Effects of RNA binding protein Ars2 on multipotent hematopoietic and pluripotent embryonic stem cells
MetadataShow full item record
Recent biochemical characterization of Arsenic resistance protein 2 (Ars2) had established Ars2 as a central factor in determining the fate of nascent RNA polymerase II (RNA pol II) transcripts. Through interactions with the nuclear 5’-7-methylguanosine (7mG) cap binding complex (CBC), Ars2 promotes co-transcriptional processing coupled with nuclear export or degradation of several classes of RNA pol II transcripts, allowing gene expression programs to facilitate rapid and sustained proliferation of immortalized cells in culture. However, rapidly dividing cells in culture do not represent the physiological condition of the vast majority of cells in an adult mammal. To examine functions of Ars2 in a physiological setting, we generated inducible Ars2 knockout mice and found that deletion of Ars2 from adult mice resulted in defective hematopoiesis in bone marrow and thymus. Importantly, only some of this defect could be explained by the requirement of Ars2 for rapid proliferation, which we found to be cell-type specific in vivo. Rather Ars2 was required for survival of developing thymocytes and for limiting differentiation of bone marrow resident long-term hematopoietic stem cells (LT-HSCs). As a result, Ars2 knockout led to rapid thymic involution and loss of the ability of mice to regenerate peripheral blood following myeloablation. These in vivo data demonstrated that Ars2 expression is important at several steps of hematopoiesis, likely because Ars2 acts on gene expression programs underlying essential cell fate decisions such as the decision to die, to proliferate, or to differentiate. Maintaining gene expression fidelity during hyper-transcription state of embryogenesis is another physiological phenomenon that requires precise sorting of newly synthesized RNA. Ars2 as a sorter of nascent transcripts is highly expressed in embryonic stem cells (ESC) and is essential for early embryogenesis. Even though Ars2 is necessary for survival of mouse embryo, little is known about its influence on ESCs and early development.To identify roles of Ars2 in early stage mammalian development, we generated inducible Ars2 knockout mouse ESC. Upon Ars2 deletion, early S phase cell-cycle progression slowed, frequency of pluripotent colonies (AP+) decreased, and expression of Oct4, Sox2, and Nanog was reduced and could not be fully rescued by culture under ground-state conditions (2i medium). To investigate effects of Ars2 on differentiation capacity of ESCs, embryoid bodies (EB), the 3D models for recapitulating early development, were formed. Ars2 KO EBs at the time of lineage commitment (day 4), drastically upregulated mesodermal genes. GSEA analysis of RNA-seq data revealed increased epithelial-to-mesenchymal transition (EMT) gene expression in Ars2 KO EBs. EMT was also confirmed by examining the E- to N-cadherin switch. Furthermore, embryonic hematopoiesis genes were highly enriched in Ars2 KO EBs. Using RNAi to reduce Ars2 expression during differentiation of ESCs to hematopoietic stem cells (ESC-HSC) dramatically increased the number of ESC-HSCs with phenotype and gene expression pattern of the most potent HSCs found in vivo. These ESC-HSCs were capable of advanced differentiation and formed colonies in methylcellulose, indicating HSC function in vitro. Overall, data establish Ars2 as a suppressor of ESC differentiation toward mesoderm; a role that may be exploited to increase efficiency of in vitro generation of ESC derived HSC for purpose of regenerative medicine.