Elucidating the immune function of the RNA helicase DDX6

PROJECT SUMMARY RNA helicases play essential roles in post-transcriptional gene regulation and have been implicated in immune regulation. However, the role of RNA helicases in hematopoietic stem cell (HSC) function and immune cell fate specification remains poorly understood. Recently, we discovered that the RNA helicase DDX6 sustains leukemia by orchestrating the translational repression and sequestration of mRNAs in RNA condensates known as P-bodies. Whether DDX6 is also crucial for HSC function and immune homeostasis remains unknown. To address this gap, we generated a novel transgenic mouse model to study the loss of DDX6 in HSCs. Preliminary data suggest that DDX6 regulates HSC quiescence and function. Specifically, the loss of DDX6 leads to extramedullary hematopoiesis and induces HSCs to exit quiescence, increase metabolic activity, and undergo cell division. These changes led to the expansion of HSCs in vivo under steady-state conditions but caused their premature exhaustion during competitive transplantation. Mechanistically, our initial analysis of DDX6-bound transcripts within P-bodies of hematopoietic progenitors revealed enrichment of untranslated mRNAs encoding critical regulators of quiescence exit. While these insights are significant, the mechanisms by which DDX6 enables HSCs to maintain longevity and function under regenerative and infectious stress remain unclear. Additionally, how DDX6 regulates the translational landscape of HSCs is yet to be fully understood. To address these questions, this application proposes two complementary aims. In SPECIFIC AIM 1, we will use novel genetic mouse models to delete DDX6 in vivo in a cell- and time-specific manner to rigorously dissect the roles of DDX6-mediated RNA sequestration in HSCs following regenerative stress and infection. In SPECIFIC AIM 2, we will explore how DDX6-mediated RNA sequestration in cytoplasmic condensates controls HSC function. We will characterize the molecular landscape of P-bodies in HSCs under regenerative and infectious stress and elucidate DDX6's widespread role in suppressing the translation of target mRNAs using Ribo-ITP, a highly sensitive and quantitative method for single-cell and low-input ribosome profiling. Additionally, we will identify factors regulated by DDX6 and examine their functional roles in HSC function. Collectively, our study will advance the understanding of RNA helicases and their underlying mechanisms in immune homeostasis, paving the way for innovative therapeutic strategies targeting DDX6 and its downstream effectors to combat infectious diseases.