Mid-Term Report Summary - GENEMIT (Regulation of gene expression in mammalian mitochondria)
The mitochondrial network produces the bulk part of the energy mammalian cells need through the oxidative phosphorylation process. The importance of mitochondria is underscored by the fact that deficient oxidative phosphorylation causes a wide number of genetic diseases. It is also heavily implicated in age-associated disease and aging. Expression of mammalian mtDNA is of key importance for maintaining mitochondrial oxidative phosphorylation, however, studies of the poorly understood regulation of mammalian mitochondrial gene expression offer substantial experimental challenges.
In the late 1990s, we felt that the challenges associated with understanding mammalian mtDNA gene expression could only be tackled by a broad interdisciplinary scientific approach. We therefore started collaboration between the Larsson (expertise in mouse genetics, in vivo physiology and cell biology) and Gustafsson laboratories (expertise in biochemistry, recombinant in vitro systems, structural biology) that has been ongoing for more than a decade. The success of this collaborative effort is documented by many joint publications in high-profile journals.
In this joint ERC project, we have made a joint effort to investigate how mtDNA expression is regulated in mammalian cells. An important part of our work has been to establish how the different levels of regulation of mtDNA gene expression communicate to achieve a coordinated response to physiological stimuli.
The work completed so far has been very successful. We have established a new saturation mutagenesis approach to identify mtDNA sequence variants that are essential for germ line transmission. This approach has helped us address the hotly debated subject of the mechanisms of mitochondrial DNA replication (Wanrooij et al, EMBO reports, 2012.). By studying mice transmitting a heteroplasmic single-base-pair deletion in the mitochondrial tRNAMet gene, we have also demonstrated that the extent of mammalian mtDNA heteroplasmy is principally determined prenatally within the developing female germline (Freyer et al, Nat Genet, 2012). We have also demonstrated that there is no recombination of mammalian mtDNA (Hagstrom et al, Nucleic Acids Res 2013).
Our work has also led to important insights into the mechanisms that regulate mtDNA gene expression in vivo. We are currently finalizing our analysis of a mitochondrial RNA polymerase knockout mouse model (Kühl et al, manuscript in preparation, Kühl et al, manuscript submitted). We have also demonstrated that the mitochondrial transcription factor TFAM is absolutely required to recruit the transcription machinery during initiation of transcription (Shi et al, PNAS 2012; Posse et al, Nucleic Acids Res 2013). Furthermore, we have demonstrated that a mitochondrial transcription factor, MTERF1, blocks antisense transcription of the ribosomal transcription unit and prevents transcription interference at the light strand promoter (Terzioglu et al, 2012, Cell Metabolism). We have also established a new mechanism for transcription termination in mammalian mitochondria. This work has been successful and resulted in two published papers of which the latest was supported by the GENEMIT grant (Wanrooij et al, PNAS 2010; Wanrooij et al, NAR 2012).
In other work, we have found that there is a molecular crosstalk between transcription and translation for the regulation of mammalian mtDNA gene expression and that this crosstalk, at least in part, is mediated by the mitochondrial transcription and translation factor MTERF3 (Wredenberg et al, 2013, PLoS Genetics). Another component of this crosstalk machinery is MTERF4 and our work has led to an atomic structure of MTERF4 in complex with a ribosomal RNA modifying factor, NSUN4. The MTERF4-NSUN4 structure has provided a molecular basis for future characterization of the highly conserved function of MTERF4 in ribosomal biogenesis (Spåhr et al, PNAS 2012).
Finally, we have gained important insights into the molecular mechanisms that regulate mRNA stability in human mitochondria. We have identified the LRPPRC protein as an key regulator of mitochondrial mRNA (Ruzzente et al, EMBO J 2011; Bratic et al, PLoS Genet 2011; Harmel et al., J Biol Chem, 2013).
In the late 1990s, we felt that the challenges associated with understanding mammalian mtDNA gene expression could only be tackled by a broad interdisciplinary scientific approach. We therefore started collaboration between the Larsson (expertise in mouse genetics, in vivo physiology and cell biology) and Gustafsson laboratories (expertise in biochemistry, recombinant in vitro systems, structural biology) that has been ongoing for more than a decade. The success of this collaborative effort is documented by many joint publications in high-profile journals.
In this joint ERC project, we have made a joint effort to investigate how mtDNA expression is regulated in mammalian cells. An important part of our work has been to establish how the different levels of regulation of mtDNA gene expression communicate to achieve a coordinated response to physiological stimuli.
The work completed so far has been very successful. We have established a new saturation mutagenesis approach to identify mtDNA sequence variants that are essential for germ line transmission. This approach has helped us address the hotly debated subject of the mechanisms of mitochondrial DNA replication (Wanrooij et al, EMBO reports, 2012.). By studying mice transmitting a heteroplasmic single-base-pair deletion in the mitochondrial tRNAMet gene, we have also demonstrated that the extent of mammalian mtDNA heteroplasmy is principally determined prenatally within the developing female germline (Freyer et al, Nat Genet, 2012). We have also demonstrated that there is no recombination of mammalian mtDNA (Hagstrom et al, Nucleic Acids Res 2013).
Our work has also led to important insights into the mechanisms that regulate mtDNA gene expression in vivo. We are currently finalizing our analysis of a mitochondrial RNA polymerase knockout mouse model (Kühl et al, manuscript in preparation, Kühl et al, manuscript submitted). We have also demonstrated that the mitochondrial transcription factor TFAM is absolutely required to recruit the transcription machinery during initiation of transcription (Shi et al, PNAS 2012; Posse et al, Nucleic Acids Res 2013). Furthermore, we have demonstrated that a mitochondrial transcription factor, MTERF1, blocks antisense transcription of the ribosomal transcription unit and prevents transcription interference at the light strand promoter (Terzioglu et al, 2012, Cell Metabolism). We have also established a new mechanism for transcription termination in mammalian mitochondria. This work has been successful and resulted in two published papers of which the latest was supported by the GENEMIT grant (Wanrooij et al, PNAS 2010; Wanrooij et al, NAR 2012).
In other work, we have found that there is a molecular crosstalk between transcription and translation for the regulation of mammalian mtDNA gene expression and that this crosstalk, at least in part, is mediated by the mitochondrial transcription and translation factor MTERF3 (Wredenberg et al, 2013, PLoS Genetics). Another component of this crosstalk machinery is MTERF4 and our work has led to an atomic structure of MTERF4 in complex with a ribosomal RNA modifying factor, NSUN4. The MTERF4-NSUN4 structure has provided a molecular basis for future characterization of the highly conserved function of MTERF4 in ribosomal biogenesis (Spåhr et al, PNAS 2012).
Finally, we have gained important insights into the molecular mechanisms that regulate mRNA stability in human mitochondria. We have identified the LRPPRC protein as an key regulator of mitochondrial mRNA (Ruzzente et al, EMBO J 2011; Bratic et al, PLoS Genet 2011; Harmel et al., J Biol Chem, 2013).