Mechanisms of post-transcriptional regulation of splicing factors

项目摘要 选择性RNA剪接能够产生不同的剪接mRNA同种型，其可以在功能上编码 不同的蛋白质剪接因子（SF）是RNA结合蛋白，其以剂量依赖性方式调节剪接。 在疾病中经常失调。富含丝氨酸/丝氨酸（SR）的蛋白质（SRSF 1至12，和SR-1） 类成员TRA 2 α，TRA 2 β）是一个重要的SF家族，与广泛的人类疾病有关。 病理学阐明SR蛋白是如何调节的对于促进我们对SR蛋白的理解至关重要。 这是控制真核生物中基因表达的基本过程，也是SF缺陷疾病的靶向过程。在这里， 我们将集中在转录后调节作为SR蛋白表达的重要调节剂， 为工具和疗法开发提供潜在可行的途径。SR蛋白基因包含超保守的 非编码外显子，称为毒外显子（PE），其控制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，PE如何调节基因 表达和维持更广泛的SF稳态，以及它们如何有助于基本的细胞功能仍然存在。 不太了解。我们假设PE在维持SF水平的严格调节中起关键作用， 这是正常细胞功能所必需的。目的1明确SR蛋白的调控机制， 使用剪接报告子小基因、靶向SF的CRISPR/Cas9文库和长读码通过PE进行交叉调节 RNA测序。通过鉴定SR蛋白和SF，它们通过PE相互连接并共同调节， 这些发现将提供SR蛋白调控网络的全面地图，并将揭示 转录后基因调控的新原理。目的2将明确SR蛋白PE在细胞内的功能作用， 使用CRISPR/Cas9在小鼠胚胎中体内缺失PE序列， 在体外人细胞分化模型中。这些发现将揭示细胞类型和细胞状态， PE的功能正常，以及PE的目标在体内和体外。目标3将制定方法， PE剪接和SR蛋白水平。这些方法将被用来推断SR蛋白的结合规则， 相关疾病模型中的PE功能。拟议的目标利用了我们实验室在开发工具和 研究疾病中剪接失调的模型。这些目标的完成将确定新的分子 PE调节SF稳态和细胞功能的机制，并提供了新的工具来操纵 SR蛋白水平。这里揭示的调节机制可能与许多SF具有广泛的相关性， 其中大多数含有PE。通过靶向PE操纵SF水平可能会导致治疗方法 用于SF缺陷的疾病，如神经系统疾病、心肌病、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.