Developing Tools to Map and Quantify Dihydrouridine in the Mammalian Transcriptome

项目概要/摘要 最近，几种典型的tRNA修饰，包括假尿苷（pseudouridine，pUDN）、N6-甲基腺苷（m6A）、N1-甲基腺苷（N1-methyladenosine，N6-methyladenosine，N1-methyladenos 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修饰的位置， 为研究它们在转录组中的功能铺平了道路。RNA修饰具有多个 在mRNA的生命周期中，它对mRNA的影响：剪接、运输、翻译和降解都可以改变。 二氢尿苷（D）是一种普遍存在于生命之树的每个分支中的tRNA中的修饰核苷酸。 哺乳动物中与mRNA相关的保守二氢尿苷脱氢酶（DUS）酶DUS 1L和DUS 3L 细胞，这表明它们修饰mRNA。二氢尿苷通过扭曲RNA的二级结构来影响RNA的二级结构。 嘧啶环，其可能深刻地影响mRNA代谢。我的初步数据显示 在酵母中，D被安装在mRNA中，我假设在哺乳动物细胞中也是如此。我有 开发了一种方法（D-seq），以单核苷酸分辨率分析D。该方法的工作原理是通过减少D， 硼氢化钠，导致逆转录酶（RT）在穿过还原D时从模板上脱落。到 为了将这种技术扩展到转录组规模，我将开发一种两步方案，直接将生物素偶联到 还原D（eD-seq）。这将允许富集含有D的RNA，并全面检查所有 在人类转录组中，D.为了量化D位置处修饰的化学计量，我还将 开发一种改良形式的D-Seq，其使用不同的RT，其在穿过D（D-MaP）时错误掺入。 seq）。为了证明这些方法的实用性，并确定疾病相关的D位点，我将全面 在两种类型的癌症的转录组中绘制和定量D， DUS.我将开发的新的D分析工具将确定DUS依赖的D在 转录组他们可能会揭示D是人类以前未知的成分， "epitranscriptome"。

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'.