Fig. 1: DEPS-1 binds to PRG-1 and mediates piRNA-dependent transgene silencing.
a DEPS-1 and PRG-1 colocalise as peri-nuclear granules. C. elegans germlines expressing GFP-DEPS-1 were dissected and immunostained for GFP and PRG-1. PRG-1 and DEPS-1 colocalise in the proliferative zone, transition zone, pachytene, oocytes and embryos. A higher proportion of PRG-1 remains perinuclear in the loop region compared to DEPS-1. Scale bar = 3 µm. b Colocalisation of PRG-1, DEPS-1 and MUT-16. Dissected germlines co-stained for PRG-1, RFP-DEPS-1 and GFP-MUT-16 were imaged and deconvoluted with HyVolution settings. Two clusters with all three proteins present are shown. Scale bar = 0.25 µm. c Domain/fragment architecture of PRG-1 and DEPS-1. Sequence alignment (Clustal W) of Ago binding motif II on Drosophila melanogaster GW182 and DEPS-1 PBS with flanking sequences. d Recombinant full-length MBP-tagged PRG-1 and MBP-tagged DEPS-1 were purified for MST assays. A serial dilution of unlabelled MBP-PRG-1 was incubated with 10 nM of label MBP-DEPS-1 and tested for binding. The Kdapp is 855 ± 133 nM. Representative of n = 2 independent experiments. Data are presented as mean values ± SD of three technical replicates. Black square = MBP-DEPS-1 and MBP-PRG-1, black triangle = MBP-PRG and MBP, open triangle = MBP-DEPS-1 and MBP titrations. e MST measurement of fluorescently labelled DEPS-1 PBS motif peptide was incubated with unlabelled MBP-tagged PRG-1 PIWI domain. Kdapp is 1.9 µM ± 98 nM. Representative of n=2 independent experiments. Data are presented as mean values ± SD of 3 technical replicates. Black square = DEPS-1 PBS peptide and MBP-PRG-1PIWI, open square = DEPS-1 PBS peptide and MBP titrations. f Mutations in deps-1 lead to piRNA sensor transgene desilencing. Whitefield (right panel) and fluorescent (left panel) images of whole mounted animals show the piRNA sensor is efficiently silenced in wild-type animals and is desilenced in prg-1(n4357), deps-1null (bn121 and bn124) and deps-1∆PBS::rfp(mj608) mutants. Scale bar = 3 µm.
