Initiation of mtDNA transcription is followed by pausing, and diverge across human cell types and during evolution
- 1 Ben-Gurion University of the Negev;
- 2 Baker Institute for Animal Health, Cornell University;
- 3 Stanford University
- ↵* Corresponding author; email: dmishmar{at}bgu.ac.il
Abstract
Mitochondrial DNA (mtDNA) genes are long known to be co-transcribed in polycistrones, yet it remains impossible to study nascent mtDNA transcripts quantitatively in vivo using existing tools. To this end we used deep sequencing (GRO-seq and PRO-seq) and analyzed nascent mtDNA-encoded RNA transcripts in diverse human cell lines and metazoan organisms. Surprisingly, accurate detection of human mtDNA transcription initiation sites (TIS) in the heavy and light strands revealed a novel conserved transcription pausing site near the light strand TIS. This pausing site correlated with the presence of a bacterial pausing sequence motif, reduced SNP density, and with a DNase footprinting signal in all tested cells. Its location within conserved sequence block 3 (CSBIII), just upstream of the known transcription-replication transition point suggests involvement in such transition. Analysis of non-human organisms enabled de novo mtDNA sequence assembly, as well as detection of previously unknown mtDNA TIS, pausing, and transcription termination sites with unprecedented accuracy. Whereas mammals (Pan troglodytes, Macaca mulatta, Rattus norvegicus, and Mus musculus) showed a human-like mtDNA transcription pattern, the invertebrate pattern (Drosophila melanogaster and Caenorhabditis elegans) profoundly diverged. Our approach paves the path towards in vivo, quantitative, reference sequence-free analysis of mtDNA transcription in all eukaryotes.
- Received May 18, 2016.
- Accepted December 29, 2016.
- Published by Cold Spring Harbor Laboratory Press
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