The Notch signalling pathway is conserved in all metazoan species and plays a crucial role in development and tissue maintenance. Canonical Notch signalling requires cell-cell contact to allow the interaction between Drosophila Notch receptor and its ligands, Serrate and Delta. The Notch Abruptex (Ax) region comprises 24-29 of the 36 epidermal growth factor-like (EGF) repeats in the Notch extracellular domain. Mutations in the Ax region give rise to three distinct phenotypes in Drosophila. Notch EGF repeats 11-12 form the ligand binding region (LBR). The recently solved structure of the module at the N-terminal of Notch ligands (MNNL) of a human Notch ligand, Jagged1, revealed that the domain was a calcium-binding C2 domain with Ca²⁺-dependent lipid binding. This study aimed to investigate the intra- and intermolecular properties of Drosophila Notch Ax region, LBR, and the MNNL of Notch ligand Serrate. In WT Drosophila Notch EGF23-25, all three EGF domains were found to be Ca²⁺-binding, and a previously unknown Ca²⁺ binding consensus sequence was identified. Ax^N-suppressor mutations D948V and N986I were shown to impair the Ca²⁺-binding properties of the mutant EGF domain without affecting the neighbouring domains, suggesting a mechanism to explain the signalling phenotype associated with this mutation type. Notch EGF11-13 showed Ca²⁺-binding in each EGF domain and binding to ligand-expressing cells. Its C-terminal tag was found to influence the Ca²⁺-dependent fold of EGF13, suggesting a future strategy for protein expression. A Serrate fragment MNNL-EGF3 showed Ca²⁺-dependent lipid binding, which was not observed in a construct lacking MNNL. The lipid binding could be reduced by a substitution D197A in MNNL, suggesting this mutant could be used to probe functional importance of MNNL in model organism studies. Binding between Serrate and Notch was assessed with a new cell aggregation assay method using flow cytometry, and agreed with previously published studies. Binding to Delta was subsequently measured, which suggested ligand specific differences although Notch residue L504 was important for both Serrate- and Delta-binding. Collectively these studies establish that Drosophila Notch and its ligand Serrate has similar properties to mammalian homologues, which will facilitate future structural and functional studies.

Drosophila melanogaster is an excellent model system to identify genes involved in synaptic growth and function. In Drosophila, the Serrate (Ser) gene encodes a transmembrane protein that is a ligand for Notch receptor. Several previous studies implicated a role for Serrate in normal wing development and patterning. In this study, I demonstrate that Serrate is required for normal synaptic growth and function. I characterized the phenotype of a Serrate mutation (serB936) that was identified by an EMS-induced genetic screen aimed at identifying novel genes that play a role in synaptic growth and function. Co-localization studies show that Serrate protein is expressed at both the pre- and postsynaptic side of larval neuromuscular junctions (NMJs). Mutations in ser impair synaptic transmission at larval NMJs. This defect is entirely presynaptic, as nerve-evoked excitatory junction potentials (EJP) and quantal content (QC) of neurotransmitter release are significantly reduced when compared to wild-type control. Further, mutations in ser also alter the growth of the NMJ and the underlying muscle. Mutations in ser significantly reduce the size of larval body wall muscles (length and surface area) as well as the number and size of synaptic boutons, and the number of secondary axonal branches. Ubiquitous or muscle-specific expression of normal Serrate in serB936 mutants restores a normal muscle size but not a normal size and structure of the innervating NMJ. However, expression of normal Serrate in the motor axon restores a normal number of synaptic boutons and secondary branches at serB936 mutant NMJs. In addition, it restores normal neurotransmitter release. These data suggest that Serrate protein is required presynaptically for normal synaptic growth and function. Interestingly, overexpression of Serrate in a wild type background resulted in similar phenotypes than to those of loss-of-function mutants. In conclusion, these data suggest a new functional role for Serrate in synaptic growth and function.

Arabidopsis thaliana HYL1 is a nuclear doublestranded RNA-binding protein involved in the maturation of pri-miRNAs. A quantitative real-time PCR platform for parallel quantification of 176 primiRNAs was used to reveal strong accumulation of 57 miRNA precursors in the hyl1 mutant that completely lacks HYL1 protein. This approach enabled us for the first time to pinpoint particular members of MIRNA family genes that require HYL1 activity for efficient maturation of their precursors. Moreover, the accumulation of miRNA precursors in the hyl1 mutant gave us the opportunity to carry out 3’ and 5’ RACE experiments which revealed that some of these precursors are of unexpected length. The alignment of HYL1- dependent miRNA precursors to A. thaliana genomic sequences indicated the presence of introns in 12 out of 20 genes studied. Some of the characterized intron-containing pri-miRNAs undergo alternative splicing such as exon skipping or usage of alternative 5’ splice sites suggesting that this process plays a role in the regulation of miRNA biogenesis. In the hyl1 mutant intron-containing pri-miRNAs accumulate alongside spliced primiRNAs suggesting the recruitment of HYL1 into the miRNA precursor maturation pathway before their splicing occurs.

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