Transcriptome analysis during mouse testis development

In animal germline cells, Piwi-interacting RNAs (piRNAs) silence retrotransposons through post-transcriptional and transcriptional mechanisms. However, little is known, especially in mammals, about the functions of piRNAs beyond retrotransposon suppression1-5. In mammalian spermatocytes, piRNAs are known to be abundantly expressed6-10. Here, we show that a subset of coding and noncoding RNAs in mouse spermatocytes is degraded by the piRNA pathway. By analyzing the germline trasnscriptome of mice deficient in piRNA biogenesis, we identify hundreds of mRNAs as direct targets of piRNAs. Remarkably, the 3' untranslated region (UTR) of the mRNAs up-regulated in the piRNA pathway mutants are highly enriched with retrotransposon sequenes, implying that these sequences serve as regulatory elements for piRNA-mediated regulation. Furthermore, deficiencies of piRNAs derived from pseudogenes result in increased mRNA levels of their cognate genes, indicating that pseudogenes regulate their functional cognates via piRNAs. Moreover, we identify a large population of testis-enriched long intergenic noncoding RNAs (lincRNAs), some of which are also degraded by the piRNA pathway. Collectively, our results reveal that the piRNA pathway regulates the expression of both mRNAs and lincRNAs in addition to retrotransposon RNAs during meiosis and the key role of retrotransposons and pseudogenes, two major types of genomic sequences, in this regulation by acting as piRNA sources and/or regulatory elements in target RNAs. Transcriptome of 7 developmental stage during mouse testis development were analyzed by deep sequencing using Illumina HiSeq.在动物生殖细胞中，Piwi互作RNA（Piwi-interacting RNAs，piRNAs）通过转录后和转录水平机制抑制逆转录转座子。然而，目前对piRNAs在逆转录转座子抑制之外的功能所知甚少，在哺乳动物中尤其如此1-5。已有研究表明，哺乳动物精母细胞中piRNAs大量表达6-10。本研究发现，小鼠精母细胞中的一部分编码RNA与非编码RNA会被piRNA通路降解。通过分析piRNA生成缺陷小鼠的生殖系转录组，我们鉴定出数百种mRNA作为piRNAs的直接靶标。值得注意的是，在piRNA通路突变体中上调的mRNA的3'非翻译区（untranslated region，UTR）高度富集逆转录转座子序列，这提示这些序列可作为piRNA介导调控的调控元件。进一步研究显示，假基因来源的piRNAs缺陷会导致其同源基因的mRNA水平升高，表明假基因可通过piRNAs调控其功能同源基因。此外，我们还鉴定出大量睾丸富集的基因间长链非编码RNA（long intergenic noncoding RNAs，lincRNAs），其中一部分同样会被piRNA通路降解。综上，本研究结果揭示：在减数分裂过程中，piRNA通路除调控逆转录转座子RNA外，还可调控mRNA与基因间长链非编码RNA的表达；而两类主要的基因组序列——逆转录转座子与假基因，可通过作为piRNA来源和/或靶RNA中的调控元件，在该调控过程中发挥关键作用。本研究通过Illumina HiSeq平台开展深度测序，分析了小鼠睾丸发育过程中的7个发育阶段的转录组。