Concerted regulation of mitochondrial and nuclear non-coding RNAs by a dual-targeted RNase Z [sRNA]

The molecular roles of the dually targeted ElaC domain protein 2 (ELAC2) during nuclear and mitochondrial RNA processing in vivo have not been distinguished. We generated conditional knockout mice of ELAC2 to identify that it is essential for life and its activity is non-redundant. Heart and skeletal muscle-specific loss of ELAC2 causes dilated cardiomyopathy and premature death at 4 weeks. Transcriptome-wide analyses of total RNAs, small RNAs, mitochondrial RNAs and miRNAs identified the nuclear and mitochondrial molecular targets of ELAC2 in vivo. We show that ELAC2 is required for processing of nuclear and mitochondrial tRNAs and for the balanced maintenance of C/D box snoRNAs, a new class of tRNA fragments, and miRNAs. We identify that correct biogenesis of regulatory non-coding RNAs is essential for both cytoplasmic and mitochondrial protein synthesis as well as the assembly of mitochondrial ribosomes and cytoplasmic polysomes. Taken together our data show that nuclear tRNA processing is required for the balanced production of snoRNAs and miRNAs for gene expression and that 3' tRNA processing follows 5' tRNA processing but nevertheless is an essential step in the production of all mature mitochondrial RNAs and the majority of nuclear tRNAs. Total RNA from heart tissue from three control mice (L/L) and three Elac2 knockout mice (L/L, cre), was sequenced with four technical replicates each and analysed for differential expression. RNA sequencing libraries were constructed using the Illumina Small RNA Sample Prep Kit, with a size selection step for 15 - 100 nt RNAs. Paired-end deep sequencing of the mitochondrial RNAs was performed on an Illumina NextSeq 500 according to the manufacturer's instructions.在体内细胞核与线粒体RNA加工过程中，双重靶向的ElaC结构域蛋白2（ELAC2）的分子功能尚未被明确区分。本研究构建了ELAC2条件性敲除小鼠，证实该蛋白对生命活动不可或缺，且其功能不存在冗余。心脏与骨骼肌特异性缺失ELAC2会导致扩张型心肌病，并在4周龄时过早死亡。通过对总RNA、小RNA、线粒体RNA及微小RNA（miRNA）进行全转录组分析，本研究明确了体内ELAC2的细胞核与线粒体分子靶点。研究表明，ELAC2参与细胞核与线粒体转运RNA（tRNA）的加工过程，同时对维持C/D盒小核仁RNA（C/D box snoRNA）、一类新型转运RNA片段及微小RNA（miRNA）的稳态平衡不可或缺。本研究证实，调控性非编码RNA的正确生物发生对于细胞质与线粒体蛋白质合成，以及线粒体核糖体与细胞质多聚核糖体的组装均至关重要。综合本研究数据显示，细胞核tRNA加工过程可通过调控snoRNA与miRNA的稳态生成以维持基因表达，且3'端tRNA加工紧随5'端tRNA加工，但其仍是生成所有成熟线粒体RNA及绝大多数细胞核成熟tRNA的必需步骤。本研究对3只对照小鼠（L/L）及3只Elac2敲除小鼠（L/L, cre）的心脏组织总RNA各设置4次技术重复进行测序，并分析其差异表达情况。本研究采用Illumina Small RNA Sample Prep Kit构建RNA测序文库，并对15~100 nt的RNA进行尺寸筛选。随后，按照制造商操作说明，在Illumina NextSeq 500平台上对线粒体RNA进行双端深度测序。