The RNA Helicase DHX16 Is Required for the Maintenance of Hematopoietic Stem Cells

Lifelong hematopoiesis relies on the homeostasis of hematopoietic stem cells(HSCs), but the underlying mechanisms remain incompletely understood. Here, we identified a critical role for DHX16, a member of DEAH-box RNA helicase, in the maintenance of HSCs. After inducing deletion of Dhx16 gene in mice, the knockout animals developed pancytopenia within 5-7 days and died within two weeks. Flow cytometric analyses demonstrated that Dhx16-deficient mice had significantly reduced hematopoietic stem and progenitor cells, altered cell cycle dynamics with decreased G0 phase and increased G2/M phase, and increased apoptosis of HSCs. Competitive BM transplantation assays convinced the impaired self-renewal ability of Dhx16 knockout mice. To identify Dhx16-dependent genes and molecular pathways involved in the regulation of HSC function, we performed the RNA-Seq and long-read Iso-Seq analysis to compare the transcriptome between the Dhx16-deficient HSCs and Dhx16 ^fl/fl HSCs. RNA-Seq results showed that signaling pathways in Dhx16-deficient hematopoietic stem cells, including ribosome and spliceosome assembly, G2/M checkpoint, and p53 pathway were significantly affected. While long-read Iso-Seq revealed 210 genes with splicing alterations. Through further study, we confirmed that Dhx16-deficiency led to the retention of intron 4 in Emg1, a gene essential for ribosome biogenesis, resulting in a premature termination and nonsense-mediated mRNA decay. Therewith, decreased Emg1 expression in HSCs impaired ribosome biogenesis, activated the p53 pathway, and resulted in the rapid exhaustion of HSCs. In conclusion, these findings reveal a critical role for DHX16 in HSC survival and function and uncover an unsuspected link between RNA helicases, alternative splicing, ribosome biogenesis, and HSC homeostasis.