Dinucleotide regulation by REXO2 maintains promoter specificity in mammalian mitochondria

Oligoribonucleases are conserved enzymes that degrade short RNA molecules of up to 5 nt in length, and are assumed to constitute the final stage of RNA turnover. We have investigated the function of the mammalian oligoribonuclease REXO2 through a combination of biochemical, structural and animal model studies. We find that REXO2 is a specialised dinucleotide degrading enzyme that shows no preference between RNA and DNA dinucleotide substrates. This specialisation is explained by crystal structures of REXO2 bound to RNA or DNA substrates. REXO2 is an essential gene, while a heart and skeletal muscle-specific knockout mouse displays elevated dinucleotide levels and alterations in gene expression patterns indicative of aberrant dinucleotide-primed transcription initiation. We find that dinucleotides act as potent stimulators of mitochondrial transcription initiation in vitro, and conclude that efficient RNA turnover is required to maintain the promoter specificity of transcription in mammalian mitochondria. Total RNA was isolated from heart tissue from 52 week old control (Rexo2loxP/loxP) and Rexo2 knockout mice (Rexo2loxP/loxP, +/Ckmm), TruSeq libraries produced in triplicate, sequenced and analysed for differential expression. Mitochondrial RNA was isolated from heart tissue from 52 week old control (Rexo2loxP/loxP) and Rexo2 knockout mice (Rexo2loxP/loxP, +/Ckmm), PARE libraries produced in triplicate and sequenced for analysis of mitochondrial RNA processing.寡核糖核酸酶（Oligoribonucleases）是一类保守的酶类，可降解长度不超过5个核苷酸的短RNA分子，被认为是RNA周转过程的最终阶段。我们通过生物化学、结构生物学与动物模型研究相结合的手段，对哺乳动物寡核糖核酸酶REXO2的功能展开系统探究。研究发现，REXO2是一种特化的二核苷酸降解酶，对RNA与DNA二核苷酸底物无明显偏好性。这一特化特性可通过REXO2与RNA或DNA底物结合的晶体结构得到合理解释。REXO2为必需基因，心脏与骨骼肌特异性敲除的小鼠不仅表现出二核苷酸水平升高的表型，同时伴随基因表达模式的改变，该改变提示存在异常的二核苷酸介导的转录起始过程。我们证实二核苷酸在体外可作为强效的线粒体转录起始刺激因子，并由此得出结论：高效的RNA周转是维持哺乳动物线粒体转录启动子特异性的必要条件。我们从52周龄的对照（Rexo2loxP/loxP）及Rexo2敲除小鼠（Rexo2loxP/loxP, +/Ckmm）的心脏组织中提取总RNA，构建三份重复的TruSeq测序文库，进行测序并分析差异表达情况。此外，我们从相同基因型的52周龄对照及Rexo2敲除小鼠的心脏组织中分离线粒体RNA，构建三份重复的PARE文库并测序，用于线粒体RNA加工的分析。