Detection and functional characterization of queuosine and m5C modification in RNA

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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.