Purification of mitochondria from skeletal muscle tissue for transcriptomic analyses reveals localization of nuclear-encoded non-coding RNAs [Mitoplasts]

Mitochondria are central to cellular function, particularly in metabolically active tissues such as skeletal muscle. Nuclear-encoded RNAs typically localise within the nucleus and cytosol but a small population may also translocate to subcellular compartments such as mitochondria. We aimed to investigate the nuclear-encoded RNAs that localise within the mitochondria of skeletal muscle cells and tissue. Intact mitochondria were isolated via immunoprecipitation (IP) followed by enzymatic treatments (RNase-A and proteinase-K) to remove transcripts located exterior to mitochondria, making it amenable for high-throughput transcriptomic sequencing. Whole-transcriptome RNA sequencing of enzymatically-purified mitochondria isolated by IP from skeletal muscle tissue showed a striking similarity in the degree of purity compared to mitoplast preparations which lack an outer mitochondrial membrane. In summary, we describe a novel, powerful sequencing approach applicable to animal and human tissues and cells that can facilitate the discovery of nuclear-encoded RNA transcripts localised within skeletal muscle mitochondria.线粒体是细胞功能的核心枢纽，在骨骼肌等代谢活性旺盛的组织中尤为关键。核编码RNA通常定位于细胞核与细胞质内，但少量转录本也可转位至线粒体等亚细胞区室。本研究旨在探究定位于骨骼肌细胞及组织线粒体中的核编码RNA。实验通过免疫沉淀（immunoprecipitation，IP）技术分离完整线粒体，随后辅以核糖核酸酶A（RNase-A）与蛋白酶K（proteinase-K）酶解处理，以去除线粒体外侧的转录本，使样本适配高通量转录组测序流程。对从骨骼肌组织中经IP分离的酶解纯化线粒体开展全转录组RNA测序，结果显示其纯度与缺乏线粒体外膜的去外膜线粒体（mitoplast）制剂高度相似。综上，本研究报道了一种全新且高效的测序方法，可应用于动物及人类的组织与细胞样本，能够助力定位于骨骼肌线粒体的核编码RNA转录本的发现工作。