Neural Differentiation Modulates the Vertebrate Brain Specific Splicing Program

Alternative splicing patterns are known to vary between tissues but these patterns have been found to be predominantly peculiar to one species or another, implying only a limited function in fundamental neural biology. Here we used high-throughput RT-PCR to monitor the expression pattern of all the annotated simple alternative splicing events (ASEs) in the Reference Sequence Database, in different mouse tissues and identified 93 brain-specific events that shift from one isoform to another (switch-like) between brain and other tissues. Consistent with an important function, regulation of a core set of 9 conserved switch-like ASEs is highly conserved, as they have the same pattern of tissue-specific splicing in all vertebrates tested: human, mouse and zebrafish. Several of these ASEs are embedded within genes that encode proteins associated with the neuronal microtubule network, and show a dramatic and concerted shift within a short time window of human neural stem cell differentiation. Similarly these exons are dynamically regulated in zebrafish development. These data demonstrate that although alternative splicing patterns often vary between species, there is nonetheless a core set of vertebrate brain-specific ASEs that are conserved between species and associated with neural differentiation.     

Lack of Dietary Polyunsaturated Fatty Acids Causes Synapse Dysfunction in the Drosophila Visual System

Polyunsaturated fatty acids (PUFAs) are essential nutrients for animals and necessary for the normal functioning of the nervous system. A lack of PUFAs can result from the consumption of a deficient diet or genetic factors, which impact PUFA uptake and metabolism. Both can cause synaptic dysfunction, which is associated with numerous disorders. However, there is a knowledge gap linking these neuronal dysfunctions and their underlying molecular mechanisms. Because of its genetic manipulability and its easy, fast, and cheap breeding, Drosophila melanogaster has emerged as an excellent model organism for genetic screens, helping to identify the genetic bases of such events. As a first step towards the understanding of PUFA implications in Drosophila synaptic physiology we designed a breeding medium containing only very low amounts of PUFAs. We then used the fly’s visual system, a well-established model for studying signal transmission and neurological disorders, to measure the effects of a PUFA deficiency on synaptic function. Using both visual performance and eye electrophysiology, we found that PUFA deficiency strongly affected synaptic transmission in the fly’s visual system. These defects were rescued by diets containing omega-3 or omega-6 PUFAs alone or in combination. In summary, manipulating PUFA contents in the fly’s diet was powerful to investigate the role of these nutrients on the fly´s visual synaptic function. This study aims at showing how the first visual synapse of Drosophila can serve as a simple model to study the effects of PUFAs on synapse function. A similar approach could be further used to screen for genetic factors underlying the molecular mechanisms of synaptic dysfunctions associated with altered PUFA levels.     

Fine‐scale refuges can buffer demographic and genetic processes against short‐term climatic variation and disturbance: a 22‐year case study of an arboreal marsupial

Ecological disturbance and climate are key drivers of temporal dynamics in the demography and genetic diversity of natural populations. Microscale refuges are known to buffer species’ persistence against environmental change, but the effects of such refuges on demographic and genetic patterns in response to short‐term environmental variation are poorly understood. We quantified demographic and genetic responses of mountain brushtail possums (Trichosurus cunninghami) to rainfall variability (1992–2013) and to a major wildfire. We hypothesized that there would be underlying differences in demographic and genetic processes between an unburnt mesic refuge and a topographically exposed zone that was burnt in 2009. Fire caused a 2‐year decrease in survival in the burnt zone, but the population grew after the fire due to immigration, leading to increased expected heterozygosity. We documented a fire‐related behavioural shift, where the rate of movement by individuals in the unburnt refuge to the burnt zone decreased after fire. Irrespective of the fire, there were long‐term differences in demographic and genetic parameters between the mesic/unburnt refuge and the nonmesic/burnt zone. Survival was high and unaffected by rainfall in the refuge, but lower and rainfall‐dependent in the nonmesic zone. Net movement of individuals was directional, from the mesic refuge to the nonmesic zone, suggesting fine‐scale source–sink dynamics. There were higher expected heterozygosity (H_E) and temporal genetic stability in the refuge, but lower H_E and marked temporal genetic structure in the exposed habitat, consistent with reduced generational overlap caused by elevated mortality and immigration. Thus, fine‐scale refuges can mediate the short‐term demographic and genetic effects of climate and ecological disturbance.     

Mapping the genetic and tissular diversity of 64 phenolic compounds in Citrus species using a UPLC–MS approach

Background and Aims Phenolic compounds contribute to food quality and have potential health benefits. Consequently, they are an important target of selection for Citrus species. Numerous studies on this subject have revealed new molecules, potential biosynthetic pathways and linkage between species. Although polyphenol profiles are correlated with gene expression, which is responsive to developmental and environmental cues, these factors are not monitored in most studies. A better understanding of the biosynthetic pathway and its regulation requires more information about environmental conditions, tissue specificity and connections between competing sub-pathways. This study proposes a rapid method, from sampling to analysis, that allows the quantitation of multiclass phenolic compounds across contrasting tissues and cultivars.Methods Leaves and fruits of 11 cultivated citrus of commercial interest were collected from adult trees grown in an experimental orchard. Sixty-four phenolic compounds were simultaneously quantified by ultra-high-performance liquid chromatography coupled with mass spectrometry.Key Results Combining data from vegetative tissues with data from fruit tissues improved cultivar classification based on polyphenols. The analysis of metabolite distribution highlighted the massive accumulation of specific phenolic compounds in leaves and the external part of the fruit pericarp, which reflects their involvement in plant defence. The overview of the biosynthetic pathway obtained confirmed some regulatory steps, for example those catalysed by rhamnosyltransferases. The results suggest that three other steps are responsible for the different metabolite profiles in ‘Clementine’ and ‘Star Ruby’ grapefruit.Conclusions The method described provides a high-throughput method to study the distribution of phenolic compounds across contrasting tissues and cultivars in Citrus, and offers the opportunity to investigate their regulation and physiological roles. The method was validated in four different tissues and allowed the identification and quantitation of 64 phenolic compounds in 20 min, which represents an improvement over existing methods of analysing multiclass polyphenols.     

Regulation of Stress-Inducible Phosphoprotein 1 Nuclear Retention by Protein Inhibitor of Activated STAT PIAS1

Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450–480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity.Stress-inducible phosphoprotein I (STI1)¹ is a conserved cochaperone protein that assists Hsp90 in managing client proteins, by mediating the transfer of proteins between Hsp70 and Hsp90 (1–3). STI1 contains several tetratricopeptide-repeat domains (TRP) that can serve as interaction modules with Hsp90 and Hsp70 (4). STI1 helps to drive the sequential steps involved in the Hsp90 chaperone machinery (5) and regulates the ATPase activity of Hsp90 (6, 7). STI1 is also secreted by distinct cells (8–12), using a noncanonical mechanism involving extracellular vesicles (11). Secreted STI1 can activate multiple signaling pathways in distinct cell types (8–10, 13–18).Elimination of STI1 in yeast sensitizes cells to Hsp90 inhibitors, but it is not by itself lethal (19). STI1 can also be eliminated in C. elegans, although it results in decreased life span (20). In contrast, STI1 mutant mice do not survive E10.5 and present several morphological defects, owing to decreased levels of several Hsp90-client proteins (21). Mouse embryonic fibroblasts obtained from STI1-deficient embryos also fail to thrive and present increased levels of the DNA damage marker γ-H2AX, suggestive of increased cellular stress (21). Hence, in mammals STI1 seems to play additional roles in cellular survival that are not yet fully understood.STI1 is abundantly expressed in the cytoplasm of cells, but can also be found in the Golgi (22), in vesicles and in multivesicular bodies (11). Moreover, this cochaperone has been shown to shuttle between the cytoplasm and the nucleus in cell lines (23). Cellular stress, arrest in G1/S phase of the cell cycle and phosphorylation are factors that seem to regulate STI1 nuclear localization (23, 24). Presumably nuclear STI1 can regulate chaperone activity, but whether it can interact with nuclear proteins is unknown.Previous experiments using cell lines have shown that knockdown of STI1 increases susceptibility of cells to irradiation (25). Whether changes in STI1 levels in primary differentiated cells, such as astrocytes, may affect their response to irradiation stress is unknown. This is of interest, as astrocytes, which can give rise to distinct tumor cells, are highly radioresistant (26). Indeed, astrocytes have a noncanonical DNA damage response (DDR) to irradiation (26). Here we show that STI1 undergoes nuclear translocation in astrocytes after γ-radiation-induced DNA damage. Moreover, astrocytes haploinsufficient for STI1 are more susceptible to cell death induced by irradiation. To understand potential mechanisms involved with STI1 nuclear retention, we have performed yeast-two hybrid screenings to identify STI1 nuclear partners. We identified protein inhibitor of activated STAT (PIAS1) as a direct interactor of STI1 and provide evidence that it acts as a small ubiquitin-like modifier (SUMO) E3 ligase for STI1. We show this interaction is involved with STI1 nuclear retention after irradiation. Interestingly, tissue microarray analysis demonstrated that higher PIAS1 levels are found in glioblastoma multiforme (GBM) when compared with non-neoplastic tissue. Furthermore, we uncovered a positive relationship between increased PIAS1 expression in GBMs and augmented STI1 nuclear localization. Our results reveal a novel mechanism by which increased expression of PIAS1, as observed in GBM, can increase the retention of nuclear STI1, a critical regulator of the chaperone machinery.     

Drosophila S2 Cells Secrete Wingless on Exosome‐Like Vesicles but the Wingless Gradient Forms Independently of Exosomes

Wingless acts as a morphogen in Drosophila wing discs, where it specifies cell fates and controls growth several cell diameters away from its site of expression. Thus, despite being acylated and membrane associated, Wingless spreads in the extracellular space. Recent studies have focussed on identifying the route that Wingless follows in the secretory pathway and determining how it is packaged for release. We have found that, in medium conditioned by Wingless‐expressing Drosophila S2 cells, Wingless is present on exosome‐like vesicles and that this fraction activates signal transduction. Proteomic analysis shows that Wingless‐containing exosome‐like structures contain many Drosophila proteins that are homologous to mammalian exosome proteins. In addition, Evi, a multipass transmembrane protein, is also present on exosome‐like vesicles. Using these exosome markers and a cell‐based RNAi assay, we found that the small GTPase Rab11 contributes significantly to exosome production. This finding allows us to conclude from in vivo Rab11 knockdown experiments, that exosomes are unlikely to contribute to Wingless secretion and gradient formation in wing discs. Consistent with this conclusion, extracellularly tagged Evi expressed from a Bacterial Artificial Chromosome is not released from imaginal disc Wingless‐expressing cells.     

A core seed endophytic bacterial community in the hyperaccumulator Noccaea caerulescens across 14 sites in France

Plant and Soil - Non-mycorrhizal species such as Banksia (Proteaceae) that depend on root exudates to acquire phosphorus (P) are prominent in south-western Australia, a biodiversity hotspot on...