Genome-wide Investigation of cis-splicing between Adjacent Genes

相邻基因之间顺式剪接的全基因组研究

• 批准号: 10006886
• 负责人: HUI LI
• 金额: $ 32.3万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-03 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10006886
• 关键词: 3-Dimensional; Alternative Splicing; Animal Model; Animals; Binding; Bioinformatics; Biological; Biological Markers; Cell Culture Techniques; Cells; Chimera organism; Chimeric Proteins; Code; DNA Methylation; DNA Sequence; Data; Data Set; Databases; Development; Dideoxy Chain Termination DNA Sequencing; Disease; Epigenetic Process; Event; Exons; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Genotype; Guide RNA; Hematology; Human; Human Genome; Human Genome Project; Intercistronic Region; Introns; Investigation; Knowledge; Libraries; Luciferases; Malignant Neoplasms; Mass Spectrum Analysis; Methods; Modeling; Morphologic artifacts; Multiomic Data; Names; Noise; Normal Cell; Normal tissue morphology; Nuclear; Online Systems; Ontology; Pattern; Peptides; Phenotype; Physiology; Process; Proteins; Publishing; RNA; RNA Interference; RNA Splicing; RNA analysis; RNase protection assay; Renilla Luciferases; Reporter; Research; Resources; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Sagittaria; Sampling; Side; Site; Solid; Source; Spliced Genes; System; Techniques; Testing; Therapeutic; Tissues; Trans-Splicing; Transcript; Transcriptional Regulation; Variant; Western Blotting; Work; base; cleavage factor; epigenomics; gene product; genome-wide; genome-wide analysis; high throughput screening; histone modification; in vivo; loss of function; mouse model; nano-string; screening; small hairpin RNA; termination factor; therapeutic target; transcription factor; transcriptome; transcriptome sequencing; web page; web site

PROJECT SUMMARY Genes and their products (RNA and protein) are not expected to intermingle, except in the situation of cancer. However, this traditional dogma is being challenged when more and more chimeric RNAs being identified in non-cancer samples. Our work on trans-splicing and cis-splicing between adjacent genes (cis-SAGe) have provided evidence that the intergenic splicings may be another mechanism to expand functional genome. In this proposal, we are focusing on the genome-wide study of the transcriptional read-through fusions (we named them cis-SAGe to differentiate from other mechanisms). Traditionally, they were believed to be rare, or artifacts. Even when proven true, they were thought to be transcriptional noise or side products. However, in our preliminary studies, we have identified thousands of such fusion RNAs in non-cancer tissues and cells. Some have been validated, and proven functional. We hypothesize that cis-SAGe chimeric fusion RNAs are a widely spread phenomenon in normal physiology, and represent a means to diversify our transcriptome. To test this hypothesis, and to gain knowledge about these fusions at the genome level, we propose the following four aims: In Aim1, we will characterize the fusion RNAs and parental genes including junction sequence, protein-coding potential, expression, and gene ontology. The fusion RNAs will also be analyzed at multiple levels with multiple techniques including non RT-based Nanostring platform, and high throughput mass spectrometry. In Aim2, we will investigate the biological significance of the cis-SAGe fusions. A high throughput screening will be used followed by candidate approaches with both gain- and loss-of-function systems. For the loss-of-function system, we will use RNAi method, which is more mature in our hands. In addition, we will use our newly adapted dCAS9-KRAB method to achieve fusion-specific silencing. We will apply the perturbation in both cell culture and animal systems. One of the fusions will be examined in more detail in a “retrogenic” mice model. In Aim3, we will investigate the generating mechanisms of cis-SAGe with two approaches, a bioinformatics approach and a reporter approach. Bioinformatically, we will interrogate multi-omics data from ENCODE and Roadmap Epigenomics to investigate epigenetic signatures, transcriptional regulatory patterns, DNA methylation as well as the three-dimensional proximity of the cis-SAGe fusion sites. In the reporter system, we have built a two-part renilla luciferase separated by introns, exons and termination site of a model fusion RNA, and we are using the system to screen candidate factors. In Aim4, we will develop an interactive web-based database to allow end users to search for fusion RNAs in normal tissues and cells. The findings from the proposed study will have the potential of not only challenging traditional dogmas that chimeric RNAs are cancer-specific features, but also enhancing our understanding of the human genome and transcriptome. The chimeric RNAs may be misregulated in disease situations, thus expanding the repertoire for biomarker and therapeutic target discovery.

项目摘要 基因和它们的产物（RNA和蛋白质）不会混合在一起，除非是在癌症的情况下。 然而，当越来越多的嵌合RNA被发现时，这一传统的教条正在受到挑战。 非癌症样本。我们在相邻基因间的反式剪接和顺式剪接（cis-SAGe）方面的工作， 为基因间剪接可能是功能基因组扩展的另一种机制提供了证据。在 这个建议，我们正集中在全基因组的研究转录通读融合（我们 将其命名为顺式SAGe以区别于其他机制）。传统上，他们被认为是罕见的，或 藏物即使被证明是真的，它们也被认为是转录噪音或副产物。但在 在我们的初步研究中，我们已经在非癌组织和细胞中鉴定出数千种这样的融合RNA。 其中一些已经过验证，并被证明是可行的。我们假设顺式-SAGe嵌合融合RNA是一种 在正常生理学中广泛传播的现象，并代表了一种使我们的转录组多样化的手段。到 为了验证这一假设，并在基因组水平上获得有关这些融合的知识，我们提出以下建议 四个目标：在Aim 1中，我们将表征融合RNA和亲本基因，包括连接序列， 蛋白质编码潜力、表达和基因本体。融合RNA也将在多个细胞中进行分析。 水平与多种技术，包括非基于RT的Nanostring平台，和高通量质量 光谱法在Aim 2中，我们将研究顺式SAGe融合的生物学意义。高通量 将使用筛选，然后使用具有功能增益和功能丧失系统的候选方法。为 功能丧失系统，我们将使用RNAi方法，这在我们手中是比较成熟的。此外，我们将使用 我们新调整的dCAS 9-KRAB方法以实现融合特异性沉默。我们将应用微扰， 细胞培养和动物系统。其中一个融合体将在一个“逆转录”小鼠中进行更详细的研究 模型在Aim 3中，我们将用两种方法研究顺式SAGe的产生机制， 生物信息学方法和报告子方法。生物信息学上，我们将询问来自 ENCODE和路线图表观基因组学研究表观遗传签名，转录调控模式， DNA甲基化以及顺式-SAGe融合位点的三维邻近性。在记者 系统中，我们构建了由内含子、外显子和终止位点分隔的两部分海肾荧光素酶模型 融合RNA，我们正在使用该系统筛选候选因子。在Aim 4中，我们将开发一个交互式 基于网络的数据库，允许最终用户搜索正常组织和细胞中的融合RNA。这些发现 这项研究不仅有可能挑战传统的教条， 是癌症特有的特征，而且也增强了我们对人类基因组和转录组的理解。 嵌合RNA可能在疾病情况下被错误调节，从而扩大了生物标志物的库。 和治疗靶点发现。