Fig. 2

Distribution of small noncoding RNAs (sncRNAs) in RNP and polysome fractions in pachytene spermatocytes, round spermatids, and elongating spermatids. a–c sncRNAs preferentially enriched in RNP or polysome fractions in pachytene spermatocytes (a), round spermatids (b), and elongating spermatids (c). Upper panels: RNP-enriched sncRNAs were defined by log₂(Levels in polysome/Levels in RNP) <0 (Student’s t-test, p < 0.05), whereas polysome-enriched sncRNAs were those with log₂(Levels in polysome/Levels in RNP) >0 (Student’s t-test, p < 0.05). The y-axis represents the total number of fraction-enriched sncRNA species. Lower panels: dot plots showing correlations between actual expression levels and the ratio of polysome levels to RNP levels of sncRNAs. The y-axis shows the log₁₀ values of all sncRNA expression counts. The x-axis represents the log₂ values of the ratio of polysome levels to RNP levels. The sncRNAs with higher expression levels in the RNP fraction tend to locate to the left, whereas sncRNAs with higher expression levels in the polysome fraction are clustered towards the right. Linear regression lines for each group are plotted over the data points. Note the angle of the regression lines of miRNAs are ~30° in pachytene spermatocytes (a) and round spermatids (b), whereas it decreased to ~15 ° in elongating spermatids (c), suggesting a shift of miRNAs from RNP to polysome fractions in elongating spermatids. d Boxplots showing average levels of the miRNAs shifting from RNP to polysome fractions in elongating spermatids. The average counts (y-axis) of those miRNAs increased in the polysome fraction but decreased in the RNP fraction from round to elongating spermatids. e Heat map showing levels of the miRNAs shifting from RNP to polysome fractions in elongating spermatids. Values of log₂ (RPKM) are presented as variable colors from red to blue. These miRNAs were upregulated in the polysome fraction and simultaneously downregulated in the RNP fraction from round to elongating spermatids. f Expression profiles of the five miRNAs displaying a shift from RNP to polysome in elongating spermatids. All five miRNAs were upregulated in the polysome fraction but downregulated in the RNP fraction from round to elongating spermatids. Student’s t-test was used to evaluate statistical significance, and p values and fold changes (FC) are marked. Asterisks indicate statistically significant p values