Yale Stem Cell Center, Yale University, New Haven, CT 06519, USA.
EMBO J. 2013 Jul 3;32(13):1869-85. doi: 10.1038/emboj.2013.121. Epub 2013 May 28.
Piwi proteins and Piwi-interacting RNAs (piRNAs) repress transposition, regulate translation, and guide epigenetic programming in the germline. Here, we show that an evolutionarily conserved Tudor and KH domain-containing protein, Tdrkh (a.k.a. Tdrd2), is required for spermatogenesis and involved in piRNA biogenesis. Tdrkh partners with Miwi and Miwi2 via symmetrically dimethylated arginine residues in Miwi and Miwi2. Tdrkh is a mitochondrial protein often juxtaposed to pi-bodies and piP-bodies and is required for Tdrd1 cytoplasmic localization and Miwi2 nuclear localization. Tdrkh mutants display meiotic arrest at the zygotene stage, attenuate methylation of Line1 DNA, and upregulate Line1 RNA and protein, without inducing apoptosis. Furthermore, Tdrkh mutants have severely reduced levels of mature piRNAs but accumulate a distinct population of 1'U-containing, 2'O-methylated 31-37 nt RNAs that largely complement the missing mature piRNAs. Our results demonstrate that the primary piRNA biogenesis pathway involves 3'→5' processing of 31-37 nt intermediates and that Tdrkh promotes this final step of piRNA biogenesis but not the ping-pong cycle. These results shed light on mechanisms underlying primary piRNA biogenesis, an area in which information is conspicuously absent.
Piwi 蛋白和 Piwi 相互作用 RNA（piRNA）在生殖细胞中抑制转座、调节翻译和指导表观遗传编程。在这里，我们表明，一个进化上保守的含有 Tudor 和 KH 结构域的蛋白质 Tdrkh（也称为 Tdrd2），是精子发生所必需的，并参与 piRNA 的生物发生。Tdrkh 通过 Miwi 和 Miwi2 中的对称二甲基精氨酸残基与 Miwi 和 Miwi2 结合。Tdrkh 是一种线粒体蛋白，通常与 pi 体和 piP 体并列，并且需要 Tdrd1 细胞质定位和 Miwi2 核定位。Tdrkh 突变体在合线期表现出减数分裂停滞，减弱了 LINE1 DNA 的甲基化，并上调了 LINE1 RNA 和蛋白质，而没有诱导细胞凋亡。此外，Tdrkh 突变体的成熟 piRNA 水平严重降低，但积累了大量独特的含有 1'U、2'O-甲基化的 31-37nt RNA，这些 RNA 在很大程度上弥补了缺失的成熟 piRNA。我们的结果表明，初级 piRNA 生物发生途径涉及 31-37nt 中间产物的 3'→5'加工，并且 Tdrkh 促进了 piRNA 生物发生的最后一步，但不促进 ping-pong 循环。这些结果揭示了初级 piRNA 生物发生的机制，在这一领域信息明显缺失。
