Mapping the regulatory landscape of RNA binding proteins and their causal roles in tumorigenesis and patient survival

摘要 我们的初步结果支持RNA结合蛋白(RBPs)的失调在癌症中的突出作用 进步。事实上，我们对癌症基因组图谱(TCGA)转录组数据的分析表明，限制性商业惯例、AS A组，在癌症中的调控明显比转录因子更失调。我们提出了一个多方面的 一组计算和实验研究，以系统地确定导致 有助于癌症的进展，并表征其下游效应机制。在一种策略中， 我们建议通过首先在3‘’中发现它们的顺式调节识别元件来识别调节不良的限制性商业惯例 以及显示动态mRNA表达的基因的5‘非编码区--在肿瘤和正常样本之间，以及 在TCGA的25个癌症队列中的每一个。这将使用信息论来实现。 高灵敏度、低误判性的从头发现线性和结构RNA基序的算法 发现率。我们以前已经证明，这样的RNA基序是限制性商业惯例的结合位点，可以调节 信使核糖核酸的稳定性，其子集调节肿瘤的发生和转移。含有这些基序的基因 构成一个孤立的RBP调节子(或RBP模块)，在癌症进展中起可疑作用。为了 确定临床上有意义的RBP模块，我们建议开发一个量化的计算框架 每个模块的表达在多大程度上影响患者在TCGA原发肿瘤中的生存 样本。我们的初步结果导致发现了许多具有显著分层的此类模块 多种癌症类型的患者存活率。对于最具临床预后的模块子集，我们 将使用生化和基于CRISPR的平行遗传筛查来确定其同源限制性商业惯例。在一个 补充策略，我们将通过计算从一个 在~250个限制性商业惯例中每一个的shRNA被敲除后获得的ENCODE转录组数据概要。在……里面 为了确定导致癌症进展的限制性商业惯例，我们建议开发一种平行的 在小鼠肿瘤形成和转移的异种移植模型中对所有RBPs进行CRISPR功能丧失筛查。 然后，我们将在显示两个重要患者的前20个RBP上进行更有针对性的CRISPR筛查 在我们的初步全面筛查中，生存分层和小鼠体内肿瘤效应。排名靠前的经验证 然后，将对RBPs在各种体外和体内癌细胞中的作用进行单独表征 表型。最后，我们建议开发一个并行的小鼠体内CRISPR上位平台，以高效地 确定特定的下游基因，RBP通过这些基因在肿瘤形成和发展中发挥作用 转移。我们的集成计算/实验策略将扩展我们对 在很大程度上还没有被探索的癌症途径失调领域，并可能揭示新的工作原理。 此外，我们对癌症进展的因果途径的关注将影响诊断、预后和 临床肿瘤学的治疗精确度。

Summary Our preliminary results support a prominent role for dysregulation of RNA binding proteins (RBPs) in cancer progression. In fact, our analysis of the cancer genome atlas (TCGA) transcriptome data shows that RBPs, as a group, are significantly more dysregulated in cancer than transcription factors. We propose a multi-faceted set of computational and experimental studies to systematically identify the set of RBPs that causally contribute to cancer progression and to characterize their downstream effector mechanisms. In one strategy, we propose to identify dysregulated RBPs by first discovering their cis-regulatory recognition elements in the 3’ and 5’ UTR of genes that show dynamic mRNA expression—both between tumor vs. normal samples, and across each of the 25 cancer cohorts in TCGA. This will be accomplished using information-theoretic algorithms that discover de novo linear and structural RNA motif elements with high sensitivity and low false discovery rates. We have previously shown that such RNA motifs are the binding sites for RBPs that modulate mRNA stability, a subset of which regulate tumorigenesis and metastasis. The genes harboring these motifs constitute an orphan RBP regulon (or RBP module) with a suspected role in cancer progression. In order to identify clinically significant RBP modules, we propose to develop a computational framework that quantifies the degree to which the expression of each module stratifies patient survival across the TCGA primary tumor samples. Our preliminary results have led to the discovery of many such modules with remarkable stratification of patient survival across multiple cancer types. For the subset of the most clinically prognostic modules, we will identify their cognate RBPs using both biochemical and CRISPR-based parallel genetic screens. In a complementary strategy, we will computationally identify such clinically prognostic RBP modules from a compendium of ENCODE transcriptome data obtained following shRNA knockdowns of each of ~250 RBPs. In order to identify RBPs that causally contribute to cancer progression, we propose to develop a parallel CRISPR loss-of-function screen for all RBPs in mouse xenograft models of tumor formation and metastasis. We will then conduct a more focused CRISPR screen on the top ~20 RBPs that show both significant patient survival stratification and mouse in vivo tumor effects in our primary comprehensive screen. The top validated RBPs will then be individually characterized for their roles in a variety of in vitro and in vivo cancer cell phenotypes. Finally, we propose to develop a parallel mouse in vivo CRISPR epistasis platform to efficiently determine the specific downstream genes through which the RBP exerts its effects on tumor formation and metastasis. Our integrated computational/experimental strategy will expand our molecular understanding of a largely unexplored domain of cancer pathway dysregulation and potentially reveal new principles at work. Furthermore, our focus on causal pathways of cancer progression will impact the diagnostic, prognostic, and therapeutic precision with which we approach clinical oncology.