"Ribonomics" of Gene Regulation to predict Innate Immune Responses

描述（由申请人提供）：基因的表达涉及一系列酶促事件，如转录、mRNA加工、mRNA衰变和翻译，这些事件受蛋白质-核酸相互作用的基因调控网络（grn）的影响。众所周知，通过调节增强子和启动子活性的调控网络来控制转录并不是基因产物产生的唯一决定因素，但外显子跳变是普遍存在的，转录后机制（如mRNA剪接和衰变）决定了mRNA诱导的动力学和丰度。事实上，在我们对巨噬细胞对病原体反应的初步研究中，我们发现大多数诱导的基因表达事件导致mRNA与基因组浏览器预测的mRNA显著偏离，mRNA的衰减受到蛋白核酸和miRNA调节网络的控制。拟议的基因调控“核糖组学”中心利用并开创了下一代测序和计算建模方法，以开发一种预测模型，用于mRNA同种异构体的表达以及给定启动子活性和转录起始率的水平。我们将开发普遍适用的工具，结合或深入和定量的实验分析巨噬细胞对病原体相关内毒素的反应，这将导致超过1000个基因的急剧上调。引人注目的是，我们的初步数据发现了900多个外显子跳跃事件，以及许多5‘或3’剪接位点使用的替代案例，强调了起始后事件的重要贡献。此外，这些剪接模式依赖于巨噬细胞亚型特异性染色质景观，并在启动或耐受状态下被诱导剪接因子改变。因此，我们将利用描述良好的巨噬细胞生物学和相关的实验模型系统，检查基因结构和序列（Aim 1），染色质修饰（Aim 2）和反式剪接因子（Aim 3）在确定成熟mRNA的身份及其相关合成速率方面的作用，然后添加刺激响应性调节网络，该网络赋予mRNA半衰期控制，从而确定每种mRNA亚型的丰度（Aim 4）。

DESCRIPTION (provided by applicant): The expression of genes involves a sequence of enzymatic events, such as transcription, mRNA processing, mRNA decay, and translation, that are subject to gene regulatory networks (GRNs) of protein-nucleic acid interactions. It is well appreciated that the control of transcription via regulatory networks that regulate enhancer and promoter activities are not the sole determinant of what gene products result, but that exon skipping is pervasive and post-transcriptional mechanisms such as mRNA splicing and decay determine the kinetics of mRNA induction and abundance. Indeed, in our preliminary studies of the macrophage response to pathogens, we find that a majority of induced gene expression events result in mRNAs that deviate substantially from those predicted by the genome-browser, and that mRNA decay is controlled by both protein- nucleic acid and miRNA regulatory networks. The proposed Center for the "Ribonomics" of Gene Regulation leverages and pioneers Next Gen Sequencing and computational modeling approaches to develop a predictive model for which mRNA isoforms are expressed and at what level given a given promoter activity and transcription initiation rate. We will develop generally applicable tools in conjunction with or in depth and quantitative experimental analysis of the macrophage response to pathogen-associated endotoxin, which results in the dramatic up regulation of more than 1000 genes. Strikingly, our preliminary data identified more than 900 exon skipping events in addition to numerous cases of alternative 5' or 3' splice site use, emphasizing the essential contribution of post-initiation events. Further, these splice patterns are dependent on the macrophage subtype-specific chromatin landscape and are altered by inducible splice factors in primed or tolerated states. Thus we will leverage the well-described macrophage biology and associated experimental model systems, to examine the role of gene structure and sequence (Aim 1), the role of chromatin modifications (Aim 2), and of trans-acting splicing factors (Aim 3) in determining the identity of mature mRNAs and their associated synthesis rates, before adding the stimulus-responsive regulatory networks that confer mRNA half-life control and thus determine the abundance of each mRNA isoform (Aim 4).