Transcriptome analysis in mouse early round spermatids deficient in pachytene piRNAs (Miwi+/- and -/-)

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. Early round spermatid mRNA profiles of Miwi+/- and -/- were analyzed by deep sequencing, in triplicate, using Illumina HiSeq.在动物生殖细胞中，Piwi互作RNA（Piwi-interacting RNAs，piRNAs）通过转录后与转录水平的机制沉默反转录转座子（retrotransposons）。然而，人们对piRNAs在反转录转座子抑制之外的功能知之甚少，在哺乳动物中尤为如此1-5。已知在哺乳动物精母细胞中，piRNAs呈高丰度表达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可被piRNA通路降解。通过分析piRNA生成缺陷小鼠的生殖细胞转录组，我们鉴定出数百个mRNA作为piRNAs的直接靶标。值得注意的是，piRNA通路突变体中上调的mRNA的3'非翻译区（3' untranslated region，UTR）富含反转录转座子序列，提示这些序列可作为piRNA介导调控的调控元件。此外，源自伪基因的piRNAs缺失会导致其同源基因的mRNA水平升高，表明伪基因可通过piRNAs调控其功能性同源基因。此外，我们鉴定出大量睾丸富集的长基因间非编码RNA（long intergenic noncoding RNAs，lincRNAs），其中一部分同样可被piRNA通路降解。综上，我们的研究结果揭示，在减数分裂过程中，piRNA通路除了调控反转录转座子RNA外，还可调控mRNA与lincRNAs的表达；同时阐明了两类主要基因组序列——反转录转座子与伪基因，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的关键作用。本研究采用Illumina HiSeq测序平台，对Miwi杂合（Miwi+/-）与纯合敲除（Miwi-/-）小鼠的早期圆形精子细胞mRNA谱进行了三次生物学重复的深度测序分析。