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Summary: Pseudouridylation plays a regulatory role in various physiological and pathological processes. A prime example is the mitochondrial myopathy, lactic acidosis, and sideroblastic anemia syndrome (MLASA), characterized by defective pseudouridylation resulting from genetic mutations in pseudouridine synthase 1 (PUS1). However, the roles and mechanisms of pseudouridylation in normal erythropoiesis and MLASA-related anemia remain elusive. We established a mouse model carrying a point mutation (R110W) in the enzymatic domain of PUS1, mimicking the common mutation in human MLASA. Pus1-mutant mice exhibited anemia at 4 weeks old. Impaired mitochondrial oxidative phosphorylation was also observed in mutant erythroblasts. Mechanistically, mutant erythroblasts showed defective pseudouridylation of targeted tRNAs, altered tRNA profiles, decreased translation efficiency of ribosomal protein genes, and reduced globin synthesis, culminating in ineffective erythropoiesis. Our study thus provided direct evidence that pseudouridylation participates in erythropoiesis in vivo. We demonstrated the critical role of pseudouridylation in regulating tRNA homeostasis, cytoplasmic translation, and erythropoiesis.

PDK1 (phosphoinositide dependent kinase-1) plays an important regulatory role in B cells, T cells and platelets. Less is known about how PDK1 acts in hematopoietic stem cells (HSCs), especially in the fetal liver (FL) during embryonic hematopoiesis, as the FL is the primary fetal hematopoietic organ and the main site of HSC expansion and differentiation. Here, we deleted the PDK1 gene in hematopoietic cells by crossing Vav-Cre transgenic mice with PDK1f/f mice. Using a transplantation assay, we found that HSCs from the E15.5 FL of Vav-Cre;PDK1f/f embryos are severely impaired compared when compared with HSCs from PDK1f/f or PDK1f/+ FLs. Additionally, we found that there were more FL HSCs in an apoptotic state and active cell cycle in PDK1-deficient embryos than in control embryos. By comparing the expression profiles of FL-derived LSKs in Vav-Cre;PDK1f/f embryos to the controls, we found that the BH3-only protein PUMA and the cyclin family proteins were expressed higher in the Vav-Cre;PDK1f/f group, which may account for the increased apoptosis and activated cell cycle in the deficient HSCs. Furthermore, we demonstrated that the expression of FoxO3a was higher in PDK1-deficient LSKs, indicating that the Akt-FoxO3a-PUMA axis may participate in regulating LSKs apoptosis in the E15.5 FL. In contrast, FoxO1 expression was lower in PDK1-deficient LSK cells, suggesting that Akt-FoxO1-CCND may regulate the HSC cell cycle. Taken together, our findings support a critical role for PDK1 in maintaining FL hematopoiesis via regulating apoptosis and cell cycle of definitive hematopoiesis by the Akt-FOXO signaling pathways. Keywords: PDK1, Akt, HSC, FOXO, Fetal liver, Embryonic hematopoiesis