In preprints: illuminating germline dark matter – pachytene piRNA biogenesis and function

You and I both descend from a long lineage of immortal germline. Myriad mechanisms are in place to preserve germline integrity, some of which involve non-coding small RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs). These small RNAs are loaded onto Argonautes (AGOs) – AGO1-4 for miRNAs and siRNAs, and PIWIL1-4 for piRNAs – forming RNA-induced silencing complexes (RISCs). Their regulatory outcomes, ranging from transcriptional gene silencing to translational repression, largely depend on the extent of complementary pairing of RISCs with their targets (Wu et al., 2020).In this article, we focus on piRNAs, which are 24- to 32-nucleotide-long RNAs transcribed from intergenic DNA elements termed piRNA clusters. They play a conserved role in maintaining genomic integrity by silencing transposable elements (TEs). Indeed, removing individual PIWI proteins in Drosophila leads to transposon mobilization and infertility (Yamashiro and Siomi, 2018). Similarly, in the nematode Caenorhabditis elegans, removing a PIWI homolog leads to de-repression of the Tc3 transposon (Das et al., 2008).Mice encode three PIWI proteins: cytoplasmic PIWIL1 (MIWI), PIWIL2 (MILI) and nuclear PIWIL4 (MIWI2). Pre-pachytene piRNAs are derived from TEs and, together with MILI and MIWI2, they maintain genomic stability in pre-meiotic germ cells (Carmell et al., 2007; Kuramochi-Miyagawa et al., 2004). Interestingly, an abundance of non-TE piRNAs are expressed during the pachytene stage of meiosis. These enigmatic piRNAs associate with MIWI and MILI and, despite lacking discernible targets, are essential for spermatogenesis (Deng and Lin, 2002). In MIWI-deficient mice, which lack pachytene piRNA pathways, spermatogenesis is arrested at the round spermatid stage and is accompanied by defects at earlier stages, including chromosome mis-segregation (Fu and Wang, 2014).Although it is clear that tight spatial and temporal regulation of piRNAs is crucial for TE silencing and proper meiosis, what controls the localization of MIWI and MILI to germ granules is not well understood. In addition, because pachytene piRNAs exhibit extreme sequence diversity, rapid evolution, lack of obvious complementarity and elusive pairing rules, the molecular mechanisms by which they regulate meiosis during spermatogenesis are unknown. Two recent preprints begin to close these important knowledge gaps.During pachytene piRNA biogenesis, MILI and MIWI concentrate in the intermitochondrial cement (IMC) – germ granules located among clustered mitochondria in spermatocytes. Yan, Wei, et al. sought to investigate the localization and dynamics of two PIWI proteins, MIWI and MILI, during the pachytene and diplotene stages (Yan et al., 2025 preprint). The preprint authors found that MILI and MIWI are recruited to the IMC by distinct mechanisms involving mitochondria-anchored proteins, ASZ1 and TDRKH, respectively. They further found that conditional loss of ASZ1 specifically in postnatal male germ cells (Asz1cKO) causes pronounced mitochondrial aggregation and nearly abolishes the pachytene piRNA pool, phenocopying animals lacking MOV10L1, an RNA helicase involved in pachytene piRNA processing. Indeed, ASZ1 was required to recruit MOV10L1 to the IMC. Of note, the levels of piRNA precursors were elevated in both Asz1cKO and Mov10l1cKO testes. These results suggest that the drastic loss of pachytene piRNAs observed in Asz1cKO testes may be due to the incorrect localization of piRNA precursors that prevents their maturation and function. Importantly, despite severe depletion of piRNAs in Asz1cKO testes, LINE1 transposon silencing remained functional. Their differential recruitment to the IMC may allow divergent roles for MIWI and MILI, with MILI functioning in pre-meiotic TE silencing and both MIWI and MILI contributing to meiotic gene regulation (Ding et al., 2019).Curiously, Yan et al. (2025 preprint) reported that, during late pachytene and diplotene stages, MIWI and MILI – together with TDRD1 and TDRD5, which are crucial for piRNA biogenesis – segregate away from ASZ1- and TDRKH-containing IMC and re-localize to the chromatoid body (CB). Hence, the authors proposed three classes of piRNA factors that show coordinated spatiotemporal regulation during spermatogenesis: (1) mitochondria-associated factors, ASZ1 and TDRKH, required for the recruitment of PIWI proteins, ensuring processing and loading of piRNA precursors in the IMC; (2) transitional factors, including MILI, MIWI, TDRD1 and TDRD5, which translocate from the IMC to CB precursors upon piRNA maturation; and (3) CB precursor-specific factors, TDRD6 and TDRD7 Tudor proteins. Loss of ASZ1 blocked recruitment of MILI to the IMC and subsequent translocation of transitional piRNA factors (MIWI, TDRD1 and TDRD5) from the IMC to CB precursors, resulting in malformation of CB in round spermatids, highlighting the crucial role of mitochondria-anchored proteins in regulating the pachytene piRNA pathway during spermatogenesis.Building on spatiotemporal regulation of piRNA factors, in this section, we discuss how pachytene piRNAs regulate gene expression to ensure proper meiosis – a long-standing question given that pachytene piRNAs lack apparent targets.Recently, Loubalova, Ahrend, Stoyko, et al. reported a surprising finding that, in mice, a small fraction of pachytene piRNAs – ∼1.6% – target genes in a precise manner, with near-perfect complementarity (Loubalova et al., 2025 preprint). This remarkable complementarity across the coding sequence and 3′ untranslated region (UTR) resembles siRNAs-mediated silencing, which typically engages Dicer and AGO, rather than PIWI, suggesting that distinct small RNA silencing pathways share conserved mechanistic principles.Two genes most strongly targeted by this siRNA-like mechanism are Ago2, an AGO essential for RNAi-mediated gene silencing, and Spin1, a histone reader that recognizes methylation on specific residues and facilitates expression of ribosomal genes. These mRNA-targeting pachytene piRNAs are apparently derived from pseudogene fragments embedded in the piRNA clusters and act in trans. Deleting Ago-2-targeting piRNA clusters led to specific upregulation of Ago2, highlighting the one-to-one regulatory relationship between individual piRNA clusters and their cognate mRNA targets. Notably, although deleting Ago-2-targeting cluster had no overt effects on spermatogenesis and fertility, the loss of Spin1-targeting piRNAs resulted in various spermatogenic defects, and many germ cells showed chromosome mis-segregation previously observed in MIWI-deficient testes, suggesting that misregulation of Spin1 may partially account for the infertility of pachytene piRNA mutants.By extending the analysis to other mammalian species, the authors found that ∼2.7% of human pachytene piRNAs exhibit near-perfect complementarity to mRNAs, with the most targeted gene being GOLGA2 (also known as GM130), a peripheral membrane protein associated with the Golgi network and involved in the regulation of cell division, polarization and migration (Nakamura, 2010). GOLGA2-targeting piRNAs are derived from a single cluster containing pseudogene fragments. Remarkably, despite rapid genetic drift and extensive sequence heterogeneity across most pachytene piRNAs, the GOLGA2-targeting cluster is conserved in primates, including rhesus macaque and marmoset, suggesting functional importance of this regulon. Supporting this, previous work in mice has linked sperm defects to Golga2 overexpression caused by depletion of specific piRNA clusters (Choi et al., 2021). Interestingly, the GOLGA2 cluster is syntenic with the Spin1 cluster; however, whereas the GOLGA2 cluster is conserved across primates, the Spin1 cluster appears to be mouse-specific and could not be detected in cows and rats. Together, these results indicate that the mechanism underlying direct piRNA-guided gene silencing during meiosis is highly conserved, although the specific mRNA targets differ between mice and primates.The two preprints discussed here beautifully illustrate the intricate regulation of the pachytene piRNA pathway in safeguarding germline integrity and promoting proper meiosis. Yet many questions remain. (1) How are MILI and MIWI recognized by different mitochondria-anchored scaffold proteins? (2) Do MILI, MIWI or both contribute to piRNA-guided mRNA silencing? (3) What are the functions of the vast majority of pachytene piRNAs without cognate targets? It has recently been proposed that most pachytene piRNAs lacking cleavage targets propagate selfishly alongside a small number of functional piRNAs important for spermatogenesis (Cecchini et al., 2026). It is tempting to speculate that the RNAi-like mode of gene regulation discussed above may, at least in part, contribute to the evolutionary retention of otherwise dispensable piRNAs.