Uncovering the functions of novel m6A and Nm writers in Drosophila melanogaster

Abstract

RNA modifications have emerged as an important regulatory layer of gene expression, decorating all RNA species in the three kingdoms of life. N6-methyladenosine (m6A), the most prevalent modification in messenger RNA (mRNA), and its methyltransferase, the METTL3-METTL14 complex, is to date the best characterized, known to fulfill important regulatory functions during development through the modulation of mRNA biogenesis. For non-coding RNAs, on the other hand, despite being abundant and heavily modified, the functions and the identity of writers of m6A and some other modifications are less characterized. One of such modifications, 2’O-methylation of the ribose (Nm), is highly enriched on ribosomal RNA (rRNA) and transfer RNA (tRNA). The improvement of mapping methodologies has unveiled its dynamic nature during development and upon stress, suggesting its importance in these processes, yet the knowledge about its writers is still incomplete. The goal of my PhD is to characterize novel m6A and Nm methyltransferases in Drosophila melanogaster in order to get more insights into these 2 modifications and their impact on non-coding RNAs. To achieve this goal, the CRISPR/Cas9 system was employed to generate loss-of-functions fly lines for putative m6A and Nm writers: CG7544 (ortholog of METTL16), CG9666 (ortholog of METTL5, identified as novel m6A writer in this study), and the novel Nm writer CG33964 (ortholog of human METTL25) The obtained mutant lines were used for molecular studies, proteomic and transcriptomic analysis, as well as behavioural assays. We identified Mettl5 as a m6A writer acting in concert with Trmt112, a conserved partner, to methylate the 18S rRNA, and show that m6A loss upon Mettl5 depletion has a significant impact on brain development and fly behaviour, providing molecular insight to the METTL5-dependent intellectual disability observed in human (Leismann, Spagnuolo et al., 2020). Our data also confirm the binding of Mettl16, another m6A methyltransferase, to spliceosomal RNA U6 and prove its essential role for fly ovarian development and fertility. The second part of my PhD, dedicated to the study of novel Nm writers, uncovers tRNAGlyGCC as target of Mettl25 ortholog in flies and shows important translational and metabolic impairments, as well as a significant lifespan reduction, upon depletion of the enzyme. Lastly, the functions of another Nm writer: CG8939, orthologous of human FTSJ3, were addressed by RNAi, nanopore and transcriptomic analysis, uncovering a methylated target in the RNase MRP, essential for ribosome biogenesis. Altogether, our findings expand the repertoire of m6A and Nm writers, and demonstrate their specialization and their significance in the context of gene expression and animal health, providing important resource data for future studies.