Mitochondrial Transcription Factor B2 Is Essential for Metabolic Function in Drosophila melanogaster Development

Self Cite

Section: Discussionmentioning

confidence: 99%

Coiled Coil Domain-containing Protein 56 (CCDC56) Is a Novel Mitochondrial Protein Essential for Cytochrome c Oxidase Function

Peralta

Clemente

Sánchez-Martínez

et al. 2012

Self Cite

“…The diminished PRC mRNA expression is associated with a reduction in the mRNAs encoding the mitochondrial transcription factors (TFAM, TFB1M, and TFB2M). The biggest decrease is in TFB2M, whose expression has recently been correlated with the transcription and replication of mtDNA in both human (34) and Drosophila (35) cells. Here, we observe the down-regulation of three mitochondrial transcripts ( Fig.…”

Section: Isolation Of Lentiviral Transductants Of U2os Cells Exhibitimentioning

confidence: 99%

Short Hairpin RNA-mediated Silencing of PRC (PGC-1-related Coactivator) Results in a Severe Respiratory Chain Deficiency Associated with the Proliferation of Aberrant Mitochondria

Vercauteren

Gleyzer

Scarpulla

2009

PRC, a member of the PGC-1 coactivator family, is responsive to serum growth factors and up-regulated in proliferating cells. Here, we investigated its in vivo role by stably silencing PRC expression with two different short hairpin RNAs (shRNA1 and shRNA4) that were lentivirally introduced into U2OS cells. shRNA1 transductants exhibited nearly complete knockdown of PRC protein, whereas shRNA4 transductants expressed PRC protein at ϳ15% of the control level. Complete PRC silencing by shRNA1 resulted in a severe inhibition of respiratory growth; reduced expression of respiratory protein subunits from complexes I, II, III, and IV; markedly lower complex I and IV respiratory enzyme levels; and diminished mitochondrial ATP production. Surprisingly, shRNA1 transductants exhibited a striking proliferation of abnormal mitochondria that were devoid of organized cristae and displayed severe membrane abnormalities. Although shRNA4 transductants had normal respiratory subunit expression and a moderately diminished respiratory growth rate, both transductants showed markedly reduced growth on glucose accompanied by inhibition of G 1 /S cell cycle progression. Microarray analysis revealed striking overlaps in the genes affected by PRC silencing in the two transductants, and the functional identities of these overlapping genes were consistent with the observed mitochondrial and cell growth phenotypes. The consistency between phenotype and PRC expression levels in the two independent transductant lines argues that the defects result from PRC silencing and not from off target effects. These results support a role for PRC in the integration of pathways directing mitochondrial respiratory function and cell growth.Mitochondria are semiautonomous organelles that function as important sites of biological oxidation and ATP production. Central to this function is the electron transport chain and oxidative phosphorylation system, which is composed predominantly of five multisubunit protein complexes embedded in the mitochondrial inner membrane (1, 2). Mitochondria contain their own genetic system directed by a covalently closed circular DNA genome whose entire protein coding capacity is devoted to the production of 13 protein subunits of respiratory complexes I, III, IV, and V. Although essential, these subunits account for only a fraction of the protein composition with the majority of the respiratory subunits of nuclear origin (3, 4). Its bigenomic expression makes the respiratory apparatus unique among mitochondrial oxidative functions and poses an important biological problem in understanding the coordination of nuclear and mitochondrial gene expression.A number of nucleus-encoded regulatory factors have been associated with the transcriptional control of both nuclear and mitochondrial genes that specify the respiratory apparatus in mammalian systems. Initial work identified nuclear respiratory factors, NRF-1 and NRF-2(GABP), as activators of nuclear cytochrome c and cytochrome oxidase genes. These factors have subsequently been ass...

“…h-mtTFB2 has been reported to have substantially higher, transcription-stimulating activity in vitro than h-mtTFB1 (21). However, studies in vivo only support a role for mtTFB2 in transcription, with mtTFB1 contributing to translation (23)(24)(25)(26). Recently, it was shown that the amino-terminal region of h-mtTFB2 interacts with the TSS, stabilizing the melted promoter and the initiating nucleotide (27).…”

mentioning

confidence: 99%

Identification of Multiple Rate-limiting Steps during the Human Mitochondrial Transcription Cycle in Vitro

Lodeiro

Uchida

Arnold

et al. 2010

We have reconstituted human mitochondrial transcription in vitro on DNA oligonucleotide templates representing the light strand and heavy strand-1 promoters using protein components (RNA polymerase and transcription factors A and B2) isolated from Escherichia coli. We show that 1 eq of each transcription factor and polymerase relative to the promoter is required to assemble a functional initiation complex. The light strand promoter is at least 2-fold more efficient than the heavy strand-1 promoter, but this difference cannot be explained solely by the differences in the interaction of the transcription machinery with the different promoters. In both cases, the rate-limiting step for production of the first phosphodiester bond is open complex formation. Open complex formation requires both transcription factors; however, steps immediately thereafter only require transcription factor B2. The concentration of nucleotide required for production of the first dinucleotide product is substantially higher than that required for subsequent cycles of nucleotide addition. In vitro, promoter-specific differences in post-initiation control of transcription exist, as well as a second rate-limiting step that controls conversion of the transcription initiation complex into a transcription elongation complex. Rate-limiting steps of the biochemical pathways are often those that are targeted for regulation. Like the more complex multisubunit transcription systems, multiple steps may exist for control of transcription in human mitochondria. The tools and mechanistic framework presented here will facilitate not only the discovery of mechanisms regulating human mitochondrial transcription but also interrogation of the structure, function, and mechanism of the complexes that are regulated during human mitochondrial transcription.Expression and maintenance of the mitochondrial genome (mtDNA) are essential for mitochondria to produce energy, function in other aspects of intermediary metabolism, contribute to pathogen sensing, and integrate into signal-transduction pathways, especially those regulated by circadian rhythm and nutritional status (1, 2). Mitochondrial dysfunction caused by defects in mtDNA replication and translation attributable to mutations in mitochondrial and nuclear genes contributes to numerous diseases, including muscular dystrophies, cardiac diseases, neurodegenerative disorders, diabetes, and certain cancers (3)(4)(5)(6)(7)(8). A link between the cis-and trans-acting factors required for mitochondrial transcription and disease has yet to be established, likely because of limited information on the mechanism and regulation of mitochondrial transcription (1, 4, 9 -11).