海鞘胚胎发育中长非编码RNA的基因网络研究

In higher eukaryotes, large portion of the genome is transcribed, however, only a small percentage encodes protein-coding genes. The rest of them are consisted of the so called non-coding genes, which include small non-coding RNAs like microRNA, piwiRNA, siRNA, snoRNA, and long non-coding RNAs. The latter are typically longer than 200nt, transcribed by RNA polymerase II, have exon-intron structures and are polyadenylated. Thus they are commonly referred to as long intergenic non coding RNAs, or lincRNAs. Vertebrate genomes typically contain thousands of lincRNAs, however, very little functional information is known for these genes. This is due to several factors. First, lincRNAs do not simply function by base-pairing, so there is no clear technique to predict function. Second, there is very little sequence conservation among lincRNA from different animal groups, making functional prediction by conservation difficulty. Finally, there lacks efficient ways for perturbing lincRNA function. In this grant, we propose to systematically study lincRNA in a marine invertebrate model system, the ascidian species Ciona intestinalis. Ascidians belong to the subphylum Urochordate, the closet relative to vertebrates. Ciona has a very compact genome, few cell number and fast embryonic cycle, making it appealing to developmental studies. In addition, the electroporation technique can generate transient transgenic animals very efficiently, allowing fast reporter assays and manipulation of endogenous gene function. Finally, ascidians are important marine biofouling animals. In this research, we will systematically predict Ciona lincRNAs, use network analysis to study the mRNA-lincRNA gene network in embryogenesis and tissue development, predict lincRNA function through gene network models, and experimentally verify the function using overexpression and knockdown assays. For this, we will use our recently developed magnetic bead based cell sorting technique to isolate major tissue types of Ciona embryo and use RNA-seq to determine the transcriptome of each tissue. We will use bioinformatics tools to systematically identify lincRNAs from the RNA-seq data and determine their expression profiles. We will then use network analysis tools like Bayesian network to identify network structures of mRNA and lincRNA, construct network modules and predict regulatory interactions between them. We will then use tissue specific overexpression and siRNA knockdown to study the roles of lincRNAs in Ciona embryonic development. Together, these studies will provide the first picture of the role of lincRNA in Ciona.

在高等生物基因组中，除了蛋白质基因，还有大量的非编码RNA参与基因调控等生物过程。非编码RNA包括小非编码RNA及长度大于200碱基的长非编码RNA。后者的物种间保守度低，生物功能所知甚少。我们计划研究玻璃海鞘的长非编码RNA在胚胎发育中的功能。海鞘属于尾索动物亚门，是和脊椎动物亲缘关系最近的无脊椎动物。由于其基因组紧凑、胚胎细胞数少、发育迅速，是一个很好的研究基因调控和功能的模型。海鞘还是一种重要的污损生物，其发育生物学研究对理解附着变态机制和抑制生物污损也有重要意义。为此我们提出用网络分析的方法，在全基因组层面研究mRNA和lincRNA组成的基因网络在发育中的变化，预测lincRNA的调控与功能。为此我们将用RNA-seq技术确定海鞘胚胎组织特异的转录组，预测lincRNA基因，构建基因网络，预测基因功能并用胚胎实验验证。这将在一个海洋模式生物中首次揭示lincRNA的功能

海鞘是一种重要的模式生物，具有独特的分类学地位，即和脊椎动物最接近的生物类别。另一方面，海鞘胚胎具有细胞数目少，细胞谱系固定的特点，对大部分生物学过程，可以认为海鞘是一个简化的近似脊椎动物的模型，在研究基因网络调控，细胞谱系发育和命运决定等方面有独特的优势。在本研究中，我们使用单细胞转录组测序技术，系统研究了海鞘胚胎发育过程中各种细胞类型的基因表达。第一次在模式生物中描绘了单个胚胎中每个细胞的基因表达与发育过程中的动态变化。这为系统研究细胞特异的基因调控网络提供了基础。同时，我们系统预测了海鞘胚胎发育中的非编码RNA，并结合单细胞转录组数据，揭示了细胞特异性的非编码RNA表达模式。这一研究证明了单细胞技术在海洋模式生物发育生物学中的重要作用，为海鞘研究领域提供了重要的系统组学信息。同时也为研究非编码RNA在发育过程中的细胞特异性及功能提供了基础