Samenvatting

RNA metabolism is governed by RNA-binding proteins (RBPs), which regulate gene expression in cells, including immune system cells. Cells overcome stressing factors to ensure survival by employing a wide range of mechanisms that mitigate damage and restore homeostasis. One of the mechanisms is the formation of stress granules (SGs), which contain RBPs that preserve mRNA stability and regulate translation. A well known core protein in the assembly of SGs is GTPase-activating protein SH3 domain binding protein 1 (G3Bp1). Understanding its role in forming SGs could lead to a better understanding of the formation of RNA granules in homeostasis and their function in normal cell physiology.
Proteomic analysis of precursor (pre)-B cells revealed that G3Bp1 exhibited decreased binding to RNA in cells with DNA double-strand breaks (DSBs), lacking signalling through ataxia telangiectasia mutated (Atm) serine threonine kinase. This finding points to the possibility that SG formation may play a functional role in B cells during physiological DSBs, such as those occurring in V(D)J recombination. Whether this finding reflects RNA granules disassembly or some other function of G3Bp1 and whether the dynamic regulation of these structures is required for normal B cell development is to be studied.
The choice of SGs markers and their optimal working concentrations for imaging analysis was validated as well as the best stress inducing molecule to trigger SGs formation. Then, reduced SG assembly in v-abl cloned cells lacking G3Bp1 wasconfirmed. With this information, the role of G3Bp1 was further studied in B cell development and DSBs repair by treating cells lacking Artemis (ART) gene with imatinib to trigger cell cycle arrest and observe the behaviour of these RNA granules, with the result highlighting the usefulness of profiling of SGs in B cells for deeper understanding of SG biology in immune cells and improve experiment design, as well as the benefit of assessing the number of SGs in other cell lines which lack crucial elements for V(D)J recombination and DSBs repair, such as RAG2 KO and ART/ATM double KO.