Purification of mitochondria from skeletal muscle tissue for transcriptomic analyses reveals localisation of nuclear-encoded non-coding RNAs

Abstract

Mitochondria are central to cellular function, particularly in metabolically active tissues such as skeletal muscle. Non-coding RNAs (ncRNAs) typically localise within the nucleus and cytosol but may also translocate to subcellular compartments such as mitochondria. We aimed to investigate the nuclear-encoded ncRNAs that localise within the mitochondria of skeletal muscle cells and tissue. Intact mitochondria were isolated via immunoprecipitation and an enzymatic digestion approach was optimised to remove transcripts located exterior to mitochondria, making it amenable for high-throughput transcriptomic sequencing. Small-RNA sequencing libraries were successfully constructed from as little as 1.8ng mitochondrial RNA input. Small-RNA and whole transcriptome sequencing of mitochondria reveals the enrichment of over 200 miRNAs and 200 lncRNAs that have not previously been observed within skeletal muscle mitochondria. In summary, we describe a novel, powerful sequencing approach applicable to animal and human tissues and cells that reveals the unexpected diversity of nuclear-encoded ncRNA transcripts localised within skeletal muscle mitochondria.