A queuosine-dependent tRNA modification loop in tRNA-Tyr in bacteria and yeast

• Applicant: Professorin Dr. Ann Elizabeth Ehrenhofer-Murray
• Subject Area: Biochemistry ; Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 550421281

Transfer RNAs (tRNAs) interpret the genetic code in mRNA sequences for the synthesis of polypeptides by the ribosome. They recognize the codons in the mRNA with the anticodon, and the respective amino acid that is loaded on the 3’ CCA tail of the tRNA is incorporated into the growing polypeptide chain. tRNAs are richly decorated with chemical modifications that regulate multiple aspects of their biology, including stability, structure and their function in decoding. In eukaryotes, each tRNA carries on average 13 modifications. Some modifications are chemically relatively simple, whereas others are chemically complex and require multiple enzymatic steps and complex biosynthetic pathways (e.g. N6-isopentenyl-adenosine, i6A, 2-methylthio-N6-isopentenyladenosine, ms2i6A, and queuosine, Q). A large number of modifications on the tRNAs are in close proximity to each other, begging the question whether there is interdependence among them. In this project, we will investigate a novel tRNA modification circuit that we have identified between two complex tRNA modifications, queuosine modification at the Wobble position 34 and 2-methylthio-N6-isopentenyladenosine (ms2i6A) modification at position 37 in the anticodon loop of tRNATyr. The mutual dependence of these modifications will be investigated in vivo in Escherichia coli and Schizosaccharomyces pombe. The effect of prior modification on the in vitro activity of the respective modification enzymes will be assessed. Furthermore, the functional implications of this cross-talk will be investigated in vivo by determining the global effects of the modifications on translation by ribosome profiling and assessing how they affect translational accuracy. Altogether, this work will provide important novel mechanistic insights into the cross-talk between tRNA modifications and will thus contribute to our understanding of how networks of tRNA modifications in the anticodon loop jointly regulate translation. Defects in tRNA modifications are implicated in human diseases, for instance in mitochondrial and neurological disorders and in cancer. This work will therefore increase to our understanding of the etiology of these diseases.

• DFG Programme: Research Grants