Abstract
Single-stranded DNA or RNA sequences rich in guanine (G) can adopt non-canonical structures known as G-quadruplexes (G4). Mitochondrial DNA (mtDNA) sequences that are predicted to form G4 are enriched on the heavy-strand and have been associated with formation of deletion breakpoints. Increasing evidence supports the ability of mtDNA to form G4 in cancer cells; however, the functional roles of G4 structures in regulating mitochondrial nucleic acid homeostasis in non-cancerous cells remain unclear. Here, we demonstrate by live cell imaging that the G4-ligand RHPS4 localizes primarily to mitochondria at low doses. We find that low doses of RHPS4 do not induce a nuclear DNA damage response but do cause an acute inhibition of mitochondrial transcript elongation, leading to respiratory complex depletion. We also observe that RHPS4 interferes with mtDNA levels or synthesis both in cells and isolated mitochondria. Importantly, a mtDNA variant that increases G4 stability and anti-parallel G4-forming character shows a stronger respiratory defect in response to RHPS4, supporting the conclusion that mitochondrial sensitivity to RHPS4 is G4-mediated. Taken together, our results indicate a direct role for G4 perturbation in mitochondrial genome replication, transcription processivity, and respiratory function in normal cells.
Highlights
Mitochondria are flexible organelles that play a crucial metabolic role in stress and disease resilience
Supporting the notion that G4 stabilization would interfere with replication, we found that 2 μM RHPS4 treatment reduced the ratio of circular to linear Mitochondrial DNA (mtDNA) content (Fig. 1d,e; Supplementary Dataset S2), a feature previously observed in dideoxycytosine and hydrogen peroxide-induced mtDNA damage and depletion[27]
To exclude that the mtDNA depletion observed at 2 μM RHPS4 was caused by induction of nuclear DNA damage response (DDR), we investigated the effect of RHPS4 on mouse embryonic fibroblast cell lines (MEFs) viability (Fig. 1f), telomere length (Supplementary Fig. S2a) and γ-H2AX abundance; the latter is a well-established marker of nuclear DNA damage[29] (Fig. 1g, Supplementary Fig. S2b and Supplementary Dataset S3)
Summary
Mitochondria are flexible organelles that play a crucial metabolic role in stress and disease resilience. In mtDNA, there are many other non-randomly distributed and well-conserved sequences with G4-forming potential, which compelled further investigation into their potential biological impact on mitochondrial function To this end, we sought to identify approaches that alter mitochondrial G-quadruplex stability and assess the effect on mtDNA metabolism and respiratory function in normal cells[15]. Leveraging the ability of RHPS4 to cause transcription or replication defects depending on concentration, we identify novel gene expression pathways that distinguish between these processes Together, these results strengthen the notion that mitochondrial G-quadruplex dysregulation affects mitochondrial nucleic acid synthesis and mitochondrial function beyond association with mtDNA deletion formation
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