Role of pseudouridines in pre-mRNA processing

假尿苷在前 mRNA 加工中的作用

• 批准号: 10558697
• 负责人: Nicole Martinez
• 金额: $ 24.9万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10558697
• 关键词: 3' Splice Site; Affect; Alternative Splicing; Area; Award; Binding; Binding Sites; Biochemical; Biological Assay; Cancer Biology; Cells; Cellular Stress; Chromatin; Co-Immunoprecipitations; Coupling; DNA Polymerase II; Deposition; Detection; Development; Disease; Enhancers; Enzymes; Event; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; HepG2; Hepatocyte; Human; In Vitro; Individual; Introns; Learning; Link; Location; Malignant Epithelial Cell; Malignant Neoplasms; Measures; Mediating; Mentors; Messenger RNA; Methods; Modification; Molecular; Nuclear; Nucleic Acid Regulatory Sequences; Nucleotides; Pathogenesis; Pathology; Pathway interactions; Phase; Play; Positioning Attribute; Postdoctoral Fellow; Primary carcinoma of the liver cells; Prognosis; Proliferating; Protein Splicing; Proteins; Pseudouridine; RNA; RNA Splicing; RNA-Protein Interaction; Regulation; Research; Role; Site; Techniques; Training; Translations; United States National Institutes of Health; Universities; Work; cell type; epitranscriptome; experimental study; genetic manipulation; hepatocellular carcinoma cell line; human disease; human tissue; in vivo; liver function; mRNA Precursor; novel; novel diagnostics; prognostic; promoter; recruit; response; skills; spatiotemporal; technology development; therapeutic target; transcriptome; tumor growth

PROJECT SUMMARY Recent development of transcriptome-wide approaches to detect modified nucleotides has revealed an expanding repertoire of modified nucleotides in messenger RNA. RNA modifications represent a new layer of eukaryotic gene regulation. Pseudouridine (Ψ) is a modified nucleotide that is prevalent in human mRNAs and can be dynamically regulated in different conditions. However, the endogenous functions of Ψ in mRNA are not currently understood. We have shown that nascent pre-mRNA is pseudouridylated co-transcriptionally at thousands of locations by multiple Pseudouridine Synthases (PUSs). We found that pre-mRNA Ψs are enriched in regions important for splicing regulation and overlap splicing factor binding sites. Genetic manipulation of Ψ synthases leads to widespread alternative splicing and individual Ψ directly affect splicing in vitro. PUSs have been linked to a wide range of diseases, including cancer. High expression of pre-mRNA Ψ synthases correlates with decreased survival in hepatocellular carcinoma cells (HCC) and rewiring of gene expression by alternative splicing is important for HCC pathology. NIH K99/R00 Pathway to Independence Award seeks to define PUS-dependent alternative splicing networks in HCC cells (Aim 1), identify molecular mechanisms by which Ψs regulates pre-mRNA splicing (Aim 2) and investigate mechanisms by which Ψ is deposited in pre-mRNA co-transcriptionally (Aim 3). During the mentored phase of this award Dr. Martinez will identify PUS-dependent alternative splicing in HepG2 cells by sequencing based methods for Ψ detection and alternative splicing analysis. splicing Dr. Martinez will determine the effect of Ψ on the binding of candidate Ψ-sensitive factors, whose binding sites are enriched for overlapping Ψ locations, using biochemical approaches. During the independent phase, Dr. Martinez will define the HCC specific pre-mRNA Ψ landscape and identify PUS-dependent HCC specific alternative splicing events by profiling Ψ, and corresponding splicing changes in primary normal and HCC cells. Her elucidation of mechanisms of Ψ-mediated splicing regulation in HCC cells will uncover potential disease related modes of splicing regulation. During the K99 and R00 portion of the award Dr. Martinez will determine how Ψ synthases are recruited to nascent pre-mRNA by investigating mechanisms of recruitment to chromatin and association with Pol II by a combination of chromatin- and co- immunoprecipitation experiments and measuring extent of pseudouridylation in relation to transcription dynamics using a novel sequencing approach. The pathology of disease-linked Ψ synthases may be due to dysregulation of their pre-mRNA targets. Therefore, defining and characterizing the effect of PUS-dependent pre-mRNA Ψs in pre-mRNA processing will help explain how these enzymes contribute to disease. The Pathway to Independence Award will allow Dr. Martinez to gain the necessary expertise (e.g. technology development and computational approaches to discover RNA modifications, techniques to study transcriptional coupling) and professional skills (e.g. mentoring and management) to become a competitive candidate for an academic position at a major research university.

项目总结 最近转录组范围内检测修饰核苷酸的方法的发展揭示了 扩充信使核糖核酸中修饰核苷酸的资料库。RNA修饰代表了新的一层 真核基因调控。假尿苷(Ψ)是一种修饰的核苷酸，广泛存在于人类mRNAs和 可在不同条件下动态调节。然而，Ψ在基因中的内源性功能不是 目前已被理解。我们已经证明，新生的Pre-mrna在转录水平上是假尿路连接的。 由多个假尿苷合成酶(PUSS)决定的数千个位置。我们发现前信使核糖核酸ΨS是 富含对剪接调控和重叠剪接因子结合位点重要的区域。遗传 Ψ合酶的操纵导致广泛的选择性剪接，单个Ψ直接影响剪接。 体外培养。猫被认为与多种疾病有关，包括癌症。前信使核糖核酸Ψ高表达 合酶与肝细胞癌存活率降低和基因重排有关 选择性剪接表达对肝细胞癌的病理意义重大。美国国立卫生研究院K99/R00独立之路 AWARD致力于定义肝癌细胞中PUS依赖的选择性剪接网络(目标1)，识别分子 ΨS调控前信使核糖核酸剪接的机制(目标2)和研究Ψ的作用机制 共转录沉积在前信使核糖核酸中(目标3)。在这个奖项的指导阶段，马丁内斯博士将 用基于测序的Ψ检测方法鉴定肝癌细胞中PUS依赖的选择性剪接 选择性剪接分析。 拼接 马丁内斯博士将确定Ψ对候选Ψ敏感分子结合的影响 因子，其结合位点因重叠的Ψ位置而丰富，使用生化方法。 在独立阶段，马丁内斯博士将确定肝细胞癌特定的前信使核糖核酸Ψ图谱，并确定 PUS依赖的肝癌特异性选择性剪接事件通过分析Ψ，以及相应的剪接变化 原代正常细胞和肝癌细胞。她对Ψ介导的肝癌细胞剪接调控机制的阐明 将揭示潜在的疾病相关剪接调控模式。在.期间 这个 K99 和 R00 部分 的 这个 奖项 Dr。 马丁内斯 将要 确定 通过研究Ψ合酶是如何被招募到新生的前信使核糖核酸的 染色质-和辅染色质-的结合对染色质募集和与POL II结合的机制 免疫沉淀实验和假尿苷酸化程度与转录的关系 使用一种新的测序方法进行动力学分析。疾病相关Ψ合成酶的病理可能是由于 他们的前信使核糖核酸靶向失调。因此，定义和表征PUS依赖的作用 前信使核糖核酸ΨS在前信使核糖核酸的处理中将有助于解释这些酶是如何导致疾病的。这个 独立之路奖将使马丁内斯博士获得必要的专业知识(例如技术 发现RNA修饰的开发和计算方法，研究转录的技术 )和专业技能(例如指导和管理)，以成为具有竞争力的候选人 在一所主要研究型大学的学术职位。