Hematopoietic stem cells (HSCs) are vital for the differentiation of all mature blood cells, with their homeostasis being tightly regulated by intrinsic and extrinsic factors. Alternative splicing, mediated by the spliceosome complex, plays a crucial role in regulating HSC homeostasis by increasing protein diversity. This study focuses on the ATP-dependent RNA helicase DHX16, a key spliceosome component, and its role in HSC regulation. Using conditional knockout mice, we demonstrate that loss of Dhx16 in the hematopoietic system results in significant depletion of hematopoietic stem and progenitor cells, bone marrow failure, and rapid mortality. Dhx16-deficient HSCs exhibit impaired quiescence, G2-M phase cell cycle arrest, reduced protein synthesis, abnormal ribosome assembly, increased apoptosis, and decreased self-renewal capacity. Multi-omics analysis identified intron 4 retention in Emg1 mRNA in Dhx16 knockout HSCs, leading to reduced EMG1 protein expression, disrupted ribosome assembly, and nucleolar stress, activating the p53 pathway. Overexpression of Emg1 in Dhx16-deficient HSCs partially restored ribosome assembly and HSC function, suggesting Emg1 as a potential therapeutic target for ribosomopathies. Our findings reveal the critical role of Dhx16 in HSC homeostasis through the regulation of alternative splicing and ribosome assembly, providing insights into the molecular mechanisms underlying hematopoietic diseases and potential therapeutic strategies.

造血干细胞（HSC）对于所有成熟血细胞的分化至关重要，其稳态受到内在和外在因素的严格调节。由剪接体复合物介导的选择性剪接通过增加蛋白质多样性在调节 HSC 稳态中发挥着至关重要的作用。本研究重点关注 ATP 依赖性 RNA 解旋酶 DHX16（一种关键的剪接体成分）及其在 HSC 调节中的作用。使用条件敲除小鼠，我们证明造血系统中Dhx16的缺失会导致造血干细胞和祖细胞显着耗竭、骨髓衰竭和快速死亡。 Dhx16缺陷的 HSC 表现出静止受损、G2-M 期细胞周期停滞、蛋白质合成减少、核糖体组装异常、细胞凋亡增加和自我更新能力降低。多组学分析发现， Dhx16敲除 HSC 中Emg1 mRNA 中的内含子 4 保留，导致 EMG1 蛋白表达减少、核糖体组装破坏和核仁应激，从而激活 p53 通路。 Dhx16缺陷型 HSC 中Emg1的过度表达部分恢复了核糖体组装和 HSC 功能，表明Emg1作为核糖体病的潜在治疗靶点。我们的研究结果揭示了Dhx16通过调节选择性剪接和核糖体组装在 HSC 稳态中的关键作用，为了解造血疾病的分子机制和潜在的治疗策略提供了见解。