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G-quadruplex dynamics contribute to regulation of mitochondrial gene expression.

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.

SUBMITTER: Falabella M

PROVIDER: S-EPMC6447596 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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G-quadruplex dynamics contribute to regulation of mitochondrial gene expression.

Falabella M M Kolesar J E JE Wallace C C de Jesus D D Sun L L Taguchi Y V YV Wang C C Wang T T Xiang I M IM Alder J K JK Maheshan R R Horne W W Turek-Herman J J Pagano P J PJ St Croix C M CM Sondheimer N N Yatsunyk L A LA Johnson F B FB Kaufman B A BA

Scientific reports 20190403 1

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 unclea ...[more]

PMID: 30944353

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Project description:Guanine (G)-rich sequences in RNA can fold into diverse RNA G-quadruplex (rG4) structures to mediate various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4s in prokaryotes are still elusive. We used QUMA-1, an rG4-specific fluorescent probe, to detect rG4 structures in a wide range of bacterial species both in vitro and in live cells and found rG4 to be an abundant RNA secondary structure across those species. Subsequently, to identify bacterial rG4 sites in the transcriptome, the model Escherichia coli strain and a major human pathogen, Pseudomonas aeruginosa, were subjected to recently developed high-throughput rG4 structure sequencing (rG4-seq). In total, 168 and 161 in vitro rG4 sites were found in E. coli and P. aeruginosa, respectively. Genes carrying these rG4 sites were found to be involved in virulence, gene regulation, cell envelope synthesis, and metabolism. More importantly, biophysical assays revealed the formation of a group of rG4 sites in mRNAs (such as hemL and bswR), and they were functionally validated in cells by genetic (point mutation and lux reporter assays) and phenotypic experiments, providing substantial evidence for the formation and function of rG4s in bacteria. Overall, our study uncovers important regulatory functions of rG4s in bacterial pathogenicity and metabolic pathways and strongly suggests that rG4s exist and can be detected in a wide range of bacterial species.IMPORTANCE G-quadruplex in RNA (rG4) mediates various biological functions and cellular processes in eukaryotic organisms. However, the presence, locations, and functions of rG4 are still elusive in prokaryotes. Here, we found that rG4 is an abundant RNA secondary structure across a wide range of bacterial species. Subsequently, the transcriptome-wide rG4 structure sequencing (rG4-seq) revealed that the model E. coli strain and a major human pathogen, P. aeruginosa, have 168 and 161 in vitro rG4 sites, respectively, involved in virulence, gene regulation, cell envelope, and metabolism. We further verified the regulatory functions of two rG4 sites in bacteria (hemL and bswR). Overall, this finding strongly suggests that rG4s play key regulatory roles in a wide range of bacterial species.

Project description:Cellular phenotypes underpinned by regulatory networks need to respond to evolutionary pressures to allow adaptation, but at the same time be robust to perturbations. This creates a conflict in which mutations affecting regulatory networks must both generate variance but also be tolerated at the phenotype level. Here, we perform mathematical analyses and simulations of regulatory networks to better understand the potential trade-off between robustness and evolvability. Examining the phenotypic effects of mutations, we find an inverse correlation between robustness and evolvability that breaks only with nonlinearity in the network dynamics, through the creation of regions presenting sudden changes in phenotype with small changes in genotype. For genotypes embedding low levels of nonlinearity, robustness and evolvability correlate negatively and almost perfectly. By contrast, genotypes embedding nonlinear dynamics allow expression levels to be robust to small perturbations, while generating high diversity (evolvability) under larger perturbations. Thus, nonlinearity breaks the robustness-evolvability trade-off in gene expression levels by allowing disparate responses to different mutations. Using analytical derivations of robustness and system sensitivity, we show that these findings extend to a large class of gene regulatory network architectures and also hold for experimentally observed parameter regimes. Further, the effect of nonlinearity on the robustness-evolvability trade-off is ensured as long as key parameters of the system display specific relations irrespective of their absolute values. We find that within this parameter regime genotypes display low and noisy expression levels. Examining the phenotypic effects of mutations, we find an inverse correlation between robustness and evolvability that breaks only with nonlinearity in the network dynamics. Our results provide a possible solution to the robustness-evolvability trade-off, suggest an explanation for the ubiquity of nonlinear dynamics in gene expression networks, and generate useful guidelines for the design of synthetic gene circuits.

Dataset Information

G-quadruplex dynamics contribute to regulation of mitochondrial gene expression.

Publications

G-quadruplex dynamics contribute to regulation of mitochondrial gene expression.

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