Circular RNAs (circRNAs) are a novel class of noncoding RNAs present in all eukaryotic cells investigated so far and generated by a special mode of alternative splicing of pre-mRNAs. Thereby, single exons, or multiple adjacent and spliced exons, are released in a circular form. CircRNAs are cell-type specifically expressed, are unusually stable, and can be found in various body fluids such as blood and saliva. Here we analysed circRNAs and the corresponding linear splice isoforms from human platelets, where circRNAs are particularly abundant, compared with other hematopoietic cell types. In addition, we isolated extracellular vesicles from purified and in vitro activated human platelets, using density-gradient centrifugation, followed by RNA-seq analysis for circRNA detection. We could demonstrate that circRNAs are packaged and released within both types of vesicles (microvesicles and exosomes) derived from platelets. Interestingly, we observed a selective release of circRNAs into the vesicles, suggesting a specific sorting mechanism. In sum, circRNAs represent yet another class of extracellular RNAs that circulate in the body and may be involved in signalling pathways. Since platelets are essential for central physiological processes such as haemostasis, wound healing, inflammation and cancer metastasis, these findings should greatly extend the potential of circRNAs as prognostic and diagnostic biomarkers.
Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring (U1, U5, spliced leader snRNPs), or variant Sm cores with snRNA-specific Sm subunits (U2, U4 snRNPs). Searching for specificity factors, we identified SMN and Gemin2 proteins that are highly divergent from known orthologs. SMN is splicing-essential in trypanosomes and nuclear-localized, suggesting that Sm core assembly in trypanosomes is nuclear. We demonstrate in vitro that SMN is sufficient to confer specificity of canonical Sm core assembly and to discriminate against binding to nonspecific RNA and to U2 and U4 snRNAs. SMN interacts transiently with the SmD3B subcomplex, contacting specifically SmB. SMN remains associated throughout the assembly of the Sm heteroheptamer and dissociates only when a functional Sm site is incorporated. These data establish a novel role of SMN, mediating snRNP specificity in Sm core assembly, and yield new biochemical insight into the mechanism of SMN activity. Before pre-mRNA splicing catalysis can occur, each intron-exon unit has to assemble into a large spliceosome complex, composed of small nuclear ribonucleoproteins (snRNPs) and many additional non-snRNP proteins. This occurs in an ordered multistep process with the spliceosome undergoing several conformational transitions (Brow 2002;Will and Lü hrmann 2006). Each of the snRNPs is assembled from snRNA and proteins that are characterized very well in their biochemical composition for the mammalian system (Will and Lü hrmann 2001). The protein components can be divided into the Sm core; a heteroheptameric, ring-like complex of seven Sm proteins (SmD3B, D1D2, EFG in the U1, U2, U4, and U5 snRNPs; LSm2-8 in the U6 snRNP); and additional, snRNA-specific proteins (Kambach et al. 1999; for review, see Khusial et al. 2005).In contrast to the mammalian and yeast systems, only a few protein components of the splicing machinery have been identified in trypanosomes. Trypanosomes are particularly interesting since their expression of proteincoding genes requires trans splicing, and in addition-at least for a small number of genes-cis splicing (for review, see Liang et al. 2003): The U2, U4/U6, and U5 snRNPs are considered to be general and essential splicing factors, whereas the SL RNP and the U1 snRNP represent transand cis-splicing-specific components, respectively.Recently, first examples of what we called ''Sm core variation'' were described in spliceosomal snRNPs from trypanosomes: First, in the U2 snRNP, two of the canonical Sm polypeptides, SmD3B, are replaced by two U2-specific Sm proteins, Sm16.5K/15K (Wang et al. 2006). Second, at least in a fraction of the U4 snRNP, the U4-specific Ssm4/LSm2 substitutes for the canonical SmD3 (Wang et al. 2006;Tkacz et al. 2007; N. Jaé and A. Bindereif, unpubl.). In sum, the U2 and U4 Sm core variants differ in one or two of the canonical Sm subunits. As shown so far for U2, this Sm core variation mediates RNA-binding specificity for the unusual Sm site in the U2 snRNA (Wang ...
Trypanosoma brucei brucei trypomastigotes are classical blood parasites of cattle, these stages might become potential targets for circulating polymorphonuclear neutrophils (PMN). We here investigated NETs extrusion and related oxygen consumption in bovine PMN exposed to motile T. b. brucei trypomastigotes in vitro. Parasite exposure induced PMN activation as detected by enhanced oxygen consumption rates (OCR), extracellular acidification rates (ECAR), and production of total and extracellular reactive oxygen species (ROS). Scanning electron microscopy (SEM) showed that co-cultivation of bovine PMN with motile trypomastigotes resulted in NETs formation within 120 min of exposure. T. b. brucei-induced NETs were confirmed by confocal microscopy demonstrating co-localization of extruded DNA with neutrophil elastase (NE) and nuclear histones. Immunofluorescence analyses demonstrated that trypomastigotes induced different phenotypes of NETs in bovine PMN, such as aggregated NETs (aggNETs), spread NETs (sprNETs), and diffuse NETs (diffNETs) with aggNETs being the most abundant ones. Furthermore, live cell 3D-holotomographic microscopy unveiled detailed morphological changes during the NETotic process. Quantification of T. b. brucei-induced NETs formation was estimated by DNA and nuclear area analysis (DANA) and confirmed enhanced NETs formation in response to trypomastigote stages. Formation of NETs does not result in a decrease of T. b. brucei viability, but a decrease of 26% in the number of motile parasites. Referring the involved signaling pathways, trypomastigoteinduced NETs formation seems to be purinergic-dependent, since inhibition via NF449 treatment resulted in a significant reduction of T. b. brucei-triggered DNA extrusion. Overall, future studies will have to analyze whether the formation of aggNETs indeed plays a role in the outcome of clinical disease and bovine African trypanosomiasisrelated immunopathological disorders, such as increased intravascular coagulopathy and vascular permeability, often reported to occur in this disease.
The generation of mature mRNA in the protozoan parasite Trypanosoma brucei requires coupled polyadenylation and trans splicing. In contrast to other eukaryotes, we still know very little on components, mechanisms, and dynamics of the 3 0 end-processing machinery in trypanosomes. To characterize the catalytic core of the polyadenylation complex in T. brucei, we first identified the poly(A) polymerase [Tb927.7.3780] as the major functional, nuclear-localized enzyme in trypanosomes. In contrast, another poly(A) polymerase, encoded by an intron-containing gene [Tb927.3.3160], localizes mainly in the cytoplasm and appears not to be functional in general 3 0 end processing of mRNAs. Based on tandemaffinity purification with tagged CPSF160 and mass spectrometry, we identified ten associated components of the trypanosome polyadenylation complex, including homologues to all four CPSF subunits, Fip1, CstF50/64, and Symplekin, as well as two hypothetical proteins. RNAi-mediated knockdown revealed that most of these factors are essential for growth and required for both in vivo polyadenylation and trans splicing, arguing for a general coupling of these two mRNA-processing reactions.
Messenger RNA processing in trypanosomes by cis and trans splicing requires spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6, and U5, as well as the spliced leader (SL) RNP. As in other eukaryotes, these RNPs share a core structure of seven Sm polypeptides. Here, we report that the identity of the Sm protein constituents varies between spliceosomal snRNPs: specifically, two of the canonical Sm proteins, SmB and SmD3, are replaced in the U2 snRNP by two novel, U2 snRNPspecific Sm proteins, Sm15K and Sm16.5K. We present a model for the variant Sm core in the U2 snRNP, based on tandem affinity purification-tagging and in vitro proteinprotein interaction assays. Using in vitro reconstitutions with canonical and U2-specific Sm cores, we show that the exchange of two Sm subunits determines discrimination between individual Sm sites. In sum, we have demonstrated that the heteroheptameric Sm core structure varies between spliceosomal snRNPs, and that modulation of the Sm core composition mediates the recognition of small nuclear RNA-specific Sm sites.
Mesenchymal stromal cells isolated from menstrual blood (MenSCs) exhibit a potent pro-angiogenic and immunomodulatory capacity. Their therapeutic effect is mediated by paracrine mediators released by their secretomes. In this work, we aimed to evaluate the effect of a specific priming condition on the phenotype and secretome content of MenSCs. Our results revealed that the optimal condition for priming MenSCs was the combination of interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) that produced a synergistic and additive effect on IDO1 release and immune-related molecule expression. The analyses of MenSC-derived secretomes after IFNγ and TNFα priming also revealed an increase in EV release and in the differentially expressed miRNAs involved in the immune response and inflammation. Proliferation assays on lymphocyte subsets demonstrated a decrease in CD4+ T cells and CD8+ T cells co-cultured with secretomes, especially in the lymphocytes co-cultured with secretomes from primed cells. Additionally, the expression of immune checkpoints (PD-1 and CTLA-4) was increased in the CD4+ T cells co-cultured with MenSC-derived secretomes. These findings demonstrate that the combination of IFNγ and TNFα represents an excellent priming strategy to enhance the immunomodulatory capacity of MenSCs. Moreover, the secretome derived from primed MenSCs may be postulated as a therapeutic option for the regulation of adverse inflammatory reactions.
Arachidonic acid (AA) is a polyunsaturated fatty acid present at high concentrations in the ovarian cancer (OC) microenvironment and associated with a poor clinical outcome. In the present study, we have unraveled a potential link between AA and macrophage functions. Methods: AA-triggered signal transduction was studied in primary monocyte-derived macrophages (MDMs) by phosphoproteomics, transcriptional profiling, measurement of intracellular Ca 2+ accumulation and reactive oxygen species production in conjunction with bioinformatic analyses. Functional effects were investigated by actin filament staining, quantification of macropinocytosis and analysis of extracellular vesicle release. Results: We identified the ASK1 - p38δ/α (MAPK13/14) axis as a central constituent of signal transduction pathways triggered by non-metabolized AA. This pathway was induced by the Ca 2+ -triggered activation of calmodulin kinase II, and to a minor extent by ROS generation in a subset of donors. Activated p38 in turn was linked to a transcriptional stress response associated with a poor relapse-free survival. Consistent with the phosphorylation of the p38 substrate HSP27 and the (de)phosphorylation of multiple regulators of Rho family GTPases, AA impaired actin filament organization and inhibited actin-driven macropinocytosis. AA also affected the phosphorylation of proteins regulating vesicle biogenesis, and consistently, AA enhanced the release of tetraspanin-containing exosome-like vesicles. Finally, we identified phospholipase A 2 group 2A (PLA2G2A) as the clinically most relevant enzyme producing extracellular AA, providing further potentially theranostic options. Conclusion: Our results suggest that AA contributes to an unfavorable clinical outcome of OC by impacting the phenotype of tumor-associated macrophages. Besides critical AA-regulated signal transduction proteins identified in the present study, PLA2G2A might represent a potential prognostic tool and therapeutic target to interfere with OC progression.
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