萨氏海鞘（Ciona savignyi） miRNA的生物信息学预测、实验验证与功能分析
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摘要
本论文用生物信息学方法分析萨氏海鞘（C. savignyi）、玻璃海鞘（C.intestinalis）和尾海鞘（Oikopleura dioica）的miRNA，共预测了52个Ciona属海鞘特有miRNA、527个保守性miRNA、37个miRNA簇。首次实验验证了6个萨氏海鞘miRNA的表达。
通过聚类分析将萨氏海鞘的180个预测miRNA归纳为140个家族，发现海鞘miRNA家族的构成与其他后口动物之间有很大的差异，80.00%的萨氏海鞘miRNA家族为尾索动物所特有，与其他几种脊索动物共有的只有16.43%，与其他几种后口动物共有的只有3.57%。海鞘系统发生中miRNA的显著变化可能推动了其机体结构的重大变化以适应固着生活的新生活方式和新环境。
对表达实验阳性的miRNA进行了生物信息学功能预测，GO功能分类表明miRNA可能调控多种生物学过程；其中csa_2-com和csa_7可能调控心脏发育，而csa_m-27c和csa_9-com可能调控胚胎发育中的多种过程。
对萨氏海鞘csa_m-27c的表达检测发现，它在精卵细胞发育过程中表达量较低，在受精卵进入胚胎发育过程之后表达显著上调，胚胎发育结束之后明显下降，提示它对胚胎发育过程的基因表达调控可能具有重要作用。
萨氏海鞘与非尾索动物之间具有数量众多的同序列异源miRNA，暗示着miRNA进化中可能存在具有序列特异性的选择压力。靶基因的序列特异性可能是miRNA趋同进化的动力。
保守性miRNA靶基因与其他物种已有的靶基因实验证据的比较分析表明，保守性miRNA家族的靶基因序列可能发生变异而脱离调控，然而miRNA通过调控与原靶基因密切相关的基因（例如其受体）仍可能保持对原生物学过程的调控。Ciona属海鞘特有的csa_m-27c是脊椎动物miR-27家族的异源同功miRNA——具有相似的调控作用，但不是同源基因。这暗示miRNA介导的转录后调控体系可能超越序列水平而在生物学过程水平上的具有保守性。
Prediction of miRNAs for3Urochordates (C. savignyi, C. intestinalis andOikopleura dioica) were carried out with bioinformatic approaches. We predicted527conservative miRNA genes from the3species, including52miRNA genesspecific to the Urochordata family Ciona and37miRNA gene clusters. Wereported the first collection of6experimentally validated miRNAs in C. savignyi.
We clustered the180predicted C savignyi miRNAs into140families by PBCbootstrap analysis among6Deuterostomias, and found that80.00%of them arespecific to family Ciona, only16.43%and3.57%are conserved with otherChordates and Deuterostomias, respectively. The profound change in miRNArepertoire might have played an important role in the dramatic change of life styleand morphology during Ascidians phylogenesis.
Function of6experimentally validated C. savignyi miRNAs are analyzed bycomputational prediction of their potential target genes. Gene ontology analysissuggests that multiple biological progresses are regulated by these miRNAs, amongwhich embryonic development may be regulated by csa_m-27c and csa_9_com, andheart development may be the targeted progress of csa_2-com and csa_7.
Temporal and spacial expression profile of the novel miRNA csa_m-27c showsthat its expression was low in the gonad, but increased significantly duringembryonic development before dropped abruptly while the C. savignyi developedinto tadpole larva, indicting an important role of csa_m-27c in the embryonicdevelopment, which is accordant to the bioinformatic function analysis.
Ciona savignyi shares many sequencially similar miRNAs with non-Urochordataanimals, but only a third of them are homologous. Numerous analogous miRNAssuggests sequencially selective pressure from miRNA targets.
By comparison between predicted targets of conservative C. savignyi miRNAsand reported experimental evidences, we found that conservative miRNAs maysometime maintain their regulation of a specific biological progress even if they lostthe control of a target gene. csa_m-27c is an analogy of miR-27c family. Itfunctions similar to miR-27but have a different origin, like a miRNA equivalent ofan isozyme. This suggests that conservation of miRNA mediated transcriptionalregulation system may exceed sequence level and live up to progress level.
通过聚类分析将萨氏海鞘的180个预测miRNA归纳为140个家族，发现海鞘miRNA家族的构成与其他后口动物之间有很大的差异，80.00%的萨氏海鞘miRNA家族为尾索动物所特有，与其他几种脊索动物共有的只有16.43%，与其他几种后口动物共有的只有3.57%。海鞘系统发生中miRNA的显著变化可能推动了其机体结构的重大变化以适应固着生活的新生活方式和新环境。
对表达实验阳性的miRNA进行了生物信息学功能预测，GO功能分类表明miRNA可能调控多种生物学过程；其中csa_2-com和csa_7可能调控心脏发育，而csa_m-27c和csa_9-com可能调控胚胎发育中的多种过程。
对萨氏海鞘csa_m-27c的表达检测发现，它在精卵细胞发育过程中表达量较低，在受精卵进入胚胎发育过程之后表达显著上调，胚胎发育结束之后明显下降，提示它对胚胎发育过程的基因表达调控可能具有重要作用。
萨氏海鞘与非尾索动物之间具有数量众多的同序列异源miRNA，暗示着miRNA进化中可能存在具有序列特异性的选择压力。靶基因的序列特异性可能是miRNA趋同进化的动力。
保守性miRNA靶基因与其他物种已有的靶基因实验证据的比较分析表明，保守性miRNA家族的靶基因序列可能发生变异而脱离调控，然而miRNA通过调控与原靶基因密切相关的基因（例如其受体）仍可能保持对原生物学过程的调控。Ciona属海鞘特有的csa_m-27c是脊椎动物miR-27家族的异源同功miRNA——具有相似的调控作用，但不是同源基因。这暗示miRNA介导的转录后调控体系可能超越序列水平而在生物学过程水平上的具有保守性。
Prediction of miRNAs for3Urochordates (C. savignyi, C. intestinalis andOikopleura dioica) were carried out with bioinformatic approaches. We predicted527conservative miRNA genes from the3species, including52miRNA genesspecific to the Urochordata family Ciona and37miRNA gene clusters. Wereported the first collection of6experimentally validated miRNAs in C. savignyi.
We clustered the180predicted C savignyi miRNAs into140families by PBCbootstrap analysis among6Deuterostomias, and found that80.00%of them arespecific to family Ciona, only16.43%and3.57%are conserved with otherChordates and Deuterostomias, respectively. The profound change in miRNArepertoire might have played an important role in the dramatic change of life styleand morphology during Ascidians phylogenesis.
Function of6experimentally validated C. savignyi miRNAs are analyzed bycomputational prediction of their potential target genes. Gene ontology analysissuggests that multiple biological progresses are regulated by these miRNAs, amongwhich embryonic development may be regulated by csa_m-27c and csa_9_com, andheart development may be the targeted progress of csa_2-com and csa_7.
Temporal and spacial expression profile of the novel miRNA csa_m-27c showsthat its expression was low in the gonad, but increased significantly duringembryonic development before dropped abruptly while the C. savignyi developedinto tadpole larva, indicting an important role of csa_m-27c in the embryonicdevelopment, which is accordant to the bioinformatic function analysis.
Ciona savignyi shares many sequencially similar miRNAs with non-Urochordataanimals, but only a third of them are homologous. Numerous analogous miRNAssuggests sequencially selective pressure from miRNA targets.
By comparison between predicted targets of conservative C. savignyi miRNAsand reported experimental evidences, we found that conservative miRNAs maysometime maintain their regulation of a specific biological progress even if they lostthe control of a target gene. csa_m-27c is an analogy of miR-27c family. Itfunctions similar to miR-27but have a different origin, like a miRNA equivalent ofan isozyme. This suggests that conservation of miRNA mediated transcriptionalregulation system may exceed sequence level and live up to progress level.
引文
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