Fig. 4: Ψ and Exportin-t mediate epigenetic inheritance and the triploid block.
From: Pseudouridine guides germline small RNA transport and epigenetic inheritance
a, Mean Ψ enrichment (log2 Ψ-IP/unbound) of 20–22-nt siRNAs and 23–25-nt siRNAs surrounding imprinted genes (−2.5 kb upstream of start; CDS; +2.5 kb downstream of stop) targeted by DME in WT (blue) and psd-13 (orange) pollen (P = <0.0001, 0.4528, <0.0001, 0.0002, 0.0428 and 0.0003, determined by two-way ANOVA with Bonferroni correction for multiple comparisons; n = 3 biological replicates). Error bars indicate the s.e.m. CDS, coding sequence. b,c, Metaplots of sperm cell abundance of 20–22-nt siRNAs (red) and 23–25-nt siRNAs (black) surrounding imprinted genes targeted by DME in WT (b) and psd-13 (c) pollen. d–g, Images of representative seeds from crosses between WT Col-0 female and osd1 diploid pollen (d), double-mutant osd1;psd-13−/− diploid pollen (e), double-mutant osd1;psd-13+/− diploid pollen (f) and double-mutant osd1;nrpd1a-3 diploid pollen (g). NRPD1a encodes the large subunit of Pol IV. h, Frequency of aborted seeds from the same crosses. i, Model for transport of small RNA during pollen development. As the pollen matures, 21–22-nt easiRNAs and 24-nt siRNAs are produced by Pol IV in the VN and pseudouridylated along with some miRNAs. Pseudouridylated miRNAs and easiRNAs are exported by PSD (Exportin-t) into sperm cells, where they are loaded onto AGO1, AGO5 and AGO9, while unmodified miRNAs are transported by HST (Exportin-5) and loaded onto AGO5 and AGO1. Pseudouridylated sperm cell easiRNAs mediate dose-dependent lethality (the triploid block) by targeting MEGs in the seed. j, mCherry::AGO9 fusion protein expressed from the AGO9 promoter (pAGO9) in developing pollen. DAPI was used to stain nucleic acids (blue). AGO9 is present in the VN at the bicellular stage but is enriched in the sperm cells in mature pollen. k, AGO9 IP from WT and psd-13 pollen shows increased binding of VN-derived 20–22-nt siRNAs and decreased binding of 23–25-nt sperm-cell-derived siRNAs to AGO9 in psd-13. Numbers on bars indicate the number of TEs analyzed; error bars indicate the s.e.m. P = <0.0001, 0.99, 0.99 and <0.0001, determined by one-way ANOVA with Bonferroni correction for multiple comparisons. In a,k, *P < 0.05, **P < 0.01 and ***P < 0.001. NS, not significant.
