The PPR domain of mitochondrial RNA polymerase is an exoribonuclease required for mtDNA replication in Drosophila melanogaster

A transcription network underlies the dual genomic coordination of mitochondrial biogenesis

“…After a serial of washes and incubations with primary antibodies, TBST and secondary antibodies, the immunoreactivity was visualized using SuperSignal West Dura Chemiluminescent Substrate (Thermo Fisher Scientific) and Amersham ImageQuant 800 system (Cytiva). The antibodies used were: Mouse anti-Actin antibody (C4, MilliporeSigma), Mouse anti-ATP5A antibody (15H4C4, abcam), Mouse anti-ND30 antibody (17D95, abcam), rabbit anti-TFAM antibody (Liu et al ., 2022), rabbit anti-HSP60 antibody (#4870, Cell Signaling), Anti-rabbit IgG, HRP-linked Antibody (#7074, Cell Signaling) and Anti-mouse IgG, HRP-linked Antibody (#7076, Cell Signaling).…”

“…Hoechst 33342 and DAPI (5 μg/mL, Thermo Fisher Scientific) incubation in PBS for 5 min was used for nuclear staining of live tissues and fixed tissues, respectively. The image processing and quantification were performed by Volocity (Perkin Elmer, for image acquisition), Zen (Zeiss, for image acquisition), Imaris (Oxford Instruments, https://imaris.oxinst.com/, for 3D surface, voxels and intensity statistics) and Fiji / Image J software (NIH, https://fiji.sc/, for image processing and statistics) based on the previous published methods (Liu et al ., 2022; Wang et al ., 2023). The relative TFAM-GFP or TFAM -mNeonGreen level, or relative SDHA-mNeonGreen level was calculated as the ratio of the mean fluorescence intensity in the RNAi or mutant clone to that of its neighboring control, with background correction.…”

“…TFAM, together with other auxiliary factors including mtTFB1 and mtTFB2, promotes the transcription of mtDNA by mitochondrial RNA polymerase (POLRMT) into long polycistronic precursor RNAs, which are further processed into individual RNAs (Chen et al ., 2019; Falkenberg et al, 2002). POLRMT can also generate short RNA oligos, owning to its exoribonuclease activity, to prime mtDNA replication by polymerase γ (Liu et al, 2022). Nuclear-encoded mitochondrial proteins are synthesized in cytoplasm and imported into mitochondria (Wiedemann and Pfanner, 2017).…”

Structural and Molecular Basis for Mitochondrial DNA Replication and Transcription in Health and Antiviral Drug Toxicity

“…However, primase has not been found in mammalian mitochondria, thus making the short RNA primer transcribed by the mitochondrial RNA polymerase (POLRMT) serve as a primer for DNA replication, similar to that of the bacteriophage systems. Although the exact switch mechanism between transcription and replication priming is unknown, it has been suggested that the exonuclease activity in POLRMT is required for replication priming in a fruit fly [ 47 ]. Pol γ begins DNA replication on this RNA primer, creating an RNA-DNA hybrid which has been found in both human and mouse cells [ 48 ].…”