Dynamic Alternative Polyadenylation during Litopenaeus Vannamei Metamorphosis Development

Abstract

As an important mechanism in the post-transcriptional regulation of eukaryotic gene expression, alternative polyadenylation (APA) plays a key role in biological processes such as cell proliferation and differentiation. However, the role and dynamic pattern of APA during Litopenaeus vannamei metamorphosis are poorly understood. Here, RNA-seq data covering from the embryo to the maturation (16 time points) of L. vannamei were utilized. We identified 247 differentially expressed APA events between early and adult stages, and through fuzzy mean clustering analysis, we discovered five dynamic APA patterns. Among them, the gradual elongation of the 3'UTR is the major APA pattern that changes over time, and its genes are enriched in the pathways of protein and energy metabolism. Finally, we constructed mRNA-miRNA and PPI networks and detected several central miRNAs that may regulate L. vannamei development. Our results revealed the complex APA mechanisms in L. vannamei metamorphosis, shedding new light on post-transcriptional regulation of crustacean metamorphosis.

Keywords: Litopenaeus vannamei; alternative polyadenylation; dynamic regulation; metamorphosis; miRNA.

Figure 1

Early and adult stages of L. vannamei development.

Figure 2

Global landscape of alternative polyadenylation (APA) during L. vannamei development. (A) Enriched motifs around proximal and distal polyA sites of APA events. The barplot shows the percentage of each motif feature. (B) PCA analysis result. (C) 3′UTR size (x-axis) and aUTR size (y-axis) of each APA event. The correlation coefficient (r) and p-value were calculated by Pearson analysis. (D) Median-centered |ΔPDUI| values between early and adult stages for L. vannamei to represent global APA variation. |ΔPDUI| values were classified into different bins of 3′UTR sizes. (E) Density plot of aUTR sizes classified by different bins of 3′UTR sizes.

Figure 3

Characterization of APA events during L. vannamei development. (A) Scatterplot of PDUIs in early (x-axis) and adult (y-axis) samples from the L. vannamei cohort. Significantly (adjusted p-value < 0.05 and |ΔPDUI| > 0.1) shortened and lengthened transcripts are indicated in red and blue, respectively, whereas those below the threshold are gray. (B) Volcano plot showing the significantly altered APA events in the L. vannamei cohort. (C) Function annotation of differential APA events. (D) Differential APA events of KEGG enrichment analysis.

Figure 4

Dynamics of APA changes during L. vannamei development. (A) Heatmap of differential APA events. Each APA event was normalized by Z-score. (B) Tracks of different APA patterns during L. vannamei development. (C) Heatmap of gene enrichment results of APA patterns. (D) Read coverage of LOC113808533 and LOC113824982 visualized by IGV. The y-axis represents time points during cardiomyocyte differentiation. The proximal polyA site was obtained from DaPars results.

Figure 5

Differential expression and cleavage and polyadenylation factors during L. vannamei development. (A) Pie chart displaying expression changes of APA genes in L. vannamei development. (B) Differential expression volcano plots of key transcription factors such as CPSF, CstF, CFI, and CFII at early developmental and adult stages. (C) Heatmap displaying expression changes of core polyA regulators. (D) APA differential events and overlapping genes in DEGs. Overlapping genes shared by more than one are represented in the area of intersection between 2 circles. (E) The expression of overlapping genes between DEGs and APA differential events.

Figure 6

Statistics of miRNAs in aUTRs and network. (A) Distribution of miRNA binding sites in aUTRs of differential APA events. (B) Differential APA event mRNA-miRNA interaction network analysis. (C) The PPI network constructed from the homologous genes of genes with differential APA events. The lines represent the APA gene–gene with STRING PPI > 0.7.