Developing Tools to Map and Quantify Dihydrouridine in the Mammalian Transcriptome

项目摘要/摘要 最近，一些经典的tRNA修饰，包括假尿苷(Ψ)，N6-甲基腺苷(M6A)，N1- 在信使RNA中发现了甲基腺苷(M1A)和5-甲基胞嘧啶(M5C)。的发展。 新的高通量测序方法揭示了单个mRNA修饰和 为通过转录组审问它们的功能铺平了道路。RNA修饰有多个 对其寿命的影响：剪接、运输、翻译和降解都是可以改变的。 二氢尿苷(D)是一种普遍存在的修饰核苷酸，存在于生命树每一分支的tRNA中。 哺乳动物保守的二氢尿苷合成酶(DUS)酶DUS1L和DUS3L，与mRNA相关 细胞，表明它们修改了信使核糖核酸。二氢尿苷通过扭曲RNA二级结构影响RNA二级结构 嘧啶环，这可能会深刻地影响mRNA的代谢。我的初步数据显示 D被安装在酵母的mRNA中，我推测在哺乳动物细胞中也是如此。我有过 开发了一种在单核苷酸分辨率下分析D的方法(D-SEQ)。该方法的工作原理是将D与 硼氢化钠，导致逆转录酶(RT)从模板上脱落，同时穿越还原的D。 将这项技术扩展到转录组规模，我将开发一个两步方案，将生物素直接偶联到 减少D(ED-SEQ)。这将允许对含有D的RNA进行浓缩，并对所有 人类转录组中D的RNA为了量化D位修饰的化学计量，我还将 开发一种修改形式的D-Seq，它使用不同的RT，在遍历D(D-MAP- SEQ)。为了展示这些方法的实用性，并确定与疾病相关的D部位，我将全面 定位和定量D在两种具有特征性过表达的癌症的转录组中 杜斯。我将开发的新D分析工具将识别依赖于DUS的D在整个 转录组。他们可能会揭示D是人类以前未知的组成部分 ‘墓志铭’。

Project Summary/Abstract Recently, several canonical tRNA modifications, including pseudouridine (Ψ ), N6-methyladenosine (m6A), N1- methyladenosine (m1A), and 5-methylcytosine (m5C) have been found in messenger RNA. The development of new high-throughput sequencing methods has revealed the location of individual mRNA modifications and paved the way for the interrogation of their function across the transcriptome. RNA modifications have multiple effects on mRNA during its lifespan: splicing, trafficking, translation and degradation can all be altered. Dihydrouridine (D) is a ubiquitous modified nucleotide found in tRNAs in every branch of the tree of life. Conserved dihydrouridine synthases (DUS) enzymes DUS1L and DUS3L, associate with mRNA in mammalian cells, suggesting that they modify mRNA. Dihydrouridine affects RNA secondary structure by distorting the pyrimidine ring, which is likely to profoundly influence mRNA metabolism. My preliminary data demonstrate that D is installed in mRNAs in yeast, and I hypothesize that this is also true in mammalian cells. I have developed a method (D-seq) to profile D at single-nucleotide resolution. The method works by reducing D with sodium borohydride, causing reverse transcriptase (RT) to fall off the template while traversing reduced D. To expand this technique to transcriptome scale, I will develop a two-step protocol to directly couple biotin to reduced D (eD-seq). This will permit enrichment of RNAs containing D, and comprehensive inspection of all RNA in the human transcriptome for D. To quantify the stoichiometry of modification at D positions, I will also develop a modified form of D-Seq that uses a different RT which misincorporates while traversing D (D-MaP- seq). To demonstrate the utility of these methods, and identify disease relevant D sites, I will comprehensively map and quantify D in the transcriptome of two types of cancer that have characteristic overexpression of DUS. The new D profiling tools that I will develop will identify the locations of DUS-dependent Ds across the transcriptome. They are likely to reveal that D is a previously unknown component of the human `epitranscriptome'.