Fig. 3: Transport of pseudouridylated siRNAs into sperm cells depends on PAUSED (Exportin-t).

a, Ψ enrichment of miRNAs in WT and psd-13 pollen. A modest reduction in Ψ enrichment of miRNAs was observed in psd pollen (P = 0.0019, determined by two-tailed paired t-test; n = 3 biological replicates; 64 miRNAs are plotted). b, Ψ enrichment of siRNAs was determined by Ψ-IP and small RNA-seq in WT and psd-13 mutant pollen and plotted by size class (20–22 nt or 23–25 nt) for individual TEs belonging to each group, namely, miR845 targets, Pol IV and non-Pol IV targets and TEs whose siRNAs were derived from sperm cells (SC) or from the VN in ref. ⁴⁴. P values show differences between group means, determined by one-way ANOVA with Bonferroni correction for multiple comparisons (P = 0.002, 0.015, <0.0001, 0.234, 0.5964, 0.5153, <0.0001, 0.0009, 0.0002 and 0.0049; n = 3 biological replicates). Numbers above violins indicate the number of individual TEs plotted (including replicates). c, siRNA abundance in SC was determined by sequencing small RNA from FACS-sorted WT, psd-13 and hst-6 SC. VN-derived 20–22-nt siRNAs were depleted from psd SC but enriched in hst SC relative to WT, while VN-derived 23–25-nt siRNAs and SC-derived siRNAs were at levels similar to WT. Numbers on bars indicate the number of individual TEs plotted (including replicates); error bars indicate the s.e.m. d,e, Abundance of highly prevalent siRNA from 30 Gypsy and 2 Copia TE families in psd and hst sperm relative to WT for 20–22 nt (d) and 23–25 nt (e) size classes. f, Abundance of highly prevalent miRNAs (reads per million mapped reads (RPM) > 100) in psd and hst sperm relative to WT. miRNAs with terminal Ψ are indicated with a black bar; miRNAs presented in h are shown in bold. g, Abundance of miRNAs with and without terminal Ψ in psd and hst mutants relative to WT in sperm cells sorted by FACS. miRNAs without terminal Ψ were lost in hst, but not in psd sperm cells (P < 0.0001, 0.6571 and <0.0001, determined by two-way ANOVA; n = 3 biological replicates). Numbers above violins indicate the number of individual miRNAs plotted. h, Ψ enrichment of miRNAs in WT and psd-13 pollen for those miRNAs dependent on PSD for pseudouridylation (P = 0.02, 0.02, 0.021, 0.037 and 0.039, determined by two-sided Student’s t-test; n = 3 biological replicates). Error bars indicate the estimated s.e.m. of log₂(fold change) calculated by DESeq2. In a,b,g,h, *P < 0.05, **P < 0.01 and ***P < 0.001.

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