In Drosophila germ cells, piRNAs are produced from specific heterochromatic loci, composed of numerous TE fragments, called piRNA clusters [74,75]. Those clusters are enriched in the H3K9me3 mark and Rhino HP1-like protein [76] (Figure 1). Rhino interacts first with the co-factors Deadlock and Cutoff, then Deadlock recruits the transcriptional initiation machinery via the large subunit of transcription initiation factor II A (TFIIA-L) paralog called Moonshiner, as well as Bootlegger, UAP56, the THO complex, and nuclear export factors Nxt1 and Nxf3 [76,77,78,79]. The piRNA precursor transcripts emerging from piRNA clusters are then exported by the Crm1 (chromosomal maintenance 1) exportin to the cytoplasmic piRNA biogenesis site, where they undergo a single strand hydrolysis by the Zucchini RNase to finally give rise to piRNAs [78,79,80,81,82,83] (Figure 1). In contrast to si- and miRNA, piRNAs are charged directly as single guide RNA onto PIWI proteins to form piRNA-induced silencing complexes (piRISC) [74]. After nuclear translocation, piRISC complexes recognize nascent TE transcripts and perform transcriptional gene silencing (TGS) by local heterochromatinization of TEs. This silencing requires nuclear Piwi-piRNAs that direct Panoramix, Nxf2, its co-factor, Nxt1, and the histone methyltransferase Eggless to the TE loci [84,85,86,87,88,89,90,91] (Figure 1). In the cytoplasm, mRNAs of TEs are cleaved by piRISC complexes, leading to post-transcriptional gene silencing (PTGS) of mobile elements. Cleaved-transcripts are not degraded; they are charged by PIWI protein to form new piRISC complexes, corresponding to an amplification mechanism called the ‘Ping-Pong cycle’ [74] (Figure 1). piRNA-dependent silencing occurs at each gonadal developmental stage [92], and disruption of the piRNA pathway leads to TE mobilization [93], which induces DNA damage [94,95].

PIWI protein distributions reveal that the piRNA pathway is highly conserved in animals, but they exist neither in plants nor in prokaryotes [96]. Interestingly, recent studies show that piRNAs are also found in somatic tissues of mammalian, arthropods, and mollusks [97,98,99,100]. In Drosophila melanogaster, however, piRNA expression is restricted to the gonads, including the germline nurse cells and the somatic follicular cells [101,102]. These findings might suggest that the ancestral capacity to synthesize piRNAs in somatic tissues (outside of gonads) has been lost in D. melanogaster. However, this is still subject to debate [103,104,105].

Genomic annotations of small RNA libraries isolated from various metazoans reveal that piRNAs are not restricted to TE regulation but can also target coding genes. In Drosophila, piRNAs derived from the 3′UTR of traffic-jam (tj) matching Fasciclin III (FasIII) can be identified in the ovarian somatic cells (OSC) line. This finding suggests that FasIII is regulated by piRNAs originated from the tj 3′UTR [102,106]. In Drosophila early embryos, nanos (nos) is regulated by piRNAs. Evidence suggests that maternal nos mRNAs are targeted by a complex composed of piRNAs, Aub, and CCR4-NOT deadenylation factors in the soma, whereas in the germ plasm, those transcripts are stabilized by the interaction with Aub [107,108]. In Aplysia, piRNAs seem to be implicated in CREB2 promoter methylation, a transcriptional repressor involved in long-term memory [109]. As the last example, sex determination in silkworm is dependent on the piRNA pathway that regulates Masculinizer, a locus required for the production of the doublesex female-specific isoform [110]. Taken together, these data highlight the idea that piRNAs are required in gene regulation of a broad range of cellular functions.

In Drosophila, the insertion of exogenous lacZ sequences into a subtelomeric piRNA cluster can functionally repress a euchromatic lacZ transgene in trans [111]. This epigenetic silencing mechanism, referred to as the trans-silencing effect (TSE), requires maternally but not paternally inherited piRNAs [71,112,113,114,115,116]. These results strongly suggest an active role for piRNAs in TSE and show that a piRNA cluster, without maternal piRNA inheritance, is not sufficient to promote this silencing process [112]. Involvement of piRNAs in epigenetic memory has been associated with epiallele emergence through a paramutation phenomenon in D. melanogaster and in C. elegans [71,117]. Paramutation is defined as a modification of one allele induced by another allele without DNA sequence modification and corresponds to an epigenetic conversion (for reviews see [118,119,120].

In Drosophila, using a locus made of repeated P-lacZ-white transgenes mimicking a piRNA cluster (called BX2 cluster), but inactive for piRNA synthesis, we have shown that homologous maternally-inherited piRNAs can stably convert this locus into an active piRNA cluster for over 200 generations [71] (Figure 2). This newly-activated cluster has the same properties as other Drosophila piRNA clusters. This piRNA-induced paramutation process requires molecular actors of the piRNA pathway [72] and performs TE-derived sequence control generation after generation. Finally, the reversibility of paramutation occurs when maternal piRNA inheritance is interrupted, underscoring the idea that this is an epigenetic process [26,71]. Taken together, based on sequence complementarity, these data show that maternally-inherited piRNAs are competent to initiate new piRNA production in the next generation and suggest that piRNAs are responsible for their own maintenance across generations. Two non-exclusive molecular mechanisms have been proposed to explain this case of epigenetic memory in which piRNA production requires piRNA inheritance [121]. In the first, inherited-piRNAs would induce heterochromatin formation on the piRNA cluster by leading to H3K9me3 deposition, inducing local Rhino enrichment [26]. The piRNA cluster-specific transcription machinery will then assemble at the locus to promote piRNA synthesis [77]. In the second, inherited piRNAs could trigger their production by Ping-Pong amplification from piRNA cluster precursors and TE transcripts [74,75].

In C. elegans, piRNAs correspond to 21 nucleotide (nt) long small RNAs with a uridine bias at the 5′-end (21U RNA) distinct from the endo-siRNAs that are 22 nt long small RNAs with a guanosine bias at the 5′-end (22G RNA) [122]. The biogenesis of nematode piRNAs depends on the Piwi protein, PRG-1 [66,123,124]. Like in Drosophila, transgenes are used to decipher the molecular mechanisms of germline repression. Analysis of the piRNA sensor indicates that piRNAs are required to trigger silencing through imperfect base-pairing [125,126,127]. This silencing is then transgenerationally propagated by the 22G RNA dependent on numerous factors, such as RdRp, NRDE-2, the Argonaute proteins WAGO-9/HRDE-1 and WAGO-4 and chromatin components, such as the histone methyltransferases SET-25, SET32, and HPL-2, a Heterochromatin 1 Protein (HP1) orthologue [45,128,129,130,131,132,133]. Indeed, specific H3K9 methyltransferases, SET-25 and SET-32, and endo-siRNAs have been shown to work together to promote silencing inheritance [134]. In addition, neuronal RDE-4 and germline HRDE-1 have been implicated in transgenerational regulation of endo-siRNAs and mRNAs controlling chemotaxis [135]. The authors proposed the existence of an intricate interaction between the neuronal system and the germline to control transgenerational behavior. Finally, precise dissection of the silencing mechanism determines that piRNAs lead to the production of secondary and tertiary siRNAs, necessary for full repression and leading to an epigenetic conversion form of paramutation [67].

Altogether, the piRNA silencing pathway in Drosophila involves piRNAs for the initiation and maintenance through generations [26,71,72], while in worms piRNAs are involved in triggering the silencing that is then maintained by siRNAs in the progeny, independent from piRNAs [124,136,137,138].

In F0 mice, an early maternal separation causes downregulation of piRNA cluster 110 [139], and a Western-like diet leads to differential expression of 190 piRNAs distributed on 63 piRNA clusters [140]. In rat, a high-fat diet induces deregulation of 1092 piRNAs, and only three of them are still differentially expressed at the next generation [141]. In C. elegans, high temperature (25 °C) induces motif-dependent piRNA biogenesis downregulation [142]. This downregulation is associated with differential gene expression and fitness reduction, which persist at the next generation. However, in those studies, piRNA expression in subsequent generations, as well as their roles in a phenotypic response and the potential consequences on the epigenome, was not directly addressed. In the case of rat stressed with vinclozolin or DDT, differentially expressed piRNAs were identified in F3, but the mechanism and role of piRNAs were not questioned [20,21,22,23,25].

In 2019, two cases of environmental stresses inducing a genomic response dependent on piRNAs have been published in C. elegans and in D. melanogaster [26,46].