Abstract: TH-PO308
Guanine Quadruplex-Mediated Pausing of Mitochondrial RNA Polymerase Regulates ATP Generation in Renal Proximal Tubule Cells
Session Information
- Fluid, Electrolyte, and Acid-Base Disorders: Basic
November 03, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid‚ Electrolyte‚ and Acid-Base Disorders
- 1001 Fluid‚ Electrolyte‚ and Acid-Base Disorders: Basic
Authors
- Watts, Jason A., National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States
- Delker, Don, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States
- Snyder, Ryan J., National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States
Background
This project identifies the role of mitochondrial RNA polymerase (mtRNAP) pausing in the regulation of ATP production and transporter function in renal proximal tubule cells (RPTECs). In the nucleus, RNA polymerase II transcribes with punctuated pauses that are coupled to regulation of gene expression; however, RNA polymerase pausing has not been studied in the regulation of mitochondrial gene expression. Here, we sought to characterize the role of nucleic acid sequence and secondary structure in the regulation of mtRNAP pausing. Guanine quadruplexes (G4) are secondary structures formed by non-canonical base-pairing between guanine residues. We found that stabilization of G4 in RPTECs results in increased mtRNAP pausing and mitochondrial dysfunction.
Methods
We utilized the precision nuclear run-on assay (PRO-seq) to characterize the location of mtRNAP in cultured human fibroblasts and RPTECs. We assessed ATP production using the extracellular flux assay and quantified transporter function in RPTECs by measuring transport of a glucose analog (2-NBDG).
Results
Using fibroblasts from different individuals, we identified over 400 locations where mtRNAP pauses at precise locations on mtDNA. These brief stops occur most often after mtRNAP has transcribed through guanine-rich regions predicted to form G4 structures. We experimentally validated G4 formation at sites where mtRNAP pauses and show that G4-stabilization with a small-molecule (RHPS4) results in more mtRNAP pausing, impaired transcription, and decreased ATP production. As RPTECs are dependent on ATP from oxidative phosphorylation to drive solute reabsorption, we asked if impaired mitochondrial transcription affects transporter function. Using differentiated RPTECs grown on transwells, we show treatment with RHPS4 results in significantly decreased glucose transport.
Conclusion
Mitochondria dysfunction is implicated in acute and chronic kidney disease. The results presented here demonstrate that G4 regulate pausing of mtRNAP and that stabilization of G4s impedes mitochondrial transcription, compromises oxidative phosphorylation, and impairs proximal tubule function. In kidney disease, the role of G4-mediated mitochondria dysfunction warrants further study.
Funding
- Other NIH Support