WAGO-4 Localization to Germ Granules Is Important for Its Accurate Small RNA Loading

There were several MS experiments used in this study: 1) Proximity labeling of the P granules 2) Protein levels estimation from whole worm lysates and 3) wago-4 KO expression check (PRM analysis). 1) Proximity labeling strategy was used to estimate P granule composition in 3xFG mutant (FG domains of GLH-1, GLH-2, and GLH-4 mutated) and compare it to the WT. PGL-3 was tagged with TurboID at its native locus using CRISPR-Cas9. Biotinylation level was determined after streptavidin pull-down followed by MS analysis, serving as a proxy for protein enrichment in P granules. 2) Whole-worm lysate MS analysis was performed on 3xFG mutant worms to assess the worm proteome at an elevated temperature (26 °C), where these mutants exhibit a fecundity phenotype. 3) A STOP codon was introduced in the second exon of the WAGO-4 gene at its native locus in C. elegans (WAGO-4 KO). To assess whether the WAGO-4 protein is expressed in this strain, lysates from WAGO-4 KO (in triplicate) and N2 (WT control) were analyzed using PRM-MS.