Mitochondria are the major energy-generating cellular organelles and as such are essential to the growth and differentiation of mammalian cells. They are semiautonomous in that they contain their own DNA genomes (mtDNA) which are replicated, transcribed and translationally expressed within the mitochondrial matrix. However, the coding capacity of mtDNA is limited to 13 protein subunits of the respiratory complexes and the rRNAs and tRNAs required for their translation. Thus, the nuclear genome, in addition to specifying the majority of respiratory subunits, provides nearly all of the constituents needed for all other mitochondrial functions including the replication and transcription, of mtDNA. This arrangement necessitates the interplay of nuclear and mitochondrial genetic systems in meeting cellular energy demands. The importance of this interplay is underscored by the numerous human diseases described in recent years that involve defects in mitochondrial respiratory function. The most well-characterized of these result from mutations in the mtDNA itself. In addition, nuclear genes have been implicated in fatal infantile mitochondrial myopathies that result from the depletion of mtDNA. The long-term objectives of this proposal are to understand how two, newly discovered nuclear transcription factors (nuclear respiratory factors 1 and 2; NRF-1 and NRF-2), that act on nuclear genes required for mitochondrial respiratory function, govern nuclear-mitochondrial interactions in mammalian cells. These factors have been implicated in the synthesis of numerous respiratory subunits and in the expression of key components of the mitochondrial transcription and replication machinery. In fact, NRF-1 and NRF-2 are required for expression of the gene encoding mitochondrial transcription factor A, an activator of mtDNA transcription and replication that is essential for the maintenance of mtDNA and respiratory function.
The specific aims are: 1) To define the structural features that govern the biological functions of NRF-1 and NRF-2. The focus will be on identifying the transcription activation domains, nuclear localization signals, and in the case of NRF-1 the in vitro and in vivo sites of phosphorylation. 2) To determine whether the activities or steady-state levels of these factors are altered by agents and conditions known to affect mitochondrial biogenesis or the maintenance of mtDNA. 3) To complete the isolation and characterization of NRF-1 and NRF-2 genes and investigate mechanisms of transcriptional regulation. The primary objectives will be on investigating the use of alternative promoters for transcriptional expression and on the induction of NRF-1 transcription by thyroid hormones. 4) To explore post-transcriptional mechanisms of NRF-1 regulation. NRF-1 is a phosphoprotein in vivo and phosphorylated by a potent kinase activity in vitro. A major focus will be on the functional consequences of these modifications. 5) To determine the physiological consequences of inhibiting NRF expression or biological function in cultured cells. Over-expression of antisense constructs or dominant negative mutants should result in the down regulation of NRF-responsive genes allowing an assessment of cellular phenotype.
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