Cancer-associated alterations of the dihydrouridine landscape in kidney cancer

肾癌中二氢尿苷景观的癌症相关改变

• 批准号: 9979467
• 负责人: Wendy Victoria Gilbert
• 金额: $ 22.71万
• 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: 2020-03-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9979467
• 关键词: Affect; Benchmarking; Binding; Biotin; Cancer Patient; Cell Line; Cells; Chemicals; Clear cell renal cell carcinoma; Complex; Computing Methodologies; Conventional (Clear Cell) Renal Cell Carcinoma; Data; Disease; Enzymes; Epithelial Cells; Genomics; Goals; Health; Human; Kidney; Knock-out; Knowledge; Laboratories; Link; Location; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of urinary bladder; Maps; Measures; Messenger RNA; Methods; Modification; Molecular Conformation; Mutation; Nucleosides; Nucleotides; Outcome; Output; Patient-Focused Outcomes; Patients; Poly(A)+ RNA; Probability; Proteins; Proximal Kidney Tubules; RNA; RNA Splicing; RNA metabolism; RNA-Directed DNA Polymerase; Regulation; Renal carcinoma; Resolution; Ribose; Site; Technology; Transcript; Transfer RNA; Translations; Untranslated RNA; Vertebral column; Woman; Work; Yeasts; base; cancer cell; epitranscriptome; genetic analysis; genome-wide; interest; mRNA Expression; men; novel; outcome forecast; overexpression; stoichiometry; success; transcriptome

PROJECT SUMMARY Significance: Numerous RNA modifying enzymes are critical for human health but the locations and stoichiometry of most RNA modifications are currently unknown. This is primarily due to the lack of high- throughput methods to detect the majority of modified nucleosides. Dihydrouridine (D) is an intriguing and understudied RNA modification that profoundly affects RNA backbone conformation, and, as a result, RNA metabolism. The RNA modifying enzymes that install dihydrouridine (D), called dihydrouridine synthases (DUS), are overexpressed and predictive of worse patient outcomes in lung, kidney and bladder cancers. The association of elevated DUS expression with poorer prognosis is consistent with a pathophysiological effect of increased dihydrouridine at some sites, which could include increased stoichiometry of D at known target sites or modification of new sites, or both. The specific RNA targets of DUS enzymes in cancer cells are unknown. Approach: Here, we propose to develop technology for comprehensive genomic analysis of dihydrouridine (D) (Aim 1) and use it to determine how the D landscape is altered in kidney cancer cells (Aim 2). Our approach exploits unique chemical features of dihydrouridine to derivatize D nucleotides, enrich for D containing RNA, and determine the locations of D with single-nucleotide resolution. Our preliminary data establish selectivity for D and the ability to generate precise modification-dependent blocks to reverse transcriptase, which we will analyze by Illumina sequencing. The probability of technical success is high: our laboratory is a technological pioneer in the discovery of RNA modification sites by developing experimental and computational methods to map the locations and quantify the relative abundance of novel mRNA modifications on a transcriptome-wide scale with single- nucleotide resolution. This proposal leverages these accomplishments to develop new methods to study the dihydrouridine landscape in human cells and reveal its dysregulation in cancer. The goal of this exploratory project is to discover the specific RNA targets of the DUS1L and DUS3L enzymes whose overexpression predicts poor prognosis in kidney cancer. Notably, DUS1L and DUS3L associate with polyadenylated RNA in cells and so the D landscape is likely to be complex and include sites in mRNA that are currently undiscovered. Aim 1 will develop high-throughput methods to define comprehensive targets of human DUS (1A) and medium-throughput methods to measure absolute dihydrouridine stoichiometry at hundreds of sites in parallel (1B). In Aim 2, we will use these methods to discover the targets of DUS1L and DUS3L and determine how the locations and stoichiometry of D modifications are altered in clear cell renal cell carcinoma cell lines that express high levels of DUS1L and DUS3L compared with matched DUS knockout cells (2A) and with non-transformed renal proximal tubule epithelial cells (2B). We anticipate that the methods developed here will be broadly useful for charting alterations in the epitranscriptome during initiation and progression of multiple malignancies where altered expression of dihydrouridine ‘writers’ is implicated in disease.

项目摘要 重要性：许多RNA修饰酶对人类健康至关重要，但它们的位置和功能对人类健康至关重要。 大多数RNA修饰的化学计量目前是未知的。这主要是因为缺乏高... 通量方法来检测大多数修饰的核苷。二氢尿苷（D）是一种有趣的， 深入研究RNA修饰，深刻影响RNA骨架构象，因此，RNA 新陈代谢.安装二氢尿苷（D）的RNA修饰酶，称为二氢尿苷脱氢酶（DUS）， 在肺癌、肾癌和膀胱癌中过度表达并预测更差的患者结果。的 升高的DUS表达与较差的预后相关，这与以下病理生理学效应一致： 某些位点的二氢尿苷增加，可能包括已知靶位点D的化学计量增加 或修改新站点，或两者兼有。癌细胞中DUS酶的特异性RNA靶点尚不清楚。 方法：在这里，我们建议开发二氢尿苷（D）的综合基因组分析技术。 (Aim 1）并使用它来确定D景观如何在肾癌细胞中改变（目的2）。我们的方法 利用二氢尿苷独特的化学特性衍生D核苷酸，富集含D的RNA， 以单核苷酸分辨率确定D的位置。我们的初步数据建立了D和 产生精确的依赖于修饰的逆转录酶阻断的能力，我们将通过以下方法进行分析： Illumina测序。技术成功的可能性很高：我们的实验室是技术领域的先驱。 通过开发实验和计算方法来定位RNA修饰位点， 并在转录组范围内用单克隆抗体定量新mRNA修饰的相对丰度。 核苷酸分辨率。该提案利用这些成就来开发新的方法来研究 二氢尿苷在人类细胞中的景观，并揭示其在癌症中的失调。这次探索的目标 该项目旨在发现DUS1L和DUS3L酶的特异性RNA靶点，其过表达预测 肾癌预后不良。值得注意的是，DUS1L和DUS3L与细胞中的多腺苷酸化RNA相关， 因此，D景观可能是复杂的，包括目前尚未发现的mRNA位点。目标1将 开发高通量方法，以确定人类DUS（1A）和中等通量的综合目标 在数百个位点平行测量绝对二氢尿苷化学计量的方法（1B）。在目标2中，我们将 使用这些方法来发现DUS1L和DUS3L的目标，并确定它们的位置和 D修饰的化学计量在表达高水平的透明细胞肾细胞癌细胞系中改变 DUS 1L和DUS 3L与匹配的DUS敲除细胞（2A）和非转化的肾近端细胞（2B）相比的差异 肾小管上皮细胞（2B）。我们预计，这里开发的方法将广泛用于制图 在多种恶性肿瘤的发生和发展过程中， 二氢尿苷“writers”的表达与疾病有关。