Cancer-associated alterations of the dihydrouridine landscape in kidney cancer

项目总结 意义：大量的RNA修饰酶对人类健康至关重要，但位置和 大多数RNA修饰的化学计量学目前尚不清楚。这主要是由于缺乏高- 检测大多数修饰核苷的通过量方法。二氢尿苷(D)是一种耐人寻味的 未被充分研究的RNA修饰深刻影响了RNA骨架构象，从而影响了RNA 新陈代谢。安装二氢尿苷(D)的RNA修饰酶称为二氢尿苷合成酶(DUS)， 在肺癌、肾癌和膀胱癌中过度表达，预示着患者预后更差。这个 DUS表达升高与预后较差的关联与 一些部位的二氢尿苷增加，这可能包括在已知目标部位D的化学计量比增加 或修改新网站，或两者兼而有之。癌细胞中DUS酶的特定RNA靶标尚不清楚。 方法：在这里，我们建议开发二氢尿苷(D)的全面基因组分析技术。 (目标1)，并用它来确定肾癌细胞的D景观是如何改变的(目标2)。我们的方法 利用二氢尿苷的独特化学特征来衍生D核苷酸，富含D的RNA，以及 用单核苷酸分辨确定D的位置。我们的初步数据确定了D和 生成精确的依赖于修饰的块来逆转录酶的能力，我们将通过以下方式进行分析 Illumina测序。技术成功的可能性很高：我们的实验室是 通过开发实验和计算方法来绘制位置图来发现RNA修饰位点 并在转录组范围内用单个-RNA修饰来量化新的mRNA修饰的相对丰度 核苷酸解析。这项建议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分布，并揭示其在癌症中的失调。这一探索性的目标是 该项目是发现DUS1L和DUS3L酶的特定RNA靶标，其过度表达预测 肾癌预后不良。值得注意是，DUS1L和DUS3L与细胞和 因此，D基因的格局可能是复杂的，包括目前未被发现的mRNA中的位置。目标1将 开发高通量方法，以确定人类DUS(1A)和中等吞吐量的综合目标 方法平行测量数百个点的绝对二氢尿苷化学计量比(1B)。在目标2中，我们将 使用这些方法发现DUS1L和DUS3L的目标，并确定位置和 在表达高水平的透明细胞肾癌细胞系中，D修饰的化学计量学改变 DUS1L和DUS3L与匹配的DUS基因敲除细胞(2A)和未转化的肾脏近端的比较 小管上皮细胞(2B)。我们预计，这里开发的方法将广泛用于绘制图表 多种恶性肿瘤发生和发展过程中表位转录组的改变 二氢尿苷“编写者”的表达与疾病有关。

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.