Paola Loguercio Polosa

Efficient mitochondrial biogenesis drives incomplete penetrance in Leber’s hereditary optic neuropathy

The mechanisms of incomplete penetrance in Leber’s hereditary optic neuropathy are elusive. Giordano et al. show that mitochondrial DNA content and mitochondrial mass are both increased in tissues and cells from unaffected mutation carriers relative to affected relatives and control individuals. Upregulation of mitochondrial biogenesis may represent a therapeutic target.

The MTERF family proteins: mitochondrial transcription regulators and beyond.

The MTERF family is a wide protein family, identified in Metazoa and plants, which consists of 4 subfamilies named MTERF1-4. Proteins belonging to this family are localized in mitochondria and show a modular architecture based on repetitions of a 30 amino acid module, the mTERF motif, containing leucine zipper-like heptads. The MTERF family includes the characterized transcription termination factors human mTERF, sea urchin mtDBP and Drosophila DmTTF. In vitro and in vivo studies show that these factors play different roles which are not restricted to transcription termination, but concern also transcription initiation and the control of mtDNA replication. The multiplicity of functions could be related to the differences in the gene organization of the mitochondrial genomes. Studies on the function of human and Drosophila MTERF3 factor showed that the protein acts as negative regulator of mitochondrial transcription, possibly in cooperation with other still unknown factors. The complete elucidation of the role of the MTERF family members will contribute to the unraveling of the molecular mechanisms of mtDNA transcription and replication.

NSUN4 Is a Dual Function Mitochondrial Protein Required for Both Methylation of 12S rRNA and Coordination of Mitoribosomal Assembly

Biogenesis of mammalian mitochondrial ribosomes requires a concerted maturation of both the small (SSU) and large subunit (LSU). We demonstrate here that the m5C methyltransferase NSUN4, which forms a complex with MTERF4, is essential in mitochondrial ribosomal biogenesis as mitochondrial translation is abolished in conditional Nsun4 mouse knockouts. Deep sequencing of bisulfite-treated RNA shows that NSUN4 methylates cytosine 911 in 12S rRNA (m5C911) of the SSU. Surprisingly, NSUN4 does not need MTERF4 to generate this modification. Instead, the NSUN4/MTERF4 complex is required to assemble the SSU and LSU to form a monosome. NSUN4 is thus a dual function protein, which on the one hand is needed for 12S rRNA methylation and, on the other hand interacts with MTERF4 to facilitate monosome assembly. The presented data suggest that NSUN4 has a key role in controlling a final step in ribosome biogenesis to ensure that only the mature SSU and LSU are assembled.

Cross-strand binding of TFAM to a single mtDNA molecule forms the mitochondrial nucleoid

Significance Altered expression of mitochondrial DNA (mtDNA) is heavily implicated in human disease and aging, but the basic organizational unit of mtDNA, the mitochondrial nucleoid, is poorly understood. Here, we have used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mammalian mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Furthermore, we show that the nucleoid ultrastructure is independent of cellular mtDNA copy number and that the core nucleoid structure is formed by cross-strand binding of mitochondrial transcription factor A (TFAM) to a single copy of mtDNA. The clarification of the ultrastructure of the mammalian mitochondrial nucleoid provides the fundamental basis for the understanding of regulation of mtDNA maintenance and expression in mammals. Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.

MTERF3 Regulates Mitochondrial Ribosome Biogenesis in Invertebrates and Mammals

Regulation of mitochondrial DNA (mtDNA) expression is critical for the control of oxidative phosphorylation in response to physiological demand, and this regulation is often impaired in disease and aging. We have previously shown that mitochondrial transcription termination factor 3 (MTERF3) is a key regulator that represses mtDNA transcription in the mouse, but its molecular mode of action has remained elusive. Based on the hypothesis that key regulatory mechanisms for mtDNA expression are conserved in metazoans, we analyzed Mterf3 knockout and knockdown flies. We demonstrate here that decreased expression of MTERF3 not only leads to activation of mtDNA transcription, but also impairs assembly of the large mitochondrial ribosomal subunit. This novel function of MTERF3 in mitochondrial ribosomal biogenesis is conserved in the mouse, thus we identify a novel and unexpected role for MTERF3 in coordinating the crosstalk between transcription and translation for the regulation of mammalian mtDNA gene expression.

The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription

DmTTF is a Drosophila mitochondrial DNA-binding protein, which recognizes two sequences placed at the boundary of clusters of genes transcribed in opposite directions. To obtain in vivo evidences on the role of DmTTF, we characterized a DmTTF knock-down phenotype obtained by means of RNA interference in D.Mel-2 cells. By a combination of RNase protection and real-time RT–PCR experiments we found that knock-down determines remarkable changes in mitochondrial transcription. In particular, protein depletion increases not only the level of (+) and (−)strand RNAs mapping immediately after of the two protein-binding site, but also that of transcripts located further downstream. Unexpectedly, depletion of the protein also causes the decrease in the content of those transcripts mapping upstream of the protein target sites, including the two rRNAs. The changes in transcript level do not depend on a variation in mitochondrial DNA (mtDNA) content, since mtDNA copy number is unaffected by DmTTF depletion. This work shows conclusively that DmTTF arrests in vivo the progression of the mitochondrial RNA polymerase; this is the first ever-obtained evidence for an in vivo role of an animal mitochondrial transcription termination factor. In addition, the reported data provide interesting insights into the involvement of DmTTF in transcription initiation in Drosophila mitochondria.

Nuclear respiratory factor 2 induces the expression of many but not all human proteins acting in mitochondrial DNA transcription and replication.

In mammals, NRF-2 (nuclear respiratory factor 2), also named GA-binding protein, is an Ets family transcription factor that controls many genes involved in cell cycle progression and protein synthesis as well as in mitochondrial biogenesis. In this paper, we analyzed the role of NRF-2 in the regulation of human genes involved in mitochondrial DNA transcription and replication. By a combination of bioinformatic and biochemical approaches, we found that the factor binds in vitro and in vivo to the proximal promoter region of the genes coding for the transcription termination factor mTERF, the RNA polymerase POLRMT, the B subunit of the DNA polymerase-γ, the DNA helicase TWINKLE, and the single-stranded DNA-binding protein mtSSB. The role of NRF-2 in modulating the expression of those genes was further established by RNA interference and overexpression strategies. On the contrary, we found that NRF-2 does not control the genes for the subunit A of DNA polymerase-γ and for the transcription repressor MTERF3; we suggest that these genes are under regulatory mechanisms that do not involve NRF proteins. Since NRFs are known to positively control the expression of transcription-activating proteins, the novelty emerging from our data is that proteins playing antithetical roles in mitochondrial DNA transcription, namely activators and repressors, are under different regulatory pathways. Finally, we developed a more stringent consensus with respect to the general consensus of NRF-2/GA-binding protein when searching for NRF-2 binding sites in the promoter of mitochondrial proteins.

Mapping and characterization of Paracentrotus lividus mitochondrial transcripts: multiple and overlapping transcription units.

SummaryThis paper reports the mapping of both mature and precursor Paracentrotus lividus mitochondrial transcripts. Several mtRNAs were found to have 5′ and 3′ termini which differ from those inferred through DNA sequencing (Cantatore et al. 1989). The 3′ ends of the two rRNAs (12S and 16S) overlap with the downstream transcripts (tRNAGlu and CoI mRNA) by 5 and 10 nt respectively. The 132nt non-coding region is extensively transcribed: in particular it contains a 124nt RNA and the 5′ end of a possible precursor of 13 clustered tRNAs. This latter overlaps by 7nt with the 3′ end of the 124nt RNA. In addition to the mature RNAs, 32 high molecular weight RNAs, which are probably the precursors of the smaller more abundant mature species, were detected by Northern blotting. The mapping of these transcripts indicates that they are processed at the level of tRNA or tRNA-like sequences and suggests the existence of two transcription initiation sites upstream of the ND1 and the cytochrome b genes respectively. In the light of these results it appears that P. lividus mitochondrial DNA transcription takes place via multiple and probably overlapping transcription units. Moreover, the wide variation in the steady-state levels of the mature mRNAs indicates that sea urchin mitochondrial DNA expression is also regulated at the level of RNA decay.

Quantitation of mitochondrial RNA species during rat liver development: the concentration of cytochrome oxidase subunit I (CoI) mRNA increases at birth.

A quantitative study on the concentration of mitochondrial DNA and two species of mtRNA, the ribosomal (16S rRNA) and messenger (CoI mRNA) has been carried out in rat liver between -3 and 14 days of age. The cellular content of mitochondrial DNA begins to increase at one day of life and goes up linearly until 14 days of age. The cellular level of 16S rRNA and CoI mRNA changes during development: the 16S rRNA increases linearly after birth, whereas CoI mRNA shows a peak at birth and thereafter remains more or less constant. The concentration of 16S rRNA per mitochondrial DNA molecule remains substantially unchanged during development, whereas that of CoI mRNA increases before birth and, at birth, reaches values higher than in adults. These results support an independent regulation of mitochondrial rRNA and mRNA level in rat liver mitochondria during development.

Contrahelicase activity of the mitochondrial transcription termination factor mtDBP

The sea urchin mitochondrial D-loop binding protein (mtDBP) is a transcription termination factor that is able to arrest bidirectionally mitochondrial RNA chain elongation. The observation that the mtDBP binding site in the main non-coding region is located in correspondence of the 3′ end of the triplex structure, where the synthesis of heavy strand mitochondrial (mt) DNA is either prematurely terminated or allowed to continue, raised the question whether mtDBP could also regulate mtDNA replication. By using a helicase assay in the presence of the replicative helicase of SV40, we show that mtDBP is able to inhibit the enzyme thus acting as a contrahelicase. The impairing activity of mtDBP is bidirectional as it is independent of the orientation of the protein binding site. The inhibition is increased by the presence of the guanosine-rich sequence that flanks mtDBP binding site. Finally, a mechanism of abrogation of mtDBP contrahelicase activity is suggested that is based on the dissociation of mtDBP from DNA caused by the passage of the RNA polymerase through the protein–DNA complex. All these findings favour the view that mtDBP, besides serving as transcription termination factor, could also act as a negative regulator of mtDNA synthesis at the level of D-loop expansion.