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Fig. 4 | Genome Biology

Fig. 4

From: MicroRNAs control mRNA fate by compartmentalization based on 3′ UTR length in male germ cells

Fig. 4

RNP-enriched miRNAs tend to target RNP-enriched mRNAs and the position of miRNA targeting sites correlates with the subcytoplasmic compartmentalization of miRNA targets. a Density plots showing the distribution of 3′ UTR length between RNP and polysome fractions in pachytene spermatocytes, round spermatids, and elongating spermatids. The RNP- and polysome-enriched transcripts are defined as those significantly upregulated in RNP or polysome fractions, respectively (Student t-test, p < 0.1). Boxplots in the inset show the average 3′ UTR lengths in RNP and polysome fractions. Overall, the 3′ UTRs are significantly shorter in RNP fractions than in polysome fractions among all three spermatogenic cell types. The Student’s t-test was performed with cross-validation through Wilcoxon rank sum test (p = 2.8e-05, 2.2e-16 and 2.2e-16). b Histograms showing the relationship between RNP-enriched miRNAs and RNP-enriched mRNAs. The y-axis represents the frequency at which miRNAs target the 3′ UTRs of their target mRNAs, while the x-axis shows the log2 values of ratios of polysome expression levels to RNP expression levels of the target mRNAs (polysome/RNP). RNP-enriched mRNAs are located to the left (of zero), whereas those polysome-enriched ones are on the right (of zero) along the x-axis. c Scatter and density plots showing the relationship between miRNA binding sites on the 3′ UTRs of miRNA targets (i.e., mRNAs) and miRNA binding energy. The y-axis represents the binding energy of the RNP-enriched miRNAs, whereas the x-axis displays the miRNA target position, i.e., the distance between miRNA binding sites and the stop codon. The scatter plots show the distributions of targeting energy versus targeting position of all RNP-enriched (orange dots) or polysome-enriched (blue dots) miRNAs. The density plots summarize the distribution of the targeting energy versus targeting positions between RNP-enriched (orange lines) and polysome-enriched miRNAs (blue lines). Note that RNP-enriched miRNAs tend to bind their targets at positions proximal to the stop codon in RNP-enriched mRNAs, whereas those polysome-enriched miRNAs appear to often target the positions distal to the stop codon. The Student’s t-test was performed with cross-validation through Wilcoxon rank sum test (p = 0.0029, 2.2e-16, and 0.0024). d Density plots showing the miRNA target site distribution between RNP up- and downregulated mRNAs from pachytene spermatocytes to round spermatids. The up- or downregulated mRNAs in RNP fractions were compared between pachytene spermatocytes and round spermatids (Student’s t-test, p < 0.1), and these mRNAs were all those that could be targeted by the miRNAs that were significantly upregulated in RNP fractions from pachytene spermatocytes to round spermatids. Note that the downregulated transcripts tend to contain miRNA target sites more distal to the stop codon (an average of 436 bp), whereas the upregulated RNP transcripts usually have miRNA targeting sites more proximal to the stop codon (an average of 291 bp) (Student’s t-test, p < 2.2e−16). e Density plots showing the miRNA target site distribution between polysome up- and downregulated mRNAs from round spermatids to elongating spermatids. The up- or downregulated mRNAs in the polysome fractions were compared between round spermatids and elongating spermatids (Student’s t-test, p < 0.1), and these mRNAs were all those that could be targeted by the miRNAs that were significantly upregulated in the polysome fractions from round spermatids to elongating spermatids. Note that the downregulated transcripts tend to contain miRNA target sites more distal to the stop codon, whereas the upregulated polysome transcripts usually have miRNA targeting sites more proximal to the stop codon. f, g The shorter (upregulated in red) and longer (downregulated in blue) 3′ UTR transcript isoforms of Sgsm2, Thoc7, Akap1, and Ankef1 are presented as dotted lines. The absolute binding energy of upregulated miRNAs in round spermatid RNP fractions is shown on the y-axis; the x-axis shows the miRNA target positions, i.e., the distance between the miRNA binding sites to the stop codon. The upregulated RNP miRNAs appear to target more distal sites in the longer 3′ UTR transcripts compared to the shorter 3′ UTR isoforms in the round spermatid RNP fractions. Asterisks indicate statistically significant p values

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