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  - 
TY  - CHAP
A1  - Liaqat, Anam
A1  - Sednev, Maksim V.
A1  - Höbartner, Claudia
T1  - In Vitro Selection of Deoxyribozymes for the Detection of RNA Modifications
T2  - Ribosome Biogenesis: Methods and Protocols
N2  - Deoxyribozymes are artificially evolved DNA molecules with catalytic abilities. RNA-cleaving deoxyribozymes have been recognized as an efficient tool for detection of modifications in target RNAs and provide an alternative to traditional and modern methods for detection of ribose or nucleobase methylation. However, there are only few examples of DNA enzymes that specifically reveal the presence of a certain type of modification, including N6-methyladenosine, and the knowledge about how DNA enzymes recognize modified RNAs is still extremely limited. Therefore, DNA enzymes cannot be easily engineered for the analysis of desired RNA modifications, but are instead identified by in vitro selection from random DNA libraries using synthetic modified RNA substrates. This protocol describes a general in vitro selection stagtegy to evolve new RNA-cleaving DNA enzymes that can efficiently differentiate modified RNA substrates from their unmodified counterpart.
KW  - RNA
KW  - deoxyribozymes
KW  - modified RNA nucleotides
KW  - catalytic DNA
KW  - epitranscriptomics
KW  - in vitro selection
KW  - RNA cleavage
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-279208
SN  - 978-1-0716-2501-9
PB  - Humana Press
ER  - 
TY  - INPR
A1  - Scheitl, Carolin P. M.
A1  - Mieczkowski, Mateusz
A1  - Schindelin, Hermann
A1  - Höbartner, Claudia
T1  - Structure and mechanism of the methyltransferase ribozyme MTR1
T2  - Nature Chemical Biology
N2  - RNA-catalysed RNA methylation was recently shown to be part of the catalytic repertoire of ribozymes. The methyltransferase ribozyme MTR1 catalyses the site-specific synthesis of 1-methyladenosine (m\(^1\)A) in RNA, using O\(^6\)-methylguanine (m\(^6\)G) as methyl group donor. Here we report the crystal structure of MTR1 at a resolution of 2.8 Å, which reveals a guanine binding site reminiscent of natural guanine riboswitches. The structure represents the postcatalytic state of a split ribozyme in complex with the m1A-containing RNA product and the demethylated cofactor guanine. The structural data suggest the mechanistic involvement of a protonated cytidine in the methyl transfer reaction. A synergistic effect of two 2'-O-methylated ribose residues in the active site results in accelerated methyl group transfer. Supported by these results, it seems plausible that modified nucleotides may have enhanced early RNA catalysis and that metabolite-binding riboswitches may resemble inactivated ribozymes that have lost their catalytic activity during evolution.
KW  - Methyltransferase Ribozyme MTR1
KW  - Crystal structure of MTR1
KW  - RNA-catalyzed RNA methylation
KW  - X-ray crystallography
KW  - RNA
Y1  - 2022
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-272170
ET  - submitted version
ER  -