Extended Data Fig. 5: Pseudouridine in miRNAs and TE-derived siRNAs in pollen from WT and paused (psd-13)/exportin-t mutants.
From: Pseudouridine guides germline small RNA transport and epigenetic inheritance
(a) Metaplots of read coverage at 42 known pseudouridylated sites in 18S and 25S rRNA, relative to untreated controls, in CMC-treated WT (magenta) and Ψ-IP WT (blue) and psd-13 (orange) small RNA sequencing libraries from pollen. (b) Read coverage from the same libraries at 17 rRNA pseudouridylation sites in 18S rRNA. (c, d) metaplot (c) and browser (d) analysis of predicted sites in rRNA from CMC/Mn2+ analysis relative to mock treated controls. (e, f) Metaplots (e) and read coverage (f) relative to untreated controls in WT (blue) and psd-13 (orange) Ψ-IP small RNA sequencing libraries from inflorescence. Known positions of Ψ in b, d and f are marked with dotted lines. (g) Ψ-enrichment of individual miRNAs detected by Ψ-IP and small RNA sequencing in WT and psd mutant inflorescence (L2FC calculated by DESeq2 with estimate of SE; n = 5 and 2 biological replicates for WT and psd, respectively; miR403 p = 0.01027, two-tailed students t-test). (h) Ψ-enrichment of miRNAs overall in WT and psd mutant inflorescence (two-way ANOVA, p = 0.2747; n = 153 miRNAs) (i) Violin plot of Ψ-enrichment of siRNAs from individual TEs based on IP-enrichment (Log2 IP/Unbound) in flower buds (inflorescence) and pollen from wild-type and psd mutants. Significant Ψ enrichment was observed in pollen, but not in inflorescence, and depended on PSD (p < 0.0001; p = 0.9181, one-way ANOVA; numbers under violins indicate number of TEs analyzed). Asterisks in g and i indicate significance (p < 0.05 (*); p < 0.01 (**); p < 0.001 (***)).
