We previously revealed that a strong selection process exists that limits the transmission of deleterious mtDNA mutation to the next generation. We also discovered that the translational boost on the mitochondrial outer membrane supports the prodigious mtDNA replication in the ovary, which is essential for mtDNA selective inheritance. Based on these studies, we proposed that a series of mitochondrial behaviors in developing oocytes are critical for mtDNa selective inheritance. (1), mtDNA segregation i.e. mtDNA must be effectively segregated and sorted into individual organelles to minimize the intra-organelle complementation. (2), Mitochondrial activation, namely germ cells use the OXPHOS system as a stress test for the integrity of mitochondrial genome, to distinguish healthy mitochondria containing wild type mtDNA from defective ones containing mtDNA mutants. (3) The selective replication of mtDNA. In particular, germ cells have to recognize healthy mitochondria and selectively promote mtDNA replication within these organelles. We have been testing these three predictions in order to further our understanding of mtDNA inheritance and selection. We previously demonstrated that the local protein synthesis mediated by MDI-Larp complex on the mitochondrial surface promotes mtDNA replication in ovary. We also found the local protein synthesis, just like mtDNA replication, depends on mitochondrial fitness as well. Additionally, mtDNA selection was greatly compromised in mdi mutant ovary that lack the local protein synthesis. These observations suggest that MDI-Larp complexes may sense the mitochondrial fitness and selectively boosts protein synthesis on healthy mitochondria specifically. Interestingly, over expression of Tom20-Larp, that constitutively localized on mitochondrial outer membrane partially restored the local protein synthesis and mtDNA replication in mt:CoIT300I background. More importantly, this promiscuous translational boost blunted the selective inheritance. Mechanistically, Larp is phosphorylated and inhibited by PINK1 that accumulates on de-energized mitochondria in mt:CoIT300I ovary. The selective mtDNA inheritance was impaired in PINK1 mutant ovary. Furthermore, overexpression of PINK1 restored the decreased mtDNA selection in Tom20-Larp overexpression but not the Tom20-LarpS1119A overexpression ovary. Our results suggest that PINK1 inhibits MDI/Larps activity and suppresses the local protein synthesis on the outer membrane of de-energized mitochondria. This regulation allows selectively propagation of healthy mitochondria carrying wild type genome and thereby limits the transmission of deleterious mtDNA mutations. Several mechanisms have been proposed to limit the transmission of mtDNA mutations. Besides the model of selective propagation of healthy organelle, mitochondrial bottleneck mediated selection on the organismal level remains to be the dominant dogma. Additoanlly, selective transportation of healthy mitochondria to Balbiani body has also been considered as mechanism of purifying selection. It remains to be clarified which of these mechanisms are truly invloved in the mtDNA selective inheritance. We determined the mtDNA segregation unit based on both the mathematic modeling and direct quantification of mtDNA nucleoids number through the germline development. We concluded that there is no effective mitochondrial bottleneck in Drosophila, nor the pattern of mtDNA transmission supports the selection on the organismal level. On the contrary, the purifying selection acts at organelle level. We provided direct experimental evidences that selective propagation of functional mtDNA at specific stage of Drosophila oogenesis plays a major role in mtDNA selective inheritance. Another purifying selection mechanism, which involves the selective transportation of healthy mitochondria to the structure of Balbiani body is also existed, but play a limited role in mtDNA selection. These two purifying selection mechanisms could act synergistically to ensure the transmission of healthy mitochondria to the next generation. mtDNA mutations often disrupt the ETC complexes and consequently render mitochondria less active. The functional expression of mtDNA might therefore be a prerequisite for cells to recognize mtDNA mutations. We found that mitochondria in germline stem cells and proliferating cysts were not active, judging from a reduced mitochondrial membrane potential and a lack of cytochrome C oxidase (COX) activity. Interestingly, mitochondria became active in the 16-cell cyst in the germarium, prior to the initiation of selective mtDNA replication of healthy mitochondria. We hypothesized that mitochondrial activation (or mtDNA expression) might serve as a stress test, to distinguish healthy mitochondria carrying the wild type genome from defective ones carrying mutated mtDNA. We conducted a candidate RNAi screen in the to identify genes required for mitochondrial activation prior to oocyte determination. We found that dMyc, downstream insulin/TORC signaling is required for mitochondrial activation in region 2B. Importantly, ectopic expression of dMyc in region 2A induce mtDNA replication and the activation of ETC complexes. We further demonstrated dPGC-1 and CG12909, a novel transcriptional factor is downstream of dMyc. and required for mitochondrial biogenesis. We are now trying to identify the targets of CG12909 and to order a hierarchic scheme for a transcriptional regulation of mitochondrial biogenesis in this highly proliferative and growing tissue. To identify novel genes involved in the regulation of mtDNA replication, we conducted an in vivo RNAi screen in Drosophila. We recoverd a novel gene, CG3862 that is essential for mtDNA replication and transcription. CG3862 mutant has severe developmental delay and is lethal at a late larva stage. Loss of CG3862 results in impaired mitochondrial oxidative phosphorylation activity, and causes a severe reduction in cell proliferation, but does not affect cell growth or apoptosis. The human homolog, WBSCR16, appears to be required for mtDNA replication and transcription as well. Knocking down WBSCR16 expression in human tissue culture significantly slows down the mtDNA replication rate and blocks the mtDNA transcription. Our work identifies CG3862/WBSCR16 as a novel factor that is essential for mitochondrial genome replication and transcription. Further characterization of CG3862 mutant and its human homolog may provide insight into the mechanism of regulation of mtDNA replication and transcription.
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