A competitive formation of DNA:RNA hybrid G-quadruplex is responsible to the mitochondrial transcription termination at the DNA replication priming site

Abstract

Human mitochondrial DNA contains a distinctive guanine-rich motif denoted conserved sequence block II (CSB II) that stops RNA transcription, producing prematurely terminated transcripts to prime mitochondrial DNA replication. Recently, we reported a general phenomenon that DNA:RNA hybrid G-quadruplexes (HQs) readily form during transcription when the non-template DNA strand is guanine-rich and such HQs in turn regulate transcription. In this work, we show that transcription of mitochondrial DNA leads to the formation of a stable HQ or alternatively an unstable intramolecular DNA G-quadruplex (DQ) at the CSB II. The HQ is the dominant species and contributes to the majority of the premature transcription termination. Manipulating the stability of the DQ has little effect on the termination even in the absence of HQ; however, abolishing the formation of HQs by preventing the participation of either DNA or RNA abolishes the vast majority of the termination. These results demonstrate that the type of G-quadruplexes (HQ or DQ) is a crucial determinant in directing the transcription termination at the CSB II and suggest a potential functionality of the co-transcriptionally formed HQ in DNA replication initiation. They also suggest that the competition/conversion between an HQ and a DQ may regulate the function of a G-quadruplex-forming sequence.

The human mitochondrial transcription factor A is a versatile G-quadruplex binding protein

“…An RNA/DNA G4 at CSBII202122 suggests a functional role in contributing to R-loop stabilization, similar to that observed in nuclear DNA51. Numerous additional G4s are predicted along the mtDNA sequence151617 and may form during replication, leading to genomic instability5253.…”

Evolution of Nucleotide Punctuation Marks: From Structural to Linear Signals

“…They guide RNA processing in mitochondria (Ojala et al, 1981), and in giant viruses and their virophages (Byrne et al, 2009; Claverie and Abergel, 2009). This ubiquitous structural signaling also applies to RNA:DNA hybrids, which play a role in the origin-independent replication priming in eukaryotic cells (Stuckey et al, 2015) and in transcription termination in human mitochondria (Zheng et al, 2014). …”

Multiplexed detection of RNA using MERFISH and branched DNA amplification

“…In particular, the RNAdependent background was unexpected because the sequences were blasted to avoid homology with the human transcriptome. We found that these specific amplifiers contained guanine nucleotides in the 20-mer repeating sequences that might form hybrid G-quadruplex structures, which have been reported to bind to mitochondrial RNA 33 . The specific spatial distribution observed with the staining of these amplifiers (Fig.…”

“…3c). Given the cellular distribution of this binding, we suspected these guanine-rich secondary amplifier sequences might form Gquadruplex structures with mitochondrial RNAs 33 . In parallel, we observed a small number of amplifier pairs that assembled with much lower efficiency, producing only small degrees of amplification, perhaps due to low melting temperature or the formation of G-quadruplex structures that inhibit proper amplifier assembly ( Fig.…”