Thesis presented February 10, 2023

Abstract:
A huge number of epigenetic modifications occur on eukaryotic messenger RNAs such as the addition of a 5' cap, a polyA tail or of internal marks including the methylation of adenosine ring at position 6 (m⁶A). These modifications tightly regulate mRNAs stability and translational activity, therefore providing crucial additional levels of gene expression regulation.
The first chapter of my thesis focuses on the study of the PCIF1 protein using Drosophila melanogaster as a model. PCIF1 is a RNA cap-specific m6A methyltransferase that binds to RNA polymerase II (RNA Pol II) and deposits a methyl group on the adenosine ring if the first transcribed nucleotide is an adenosine (m⁶Am). In knock-out mice, the lack of PCIF1 results in reduced body weight and deregulation of a pool of genes in several tissues, particularly in testis. In Drosophila, Pcif1 is conserved but a single amino acid substitution inactivates the methyltransferase activity. I used CRISPR-Cas9 to create Pcif1 Drosophila mutants and performed their phenotypic and molecular analysis in order to determine the non-catalytic functions of Pcif1. I showed that Pcif1 mutants displayed reduced fertility. Moreover, RNA sequencing revealed a set of dysregulated genes in Pcif1 mutant ovaries indicating a role in gene expression regulation. I also observed that reduced Pcif1 dosage in heterozygous flies induced a suppression of the white gene silencing due to the position-effect variegation (PEV), in which the euchromatic white gene is translocated near the heterochromatin rendering it sensitive to slight modifications of gene expression regulation. Finally, experiments performed by our collaborators in Pr. E. Brasset laboratory revealed that Pcif1 binds to euchromatin at interphasic chromosomal bands, indicating that it is associated with actively transcribed genes. Taken together, our results suggest that despite the lack of catalytic activity, Pcif1 is involved in gene expression regulation, putatively acting as a scaffolding protein recruited in the RNA Pol II complex, a hypothesis reinforced by the fact that Pcif1 is known to bind to the C-term of Pol II.
The second chapter of my thesis is centred on the identification of alternative RNA cap structures and decapping enzymes that ensure both the degradation of mRNA and their recycling into nucleotides, in Drosophila. The canonical cap is defined as the N7-methylguanine (m⁷G-cap) that is attached to the first transcribed nucleotide of eukaryotic mRNAs and hydrolysed by the conserved DCP2 decapping enzyme. DCP2 belongs to a large family of evolutionary conserved hydrolases, Nudix, which consists of 22 members in mammals that all share the ability to hydrolyse different metabolite substrates. In addition, several alternative cap structures have been identified so far in various bacteria or eukaryotes such as: nicotinamide adenine dinucleotide (NAD), flavine dinucleotide (FAD), diphospho-coenzymeA (dpCoA) and diadenosine-tetraphosphate (Ap4A), uridine diphosphate glucose (UDP-Glc), and uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Since little is known about the role of the Nudix family members and the putative presence of alternative cap structures in higher eukaryotes, my goal was to perform a functional analysis of the Nudix family in Drosophila. By using an RNAi-based genetic screen approach, I induced the silencing of each of the 12 Nudix genes in a tissue specific manner in Drosophila. My work pointed out that the closely related Nudix proteins Nudt19A and Nudt19B are required for full fly fertility. These proteins are predicted to hydrolyse Coenzyme A but nothing is known about their putative decapping activity. We generated CRISPR/Cas9 mutants of Nudt19A and Nudt19B, in order to further investigate their specific function in fly fertility and potential role in mRNA decapping in eukaryotes.

Keywords:
Epigentics, molecular biology, Drosophila

On-line thesis. ​