TY - INPR A1 - Seitz, Florian A1 - Jungnickel, Tina A1 - Kleiber, Nicole A1 - Kretschmer, Jens A1 - Dietzsch, Julia A1 - Adelmann, Juliane A1 - Bohnsack, Katherine E. A1 - Bohnsack, Markus T. A1 - Höbartner, Claudia T1 - Atomic mutagenesis of N\(^6\)-methyladenosine reveals distinct recognition modes by human m\(^6\)A reader and eraser proteins T2 - Journal of the American Chemical Society N2 - N\(^6\)-methyladenosine (m\(^6\)A) is an important modified nucleoside in cellular RNA associated with multiple cellular processes and is implicated in diseases. The enzymes associated with the dynamic installation and removal of m\(^6\)A are heavily investigated targets for drug research, which requires detailed knowledge of the recognition modes of m\(^6\)A by proteins. Here, we use atomic mutagenesis of m\(^6\)A to systematically investigate the mechanisms of the two human m\(^6\)A demethylase enzymes FTO and ALKBH5 and the binding modes of YTH reader proteins YTHDF2/DC1/DC2. Atomic mutagenesis refers to atom-specific changes that are introduced by chemical synthesis, such as the replacement of nitrogen by carbon atoms. Synthetic RNA oligonucleotides containing site-specifically incorporated 1-deaza-, 3-deaza-, and 7-deaza-m\(^6\)A nucleosides were prepared by solid-phase synthesis and their RNA binding and demethylation by recombinant proteins were evaluated. We found distinct differences in substrate recognition and transformation and revealed structural preferences for the enzymatic activity. The deaza m\(^6\)A analogues introduced in this work will be useful probes for other proteins in m\(^6\)A research. KW - modified nucleosides KW - N6-methyladenosine (m6A) KW - atomic mutagenesis KW - YTH reader proteins KW - demethylase enzymes FTO and ALKBH5 Y1 - 2024 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-352376 ER - TY - INPR A1 - Sednev, Maksim V. A1 - Mykhailiuk, Volodymyr A1 - Choudhury, Priyanka A1 - Halang, Julia A1 - Sloan, Katherine E. A1 - Bohnsack, Markus T. A1 - Höbartner, Claudia T1 - N\(^6\)-methyladenosine-sensitive RNA-cleaving deoxyribozymes T2 - Angewandte Chemie, International Edition N2 - Deoxyribozymes are synthetic enzymes made of DNA that can catalyze the cleavage or formation of phosphodiester bonds and are useful tools for RNA biochemistry. Here we report new RNA-cleaving deoxyribozymes to interrogate the methylation status of target RNAs, thereby providing an alternative method for the biochemical validation of RNA methylation sites containing N\(^6\)-methyladenosine, which is the most wide-spread and extensively investigated natural RNA modification. Using in vitro selection from random DNA, we developed deoxyribozymes that are sensitive to the presence of N\(^6\)-methyladenosine in RNA near the cleavage site. One class of these DNA enzymes shows faster cleavage of methylated RNA, while others are strongly inhibited by the modified nucleotide. The general applicability of the new deoxyribozymes is demonstrated for several examples of natural RNA sequences, including a lncRNA and a set of C/D box snoRNAs, which have been suggested to contain m\(^6\)A as a regulatory element that influences RNA folding and protein binding. KW - N6-methyladenosine KW - RNA modification KW - deoxyribozymes KW - in vitro selection KW - DNA catalyst Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171753 N1 - This is the pre-peer reviewed version of the following article: M.V. Sednev, V. Mykhailiuk, P. Choudhury, J. Halang, K. E. Sloan, M. T. Bohnsack, C. Höbartner, Angew. Chem. Int. Ed. 2018, 57, 15117-15121, which has been published in final form at https://doi.org/10.1002/anie.201808745. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. ER - TY - JOUR A1 - Kleiber, Nicole A1 - Lemus-Diaz, Nicolas A1 - Stiller, Carina A1 - Heinrichs, Marleen A1 - Mong-Quyen Mai, Mandy A1 - Hackert, Philipp A1 - Richter-Dennerlein, Ricarda A1 - Höbartner, Claudia A1 - Bohnsack, Katherine E. A1 - Bohnsack, Markus T. T1 - The RNA methyltransferase METTL8 installs m\(^3\)C\(_{32}\) in mitochondrial tRNAs\(^{Thr/Ser(UCN)}\) to optimise tRNA structure and mitochondrial translation JF - Nature Communication N2 - Modified nucleotides in tRNAs are important determinants of folding, structure and function. Here we identify METTL8 as a mitochondrial matrix protein and active RNA methyltransferase responsible for installing m\(^3\)C\(_{32}\) in the human mitochondrial (mt-)tRNA\(^{Thr}\) and mt-tRNA\(^{Ser(UCN)}\). METTL8 crosslinks to the anticodon stem loop (ASL) of many mt-tRNAs in cells, raising the question of how methylation target specificity is achieved. Dissection of mttRNA recognition elements revealed U\(_{34}\)G\(_{35}\) and t\(^6\)A\(_{37}\)/(ms\(^2\))i\(^6\)A\(_{37}\), present concomitantly only in the ASLs of the two substrate mt-tRNAs, as key determinants for METTL8-mediated methylation of C\(_{32}\). Several lines of evidence demonstrate the influence of U\(_{34}\), G\(_{35}\), and the m\(^3\)C\(_{32}\) and t\(^6\)A\(_{37}\)/(ms\(^2\))i\(^6\)A\(_{37}\) modifications in mt-tRNA\(^{Thr/Ser(UCN)}\) on the structure of these mt-tRNAs. Although mt-tRNA\(^{Thr/Ser(UCN)}\) lacking METTL8-mediated m\(^3\)C\(_{32}\) are efficiently aminoacylated and associate with mitochondrial ribosomes, mitochondrial translation is mildly impaired by lack of METTL8. Together these results define the cellular targets of METTL8 and shed new light on the role of m\(^3\)C\(_{32}\) within mt-tRNAs. KW - Modified Nucleotides in tRNAs KW - METTL8 KW - Mitochondrial Matrix Protein KW - RNA Methyltransferase KW - RNA KW - Enzymes KW - Organelles Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-254592 VL - 13 ER -