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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要基因及其产物（RNA 和蛋白质）预计不会混合，除非发生癌症。然而，当越来越多的嵌合 RNA 被鉴定出来时，这一传统教条正在受到挑战。我们在相邻基因之间的反式剪接和顺式剪接（cis-SAGe）方面的工作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。在这个提案中，我们专注于转录通读融合的全基因组研究（我们将它们命名为 cis-SAGe 以区别于其他机制）。即使被证明是真实的，它们也被认为是转录噪音或副产品。我们的初步研究已经在非癌组织和细胞中鉴定出数千个这样的融合RNA。有些已被验证并被证明具有功能性，我们认为 cis-SAGe 嵌合融合 RNA 是一种功能性的RNA。正常生理学中广泛传播的现象，代表了使我们的转录组多样化的一种手段。检验这个假设，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 Aim1 中，我们将表征融合 RNA 和亲本基因，包括连接序列，还将对融合 RNA 的蛋白质编码潜力、表达和基因本体进行分析。具有多种技术水平，包括非基于 RT 的 Nanostring 平台和高通量质量在 Aim2 中，我们将研究 cis-SAGe 融合的生物学意义。筛选后将使用功能获得和丧失系统的候选方法。功能缺失系统，我们会使用RNAi方法，这个方法我们手中比较成熟，另外我们会使用。我们新采用的 dCAS9-KRAB 方法来实现融合特异性沉默，我们将在中应用扰动。细胞培养和动物系统中的一种融合将在“逆转录”小鼠中进行更详细的检查。在 Aim3 模型中，我们将通过两种方法研究 cis-SAGe 的生成机制：生物信息学方法和报告者方法从生物信息学角度来看，我们将询问来自多组学的数据。 ENCODE 和路线图表观基因组学，用于研究表观遗传特征、转录调控模式、 DNA 甲基化以及 cis-SAGe 融合位点的三维邻近度。系统，我们构建了由模型的内含子、外显子和终止位点分隔的两部分海肾荧光素酶融合RNA，我们正在使用该系统来筛选候选因子，在Aim4中，我们将开发一个交互式的。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。拟议的研究不仅有可能挑战嵌合 RNA 的传统教条是癌症特有的特征，同时也增强了我们对人类基因组和转录组的理解。嵌合 RNA 在疾病情况下可能会被错误调节，从而扩大了生物标志物的范围和治疗靶点发现。