title: Initiation of mtDNA transcription is followed by pausing, and diverges across human cell types and during evolution.
abstract: Mitochondrial DNA (mtDNA) genes are long known to be cotranscribed 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 (TISs) 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, with 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 nonhuman 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, andMus musculus) showed a human-like mtDNA transcription pattern, the invertebrate pattern (Drosophila melanogasterandCaenorhabditis elegans) profoundly diverged. Our approach paves the path toward in vivo, quantitative, reference sequence-free analysis of mtDNA transcription in all eukaryotes.
authors.affiliation: Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva, 84105 Israel.
authors.forename: Amit
authors.initials: A
authors.lastname: Blumberg
authors.affiliation: Baker Institute for Animal Health, Cornell University, Ithaca, New York 14853, USA.
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Rice
authors.affiliation: Department of Genetics, Stanford University, Stanford, California 94305-5120, USA.
authors.forename: Anshul
authors.identifier: 0000-0003-3084-2287
authors.initials: A
authors.lastname: Kundaje
authors.affiliation: Baker Institute for Animal Health, Cornell University, Ithaca, New York 14853, USA.
authors.forename: Charles G
authors.identifier: 0000-0002-1999-7125
authors.initials: CG
authors.lastname: Danko
authors.affiliation: Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva, 84105 Israel.
authors.forename: Dan
authors.identifier: 0000-0003-3488-8792
authors.initials: D
authors.lastname: Mishmar
mesh_terms: D000818:Animals; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics*; D019143:Evolution, Molecular; D006801:Humans; D007448:Invertebrates / Q000235:genetics; D009928:Organ Specificity; D020641:Polymorphism, Single Nucleotide; D011323:Primates / Q000235:genetics; D012377:Rodentia / Q000235:genetics; D024363:Transcription Initiation Site; D061785:Transcription Initiation, Genetic; D059467:Transcriptome
chemical_list: D004272:DNA, Mitochondrial