Mutator foci 22G-RNA amplification in Caenorhabditis elegans

In the Caenorhabditis elegans germline, silencing guided by primary small RNAs is weak, and is made effective by a second round of small RNA synthesis. A primary Argonaute such as RDE-1 recognizes a target transcript and recruits the NYN-domain endoribonuclease RDE-8, which cuts it. The ribonucleotidyltransferase RDE-3 (MUT-2) gives the 5' fragment a poly(UG) tail, and RNA-dependent RNA polymerases (RdRPs) then use the tailed RNA as a template to synthesize abundant 22-nucleotide, 5'-guanosine secondary siRNAs (22G-RNAs). Most of this amplification takes place in Mutator foci. These are perinuclear, phase-separated condensates that lie next to P granules at nuclear pores. The intrinsically disordered Q/N-rich protein MUT-16 is the scaffold of the foci. Distinct regions of MUT-16 recruit the RdRP RRF-1; MUT-15 (RDE-5), which in turn recruits RDE-8; the DEAD-box RNA helicase MUT-14, which acts redundantly with SMUT-1 to start amplification; RDE-3; and, through the adaptor RDE-2 (MUT-8), the 3'-5' exoribonuclease MUT-7. The 22G-RNAs made in this way are loaded into worm-specific Argonautes (WAGOs) that lack slicer residues, such as WAGO-1, and silence transposons, pseudogenes and other germline-repressed genes. A second germline RdRP, EGO-1, makes most of the 22G-RNAs bound by the catalytic Argonaute CSR-1, which target most germline-expressed genes and protect them from WAGO-mediated silencing instead of silencing them. The two RdRP systems are not fully separate: EGO-1 also contributes, partly redundantly with RRF-1, to WAGO-class 22G-RNAs.

Boundary. This module is the amplification machinery: the steps between target recognition by a primary Argonaute and the loading of secondary 22G-RNAs into effector Argonautes. It does not cover what selects targets. In the germline the main trigger is PRG-1/21U-RNA scanning; for exogenous RNAi it is RDE-1. It also does not cover how silencing is made heritable. Both are modeled in MODULE:pirna_mediated_germline_silencing, which links to this module for its amplification step. The primary Argonaute itself is outside the boundary, but the RDE-8 cut it directs is inside, because the cut produces the RNA that is tailed and copied. Order of steps: target cleavage (RDE-8) precedes pUG tailing (RDE-3), which precedes RdRP synthesis (RRF-1). The 26G-RNA (ERGO-1, ALG-3/4) and somatic RRF-1 pathways are outside the germline boundary. Initiation part. MUT-14 and SMUT-1 are redundant DEAD-box helicases. Only MUT-14 has a gene review, so only MUT-14 is modeled; SMUT-1 is named in prose. Where the helicase step falls relative to RDE-8 cleavage is not known, so MUT-14 is grouped with cleavage in one initiation part rather than given its own ordered step. MUT-15 (RDE-5) has no molecular function term in its review or here: it recruits RDE-8 (and NYN-1/NYN-2) to MUT-16, but whether it is a direct adaptor is not established. NYN-1 and NYN-2 have no gene reviews and are not modeled. Effector Argonautes. WAGO-1 is the representative cytoplasmic WAGO effector. The nuclear WAGO HRDE-1 and the Z granule WAGO-4 also bind Mutator-derived 22G-RNAs. They are modeled in the piRNA/inheritance module because their defining role is heritability, not amplification. CSR-1 branch. The final part has two variants (ONE_OR_MORE). Both are germline RdRP-derived 22G-RNA systems, but only the WAGO branch uses the Mutator machinery. CSR-1 22G-RNAs are made by EGO-1, are not enriched at Mutator target genes, and license or protect germline-expressed genes instead of silencing them. The CSR-1 variant is included to make that contrast explicit. It is not a claim that CSR-1 acts in Mutator foci. Its MF follows the csr-1 review's licensing core function (single-stranded RNA binding). The review's separate slicer core function (GO:0070551) is not modeled here, because this module concerns 22G-RNA production and partition, not target cleavage. RdRP overlap. EGO-1 is modeled in the CSR-1 branch, where it makes most of the CSR-1 22G-RNAs, with an added connection to the WAGO branch because EGO-1 and RRF-1 are redundantly required for piRNA-driven 22G-RNA biogenesis. RRF-1 has three UniProtKB entries (G5ECM1, G3MU40, Q7JLA0); G5ECM1, the one cross-referenced to WormBase isoform a, is the accession used by the rrf-1 gene review.