Detection and functional characterization of queuosine and m5C modification in RNA

tRNA经过多种化学修饰，调节其翻译功能。在这个项目中，我们研究了两个这样的修饰，胞嘧啶（Q）和胞嘧啶-5甲基化（m5 C）。我们早期在粟酒裂殖酵母中的工作表明，TRNA-Asp中C38的Dnmt 2依赖性甲基化受到Q的强烈刺激，Q取代了所选tRNA反密码子中摆动位置（Q34）的鸟苷。有趣的是，真核生物中的Q最初来自营养中的细菌来源和肠道微生物区系，从而提供了宿主中营养供应和翻译之间的联系。在过去的SPP 1784授权期内，我们利用核糖体分析研究了Q和Dnmt 2对全局翻译的影响，发现Q密码子在S中的翻译速度受到影响。粟酒和人类细胞。此外，S. pombe抑制了所选近同源密码子的错误阅读，揭示了Q修饰在翻译准确性中的作用。此外，我们建立了全转录组亚硫酸氢盐测序的计算工具，并确定了S. pombe及其对Q、Dnmt 2和两个S. pombe Trm 4/NSun 2同源物。这表明Dnmt 2对tRNA-Asp具有显著的选择性，并且在tRNA中产生其他m5 C位点的两个Trm 4同源物之间存在分工。我们的工作进一步表明，通过tRNA的先前Q修饰对Dnmt 2的刺激在进化过程中是保守的，从而揭示了高等真核生物中营养控制翻译的途径。这些见解是通过Ann Escherhofer-Murray的实验室之间的密切合作获得的，用于“湿”工作和Frank Lyko实验室的生物信息学工具的开发。在这个后续计划中，我们将开发新的方法来检测RNA中的Q和m5 C。第三代测序技术，如纳米孔和单分子实时（SMRT）和测序将被测试其适用性的目的。此外，逆转录和深度测序以及修饰特异性RNA核酸内切酶将朝着这一目标发展。一旦成功，这些方法将被用于寻找携带Q或m5 C的tRNA以外的RNA。这是重要的，因为mRNA中m5 C的存在是有争议的，并且Q已经被认为存在于人类病原体的mRNA中。总而言之，这个项目将产生新的工具，用于检测这些修改，并将进一步我们的真核生物中的epitranscriptome的理解。

tRNAs are subject to a variety of chemical modifications that modulate their function in translation. In this project, we have investigated two such modifications, queuosine (Q) and cytosine-5 methylation (m5C). Our earlier work in Schizosaccharomyces pombe showed that Dnmt2-dependent methylation of C38 in tRNA-Asp is strongly stimulated by Q, which replaces guanosine at the wobble position (Q34) in the anticodon of selected tRNAs. Interestingly, Q in eukaryotes originally comes from bacterial sources in nutrition and from the gut microflora, thus providing a connection between nutritional supply and translation in the host. In the past grant period of SPP1784, we have investigated the effect of Q and Dnmt2 on global translation using ribosome profiling and found effects on translational speed of Q codons in S. pombe and human cells. Furthermore, Q modification in S. pombe suppressed erroneous reading of selected near-cognate codons, revealing a role for Q modification in translational accuracy. Also, we established computational tools for whole-transcriptome bisulfite sequencing and determined the full m5C pattern in S. pombe and its dependence on Q, Dnmt2 and the two S. pombe Trm4/ NSun2 homologs. This showed a remarkable selectivity of Dnmt2 for tRNA-Asp and a division of labour between the two Trm4 homologs in generating other m5C sites in tRNA. Our work furthermore shows that the stimulation of Dnmt2 by prior Q modification of the tRNA is conserved across evolution, thus revealing a pathway of nutritional control of translation in higher eukaryotes. These insights were obtained through a close collaboration between the labs of Ann Ehrenhofer-Murray for the “wet” work and the development of bioinformatics tools in the lab of Frank Lyko. In this follow-up proposal, we will develop novel methods to detect Q and m5C in RNA. Third-generation sequencing technologies such as nanopore and single-molecule real-time (SMRT) and sequencing will be tested for their suitability for the purpose. Also, reverse transcription and deep sequencing as well as a modification-specific RNA endonuclease will be developed towards this goal. Once successful, such methods will be employed in order to seek for RNAs other than tRNAs that carry Q or m5C. This is important, because the existence of m5C in mRNA is controversial, and Q has been suggested to be present in mRNA of a human pathogen. Altogether, this project will yield novel tools for the detection of these modifications and will further our understanding of the epitranscriptome in eukaryotes.