Project description:Wiskott-Aldrich syndrome (WAS), which is caused by mutations in the gene encoding WASP, manifests in a wide range of hematologic and immune dysfunctions and predisposition to cancer development 1. WASP is most known as a cytoplasmic effector of actin cytoskeleton rearrangement. However, defective actin polymerization cannot explain many aspects of WAS pathogenesis. Incomplete knowledge of WASP function precludes in-depth understanding of the underlying mechanisms of WAS, and therefore hampers the development of effective therapies. Here we generated induced pluripotent stem cells (iPSCs) from WAS patients (WAS-iPSC) and corresponding isogenic iPSCs wherein the mutations were corrected by targeted genome editing. Hematopoietic cells differentiated from WAS-iPSCs not only recapitulated known disease phenotypes, but also revealed novel nuclear functions of WASP. WASP deficiency causes differential expression of genes involved in many aspects of immune cell function, most prominently cell proliferation. It also leads to a large number of alternative splicing events that are highly enriched in cell cycle regulators. Proteomic analyses revealed that WASP physically interacted with nuclear body components, nuclear structural proteins, chromatin modifying complexes, and many RNA-binding proteins including multiple splicing factors. We show that WASP physically interacts with SRSF2 and is important for nuclear speckle organization. WASP regulates cell cycle progression by physically interacting with EZH2 and thereby influencing epigenetic silencing of its target gene CDKN2A (aka p16). Together, these observations unveil novel functions of WASP in regulation of RNA splicing and cell cycle. The binding of WASP to SRSF2 and EZH2 and the misregulation of these two frequently mutated genes in hematologic malignancies2 in WAS cells provide a possible mechanism for the frequent occurance of malignancy in WAS patients. Furthermore, WAS-associated splicing and proliferation abnormalities could serve as diagnosis tools for patients at risk for cancer and may be exploited as therapeutic targets.

Project description:Phagocytosis requires the activation of a plethora of mechanisms that include the activation of the actin cytoskeleton guided by the Arp2/3 complex. These are promoted by activators such as the Wiskott Aldrich Syndrome Protein (WASP) family members. In order to further understand the molecular mechanisms involved in the early events leading the phagocytosis of the pathogenic Mycobacterium tuberculosis, we set out to examine potential roles of miRNAs in phagocytosis using genome-wide expression profiling to identify miRNAs differentially regulated following mycobacterial infection. One of the miRNAs activated upon infection of mouse macrophages with the non-pathogenic Mycobacterium smegmatis, the widely conserved miR-142-3p, was predicted and confirmed to target the Neural-WASP (N-WASP). Upregulating of miR-142-3p in mouse macrophages inversely correlated with levels of N-WASP, upon infection with live pathogenic and non-pathogenic mycobacteria, suggesting an active role of Mycobacterium tuberculosis on the regulation of phagocytosis, at the post-transcriptional level, in host cells. The reduction of N-WASP correlated with a reduced internalization of bacteria per macrophage, independently of the phagocytosis index. Furthermore, the downregulation of WASP levels accompanied those of N-WASP, at early but not at late time points, suggesting a closely regulatory mechanism among both family members, dependent on the time frame of the phagocytosis. Additionally, upregulating of miR-142-3p promoted the change in the protein levels of another predicted and confirmed target, the Cofilin2 protein, in a phagocytosis-independent fashion. Downregulation experiments promoted aberrant morphologic phenotypes in macrophages, similar to observed by others in PBMCs of humans with Wiskott Aldrich Syndrome, suggesting the strong involvement of miR-142-3p on the regulation of the actin machinery in macrophages. Altogether these results show for the first time that miRNAs are involved in the regulation of actin-mediated phagocytosis of pathogenic bacteria and that these are direct targets of Mycobacterium tuberculosis.

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IdChrBici1

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