Transcriptome analysis in mouse late spermatocyte 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. Late spermatocyte 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）可通过转录后与转录机制沉默逆转录转座子。然而，除逆转录转座子沉默功能外，人们对piRNAs的其他生物学作用仍知之甚少，在哺乳动物中更是如此[1-5]。已知在哺乳动物精母细胞中，piRNAs呈高表达状态[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，在piRNA通路突变体中上调的mRNA的3'非翻译区（3' untranslated region，UTR）中，逆转录转座子序列高度富集，这表明这些序列可作为piRNA介导调控的调控元件。此外，假基因来源的piRNAs缺失会导致其对应功能基因的mRNA水平升高，这表明假基因可通过piRNAs调控其功能性同源基因。不仅如此，我们还鉴定出大量睾丸富集型基因间长链非编码RNA（long intergenic noncoding RNAs，lincRNAs），其中一部分同样可被piRNA通路降解。综上，我们的研究结果表明，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lincRNAs的表达；同时，两种主要基因组序列类型——逆转录转座子与假基因，可通过充当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。本研究采用Illumina HiSeq平台对Miwi+/-与Miwi-/-小鼠的晚期精母细胞mRNA谱进行了三次重复深度测序分析。