European Molecular Biology Laboratory, Mouse Biology Unit, Via Ramarini 32, Monterotondo Scalo 00015, Italy.
Nature. 2011 Oct 23;480(7376):259-63. doi: 10.1038/nature10547.
Piwi proteins and Piwi-interacting RNAs (piRNAs) have conserved functions in transposon silencing. The murine Piwi proteins Mili and Miwi2 (also called Piwil2 and Piwil4, respectively) direct epigenetic LINE1 and intracisternal A particle transposon silencing during genome reprogramming in the embryonic male germ line. Piwi proteins are proposed to be piRNA-guided endonucleases that initiate secondary piRNA biogenesis; however, the actual contribution of their endonuclease activities to piRNA biogenesis and transposon silencing remain unknown. To investigate the role of Piwi-catalysed endonucleolytic activity, we engineered point mutations in mice that substitute the second aspartic acid to an alanine in the DDH catalytic triad of Mili and Miwi2, generating the Mili(DAH) and Miwi2(DAH) alleles, respectively. Analysis of Mili-bound piRNAs from homozygous Mili(DAH) fetal gonadocytes revealed a failure of transposon piRNA amplification, resulting in the marked reduction of piRNA bound within Miwi2 ribonuclear particles. We find that Mili-mediated piRNA amplification is selectively required for LINE1, but not intracisternal A particle, silencing. The defective piRNA pathway in Mili(DAH) mice results in spermatogenic failure and sterility. Surprisingly, homozygous Miwi2(DAH) mice are fertile, transposon silencing is established normally and no defects in secondary piRNA biogenesis are observed. In addition, the hallmarks of piRNA amplification are observed in Miwi2-deficient gonadocytes. We conclude that cycles of intra-Mili secondary piRNA biogenesis fuel piRNA amplification that is absolutely required for LINE1 silencing.
Piwi 蛋白和 Piwi 相互作用的 RNA（piRNAs）在转座子沉默中具有保守功能。小鼠的 Piwi 蛋白 Mili 和 Miwi2（也分别称为 Piwil2 和 Piwil4）在雄性胚胎生殖细胞的基因组重编程过程中指导表观遗传的 LINE1 和内粒 A 颗粒转座子沉默。Piwi 蛋白被提议为 piRNA 指导的内切核酸酶，可启动次级 piRNA 生物发生；然而，它们的内切核酸酶活性对 piRNA 生物发生和转座子沉默的实际贡献仍然未知。为了研究 Piwi 催化的内切核酸酶活性的作用，我们在小鼠中设计了点突变，将 Mili 和 Miwi2 的 DDH 催化三联体中的第二个天冬氨酸突变为丙氨酸，分别产生 Mili(DAH)和 Miwi2(DAH)等位基因。来自纯合 Mili(DAH)胎儿生殖细胞的 Mili 结合 piRNA 的分析显示转座子 piRNA 扩增失败，导致 Miwi2 核糖核蛋白颗粒内结合的 piRNA 显著减少。我们发现 Mili 介导的 piRNA 扩增选择性地需要 LINE1，但不需要内粒 A 颗粒沉默。Mili(DAH) 小鼠中缺陷的 piRNA 途径导致精子发生失败和不育。令人惊讶的是，纯合 Miwi2(DAH) 小鼠是可育的，转座子沉默正常建立，并且没有观察到次级 piRNA 生物发生的缺陷。此外，在 Miwi2 缺陷的生殖细胞中观察到 piRNA 扩增的特征。我们得出结论，Mili 内的次级 piRNA 生物发生循环为绝对需要 LINE1 沉默的 piRNA 扩增提供燃料。
