Search

SUMMARYThe polarization of cells often involves the transport of specific mRNAs and their localized translation in distal projections. Neurons and glia both contain long cytoplasmic processes with important functions. mRNA localization has been studied extensively in neurons, but very little in glia, especially in intact nervous systems. Here, we predicted 1,700 localizedDrosophilaglial transcripts by extrapolating from our meta-analysis of 8 existing studies characterizing the localized transcriptomes and translatomes of synaptically-associated mammalian glia. We tested these predictions in glia of the neuromuscular junction ofDrosophilalarvae and found that localization to mammalian glia is a strong predictor of mRNA localization of the high confidenceDrosophilahomologues. We further showed that some of these localized transcripts are required in glia for plasticity of the neuromuscular junction synapses. We conclude that peripheral glial mRNA localization is a common and conserved phenomenon and propose that it is likely to be functionally important.

SummaryWhile post-transcriptional control is thought to be required at the periphery of neurons and glia, its extent is unclear. Here, we investigate systematically the spatial distribution and expression of mRNA at single molecule sensitivity and their corresponding proteins of 200 YFP trap protein trap lines across the intact Drosophila nervous system. 98% of the genes studied showed discordance between the distribution of mRNA and the proteins they encode in at least one region of the nervous system. These data suggest that post-transcriptional regulation is very common, helping to explain the complexity of the nervous system. We also discovered that 68.5% of these genes have transcripts present at the periphery of neurons, with 9.5% at the glial periphery. Peripheral transcripts include many potential new regulators of neurons, glia and their interactions. Our approach is applicable to most genes and tissues and includes powerful novel data annotation and visualisation tools for post-transcriptional regulation.Brief outlineA novel high resolution and sensitive approach to systematically co-visualise the distribution of mRNAs and proteins in the intact nervous system reveals that post-transcriptional regulation of gene expression is very common. The rich data landscape is provided as a browsable resource (link), using Zegami, a cloud-based data exploration platform (link). Our solution provides a paradigm for the characterisation of post-transcriptional regulation of most genes and model systems.Highlights196/200 (98%) Drosophila genes show discordant RNA and protein expression in at least one nervous system region137/200 (68.5%) mRNAs are present in at least one synaptic compartmentNovel localised mRNA and protein discovered in periphery of glial processesNew paradigm for analysis of post-transcriptional regulation and data exploration

Despite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single-molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, subgenomic RNAs, and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications.

Summary Microglia are implicated in neurodegeneration, potentially by phagocytosing neurons, but it is unclear how to block the detrimental effects of microglia while preserving their beneficial roles. The microglial P2Y6 receptor (P2Y6R) – activated by extracellular UDP released by stressed neurons – is required for microglial phagocytosis of neurons. We show here that injection of amyloid beta (Aβ) into mouse brain induces microglial phagocytosis of neurons, followed by neuronal and memory loss, and this is all prevented by knockout of P2Y6R. In a chronic tau model of neurodegeneration (P301S TAU mice), P2Y6R knockout prevented TAU-induced neuronal and memory loss. In vitro, P2Y6R knockout blocked microglial phagocytosis of live but not dead targets and reduced tau-, Aβ-, and UDP-induced neuronal loss in glial-neuronal cultures. Thus, the P2Y6 receptor appears to mediate Aβ- and tau-induced neuronal and memory loss via microglial phagocytosis of neurons, suggesting that blocking this receptor may be beneficial in the treatment of neurodegenerative diseases., Graphical abstract, Highlights • P2Y6R knockout prevents microglial phagocytosis of stressed-but-viable cells • P2Y6R knockout does not alter microglial phagocytosis of healthy or dead cells • P2Y6R knockout prevents microglial phagocytosis of neurons induced by Aβ in vivo • P2Y6R knockout reduces neuronal loss and memory deficits induced by Aβ or Tau in mice, Puigdellívol et al. find that the knockout of the microglial P2Y6 receptor, required for microglial engulfment of neurons, prevents neuronal and memory loss in two different mouse models of neurodegeneration, suggesting that neuronal loss in neurodegeneration is due to microglia eating neurons, which may be prevented by blocking the P2Y6 receptor.

SummaryDespite an unprecedented global research effort on SARS-CoV-2, early replication events remain poorly understood. Given the clinical importance of emergent viral variants with increased transmission, there is an urgent need to understand the early stages of viral replication and transcription. We used single molecule fluorescence in situ hybridisation (smFISH) to quantify positive sense RNA genomes with 95% detection efficiency, while simultaneously visualising negative sense genomes, sub-genomic RNAs and viral proteins. Our absolute quantification of viral RNAs and replication factories revealed that SARS-CoV-2 genomic RNA is long-lived after entry, suggesting that it avoids degradation by cellular nucleases. Moreover, we observed that SARS-CoV-2 replication is highly variable between cells, with only a small cell population displaying high burden of viral RNA. Unexpectedly, the B.1.1.7 variant, first identified in the UK, exhibits significantly slower replication kinetics than the Victoria strain, suggesting a novel mechanism contributing to its higher transmissibility with important clinical implications.Graphical AbstractIn briefBy detecting nearly all individual SARS-CoV-2 RNA molecules, we quantified viral replication and defined cell susceptibility to infection. We discovered that a minority of cells show significantly elevated viral RNA levels and observed slower replication kinetics for the Alpha variant relative to the Victoria strain.HighlightsSingle molecule quantification of SARS-CoV-2 replication uncovers early infection kineticsThere is substantial heterogeneity between cells in rates of SARS-CoV-2 replicationGenomic RNA is stable and persistent during the initial stages of infectionB.1.1.7 variant replicates more slowly than the Victoria strain

COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 2 million fatalities to date. Viral replication is shaped by the cellular microenvironment and one important factor to consider is oxygen tension, where hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its Spike glycoprotein binding to angiotensin-converting enzyme (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via a HIF-1α dependent pathway. Hypoxia and Roxadustat inhibit SARS-CoV-2 RNA replication showing that post-entry steps in the viral life cycle are oxygen-sensitive. This study highlights the importance of HIF signalling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention or treatment of COVID-19., Graphical Abstract, Wing et al. demonstrate that key aspects of the SARS-CoV-2 life cycle are dependent on cellular oxygen tension. Activation of the cellular oxygen sensing pathway inhibits SARS-CoV-2 infection, highlighting a key cellular pathway that could be exploited as a potential therapeutic avenue for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19. SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control its life cycle remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively the cellular and viral RBPs that are involved in SARS-CoV-2 infection. We reveal that SARS-CoV-2 infection profoundly remodels the cellular RNA-bound proteome, which includes wide-ranging effects on RNA metabolic pathways, non-canonical RBPs and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Amongst them, several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19., Graphical Abstract, Kamel, Noerenberg, Cerikan and colleagues apply a multi-omic approach to identify the RNA-binding proteins that regulate SARS-CoV-2 infection. They discovered that the complement of RNA-binding proteins heavily remodels upon SARS-CoV-2 infection. They also show that the viral RNA interacts with dozens of cellular and six viral RNA-binding proteins. These host-virus interactions are fundamental for SARS-CoV-2 infection and have great potential for new therapeutic approaches against COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19. SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control SARS-CoV-2 infection remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively which cellular and viral RBPs are involved in SARS-CoV-2 infection. We reveal that the cellular RNA-bound proteome is remodelled upon SARS-CoV-2 infection, having widespread effects on RNA metabolic pathways, non-canonical RBPs and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Amongst them, several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.

During Drosophila and vertebrate brain development, the conserved transcription factor Prospero/Prox1 is an important regulator of the transition between proliferation and differentiation. Prospero level is low in neural stem cells and their immediate progeny, but is upregulated in larval neurons and it is unknown how this process is controlled. Here, we use single molecule fluorescent in situ hybridisation to show that larval neurons selectively transcribe a long prospero mRNA isoform containing a 15 kb 3′ untranslated region, which is bound in the brain by the conserved RNA-binding protein Syncrip/hnRNPQ. Syncrip binding increases the stability of the long prospero mRNA isoform, which allows an upregulation of Prospero protein production. Adult flies selectively lacking the long prospero isoform show abnormal behaviour that could result from impaired locomotor or neurological activity. Our findings highlight a regulatory strategy involving alternative polyadenylation followed by differential post-transcriptional regulation. This article has an associated First Person interview with the first author of the paper., Summary: Different prospero mRNA isoforms have different stabilities, depending on an interaction with the RNA binding protein Syncrip. Isoform choice in different cell types controls the expression level of Prospero protein.

COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 1 million fatalities to date. Understanding how host factors modify the viral life cycle could inform susceptibility to viral infection and the design of new therapies. Viral replication is shaped by the cellular microenvironment and one important factor is local oxygen tension, where hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its Spike glycoprotein binding to angiotensin-converting enzyme (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat (FG-4592) reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via a HIF-1α dependent signalling pathway. Further, hypoxia and Roxadustat inhibit viral replication in SARS-CoV-2 infected cells, showing that post-entry steps in the viral life cycle are oxygen-sensitive. This study highlights the importance of hypoxia and HIF signalling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention and/or treatment of COVID-19. Funding: The McKeating laboratory is funded by a Wellcome Investigator Award (IA) 200838/Z/16/Z, UK Medical Research Council (MRC) project grant MR/R022011/1 and Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China (grant number: 2018-I2M-2-002). The Ratcliffe laboratory is funded by the Oxford Branch of the Ludwig Institute for Cancer Research; Wellcome IA 106241/Z/14/Z; the Francis Crick Institute, which receives core funding from Cancer Research UK (FC001501), UK MRC (FC001501) and Wellcome (FC001501); the Paradifference Foundation. PJR, EJH and TB are additionally funded by the COVID-19 Research Response Fund, University of Oxford. SK is funded by the Clarendon Scholarships Fund and the Christopher Welch Trust. The Davis laboratory is funded by Wellcome IA 209412/Z/17/Z and Wellcome Strategic Awards 091911/B/10/Z and 107457/Z/15/Z. JYL is funded by the Medial Sciences Graduate Studentship, University of Oxford. The Hinks laboratory is funded by grants from the Wellcome (104553/z/14/z, 211050/Z/18/z) and the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre; the views expressed are those of the authors and not those of the NHS or NIHR. Conflict of Interest: EJH is employed under the Cambridge Experimental Medicine Initiative, which is partly funded by AstraZeneca although they have not been involved in this project. The other authors declare no financial interests. Ethical Approval: The study was reviewed by the Oxford Research Ethics Committee B (18/SC/0361).

The current study has been undertaken by studying the data from the hospital registers at the Kasugu District Hospital, Malawi. The malaria cases of 2014 were chosen for the study. They typically represent disease distribution based on the ages and genders of the patients. The data suggest that the cure rates among patients under age 5 and above do not have any significant difference, contrary to the commonly anticipated outcome that the older children should have better developed immune systems and should have higher cure rates. A notable point is there are huge dichotomies between genders in terms of the reported registered cases, the cure and mortality rates. The gender differentiation in the reporting and management of malaria is understood under the contemporary socio-cultural beliefs and economic realities of the Malawian society. This analysis has been carried out from a qualitative perspective, but with an attempt to identify a pattern from a year's length of statistical data. Key words: Malaria epidemiology, malaria cases, mortality, case fatality rates.

Publisher Summary At the microscopic level, tissues are composed of complicated interacting and interdependent cell populations, which are regulated by the local extracellular matrix. Thus, analyzing critical gene expression patterns in development, normal function, and disease progression depends on the extraction of specific cells from their complex tissue milieu. Laser capture microdissection (LCM) is developed to provide a rapid, reliable method to procure pure populations of selected cells from specific microscopic regions of tissue sections for molecular analysis. This chapter discusses the molecular analysis of microdissected tissue. Further, it describes protocols for preparing the tissue before microdissection and conducting the PCR amplification after microdissection. Finally, it outlines the applications. DNA obtained from cells procured by LCM from clinical specimens is analyzed by a variety of methods, including loss of heterozygosity, clonal analysis, and direct sequencing. Similarly, LCM is used to study expression differences in various tissues using RNA-based techniques, such as cDNA library construction, microarray hybridization, and differential gene expression.