Role of pseudouridines in pre-mRNA processing

假尿苷在前 mRNA 加工中的作用

• 批准号: 10055153
• 负责人: Nicole Martinez
• 金额: $ 9.68万
• 依托单位: YALE 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 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10055153
• 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; Protein Splicing; Proteins; Pseudouridine; RNA; RNA I; RNA Splicing; RNA-Protein Interaction; Regulation; Research; Role; Site; Structure; Techniques; Training; Translations; United States National Institutes of Health; Universities; Work; base; cell type; chromatin immunoprecipitation; epitranscriptome; experimental study; genetic manipulation; hepatocellular carcinoma cell line; human disease; human tissue; in vivo; liver function; mRNA Precursor; novel; novel diagnostics; outcome forecast; 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.

项目摘要 最新的全转录组方法检测改良的核苷酸的方法表明 扩大了信使RNA中修饰的核苷酸的曲目。 RNA修饰代表了一个新层 真核基因调节。伪苷（ψ）是一种修饰的核苷酸，在人类mRNA和 可以在不同条件下动态调节。但是，mRNA中ψ的内源性不是 目前了解。我们已经表明，新生的前mRNA在 数千个位置由多种假氨酸合酶（Puss）。我们发现前mRNAψ是 富含对剪接调节和重叠剪接因子结合位点重要的区域。遗传 操纵ψ合酶会导致宽度替代剪接，单个ψ直接影响剪接 体外。猫与包括癌症在内的多种疾病有关。前MRNAψ的高表达 合成酶与肝细胞癌细胞（HCC）中的生存率提高和基因的重新布线相关 通过替代剪接表达对于HCC病理很重要。 NIH K99/R00独立途径 奖项旨在定义HCC细胞中的PU依赖性替代剪接网络（AIM 1），确定分子 ψs调节前mRNA剪接的机制（AIM 2）并研究了ψ为 共同转录保存在前mRNA中（AIM 3）。在该奖项的修订阶段，马丁内斯博士将 通过基于测序的检测和 替代剪接分析。 剪接 马丁内斯博士将确定ψ对候选ψ敏感的结合的影响 通过生化方法，其结合位点富含重叠位置的因素。 在独立阶段，Martinez博士将定义HCC特定的MRNAψ景观并识别 通过分析ψ以及相应的剪接变化 主要正常和HCC细胞。她阐明了HCC细胞中ψ介导的剪接调节机制 将发现剪接调节的潜在疾病相关模式。期间 这 K99 和 R00 部分 的 这 奖 博士 马丁内斯 将要 决定 如何通过研究将ψ合成酶募集到新生的前mRNA 通过染色质和共同培养的机理和与POL II相关的机制 与转录有关 使用新型测序方法的动力学。疾病连接ψ合酶的病理可能是由于 其前MRNA靶标的失调。因此，定义和表征脓液依赖性的效果 前MRNA加工中的mRNAψ将有助于解释这些酶如何对疾病有效。这 获得独立奖的途径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 耦合）和专业技能（例如心理和管理）成为竞争性的候选人 一所大型研究学院的学术职位。