Kuznetsova Irina, Siira Stefan J, Shearwood Anne-Marie J, Ermer Judith A, Filipovska Aleksandra, Rackham Oliver
Harry Perkins Institute of Medical Research and Centre for Medical Research, The University of Western Australia, Nedlands 6009, Australia.
School of Molecular Sciences, The University of Western Australia, Crawley 6009, Australia.
Nucleic Acids Res. 2017 May 19;45(9):5487-5500. doi: 10.1093/nar/gkx104.
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.
哺乳动物线粒体RNA很独特，因为它们源自几乎涵盖整个线粒体基因组的初级转录本。这就需要进行广泛的加工，以释放基因表达所需的单个mRNA、rRNA和tRNA。最近的研究已经揭示了线粒体RNA加工所需的许多蛋白质，然而前体RNA的快速周转使得分析它们的组成和加工层次变得不可能。在这里，我们发现深度测序之前RNA的环化能够发现和表征未加工的RNA。使用这种方法，我们鉴定出最稳定的加工中间体以及部分降解和多聚腺苷酸化的中间加工产物的存在。对来自缺乏线粒体核糖核酸酶P核酸酶亚基的小鼠的RNA构建的文库进行分析，揭示了停滞加工中间体的身份、它们的切割顺序，并证实了核糖核酸酶P在产生成熟线粒体RNA中的重要性。在文库制备之前使用RNA环化应该为研究许多不同生物系统中的RNA加工提供一种普遍有用的方法。
