piRNA-mediated germline silencing

Animal germ cells use PIWI-clade Argonautes loaded with PIWI-interacting RNAs (piRNAs) to recognize transposon transcripts and other foreign or unwanted RNAs and to silence them. The module has three conserved steps. First, a cytoplasmic PIWI-piRNA complex in perinuclear nuage recognizes a target RNA by base pairing. Second, recognition is converted into an amplified pool of secondary small RNAs. Third, a nuclear Argonaute carries small RNAs into the nucleus and silences complementary loci co-transcriptionally. How the second and third steps are carried out differs between lineages. In insects and vertebrates, amplification is the ping-pong cycle: piRNA-guided slicing by cytoplasmic PIWI proteins (fly Aubergine and Ago3, mammalian PIWIL2/MILI) cuts target and precursor transcripts, and the cleavage products become new piRNAs. Nematodes lack ping-pong. In Caenorhabditis elegans the PIWI protein PRG-1 does not need to slice; its targets are instead used as templates by RNA-dependent RNA polymerase to make secondary 22G-RNAs, which are loaded into nematode-specific WAGO Argonautes. The nuclear step is also carried out by different, non-slicing effectors: fly Piwi directs H3K9me3 heterochromatin, mammalian PIWIL4/MIWI2 directs de novo DNA methylation of transposons, and in nematodes the WAGO HRDE-1 recruits the nuclear RNAi factor NRDE-2 and directs H3K9me3. In C. elegans the silenced state can be inherited for many generations without the piRNA trigger, through the nuclear HRDE-1 loop and a cytoplasmic loop in which the helicase ZNFX-1 and WAGO-4 form Z granules. Loss of the core PIWI proteins causes transposon derepression and sterility in flies and mice, and progressive loss of fertility in nematodes.

Renamed and generalized from the C. elegans-only module c_elegans_pirna_transgenerational_silencing. Slug choice. The slug says germline silencing rather than transposon silencing because, in nematodes, piRNAs scan the whole germline transcriptome for non-self sequences (transgenes and other foreign RNAs as well as transposons), and the nematode silenced state is heritable. Transposable element silencing remains the main conserved output and is a module concept. Boundary. The module covers how piRNA-guided silencing is targeted, amplified and imposed on chromatin, plus the nematode transgenerational memory. Primary piRNA biogenesis (piRNA cluster transcription, Zucchini/ PLD6 phasing, PARN-1/PNLDC1 trimming, HENN1 2'-O-methylation, and the nematode USTC complex) is upstream and excluded. The pachytene piRNA functions of mammalian PIWIL1/MIWI in spermiogenic mRNA turnover, and the maternal germ plasm roles of Aubergine, are outside the boundary. Lineage choices. Amplification is a variant set: ping-pong (insects, vertebrates) versus RdRP-driven 22G-RNA synthesis (nematodes). The nematode amplification machinery itself is modeled in MODULE:c_elegans_mutator_22g_rna_amplification and is not repeated here; the nematode variant holds DEPS-1 (the PRG-1 partner) and the WAGO-1 effector. Nuclear silencing is a second variant set with three lineage effectors (fly Piwi, mammalian PIWIL4, nematode HRDE-1 with NRDE-2). None of the three nuclear effectors is given an endonuclease function here: fly Piwi and mouse Miwi2 slicer mutants silence transposons normally, and HRDE-1 is a WAGO without the catalytic residues. PIWIL2 contributes to DNA methylation only upstream, by supplying the secondary piRNAs loaded into PIWIL4; it is therefore placed in the ping-pong variant, with a connection to PIWIL4, and not in the nuclear step. The Z granule loop is an optional, nematode-specific part. PIWI family. Recognition is a FAMILY role over PIWI-clade Argonautes. PANTHER puts all Argonautes, AGO and PIWI clades alike, in PTHR22891, so no PANTHER family id is asserted for the PIWI clade. The PAINT node PTN007716312 (piRNA binding IBD) is inherited by every PIWI protein in this module (PRG-1, PIWIL1, PIWIL2, PIWIL4, Piwi, Aub, Ago3) but not by HRDE-1 or the other WAGOs. Named but not modeled. Mammalian PIWIL1 is a pachytene effector and is only a representative of recognition. Ping-pong cofactors (Vasa/DDX4, Krimper, Qin, TDRD proteins, GTSF1, the EXD1-TDRD12 PET complex), the fly nuclear factors (Panoramix/SFiNX, Asterix/Gtsf1, Eggless/SetDB1, HP1a), the mammalian methylation machinery (SPOCD1, DNMT3C, DNMT3L), the nematode NRDE-1/3/4 factors, SET-25, SET-32, HPL-2 and SIMR-1, and the nearly inactive PRG-1 paralog PRG-2 are named only.