Mechanisms of post-transcriptional regulation of splicing factors

项目总结 替代的RNA剪接能够产生不同的剪接的mRNA亚型，这些剪接的mRNA亚型可以在功能上编码 不同的蛋白质。剪接因子(SFS)是一种rna结合蛋白，以剂量依赖的方式调节剪接。 在疾病中经常表现出失调的状态。富含丝氨酸/精氨酸(SR)蛋白(SRSF1至12和SR- 与成员TRA2α、TRA2β一样，TRA2 SFS家族在多种人类疾病中都有致病作用 病理学。阐明SR蛋白是如何调控的，这对于促进我们对SR蛋白的理解至关重要 在真核生物中控制基因表达和针对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器，以及 潜在的可操作的工具和疗法开发途径。SR蛋白基因含有超保守性 非编码外显子，称为毒外显子(PEs)，控制SR蛋白的自动调节。体育的守恒 跨物种的序列表明它们在调节SFS方面的重要性。然而，PE是如何调控基因的 表达和维持更广泛的SF动态平衡，以及它们对基本细胞功能的贡献仍然存在 人们对此知之甚少。我们假设PES在维持对SF水平的严格调控方面起着关键作用， 这是正常细胞功能所必需的。目标1将定义SR蛋白的调节机制和 通过使用剪接报告迷你基因的PE进行交叉调控，针对SFS的CRISPR/Cas9文库，以及长期阅读 RNA测序。通过鉴定通过PE相互连接和共同调节的SR蛋白和SFS 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PES在 利用CRISPR/Cas9在小鼠胚胎和小鼠体内删除PE序列的发育和细胞分化 在体外建立人类细胞分化模型。这些发现将揭示细胞类型和细胞状态，需要 PES功能正常，以及体内和体外的PE靶点。目标3将开发调制方法 PE剪接和SR蛋白水平。这些方法将被用来推断SR蛋白的结合规律，并用于探测 PE在相关疾病模型中的作用。建议的目标是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。完成这些目标将发现新的分子 PES调节SF动态平衡和细胞功能的机制，并提供了新的操纵工具 SR蛋白水平。这里揭示的监管机制很可能与许多SFS具有广泛的相关性， 其中大部分含有PE。通过靶向PE来操纵SF水平可能导致治疗方法 适用于有SF缺陷的疾病，如神经系统疾病、心脏肌病、糖尿病、狼疮或癌症。

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.