Mechanisms of post-transcriptional regulation of splicing factors

剪接因子转录后调控机制

• 批准号: 10210414
• 负责人: OLGA ANCZUKOW-CAMARDA
• 金额: $ 42.53万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-06 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10210414
• 关键词: Affect; Alternative Splicing; Apoptosis; Arginine; Binding; Binding Proteins; Binding Sites; Biological Assay; CRISPR library; CRISPR screen; CRISPR/Cas technology; Cardiac; Cell Differentiation process; Cell Line; Cell model; Cell physiology; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Data; Defect; Development; Diabetes Mellitus; Differentiation Antigens; Disease; Disease model; Dose; Embryo; Embryonic Development; Engineering; Epithelial; Eukaryota; Exons; Family; Gene Expression; Gene Proteins; Generations; Genes; Homeostasis; Human; Human Pathology; In Vitro; Lead; Lupus; Malignant Neoplasms; Maps; Measurement; Measures; Messenger RNA; Modeling; Molecular; Molecular Target; Morphology; Mus; Myopathy; Neurons; Nonsense Codon; Normal Cell; Normal tissue morphology; Outcome; Phenotype; Play; Poison; Positioning Attribute; Post-Transcriptional Regulation; Process; Protein Isoforms; Protein Splicing; Proteins; RNA; RNA Recognition Motif; RNA Splicing; RNA-Binding Proteins; Regulation; Reporter; Role; Serine; Spliced Genes; Therapeutic; Untranslated RNA; cell type; genetic regulatory protein; human disease; in vivo; knock-down; mRNA Decay; member; nervous system disorder; novel; overexpression; pathway tools; protein expression; therapeutic development; tool; tool development; transcriptome sequencing; tumor

PROJECT SUMMARY Alternative RNA splicing enables generation of different spliced mRNA isoforms that can encode functionally distinct proteins. Splicing factors (SFs) are RNA-binding proteins that regulate splicing in a dose-dependent manner and are frequently dysregulated in diseases. Serine/arginine-rich (SR) proteins (SRSF1 to 12, and SR- like members TRA2α, TRA2β) are a family of essential SFs causatively implicated in a wide range of human pathologies. Elucidating how SR proteins are regulated is crucial to advance our understanding of the fundamental processes that control gene expression in eukaryotes and to target diseases with SF defects. Here, we will focus on post-transcriptional regulation as an important modulator of SR protein expression, and a potentially actionable pathway for tool and therapeutics development. SR protein genes contain ultra-conserved non-coding exons, called poison-exons (PEs), which control SR protein auto-regulation. Conservation of PE sequences across species suggests their importance in regulating SFs. However, how PEs regulate gene expression and maintain broader SF homeostasis, and how they contribute to fundamental cell functions remain poorly understood. We hypothesize that PEs play a critical role in maintaining a tight regulation of SF levels, which is necessary for normal cell functions. Aim 1 will define the mechanisms of SR protein regulation and cross-regulation via PEs using splicing reporter minigenes, a CRISPR/Cas9 library targeting SFs, and long-read RNA sequencing. By identifying the SR proteins and SFs that are interconnected and co-regulated through PE splicing, these findings will provide a comprehensive map of the SR protein regulatory network and will uncover novel principles of post-transcriptional gene regulation. Aim 2 will define the functional role of SR protein PEs in development and cell differentiation using CRISPR/Cas9 to delete PE sequences in vivo in mouse embryos and in vitro in human cell differentiation models. These findings will reveal cell types and cellular states that require PEs to function normally, as well as PE targets in vivo and in vitro. Aim 3 will develop approaches to modulate PE splicing and SR protein levels. These approaches will be used to infer SR protein binding rules, and to probe PE function in relevant disease models. The proposed aims leverage our lab’s expertise in developing tools and models to study splicing dysregulation in diseases. Completion of these aims will identify novel molecular mechanisms by which PEs regulate SF homeostasis and cellular functions, and provide new tools to manipulate SR protein levels. The regulatory mechanisms uncovered here are likely to have broad relevance to many SFs, the majority of which contain PEs. Manipulating SF levels by targeting PEs could lead to therapeutic approaches for diseases with SF defects, such as neurological disorders, cardiac myopathies, diabetes, lupus, or cancer.

项目概要 替代 RNA 剪接能够生成可进行功能编码的不同剪接 mRNA 亚型 不同的蛋白质。剪接因子 (SF) 是 RNA 结合蛋白，以剂量依赖性方式调节剪接 方式，并且在疾病中经常失调。富含丝氨酸/精氨酸 (SR) 蛋白（SRSF1 至 12 和 SR- 像成员 TRA2α、TRA2β 一样）是一个重要 SF 家族，与广泛的人类 病理学。阐明 SR 蛋白的调控机制对于加深我们对 SR 蛋白的理解至关重要 控制真核生物基因表达和针对 SF 缺陷疾病的基本过程。这里， 我们将重点关注转录后调控作为 SR 蛋白表达的重要调节剂，以及 工具和疗法开发的潜在可行途径。 SR蛋白基因含有超保守的 非编码外显子，称为毒外显子 (PE)，控制 SR 蛋白的自动调节。保护PE 跨物种的序列表明它们在调节 SF 中的重要性。然而，PEs如何调控基因 表达并维持更广泛的 SF 稳态，以及它们如何促进基本细胞功能仍然存在 不太了解。我们假设 PE 在维持 SF 水平的严格调节方面发挥着关键作用， 这是正常细胞功能所必需的。目标 1 将定义 SR 蛋白调节的机制和 使用剪接报告小基因、针对 SF 的 CRISPR/Cas9 文库和长读长通过 PE 进行交叉调节 RNA测序。通过识别通过 PE 互连和共同调节的 SR 蛋白和 SF 剪接，这些发现将提供 SR 蛋白调控网络的全面图谱，并将揭示 转录后基因调控的新原理。目标 2 将定义 SR 蛋白 PE 的功能作用 使用 CRISPR/Cas9 删除小鼠胚胎体内的 PE 序列进行发育和细胞分化 体外人类细胞分化模型。这些发现将揭示需要的细胞类型和细胞状态 PE 正常发挥作用，以及 PE 在体内和体外发挥作用。目标 3 将开发调节方法 PE 剪接和 SR 蛋白水平。这些方法将用于推断 SR 蛋白结合规则，并探测 PE在相关疾病模型中的功能。拟议的目标利用我们实验室在开发工具和 研究疾病中剪接失调的模型。完成这些目标将确定新的分子 PEs 调节 SF 稳态和细胞功能的机制，并提供新的操纵工具 SR 蛋白水平。这里揭示的监管机制可能与许多 SF 具有广泛的相关性， 其中大部分包含 PE。通过靶向 PE 来控制 SF 水平可能会带来治疗方法 用于具有 SF 缺陷的疾病，例如神经系统疾病、心肌病、糖尿病、狼疮或癌症。