@article{KleiberLemusDiazStilleretal.2022, author = {Kleiber, Nicole and Lemus-Diaz, Nicolas and Stiller, Carina and Heinrichs, Marleen and Mong-Quyen Mai, Mandy and Hackert, Philipp and Richter-Dennerlein, Ricarda and H{\"o}bartner, Claudia and Bohnsack, Katherine E. and Bohnsack, Markus T.}, title = {The RNA methyltransferase METTL8 installs m\(^3\)C\(_{32}\) in mitochondrial tRNAs\(^{Thr/Ser(UCN)}\) to optimise tRNA structure and mitochondrial translation}, series = {Nature Communication}, volume = {13}, journal = {Nature Communication}, doi = {10.1038/s41467-021-27905-1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-254592}, year = {2022}, abstract = {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cellular targets of METTL8 and shed new light on the role of m\(^3\)C\(_{32}\) within mt-tRNAs.}, language = {en} } @unpublished{ScheitlMieczkowskiSchindelinetal.2022, author = {Scheitl, Carolin P. M. and Mieczkowski, Mateusz and Schindelin, Hermann and H{\"o}bartner, Claudia}, title = {Structure and mechanism of the methyltransferase ribozyme MTR1}, series = {Nature Chemical Biology}, journal = {Nature Chemical Biology}, edition = {submitted version}, doi = {10.1038/s41589-022-00976-x}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-272170}, year = {2022}, abstract = {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 {\AA}, 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.}, language = {en} } @incollection{LiaqatSednevHoebartner2022, author = {Liaqat, Anam and Sednev, Maksim V. and H{\"o}bartner, Claudia}, title = {In Vitro Selection of Deoxyribozymes for the Detection of RNA Modifications}, series = {Ribosome Biogenesis: Methods and Protocols}, booktitle = {Ribosome Biogenesis: Methods and Protocols}, publisher = {Humana Press}, isbn = {978-1-0716-2501-9}, doi = {10.1007/978-1-0716-2501-9_10}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-279208}, publisher = {Universit{\"a}t W{\"u}rzburg}, pages = {167-179}, year = {2022}, abstract = {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.}, language = {en} } @phdthesis{Matera2022, author = {Matera, Gianluca}, title = {Global mapping of RNA-RNA interactions in \(Salmonella\) via RIL-seq}, doi = {10.25972/OPUS-26877}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-268776}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2022}, abstract = {RNA represents one of the most abundant macromolecules in both eukaryotic and prokaryotic cells. Since the discovery that RNA could play important gene regulatory functions in the physiology of a cell, small regulatory RNAs (sRNAs) have been at the center of molecular biology studies. Functional sRNAs can be independently transcribed or derived from processing of mRNAs and other non-coding regions and they often associate with RNA-binding proteins (RBPs). Ever since the two major bacterial RBPs, Hfq and ProQ, were identified, the way we approach the identification and characterization of sRNAs has drastically changed. Initially, a single sRNA was annotated and its function studied with the use of low-throughput biochemical techniques. However, the development of RNA-seq techniques over the last decades allowed for a broader identification of sRNAs and their functions. The process of studying a sRNA mainly focuses on the characterization of its interacting RNA partner(s) and the consequences of this binding. By using RNA interaction by ligation and sequencing (RIL-seq), the present thesis aimed at a high-throughput mapping of the Hfq-mediated RNA-RNA network in the major human pathogen Salmonella enterica. RIL-seq was at first performed in early stationary phase growing bacteria, which enabled the identification of ~1,800 unique interactions. In- depth analysis of such complex network was performed with the aid of a newly implemented RIL-seq browser. The interactome revealed known and new interactions involving sRNAs and genes part of the envelope regulon. A deeper investigation led to the identification of a new RNA sponge of the MicF sRNA, namely OppX, involved in establishing a cross-talk between the permeability at the outer membrane and the transport capacity at the periplasm and the inner membrane. Additionally, RIL-seq was applied to Salmonella enterica grown in SPI-2 medium, a condition that mimicks the intracellular lifestyle of this pathogen, and finally extended to in vivo conditions during macrophage infection. Collectively, the results obtained in the present thesis helped unveiling the complexity of such RNA networks. This work set the basis for the discovery of new mechanisms of RNA-based regulation, for the identification of a new physiological role of RNA sponges and finally provided the first resource of RNA interactions during infection conditions in a major human pathogen.}, subject = {Small RNA}, language = {en} }