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: 10217201
• 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/10217201
• 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 除了在癌症的情况下，基因及其产物（RNA和蛋白质）不会混合。 但是，当在越来越多的嵌合RNA中识别出来时，这种传统的教条正受到挑战 非癌症样品。我们在相邻基因之间（顺式）之间进行跨剪接和顺式切割的工作已有 提供的证据表明，基因间剪接可能是扩展功能基因组的另一种机制。 这项建议，我们重点关注整个基因组的转录读取融合研究（我们 将它们命名为顺式，以与其他机制区分开）。传统上，它们被认为很少见，或者 文物。即使被证明是真实的，它们也被认为是转录噪声或副产品。但是，在 我们的初步研究，我们已经确定了非癌组织和细胞中数千种此类融合RNA。 有些已得到验证，并证明了功能。我们假设CIS-SAGE嵌合融合RNA是一个 在正常生理学中广泛传播现象，代表了多样化我们转录组的一种手段。到 检验这一假设，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 四个目标：在AIM1中，我们将表征融合RNA和父母基因，包括连接序列， 蛋白质编码的潜力，表达和基因本体论。融合RNA也将在多个 具有多种技术的水平，包括非基于RT的纳米弦平台和高吞吐量质量 光谱法。在AIM2中，我们将研究顺式融合的生物学性。高通量 将使用筛查，然后使用具有功能丧失系统的候选方法。为了 功能丧失系统，我们将使用RNAi方法，这在我们手中更成熟。此外，我们将使用 我们新适应的DCAS9-KRAB方法，以实现融合特异性的沉默。我们将在 细胞培养和动物系统。其中一种融合将在“后源性”小鼠中更详细地检查 模型。在AIM3中，我们将通过两种方法研究顺式平衡的生成机制 生物信息学方法和记者方法。从生物信息上讲，我们将从 编码和路线图表观基因组学研究表观遗传学签名，转录调节模式， DNA甲基化以及顺式融合位点的三维接近度。在记者中 系统，我们建造了一个由内含子，外显子和模型的终止位点隔开的两部分肾素荧光素酶 融合RNA，我们正在使用该系统筛选候选因素。在AIM4中，我们将发展一个互动性 基于Web的数据库允许最终用户在正常组织和细胞中搜索融合RNA。发现 从拟议的研究中，不仅有可能挑战嵌合RNA的传统教条 是癌症特异性的特征，但也增强了我们对人类基因组和转录组的理解。 在疾病情况下可能会错过嵌合RNA，从而扩大生物标志物的曲目 和治疗目标发现。