ABSTRACT
Mitochondrial genomes of many eukaryotic organisms do not code for the full tRNA set necessary for organellar translation. Missing tRNA species are imported from the cytosol. In particular, one out of two cytosolic lysine tRNAs of the yeast Saccharomyces cerevisiae is partially internalized by mitochondria. The key protein factor of this process is the precursor of mitochondrial lysyl-tRNA synthetase, preMsk1p. In this work, we show that recombinant preMsk1p purified from E. coli in native conditions, when used in an in vitro tRNA import system, demonstrates some properties different from those shown by the renatured protein purified from E. coli in the denatured state. We also discuss the possible mechanistic reasons for this phenomenon.
Subject(s)
Lysine-tRNA Ligase , Mitochondria , Mitochondrial Proteins , RNA, Fungal , RNA, Transfer, Lys , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Biological Transport, Active , Escherichia coli/genetics , Escherichia coli/metabolism , Lysine-tRNA Ligase/chemistry , Lysine-tRNA Ligase/genetics , Lysine-tRNA Ligase/isolation & purification , Lysine-tRNA Ligase/metabolism , Mitochondria/chemistry , Mitochondria/genetics , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/genetics , Mitochondrial Proteins/isolation & purification , Mitochondrial Proteins/metabolism , RNA, Fungal/chemistry , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Transfer, Lys/chemistry , RNA, Transfer, Lys/genetics , RNA, Transfer, Lys/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/isolation & purification , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Small non-coding RNAs are today a topic of great interest for molecular biologists because they can be regarded as relicts of a hypothetical "RNA world" which, apparently, preceded the modern stage of organic evolution on Earth. The small molecule of 5S rRNA (approximately 120 nucleotides) is a component of large ribosomal subunits of all living beings (5S rRNAs are not found only in mitoribosomes of fungi and metazoans). This molecule interacts with various protein factors and 23S (28S) rRNA. This review contains the accumulated data to date concerning 5S rRNA structure, interactions with other biological macromolecules, intracellular traffic, and functions in the cell.
Subject(s)
Macromolecular Substances/metabolism , RNA, Ribosomal, 5S/chemistry , RNA, Ribosomal, 5S/metabolism , Animals , Base Sequence , Humans , Mitochondria/metabolism , Proteins/metabolism , RNA, Ribosomal, 23S/chemistry , RNA, Ribosomal, 23S/genetics , RNA, Ribosomal, 23S/metabolism , RNA, Ribosomal, 5S/geneticsABSTRACT
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.
Subject(s)
Mitochondria/metabolism , RNA, Ribosomal, 5S/metabolism , RNA, Transfer, Lys/metabolism , Amino Acid Sequence , Base Sequence , Biological Transport , DNA Primers , Humans , In Vitro Techniques , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Ribosomal, 5S/chemistry , RNA, Transfer, Lys/chemistry , Saccharomyces cerevisiae/metabolism , Sequence Homology, Amino AcidABSTRACT
Mitochondria, though 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. This targeting, if not universal, is widely spread among species. The origin and the mechanisms of RNA import seem to differ from one system to another and striking differences are observed even in closely related species. We describe data concerning the various experimental systems of studying RNA import with emphasis on the model of the yeast Saccharomyces cerevisiae, which was studied in our laboratory. We compare various requirements of RNA import into mitochondria in different species and demonstrate that this pathway can be transferred from yeast to human cells, in which tRNAs normally are not imported. We speculate on the possibility to use RNA import for biomedical purposes.
Subject(s)
Intracellular Membranes/metabolism , Mitochondria/metabolism , RNA/metabolism , Animals , Humans , Intracellular Membranes/drug effects , Mitochondria/drug effects , Mitochondria/genetics , Mitochondrial Myopathies/drug therapy , Mitochondrial Myopathies/genetics , Mitochondrial Myopathies/metabolism , Mutation/drug effects , Mutation/physiology , Plants/drug effects , Plants/metabolism , RNA/administration & dosage , RNA/genetics , RNA, Mitochondrial , RNA, Transfer/administration & dosage , RNA, Transfer/genetics , RNA, Transfer/metabolism , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/metabolism , Translocation, Genetic/drug effects , Translocation, Genetic/physiologyABSTRACT
Mitochondrial import of a cytoplasmic transfer RNA (tRNA) in yeast requires the preprotein import machinery and cytosolic factors. We investigated whether the tRNA import pathway can be used to correct respiratory deficiencies due to mutations in the mitochondrial DNA and whether this system can be transferred into human cells. We show that cytoplasmic tRNAs with altered aminoacylation identity can be specifically targeted to the mitochondria and participate in mitochondrial translation. We also show that human mitochondria, which do not normally import tRNAs, are able to internalize yeast tRNA derivatives in vitro and that this import requires an essential yeast import factor.
Subject(s)
DNA, Mitochondrial/metabolism , Mitochondria/metabolism , Acylation , Base Sequence , Biological Transport , Cytoplasm/metabolism , DNA, Mitochondrial/genetics , Genes, Fungal , Humans , In Vitro Techniques , Molecular Sequence Data , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Suppression, GeneticABSTRACT
Yeast tRNA(Lys)CUU is nucleus-encoded and is partially imported into the mitochondria. Another lysine isoacceptor, tRNA(Lys)SUU, is also nucleus-encoded but is not imported. These two tRNAs differ in 21 bases. We have previously localised import selectivity determinants in the anticodon arm. By in vitro import of mutant transcripts and by expression of mutant tRNA genes in vivo we show here that the first base pair (1:72) and the discriminator base 73 are also relevant to import selectivity. Replacement of bases 1:72 in tRNA(Lys)SUU by those of tRNA(Lys)CUU makes it importable with a transport efficiency similar to natural.
Subject(s)
Mitochondria/metabolism , Mutation , RNA, Transfer, Lys/metabolism , Saccharomyces cerevisiae/genetics , Anticodon/genetics , Anticodon/metabolism , Base Pairing/genetics , Base Pairing/physiology , Base Sequence , Cell Nucleus/genetics , Cell Nucleus/metabolism , Mitochondria/genetics , Nucleic Acid Conformation , RNA, Transfer, Lys/chemistry , RNA, Transfer, Lys/genetics , Saccharomyces cerevisiae/metabolismABSTRACT
In the yeast Saccharomyces cerevisiae, one of the two cytoplasmic lysine tRNAs, tRNACUULys, 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 tRNAmnmLys5s2UUU [where 5-[(methylamino)-methyl]-2-thiouridine is mnm5s2U] is exclusively localized in the cytoplasm. To study import determinants within the tRNACUULys 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 tRNACUULys with that of tRNAmnmLys5s2UUU abolished import without affecting aminoacylation. A version of tRNAmnmLys5s2UUU 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 tRNACUULys transcript is still imported but its import requires reannealing of the two tRNA moieties, which implies that tRNACUULys is imported as a folded molecule.
Subject(s)
Mitochondria/metabolism , RNA, Fungal/metabolism , RNA, Transfer, Lys/metabolism , Anticodon , Base Sequence , Biological Transport , Cytoplasm/enzymology , Genetic Variation , Lysine-tRNA Ligase/metabolism , Molecular Sequence Data , Mutation , Protein Binding , Protein Precursors/metabolism , RNA Precursors/metabolism , RNA, Fungal/genetics , RNA, Transfer, Lys/genetics , Saccharomyces cerevisiae , Structure-Activity RelationshipABSTRACT
The yeast tRNA(CUU)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.
Subject(s)
Mitochondria/metabolism , RNA, Transfer, Lys/metabolism , Saccharomyces cerevisiae/metabolism , Cell Fractionation , Cell Nucleus/metabolism , Cytosol/metabolism , Fungal Proteins/metabolism , Intracellular Membranes/metabolism , Kinetics , RNA Processing, Post-Transcriptional , RNA, Transfer, Lys/biosynthesis , Transcription, GeneticABSTRACT
Mitochondrial import of tRNA is now considered as a quasi-universal phenomenon. In the yeast Saccharomyces cerevisiae, one of the three lysine isoacceptors, the tRNA(Lys)1 with the anticodon CUU (tRNA-K1), is encoded by the nuclear genome and distributed between the cytoplasmic (> 95%) and mitochondrial (< 5%) compartments. In vivo and in vitro import assays were developed to study the mechanisms of tRNA-K1 mitochondrial import. Transmembrane translocation of the tRNA requires the intactness of at least two of the components of the mitochondrial import machinery of pre-proteins, MOM19 and MIM44, as well as energy of ATP hydrolysis and an electrochemical potential across the inner membrane. The import of tRNA-K1 involves formation of an RNP complex on the mitochondrial outer membrane. tRNA-K1 import is also dependent upon cytosolic protein factors, one of which was identified as the precursor of the mitochondrial lysyl-tRNA synthetase (MSK). Although essential for tRNA-K1 import in vitro and in vivo, pre-MSK is however not sufficient to direct the import in vitro, which suggests the need of additional cytosolic factor(s). The tRNA can be imported in its mature form and nucleoside modification is not essential. Aminoacylation of the imported tRNA by the cytoplasmic lysyl-tRNA synthetase is a prerequisite for import. Possible mechanisms of intracellular partitioning and mitochondrial membrane translocation of tRNA-K1 are discussed.
Subject(s)
Mitochondria/metabolism , RNA, Transfer, Lys/metabolism , Saccharomyces cerevisiae/metabolism , Amino Acyl-tRNA Synthetases/metabolism , Base Sequence , Biological Transport , Electrophoresis, Gel, Two-Dimensional , Membrane Proteins/metabolism , Models, Biological , Molecular Sequence Data , Nucleic Acid Conformation , Ribonucleoproteins/chemistryABSTRACT
The cytoplasmic tRNA(Lys)(CUU) (tRNA(1Lys)) is the single yeast tRNA species to be traffiked from the cytoplasm into the mitochondrial compartment of the cell. To study mechanisms of this targetting we worked out two test systems. The in vivo system based on the electroporation of intact yeast cells was used to introduce labelled tRNAs into the cytoplasm. All tRNA species tested were effectively introduced into the cytoplasm, but only the cytoplasmic tRNA(1Lys) was found in the mitochondrial compartment within 1-2 hours after the electroporation procedure. The in vitro system permits specific transfer of the tRNA(1Lys) into isolated mitochondria. Contrary to the known systems for protein transport into isolated mitochondria, mitochondrial import of tRNA(1Lys) in vitro requires the presence of soluble cellular proteins in the reaction mixture. The translocation proved to be ATP-dependent and to require the presence of an ATP-generation system in the reaction. Preincubation of the tRNA with the total cellular extract of the cell markedly increases the rate of the translocation. Two protein fractions are necessary to direct the import in vitro. The first one has high heparin-binding affinity, while the other protein fraction is not retained by heparin-Sepharose.
