Mitochondrial transport of catalytic RNAs and targeting of the organellar transcriptome in human cells.

Mutations in the small genome present in mitochondria often result in severe pathologies. Different genetic strategies have been explored, aiming to contribute to rescue such mutations. A number of these were based on the capacity of human mitochondria to import RNAs from the cytosol and were designed to repress the replication of the mutated genomes or to provide the organelles with wild-type versions of mutant transcripts. However, the mutant RNAs present in mitochondria turned out to be an obstacle to therapy and little attention has been devoted so far to their elimination. Here, we present the development of a strategy to knockdown mitochondrial RNAs in human cells using the transfer RNA-like structure of the Brome mosaic virus or the Tobacco mosaic virus as a shuttle to drive trans-cleaving ribozymes into the organelles in human cell lines. We obtained a specific knockdown of the targeted mitochondrial ATP6 mRNA, followed by a deep drop in ATP6 protein and a functional impairment of the oxidative phosphorylation chain. Our strategy opens a powerful approach to eliminate mutant organellar transcripts and to analyze the control and communication of the human organellar genetic system.

RADA-dependent branch migration has a predominant role in plant mitochondria and its defect leads to mtDNA instability and cell cycle arrest.

Mitochondria of flowering plants have large genomes whose structure and segregation are modulated by recombination activities. The post-synaptic late steps of mitochondrial DNA (mtDNA) recombination are still poorly characterized. Here we show that RADA, a plant ortholog of bacterial RadA/Sms, is an organellar protein that drives the major branch-migration pathway of plant mitochondria. While RadA/Sms is dispensable in bacteria, RADA-deficient Arabidopsis plants are severely impacted in their development and fertility, correlating with increased mtDNA recombination across intermediate-size repeats and accumulation of recombination-generated mitochondrial subgenomes. The radA mutation is epistatic to recG1 that affects the additional branch migration activity. In contrast, the double mutation radA recA3 is lethal, underlining the importance of an alternative RECA3-dependent pathway. The physical interaction of RADA with RECA2 but not with RECA3 further indicated that RADA is required for the processing of recombination intermediates in the RECA2-depedent recombination pathway of plant mitochondria. Although RADA is dually targeted to mitochondria and chloroplasts we found little to no effects of the radA mutation on the stability of the plastidial genome. Finally, we found that the deficient maintenance of the mtDNA in radA apparently triggers a retrograde signal that activates nuclear genes repressing cell cycle progression.

Evaluating malformations of the lacrimal drainage system in brachycephalic dog breeds: A comparative computed tomography analysis.

OBJECTIVES: This study aimed to investigate and compare the anatomical features of the nasolacrimal drainage system (NDS) in three brachycephalic dog breeds with those of normocephalic dogs, taking into account how the NDS was related to the malformed brachycephalic head. ANIMALS: Fifty-one brachycephalic dogs were examined, comprising 23 Pugs, 18 French Bulldogs, and 10 English Bulldogs. Six normocephalic dogs of different breeds served as a comparison. METHODS: Computed tomographic dacryocystography was performed. Parameters such as length, angulation, and gradient were determined. Crossing of the nasolacrimal duct (NLD) beneath the maxillary canine root, as well as the incidence of an accessory opening, were also analyzed. RESULTS AND CONCLUSIONS: In all three brachycephalic breeds, the NDS was grossly malformed. We regard this as a further consequence of exaggerated breeding for a short head conformation. While the length of the NLD was substantially reduced by 41 to 57 percent in brachycephalic dogs, their lacrimal canaliculi were two to three times as long as those of normocephalic dogs. Varying parts of the nasolacrimal drainage system followed an inverse direction in short-headed dogs, giving the entire nasolacrimal apparatus an anomalous U- or V-shaped appearance. The NLD exhibited a three to five times steeper alignment in brachycephalic dogs than in normocephalic ones. Obviously, this strong slope did not cause clinical symptoms only because there was an aberrant outflow pathway. The brachycephalic dogs consistently exhibited an accessory opening, through which most of fluid escaped into the posterior nasal cavity instead of through the common route into the nasal vestibule via the nasolacrimal ostia.

Plant mitochondria import DNA via alternative membrane complexes involving various VDAC isoforms.

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.

Mitochondrial Transcriptome Control and Intercompartment Cross-Talk During Plant Development.

We address here organellar genetic regulation and intercompartment genome coordination. We developed earlier a strategy relying on a tRNA-like shuttle to mediate import of nuclear transgene-encoded custom RNAs into mitochondria in plants. In the present work, we used this strategy to drive trans-cleaving hammerhead ribozymes into the organelles, to knock down specific mitochondrial RNAs and analyze the regulatory impact. In a similar approach, the tRNA mimic was used to import into mitochondria in Arabidopsis thaliana the orf77, an RNA associated with cytoplasmic male sterility in maize and possessing sequence identities with the atp9 mitochondrial RNA. In both cases, inducible expression of the transgenes allowed to characterise early regulation and signaling responses triggered by these respective manipulations of the organellar transcriptome. The results imply that the mitochondrial transcriptome is tightly controlled by a "buffering" mechanism at the early and intermediate stages of plant development, a control that is released at later stages. On the other hand, high throughput analyses showed that knocking down a specific mitochondrial mRNA triggered a retrograde signaling and an anterograde nuclear transcriptome response involving a series of transcription factor genes and small RNAs. Our results strongly support transcriptome coordination mechanisms within the organelles and between the organelles and the nucleus.

External Human-Machine Interfaces on Automated Vehicles: Effects on Pedestrian Crossing Decisions.

OBJECTIVE: In this article, we investigated the effects of external human-machine interfaces (eHMIs) on pedestrians' crossing intentions. BACKGROUND: Literature suggests that the safety (i.e., not crossing when unsafe) and efficiency (i.e., crossing when safe) of pedestrians' interactions with automated vehicles could increase if automated vehicles display their intention via an eHMI. METHODS: Twenty-eight participants experienced an urban road environment from a pedestrian's perspective using a head-mounted display. The behavior of approaching vehicles (yielding, nonyielding), vehicle size (small, medium, large), eHMI type (1. baseline without eHMI, 2. front brake lights, 3. Knightrider animation, 4. smiley, 5. text [WALK]), and eHMI timing (early, intermediate, late) were varied. For yielding vehicles, the eHMI changed from a nonyielding to a yielding state, and for nonyielding vehicles, the eHMI remained in its nonyielding state. Participants continuously indicated whether they felt safe to cross using a handheld button, and "feel-safe" percentages were calculated. RESULTS: For yielding vehicles, the feel-safe percentages were higher for the front brake lights, Knightrider, smiley, and text, as compared with baseline. For nonyielding vehicles, the feel-safe percentages were equivalent regardless of the presence or type of eHMI, but larger vehicles yielded lower feel-safe percentages. The Text eHMI appeared to require no learning, contrary to the three other eHMIs. CONCLUSION: An eHMI increases the efficiency of pedestrian-AV interactions, and a textual display is regarded as the least ambiguous. APPLICATION: This research supports the development of automated vehicles that communicate with other road users.

The Reverse Transcriptase/RNA Maturase Protein MatR Is Required for the Splicing of Various Group II Introns in Brassicaceae Mitochondria.

Group II introns are large catalytic RNAs that are ancestrally related to nuclear spliceosomal introns. Sequences corresponding to group II RNAs are found in many prokaryotes and are particularly prevalent within plants organellar genomes. Proteins encoded within the introns themselves (maturases) facilitate the splicing of their own host pre-RNAs. Mitochondrial introns in plants have diverged considerably in sequence and have lost their maturases. In angiosperms, only a single maturase has been retained in the mitochondrial DNA: the matR gene found within NADH dehydrogenase 1 (nad1) intron 4. Its conservation across land plants and RNA editing events, which restore conserved amino acids, indicates that matR encodes a functional protein. However, the biological role of MatR remains unclear. Here, we performed an in vivo investigation of the roles of MatR in Brassicaceae. Directed knockdown of matR expression via synthetically designed ribozymes altered the processing of various introns, including nad1 i4. Pull-down experiments further indicated that MatR is associated with nad1 i4 and several other intron-containing pre-mRNAs. MatR may thus represent an intermediate link in the gradual evolutionary transition from the intron-specific maturases in bacteria into their versatile spliceosomal descendants in the nucleus. The similarity between maturases and the core spliceosomal Prp8 protein further supports this intriguing theory.

The RECG1 DNA Translocase Is a Key Factor in Recombination Surveillance, Repair, and Segregation of the Mitochondrial DNA in Arabidopsis.

The mitochondria of flowering plants have considerably larger and more complex genomes than the mitochondria of animals or fungi, mostly due to recombination activities that modulate their genomic structures. These activities most probably participate in the repair of mitochondrial DNA (mtDNA) lesions by recombination-dependent processes. Rare ectopic recombination across short repeats generates new genomic configurations that contribute to mtDNA heteroplasmy, which drives rapid evolution of the sequence organization of plant mtDNAs. We found that Arabidopsis thaliana RECG1, an ortholog of the bacterial RecG translocase, is an organellar protein with multiple roles in mtDNA maintenance. RECG1 targets to mitochondria and plastids and can complement a bacterial recG mutant that shows defects in repair and replication control. Characterization of Arabidopsis recG1 mutants showed that RECG1 is required for recombination-dependent repair and for suppression of ectopic recombination in mitochondria, most likely because of its role in recovery of stalled replication forks. The analysis of alternative mitotypes present in a recG1 line and of their segregation following backcross allowed us to build a model to explain how a new stable mtDNA configuration, compatible with normal plant development, can be generated by stoichiometric shift.

Nucleic acid import into mitochondria: New insights into the translocation pathways.

Mitochondria have retained indispensable but limited genetic information and they import both proteins and nucleic acids from the cytosol. RNA import is essential for gene expression and regulation, whereas competence for DNA uptake is likely to contribute to organellar genome dynamics and evolution. Contrary to protein import mechanisms, the way nucleic acids cross the mitochondrial membranes remains poorly understood. Using proteomic, genetic and biochemical approaches with both plant and yeast organelles, we develop here a model for DNA uptake into mitochondria. The first step includes the voltage-dependent anion channel and an outer membrane-located precursor fraction of a protein normally located in the inner membrane. To proceed, the DNA is then potentially recruited in the intermembrane space by an accessible subunit of one of the respiratory chain complexes. Final translocation through the inner membrane remains the most versatile but points to the components considered to make the mitochondrial permeability transition pore. Depending on the size, DNA and RNA cooperate or compete for mitochondrial uptake, which shows that they share import mechanisms. On the other hand, our results imply the existence of more than one route for nucleic acid translocation into mitochondria.

Organellar non-coding RNAs: emerging regulation mechanisms.

Originally focused on the nuclear and cytosolic compartments, the concept of regulation driven by non-coding RNAs (ncRNAs) is extending to mitochondria and chloroplasts. These organelles have distinct genetic systems that need coordination with cellular demands. In mammals, nuclear-encoded microRNAs were found associated with the mitochondria. Some of these contribute to the regulation of mitochondrial transcription and translation. Others were proposed to be stored in the organelles and to be released for regulation of nuclear transcripts. Further ncRNAs of various sizes derive from the mitochondrial genome and it was speculated that organelles host antisense or RNA interference pathways. Long ncRNAs mapping to the mitochondrial DNA seem to operate in the nucleus. Altogether, the origin and trafficking of ncRNAs categorized as mitochondrial in mammals raise questions far beyond the current knowledge. In protozoa, hundreds of guide RNAs specify editing events needed to generate functional messenger RNAs. Only few ncRNAs have been reported in plant mitochondria, but editing sites were revealed in non-coding regions of the organellar genome, suggesting that the corresponding transcripts have a function. Conversely, numerous ncRNA candidates were identified in chloroplasts, essentially mapping to the plastid genome. A synthetic view of the data with their functional implications is given here.

Mitochondrial targeting of catalytic RNAs.

Genetic transformation of mitochondria in multicellular eukaryotes has remained inaccessible, hindering fundamental investigations and applications to gene therapy or biotechnology. In this context, we have developed a strategy to target nuclear transgene-encoded RNAs into mitochondria in plants. We describe here mitochondrial targeting of trans-cleaving ribozymes destined to knockdown organelle RNAs for regulation studies and inverse genetics and biotechnological purposes. The design and functional assessment of chimeric RNAs combining the ribozyme and the mitochondrial shuttle are detailed, followed by all procedures to prepare constructs for in vivo expression, generate stable plant transformants, and establish target RNA knockdown in mitochondria.

The plant mitochondrial genome: dynamics and maintenance.

Plant mitochondria have a complex and peculiar genetic system. They have the largest genomes, as compared to organelles from other eukaryotic organisms. These can expand tremendously in some species, reaching the megabase range. Nevertheless, whichever the size, the gene content remains modest and restricted to a few polypeptides required for the biogenesis of the oxidative phosphorylation chain complexes, ribosomal proteins, transfer RNAs and ribosomal RNAs. The presence of autonomous plasmids of essentially unknown function further enhances the level of complexity. The physical organization of the plant mitochondrial DNA includes a set of sub-genomic forms resulting from homologous recombination between repeats, with a mixture of linear, circular and branched structures. This material is compacted into membrane-bound nucleoids, which are the inheritance units but also the centers of genome maintenance and expression. Recombination appears to be an essential characteristic of plant mitochondrial genetic processes, both in shaping and maintaining the genome. Under nuclear surveillance, recombination is also the basis for the generation of new mitotypes and is involved in the evolution of the mitochondrial DNA. In line with, or as a consequence of its complex physical organization, replication of the plant mitochondrial DNA is likely to occur through multiple mechanisms, potentially involving recombination processes. We give here a synthetic view of these aspects.

Targeting nucleic acids into mitochondria: progress and prospects.

Given the essential functions of these organelles in cell homeostasis, their involvement in incurable diseases and their potential in biotechnological applications, genetic transformation of mitochondria has been a long pursued goal that has only been reached in a couple of unicellular organisms. The challenge led scientists to explore a wealth of different strategies for mitochondrial delivery of DNA or RNA in living cells. These are the subject of the present review. Targeting DNA into the organelles currently shows promise but remarkably a number of alternative approaches based on RNA trafficking were also established and will bring as well major contributions.

Transfection of plant mitochondria and in organello gene integration.

Investigation and manipulation of mitochondrial genetics in animal and plant cells remains restricted by the lack of an efficient in vivo transformation methodology. Mitochondrial transfection in whole cells and maintenance of the transfected DNA are main issues on this track. We showed earlier that isolated mitochondria from different organisms can import DNA. Exploiting this mechanism, we assessed the possibility to maintain exogenous DNA in plant organelles. Whereas homologous recombination is scarce in the higher plant nuclear compartment, recombination between large repeats generates the multipartite structure of the plant mitochondrial genome. These processes are under strict surveillance to avoid extensive genomic rearrangements. Nevertheless, following transfection of isolated organelles with constructs composed of a partial gfp gene flanked by fragments of mitochondrial DNA, we demonstrated in organello homologous recombination of the imported DNA with the resident DNA and integration of the reporter gene. Recombination yielded insertion of a continuous exogenous DNA fragment including the gfp sequence and at least 0.5 kb of flanking sequence on each side. According to our observations, transfection constructs carrying multiple sequences homologous to the mitochondrial DNA should be suitable and targeting of most regions in the organelle genome should be feasible, making the approach of general interest.

Organelle trafficking of chimeric ribozymes and genetic manipulation of mitochondria.

With the expansion of the RNA world, antisense strategies have become widespread to manipulate nuclear gene expression but organelle genetic systems have remained aside. The present work opens the field to mitochondria. We demonstrate that customized RNAs expressed from a nuclear transgene and driven by a transfer RNA-like (tRNA-like) moiety are taken up by mitochondria in plant cells. The process appears to follow the natural tRNA import specificity, suggesting that translocation indeed occurs through the regular tRNA uptake pathway. Upon validation of the strategy with a reporter sequence, we developed a chimeric catalytic RNA composed of a specially designed trans-cleaving hammerhead ribozyme and a tRNA mimic. Organelle import of the chimeric ribozyme and specific target cleavage within mitochondria were demonstrated in transgenic tobacco cell cultures and Arabidopsis thaliana plants, providing the first directed knockdown of a mitochondrial RNA in a multicellular eukaryote. Further observations point to mitochondrial messenger RNA control mechanisms related to the plant developmental stage and culture conditions. Transformation of mitochondria is only accessible in yeast and in the unicellular alga Chlamydomonas. Based on the widespread tRNA import pathway, our data thus make a breakthrough for direct investigation and manipulation of mitochondrial genetics.

DNA delivery to mitochondria: sequence specificity and energy enhancement.

PURPOSE: Mitochondria are competent for DNA uptake in vitro, a mechanism which may support delivery of therapeutic DNA to complement organelle DNA mutations. We document here key aspects of the DNA import process, so as to further lay the ground for mitochondrial transfection in intact cells. METHODS: We developed DNA import assays with isolated mitochondria from different organisms, using DNA substrates of various sequences and sizes. Further import experiments investigated the possible role of ATP and protein phosphorylation in the uptake process. The fate of adenine nucleotides and the formation of phosphorylated proteins were analyzed. RESULTS: We demonstrate that the efficiency of mitochondrial uptake depends on the sequence of the DNA to be translocated. The process becomes sequence-selective for large DNA substrates. Assays run with a natural mitochondrial plasmid identified sequence elements which promote organellar uptake. ATP enhances DNA import and allows tight integration of the exogenous DNA into mitochondrial nucleoids. ATP hydrolysis has to occur during the DNA uptake process and might trigger phosphorylation of co-factors. CONCLUSIONS: Our data contribute critical information to optimize DNA delivery into mitochondria and open the prospect of targeting whole mitochondrial genomes or complex constructs into mammalian organelles in vitro and in vivo.

Mitochondrial transfection for studying organellar DNA repair, genome maintenance and aging.

Maintenance of the mitochondrial genome is a major challenge for cells, particularly as they begin to age. Although it is established that organelles possess regular DNA repair pathways, many aspects of these complex processes and of their regulation remain to be investigated. Mitochondrial transfection of isolated organelles and in whole cells with customized DNA synthesized to contain defined lesions has wide prospects for deciphering repair mechanisms in a physiological context. We document here the strategies currently developed to transfer DNA of interest into mitochondria. Methodologies with isolated mitochondria claim to exploit the protein import pathway or the natural competence of the organelles, to permeate the membranes or to use conjugal transfer from bacteria. Besides biolistics, which remains restricted to yeast and Chlamydomonas reinhardtii, nanocarriers or fusion proteins have been explored as methods to target custom DNA into mitochondria in intact cells. In further approaches, whole mitochondria have been transferred into recipient cells. Repair failure or error-prone repair leads to mutations which potentially could be rescued by allotopic expression of proteins. The relevance of the different approaches for the analysis of mitochondrial DNA repair mechanisms and of aging is discussed.

DNA repair in organelles: Pathways, organization, regulation, relevance in disease and aging.

Both endogenous processes and exogenous physical and chemical sources generate deoxyribonucleic acid (DNA) damage in the nucleus and organelles of living cells. To prevent deleterious effects, damage is balanced by repair pathways. DNA repair was first documented for the nuclear compartment but evidence was subsequently extended to the organelles. Mitochondria and chloroplasts possess their own repair processes. These share a number of factors with the nucleus but also rely on original mechanisms. Base excision repair remains the best characterized. Repair is organized with the other DNA metabolism pathways in the organelle membrane-associated nucleoids. DNA repair in mitochondria is a regulated, stress-responsive process. Organelle genomes do not encode DNA repair enzymes and translocation of nuclear-encoded repair proteins from the cytosol seems to be a major control mechanism. Finally, changes in the fidelity and efficiency of mitochondrial DNA repair are likely to be involved in DNA damage accumulation, disease and aging. The present review successively addresses these different issues.

Membrane association of mitochondrial DNA facilitates base excision repair in mammalian mitochondria.

Mitochondrial DNA encodes a set of 13 polypeptides and is subjected to constant oxidative stress due to ROS production within the organelle. It has been shown that DNA repair in the mitochondrion proceeds through both short- and long-patch base excision repair (BER). In the present article, we have used the natural competence of mammalian mitochondria to import DNA and study the sub-mitochondrial localization of the repair system in organello. Results demonstrate that sequences corresponding to the mtDNA non-coding region interact with the inner membrane in a rapid and saturable fashion. We show that uracil containing import substrates are taken into the mitochondrion and are used as templates for damage driven DNA synthesis. After further sub-fractionation, we show that the length of the repair synthesis patch differs in the soluble and the particulate fraction. Bona fide long patch BER synthesis occurs on the DNA associated with the particulate fraction, whereas a nick driven DNA synthesis occurs when the uracil containing DNA accesses the soluble fraction. Our results suggest that coordinate interactions of the different partners needed for BER is only found at sites where the DNA is associated with the membrane.

Plant mitochondria possess a short-patch base excision DNA repair pathway.

Despite constant threat of oxidative damage, sequence drift in mitochondrial and chloroplast DNA usually remains very low in plant species, indicating efficient defense and repair. Whereas the antioxidative defense in the different subcellular compartments is known, the information on DNA repair in plant organelles is still scarce. Focusing on the occurrence of uracil in the DNA, the present work demonstrates that plant mitochondria possess a base excision repair (BER) pathway. In vitro and in organello incision assays of double-stranded oligodeoxyribonucleotides showed that mitochondria isolated from plant cells contain DNA glycosylase activity specific for uracil cleavage. A major proportion of the uracil-DNA glycosylase (UDG) was associated with the membranes, in agreement with the current hypothesis that the DNA is replicated, proofread and repaired in inner membrane-bound nucleoids. Full repair, from uracil excision to thymidine insertion and religation, was obtained in organello following import of a uracil-containing DNA fragment into isolated plant mitochondria. Repair occurred through single nucleotide insertion, which points to short-patch BER. In vivo targeting and in vitro import of GFP fusions showed that the putative UDG encoded by the At3g18 630 locus might be the first enzyme of this mitochondrial pathway in Arabidopsis thaliana.