Implication of G-quadruplexes in mitochondrial gene expression and genome replication

Single-stranded DNA or RNA sequences rich in guanine (G) can adopt non-canonical structures known as G-quadruplexes (G4). G4 in the mitochondrial genome are heavy-strand enriched and have been associated with the formation of deletion breakpoints that cause mitochondrial diseases. However, the functional role of G4 structures in mitochondria remains unclear. Here, we have identified RHPS4 as a G4-specific ligand that localizes to mitochondria and causes replication pausing, with mitochondrial DNA (mtDNA) depletion occurring at higher dosage. We further show that RHPS4 interferes with mitochondrial transcript elongation at low doses, leading to respiratory complex depletion. These unprecedented observations suggest that G4 motifs modulate mitochondrial transcription and replication efficiency. Using the differential effects of high vs low RHPS4 dosing, we characterized gene expression pathway responses to mitochondrial transcription inhibition or mitochondrial genome depletion. Importantly, a human mtDNA mutation that increases G4 formation potential strongly enhanced the RHPS4-mediated mitochondrial respiratory defect. We propose that abnormal G4 dynamics may contribute to mtDNA instability and gene expression defects, particularly in the presence of mitochondrial mutations that enhance the G4 formation. Total RNA was extracted from the mouse embryonic fibroblasts (MEFs) stimulated with 0um (n=3), 2um (n=3), and 10um (n=2) RHPS4. Total stranded RNA libraries (ribo-depleted) were generated and sequenced on the Illumina NextSeq 500 NGS platform. RNA-seq data was analyzed for differentially expressed genes between groups of samples.

富含鸟嘌呤（G）的单链DNA或RNA序列可形成一类被称为G-四链体（G-quadruplexes, G4）的非经典二级结构。线粒体基因组中的G4富集于重链，且与引发线粒体疾病的缺失断点形成密切相关。然而，线粒体中G4结构的功能角色仍未明确。本研究鉴定出RHPS4是一种G4特异性配体，其可定位于线粒体并引发复制停滞；高剂量使用时会导致线粒体DNA（mitochondrial DNA, mtDNA）耗竭。进一步研究表明，低剂量RHPS4会干扰线粒体转录延伸过程，进而引发呼吸链复合体耗竭。这些前所未有的观测结果提示，G4基序可调控线粒体的转录与复制效率。研究人员借助高低剂量RHPS4产生的差异化效应，表征了线粒体转录抑制或线粒体基因组耗竭状态下的基因表达通路响应。值得注意的是，一种可增强G4形成潜力的人类mtDNA突变，会显著加剧RHPS4介导的线粒体呼吸功能缺陷。本研究提出，异常的G4动态变化可能会促成mtDNA不稳定与基因表达缺陷，尤其在存在可增强G4形成的线粒体突变的场景中。研究团队从经0μM（n=3）、2μM（n=3）及10μM（n=2）RHPS4处理的小鼠胚胎成纤维细胞（mouse embryonic fibroblasts, MEFs）中提取总RNA。随后构建链特异性总RNA文库（经核糖体RNA去除处理），并在Illumina NextSeq 500下一代测序（next-generation sequencing, NGS）平台上完成测序。针对各组样本间的差异表达基因，开展了RNA-seq数据分析。