Fig. 4
TUT4/7-deficiency has a minor impact on pachytene small RNA pathways. a Frequency of 3′ terminal uridylation (blue), adenylation (orange), cytidylation (green), and guanylation (purple) for all pachytene miRNAs or let-7 miRNAs from Tut4/7CTL and Tut4/7cKO animals. For each group, the frequency of mono- (light) and oligo-nucleotide additions (dark) is shown. The fold change between the different groups is indicated together with the significance (*P < 0.05, **P < 0.01, ***P < 0.001, t-test two-sided; Tut4/7CTL, n = 3; Tut4/7cKO, n = 3). Each dot represents a biological replicate. The heights of the bars indicate the mean value for the different replicates, and the error bars show the standard deviation. b Scatter plot of miRNA expression levels in Tut4/7CTL vs Tut4/7cKO pachytene cells. Let-7 miRNA significantly changing more than two-fold (P < 0.05, Wald test) are highlighted in red. Other miRNAs significantly changing more than two-fold (P < 0.05, Wald test) are highlighted in blue. (Tut4/7CTL, n = 3; Tut4/7cKO, n = 3). The linear regression and the identity line are indicated in red and black, respectively. c Read count frequency of different miRNA families from Tut4/7CTL and Tut4/7cKO pachytene spermatocytes. d Read count frequency of different piRNA deriving from retrotransposons, DNA transposons, coding genes, and other non-coding RNAs (ncRNA) in Tut4/7CTL and Tut4/7cKO pachytene cells. e Scatter plot of all piRNA expression levels in Tut4/7CTL vs Tut4/7cKO pachytene spermatocytes. The identity line is shown in red. f Normalized length distribution of pachytene piRNA from Tut4/7CTL and Tut4/7cKO males. Each bar corresponds to a biological replicate. (*P < 0.05, **P < 0.01, t-test two-sided; Tut4/7CTL, n = 3; Tut4/7cKO, n = 3). g Terminal U frequency for 30 and 31 nucleotide long pachytene piRNAs from Tut4/7CTL and Tut4/7cKO males. Each dot corresponds to a biological replicate. The center value represents the median, and the error bars the range (*P < 0.05, t-test one-sided; Tut4/7CTL, n = 3; Tut4/7cKO, n = 3). h Confocal micrographs of testes sections from Tut4/7CTL, Tut4/7cKO, and MiliKO animals stained with an anti-ORF1 LINE1 antibody (green) are shown. DNA was stained with Hoechst 33342 (blue). Scale bar, 40 μm
