Transcription profiling suggests that mitochondrial topoisomerase IB acts as a topological barrier and regulator of mitochondrial DNA transcription

Abstract

Mitochondrial DNA (mtDNA) is essential for cell viability because it encodes subunits of the respiratory chain complexes. Mitochondrial topoisomerase IB (TOP1MT) facilitates mtDNA replication by removing DNA topological tensions produced during mtDNA transcription, but it appears to be dispensable. To test whether cells lacking TOP1MT have aberrant mtDNA transcription, we performed mitochondrial transcriptome profiling. To that end, we designed and implemented a customized tiling array, which enabled genome-wide, strand-specific, and simultaneous detection of all mitochondrial transcripts. Our technique revealed that KO mouse cells process the mitochondrial transcripts normally but that protein-coding mitochondrial transcripts are elevated. Moreover, we found discrete long noncoding RNAs produced by H-strand transcription and encompassing the noncoding regulatory region of mtDNA in human and murine cells and tissues. Of note, these noncoding RNAs were strongly up-regulated in the absence of TOP1MT. In contrast, 7S DNA, produced by mtDNA replication, was reduced in the KO cells. We propose that the long noncoding RNA species in the D-loop region are generated by the extension of H-strand transcripts beyond their canonical stop site and that TOP1MT acts as a topological barrier and regulator for mtDNA transcription and D-loop formation.

The mitochondrial type IB topoisomerase drives mitochondrial translation and carcinogenesis

“…4b, Supplementary Fig. 4b), which is consistent with our recent findings in Top1mt -KO MEFs 31 . Although mitochondrial transcripts were elevated, steady-state levels of the respiratory chain proteins, encoded by both mitochondrial and nuclear DNA, were significantly diminished in TOP1MT -KO tumors (Fig.…”

“…4b, Supplementary Fig. 5f), which is in line with our recent genomic analyses of mitochondrial transcription 31 . The upregulation of mitochondrial transcripts could result from a compensatory mechanism in the context of impaired mitochondrial translation.…”

“…It has been previously reported that defective mitochondrial protein synthesis is often associated with increased transcript levels as part of a compensatory mechanism to overcome impaired oxidative phosphorylation 32,33 . Thus, our results reveal a function of TOP1MT in mitochondrial translation in addition to its roles in the release of DNA torsional stress 18,29 and mitochondrial transcription 31 .…”

“…a Reduced mtDNA copy number was determined by RT-qPCR in TOP1MT -KO and WT HCT116 tumor xenografts ( n = 9, each genotype, each performed in triplicates). b Conserved mitochondrial transcription profiles of TOP1MT -KO vs. WT tumor xenografts determined by tiling array 31 ( n = 4, each genotype). c Representative western blots showing reduced levels of mitochondrial OXPHOS proteins in TOP1MT -KO tumor xenografts (lane 5–8).…”

Does mitochondrial DNA evolution in metazoa drive the origin of new mitochondrial proteins?

“…There is indeed proof that TOP1MT interacts with the control region. Absence of TOP1MT effects non‐coding RNAs of the Dloop region, 7S DNA and negative supercoiling . In addition, trapping of TOP1MT with camptothecin showed a cluster of TOP1mt sites confined to a 150 bp region downstream from the site at which replication is prematurely terminated.…”