Mettl3 is required cell intrinsically in both muscle and neurons for development of functional flight muscle

Heemken, Jakob (2025): Mettl3 is required cell intrinsically in both muscle and neurons for development of functional flight muscle. Dissertation, LMU München: Medizinische Fakultät

Abstract

Muscle development is a highly organized and complex process. Through the precise regulation of gene expression and alternative splicing, muscles define temporal and spatial patterns of gene isoform expression that promote sarco-mere assembly and fine-tune contractile properties. Misregulation of gene iso-form expression is observed in diseases ranging from muscular dystrophies to cardiomyopathies, illustrating the importance of understanding this process. N6-Methyladenosine (m6A) is an RNA modification that is suggested to regulate multiple steps in RNA processing, including alternative splicing, mRNA stability, trafficking and translation efficiency, but many of its cellular functions still re-main elusive. Core enzymes in the m6A-pathway such as Mettl3 and Mettl14 are highly conserved, providing the opportunity to utilize powerful genetic tools available in model organisms such as Drosophila melanogaster to understand in vivo functions of this ancient pathway. In this thesis, I show that Mettl3 plays an important role in the development of both the indirect flight muscles (IFMs) and their corresponding motor neurons. Complete loss of the essential enzyme Mettl3 in mutant flies results in flight-lessness and impaired climbing ability. Confocal imaging revealed altered sar-comere dimensions in IFM, as well as extensive over-branching of motor neu-rons. Using tissue-specific RNAi knockdown, we found that muscle-specific Mettl3 knockdown flies are flightless and have defects in sarcomere length, while neuronal-specific Mettl3 knockdown flies have defects in motor neuron branching. Sarcomere length defects could be rescued selectively by expres-sion of Mettl3 with Mef2-Gal4 in muscle, while neuronal defects could be res-cued selectively by expression of Mettl3 with Elav-Gal4 in neurons. This indi-cates muscle and neuron-intrinsic Mettl3 function. Using mRNA-Sequencing and qPCR, we show that IFM lacking Mettl3 has changes in gene expression of sarcomere, mitochondrial and synapse-associated genes, many of which are also misexpressed on the protein level as detected by whole-proteome mass spectrometry and GFP-tagged reporters. Strikingly, our experiments revealed significant changes in muscle specific al-ternative splicing, including isoform switches in key sarcomere proteins such as the Titin-like gene bent (bt, Projectin), Myofilin (Mf), Zasp52 and Unc-89. This data confirms a muscle-intrinsic function for m6A modification in regulating al-ternative splicing and protein expression levels during myofibrillogenesis that is necessary for proper growth in sarcomere length and muscle function.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Genetik, RNA-Signalweg, Muskelgewebe, Drosophila
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 19. September 2025
• 1. Bericht­erstatter:in: Ladurner, Andreas