Hakai is required for stabilization of core components of the m6A mRNA methylation machinery

N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex

Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila

Cell number plasticity is coupled to circuitry in the nervous system, adjusting cell mass to functional requirements. In mammals, this is achieved by neurotrophin (NT) ligands, which promote cell survival via their Trk and p75 receptors and cell death via p75 and Sortilin. NTs (DNTs) bind Toll receptors instead to promote neuronal survival, but whether they can also regulate cell death is unknown. In this study, we show that DNTs and Tolls can switch from promoting cell survival to death in the central nervous system (CNS) via a three-tier mechanism. First, DNT cleavage patterns result in alternative signaling outcomes. Second, different Tolls can preferentially promote cell survival or death. Third, distinct adaptors downstream of Tolls can drive either apoptosis or cell survival. Toll-6 promotes cell survival via MyD88-NF-κB and cell death via Wek-Sarm-JNK. The distribution of adaptors changes in space and time and may segregate to distinct neural circuits. This novel mechanism for CNS cell plasticity may operate in wider contexts

Project 1: Analysis of embryonic abnormal vision system function in neuronal development AND Project 2: Function of Drosophila neurotrophin in dendritic development

Project 1:- Cells generate numerous mRNAs from a single gene via alternative splicing, which increase the proteome and level of post-transcriptional expression. Post-transcriptional gene expression by RNA binding proteins (RBPs) is important for generating organismal complexity in eukaryotes. Brain is an organ where alternative mRNA processing is abundant, with RBPs being key regulators of alternative splicing and polyadenylation. Elav (embryonic lethal abnormal vision system) is a RBP that is expressed only in differentiated neurons in the brain, where it is involved in alternative splicing, polyadenylation, mRNA stability and translation. In here, we wanted to see what effect does the expression of Elav during early stages of neurogenesis have on the differentiating neuronal precursor cells i.e. eye primordium and neuroblasts. Therefore, we tested ectopic Elav expression in the developing eyes using various drivers in an $in-vivo$ $Drosophila$ tumour eye model $via$ the GAL4/UAS expression system. In addition, we also ectopically expressed Elav in various neuroblast, neuron and glial drivers and via immunostaining observed variations in the optic lobes and the ventral nerve cord of the larval brain. Project 2:- Neurotrophin and Toll receptors play key roles in birth, survival and death of neurons in the brain. In neurons, dendrite morphology and structure of the dendritic tree is important for proper function and connectivity between neuronal circuits. Many proteins have been involved in shaping up the synaptic connections and shapes of the dendrites, but our understanding on dendrite remodelling, arborisation and circuitry is still limited. In here, we look into how ectopic expression of $Drosophila$ neurotrophins and Toll receptors affect the structure and dendrite morphology of lobula plate tangential cell neurons and ellipsoid body (EB) associated R-neurons in the adult $Drosophila$ brain using immunostaining and confocal microscopy. In addition, we also look into identifying suitable GAL4 lines for central and peripheral nervous system that could characterise circuitry and sensory neuron dendrite arborisation. To visualize the dendrite arborisation of CNS interneurons and PNS DAIV neurons, we used mCD8GFP and DenMark to observe the dendrite branching pattern. Analysis through ImageJ showed DNTs and Toll receptors to have a potential effect on the structural organisation of dendrites in adult $Drosophila$ brain, while in the case of circuitry and sensory neurons the orientation of dendrite arborisation was found to be distinct for different neurons

CMTr mediated 2'-O-ribose methylation status of cap adjacent-nucleotides across animals

Cap methyltransferases (CMTrs) O methylate the 2′ position of the ribose (cOMe) of cap-adjacent nucleotides of animal, protist, and viral mRNAs. Animals generally have two CMTrs, whereas trypanosomes have three, and many viruses encode one in their genome. In the splice leader of mRNAs in trypanosomes, the first four nucleotides contain cOMe, but little is known about the status of cOMe in animals. Here, we show that cOMe is prominently present on the first two cap-adjacent nucleotides with species- and tissue-specific variations in Caenorhabditis elegans, honeybees, zebrafish, mouse, and human cell lines. In contrast, Drosophila contains cOMe primarily on the first cap-adjacent nucleotide. De novo RoseTTA modeling of CMTrs reveals close similarities of the overall structure and near identity for the catalytic tetrad, and for cap and cofactor binding for human, Drosophila and C. elegans CMTrs. Although viral CMTrs maintain the overall structure and catalytic tetrad, they have diverged in cap and cofactor binding. Consistent with the structural similarity, both CMTrs from Drosophila and humans methylate the first cap-adjacent nucleotide of an AGU consensus start. Because the second nucleotide is also methylated upon heat stress in Drosophila, these findings argue for regulated cOMe important for gene expression regulation