Extended Data Fig. 3: C. tropicalis PRG-1.1 and PRG-1.2 are redundant paralogs and PRG-1.2 acts maternally.
From: Selfish conflict underlies RNA-mediated parent-of-origin effects
a, Protein alignment of PRG-1.1 and PRG-1.2. The two proteins share 87% amino-acid pairwise identity with 722/828 identical sites. Conserved PAZ and PIWI domains are highlighted. b, Detection of endogenously tagged PRG-1.1::FLAG and PRG-1.2::FLAG by western blot. Black arrow indicates the expected MW. EG6180 was used as a negative control. Western blot against β-Actin serves as a sample processing control. Uncropped gels available in Supplementary Fig. 1. c, Quantification of FLAG immunofluorescence quantification of C. tropicalis PRG-1.1 and PRG-1.2 expression from embryos, (nEG6180 = 53, nprg-1.1 = 34, nprg-1.2 = 35, one-way ANOVA, p < 0.0001, Tukey post hoc test, pEG vs prg-1.1 < 0.0001, pEG vs prg-1.2 < 0.0001, pprg-1.1 vs prg-1.2 < 0.0001, mean +/−SEM is shown). d, Selfing of prg-1.1(−); prg-1.2(+/−) strain. All prg-1.1(−); prg-1.2(−) individuals were sterile, therefore the line couldn’t be propagated (mean +/−95% CI is shown). e, Cross of prg-1.2(−) hermaphrodites to NIL males (top) and NIL hermaphrodites to prg-1.2(−) males (bottom) indicates that paternal slow-1 is repressed only when prg-1.2 is maternally inherited. Percentage of F2 EG/EG delayed progeny (right) when prg-1.2 is absent from the mother or the father compared to the WT cross. When prg-1.2 null mutant mothers were crossed to wild-type NIL males, we observed that 52.1% of F2 homozygous EG/EG individuals were delayed. In contrast, when EG6180 mothers were crossed to prg-1.2 null mutant NIL males, F2 homozygous EG/EG individuals were mostly wild-type (13.8% delay), indicating that maternal prg-1.2 is necessary for slow-1 repression (np = 53, nmother prg-1.2(−) = 23, nfather prg-1.2(−) = 29, two-sided Fisher’s exact test compared to WT, pmother prg-1.2(−) = 0.0001, pfather prg-1.2(−) = 0.71, mean +/−95% CI is shown). f, PRG-1.2 is not necessary for the maintenance of slow-1 repression. In this crossing scheme mothers provide PRG-1.2 to their F1 progeny, which is sufficient for piRNA-mediated repression, and the slow-1/grow-1 is paternally inherited. Since EG6180 hermaphrodites do not provide maternal slow-1 transcripts, then the slow-1 paternal allele is epigenetically repressed in the F1. Then F2 progeny are singled, and their offspring genotyped for both the TA and the prg-1.2 locus. Hermaphrodites that are homozygous carriers for both the TA and the prg-1.2(−/−) null allele are identified (6.25% the F2 progeny) and propagated for 1 or 7 generations. Then these hermaphrodites are crossed to EG6180 prg-1.2 (−/−) males to test whether the TA is active. Inset shows the observed activity of the TA measured as percentage of delayed (EG/EG) individuals (nM = 34, nprg-1.2_3_gen = 62, two-sided Fisher’s exact test, p < 0.0001, mean +/−95% CI is shown).
