Genomics-based approaches to understanding mechanistic alterations of spliceosome function in disease states

基于基因组学的方法来了解疾病状态下剪接体功能的机制改变

• 批准号: 10183903
• 负责人: Paul Lawrence Boutz
• 金额: $ 32.34万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10183903
• 关键词: 3' Splice Site; Affect; Alleles; Amino Acids; Auxins; Binding; Biological; Biology; Catalysis; Cell Line; Cells; Code; Copy Number Polymorphism; Data; Deletion Mutation; Dependence; Development; Disease; Epitopes; Etiology; Event; Exhibits; Exons; Functional disorder; Gene Amplification; Gene Expression; Gene Mutation; Genes; Genetic; Genetic Diseases; Genetic Screening; Genetic Transcription; Genomics; Human; Individual; Introns; Light; Machine Learning; Malignant Neoplasms; Mechanics; Mediating; Methods; Missense Mutation; Modeling; Motor; Mutate; Mutation; Normal Cell; Outcome; Pathway interactions; Pattern; Poly A; Positioning Attribute; Procedures; Protein Splicing; Proteins; RNA Binding; RNA Helicase; RNA Splicing; Role; Series; Site; Specificity; Spliced Genes; Spliceosomes; Structure; Syndrome; System; Techniques; Testing; Variant; base; causal variant; cell type; cofactor; deep learning; deep neural network; experimental study; gene expression variation; genome-wide; genomic data; helicase; human disease; insight; mutant; novel; novel therapeutics; patched protein; recombinase; recombinase-mediated cassette exchange; recruit; response; tool; transcriptome; transcriptome sequencing; tumor; virtual

Splicing factors are frequently altered by mutations and copy-number changes both in cancer and in germline genetic diseases resulting in multi-system developmental syndromes. Despite the fact that virtually all genes in humans undergo splicing, spliceosomal genetic alterations tend to exhibit surprisingly specific effects on subsets of splicing events, leaving most insignificantly changed. These effects can be allele-specific, cell-type specific, and dependent on the genetic background of the afflicted cell. This makes it especially challenging to determine which affected splicing events contribute to disease etiology. The fact that a limited set of introns is responsive to any specific splicing factor alteration indicates that introns and their flanking exons have evolved in structure and sequence to confer differential sensitivity to the action of different spliceosome components. This raises a fundamental question: what are the features common to sets of introns that confer this specificity? Using naturally occurring splicing gene mutations, amplifications, and deletions, these perturbations will be modelled in a genetically stable, untransformed, isogenic cell system where it is possible to isolate the effect of a single alteration on the transcriptome and on the binding patterns of the altered protein. These studies will shed light on the mechanisms of normal spliceosome function, and provide insight into which genes and biological pathways affected by splicing dysfunction likely contribute to disease states. The proposed experiments will employ three distinct methods to model spliceosome perturbations associated with human disease, with a focus on factors that physically or functionally interact with the essential spliceosome protein SF3B1. (Specific Aim 1) Introduction of an allelic series of cancer-associated SF3B1 missense-mutations into isogenic cell lines using recombinase-mediated cassette exchange (RMCE); (Specific Aim 2) CRISPRa/i-mediated activation or inhibition of transcription to up- or down- regulate splicing factors that are amplified in cancers (PUF60, SF3B4, and U2AF2) and lost in developmental syndromes (PUF60, SF3B4); and (Specific Aim 3) rapid depletion of spliceosomal RNA helicases (DDX39B, DDX46, and DHX16) and their putative co-factors (SUGP1, RBM17, and GPKOW) at the protein level using auxin-inducible degrons. Three distinct methods of RNA sequencing will be used to quantify the changes resulting from these perturbations: poly(A)-selected RNAseq, allele-specific eCLIP, and a novel intron lariat capture sequencing approach. Lastly, we will integrate these genomic data sets into models using deep learning neural networks to interrogate our central hypothesis: the sequence and structure of individual mammalian introns have evolved to confer differential dependence on specific ‘core’ components of the spliceosome, and that mutations, amplifications, and deletions in these core components causal for human disease will uncover intron-centric gene expression regulatory circuits that are controlled though modulation of the abundance or activity of the associated splicing factors in normal cells.

在癌症和癌症中，剪接因子经常因突变和拷贝数变化而改变 生殖系遗传病导致多系统发育综合征。尽管事实上所有的 人类的基因经历剪接，剪接体的基因改变往往会显示出令人惊讶的特定效果 在剪接事件的子集上，留下了最不显著的变化。这些效应可以是等位基因特定的、细胞类型的。 特定的，并依赖于患病细胞的遗传背景。这使得它特别具有挑战性 确定哪些受影响的剪接事件与疾病病因有关。一组有限的内含子是 对任何特定剪接因子的反应改变表明内含子及其侧翼外显子已经进化 在结构和序列上对不同剪接体组件的作用赋予差异敏感性。 这提出了一个基本问题：赋予这种特异性的内含子集合的共同特征是什么？ 利用自然发生的剪接基因突变、扩增和缺失，这些扰动将被 在遗传稳定、未转化的等基因细胞系统中建模，在该系统中可以分离出一种 转录组和改变的蛋白质的结合模式上的单一改变。这些研究将使 阐明了正常剪接体功能的机制，并提供了对哪些基因和生物 受剪接功能障碍影响的通路可能会导致疾病状态。 拟议的实验将使用三种不同的方法来模拟剪接体的扰动 与人类疾病相关的，重点是与基本的生理或功能相互作用的因素 剪接体蛋白SF3B1。(特定目标1)引入一系列与癌症相关的等位基因SF3B1 利用重组酶介导盒交换(RMCE)将错义突变转化为等基因细胞系；(特异性 目的2)CRISPRA/I介导的转录激活或抑制以上调或下调剪接因子 在癌症(PUF60，SF3B4和U2AF2)中扩增，在发育综合征(PUF60， SF3B4)；以及(特定目标3)剪接体RNA解旋酶(DDX39B、DDX46和DHX16)的快速耗尽 以及它们的假定辅助因子(SUGP1、RBM17和GPKOW)在蛋白质水平上使用生长素诱导的退化。 我们将使用三种不同的RNA测序方法来量化这些变化 扰动：Poly(A)选择的RNAseq、等位基因特异的eCLIP和新的内含子套索捕获测序 接近。最后，我们将使用深度学习神经网络将这些基因组数据集集成到模型中，以 质疑我们的中心假设：单个哺乳动物内含子的序列和结构已经进化到 使剪接体的特定核心成分具有不同的依赖性，而突变， 这些导致人类疾病的核心成分的扩增和缺失将揭示内含子中心 基因表达调控电路，通过调节基因的丰度或活性来控制 正常细胞中的相关剪接因子。