Abstract
Mammalian mitochondrial RNAs are unique as they are derived from primary transcripts that encompass almost the entire mitochondrial genome. This necessitates extensive processing to release the individual mRNAs, rRNAs and tRNAs required for gene expression. Recent studies have revealed many of the proteins required for mitochondrial RNA processing, however the rapid turnover of precursor RNAs has made it impossible to analyze their composition and the hierarchy of processing. Here, we find that circularization of RNA prior to deep sequencing enables the discovery and characterization of unprocessed RNAs. Using this approach, we identify the most stable processing intermediates and the presence of intermediate processing products that are partially degraded and polyadenylated. Analysis of libraries constructed using RNA from mice lacking the nuclease subunit of the mitochondrial RNase P reveals the identities of stalled processing intermediates, their order of cleavage, and confirms the importance of RNase P in generating mature mitochondrial RNAs. Using RNA circularization prior to library preparation should provide a generally useful approach to studying RNA processing in many different biological systems.
Highlights
In the pathway from gene to protein, many different forms of regulation have been identified that can control the final amounts of individual proteins that are produced
CircLigase II only catalyzes the ligation of nucleic acids bearing a 5 -phosphate and a 3 -hydroxyl group, which is thought to be a characteristic of all mammalian mitochondrial RNAs [4,27]
We used deep sequencing of circularized RNA to discover the composition of longer-lived mitochondrial RNA precursors both in normal heart mitochondria and in mitochondria lacking proteinaceous RNase P activity
Summary
In the pathway from gene to protein, many different forms of regulation have been identified that can control the final amounts of individual proteins that are produced. An analogous situation occurs in mammalian mitochondria, where both strands of the mitochondrial genome are transcribed as two long polycistronic RNAs encompassing the whole genome sequence [3,4]. In this case, we are only beginning to understand the mechanisms controlling the levels of individual mitochondrial proteins [4,5,6]
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