Compartmentalized RNA Surveillance Through Membraneless barriers

Biomolecular condensates are thought to compartmentalize biochemical activities, yet how they control macromolecular access without membranes remains unclear. We show that C. elegans germline condensates have distinct RNA selectivity: P granules broadly admit RNA, whereas Mutator foci associate only with RNAs destined for silencing. The Mutator scaffold MUT-16 is required for this selectivity; charge-reversal substitutions in its C-terminal LOTUS-like domain disrupt RNA exclusion, reduce fertility, and elicit promiscuous RNA silencing. Synthetic RNAs bearing piRNA target sites silence a cognate reporter in a PRG-1 dependent manner, indicating both piRNA and PIWI protein facilitate targeted RNA entry into Mutator foci. These findings support a two-tier gate: P granules admit RNA while excluding translation and silencing effectors, whereas Mutator foci act as a physical RNA barrier, excluding transcripts except those marked for piRNA silencing. Thus, scaffold physical properties enable membrane-independent RNA gating. Our research broadens the concept of selective compartmentalization beyond membranes. Overall design: Small-RNAs for whole worms were cloned from WT, lambdaN tethered strains and multiple mut-16 mutants including lambdaN tethered different strains, mut-16(DE6R) and rde-3 mut-16(DE6R) worms.