Genome-wide Investigation of cis-splicing between Adjacent Genes

项目总结 基因及其产物(RNA和蛋白质)预计不会混合在一起，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，这一传统教条正受到挑战 非癌症样本。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 提供了基因间剪接可能是扩大功能基因组的另一种机制的证据。在……里面 在这项提议中，我们将重点放在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)。传统上，它们被认为是稀有的，或者 手工艺品。即使被证实是真的，它们也被认为是转录噪音或副产品。但是，在 在我们的初步研究中，我们已经在非癌症组织和细胞中发现了数千个这样的融合RNA。 其中一些已经过验证，并被证明是有效的。我们假设cis-sage嵌合融合RNA是一种 在正常生理中广泛存在的现象，代表了一种使我们的转录组多样化的手段。至 验证这一假设，并在基因组水平上获得关于这些融合的知识，我们提出以下建议 四个目标：在Aim1中，我们将鉴定融合RNA和亲本基因，包括连接序列， 蛋白质编码潜力、表达和基因本体论。还将对融合RNA进行多个 具有多种技术的级别，包括非基于RT的纳米串平台和高通量质量 光谱分析。在AIM2中，我们将研究顺式鼠尾融合的生物学意义。高吞吐量 将使用筛选，然后使用增益型和失调型系统的候选方法。对于 对于功能丧失的系统，我们将使用RNAi方法，这是我们手中比较成熟的方法。此外，我们还将使用 我们新采用的dCAS9-KRAB方法实现了融合特异性沉默。我们将把微扰应用于 无论是细胞培养还是动物系统。其中一种融合将在“回溯基因”小鼠身上进行更详细的检查。 模特。在AIM3中，我们将用两种方法来研究顺式SAGE的产生机制，一个是 生物信息学方法和记者方法。在生物信息学方面，我们将询问来自 编码和路线图表观基因组学，研究表观遗传特征，转录调控模式， DNA甲基化以及顺式SAGE融合位点的三维邻近。在记者面前 系统中，我们构建了一个由内含子、外显子和终止位点分离的两部分的肾形荧光素酶模型 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，允许终端用户在正常组织和细胞中搜索融合RNA。调查结果 这项拟议的研究不仅可能挑战嵌合RNA的传统教条 都是癌症特有的特征，但也增强了我们对人类基因组和转录组的了解。 嵌合RNA在疾病情况下可能被错误调控，从而扩大了生物标记物的谱系 和治疗靶点的发现。

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.