An overview of biochemical and imaging tools for investigating mitochondrial gene expression. Mitochondrial DNA (A) mt-ZFN and mt-TALEN use a zinc-finger DNA-binding domain and transcription activator-like elements, respectively, and each links to a DNA-cleavage domain of the restriction enzyme, Fok1. (B) EtBr or SyBr binds to double-stranded mt-DNA, whereas BrdU and EdU can incorporate into mt-DNA during mt-DNA replication, which allows intact mt-DNA to be visualized in live cells. (C) An mt-DNA specific sequence can be detected by using a FISH probe. (D) GFP-tagged TFAM is also used to visualize mt-DNA in live cells. Mitochondrial RNA (E) Three possible mechanisms of RNA transport into mitochondrial matrix are suggested: (1) PNPASE transports RNA of RNase P, RNAs of mitochondrial ribosomal proteins, and 5S rRNAs by recognizing the stem-loop structure of their RNAs; (2) miRNA can be delivered to mitochondria by Ago2; (3) PNPASE also translocates pre-miRNA into mitochondria. (F) RNA recognition motif domains of human PUMILIO1 are tagged with split fragments of EGFP. When the two split EGFPs are reassembled by binding to ND6 mRNA, EGFP signals appear and the dynamics of ND6 mRNA can be visualized. Mitochondrial protein (G) Mitochondria-specific metabolic labeling can be achieved by using isotope or non-canonical amino acids while inhibiting cytoplasmic translation. Isotope-labeled mitochondrial proteins are separated by SDS-PAGE. Azide-conjugated non-canonical amino acids are detected by biotin or a fluorescent dye through click reaction.
