Background Germ granules are large cytoplasmic ribonucleoprotein complexes that emerge in the germline to participate in RNA regulation. The two most prominent germ granules are the intermitochondrial cement (IMC) in meiotic spermatocytes and the chromatoid body (CB) in haploid round spermatids, both functionally linked to the PIWI-interacting RNA (piRNA) pathway. Aims In this study, we clarified the IMC function by identifying proteins that form complexes with a well-known IMC protein PIWIL2/MILI in the mouse testis. Results The PIWIL2 interactome included several proteins with known functions in piRNA biogenesis. We further characterized the expression and localization of two of the identified proteins, Exonuclease 3′–5′ domain-containing proteins EXD1 and EXD2, and confirmed their localization to the IMC. We showed that EXD2 interacts with PIWIL2, and that the mutation of Exd2 exonuclease domain in mice induces misregulation of piRNA levels originating from specific pachytene piRNA clusters, but does not disrupt male fertility. Conclusion Altogether, this study highlights the central role of the IMC as a platform for piRNA biogenesis, and suggests that EXD1 and EXD2 function in the IMC-mediated RNA regulation in postnatal male germ cells., We would like to thank all Kotaja lab members for their support and help. The Turku BioScience Cell Imaging and Proteomics Core Facilities are acknowledged for their services and Turku Center for Disease Modeling (TCDM) and Turku Central Animal Facility for providing expertise on animal experimentation. This study was supported by the Academy of Finland, the Sigrid Jusélius Foundation, the Novo Nordisk Foundation, the Jalmari and Rauha Ahokas Foundation, Turku Doctoral Programme of Molecular Medicine (TuDMM), and the Finnish Cultural Foundation.

[Background] Germ granules are large cytoplasmic ribonucleoprotein complexes that emerge in the germline to participate in RNA regulation. The two most prominent germ granules are the intermitochondrial cement (IMC) in meiotic spermatocytes and the chromatoid body (CB) in haploid round spermatids, both functionally linked to the PIWI-interacting RNA (piRNA) pathway., [Aims] In this study, we clarified the IMC function by identifying proteins that form complexes with a well-known IMC protein PIWIL2/MILI in the mouse testis., [Results] The PIWIL2 interactome included several proteins with known functions in piRNA biogenesis. We further characterized the expression and localization of two of the identified proteins, Exonuclease 3′–5′ domain-containing proteins EXD1 and EXD2, and confirmed their localization to the IMC. We showed that EXD2 interacts with PIWIL2, and that the mutation of Exd2 exonuclease domain in mice induces misregulation of piRNA levels originating from specific pachytene piRNA clusters, but does not disrupt male fertility., [Conclusion] Altogether, this study highlights the central role of the IMC as a platform for piRNA biogenesis, and suggests that EXD1 and EXD2 function in the IMC-mediated RNA regulation in postnatal male germ cells., This study was supported by the Academy of Finland, the Sigrid Jusélius Foundation, the Novo Nordisk Foundation, the Jalmari and Rauha Ahokas Foundation, Turku Doctoral Programme of Molecular Medicine (TuDMM), and the Finnish Cultural Foundation., Peer reviewed

9 pages. -- Figure S1. Expression of EXD2 and EXD1 during spermatogenesis. -- Figure S2. Co-localization of EXD2 and EXD1 with DDX4. -- Figure S3. Characterization of the genomic deletion in Exd2∆ mice. -- Figure S4. RNA-sequencing analysis of Exd2∆ testes., Peer reviewed