Recent advances in tRNA mitochondrial import

doi:10.1016/j.tibs.2008.04.010

Trends in Biochemical Sciences

Volume 33, Issue 7, July 2008, Pages 320-329

https://doi.org/10.1016/j.tibs.2008.04.010 Get rights and content

In many eukaryotes, tRNA import from the cytosol into mitochondria is essential for mitochondrial biogenesis and, consequently, for cell viability. Recent work has begun to unravel the molecular mechanisms involved in tRNA transport in yeast, trypanosomatids and plants. The mechanisms of tRNA targeting to, and translocation through, the double mitochondrial membrane in addition to how selectivity and regulation of these processes are achieved are the main questions that have been addressed. The characterization of both direct and co-import mechanisms involving distinct protein-import factors is in agreement with a polyphyletic origin of tRNA import. Moreover, our increased understanding of the tRNA-import pathway has been exploited recently to rescue dysfunctions associated with mitochondrial tRNA mutations.

Section snippets

tRNA transport into mitochondria

Mitochondria, organelles present in most eukaryotic cells, function in a variety of essential cellular processes including respiration, oxidative phosphorylation-mediated ATP production, cellular metabolism and apoptosis. They contain their own genome that encodes a limited number of macromolecules. Consequently, the majority of mitochondrial proteins are encoded in the nucleus and imported into the organelle (Box 1). In comparison to proteins, a low number of RNAs are imported into

Why must mitochondria import tRNAs?

In contrast to the human mitochondrial genome, which contains a minimal but complete set of tRNA genes, the number of tRNA-encoding genes in numerous mitochondrial genomes is insufficient for proper protein synthesis to occur. For example, protozoa such as Trypanosoma brucei and Leishmania tarentolae represent the most extreme situation because their mitochondrial genomes are completely devoid of tRNA genes. In the fungus Spizellomyces punctatus, only eight mitochondrially encoded tRNA genes

Divergent mechanisms for a convergent goal

As described, mitochondrial tRNA import is, in general, essential for mitochondrial protein synthesis; however, the ways to achieve this goal are clearly different. Elucidating the mechanisms for this process requires that several questions are answered (Box 2). What factors determine tRNA-import selectivity? How are tRNAs targeted from the nucleus to the mitochondrial surface? What protein factors constitute tRNA mitochondrial membrane import channels? Is the extent of tRNA import regulated?

An attractive tool for correcting disease-causing mitochondrial tRNA gene mutations

Mitochondrial DNA mutations are associated with many human diseases; more than half of these mutations are located within mitochondrial tRNA genes. The best characterized are MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and MERFF (myoclonic epilepsy and red ragged fibers) syndromes. The most prevalent point mutations associated with MELAS and MERFF syndrome are in mitochondrial trnL (Ala3243→Gly mutation) and trnK (Ala8344→Gly mutation), respectively

Concluding remarks and future perspectives

Several advances in the past several years have bolstered our understanding of how nuclear-encoded tRNAs are imported into the mitochondria of different organisms. It is remarkable to note that each organism recruits distinct housekeeping proteins to direct mitochondrial import 10, 27, 37, 45. Although the overall process of mitochondrial protein import is conserved 58, 59, it is tempting to speculate that divergent mechanisms for tRNA import have emerged to reach a convergent evolutionary

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Mitochondria possess transport mechanisms for import of RNA and DNA. Based on import into isolated Solanum tuberosum mitochondria in the presence of competitors, inhibitors or effectors, we show that DNA fragments of different size classes are taken up into plant organelles through distinct channels. Alternative channels can also be activated according to the amount of DNA substrate of a given size class. Analyses of Arabidopsis thaliana knockout lines pointed out a differential involvement of individual voltage-dependent anion channel (VDAC) isoforms in the formation of alternative channels. We propose several outer and inner membrane proteins as VDAC partners in these pathways.
Role of the translocase of the mitochondrial inner membrane in the import of tRNAs into mitochondria in Trypanosoma brucei
2020, Gene
Trypanosomatids are unicellular parasitic protozoa. Many of the species of this genera cause severe diseases in human, such as Leishmaniasis, African trypanosomiasis and Chagas disease. These parasites possess a single reticular mitochondrion with a concatenated structure of mitochondrial DNA known as kinetoplast or kDNA. kDNA encodes few essential mitochondrial proteins but no tRNAs. Therefore, trypanosomatid mitochondrion import a full set of nucleus-encoded tRNAs for mitochondrial translation. Recent advances indicated that mitochondrial protein translocases, particularly the subunits of the ATOM complex, are involved in the import of a tRNA in Trypanosoma brucei. However, the global picture and the role of the translocase components of the mitochondrial inner membrane (TbTims) are not well understood. Here we investigated the relative abundance of 16 different tRNAs in the cytosolic and mitochondrial fractions isolated from the six TbTims knockdown cell lines. We found that knockdown of TbTim17, one of the primary components of the TbTIM complex, reduced the abundance of all of these tRNAs into mitochondria and increased their abundance in the cytosol. Depletion of TbTim62, a TbTim17 associated proteins, also reduced the relative abundance of most of these tRNAs into mitochondria except for tRNA^leu, tRNA^met, and tRNA^glu. Whereas, knockdown of other TbTims, like TbTim50 and two small TbTims, TbTim10 and TbTim8/13, didn’t have any effect on tRNA abundance either in the cytosol or mitochondria. Depletion of any of these TbTims showed minimal effect on the levels of total tRNAs in T. brucei. Absolute quantification of tRNA levels revealed that TbTim17 knockdown reduced the levels of different tRNAs in mitochondria from 3–6% to 0.8–1.4%, which is equivalent to ~70% reduction in average. Whereas, TbTim62 depletion showed somewhat selective effect. Overall, our results suggest that TbTim17 and TbTim62 are essential for tRNA import that further makes a connection between the tRNA and protein import into mitochondria in T. brucei.
Validation of Gene Therapy for Mutant Mitochondria by Delivering Mitochondrial RNA Using a MITO-Porter
2020, Molecular Therapy Nucleic Acids
Here, we report on validating a mitochondrial gene therapy by delivering nucleic acids to mitochondria of diseased cells by a MITO-Porter, a liposome-based carrier for mitochondrial delivery. We used cells derived from a patient with a mitochondrial disease with a G625A heteroplasmic mutation in the tRNA^Phe of the mitochondrial DNA (mtDNA). It has been reported that some mitochondrial gene diseases are caused by heteroplasmic mutations, in which both mutated and wild-type (WT) genes are present, and the accumulation of pathological mutations leads to serious, intractable, multi-organ diseases. Therefore, the decrease of the mutated gene rate is considered to be a useful gene therapy strategy. To accomplish this, wild-type mitochondrial pre-tRNA^Phe (pre-WT-tRNA^Phe), prepared by in vitro transcription, was encapsulated in the MITO-Porter. The pre-WT-tRNA^Phe encapsulated in the MITO-Porter was transfected into diseased mitochondrial cells, and the resulting mutant levels were examined by an amplification refractory mutation system (ARMS)-quantitative PCR. The mutation rate of tRNA^Phe was decreased, and this therapeutic effect was sustained even on the 8th day after transfection. Furthermore, mitochondrial respiratory activity of the disease cells was increased after the transfection of therapeutic pre-WT-tRNA^Phe. These results support the conclusion that the mitochondrial delivery of therapeutic nucleic acids represents a viable strategy for mitochondrial gene therapy.
The single CCA-adding enzyme of T. Brucei has distinct functions in the cytosol and in mitochondria
2020, Journal of Biological Chemistry
Citation Excerpt :
Thus, all mitochondrial tRNAs must be imported from the cytosol. Mitochondrial tRNA import is not restricted to trypanosomes but has been found in many organisms including some fungi, plants, and protozoans (2, 5–7). But in essentially all other systems, at least a subset of tRNAs is still encoded in the mitochondrial genome.
tRNAs universally carry a CCA nucleotide triplet at their 3′-ends. In eukaryotes, the CCA is added post-transcriptionally by the CCA-adding enzyme (CAE). The mitochondrion of the parasitic protozoan Trypanosoma brucei lacks tRNA genes and therefore imports all of its tRNAs from the cytosol. This has generated interest in the tRNA modifications and their distribution in this organism, including how CCA is added to tRNAs. Here, using a BLAST search for genes encoding putative CAE proteins in T. brucei, we identified a single ORF, Tb927.9.8780, as a potential candidate. Knockdown of this putative protein, termed TbCAE, resulted in the accumulation of truncated tRNAs, abolished translation, and inhibited both total and mitochondrial CCA-adding activities, indicating that TbCAE is located both in the cytosol and mitochondrion. However, mitochondrially localized tRNAs were much less affected by the TbCAE ablation than the other tRNAs. Complementation assays revealed that the N-terminal 10 amino acids of TbCAE are dispensable for its activity and mitochondrial localization and that deletion of 10 further amino acids abolishes both. A growth arrest caused by the TbCAE knockdown was rescued by the expression of the cytosolic isoform of yeast CAE, even though it was not imported into mitochondria. This finding indicated that the yeast enzyme complements the essential function of TbCAE by adding CCA to the primary tRNA transcripts. Of note, ablation of the mitochondrial TbCAE activity, which likely has a repair function, only marginally affected growth.
Interchangeable parts: The evolutionarily dynamic tRNA population in plant mitochondria
2020, Mitochondrion
Transfer RNAs (tRNAs) remain one of the very few classes of genes still encoded in the mitochondrial genome. These key components of the protein translation system must interact with a large enzymatic network of nuclear-encoded gene products to maintain mitochondrial function. Plants have an evolutionarily dynamic mitochondrial tRNA population, including ongoing tRNA gene loss and replacement by both horizontal gene transfer from diverse sources and import of nuclear-expressed tRNAs from the cytosol. Thus, plant mitochondria represent an excellent model for understanding how anciently divergent genes can act as “interchangeable parts” during the evolution of complex molecular systems. In particular, understanding the integration of the mitochondrial translation system with elements of the corresponding machinery used in cytosolic protein synthesis is a key area for eukaryotic cellular evolution. Here, we review the increasingly detailed phylogenetic data about the evolutionary history of mitochondrial tRNA gene loss, transfer, and functional replacement that has created extreme variation in mitochondrial tRNA populations across plant species. We describe emerging tRNA-seq methods with promise for refining our understanding of the expression and subcellular localization of tRNAs. Finally, we summarize current evidence and identify open questions related to coevolutionary changes in nuclear-encoded enzymes that have accompanied turnover in mitochondrial tRNA populations.
Transfer RNA: From pioneering crystallographic studies to contemporary tRNA biology
2016, Archives of Biochemistry and Biophysics
Transfer RNAs (tRNAs) play a key role in protein synthesis as adaptor molecules between messenger RNA and protein sequences on the ribosome. Their discovery in the early sixties provoked a worldwide infatuation with the study of their architecture and their function in the decoding of genetic information. tRNAs are also emblematic molecules in crystallography: the determination of the first tRNA crystal structures represented a milestone in structural biology and tRNAs were for a long period the sole source of information on RNA folding, architecture, and post-transcriptional modifications. Crystallographic data on tRNAs in complex with aminoacyl-tRNA synthetases (aaRSs) also provided the first insight into protein:RNA interactions. Beyond the translation process and the history of structural investigations on tRNA, this review also illustrates the renewal of tRNA biology with the discovery of a growing number of tRNA partners in the cell, the involvement of tRNAs in a variety of regulatory and metabolic pathways, and emerging applications in biotechnology and synthetic biology.

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Trends in Biochemical Sciences

ReviewRecent advances in tRNA mitochondrial import

Section snippets

tRNA transport into mitochondria

Why must mitochondria import tRNAs?

Divergent mechanisms for a convergent goal

An attractive tool for correcting disease-causing mitochondrial tRNA gene mutations

Concluding remarks and future perspectives

J. Biol. Chem.

Trends Cell Biol.

Mol. Cell

Adv. Drug Deliv. Rev.

Biochim. Biophys. Acta

Biochimie

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

FEBS Lett.

J. Biol. Chem.

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Biochim. Biophys. Acta

Trends Cell Biol.

J. Biol. Chem.

Trends Genet.

Evidence for the presence of 5S rRNA in mammalian mitochondria

Mol. Biol. Cell

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

RNA

The RNase P associated with HeLa cell mitochondria contains an essential RNA component identical in sequence to that of the nuclear RNase P

Mol. Cell. Biol.

Import of nuclear DNA-encoded RNAs into mitochondria and mitochondrial translation

Cell Cycle

Coexistence of nuclear DNA-encoded tRNAVal(AAC) and mitochondrial DNA-encoded tRNAVal(UAC) in mitochondria of a liverwort Marchantia polymorpha

Nucleic Acids Res.

Saccharomyces cerevisiae imports the cytosolic pathway for Gln-tRNA synthesis into the mitochondrion

Genes Dev.

Elongation factor 1a mediates the specificity of mitochondrial tRNA import in T. brucei

EMBO J.

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

Genes Dev.

The D arm of tRNATyr is necessary and sufficient for import into Leishmania mitochondria in vitro

Nucleic Acids Res.

Mitochondrial RNA import in Leishmania tropica: aptamers homologous to multiple tRNA domains that interact cooperatively or antagonistically at the inner membrane

Mol. Cell. Biol.

Review
Recent advances in tRNA mitochondrial import

The D arm of tRNA^Tyr is necessary and sufficient for import into Leishmania mitochondria in vitro