Functional Diversity of Mitochondrial Peptidyl-tRNA Hydrolase ICT1 in Human Cells.

Mitochondria are energy producing organelles of the eukaryotic cell, involved in the synthesis of key metabolites, calcium homeostasis and apoptosis. Protein biosynthesis in these organelles is a relic of its endosymbiotic origin. While mitochondrial translational factors have homologues among prokaryotes, they possess a number of unique traits. Remarkably as many as four mammalian mitochondrial proteins possess a clear similarity with translation termination factors. The review focuses on the ICT1, which combines several functions. It is a non-canonical termination factor for protein biosynthesis, a rescue factor for stalled mitochondrial ribosomes, a structural protein and a regulator of proliferation, cell cycle, and apoptosis. Such a diversity of roles demonstrates the high functionality of mitochondrial translation associated proteins and their relationship with numerous processes occurring in a living cell.

Protein Biosynthesis in Mitochondria: Past Simple, Present Perfect, Future Indefinite.

Mitochondria are obligate organelles of most eukaryotic cells that perform many different functions important for cellular homeostasis. The main role of mitochondria is supplying cells with energy in a form of ATP, which is synthesized in a chain of oxidative phosphorylation reactions on the organelle inner membrane. It is commonly believed now that mitochondria have the endosymbiotic origin. In the course of evolution, they have lost most of their genetic material as a result of genome reduction and gene transfer to the nucleus. The majority of mitochondrial proteins are synthesized in the cytosol and then imported to the mitochondria. However, almost all known mitochondria still contain genomes that are maintained and expressed. The processes of protein biosynthesis in the mitochondria - mitochondrial translation - substantially differs from the analogous processes in bacteria and the cytosol of eukaryotic cells. Mitochondrial translation is characterized by a high degree of specialization and specific regulatory mechanisms. In this review, we analyze available information on the common principles of mitochondrial translation with emphasis on the molecular mechanisms of translation initiation in the mitochondria of yeast and mammalian cells.

[40 Years of Studying RNA Import into Mitochondria: From Basic Mechanisms to Gene Therapy Strategies].

Mitochondria of many living species internalize nuclear DNA-encoded ribonucleic acids. The pools of imported RNA molecules, as well as fine mechanisms of these processes, are highly species-specific. To date, baker's yeast Saccharomyces cerevisiae are the best studied in this regard. Moreover, the processes of yeast RNA mitochondrial import have been the basis of modeling several gene therapy strategies aimed to palliate negative effects of pathogenic mutations in human mitochondrial DNA. In this review, we summarize our current knowledge about the molecular events taking place in course of yeast RNA import into mitochondria. Also, we describe how this process can be used for compensation of pathogenic mutations in mitochondrial genomes of humans.

Aim23p Interacts with the Yeast Mitochondrial Ribosomal Small Subunit.

Protein synthesis in mitochondria is generally organized in a bacterial-like manner but, at the same time, possesses several unique traits. Translation initiation in mitochondria is regulated by two protein factors, mtIF2 and mtIF3. Previously we demonstrated that Saccharomyces cerevisiae Aim23 protein is an ortholog of IF3 in budding yeast. However, the data on the interactions between Aim23p and other proteins are limited. Here, we demonstrated that Aim23p interacts with the yeast mitochondrial ribosomal small subunit both in vivo and in vitro using co-immunoprecipitation and density gradient sedimentation.

Import of Proteins and Nucleic Acids into Mitochondria.

Many mitochondrial genes have been transferred to the nucleus in course of evolution. The products of expression of these genes, being still necessary for organelle function, are imported there from the cytosol. Molecular mechanisms of protein import are studied much deeper than those of nucleic acids. The latter, it seems to us, retards the development of mitochondrial genome editing technologies. In this review, we describe mechanisms of DNA, RNA, and protein import into mitochondria of different eukaryotes. The description is given for the natural processes, as well as for artificial targeting of macromolecules into mitochondria for therapy. Also, we discuss different approaches to introduce changes into the mitochondrial DNA sequence.

Activation of Yeast Mitochondrial Translation: Who Is in Charge?

Mitochondrial genome has undergone significant reduction in a course of evolution; however, it still contains a set of protein-encoding genes and requires translational machinery for their expression. Mitochondrial translation is of the prokaryotic type with several remarkable differences. This review is dedicated to one of the most puzzling features of mitochondrial protein synthesis, namely, the system of translational activators, i.e., proteins that specifically regulate translation of individual mitochondrial mRNAs and couple protein biosynthesis with the assembly of mitochondrial respiratory chain complexes. The review does not claim to be a comprehensive analysis of all published data; it is rather focused on the idea of the "core component" of the translational activator system.

"Noah's Ark" Project: Interim Results and Outlook for Classic Collection Development.

The "Noah's Ark" project, afoot at M.V. Lomonosov Moscow State University since 2015 and aimed at studying biodiversity, is the largest ongoing Russian project in life sciences. During its implementation, several hundred new species have been described; a comprehensive genetic and biochemical characterization of these species, as well as that of the pre-existing specimens in Moscow University's collections, has been performed. A consolidated IT system intended to house the knowledge generated by the project has been developed. Here, we summarize the investigations around the Moscow University classical biocollections which have taken place within the framework of the project and discuss future promise and the outlook for these collections.

Factors beyond Enolase 2 and Mitochondrial Lysyl-tRNA Synthetase Precursor Are Required for tRNA Import into Yeast Mitochondria.

In yeast, the import of tRNALys with CUU anticodon (tRK1) relies on a complex mechanism where interaction with enolase 2 (Eno2p) dictates a deep conformational change of the tRNA. This event is believed to mask the tRNA from the cytosolic translational machinery to re-direct it towards the mitochondria. Once near the mitochondrial outer membrane, the precursor of the mitochondrial lysyl-tRNA synthetase (preMsk1p) takes over enolase to carry the tRNA within the mitochondrial matrix, where it is supposed to participate in translation following correct refolding. Biochemical data presented in this report focus on the role of enolase. They show that despite the inability of Eno2p alone to form a complex with tRK1, mitochondrial import can be recapitulated in vitro using fractions of yeast extracts sharing either recombinant or endogenous yeast Eno2p as one of the main components. Taken together, our data suggest the existence of a protein complex containing Eno2p that is involved in RNA mitochondrial import.

Procedure for Purification of Recombinant preMsk1p from E. coli Determines Its Properties as a Factor of tRNA Import into Yeast Mitochondria.

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.

Biological Collections: Chasing the Ideal.

This article is based on the results of an analysis of existing biological collections in Russia and abroad set up in the framework of the project "Scientific Basis of the National Biobank -Depository of Living Systems" by M.V. Lomonosov Moscow State University [1].

Binding of DNA with Abf2p Increases Efficiency of DNA Uptake by Isolated Mitochondria.

Mutations in mitochondrial DNA often lead to severe hereditary diseases that are virtually resistant to symptomatic treatment. During the recent decades, many efforts were made to develop gene therapy approaches for treatment of such diseases using nucleic acid delivery into the organelles. The possibility of DNA import into mitochondria has been shown, but this process has low efficiency. In the present work, we demonstrate that the efficiency of DNA import can be significantly increased by preforming its complex with a mitochondria-targeted protein nonspecifically binding with DNA. As a model protein, we used the yeast protein Abf2p. In addition, we measured the length of the DNA site for binding this protein and the dissociation constant of the corresponding DNA-protein complex. Our data can serve as a basis for development of novel, highly efficient approaches for suppressing mutations in the mitochondrial genome.

Mitochondrial Matrix Processes.

Mitochondria possess their own genome that, despite its small size, is critically important for their functioning, as it encodes several dozens of RNAs and proteins. All biochemical processes typical for bacterial and nuclear DNA are described in mitochondrial matrix: replication, repair, recombination, and transcription. Commonly, their mechanisms are similar to those found in bacteria, but they are characterized by several unique features. In this review, we provide an overall description of mitochondrial matrix processes paying special attention to the typical features of such mechanisms.

Mitochondrial Fission and Fusion.

Mitochondria are key cellular organelles responsible for many different functions. The molecular biology of mitochondria is continuously subject to comprehensive studies. However, detailed mechanisms of mitochondrial biogenesis are still unclear. Fusion and fission are among the most enigmatic processes connected with mitochondria. On the other hand, it has been shown that these events are of great biological importance for functioning of living cells. In this review, we summarize existing molecular data on mitochondrial dynamics and discuss possible biological functions of fusion and fission of these organelles.

Mutations in mitochondrial DNA and approaches for their correction.

Apart from the nucleus, the mitochondrion is the only organelle of an animal cell that contains its own genome. Mitochondrial DNA is much less in size than the nuclear one and codes for only several dozens of biological macromolecules. Nevertheless, mutations in mitochondrial genes often result in the occurrence of serious hereditary neuromuscular diseases. New mitochondrial DNA mutations and their relations to clinical symptoms are continuously reported in the scientific literature. In this review, we summarize existing data about such mutations, and also about contemporary gene therapy approaches that have been developed for their suppression.

Protein biosynthesis in mitochondria.

Translation, that is biosynthesis of polypeptides in accordance with information encoded in the genome, is one of the most important processes in the living cell, and it has been in the spotlight of international research for many years. The mechanisms of protein biosynthesis in bacteria and in the eukaryotic cytoplasm are now understood in great detail. However, significantly less is known about translation in eukaryotic mitochondria, which is characterized by a number of unusual features. In this review, we summarize current knowledge about mitochondrial translation in different organisms while paying special attention to the aspects of this process that differ from cytoplasmic protein biosynthesis.

Noncanonical functions of aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases, together with their main function of covalent binding of an amino acid to a corresponding tRNA, also perform many other functions. They take part in regulation of gene transcription, apoptosis, translation, and RNA splicing. Some of them function as cytokines or catalyze different reactions in living cells. Noncanonical functions can be mediated by additional domains of these proteins. On the other hand, some of the noncanonical functions are directly associated with the active center of the aminoacylation reaction. In this review we summarize recent data on the noncanonical functions of aminoacyl-tRNA synthetases and on the mechanisms of their action.

Import of nuclear encoded RNAs into yeast and human mitochondria: experimental approaches and possible biomedical applications.

Mitochondria import from the cytoplasm the vast majority of proteins and some RNAs. Although there exists extended knowledge concerning the mechanisms of protein import, the import of RNA is poorly understood. It was almost exclusively studied on the model of tRNA import, in several protozoans, plants and yeast. Mammalian mitochondria, which do not import tRNAs naturally, are hypothesized to import other small RNA molecules from the cytoplasm. We studied tRNA import in the yeast system, both in vitro and in vivo, and applied similar approaches to study 5S rRNA import into human mitochondria. Despite the obvious divergence of RNA import systems suggested for different species, we find that in yeast and human cells this pathway involves similar mechanisms exploiting cytosolic proteins to target the RNA to the organelle and requiring the integrity of pre-protein import apparatus. The import pathway might be of interest from a biomedical point of view, to target into mitochondria RNAs that could suppress pathological mutations in mitochondrial DNA. Yeast represents a good model to elaborate such a gene therapy approach. We have described here the various approaches and protocols to study RNA import into mitochondria of yeast and human cells in vitro and in vivo.