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Mitochondrial genome dynamics and maintenance

Higher plant mitochondria possess large size genomes (up to over 1800 kb for the known sequences) that contain long intergenic regions, multiple promoters and introns (Fig. 1a). These genomes have a multipartite and dynamic structure resulting from recombination events that are tightly controlled by specific nuclear-encoded factors (Fig. 1b). The genetic information is distributed into sub-genomic DNA molecules resulting from recombination between large sequence repeats. Rare recombination across short repeats and microhomology-mediated recombination can further generate alternative configurations that contribute to mtDNA heteroplasmy and to the rapid evolution of the mtDNA structure. Maintenance of this complex genetic material in the context of oxidative pressure generated by the respiratory chain activity or by environmental conditions requires efficient DNA repair mechanisms. The latter are still poorly documented in plant organelles, in particular concerning the involvement of recombination in DNA break repair. In mammalian mitochondria, the available information is mainly restricted to base excision repair, while recombination is considered to be rare. Developing homologous recombination in human mitochondria would be of major interest to complement pathogenic mtDNA mutations.

Figure 1. Structure of a plant mitochondrial genome. (a) Organization of protein and ribosomal RNA genes in the mtDNA of Arabidopsis thaliana (Unseld et al., 1997, Nat. Genet. 15, 57-61), highlighting the low gene density and the presence of large sequence repeats (R1, R2); (b) multipartite structure resulting from homologous recombination involving the large repeats R1 and R2.

Mitochondrial transfection

Progress in the understanding and applications of mitochondrial genetics suffers from the lack of established molecular approaches to transform the mtDNA. Whereas great advances have been reported in chloroplast genome engineering, mitochondria have been amenable to regular genetic transformation by biolistics in only a couple of unicellular organisms. In humans, transformation of mitochondria would support gene therapy for diseases caused by mtDNA mutations. In plants, it would help mastering agronomical traits based on cytoplasmic inheritance. Due to the importance of these issues, a wealth of strategies have been explored to manipulate the mitochondrial genetic system. These include a variety of approaches still aiming to transfer and maintain DNA of interest in mitochondria (e.g. Fig. 2). Conversely, to circumvent the need for mtDNA transformation, nuclear expression and mitochondrial targeting of proteins or RNAs through different shuttling systems has been widely assayed (e.g. Fig. 3). A number of these methodologies were reported to be successful, for instance to rescue pathogenic mtDNA mutations in mammalian cellular models. Currently, the main challenge in the field is to get consensus biotechnological tools out of the collected data.navette

 

Figure 2. A strategy to target DNA constructs into mitochondria in mammalian cells (reviewed in D'Souza et al., 2007, Pharm. Res. 24, 228-238). The DNA is loaded into mitochondriotropic nanovesicles made of dequalinium or lipids; the loaded vesicles enter the cells through endocytosis and migrate towards mitochondria, where they collapse upon interaction with the organelle membranes; it is hypothesized that the released DNA will be imported into mitochondria thanks to the natural competence demonstrated for isolated organelles (Koulintchenko et al., 2006, Hum. Mol. Genet. 15, 143-154).

Mitochondrial regulation, cross-talk and reverse genetics

Mitochondrial genetic and functional processes must be regulated and tuned so as to ensure proper contribution to cellular homeostasis. Mitochondria talk to the nucleus through retrograde signaling. The mechanisms that relay the information have been documented in yeast, but the data is scarce for mammals and plants. Searching for putative regulation of mitochondrial RNA steady-state levels and coordination with nuclear gene expression has also been hampered by the lack of direct genetic tools. Knockdown of individual mitochondrial RNAs has recently become possible in plants, through organelle targeting of specific trans-cleaving hammerhead ribozymes driven by a shuttle RNA (Fig. 3). The approach is also suitable to target sense sequences, including RNAs associated with cytoplasmic male sterility. Such directed decrease or increase of specific transcript levels in the organelles is expected to highlight potential coordination mechanisms in mitochondrial gene expression and retrograde response. Plant mitochondrial genomes also contain conserved protein genes of unknown function and sequences that might encode regulatory RNAs. Substituting for reverse genetics, trans-ribozyme-mediated knockdown of the RNAs expressed from these putative genes is expected to reveal novel organelle functions

 

import ribozyme

Figure 3. In vivo import of customized RNAs into mitochondria in plant cells (Val et al., 2011, Nucleic Acids Res. 39, 9262-9274). The sequence of interest or "passenger sequence" (PS) is expressed from a nuclear transgene as a 5'-trailor attached to a tRNA mimic (PKTLS) through a structurally neutral linker (L); the chimeric RNA is exported from the nucleus, recognized by specific import factors and translocated into mitochondria through the natural tRNA import pathway. The PKTLS tRNA mimic corresponds to the last 120 nucleotides from the 3'-end of the genomic RNA of the Turnip yellow mosaic virus (TYMV). Both sense (e.g. short coding sequences) and antisense (e.g. trans-cleaving ribozymes) can be used as passenger sequences.

 

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