PIWI proteins and their associated piRNAs act to silence transposons and promote gametogenesis. Murine PIWI proteins MIWI, MILI, and MIWI2 have multiple arginine and glycine (RG)-rich motifs at their N-terminal domains. Despite being known as docking sites for the TDRD family proteins, the in vivo regulatory roles for these RG motifs in directing PIWI in piRNA biogenesis and spermatogenesis remain elusive. To investigate the functional significance of RG motifs in mammalian PIWI proteins in vivo, we genetically engineered an arginine to lysine (RK) point mutation of a conserved N-terminal RG motif in MIWI in mice. We show that this tiny MIWI RG motif is indispensable for piRNA biogenesis and male fertility. The RK mutation in the RG motif disrupts MIWI-TDRKH interaction and impairs enrichment of MIWI to the intermitochondrial cement (IMC) for efficient piRNA production. Despite significant overall piRNA level reduction, piRNA trimming and maturation are not affected by the RK mutation. Consequently, MiwiRK mutant mice show chromatoid body malformation, spermatogenic arrest, and male sterility. Surprisingly, LINE1 transposons are effectively silenced in MiwiRK mutant mice, indicating a LINE1-independent cause of germ cell arrest distinctive from Miwi knockout mice. These findings reveal a crucial function of the RG motif in directing PIWI proteins to engage in efficient piRNA production critical for germ cell progression and highlight the functional importance of the PIWI N-terminal motifs in regulating male fertility.

中文翻译：

---

MIWI N 端 RG 基序可促进有效的粗线期 piRNA 产生和精子发生，与 LINE1 转座子沉默无关。

PIWI 蛋白及其相关的 piRNA 可以沉默转座子并促进配子发生。鼠 PIWI 蛋白 MIWI、MILI 和 MIWI2 在其 N 端结构域具有多个富含精氨酸和甘氨酸 (RG) 的基序。尽管被称为 TDRD 家族蛋白的对接位点，但这些 RG 基序在 piRNA 生物发生和精子发生中指导 PIWI 的体内调节作用仍然难以捉摸。为了研究 RG 基序在哺乳动物 PIWI 蛋白中的体内功能意义，我们对小鼠 MIWI 中保守的 N 端 RG 基序进行了基因工程改造，将精氨酸转变为赖氨酸 (RK) 点突变。我们证明这个微小的 MIWI RG 基序对于 piRNA 生物发生和男性生育能力是不可或缺的。RG 基序中的 RK 突变破坏了 MIWI-TDRKH 相互作用，并损害了 MIWI 向线粒体间水泥 (IMC) 的富集，从而无法有效生产 piRNA。尽管整体 piRNA 水平显着降低，但 piRNA 修剪和成熟不受 RK 突变的影响。因此，MiwiRK 突变小鼠表现出类染色体畸形、生精停滞和雄性不育。令人惊讶的是，LINE1 转座子在 MiwiRK 突变小鼠中被有效沉默，这表明与 Miwi 基因敲除小鼠不同，生殖细胞停滞的原因与 LINE1 无关。这些发现揭示了 RG 基序在指导 PIWI 蛋白参与对生殖细胞进展至关重要的高效 piRNA 生产中的关键功能，并强调了 PIWI N 端基序在调节男性生育力中的功能重要性。