Unraveling the Functions of Wiskott-Aldrich Syndrome Protein: Insights into RNA Splicing, Nucleolus Regulation, and Immunosenescence

Abstract

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, autoimmunity, and predisposition to malignancy. Mutations in the WAS gene lead to dysfunction of the Wiskott-Aldrich syndrome protein (WASP), a multifunctional regulator implicated in various hematopoietic and immune processes. While some disease phenotypes have been linked to classical WASP's actin nucleation function, recent advances have unveiled additional nuclear functions, such as involvement in R-loop formation, transcriptional regulation during T helper 1 cell differentiation, and homology-directed repair. However, a comprehensive understanding of WASP's multifaceted functions remains elusive. We employ induced pluripotent stem cells (iPSCs) and Clustered Regularly Interspaced Palindromic Repeats (CRISPR) technology, specifically the CRISPR-Cas9 system, as powerful tools to investigate the mechanisms underlying WASP's functions. We first explore the consequences of WASP loss on RNA splicing. We reveal its critical role in RNA splicing. WASP-deficiency causes widespread alterations RNA splicing patterns and epigenetic activation of splicing factor gene promoters. Additionally, we uncover its involvement in liquid-liquid phase separation, forming phase-separated condensates to dynamically regulate the splicing machinery. In the second part of this thesis, our investigation uncovers the presence of WASP within the nucleolus and its interactions with key nucleolar proteins. Intriguingly, depletion of WASP leads to significant reduction in nucleolar size, disrupted nucleolar morphology, and decreased ribosomal RNA transcription, unveiling its critical role in nucleolus structure and function. Furthermore, we successfully recapitulated nucleolus changes and ribosomal RNA profile in patient samples. Lastly, we investigate immunosenescence, a crucial aspect of aging-related immune dysregulation, in the context of WAS. Through the use of WASP-deficient macrophage cells, our study revealed several distinctive features associated with immunosenescence in WASP-KO-iMPs. These include increased senescent cell proportions, heightened expression of senescence-associated secretory phenotype genes, nuclear deformation, loss of heterochromatin, and enhanced susceptibility to DNA damage. These preliminary findings offer valuable insights into our understanding of immunosenescence within the framework of WASP-deficient macrophages and its association with conditions related to WAS. In conclusion, the mechanistic study of WASP has unveiled its novel roles in regulating RNA splicing, nucleolus structure and function, as well as its potential involvement in immunosenescence.