Project description:During the bloodstream stage of the Trypanosoma brucei lifecycle, the parasite exists as the proliferative slender-form or the non-proliferative, transmissible, stumpy-form. The transition from the slender to stumpy-form is stimulated by a density-dependent mechanism and is important in infection dynamics, ordered antigenic variation and disease transmissibility. Here, we use a monomorphic reporter cell line in a whole-cell fluorescence-based assay to screen over 6000 small molecules from a kinase-focussed compound library for their ability to induce stumpy-like formation in a high-throughput screening programme. This identified one compound able to induce modest, yet specific, changes in gene expression indicative of a partial differentiation to stumpy forms. This not only provides a potential tool for the further understanding of stumpy formation, but also demonstrates the use of high throughput screening in the identification of compounds able to induce specific phenotypes, such as differentiation, in African trypanosomes. Examination of gene expression in response to treatment with DDD00015314.
Project description:Biochemical characterization of the hypoxia-induced long non coding RNA NTRAS. NTRAS was found to regulate cell cycle progression, in vitro sprouting angiogenesis, and the paracellular permeability in human umbilical vein endothelial cells (HUVECs). Using desthiobiotinylated 2’O-Me-RNA probes, we purified endogenous NTRAS-protein-complexes and identified NTRAS interacting proteins by mass spectrometry.
Project description:African trypanosomes have been recently shown to colonise the skin in a process critical for parasite transmission. However, the tissue responses to infection, especially in the lead to parasite transition from the host skin to the vector remain unresolved. Here, using a combination of spatial and single cell transcriptomics, coupled with imaging mass cytometry and genetic models, we investigated the local immune response of the skin in both a murine model of infection and in human samples from the Democratic Republic of Congo (DRC). Our results provide several novel key findings previously unappreciated in the context of parasitic infections in the skin. Firstly, we identified that the skin stromal cells, in particular interstitial preadipocytes located in the subcutis, upregulate several genes involved in inflammatory signalling and antigen presentation, including several molecules involved in T cell activation and survival. Secondly, we detected a significant expansion of a population of IL-17 producing Vg6 gdT cells in the infected murine skin compared to naïve controls, that occur mainly in the subcutis, that we further validated at the protein level by flow cytometry. In silico cell-cell communication analyses between adipocytes and T cells suggests that adipocytes trigger T cell activation locally via Il6, Il10, and Tnfsf18 signalling, amongst others. Thirdly, mice deficient of Vg6 gdT cells show extensive inflammation, increased IFNg-producing CD4+ T cells, and tissue parasite burden compared to naïve controls, indicating that Vg6 gdT cells are important to limit skin inflammation and parasite replication. Based on these observations, we proposed a model whereby interstitial preadipocytes (and potentially adipocytes) as well as Vg6 gdT cells act concertedly in the subcutis to limit tissue damage and parasite load, thus imposing an immunological barrier for transmission. These studies shed light into the mechanisms of gdT cells-mediated immunity in the skin in the context of African trypanosomes infection, as well as a potential role of immature and mature adipocytes as homeostatic regulators on the skin during chronic infection.
Project description:Echinococcus multilocularis (Em) infection and the growth and proliferation of its metacestode within the liver of hosts are related to complex host–parasite interactions at the molecular level. However, the profiles of long non-coding RNAs (lncRNAs) and mRNAs of mice in response to Em are poorly understood. In this study, we detected numerous differentially expressed lncRNAs (DELs) and mRNAs (DEMs) in the mouse liver at eight time points after Em infection. Some DEMs and DELs were found continuously dysregulated. These DEMs were notably enriched in the “antigen processing and presentation,” “Th1 and Th2 cell differentiation” and “Th17 cell differentiation” pathways.
Project description:Neurogenesis is a pro-survival process that comprises of dendritic and axonal growth, synaptogenesis, synaptic and neuronal pruning. These complex processes are determined by temporal gene expression during development, which is in turn tightly regulated by long non-coding RNAs and microRNAs. In this study, we investigated the processes implicated in the maturation of primary neuronal cultures based on RNA expression profiling. Correlation between neuron specific gene ontologies of mRNA and non-coding RNAs identified direct regulation of axonogenesis and dendritogenesis. Temporally regulated mRNA and their associated long non-coding RNAs were significantly overrepresented in proliferation and differentiation associated signalling, cell adhesion molecules and neurotrophin signalling pathways during neuronal maturation. Long non-coding RNAs associated with Axin2, Cntn1, Ncam1, Negr1, Ntrk2, Nrxn1 and Sh2b3 displayed an inverse expression profile to their mRNA whereas long non-coding RNA -mRNA pairs for Kit, Prkcb and Ralgds displayed similar expression profiles. These genes were also predicted targets of the altered miRNAs, miR-124, -128, -129-5p, -203, -218, -290-5p, -326, -329, -377 and -495. These microRNAs particularly regulate the cell adhesion molecules, Cntn1, Ncam1, Negr1 and Nrxn1 that determine axonogenesis and dendritogenesis, supporting the observed co-regulation of these biological processes by non-coding RNAs. Verification of expression of these long non-coding RNA-mRNA pairs in an in vitro model of ischemic-reperfusion injury showed an inverse expression profile, thus confirming their role(s) in maintenance of the neuronal structure and function. This neuronal transcriptome (mRNAs, lncRNAs, miRNAs) is in turn orchestrated by C/EBPM-NM-1/M-NM-2 transcription factors and CTCF, thereby governing intricate control of neuronal development. mRNA and long non-coding RNA expression profiling of maturing primary cortical neurons from E15 mouse embryos and neurons subjected to oxygen-glucose deprivation. Maturing neurons were harvested on Days 2, 4, 6 and 8. Neurons on Day 6 were subjected to oxygen-glucose deprivation for different time periods and 24 hours reperfusion before being harvested.
Project description:The eukaryotic RNA processing factor Y14 participates in double-strand break (DSB) repair via its RNA-dependent interaction with the non-homologous end-joining (NHEJ) complex. We identified the long non-coding RNA HOTAIRM1 as a candidate that mediates this interaction. HOTAIRM1 localized to DNA damage sites induced by ionizing radiation. Depletion of HOTAIRM1 delayed the recruitment of DNA damage response and repair factors to DNA lesions and reduced DNA repair efficiency. Identification of the HOTAIRM1 interactome revealed a large set of RNA processing factors including mRNA surveillance factors. The surveillance factors Upf1 and SMG6 localized to DNA damage sites in a HOTAIRM1-dependent manner. Depletion of Upf1 or SMG6 increased the level of DSB-induced non-coding transcripts at damaged sites, indicating a pivotal role for Upf1/SMG6-mediated RNA degradation in DNA repair. We conclude that HOTAIRM1 serves as an assembly scaffold for both DNA repair and RNA processing factors that act in concert to repair DSBs.
Project description:The nuclear receptor, Farnesoid X Receptor (FXR, NR1H4), is a key transcriptional regulator of metabolism and a promising drug target for non-alcoholic fatty liver disease (NAFLD), a leading cause of liver failure and death. Protein coding genes regulated by FXR are known, but it remains unknown whether FXR mediates its function through regulating expression of long non-coding RNA (lncRNA) genes. Utilizing global RNA-seq and Gro-seq analyses, we identify an FXR-induced novel long non-coding RNA (lncRNA), termed FincoR.
Project description:RNA-binding proteins are key players in coordinated post-transcriptional regulation of functionally related genes, defined as RNA regulons. RNA regulons play particularly critical roles in parasitic trypanosomes, which exhibit unregulated co-transcription of long unrelated gene arrays. In this report, we present a systematic analysis of an essential RNA-binding protein, RBP42, in the mammalian-infective bloodstream form of African trypanosome, and show that RBP42 is a key regulator of parasite’s central carbon and energy metabolism. Using individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to identify genome-wide RBP42-RNA interactions, we show that RBP42 preferentially binds within the coding region of mRNAs encoding core metabolic enzymes. Global quantitative transcriptomic and proteomic analyses reveal that loss of RBP42 reduces the abundance of target mRNA-encoded proteins, but not target mRNA, suggesting a positive translational regulatory role of RBP42. Significant changes in central carbon metabolic intermediates, following loss of RBP42, further support its critical role in cellular energy metabolism.
Project description:The evolution of brain complexity correlates with an increased expression of long, non-coding (lnc) RNAs in neuronal tissues. Although prominent examples illustrate the potential of lncRNAs to scaffold and target epigenetic regulators to chromatin loci, only few cases have been described to function during neurogenesis. We present a first functional characterization of the lncRNA LINC01322, which we term RUS for ‘RNA upstream of Slitrk3’. The RUS gene is well conserved in mammals by sequence and synteny next to the neurodevelopmental gene Slitrk3. RUS is exclusively expressed in neural cells and its expression increases along with neuronal markers during neuronal differentiation of mouse embryonic cortical neural stem cells. Depletion of RUS locks neuronal precursors in an intermediate state towards neuronal differentiation, with arrested cell cycle and increased apoptosis. RUS associates with chromatin in the vicinity of genes involved in neurogenesis, most of which change their expression upon RUS depletion. The identification of a range of epigenetic regulators as specific RUS interactors suggests that the lncRNA may mediate gene activation and repression in a highly context-dependent manner.