A few days ago, Nature Communication issued a publication entitled Secondary Structure Prediction for RNA Sequences Including N6-methyladenosine by E. Kierzek (co-author for correspondence), X. Zhang, R. Watson, S. Kennedy, M. Szabat, R. Kierzek and D.H. Mathews (OA: Nature Communication, 13, 1271, 2022). It was performed due to collaboration of Professor Elżbieta Kierzek, Doctor Marta Szabat and Professor Ryszard Kierzek with the research group of Professor David H. Mathews from the Department of Biochemistry and Biophysics of the University of Rochester in the USA. The work concerns the prediction of folding of RNAs containing N6-methyladenosine with RNAstructure software. Apart from pseudouridine, N6-methyladenosine (m6A) is the most common natural RNA modification. It is found in evolutionary conserved RNA regions (long internal exons, 3’UTR, near the stop codons) in mRNA, rRNA, tRNA and ncRNA from various organisms. N6-methyladenosine is predicted to occur approximately 200,000 times in the human genome and performs various biological functions. Some of them are associated with various cancers and diseases related with metabolic disorders, immuneregulation and neurodegradation. Almost all RNA viruses (e.g., influenza, Dengue, Zika, West-Nile, HCV, HIV, SARS-CoV-2 viruses) contain N6-methyladenosine and its function is little known.
In order to extend the range of structures predicted by RNAstructure, 15 thermodynamic parameters were determined that define the folding rules for RNAs containing N6-methyladenosine. The parameters were determined based on the thermodynamic stability of 45 model RNAs containing N6-methyladenosine in different structural arrangements. The UV-melting method was used to determine the thermodynamic stability. Next, the thermodynamic parameters concerning m6A were incorporated into the algorithm system of the RNAstructure program, which in its previous form made it possible to predict the structure of RNA containing only A, C, G and U.
The accuracy of the RNA structure containing N6-methyladenosine predicted by the modified RNAstructure program was tested on the lncRNA fragment MALAT1 (metastasis associated lung adenocarcinoma transcript). It was an RNA fragment forming a hairpin with N6-methyladenosine located in the stem. The RNA structure predicted with the m6A modified RNAstructure program did not show any significant structural changes but only a lower thermodynamic stability. These observations were confirmed by chemical mapping of the structures of the same two RNA hairpins, one containing A and the other m6A. Again, the structures of both RNAs were practically identical. NMR studies of the same RNA hairpins were also carried out, both in the presence and absence of different concentrations of magnesium cations. Again, the NMR spectra suggested only a structural weakness in the region containing N6-methyladenosine, without the m6A-induced rearrangement of the RNA hairpin.
Using the RNAstructure program modified for m6A parameters, a structural analysis of 18026 mRNAs, known to contain RNA N6-methylation sites, was carried out. Their structures were additionally mapped with S1 and V1 enzymes (PARS method). The 800-nucleotide fragments were selected for structural studies as previous studies have shown that the folding of such RNA fragments is identical to that of the entire mRNA. The conducted structural analysis showed a significantly lower probability of formation a helical structure within the N6-methylation site when N6-methyladenosine would be present instead of adenosine.
The latter results, combined with the results of chemical mapping and structural studies using NMR, correlate well with the enzymatic pathway of N6-methylation mechanism and function of N6-methyladenosine. Three types of proteins are involved in the formation and functioning if this post-transcriptional modification. The first group of proteins is involved in N6-methylation of adenosine (methyltransferases, so-called writers), the next are proteins that bind to the RNA regions containing N6-methyladenosine (the so-called readers), and the last in this group are proteins that remove methyl from the N6 position of adenosine (demethylases, so-called erasers).
Described in publication the thermodynamic and structural properties of m6A RNA, combined with current knowledge about its function, suggest that N6-methyladenosine may be a key element of a very precise and function-driven structural switch within of the natural RNAs.
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