Roles of tRNA modification cross-talk for mRNA decoding and translational fidelity

• Applicant: Professor Dr. Raffael Schaffrath, since 11/2020
• Subject Area: Biochemistry ; General Genetics and Functional Genome Biology ; Biological and Biomimetic Chemistry ; Cell Biology
• Term: from 2015 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 277253872

This research proposal takes full advantage of the momentum gained during our grant and is a logical spin-off from our expertise on ‘tRNA modification cross-talk’. It proposes to disclose consequences of cross-talk among multiple tRNA modifications in the anticodon stem and variable loops (ASL, VL) onto decoding and translational fidelity. Using Saccharomyces cerevisiae as a eukaryal model system, our chosen strategy aims to reveal how modifications in the ASL (mcm5s2U34, ct6A37, Ψ38) and VL (m5C48) cooperate to safeguard anticodon-codon interaction during decoding by tRNALysUUU and to protect from error-prone mRNA translation and proteotoxicity. To do so, we will achieve the following goals:- profile the modification landscape of tRNALysUUU with regards to its importance for translational fidelity using assays diagnostic for ribosomal +1 frameshifts in vivo- examine de-/aminoacylation and stability states of tRNAs in response to defective modifications in the ASL and/or VL- study how modification cross-talk in tRNALysUUU affects initial mRNA decoding steps/kinetics using a reconstituted ribosome system for translation in vitro- examine the role of tRNA modifications in changing the efficiency of decoding by near cognate tRNAs- analyze the potential implications of mistranslation for protein homeostasis in aggregation prone tRNA modification mutantsCollectively, we will study at atomic, molecular and cellular levels how chemical disturbances in the modification states of the ASL and the VL put tRNA decoding and translational fidelity at risk. Here, major aims are to identify whether faulty modifications cause error-prone translation and to define which non-Watson Crick base pairs are erroneously accepted for decoding in the absence of tRNA modifications. Another aim closely linked to tRNA function and performance will be to define the impact of ASL and VL modification cross-talk on tRNA stability, structural integrity, aminoacylation status and efficiency in key decoding steps in vitro. As for the latter, analysis of tRNA charging and in vitro kinetics of decoding steps in composite modification mutants will be crucial to understand at a mechanistic level, how modifications cooperate to maintain decoding fidelity. Directly linked with ribosomal inaccuracy and translational infidelity is our final goal which aims to functionally correlate erroneous decoding and mistranslation with protein aggregation and proteotoxicity. Clearly, these objectives, which lie at the heart of our yeast based project, also call for mechanistic links between tRNA decoding, translational fidelity, and cell proliferation in higher eukaryotes (including our own cells). As a result, inappropriate tRNA modification, mRNA translation and proteostasis have been shown to drive formation of tumours and neurodegenerative diseases in humans. So we anticipate our SPP1784/2 proposal has biomedical significance in the long-term.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1784: Chemical Biology of native Nucleic Acid Modifications
• Ehemaliger Antragsteller: Dr. Roland Klassen, until 10/2020