Systematic Analysis of Small RNAs Associated with Human Mitochondria by Deep Sequencing: Detailed Analysis of Mitochondrial Associated miRNA

Mitochondria are one of the central regulators of many cellular processes beyond its well established role in energy metabolism. The inter-organellar crosstalk is critical for the optimal function of mitochondria. Many nuclear encoded proteins and RNA are imported to mitochondria. The translocation of small RNA (sRNA) including miRNA to mitochondria and other sub-cellular organelle is still not clear. We characterized here sRNA including miRNA associated with human mitochondria by cellular fractionation and deep sequencing approach. Mitochondria were purified from HEK293 and HeLa cells for RNA isolation. The sRNA library was generated and sequenced using Illumina system. The analysis showed the presence of unique population of sRNA associated with mitochondria including miRNA. Putative novel miRNAs were characterized from unannotated sRNA sequences. The study showed the association of 428 known, 196 putative novel miRNAs to mitochondria of HEK293 and 327 known, 13 putative novel miRNAs to mitochondria of HeLa cells. The alignment of sRNA to mitochondrial genome was also studied. The targets were analyzed using DAVID to classify them in unique networks using GO and KEGG tools. Analysis of identified targets showed that miRNA associated with mitochondria regulates critical cellular processes like RNA turnover, apoptosis, cell cycle and nucleotide metabolism. The six miRNAs (counts >1000) associated with mitochondria of both HEK293 and HeLa were validated by RT-qPCR. To our knowledge, this is the first systematic study demonstrating the associations of sRNA including miRNA with mitochondria that may regulate site-specific turnover of target mRNA important for mitochondrial related functions.线粒体不仅是学界公认的能量代谢核心元件，更是众多细胞进程的关键调控枢纽之一。细胞器间的信号互作对于线粒体发挥最优功能至关重要。大量由细胞核编码的蛋白质与RNA会被转运至线粒体中。包括微小RNA（miRNA）在内的小RNA（small RNA, sRNA）向线粒体及其他亚细胞细胞器的转运机制目前仍未阐明。本研究通过细胞分级分离与高通量测序技术，对与人类线粒体结合的小RNA（包括miRNA）进行了系统表征。研究从HEK293与HeLa细胞中纯化线粒体以提取RNA，随后构建小RNA文库并采用Illumina测序平台进行测序。测序分析结果显示，线粒体中存在一类独特的小RNA群体，其中包含miRNA；研究人员从未注释的小RNA序列中鉴定出了潜在的新型miRNA。本研究发现，HEK293细胞的线粒体中共结合428种已知miRNA与196种潜在新型miRNA；HeLa细胞的线粒体中共结合327种已知miRNA与13种潜在新型miRNA。同时还对小RNA与线粒体基因组的序列比对情况进行了分析。研究采用DAVID数据库及基因本体（GO）、京都基因与基因组百科全书（KEGG）分析工具对miRNA的靶标基因进行注释分类并构建特异性功能网络。靶标分析结果表明，与线粒体结合的miRNA可调控RNA周转、细胞凋亡、细胞周期与核苷酸代谢等关键细胞生物学过程。针对在HEK293与HeLa细胞线粒体中均有表达且测序计数>1000的6种miRNA，本研究通过实时定量聚合酶链反应（RT-qPCR）进行了验证。据我们所知，本研究是首个系统性证实小RNA（包括miRNA）与线粒体结合的研究，这类结合的miRNA可通过调控靶mRNA的位点特异性降解，进而参与线粒体相关生物学功能的调控。