Light is a very important environmental factor that governs many cellular responses in organisms. As a consequence, organisms possess different kinds of light-sensing photoreceptors to regulate their physiological variables and adapt to a given habitat. The cryptochrome/photolyase family (CPF) includes photoreceptors that perform different functions in different organisms. Photolyases repair ultraviolet-induced DNA damage by a process known as photoreactivation using photons absorbed from the blue end of the light spectrum. On the other hand, cryptochromes act as blue light circadian photoreceptors in plants and Drosophila to regulate growth and development. In mammals, cryptochromes have lightindependent functions and are very important transcriptional regulators that act at the molecular level as negative transcriptional regulators of the circadian clock. In this review, we highlight current knowledge concerning the structural and functional relationships of CPF members.

Alternative splicing is a key feature of human genes, yet studying its regulation is often complicated by large introns. The Down Syndrome Cell Adhesion Molecule (Dscam) gene from Drosophila is one of the most complex genes generating vast molecular diversity by mutually exclusive alternative splicing. To resolve how alternative splicing in Dscam is regulated, we first developed plasmid-based UAS reporter genes for the Dscam variable exon 4 cluster and show that its alternative splicing is recapitulated by GAL4-mediated expression in neurons. We then developed gap-repair recombineering to very efficiently manipulate these large reporter plasmids in Escherichia coli using restriction enzymes or sgRNA/Cas9 DNA scission to capitalize on the many benefits of plasmids in phiC31 integrase-mediated transgenesis. Using these novel tools, we show that inclusion of Dscam exon 4 variables differs little in development and individual flies, and is robustly determined by sequences harbored in variable exons. We further show that introns drive selection of both proximal and distal variable exons. Since exon 4 cluster introns lack conserved sequences that could mediate robust long-range base-pairing to bring exons into proximity for splicing, our data argue for a central role of introns in mutually exclusive alternative splicing of Dscam exon 4 cluster.

Securing food supply for a growing population is a major challenge and heavily relies on the use of agrochemicals to maximize crop yield. It is increasingly recognized, that some neonicotinoid insecticides have a negative impact on non-target organisms, including important pollinators such as the European honeybee Apis mellifera. Toxicity of neonicotinoids may be enhanced through simultaneous exposure with additional pesticides, which could help explain, in part, the global decline of honeybee colonies. Here we examined whether exposure effects of the neonicotinoid thiamethoxam on bee viability are enhanced by the commonly used fungicide carbendazim and the herbicide glyphosate. We also analysed alternative splicing changes upon pesticide exposure in the honeybee. In particular, we examined transcripts of three genes: (i) the stress sensor gene X box binding protein-1 (Xbp1), (ii) the Down Syndrome Cell Adhesion Molecule (Dscam) gene and iii) the embryonic lethal/abnormal visual system (elav) gene, which are important for neuronal function. Our results showed that acute thiamethoxam exposure is not enhanced by carbendazim, nor glyphosate. Toxicity of the compounds did not trigger stress-induced, alternative splicing in the analysed mRNAs, thereby leaving dormant a cellular response pathway to these man-made environmental perturbations.

Unpaired ligands are secreted signals that act via a GP130-like receptor, domeless, to activate JAK/STAT signalling in Drosophila. Like many mammalian cytokines, unpaireds can be activated by infection and other stresses and can promote insulin resistance in target tissues. However, the importance of this effect in non-inflammatory physiology is unknown. Here, we identify a requirement for unpaired-JAK signalling as a metabolic regulator in healthy adult Drosophila muscle. Adult muscles show basal JAK-STAT signalling activity in the absence of any immune challenge. Plasmatocytes (Drosophila macrophages) are an important source of this tonic signal. Loss of the dome receptor on adult muscles significantly reduces lifespan and causes local and systemic metabolic pathology. These pathologies result from hyperactivation of AKT and consequent deregulation of metabolism. Thus, we identify a cytokine signal that must be received in muscle to control AKT activity and metabolic homeostasis.

Changes in gene expression are a hallmark of learning and memory consolidation. Little is known about how alternative mRNA processing, particularly abundant in neuron-specific genes, contributes to these processes. Prototype RNA binding proteins of the neuronally expressed ELAV/Hu family are candidates for roles in learning and memory, but their capacity to cross-regulate and take over each other’s functions complicate substantiation of such links. Honey bees Apis mellifera have only one elav/Hu family gene elavl2, that has functionally diversified by increasing alternative splicing including an evolutionary conserved microexon. RNAi knockdown demonstrates that ELAVL2 is required for learning and memory in bees. ELAVL2 is dynamically expressed with altered alternative splicing and subcellular localization in mushroom bodies, but not in other brain regions. Expression and alternative splicing of elavl2 change during memory consolidation illustrating an alternative mRNA processing program as part of a local gene expression response underlying memory consolidation.

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Alternative splicing of pre-mRNA is a major mechanism to diversify protein functionality in metazoans from a limited number of genes. In the Drosophila Down Syndrome Cell Adhesion Molecule (Dscam) important for neuronal wiring up to 38,016 isoforms can be generated by mutually exclusive alternative splicing in four clusters of variable exons. However, it is not understood how a specific exon is chosen from the many variables and how variable exons are prevented from being spliced together. A main role in the regulation of Dscam alternative splicing has been attributed to RNA binding proteins, but how they impact on exon selection is not well understood. Serine-arginine-rich (SR) proteins and hnRNP proteins are the two main types of RNA binding proteins with major roles in exon definition and splice site selection. Here, we analyzed the role of SR and hnRNP proteins in Dscam exon 9 alternative splicing in mutant Drosophila embryos because of their essential function for development. Strikingly, Dscam alternative exon selection is very robust against loss or overexpression of canonical SR and hnRNP proteins even when multiple proteins are depleted together. Conversely, non-canonical SR protein Serine-arginine repetitive matrix 234 (Srrm234) is a main determinant of exon inclusion in Dscam exon 9 cluster. Since long-range base-pairings are absent in the exon 9 cluster, our data argue for a small complement of regulatory factors as main determinants of exon inclusion in the Dscam exon 9 cluster. performed experiments. A. T-M and M.I. generated and validated the Srrm234 ∆ N allele. R.A. performed bioinformatic analysis. P.U. and M.S. wrote the manuscript. All authors read and approved the final manuscript. References Anko ML, Morales L, Henry I, Beyer A, Neugebauer KM. 2010. Global analysis reveals SRp20and SRp75-specific mRNPs in cycling and neural cells. Nat Struct Mol Biol 17: 962-970. Anko ML, Muller-McNicoll M, Brandl H, Curk T, Gorup C, Henry I, Ule J, Neugebauer KM. 2012. The RNA-binding landscapes of two SR proteins reveal unique functions and binding to diverse RNA classes. Genome Biol 13: R17. Appocher C, Mohagheghi F, Cappelli S, Stuani C, Romano M, Feiguin F, Buratti E. 2017. Major hnRNP proteins act as general TDP-43 functional modifiers both in Drosophila and human neuronal cells. Nucleic Acids Res 45: 8026-8045. Ustaoglu et al. 20 Bessonov S, Anokhina M, Will CL, Urlaub H, Luhrmann R. 2008. Isolation of an active step I spliceosome and composition of its RNP core. Nature 452: 846-850. Best A, Dalgliesh C, Kheirollahi-Kouhestani M, Danilenko M, Ehrmann I, Tyson-Capper A, Elliott DJ. 2014. Tra2 protein biology and mechanisms of splicing control. Biochem Soc Trans 42: 1152-1158. Black DL. 2003. Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem 72: 291-336. Blanchette M, Green RE, MacArthur S, Brooks AN, Brenner SE, Eisen MB, Rio DC. 2009. Genome-wide analysis of alternative pre-mRNA splicing and RNA-binding specificities of the Drosophila hnRNP A/B family members. Mol Cell 33: 438-449....

Running title: Long-term low dose Thiamethoxam exposure induces short ORFs AbstractMaximizing crop yields heavily relies on the use of agrochemicals to control insect pests. One of the most widely used insecticides are neonicotinoids. Here, we analysed the impact of sub-lethal chronic long-term exposure to the neonicotinoid Thiamethoxam on gene expression and alternative splicing in brains of Africanized honey bees Apis mellifera. Our results reveal a small number of differentially regulated genes showing a concentration dependent response to two low doses of chronic, 10-day Thiamethoxam exposure. Unexpectedly, most of these genes have no annotated function, but encode short Open Reading Frames (sORFs), a characteristic feature of anti-microbial peptides. Likewise, we find that Thiamethoxam exposure sensitizes bees to infection by non-pathogenic bacteria Bacillus badius and Ochrobactrum anthropi. Moreover, infection with estimated single Serratia marcescens kills bees arguing that Varroa mites may essentially contribute to colony collapse by penetrating the cuticle to spread this pathogen. Our results implicate an altered immune response to Thiamethoxam exposure compromising the immune response leading to a decline in bee populations.Understanding the risk for honey bees requires detailed knowledge of cellular and molecular effects that result from the exposure to an insecticide in order to mitigate negative effects or refine the target specificity towards pest species.Changes in gene expression and processing of RNAs, including alternative splicing, are one of the options available to an organism to respond to environmental perturbations 15,16 . Sub-lethal exposure of xenobiotics can induce modulation of splicing reactions [17][18][19] .Neonicotinoid exposure has been linked to a decline in bee health including a reduction of immune competence 20 ;; 21-23 . Insects do not have antibodies and rely on the innate immune system to fight microbial infections. The best insight on insect immunity stems from studies in the fruit fly Drosophila melanogaster, where cellular and humoral immune responses have been identified 24,25 . The cellular response is mediated by three types of hematopoetic cell lineages 24,26,27 , while the humoral immune system can be split into Toll and Imd pathways [reviewed in 28 ]. The Tollpathway is triggered following an immune challenge by Gram-positive bacteria and fungi, ultimately leading to expression of antimicrobial peptides (AMPs) that are then secreted from the fat body into the hemolymph 24,29,30 . In contrast, the Immune deficiency (Imd) pathway leads to expression of a different set of AMPs after a Gramnegative bacterial infection activates pattern recognition receptors and a complex intracellular signaling cascade 24,25,29 . AMPs are short peptides of 10-100 amino acids and are characterised by an evolutionary dynamism among insect species 31 .We have previously shown that worker-bee larvae in colonies contaminated with the neonicotinoid imidacloprid, have altered expression o...