A-To-I RNA Editing By ADAR1 Is Essential For Hematopoiesis

The role of RNA and its regulation is becoming increasingly appreciated as a vital component of hematopoietic development. RNA editing by members of the Adenosine Deaminase Acting on RNA (ADAR) gene family is a form of post-transcriptional modification which converts genomically encoded adenosine to inosine (A-to-I) in double-stranded RNA. A-to-I editing by ADAR directly converts the sequence of the RNA substrate and can alter the structure, function, processing, and localization of the targeted RNA. ADAR1 is ubiquitously expressed and we have previously described essential roles in the development of hematopoietic and hepatic organs. Germline ablation of murine ADAR1 results in a significant upregulation of interferon (IFN) stimulated genes and embryonic death between E11.5 and E12.5 associated with fetal liver disintegration and failed hemopoiesis. To determine the biological importance of A-to-I editing by ADAR1, we generated an editing dead knock-in allele of ADAR1 (ADAR1^E861A). Mice homozygous for the ADAR1^E861A allele died in utero at ∼E13.5. The fetal liver (FL) was small and had significantly lower cellularity than in controls. Analysis of hemopoiesis demonstrated increased apoptosis and a loss of hematopoietic stem cells (HSC) and all mature lineages. Most notably erythropoiesis was severely impaired with ∼7-fold reduction across all erythrocyte progenitor populations compared to controls. These data are consistent with our previous findings that ADAR1 is essential for erythropoiesis (unpublished data) and suggest that the ADAR1^E861A allele phenocopies the null allele in utero. To assess the requirement of A-to-I editing in adult hematopoiesis, we generated mice where we could somatically delete the wild-type ADAR1 allele and leave only ADAR1^E861A expressed in HSCs (hScl-Cre^ERAdar1^fl/E861A). In comparison to hScl-Cre^ERAdar1^fl/+ controls, hScl-Cre^ERAdar1^fl/E861A mice were anemic and had severe leukopenia 20 days post tamoxifen treatment. Investigation of marrow hemopoiesis revealed a significant loss of all cells committed to the erythroid lineage in hScl-Cre^ERAdar1^fl/E861A mice, despite having elevated phenotypic HSCs. Upon withdrawal of tamoxifen diet, all blood parameters were restored to control levels within 12 weeks owing to strong selection against cells expressing only the ADAR1^E861A allele. To understand the mechanism through which ADAR1 mediated A-to-I editing regulates hematopoiesis, RNA-seq was performed. Gene expression profiles showed that a loss of ADAR1 mediated A-to-I editing resulted in a significant upregulation of IFN signatures, consistent with the gene expression changes in ADAR1 null mice. To define substrates of ADAR1 we assessed A-to-I mismatches in the RNA-seq data sets. 3,560 previously known and 353 novel A-to-I editing sites were identified in our data set. However, no single editing substrate discovered could account for the IFN signature observed or the lethality of ADAR1^E861A/E861A mice. These results demonstrate that ADAR1 mediated A-to-I editing is essential for the maintenance of both fetal and adult hemopoiesis in a cell-autonomous manner and a key suppressor of the IFN response in hematopoiesis. Furthermore the ADAR1^E861A allele demonstrates the essential role of ADAR1 in vivo is A-to-I editing.