Abstract
Mitochondrial DNA (mtDNA) is maintained within nucleoprotein complexes known as nucleoids. These structures are highly condensed by the DNA packaging protein, mitochondrial Transcription Factor A (TFAM). Nucleoids also include RNA, RNA:DNA hybrids, and are associated with proteins involved with RNA processing and mitochondrial ribosome biogenesis. Here we characterize the ability of TFAM to bind various RNA containing substrates in order to determine their role in TFAM distribution and function within the nucleoid. We find that TFAM binds to RNA-containing 4-way junctions but does not bind appreciably to RNA hairpins, internal loops, or linear RNA:DNA hybrids. Therefore the RNA within nucleoids largely excludes TFAM, and its distribution is not grossly altered with removal of RNA. Within the cell, TFAM binds to mitochondrial tRNAs, consistent with our RNA 4-way junction data. Kinetic binding assays and RNase-insensitive TFAM distribution indicate that DNA remains the preferred substrate within the nucleoid. However, TFAM binds to tRNA with nanomolar affinity and these complexes are not rare. TFAM-immunoprecipitated tRNAs have processed ends, suggesting that binding is not specific to RNA precursors. The amount of each immunoprecipitated tRNA is not well correlated with tRNA celluar abundance, indicating unequal TFAM binding preferences. TFAM-mt-tRNA interaction suggests potentially new functions for this protein.
Highlights
Mitochondria are involved in numerous cellular functions, including many biosynthetic pathways, calcium signaling, apoptosis, innate immune responses, and production of ATP through oxidative phosphorylation (OXPHOS)
With regard to RNA substrates, we demonstrate that TFAM is unable to bind to linear RNA:DNA hybrids, double-stranded RNA, or the singlestranded RNA substrates poly or poly(rU) (Fig 1C–1F)
We have characterized the ability of TFAM to bind to various RNA containing substrates in order to elucidate the potential roles of this protein in both nucleoid structure and mitochondrial RNA function
Summary
Mitochondria are involved in numerous cellular functions, including many biosynthetic pathways, calcium signaling, apoptosis, innate immune responses, and production of ATP through oxidative phosphorylation (OXPHOS). Most mitochondrial proteins are encoded in the nucleus and imported into the mitochondria, this organelle contains its own genome. Mitochondrial DNA (mtDNA) encodes 13 proteins that are essential for OXPHOS, and includes genes for two rRNAs and the 22 tRNAs that are needed for protein translation on mitochondrial ribosomes. The mtDNA exists in a nucleoprotein complex known as the mitochondrial nucleoid.
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