Immortalized Human Renal Proximal Tubule Cells-hTERT

Guanine Quadruplex Mediated Pausing Regulates Mitochondrial Function

DNA G4 structures are established regulators of transcription yet roles for RNA G4s in regulating mitochondrial transcription have not been investigated. Snyder et al. sought to determine if RNA G4s regulate mitochondrial RNA polymerase (POLRMT) pausing and mitochondrial function.

They tested the functional consequences of G4 mediated POLRMT pausing in immortalized human renal proximal tubule cells (RPTEC) due to the kidney's metabolic requirements. Treatment with RHPS4 for 24h resulted in accumulation of POLRMT in mitochondria in a time dependent manner (Fig. 1A) and significantly reduced expression of all 12 heavy strand-encoded polycistronic mRNAs, including MT-CO1 (Fig. 1B). This occurred while maintaining mtDNA content (Fig. 1C, D). Expression level of each gene was negatively correlated with distance from the transcription start site, further supporting the idea that POLRMT accumulation at G4 sites inhibits distal transcription. Mitochondria-encoded proteins are required for respiration so they measured oxygen consumption. RHPS4 significantly inhibited O₂ consumption (Fig. 1E) and increased compensatory glycolysis (Fig. 1F). Thus, stabilizing G4s to induce POLRMT pausing impaired mitochondrial gene expression and cellular energy production. RHPS4 decreased mitochondria-encoded COX1 protein without changing nuclear-encoded TOMM20 or mitochondria abundance in RPTECs grown on transwell inserts (Fig. 1G). This caused an imbalance in cellular energy that activated AMPK (Fig. 1G).