Project Details
Projekt
Detection and functional characterization of queuosine and m5C modification in RNA
Subject Area
Cell Biology
General Genetics and Functional Genome Biology
General Genetics and Functional Genome Biology
Term
from 2015 to 2024
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 277246736
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.
DFG Programme
Priority Programmes
