Abstract
RNA sequencing using next-generation technologies provides comprehensive coverage of transcriptomes at a much greater depth than conventional transcriptomic methods. The human mitochondrial genome is relatively small, and sequencing its transcriptome provides a valuable method to investigate changes in RNA metabolism in great detail. Here we describe two methods that use next-generation technologies to investigate mitochondrial RNAs. Directional RNA sequencing enables the analyses of RNA abundance from each strand of the mitochondrial DNA. Parallel analysis of RNA ends enables the analyses of processing of mitochondrial transcripts, their termini, and annotation of any new transcripts.
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References
Rackham O, Mercer RT, Filipovska A (2012) The human mitochondrial transcriptome and the RNA-binding proteins that regulate its expression. Wiley Interdiscip Rev RNA 3:675–695
Rorbach J, Minczuk M (2012) The post-transcriptional life of mammalian mitochondrial RNA. Biochem J 444:357–373
Shutt TE, Shadel GS (2010) A compendium of human mitochondrial gene expression machinery with links to disease. Environ Mol Mutagen 51(5):360–379
Temperley RJ, Wydro M, Lightowlers RN et al (2010) Human mitochondrial mRNAs—like members of all families, similar but different. Biochim Biophysica Acta 1797:1081–1085
Christian EB, Spremulli LL (2012) Mechanism of protein biosynthesis in mammalian mitochondria. Biochim Biophys Acta 1819:1035–1054
Mercer TR, Neph S, Dinger ME et al (2011) The human mitochondrial transcriptome. Cell 146:645–658
German MA, Luo S, Schroth G et al (2009) Construction of parallel analysis of RNA ends (PARE) libraries for the study of cleaved miRNA targets and the RNA degradome. Nat Protoc 4:356–362
German MA, Pillay M, Jeong D-H et al (2008) Global identification of microRNA-target RNA pairs by parallel analysis of RNA ends. Nat Biotechnol 26:941–946
Lopez Sanchez MI, Mercer TR, Davies SM et al (2011) RNA processing in human mitochondria. Cell Cycle 10:2904–2916
Rackham O, Shearwood A, Mercer TR et al (2011) Long noncoding RNAs are generated from the mitochondrial genome and regulated by nuclear-encoded proteins. RNA 17:2085–2093
Rackham O, Filipovska A (2012) The role of mammalian PPR domain proteins in the regulation of mitochondrial gene expression. Biochim Biophys Acta 1819:1008–1016
Ebhardt H, Tsang H, Dai D et al (2009) Meta-analysis of small RNA-sequencing errors reveals ubiquitous post-transcriptional RNA modifications. Nucleic Acids Res 37:2461–2470
Findeiss S, Langenberger D, Stadler P et al (2011) Traces of post-transcriptional RNA modifications in deep sequencing data. Biol Chem 392:305–313
Acknowledgements
We thank the Australian Research Council and the National Health and Medical Research Council for the award of our fellowships (FT0991008 and FT0991113) and grants (APP1058442, APP1045677, APP1041582, APP1023460, APP1005030 and DP140104111). We thank the members of our groups and colleagues that have contributed to our research and GeneWorks for directional RNA-Seq library preparation and sequencing services.
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Rackham, O., Filipovska, A. (2014). Analysis of the Human Mitochondrial Transcriptome Using Directional Deep Sequencing and Parallel Analysis of RNA Ends. In: Rorbach, J., Bobrowicz, A. (eds) Polyadenylation. Methods in Molecular Biology, vol 1125. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-971-0_21
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DOI: https://doi.org/10.1007/978-1-62703-971-0_21
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Publisher Name: Humana Press, Totowa, NJ
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