Extended Data Fig. 7: Characterization of pseudouridylation of siRNA in psd-13 mutants.
From: Pseudouridine guides germline small RNA transport and epigenetic inheritance
(a,b) Ψ-enrichment of PolIV-dependent and -independent (a) or VN- and SC-derived (b) TE-siRNAs in wild-type and psd-13 pollen for each size class (average enrichment of TE-siRNAs from each classification is shown +/− s.e.m. for each biological replicate; numbers below bars indicate number of TEs analyzed). (c) siRNA abundance was determined by sequencing small RNA from FACS sorted wild-type, psd-13 and hst-6 sperm cells. SC abundance of PolIV-dependent and non-PolIV-dependent TE-siRNAs was assessed in each size class (average enrichment of TE-siRNAs relative to wild-type from each TE classification is shown +/− s.e.m.; numbers below bars indicate number of TEs analyzed). (d-i) Metaplots of 20-22nt (red) and 23-25nt (black) siRNA abundance across PolIV-TEs from biological replicates of wild-type (a,d), psd-13 (b,e) and hst-6 (c,f) sperm cells sorted by FACS. 250 bp up- and down-stream regions as well as 500 bp from each end of the TE are shown. (j-l) Scatterplots of small RNA sequencing data from two biological replicates mapped to TE families in wild-type (j), psd-13 (k) and hst-6 (l) sperm cells isolated by FACS. Simple linear regression used to determine R-squared value. (m) Ψ-enrichment of siRNAs from TEs surrounding maternally expressed imprinted genes (MEGs, n = 42) and paternally expressed imprinted genes (PEGs, n = 9) in wild type (blue) and psd-13 (orange) pollen. * = p < 0.05, two-sided paired t-test. (n) Abundance of 20-22 nt and 23-25 nt siRNA surrounding imprinted genes in wild-type (blue) and psd-13 (orange) sperm cells. siRNA abundance in sperm cells matching MEGs and PEGs was lower in psd-13 mutants for 20-22nt siRNA but relatively higher for 23-25nt siRNA. * = p < 0.05; ** p < 0.01, *** = p < 0.001, paired t-test with Bonferroni correction for multiple comparisons; numbers below boxes indicate individual TEs analyzed. Boxes indicate median, interquartile range and min/max (y-axis clipped to visualize median). (o) Levels of miR845a and miR845b were unchanged or higher in psd-13 mutant pollen (Bars show average RPM; error bars show SD of biological replicates, n = 3).
