Investigating the regulation of alternative splicing and its role in disease

项目二：绘制控制选择性剪接的基因调控网络我们对转录组复杂性的理解与大规模方法学方法的发展同步发展，这些方法能够检测和绘制整个转录组的RNA结合蛋白（RBP）。这些研究为RNA加工调控及其对疾病状态的贡献提供了机制性见解。尽管取得了显著的进展，但我们仍然缺乏对控制可变剪接的调控网络的完整了解。这些在应激相关途径的背景下尤其普遍，这些途径被癌细胞利用来产生对肿瘤生长具有有利影响的转录亚型。因此，关键是要阐明的分子途径，使压力信号，以汇聚在重塑选择性剪接程序。目的1：开发和应用大规模平行和定量的CRISPR筛选平台，用于定位剪接调控网络。为了实现大规模组合CRISPR扰动与替代性前mRNA剪接读出，我们开发了一种新的方法，将CHyMErA组合遗传扰动与剪接报告基因的深度测序读出相结合，以定量测量剪接表型并将其与每个扰动相关联。为了实现这一点，我们修饰了我们的慢病毒组合CRISPR筛选载体，以使剪接小基因报告基因能够以诱导型方式表达，同时在报告基因的3'端内携带gRNA表达盒。这些构建体在表达Cas核酸酶的细胞中的慢病毒递送导致可编程的基因组编辑，如由组成型表达的gRNA所确定的，随后是在所需时间的报告基因表达的多西环素依赖性激活。纯化来自这些细胞的mRNA，并从剪接报告cDNA产生Illumina测序文库。配对末端测序用于定量剪接报告基因表型，并将其与由gRNA序列确定的相应遗传扰动联系起来，从而实现剪接调节因子的全基因组询问。通过应用不同的剪接报告，我们已经确定了几百个基因，可以影响选择性剪接，基因是高度富集的RNA剪接和剪接体生物发生功能的条款。这证明了我们的方法识别真正的剪接调节基因的可行性和力量。目的2：选择性剪接程序的新调节子的鉴定和表征。我们的全基因组剪接报告筛选已经确定了几个基因，影响剪接的多个报告，以前没有与剪接调控。基于BIOGRID和/或STRING数据库，这些基因中的一些还显示出与剪接相关蛋白相互作用的证据。这些包括基因如PAXBP 1、ZNF 41、ZNF 207和KBTBD 2，所有这些都是核基因，而研究不足的基因大多与转录调控有关。我们假设这些因素可以直接影响特定基因的选择性剪接。为了验证这一假设，我们将使用降解决定子快速耗尽人类细胞中的这些基因，并使用Nascent-Seq评估它们对新转录的RNA的影响。我们还将应用亲和纯化结合质谱以及交联和免疫沉淀实验，以确定这些因子的直接蛋白质和RNA相互作用，并获得关于它们如何调节选择性剪接决定的机制见解。这些研究有望为人类细胞中的选择性剪接调控提供新的见解。目的3：哪些分子途径控制细胞应激的选择性剪接反应？剪接领域的一个重要挑战是了解剪接调控途径如何响应环境变化，包括渗透压或温度变化引起的压力。事实上，高渗应激和热休克都导致广泛的剪接改变。我们已经产生了对这些应激有反应的剪接报告基因，并且我们已经在经受相应应激条件的细胞中进行了如上所述的全基因组筛选。这些筛选已经确定了几个对压力依赖性剪接变化至关重要的候选基因。

Project 2: Mapping gene regulatory networks that control alternative splicing Our understanding of transcriptomic complexity has advanced in parallel with the development of large-scale methodological approaches enabling the detection and mapping of RNA binding proteins (RBPs) across the transcriptome. Such studies have provided mechanistic insights into RNA processing regulation and its contribution to disease-states. Despite this remarkable progress, we are still lacking a complete understanding of the regulatory networks that control alternative splicing. These is particularly prevalent in the context of stress-related pathways that are exploited from cancer cells to generate transcript isoform with advantageous effects on tumor growth. It is thus crucial to elucidate the molecular pathways that enable stress signaling to converge on remodeling alternative splicing programs. Aim 1: Development and application of a massively parallel and quantitative CRISPR screening platform for mapping splicing regulatory networks. To enable large-scale combinatorial CRISPR perturbations coupled with alternative pre-mRNA splicing readouts, we have developed a novel methodology combining CHyMErA combinatorial genetic perturbations with deep-sequencing readouts of splicing reporters to quantitatively measure splicing phenotypes and associate them with each perturbation. To achieve this, we modified our lentiviral combinatorial CRISPR screening vector to enable the expression of splicing minigene reporters in an inducible manner while harboring gRNA expression cassette within the 3' end of the reporter. Lentiviral delivery of these constructs in cells expressing Cas nucleases results in programable genome editing as determined by the constitutively expressed gRNAs, which is followed by the doxycycline-dependent activation of reporter expression at the desired time. mRNA from these cells is purified and Illumina sequencing libraries are generated from splicing reporter cDNA. Paired-end sequencing is used to quantify splicing reporter phenotypes and link them with the corresponding genetic perturbations determined by the gRNA sequences, thus enabling genome-wide interrogation of splicing regulators. By applying different splicing reporters, we have identified several hundred genes that can impact alternative splicing, genes that are highly enriched for RNA splicing and spliceosome biogenesis functional terms. This demonstrates the feasibility and the power of our approach to identify bona-fide splicing regulatory genes. Aim 2: Identification and characterization of novel regulators of alternative splicing programs. Our genome-wide splicing reporter screens have identified several genes to impact the splicing of multiple reporters that have not been previously associated with splicing regulation. Some of these genes also display evidence of interacting with splicing related proteins based on BIOGRID and/or STRING databases. These include genes such as PAXBP1, ZNF41, ZNF207 and KBTBD2 all of which are nuclear and rather understudied genes that have been mostly implicated with transcription regulation. We hypothesize that these factors can directly impact alternative splicing of specific genes. To test this hypothesis, we will rapidly deplete these genes in human cells using degrons and assess their impact on newly transcribed RNA using Nascent-Seq. We will also apply affinity purification coupled to mass spectrometry as well as crosslinking and immunoprecipitation experiments to identify the direct protein and RNA interactions of these factors and gain mechanistic insights on how they can regulate alternative splicing decisions. These studies are expected to provide novel insights of alternative splicing regulation in human cells. Aim 3: Which are the molecular pathways that control alternative splicing responses to cellular stress? An important challenge in the splicing field is to understand how splicing regulatory pathways respond to environmental changes including stresses imposed by alterations in osmolarity or temperature. Indeed, hyperosmotic stress and heat shock both result in widespread splicing alterations. We have generated splicing reporters that are responsive to these stresses, and we have performed genome-wide screens as described above in cells subjected to the corresponding stress condition. These screens have identified several candidate genes critical for stress-dependent splicing changes.