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.

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