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: 10360658
• 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/10360658
• 关键词: 3' Splice Site; Affect; Alleles; Amino Acids; Auxins; Binding; Biological; Biology; CRISPR-mediated transcriptional activation; 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.

剪接因子经常因突变而改变，并且在癌症和中的拷贝数变化都会改变 生殖线遗传疾病导致多系统发育综合征。尽管实际上是 人类中的基因经历剪接，剪接体遗传改变往往存在令人惊讶的特定效应 在剪接事件的子集上，使最不重要的变化。这些效果可以是等位基因特异性的细胞类型 具体，并取决于受苦细胞的遗传背景。这使得尤其挑战 确定哪些受影响的剪接事件有助于疾病病因。有限的内含子的事实是 对任何特定的剪接因子的响应敏感，表明介绍及其侧面外显子已经发展 在会议对不同剪接体成分作用的不同敏感性的结构和顺序中。 这提出了一个基本问题：会议这种特殊性的内含子集合的功能是什么？ 使用天然存在的剪接基因突变，扩增和缺失，这些扰动将是 以一般稳定的，未转换的等源性细胞系统建模，可以在其中隔离 转录组和改变蛋白质的结合模式的单一改变。这些研究将丢弃 对正常剪接体功能的机制进行了启示，并提供了有关哪些基因和生物学的洞察力 受剪接功能障碍影响的途径可能会导致疾病状态。 提出的实验将采用三种不同的方法来建模剪接扰动 与人类疾病相关，重点是与本质上的物理或功能相互作用的因素 剪接体蛋白SF3B1。 （特定目的1）引入等位基因系列癌症相关的SF3B1 使用重组酶介导的盒式盒式交换（RMCE），错义对等生细胞系进行了误解； （具体的 目标2）CRISPRA/i介导的激活或抑制转录对上调节或下调的剪接因子 在癌症（PUF60，SF3B4和U2AF2）中被放大并丢失发育综合征（PUF60，， SF3B4）; （特定目的3）快速部署剪接体RNA解旋酶（DDX39B，DDX46和DHX16） 及其推定的共同因素（SUGP1，RBM17和GPKOW）在蛋白质水平上使用生长素诱导的脱脂剂。 将使用三种不同的RNA测序方法来量化这些测序的变化 扰动：poly（a）选择的RNASEQ，等位基因特异性eclip和新型内含子套索捕获测序 方法。最后，我们将使用深度学习神经网络将这些基因组数据集整合到模型中 询问我们的中心假设：单个哺乳动物内含子的序列和结构已演变为 会议差异依赖剪接体的特定“核心”成分，以及突变， 这些核心成分的扩增和删除人类疾病因果因果 基因表达调节回路，这些回路是通过对抽象或活性的调节而受到控制的 正常细胞中的相关剪接因子。