Fig 1.
Identification and Characterization of lncRNAs in S. japonicum.
(A) The schematic diagram of schistosomes at four-time points and the bioinformatics workflow for lncRNA prediction. See the description in Methods for details. The cartoon mouse was modified from a public domain image by Creazilla (https://creazilla.com/nodes/19261-cartoon-grey-mouse-clipart). (B) Circos plot showing the chromosomal distribution of lncRNAs. The histogram indicates the abundance of lncRNAs(black) and mRNAs(orange) in physical bins of 10 Kb for each chromosome. Lines located in the innermost circle mark the genomic localization of lncRNAs. (C) The classification of lncRNAs into four categories (intergenic, antisense genic, sense genic, and isolated) according to the relationships between lncRNAs and their adjacent mRNA. (D) GC content of lncRNA transcripts (lncRNA), protein-coding sequences (CDS), and intergenic sequences (Intergenic). The respective average values for the GC content of LncRNA, CDS, and Intergenic regions are 35.39%, 36.59%, and 34.83%. Statistical analysis was performed using the Kruskal-Wallis test with Dunn’s multiple comparisons, ****, P-value < 0.0001. (E) Lengths distribution of lncRNA and mRNA transcripts. (F) Numbers of exons in lncRNA and mRNA genes.
Fig 2.
Dynamic expression profiles of lncRNAs during schistosome reproductive development.
(A-B) Principal component analysis (PCA) of 21 distinct samples across four-time points based on normalized lncRNAs (A) and mRNAs (B) expression levels. Male and female samples were mainly located in regions indicated by blue and red shading. (C) Clustering of major expression patterns of dynamic lncRNAs during reproductive development in females (up) and males (down). Lines estimated through LOESS regression; 95% confidence interval shown in grey. (D) The proportion of lncRNAs in each cluster to the total dynamic lncRNAs in male and female worms. (E) The bar plot represents the count of sex-biased lncRNAs and mRNAs at 18, 22, and 26 dpi. The categories were divided into female-biased (Log2FC ≥ 1, adjusted P-value < 0.05) and male-biased (Log2FC ≤ -1, adjusted P-value < 0.05) genes. A significant difference was observed between the counts of sex-biased lncRNAs and mRNAs (P-value < 0.001, Fisher’s exact test). (F) Sankey diagram showing the transition of female-biased lncRNAs (red), male-biased lncRNAs (blue), and non-biased lncRNAs (green) between 18, 22, and 26 dpi. The proportion of each class is represented by the size of the rectangle.
Fig 3.
Weighted gene correlation network analysis (WGCNA) identifies lncRNAs-mRNA co-expression network modules.
(A) The dendrogram shows 19 lncRNAs-mRNA co-expression modules identified using WGCNA. (B) The heatmap illustrates the correlation between the identified modules and various biological traits. Each column represents a trait, while each row corresponds to a module eigengene. The strength and direction of the association between each trait and eigengene are indicated by a Pearson’s correlation coefficient, with the corresponding P-value provided in parentheses. The color gradient within each cell reflects the correlation, with blue signifying a strong negative correlation and red indicating a strong positive correlation. (C) Heatmap of lncRNAs in the turquoise and yellow modules during 14–26 dpi in male and female worms. Red and blue represent high and low gene expression levels, respectively. (D) Gene ontology (GO)) enrichment of the coding genes in turquoise and yellow modules. Color intensity represents the magnitude of -log10(adjusted P-value) (Fisher’s exact test). Only GO terms with adjusted P-value < 10−4 are shown.
Fig 4.
Subnetwork of hub lncRNAs extracted from yellow module.
The figure shows the network of interconnections between hub lncRNAs and their co-expression mRNAs (TOM > 0.2) within the yellow module. Hub-lncRNAs are represented by orange circles, while mRNAs are depicted as blue circles. The table on the upper right corner shows the performance of S.japoniucm after RNAi of selected lncRNAs.
Fig 5.
A natural antisense lncRNA LNC3385 regulates the development of S. japonicum.
(A) Genome browser view of LNC3385 and RERE (Sjc_0009416). The gene structure model is shown below the chromosomal coordinate axis. The central red block denotes the region targeted by the LNC3385 and RERE dsRNA probes. The qRT-PCR primer lines indicate the genomic positions where primers for these genes are designed: purple represents the forward primer, and green represents the reverse primer. (B) Silencing LNC3385 led to decreased egg production of female worms in vitro. Left, the number of eggs per pair after the treatment of GFP or LNC3385 dsRNA for 8 Days; Right, qPCR analysis showing the relative expression level (mean ± SE, n = 3) of LNC3385 and RERE in males and females after RNAi which shows a 74.1% decrease in LNC3385 expression levels in females and an 89.2% decrease in males, as compared to GFP-treated controls. (C) Schematic diagram illustrating the in vivo RNAi experiments conducted post cercariae infection. Mouse image modified from Wikimedia Commons (https://commons.wikimedia.org/wiki/File:Vector_diagram_of_laboratory_mouse_(black_and_white).svg, CC BY-SA 4.0). Syringe icon modified from Wikimedia Commons (https://commons.wikimedia.org/wiki/File:Noto_Emoji_Oreo_1f489.svg, Apache License 2.0). (D) Relative expression level (mean ± SE) of LNC3385 in RNAi-treated parasites by qPCR analysis. In vivo RNAi resulted in an 81.9% decrease in LNC3385 expression in male worms, while females showed a similar reduction that was not statistically significant. Three biological replicates were performed. (E) Brightfield microscopy of male and female parasites from GFP and LNC3385 dsRNA treated groups. (F) Worm length (left) and worm burden (right) of the parasites recovered at 38 dpi in GFP and LNC3385 RNAi groups. (G) Morphological changes of male and female worms in the reproductive system after in vivo RNAi of LNC3385. * P-value < 0.05; ** P-value < 0.01; *** P-value < 0.005; **** P-value < 0.001; n.s. not statistically significant.
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Fig 6.
Transcriptomic and gene network analysis following LNC3385 RNAi in vitro.
(A) Principal component analysis (PCA) of the transcriptome of LNC3385 or GFP dsRNA treated worms. (B) Volcano plot of differentially expressed genes (DEGs). DEGs were defined as |log2 (fold change)| ≥ 1 and adjusted P-value< 0.05. (C) Gene set enrichment analysis (GSEA) showed the downregulation of genes in the LNC3385 co-expression network (TOM > 0.1) in the LNC3385 RNAi group. (D) Gene Ontology analysis of core enrichment genes, which contribute most to the enrichment result, within the LNC3385 co-expression gene set. (E) The diagram represents a hypothesized gene network regulated by LNC3385 (TOM > 0.1). Each node represents a gene and the color indicates the expression level. Genes with a fold change < -0.15 and P-value < 0.05 are labeled.