S275: pus1 regulates erythropoiesis via trna pseudouridylation and cytoplasmic translation

Topic: 28. Enzymopathies, membranopathies and other anemias Background: Pseudouridine (Ψ) is the most abundant RNA modification in cells. Growing evidences suggest that pseudouridine is critical in physiology and pathology processes. The importance of this biological function is exemplified by the clinical syndrome called mitochondrial myopathy, lactic acidosis and sideroblastic anemia syndrome (MLASA), in which genetic mutations of pseudouridine synthase 1 (PUS1) cause defective pseudouridylation that leads to MLASA. However, the roles of pseudouridylation in normal erythropoiesis and in anemia of MLASA remain poorly understood. Aims: To explore the role and the molecular mechanism underlying pseudouridine modification in erythropoiesis dysfunction and anemia. Methods: We established a mouse model carrying a point mutation R110W located in enzymatic domain of PUS1 with CRISPR-Cas9, mimicking the same mutation site mostly seen in human MLASA patients. Phenotypic studies such as the peripheral blood routine, erythroid development of bone marrow and spleen, the proportion and absolute number of cells in each stage of hematopoiesis were analyzed. Serial competitive transplantation experiments were performed to identify the ability of hematopoietic stem and progenitor cells in regeneration of erythroid cells. Furthermore, the oxidative phosphorylation of erythroid cells was measured by Seahorse experiment, and the activities mitochondrial OXPHOS complex units was determined by ELISA. The mitochondrial membrane potential, mitochondrial mass, mitochondrial superoxide and cellular Ros levels of erythroid cells were also determined by flow cytometry. In order to study the effect of R110W mutation on tRNA and translation, tRNA PCR Array were used to determine the changes of tRNA expression in mutant mouse erythroid cells, and RNA sequencing and Ribosome profiling were done to characterize the differentially translated proteins in mutant mouse erythroid cells. To clarify the effects of mitochondrial OXPHOS, cytoplasmic translation and mitochondrial translation on erythroid development, and the corresponding inhibitors were evaluated in an in vitro erythroid differentiation system. Results:Pus1 mutant mice (R110W) exhibited anemia at 4-week of age due to a blockage of erythroid maturation in both bone marrow and spleen. Whereas MEP is slightly decreased in R110W groups, the HSPC population is mainly unchanged in steady state when compared with that of the controls. During serial transplantation, the recovery of erythroid and platelet in R110W groups was continuously decreased. While lymphoid reconstitution is maintained during the first transplantation, a large decrease was observed in the secondary transplantation, indicating an cell autonomous deficiency in R110W HSCs function under transplantation stress. In consistent with mitochondrial dysfunction observed in MLASA patients, impaired mitochondrial OXPHOS, altered activity of OXPHOS complex I and III, and elevated oxidative stress were also observed in R110W BM erythroblasts. Mechanistically, BM erythroblasts of PUS1 R110W mutant mice showed defective pseudouridylation of targeted tRNAs and altered mitochondrial and cytoplasmic tRNA profiles as revealed by tRNA PCR Array and CMCT assay. Consequently, R110W erythroblasts exhibited decreased cytoplasmic translation, suppressed translation efficiency of ribosomal protein genes, and a lower level of hemoglobin protein, which led to ineffective erythropoiesis. Summary/Conclusion: Our study thus provided direct evidences that pseudouridylation participates in erythropoiesis in vivo, and demonstrated a critical role of pseudouridylation in regulating tRNA homeostasis, ribosome biogenesis, and erythropoiesis.Keywords: Erythropoieisis, Anemia