mitochondrial membranes in its aminoacylated form and remains relatively stable inside the organelle. Possible roles of aminoacylation, tRNA‐protein interactions and nucleoside modification in subcellular partitioning of the tRNA are discussed.
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