MIWI N-terminal RG motif promotes efficient pachytene piRNA production and spermatogenesis independent of LINE1 transposon silencing

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. The presence of RG-rich motifs in the N-terminal region of the PIWI family proteins is a conserved feature. However, how these RG-motifs direct PIWI proteins to participate in piRNA biogenesis and germ cell function in vivo remains unknown. Here we genetically mutate a conserved RG motif in MIWI/PIWIL1 in mice to elucidate its physiological function in piRNA regulation and spermatogenesis. For the first time, we show that an RG motif mediating crucial protein-protein interactions is essential for piRNA biogenesis and male fertility in mice. Unexpectedly, this genetic model separates the two defects of the MIWI mutant: spermiogenesis arrest and LINE1 transposon activation. These findings highlight the functional importance of this tiny protein motif in fertility regulation.