Mechanisms of post-transcriptional regulation of splicing factors

剪接因子转录后调控机制

基本信息

批准号：
10600109
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
项目类别：
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/10600109
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 Epithelium Eukaryota Exons Family Gene Expression Gene Proteins Generations Genes Homeostasis Human Human Pathology In Vitro Invaded 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 Proliferating Protein Isoforms 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

项目摘要

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.

项目摘要替代RNA剪接可以生成可以在功能上编码的不同剪接的mRNA同工型不同的蛋白质。剪接因子（SFS）是RNA结合蛋白，可调节剂量依赖性剪接方式，经常在疾病中失调。丝氨酸/精氨酸（SR）蛋白（SRSF1至12），SR- 像成员tra2α一样病理。阐明如何调节SR蛋白对于促进我们对我们的理解至关重要控制真核生物中基因表达并靶向具有SF缺陷的疾病的基本过程。这里，我们将专注于转录后调节作为SR蛋白表达的重要调节剂，A SR蛋白基因含有超保存非编码外显子，称为毒药（PES），它控制SR蛋白自动调节。 PE的保护跨物种的序列表明它们在调节SFS方面的重要性。但是，PES如何调节基因表达并保持更广泛的SF稳态，以及它们如何对基本细胞功能做出贡献理解不佳。我们假设PES在维持SF水平的严格调节中起着至关重要的作用，这是正常细胞功能所必需的。 AIM 1将定义SR蛋白调节的机制和使用剪接记者小型素，针对SFS的CRIS/CAS9库和长阅读的跨PES进行交叉调节 RNA测序。通过识别通过PE相互联系并共同调节的SR蛋白和SF 拼接，这些发现将提供SR蛋白调节网络的综合图，并将发现转录后基因调控的新原理。 AIM 2将定义SR蛋白PES在使用CRISPR/CAS9在小鼠胚胎中删除体内的PE序列的开发和细胞分化人类细胞分化模型中的体外。这些发现将揭示需要的细胞类型和细胞状态 PE正常功能以及体内和体外的PE靶标。 AIM 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，并证明相关疾病模型中的PE功能。拟议的旨在利用我们实验室的专业知识来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 PES调节SF稳态和蜂窝功能的机制，并提供了操纵的新工具 SR蛋白水平。这里发现的监管机制可能与许多SFS具有广泛的相关性，其中大多数包含PE。通过靶向PE来操纵SF水平可能会导致治疗方法对于患有SF缺陷的疾病，例如神经系统疾病，心脏肌病，糖尿病，狼疮或癌症。