PIWI proteins tether the piRNA biogenesis machinery to mitochondria during mammalian spermatogenesis.

piRNA biogenesis occurs in the intermitochondrial cement (IMC) in mammalian germ cells. The mechanisms by which IMC components engage mitochondria to form an efficient piRNA biogenesis machinery remain elusive. Here, we demonstrate that PIWI proteins orchestrate the assembly and disassembly of the piRNA biogenesis machinery in mice. The mitochondrial-anchored protein ASZ1 specifically interacts with PIWIL2 and recruits PIWIL2 to IMC granules. Sequentially, piRNAs competitively bind PIWIL2, leading to ASZ1-PIWIL2 dissociation. In fetal male germ cells, ASZ1-PIWIL2-TDRD1 forms a seed complex to initiate the assembly of the piRNA biogenesis machinery. During postnatal meiosis, the TDRKH-PIWIL1-TDRD1 complex synergizes with the ASZ1-PIWIL2-TDRD1 complex to induce substantial IMC assembly and pachytene piRNA biogenesis through TDRD1-mediated phase separation. PIWI proteins act as bridges, tethering non-mitochondrial proteins to mitochondrial-anchored proteins in IMC granules with the assistance of TDRD1. Together, our findings establish the pivotal role of PIWI proteins in governing the spatiotemporal dynamics of piRNA biogenesis machinery during mammalian spermatogenesis.