Detection and functional characterization of queuosine and m5C modification in RNA

TRNA受到各种化学修饰的影响，这些修饰调节了它们在翻译中的功能。在这个项目中，我们研究了两个这样的修饰，Queuosine(Q)和Cytosine-5甲基化(M5C)。我们早期在裂殖酵母中的工作表明，Q强烈刺激tRNA-Asp中依赖于DNMT2的c38甲基化，Q取代了所选tRNAs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，从而在营养供应和宿主体内的翻译之间提供了联系。在过去的SPP1784资助期间，我们利用核糖体图谱研究了Q和DNMT2对整体翻译的影响，发现了Q密码子在S.pombe和人类细胞中翻译速度的影响。此外，S.pombe中的Q修饰抑制了对选定的近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，我们还建立了全转录组亚硫酸氢盐测序的计算工具，并确定了pombe中完整的M5C模式及其对Q、DNMT2和两个S.pombe Trm4/NSUN2同源物的依赖。这表明DNMT2对tRNA-Asp具有显着的选择性，并且在tRNA中产生其他M5C位点时，两个Trm4同系物之间存在分工。我们的工作进一步表明，tRNA先前Q修饰对DNMT2的刺激在进化过程中是保守的，从而揭示了高等真核生物翻译的营养控制途径。这些洞察力是通过Ann Ehrenhofer-Murray的实验室与Frank Lyko实验室的生物信息学工具开发工作的密切合作获得的。在这项后续计划中，我们将开发新的方法来检测RNA中的Q和M5C。将测试纳米孔和单分子实时(SMRT)和测序等第三代测序技术是否适用于这一目的。此外，还将为实现这一目标开发反转录和深度测序以及修饰特异的RNA内切酶。一旦成功，这种方法将被用于寻找携带Q或M5C的tRNA以外的RNA。这一点很重要，因为M5C在mRNA中的存在是有争议的，而Q被认为存在于人类病原体的mRNA中。总之，该项目将为检测这些修饰提供新的工具，并将进一步加深我们对真核生物表观转录组学的理解。

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.