High-throughput phenotypic screening for functionally characterizing alt. exons

用于功能表征 alt 的高通量表型筛选。

• 批准号: 10926461
• 负责人: Thomas Gonatopoulos-Pournatzis
• 金额: $ 61.48万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10926461
• 关键词: Address; Affect; Alternative Splicing; Biological; Biomedical Research; CRISPR screen; Cells; Code; Complex; Coupled; Coupling; Cues; Data; Development; Dimensions; Disease; Essential Genes; Event; Exons; Follow-Up Studies; Gene Expression; Gene Expression Alteration; Gene Expression Profile; Genes; Genetic; Genetic Screening; Genetic Transcription; Genome; Goals; High-Throughput Nucleotide Sequencing; Human; Human Genome; Individual; Introns; Knowledge; Libraries; Link; Malignant Neoplasms; Manuscripts; Maps; Molecular Analysis; Mutation; Names; Neurodevelopmental Disorder; Nonsense Codon; Nuclear; Outcome; Output; Pathogenicity; Peptide Initiation Factors; Phenotype; Physiology; Poly A; Poly(A)+ RNA; Polyadenylation; Post-Translational Modification Site; Process; Proliferating; Protein Binding Domain; Protein Isoforms; Proteins; Proteomics; RNA Splicing; RNA library; RNA-Binding Proteins; Research; Resolution; Role; Series; Site; Splice-Site Mutation; TAF1 gene; TAF5 gene; TATA-Box Binding Protein; Tertiary Protein Structure; Tissues; Transcript; Transcription Initiation; Transcription Initiation Site; Translating; Translations; Vertebrates; Work; biological adaptation to stress; cell type; combinatorial; exome; experimental study; fitness; functional genomics; genome-wide; high dimensionality; insight; interest; mRNA Precursor; mutation screening; new technology; novel; preservation; programs; recruit; respiratory; screening; single-cell RNA sequencing; tool; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

Alterative pre-mRNA splicing greatly diversifies the human transcriptome expanding the coding potential of the genome. RNA sequencing profiling studies have revealed that almost all multi-exon human genes are subject to alternative splicing. Networks of timely coordinated alternative exons and introns are under elaborate regulatory control in different cell and tissue types and upon altering environmental cues generating diverse transcriptomic and proteomic landscapes that can support different cell fates or stress responses. It is estimated that over 10% of pathogenic mutations impact alternative splice site activation, and dysregulation of alternative splicing has been associated with complex diseases such as cancer and neurodevelopmental disorders. Major challenges in the alternative splicing field include i) the functional characterization of alternative exons, the overwhelming majority of which remain uncharacterized and, ii) understanding how splicing programs are remodeled in various cell types or environmental conditions. My research group is using a combination of functional genomics and focused molecular analyses to address these challenges as elaborated below. Project 1: High-throughput identification and characterization of phenotypically critical alternative exons Frame-preserving alternatively spliced exons that overlap coding sequences and are devoid of premature stop codons are translated and contribute to protein diversity. The functional consequences of such splice isoforms are difficult to predict, and it has been a matter of debate to what extent they represent stochastic differences in splice site selection during transcription with little biological repercussions9,10. Accordingly, a major challenge in the splicing field is to identify the full repertoire of biologically relevant alternative exons that contribute to phenotypic outcomes. Such knowledge will facilitate the prioritization of alternative splicing events for focused studies and will also provide predictive phenotypic insights of disease or disorder states characterized by widespread splicing alterations. To enable exon-resolution functional genomics, we have developed orthogonal ultra-efficient exon deletion and splice site mutation screening platforms and we have shown that can be used for the high-throughput interrogation phenotypically relevant exons in the human genome. Aim 1: Systematic identification of the human exons underlying cell fitness & proliferation. The first aim of our group has been to systematically profile the human exome to identify individual exons that can impact cell fitness and proliferation, a very important and quantitative cellular phenotype. To achieve this, we took advantage of the tools we have developed and generated guide (g)RNA libraries for exon deletion and splice site mutation screening to profile 15,000 frame-preserving exons from 2,500 genes in human HAP1 and RPE1 cells in total. Reassuringly, both screening strategies result in a significant overlap and positive correlation of exons that affect cell fitness. Our screens revealed that 20% of the interrogated exons affect cell fitness and these exons are more frequently found in highly expressed and essential genes. The cell fitness-affecting exons tend to be conserved in vertebrates and frequently overlap post-translation modification sites. Furthermore, we have shown that the fitness exons are enriched for overlapping modular protein domains and protein-protein interaction interfaces, while being devoid of low complexity regions. A manuscript describing these data is under revision. Aim 2: Development and application of exon-resolution genetic screening coupled to high-dimensional single-cell transcriptomic read-out. The transcriptome represents a very powerful and information-rich phenotype of cellular state and thus the coupling of CRISPR screens with single-cell RNA-Sequencing (scRNA-Seq) has been harnessed for examining high-dimensional phenotypic landscapes. To enable exon deletion screens with high-dimensional phenotypic profiling, we applied a series of optimization steps to have been able to combine exon deletion screens with single-cell RNA-Seq phenotypic readouts (an approach dubbed as scCHyMErA-Seq). We are currently performing a large-scale exon perturbation screen, targeting 500 exons for deletion, coupled to scRNA-Seq readouts. In these experiments, we built on our previously performed proliferation screens (see Aim 1), which identified 1,000 exons that affect cell fitness in human cells. We have generated CHyMErA libraries to excise a subset of these exons (with a focus on exons residing in gene expression regulators) and assess the gene expression changes in individual cells using scCHyMErA-Seq. This study represents the first large scale exon perturbation screening strategy with dimensional phenotypic read out and is fundamental novel insights of the exons in the human genome that affect gene expression signatures. Aim 3: In depth mechanistic characterization of alternative exons in transcription factors that control gene expression outputs. A major interest of our group is to functionally characterize alternative exons that can impact transcription, pre-mRNA processing or translation as demonstrated by our previous work. Both the cell fitness as well as the scCHyMErA screens have identified dozens of frame-preserving alternative exons in transcription factors. Focused follow-up studies of an alternative exon in the TFIID general transcription initiation factor component TAF5, uncovers that the inclusion levels of this single exon can control the overall assembly of the TFIID complex. Forced skipping of this alternatively spliced exon results in widespread gene expression alterations suggesting that this exon functions as a regulatory switch to tune TFIID formation and activity by controlling TATA-binding protein recruitment to transcription start sites. This study highlights the power of applying exon perturbation screens to interrogate phenotypically important exons at genome-scale, as a means for uncovering novel mechanisms that control gene expression and cell fitness. Preliminary analysis of our exon deletion single-cell RNA-Sequencing screens has revealed that among other exons, an alternative exon in NRF1, nuclear respiratory transcription factor 1, affects gene expression signatures. Based on these observations, we hypothesize that this exon is important, at least partially, for NRF1 function and currently performing focused studies to understand the role of this exon in regulating NRF1 activity and gene expression outputs.

选择性前mrna剪接极大地丰富了人类转录组，扩大了基因组的编码潜力。RNA测序分析研究表明，几乎所有的多外显子人类基因都受到选择性剪接。在不同的细胞和组织类型中，外显子和内含子的适时协调网络受到精心的调节控制，并在改变环境线索时产生不同的转录组学和蛋白质组学景观，从而支持不同的细胞命运或应激反应。据估计，超过10%的致病性突变影响选择性剪接位点的激活，而选择性剪接的失调与癌症和神经发育障碍等复杂疾病有关。替代剪接领域的主要挑战包括：1)替代外显子的功能表征，其中绝大多数仍未被表征；2)了解剪接程序如何在各种细胞类型或环境条件下重塑。我的研究小组正在结合功能基因组学和集中的分子分析来解决这些挑战，如下所述。项目1：表型关键可选外显子的高通量鉴定和表征保存框架的可选剪接外显子与编码序列重叠，缺乏过早终止密码子，这些外显子被翻译并有助于蛋白质多样性。这些剪接异构体的功能后果很难预测，它们在多大程度上代表了转录过程中剪接位点选择的随机差异，而几乎没有生物学影响，这一直是一个有争议的问题9,10。因此，剪接领域的一个主要挑战是确定与表型结果相关的生物学相关的替代外显子的全部曲目。这些知识将有助于重点研究备选剪接事件的优先级，也将提供以广泛剪接改变为特征的疾病或紊乱状态的预测性表型见解。为了实现外显子分辨率功能基因组学，我们开发了正交超高效外显子缺失和剪接位点突变筛选平台，我们已经证明可以用于高通量讯问人类基因组中表型相关的外显子。目的1：系统地鉴定人类外显子对细胞适应性和增殖的影响。我们小组的第一个目标是系统地分析人类外显子组，以识别可以影响细胞适应性和增殖的单个外显子，这是一种非常重要的定量细胞表型。为了实现这一目标，我们利用我们开发和生成的用于外显子缺失和剪接位点突变筛选的指导(g)RNA文库来分析人类HAP1和RPE1细胞中总计2,500个基因的15,000个保框外显子。令人欣慰的是，这两种筛选策略都会导致影响细胞适应性的外显子的显著重叠和正相关。我们的筛选显示，20%的被询问外显子影响细胞适应性，这些外显子更常出现在高表达和必需基因中。在脊椎动物中，影响细胞适应性的外显子往往是保守的，并且经常重叠翻译后修饰位点。此外，我们已经证明适应度外显子富集于重叠的模块化蛋白质结构域和蛋白质-蛋白质相互作用界面，而缺乏低复杂性区域。描述这些数据的手稿正在修订中。目的2：外显子分辨率基因筛选与高维单细胞转录组读出的发展和应用。转录组代表了一种非常强大且信息丰富的细胞状态表型，因此CRISPR筛选与单细胞rna测序（scRNA-Seq）的耦合已被用于检查高维表型景观。为了使外显子缺失筛选具有高维表型分析，我们应用了一系列优化步骤，以便能够将外显子缺失筛选与单细胞RNA-Seq表型读数相结合（一种称为scCHyMErA-Seq的方法）。我们目前正在进行大规模的外显子扰动筛选，针对500个外显子进行删除，并与scRNA-Seq读数相结合。在这些实验中，我们建立了之前进行的增殖筛选（见目的1），确定了1000个影响人类细胞适应性的外显子。我们已经生成了CHyMErA文库来切除这些外显子的一个子集（重点是居住在基因表达调控子中的外显子），并使用scCHyMErA-Seq评估单个细胞中的基因表达变化。这项研究代表了第一个具有维度表型读出的大规模外显子扰动筛选策略，是对人类基因组中影响基因表达特征的外显子的基本新见解。目标3：深入研究控制基因表达输出的转录因子中可选外显子的机制特征。我们小组的一个主要兴趣是功能表征可以影响转录，前mrna加工或翻译的其他外显子，正如我们之前的工作所证明的那样。细胞适应度和scCHyMErA筛选都在转录因子中发现了数十个保框外显子。对TFIID一般转录起始因子组分TAF5中另一个外显子的重点后续研究发现，该单个外显子的包含水平可以控制TFIID复合物的整体组装。这种选择性剪接外显子的被迫跳过导致广泛的基因表达改变，这表明该外显子作为调节开关，通过控制tata结合蛋白向转录起始位点的募集来调节TFIID的形成和活性。这项研究强调了应用外显子扰动筛选在基因组尺度上询问表型重要外显子的能力，作为揭示控制基因表达和细胞适应性的新机制的一种手段。我们的外显子缺失单细胞rna测序筛选的初步分析显示，在其他外显子中，NRF1中的另一个外显子，核呼吸转录因子1，影响基因表达特征。基于这些观察结果，我们假设该外显子对NRF1功能很重要，至少部分重要，目前正在进行重点研究，以了解该外显子在调节NRF1活性和基因表达输出中的作用。