Identification of global alternative splicing and sex-specific splicing via comparative transcriptome analysis of gonads of Chinese tongue sole (Cynoglossus semilaevis)
摘要: 半滑舌鳎（Cynoglossus semilaevis）是一种具有ZZ/ZW性别决定系统的重要海水经济鱼类，其性别决定和分化受温度的影响。可变剪切（AS）是调节性别决定和性腺分化相关基因表达的重要机制之一，但在鱼类中鲜有报道。在该研究中，我们结合全长转录组（Iso-Seq）和RNA测序（RNA-Seq），对半滑舌鳎的雌雄性腺进行了分析。在雌性和雄性中分别获得了32 341和81 883个全长转录本。共鉴定出8 279个AS基因，其中2 639个基因在雄性和雌性中存在差异可变剪切（DAS）。许多DAS基因和DEG的交集基因富集在“减数分裂细胞周期”通路，性腺分化相关基因如esrrb和wt1a等存在性别特异转录本。该研究首次揭示了半滑舌鳎雌雄性腺转录组中的AS事件，发现了精巢活跃转录的特征，为研究AS调控半滑舌鳎性腺分化的分子机制提供了候选基因。Abstract: Chinese tongue sole (Cynoglossus semilaevis) is an economically important marine fish species with a ZZ/ZW sex determination mechanism, which can be influenced by temperature. Alternative splicing (AS) is an important mechanism regulating the expression of genes related to sex determination and gonadal differentiation, but has rarely been reported in fish. In this study, to explore the molecular regulatory mechanisms of sex determination and gonadal differentiation, we combined isoform and RNA sequencing (Iso-Seq and RNA-Seq) to perform transcriptome profiling of male and female gonads in C. semilaevis. In total, 81 883 and 32 341 full-length transcripts were obtained in males and females, respectively. A total of 8 279 AS genes were identified, including 2 639 genes showing differential AS (DAS) between males and females. Many intersecting DAS genes and differentially expressed genes (DEGs) were enriched in the meiotic cell cycle pathway, and genes related to gonadal differentiation, such as esrrb and wt1a, were found to have sex-specific isoforms. Thus, this study revealed AS events in the gonadal transcriptomes of male and female C. semilaevis, described the characteristics of active transcription in the testes, and identified candidate genes for studying the regulatory mechanisms of AS during gonadal differentiation.
Figure 1. Light microscope images of C. semilaevis gonads at 6 mpf
A: Cross-section of ovary at different magnifications (left bar: 10 μm; right bar: 50 μm). B: Cross-section of testis at different magnifications (left bar: 10 μm; right bar: 50 μm). OC, ovarian cavity; OL, ovarian lobule; OG, oogonium; OO, oocyte; SL, seminal lobule; SV, seminal vesicle; SG, spermatogonium; SC, spermatocyte; SZ, spermatozoa.
Figure 3. Number distribution and enriched pathway analysis of genes showing DAS in female and male gonads of C. semilaevis
A: Pie plot showing proportions of five genes exhibiting DAS events. B: Venn diagram showing intersection of KEGG signaling pathway categories associated with five types of DAS events. C–F: Sashimi plot depicting RNA-Seq and exon junction reads from females and males above gtf2a1, ercc2, stag3, and cdk7 full-length transcripts and gene structure. Arrows indicate direction of gene transcription.
Figure 4. Analysis of AS and DEG association in female and male gonads of C. semilaevis
A: Venn diagram showing overlap between DEGs and AS genes. B: Number of DEGs in which AS occurred and proportion of DEGs showing different AS types in gonads. C: Stacked histograms showing ratio of up- and down-regulated genes and non-DEGs among AS types in gonads. D: Box plots showing average expression levels (log2FPKM) of genes showing five splicing types or no splicing. ns: No significance. ****: P<0.0001.
Figure 5. Function and expression analysis of intersecting DAS genes and DEGs
A: Scatter plots showing DEGs that underwent DAS in gonads, with each dot representing a gene. Density plots above and to the right of scatter plots illustrate distribution of dots. B: Enriched GO terms of differentially expressed DAS genes. Network of enriched terms is colored according to cluster identity. Nodes sharing the same cluster identity are typically close to each other. C: Bar plot showing expression of genes involved in gonadal differentiation (log2FC>0 indicates up-regulated gene in females, log2FC<0 indicates up-regulated gene in males). D, E: RNA-Seq reads, exon junction reads, and RNA-Seq read coverage of esrrb and wt1a. Lower box represents gene structure. Gray box indicates differentially spliced region. Arrows indicate direction of gene transcription.
Figure 6. Gene expression and validation of splicing regulators in gonads of C. semilaevis
A: Expression of splicing regulators in gonads. Heatmap showing relative expression (Z score) of splicing regulators between male and female gonads. B: qRT-PCR validation of gene expression of splicing regulators in gonads. ns: No significance; *: P<0.05; **: P<0.01; ***: P<0.001.
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