Role of pseudouridines in pre-mRNA processing

项目摘要 最近发展的全转录组方法来检测修饰的核苷酸已经揭示了一种新的方法， 扩大信使RNA中修饰核苷酸的库。RNA修饰代表了一个新的 真核基因调控假尿苷（Pseudouridine，简称DNT）是一种在人类mRNA中普遍存在的修饰核苷酸， 可在不同条件下动态调节。然而，内源性的功能， 目前了解。我们已经证明，新生的前mRNA是假尿苷化共转录， 通过多个假尿苷合酶（PUS）的数千个位置。我们发现前mRNA Ψ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 操纵剪接酶导致广泛的选择性剪接，单个剪接酶直接影响剪接， 体外PUSs与包括癌症在内的多种疾病有关。前mRNA Ψ的高表达 肝细胞癌（HCC）细胞中的凋亡酶与生存率降低和基因重排相关 通过选择性剪接的表达对于HCC病理学是重要的。NIH K99/R 00独立之路 Award旨在定义HCC细胞中的PUS依赖性可变剪接网络（Aim 1）， 调节前体mRNA剪接的机制（Aim 2），并研究其调节前体mRNA剪接的机制。 共转录沉积在前体mRNA中（Aim 3）。在该奖项的指导阶段，马丁内斯博士将 通过基于测序方法鉴定HepG 2细胞中的PUS-dependent可变剪接， 可变剪接分析。 剪接 博士Martinez将确定BMPs对候选BMPs敏感的结合的影响。 因子，其结合位点富集重叠的重叠位置，使用生物化学方法。 在独立阶段，Martinez博士将定义HCC特异性前mRNA表达谱，并确定 PUS依赖性HCC特异性选择性剪接事件的分析，以及相应的剪接变化， 原代正常和HCC细胞。她阐明了HCC细胞中β介导的剪接调控机制 将揭示潜在的疾病相关的剪接调节模式。期间 的 K99 和 R00 部分 的 的 奖 博士 马丁内斯 将 确定 通过研究β-内酰胺酶如何被募集到新生的前mRNA， 通过染色质和共染色质的结合， 免疫沉淀实验和测量与转录相关的假尿苷化程度 动态使用一种新的测序方法。疾病相关性乳腺癌的病理学可能是由于 它们的前mRNA靶点的失调。因此，定义和表征PUS-dependent 前mRNA加工中的前mRNA酶将有助于解释这些酶如何导致疾病。的 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修饰的发展和计算方法，研究转录的技术， 耦合）和专业技能（如指导和管理），成为一个有竞争力的候选人， 一所主要研究型大学的学术职位。

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.