TDRD5 facilitates piRNA biogenesis and male fertility through phase separation

piRNAs are small regulatory RNAs with critical roles in transposon silencing and regulation of gametogenesis. Phase separation is a driving force for piRNA biogenesis machinery formation; however, the specific piRNA-related proteins that mediate phase separation and how they coordinate to drive piRNA production remain incompletely understood. Through systematic screening, we show here that TDRD5 undergoes phase separation to promote piRNA biogenesis. TDRD5 condensation is mediated by its Lotus domains and an intrinsically disordered region enriched with aromatic residues. Furthermore, TDRD1 and TDRD5 colocalize and synergistically enhance each other's phase separation. TDRD5 phase separation is required for intermitochondrial cement assembly and fetal piRNA processing in fetal/neonatal gonocytes but is dispensable for pachytene piRNA biogenesis in adult mice. TDRD5 phase separation deficiency impairs male fertility in mice and may contribute to human male infertility. Our findings highlight the critical but diverse contributions of phase-separated proteins to germ granule assembly and piRNA production.