Following its endosymbiosis from an α-proteobacterial ancestor, the mitochondrial genome has been streamlined into a small, high copy, bioenergetically specialized genetic system that allows individual mitochondria to respond, by gene expression, to changes in membrane potential and maintain oxidative phosphorylation (Lane and Martin, 2010
). Given this dedicated role, it is not surprising that the mitochondrial genome is regulated and expressed in a unique manner.
Human mitochondria contain a compact circular genome that is 16,569 bp in length (Anderson et al., 1981
). Replication and transcription of mitochondrial DNA (mtDNA) is initiated from a small noncoding region, the D-loop, and is regulated by nuclear-encoded proteins that are post-translationally imported into mitochondria. Mitochondrial RNAs are transcribed as long polycistronic precursor transcripts from both strands (historically termed ‘heavy’ and ‘light’) that are processed according to the ‘tRNA punctuation model’ whereby 22 interspersed tRNAs are excised to concomitantly release individual rRNAs and mRNAs (Ojala et al., 1981
). The liberated RNAs then undergo maturation that involves polyadenylation of the 3′ ends of mRNAs and rRNAs, and specific nucleotide modifications and addition of CCA trinucleotides to the 3′ ends of tRNAs (Nagaike et al., 2005
). Together, this comprises a unique genetic system that is able to translate the mitochondria-encoded genes into 13 protein subunits of the electron transport chain.
Little is known about the fine features of the mitochondrial transcriptome, in particular about the regulation of transcript abundance, sites of RNA processing and modification, and the possible presence of noncoding RNAs. The recent advent of deep sequencing has provided a global profile of the nuclear transcriptome, revealing unforeseen transcriptional complexity that includes prevalent post-transcriptional processing and abundant noncoding RNAs (Jacquier, 2009
). We have taken a similar approach to investigate the mitochondrial transcriptome. Although the atypical features of mitochondrial gene expression require special considerations in sequencing and analysis, the small size of the genome affords a depth of coverage that provides unprecedented opportunities to sample the total RNA population and characterize even rare and transient events.
Here we provide the first comprehensive map of the human mitochondrial transcriptome by near-exhaustive deep sequencing of long and short RNA fractions from purified mitochondria. Despite their common polycistronic origin, we observe wide variation between individual tRNAs, mRNAs and rRNA amounts, attesting to the importance of post-transcriptional cleavage and processing mechanisms in the regulation of mitochondrial gene expression. By parallel analysis of RNA ends (PARE) sequencing, we provide a global profile that precisely resolves these cleavage processes, as well as indicating further unexpected non-canonical cleavage events. During this analysis we were also able to discern the contribution of nuclear RNAs to the mitochondrial transcriptome, accounting for co-purifying contaminants by a two-phase sequencing approach.
Finally, we have analyzed in vivo
DNaseI protection patterns by massively parallel sequencing to generate a profile of protein-DNA interactions across the entire mitochondrial genome at single nucleotide resolution. Analysis of these sites of DNA-protein interaction, in combination with transcriptional profiling, provides novel regulatory insight into mitochondrial genome dynamics. The integration of these maps reveals unexpected complexity of the regulation, expression and processing of the mitochondrial transcriptome, and comprises an important resource for the future study of mitochondrial function and disease. This resource comprises 10 new datasets that, combined with meta-analyses of 20 publicly available sets (; Table S1
), are accessible within the mitochondria-specific genome browser hosted at mitochondria.matticklab.com
Figure 1 Map of the human mitochondrial genome indicating the following features (from centre with outer tracks corresponding to heavy strand and inner track corresponding to light strand); DNase sensitivity profile (central black track, opacity indicates strength (more ...)