The epitranscriptome in Drosophila melanogaster
Biochemistry
Developmental Biology
Final Report Abstract
The ensemble of RNA molecules of a cell in a given biological context, known as the transcriptome, is rendered more complex by the presence of non-canonical nucleosides. Including such RNA modifications leads from the transcriptome to the epitranscriptome, where the modifications exert structural and regulatory functions of which many are yet unknown. Modifications occur in most noncoding RNAs as well as in mRNA, where the prevalent presence of N6-methyladenosine (m6A) dominates modification research in eukaryotes, despite technical shortcomings in the localization ("mapping”) of m6A. A comprehensive understanding of the epitranscriptome requires knowledge of its modification types and sites, as well as of modification enzymes and molecular mechanisms that react to modifications. Aspects of the former have been studied in different model organism, but little is known in the fruit fly drosophila melanogaster. The current projects tasks where to identify factors in the fruit fly that are involved in the catalytic mechanism of m6A deposition in mRNA, and to develop improved detection methodology that can detect quantify m6A at single nucleotide resolution. A further aim was to generate the beginnings of a collection of knockout organisms of modification enzymes to study their impact on fruit fly metabolism and behavior. Correspondingly, two proteins, namely in the course of the project, Zc3h13/Flacc and Hakai were characterized as protein components of the drosophila m6A methyltransferase complex. Furthermore, a method for detection and quantification of m6A with single nucleotide resolution was developed based on chemical deamination of exocyclic amine functions in nucleobases using nitrous acid. Application of this method was developed for defined amplicons only, since problems with reverse transcription yield of deaminated RNA prevented transcriptome wide application. Finally, knockout organisms for a number of tRNA modifications were generated and its basic properties characterized.
Publications
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Zc3h13/Flacc is required for adenosine methylation by bridging the mRNA-binding factor Rbm15/Spenito to the m6A machinery component Wtap/Fl(2)d. Genes & Development, 32(5-6), 415-429.
Knuckles, Philip; Lence, Tina; Haussmann, Irmgard U.; Jacob, Dominik; Kreim, Nastasja; Carl, Sarah H.; Masiello, Irene; Hares, Tina; Villaseñor, Rodrigo; Hess, Daniel; Andrade-Navarro, Miguel A.; Biggiogera, Marco; Helm, Mark; Soller, Matthias; Bühler, Marc & Roignant, Jean-Yves
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NOseq: amplicon sequencing evaluation method for RNA m6A sites after chemical deamination. Nucleic Acids Research, 49(4), e23-e23.
Werner, Stephan; Galliot, Aurellia; Pichot, Florian; Kemmer, Thomas; Marchand, Virginie; Sednev, Maksim V; Lence, Tina; Roignant, Jean-Yves; König, Julian; Höbartner, Claudia; Motorin, Yuri; Hildebrandt, Andreas & Helm, Mark
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Hakai is required for stabilization of core components of the m6A mRNA methylation machinery. Nature Communications, 12(1).
Bawankar, Praveen; Lence, Tina; Paolantoni, Chiara; Haussmann, Irmgard U.; Kazlauskiene, Migle; Jacob, Dominik; Heidelberger, Jan B.; Richter, Florian M.; Nallasivan, Mohanakarthik P.; Morin, Violeta; Kreim, Nastasja; Beli, Petra; Helm, Mark; Jinek, Martin; Soller, Matthias & Roignant, Jean-Yves
