NCN OPUS 22 UMO-2021/43/B/ST4/01570

Over 160 naturally occurring modified nucleosides have been identified in all RNA species. Some of them have an impact on RNA structure, folding, stability and function. Others play regulatory or signaling roles in cellular processes. The structural RNA perturbations caused by the loss of naturally existing modified nucleosides can result in severe human diseases, e.g. mitochondrial diseases associated with structurally defected mitochondrial tRNAs (mt-tRNAs). Recently, epigenetic mRNA modifications have been discovered to be dynamic and reversible, and might have critical regulatory roles within the cells, including embryonic stem cells and cancer cells. In the course of this Project we plan to tackle two complex problems related to the biological role of mammalian modified RNA nucleosides identified at the coding region of mRNA and the anticodon arm domain of mt-tRNA in the translation process and human diseases. In the first research goal, we focus on dynamic and reversible mRNA epigenetic modifications derived from 5-methylcytidine, namely 5-hydroxymethylcytidine (hm5C), 5-formylcytidine (f5C) and 5-carboxycytidine (ca5C), which are suggested to play a regulatory role in translation at the mRNA level. Although their DNA analogs have been extensively studied over the past few years, the role of rybocytidines hm5C, f5C and ca5C in biological systems and their influence on oligonucleotide properties are largery unknown. Within the Project, we plan to perform a biophysical and structural characteristic of modified oligonucleotides of biologically important sequence by UV melting experiments and NMR and CD spectroscopies to evaluate the effect of hm5C, f5C and ca5C on hybridization properties, base-pairing selectivity and conformation change. It brings us closer to answer the question about regulatory contribution of epigenetic cytidines to translation process. Furthermore, literature data regarding hm5C indicate a reduced amount of this modification in cancer cells, suggesting its significant biological or pathogenic functions in the formation of cancer cells. This intriguing observation prompted us to hypothesize the possible metabolic pathway of hm5C. In the project, we plan to verify proposed three-step enzymatic conversions of synthetically obtained hm5C-RNA oligomer involving cytidine deamination, hm5-uracil excision and RNA cleavage at abasic site mediated by A3A, hSMUG1 and APE1 enzymes, respectively. If so, obtained results will provide new insight into the role of the post-transcriptional modifications in the control of gene expression in cancer cells and shed new light on the unknown regulatory function of A3A and hSMUG1 enzymes. In the second research goal we focus on hmt-tRNAMet which contains f5C at the position 34. Two pathogenic nucleosides were identified at the position 37 of the anticodon stem-loop (ASL) domain of hmt-tRNAMet as an effect of A4435→G mutation in hmt-tRNAMet gene. The replacement of A37→G37 causes severe abnormalities in mitochondria associated with hypertension, type 2 diabetes and Leber's hereditary optic neuropathy (LHON) in humans. Further cellular conversion of G37 to 1-methylguanosine (m1G) was found to accelerate mitochondria dysfunctions. Recently, we have proved that the replacement of conserved A37 to G37 and next to m1G37 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]. We believe that biochemical and structural studies (ribosome binding assays and 3D structure determination by NMR spectroscopy) will allow us to evaluate how G37 and m1G37 affect hmt-ASLMet and establish their contribution to human diseases at the molecular level.