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Effect of pseudouridine modifications on tRNA structure

Effect of pseudouridine modifications on tRNA structure

Filip Sebest (ORCID: 0000-0002-1783-2212)
  • Grant DOI 10.55776/ESP1945924
  • Funding program ESPRIT
  • Status ongoing
  • Start October 1, 2024
  • End September 30, 2027
  • Funding amount € 340,819
  • E-mail

Disciplines

Construction Engineering (40%); Biology (15%); Chemistry (30%); Industrial Biotechnology (15%)

Keywords

    N1-methylpseudouridine, Pseudouridine, Relaxation dispersion, RNA, Molecular dynamics, Isotopic labelling

Abstract

Ribonucleic acids (RNAs) are molecules essential for all known forms of life and most biological functions. They are built from nucleosides adenosine, guanosine, cytidine, and uridine. However, all living organisms make changes to these nucleosides in a process known as RNA modification. The reason behind this is that RNA modifications can dramatically influence the stability, function, and activity of RNAs and have been linked with human diseases. Therefore, it is crucial to understand which modification has an effect on which biological function and which disease. Out of all types of RNAs, RNA modifications are most frequently present in transfer RNAs (tRNAs), which are responsible for protein production. In this ESPRIT project funded by the FWF, we will study the effects of two important uridine RNA modifications, namely pseudouridine and N1-methylpseudouridine, on tRNAs. While pseudouridine is the most abundant RNA modification, N1-methylpseudouridine is a non-natural modification for eukaryotic tRNAs but has been found in many archaeal tRNAs. These two modifications have recently gained a lot of media attention for being the key players in developing messenger RNA (mRNA) vaccines during the COVID-19 pandemic. N1-Methylpseudouridine in particular enabled high vaccine efficiency and decreased unwanted inflammatory responses. While clearly important in mRNAs, the primary goal of this project is to focus on tRNAs and find out what effects these two modifications have on their structural and functional features. Crucially, can we link this information to biological functions? So far, such studies have been lacking due to the insufficient means to analyse the complex nature of the tRNA system. Even if nuclear magnetic resonance (NMR) spectroscopy is the best method to study these effects, it requires the use of stable isotopes to simplify the NMR data. Stable isotopes are less abundant (and hence more expensive), non-radioactive variations of the same element with a different number of neutrons in their nucleus, which makes them useful for NMR applications. Here, we propose a novel, cost-effective approach in which we use relatively inexpensive reagents in a highly optimised and efficient chemo-enzymatic synthesis to first label target RNA modifications with stable isotopes of hydrogen, carbon, and nitrogen, and then incorporate them into tRNA sequences. Finally, we will study these tRNA sequences by NMR to find out what effects the target RNA modifications have on their structure and reactivity, with the aim to elucidate why nature goes to such lengths to incorporate them in tRNAs. The research will be carried out in the group of Assoc. Prof. Christoph Kreutz and will build on his expertise in the fields of isotopically-labelled synthesis of RNA modifications and biomolecular NMR.

Research institution(s)
  • Universität Innsbruck - 100%

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