Despite being one of the primary mechanisms of gene expression regulation in eukaryotes, alternative splicing is often overlooked in ecotoxicogenomic studies. The process of alternative splicing facilitates the production of multiple mRNA isoforms from a single gene thereby greatly increasing the diversity of the transcriptome and proteome. This process can be important in enabling the organism to cope with stressful conditions. Accurate identification of splice sites using RNA sequencing requires alignment to independent exonic positions within the genome, presenting bioinformatic challenges, particularly when using short read data. Although technological advances allow for the detection of splicing patterns on a genome-wide scale, very little is known about the extent of intraspecies variation in splicing patterns, particularly in response to environmental stressors. In this study, we used RNA-sequencing to study the molecular responses to acute copper exposure in three lineages of Daphnia pulex by focusing on the contribution of alternative splicing in addition to gene expression responses. By comparing the overall gene expression and splicing patterns among all 15 copper-exposed samples and 6 controls, we identified 588 differentially expressed (DE) genes and 16 differentially spliced (DS) genes. Most of the DS genes (13) were not found to be DE, suggesting unique transcriptional regulation in response to copper that went unnoticed with conventional DE analysis. To understand the influence of genetic background on gene expression and alternative splicing responses to Cu, each of the three lineages was analyzed separately. In contrast to the overall analysis, each lineage had a higher proportion of unique DS genes than DE genes suggesting that genetic background has a larger influence on DS than on DE. Gene Ontology analysis revealed that some pathways involved in stress response were jointly regulated by DS and DE genes while others were regulated by only transcription or only splicing. Our findings suggest an important role for alternative splicing in shaping transcriptome diversity in response to metal exposure in Daphnia, highlighting the importance of integrating splicing analyses with gene expression surveys to characterize molecular pathways in evolutionary and environmental studies.

中文翻译：

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响应急性铜暴露，水蚤谱系中的可变剪接是高度可变的。

尽管是真核生物中基因表达调控的主要机制之一，但在生态毒理基因组学研究中，选择性剪接常常被忽略。选择性剪接的过程促进了从单个基因产生多种mRNA同工型，从而大大增加了转录组和蛋白质组的多样性。这个过程对于使生物体适应压力条件可能很重要。使用RNA测序准确鉴定剪接位点需要对准基因组中独立的外显子位置，这对生物信息学提出了挑战，特别是在使用短读取数据时。尽管技术进步允许在全基因组范围内检测剪接模式，但对剪接模式中种内变异程度的了解甚少，特别是应对环境压力。在这项研究中，我们通过RNA测序研究了水蚤（Daphnia pulex）的三个谱系对急性铜暴露的分子反应，并着眼于除了基因表达反应以外的其他剪接作用。通过比较所有15个铜暴露样品和6个对照之间的整体基因表达和剪接模式，我们鉴定了588个差异表达（DE）基因和16个差异剪接（DS）基因。大多数DS基因（13）未发现是DE，这表明对铜的独特转录调控是常规DE分析中未注意到的。为了了解遗传背景对基因表达和对Cu的可变剪接反应的影响，分别对这三个谱系进行了分析。与总体分析相反，每个谱系的独特DS基因比例均高于DE基因，这表明遗传背景对DS的影响大于对DE的影响。基因本体论分析显示，与应激反应有关的某些途径由DS和DE基因共同调控，而另一些则仅通过转录或剪接调控。我们的发现表明，选择性剪接在水蚤对金属暴露的反应中，在形成转录组多样性中起着重要的作用，突出了将剪接分析与基因表达调查相结合以表征进化和环境研究中分子途径的重要性。基因本体论分析显示，与应激反应有关的某些途径由DS和DE基因共同调控，而另一些则仅通过转录或剪接调控。我们的研究结果表明，选择性剪接在响应水蚤金属暴露而形成转录组多样性中起着重要的作用，强调了将剪接分析与基因表达调查相结合以表征进化和环境研究中分子途径的重要性。基因本体分析表明，一些参与应激反应的途径由DS和DE基因共同调控，而另一些则仅通过转录或剪接调控。我们的研究结果表明，选择性剪接在响应水蚤中金属暴露而形成转录组多样性方面起着重要作用，强调了将剪接分析与基因表达调查相结合以表征进化和环境研究中分子途径的重要性。