PHD Student no. 43/2022/ICHB/PSD

KEY WORDS: NMR spectroscopy, nucleic acids, modified RNA units

Research topic: “”

Principal Investigator: dr Witold Andrałojć

I. Project description

Modified nucleosides present in mRNA and tRNA are important elements of the cellular machinery responsible for effective and accurate protein biosynthesis. The major players of this machinery are modified nucleosides located at the coding region of mRNAs and the anticodon stem-loop domain (ASL) of cytosolic and mitochondrial (mt) tRNAs, directly attending the codon-anticodon interactions on the ribosome. Since mitochondria produce ~90% of the energy required by the cell, the structural perturbation of mt-tRNAs were found to cause several human diseases (e.g. MERRF, MELAS, LHON). In contrast to tRNAs, mRNA molecules contain a rather small number of modifications, but their importance in the regulation of cellular processes has been clearly demonstrated. Some of them, called epigenetic, were recently discovered to be dynamic and can play a critical regulatory role in protein biosynthesis.

Within the scope of the current project, we focus on two complex problems addressing the role of mRNA and mt-tRNA modifications in translation process. The first subject of our interest is epigenetic modifications derived from 5-methylcytidine: 5- hydroxymethylcytidine (hm5C), 5-formylcytidine (f 5C) and 5-carboxycytidine (ca5C) identified in coding region of mRNA. It has been suggested that they play a regulatory role in the translation at the mRNA level, but there is no systematic research to date. Within the Project, we plan to perform the biophysical and structural characterization of suitably modified oligonucleotides to evaluate the impact of structurally distinct epigenetic cytidines on RNA properties and functionality. The second goal of our Project concerns two pathogenic nucleosides identified at the position 37 of mt-tRNAMet, resulting from mutation A4435→G in human mt-DNA and subsequent enzymatic methylation G37→m1G37. Both pathogenic mutations cause severe mitochondrial dysfunction in some patients associated with hypertension, type 2 diabetes or Leber's hereditary optic neuropathy (LHON). Recently, we have proved that the replacement of conserved A37 to G37 and next to m 1G37 alters the thermal stability of ASL hairpin motif, particularly in the case of G37-containing ASL, which was predicted to form a super-stable tetraloop hairpin [Chem.Commun., 2021, 57, 12540]. Within the project we plan to use appropriate oligonucleotide models for biochemical and structural studies to assess the properties of damaged hmt-ASLMet molecules.

The project is realized in a consortium with Łódź University of Technology (leader). The research tasks performed at IBCh will concentrate on high-resolution characterisation of the studied systems using NMR spectroscopy methods, complemented by UV and CD spectroscopies, as well as electrophoretic methods.

Additional information: