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Abstract Background miRNAs are regulatory transcripts established as repressors of mRNA stability and translation that have been functionally implicated in carcinogenesis. miR-10b is one of the key onco-miRs associated with multiple forms of cancer. Malignant gliomas exhibit particularly striking dependence on miR-10b. However, despite the therapeutic potential of miR-10b targeting, this miRNA’s poorly investigated and largely unconventional properties hamper the clinical translation. Methods We utilized Covalent Ligation of Endogenous Argonaute-bound RNAs and their high-throughput RNA sequencing to identify miR-10b interactome and a combination of biochemical and imaging approaches for target validation. They included Crosslinking and RNA immunoprecipitation with spliceosomal proteins, a combination of miRNA FISH with protein immunofluorescence in glioma cells and patient-derived tumors, native Northern blotting, and the transcriptome-wide analysis of alternative splicing. Results We demonstrate that miR-10b binds to U6 snRNA, a core component of the spliceosomal machinery. We provide evidence of the direct binding between miR-10b and U6, in situ imaging of miR-10b and U6 co-localization in glioma cells and tumors, and biochemical co-isolation of miR-10b with the components of the spliceosome. We further demonstrate that miR-10b modulates U6 N-6-adenosine methylation and pseudouridylation, U6 binding to splicing factors SART3 and PRPF8, and regulates U6 stability, conformation, and levels. These effects on U6 result in global splicing alterations, exemplified by the altered ratio of the isoforms of a small GTPase CDC42, reduced overall CDC42 levels, and downstream CDC42 -mediated effects on cell viability. Conclusions We identified U6 snRNA, the key RNA component of the spliceosome, as the top miR-10b target in glioblastoma. We, therefore, present an unexpected intersection of the miRNA and splicing machineries and a new nuclear function for a major cancer-associated miRNA.

Responsive exocytosis in neutrophil leukocytes involves actin depolymerisation-dependent sequential release of gelatinase granules, then strongly pro-inflammatory azurophilic granules. Here authors show that the actin nucleator protein WASH facilitates the initial step of innate immune activation by gelatinase granules while inhibiting release of pro-inflammatory granules.

Heterozygous mutations in the NLRP3 gene in patients with cryopyrin associated periodic syndrome (CAPS) lead to hyper-responsive inflammasome function. CAPS is a systemic auto-inflammatory syndrome characterized by the activation of the innate immune system induced by elevated pro-inflammatory cytokines, but the involvement of selective innate immune cells in this process is not fully understood. Neutrophil secretion and the toxic components of their granules are mediators of inflammation associated with several human diseases and inflammatory conditions. Here, using the Nlrp3A350V inducible mouse model (MWS CreT) that recapitulates human patients with the A352V mutation in NLRP3 observed in the Muckle-Wells sub-phenotype of CAPS, we studied the relationship between hyper-activation of the inflammasome and neutrophil exocytosis. Using a flow cytometry approach, we show that Nlrp3A350V (MWS) neutrophils express normal basal levels of CD11b at the plasma membrane and that the upregulation of CD11b from secretory vesicles in response to several plasma membrane or endocytic agonist including the bacterial-derived mimetic peptide formyl-Leu-Met-Phe (fMLF) and the unmethylated oligonucleotide CpG is normal in MWS neutrophils. Significant but modest CD11b upregulation in MWS neutrophils compared to wild type was only observed in response to GM-CSF and CpG. The same pattern was observed for the secretion of matrix metalloproteinase-9 (MMP-9) from gelatinase granules in that MMP-9 secretion in MWS neutrophils was not different from that observed in wild-type neutrophils except when stimulated with GM-CSF and CpG. In contrast, azurophilic granule secretion, whose cargoes constitute the most toxic secretory and pro-inflammatory factors of the neutrophil, was markedly dysregulated in MWS neutrophils under both basal and stimulated conditions. This could not be attributed to paracrine effects of secretory cytokines because IL-1β secretion by neutrophils was undetectable under these experimental conditions. The increased azurophilic granule exocytosis in MWS neutrophils was attenuated by treatment with the neutrophil exocytosis inhibitor Nexinhib20. In agreement with a possible neutrophil contribution to systemic inflammation in CAPS, the levels of neutrophil secretory proteins were significantly elevated in the plasma from Nlrp3A350V mice. Altogether, our data indicates an azurophilic granule-selective dysregulation of neutrophil exocytosis in CAPS.