Guanine quadruplexes mediate mitochondrial RNA polymerase pausing

Abstract

The information content within nucleic acids extends beyond the primary sequence to include secondary structures with functional roles in cells. Guanine-rich sequences form structures called guanine quadruplexes (G4) that result from non-canonical base pairing between guanine residues. These stable structures are enriched in gene promoters and have been correlated with the locations of RNA polymerase II pausing (Pol II). While promoter-proximal RNA polymerase pausing regulates gene expression, the effects of guanine quadruplexes on gene transcription have been less clear. We determined the pattern of mitochondrial RNA polymerase (mtRNAP) pausing in human fibroblasts and found that it pauses over 400 times on the mitochondrial genome. We identified quadruplexes as a mediator of mtRNAP pausing and show that stabilization of quadruplexes impeded transcription by mtRNAP. Gene products encoded by the mitochondrial genome are required for oxidative phosphorylation and the decreased transcription by mtRNAP resulted in lower expression of mitochondrial genes and significantly reduced ATP generation. Energy from mitochondria is essential for transport function in renal epithelia, and impeded mitochondrial transcription inhibits transport function in renal proximal tubule cells. These results link formation of guanine quadruplex structures to regulation of mtRNAP elongation and mitochondrial function.

Figure 1.. mtRNAP pause throughout the mitochondrial genome.

(A) Locations where mtRNAP pauses along the mitochondrial genome. From the center: PRO-seq signal from the light strand shown as a circularized bar graph (red), guanines (grey), genes on the light strand (mRNA red, tRNA dark red), genes on the heavy strand (rRNA green, mRNA blue, tRNA dark blue), guanines (grey), PRO-seq signal from the heavy strand shown in the outer circularized bar graph in blue. The location of a previously identified promoter-proximal pause is indicated (*). Y-axis is 0 to 50 RPM for both heavy and light strands. Data are average PRO-seq signal from primary fibroblasts (N=5). (B) Guanine content is significantly higher at mtRNAP pause sites (G-rich stand P<10⁻¹³, G-poor strand P<3×10⁷, Wilcoxon test). (C) Violin plots of mtRNAP abundance by guanine content upstream of the pause (*P<0.05, ****P<0.0001; Mann-Whitney U-test).

Figure 2.. G4 are enriched upstream of mtRNAP pause sites.

(A,B) G-quadruplexes were quantified by two algorithms, (A) G4Hunter (B) QGRS, and the abundance of G-quadruplexes for the 100 nucleotides up- and downstream of the 465 mtRNAP pause sites are shown in the Y-axis as the scores (error bands represent S.E.M. ). (C) The abundance of G-quadruplexes determined experimentally by BG4 ChIP-seq are also shown for 100 nucleotides up and downstream of the 465 mtRNAP pause sites (error bands represent S.E.M.), (D) G4 forming sequence (underlined) present upstream of pause site in CO1 gene. Corresponding DNA-IP shows enrichment of G4 where mtRNAP pauses (N=4; ***P<0.01; t-test). (E) NMR spectra for CO1-G4 sequences in RNA, or DNA or where the guanines are converted to uracil to prevent G4 formation. NMR chemical shifts in the range of 10–12 ppm indicate Hoogsteen base-pairing indicative of quadruplex formation. (F) Circular dichroism spectra of RNA (red) or DNA (blue) oligos corresponding to sequences in panel D. The pattern with minima at 240 nm and maxima at 260 nm is consistent with an RNA G-quadruplex.

Figure 3.. Stable G4 lead to more paused mtRNAP.

(A) Confocal image showing RHPS4 (green) localization in the cytoplasm following 24 hrs of drug treatment. Scale bar is 50 μm. (B) Bulk G4 abundance measured by immunofluorescence, expressed as percentage of intracellular area labeled, before and after RHSP4 treatment. (C) PRO-seq data from biologic replicate experiments showing mtRNAP distribution before and after treatment with RHPS4. Y-axis is RPM. RHPS4 results in a shift in mtRNAP localization with more polymerase at the 5’ end of the polycistron and decreased mtRNAP at the 3’ end of polycistron. G-rich light strand transcripts (red). G-poor heavy strand transcripts (blue) (D) Mitochondrial gene expression before and 24 hours after RHPS4 treatment normalized to vehicle expression in arbitrary units (N=3; P<0.0001, one-sided ANOVA, error bars=S.E.M.). (E) Basal oxygen consumption and ATP production before and after 24 hours of RHPS4 treatment are shown. (N=6; *P<0.05, ***P<0.001, t-test, error bars=S.E.M.).

Figure 4.. mtRNAP pausing impairs renal proximal tubule function.

(A) Schematic of renal proximal tubule culture system, where cells are maintained on the bottom of the permeable membrane. The lower chamber is apical to the cells and the upper chamber is basal. Fluorescent substrates are placed in the lower chamber and active transport measured by detection of fluorescence in the upper chamber. (B) Protein expression nuclear encoded TOMM20, mitochondria encoded CO-1 and AMPK before and after 72 hrs of RHPS4 treatment (N=1). (C) Active transport of glucose analogue 2-NBDG, expressed as a percentage of transport performed by DMSO-treated cells. (N=5; ***P<0.001, t-test, error bars=S.E.M).