Global analyses of endonucleolytic cleavage in mammals reveal expanded repertoires of cleavage-inducing small RNAs and their targets

In mammals, small RNAs are important players in post-transcriptional gene regulation. While their roles in mRNA destabilization and translational repression are well appreciated, their involvement in endonucleolytic cleavage of target RNAs is poorly understood. Very few microRNAs are known to guide RNA cleavage. Endogenous small interfering RNAs are expected to induce target cleavage, but their target genes remain largely unknown. We report a systematic study of small RNA-mediated endonucleolytic cleavage in mouse through integrative analysis of small RNA and degradome sequencing data without imposing any bias towards known small RNAs. Hundreds of small cleavage-inducing RNAs and their cognate target genes were identified, significantly expanding the repertoire of known small RNA-guided cleavage events. Strikingly, both small RNAs and their target sites demonstrated significant overlap with retrotransposons, providing evidence for the long-standing speculation that retrotransposable elements in mRNAs are leveraged as signals for gene targeting. Furthermore, our analysis showed that the RNA cleavage pathway is also present in human cells but affecting a different repertoire of retrotransposons. These results show that small RNA-guided cleavage is more widespread than previously appreciated. Their impact on retrotransposons in non-coding regions shed light on important aspects of mammalian gene regulation. rRNA depleted RNA sequencing was done for four stages of testis development.在哺乳动物中，小RNA是转录后基因调控的关键调控因子。尽管其在mRNA降解与翻译抑制中的功能已被广泛阐明，但它们在靶RNA内切切割过程中的作用却仍知之甚少。目前已知可介导RNA切割的微小RNA（microRNA）数量极少。内源性小干扰RNA（small interfering RNA, siRNA）理论上可诱导靶RNA切割，但其对应的靶基因仍尚未被广泛探明。本研究通过整合分析小RNA与降解组测序（degradome sequencing）数据，且未对已知小RNA施加任何偏倚，系统性探究了小鼠体内小RNA介导的RNA内切切割现象。研究共鉴定出数百种可诱导切割的小RNA及其对应靶基因，大幅扩充了已知的小RNA介导切割事件库。令人瞩目的是，这些小RNA及其靶位点均与逆转录转座子（retrotransposon）存在显著重叠，为长期以来的推测——即mRNA中的逆转录转座子元件可被用作基因靶向信号——提供了实证依据。此外，本研究分析还显示，RNA切割通路同样存在于人类细胞中，但仅作用于不同类别的逆转录转座子。上述结果表明，小RNA介导的切割事件分布范围比此前认知的更为广泛；其对非编码区域逆转录转座子的影响，为哺乳动物基因调控的诸多重要方面提供了新的研究视角。本研究针对睾丸发育的四个阶段开展了核糖体RNA去除RNA测序（rRNA-depleted RNA sequencing）。