In addition to our finding that ADAR1 is essential for maintenance of hematopoiesis in the fetal liver and adult bone marrow, we identified ADAR1 as an essential suppressor of interferon signaling. Stringent control of this pathway, which is involved in numerous physiological processes, including inflammation and the innate immune response, is required to prevent the deleterious effects of excessive interferon activation. Importantly, defective interferon signaling has been associated with various pathological conditions, such as chronic inflammation, autoimmune disorders and cancer. The loss of HSCs and immature progenitors in the absence of ADAR1 was most likely a consequence of failure to hold interferon activation in check.
Conversion of adenosine to inosine in nuclear transcripts is the only established function of ADAR1. It is tempting, therefore, to speculate that ADAR1 regulates interferon signaling by editing critical, currently unknown transcripts. However, given the complexity of the interferon pathway that is activated by numerous stimuli it is instructive to consider alternative possibilities. First, as ADAR1 is a dsRNA binding protein and the innate immune response to dsRNA involves activation of type-I interferons35
, we interrogated our expression data for genes induced by dsRNA. Indeed, a gene set obtained from cells treated with dsRNA correlated with the Adar−/−
signature in that interferon-induced genes are significantly upregulated. ADAR1 deficiency enhanced the expression of transcripts encoding the interferon-inducible dsRNA binding proteins PKR (EIF2AK2), IFIH1 (RIG-1) and TLR3, whereas the expression of transcripts for other dsRNA binding proteins was not appreciably altered. This observation may suggest that immunoreactive dsRNA resulting from failure of an unknown ADAR1-dependent process contributes to the interferon response induced by ADAR1 deficiency. Second, ADAR1 has been shown previously to interact with the dsRNA binding protein NF90 via dsRNA and enhance NF90-mediated expression of genes, including the IFN-β gene, in an RNA-editing independent fashion36
. Interestingly, the Adar−/−
signature correlates with profiles obtained from cells that (over)express a carboxyterminal variant of NF90 protein37
, raising the possibility that ADAR1 deficiency disrupts ADAR1-containing protein complexes involved in the regulation of interferon-induced gene expression. Third, a recent study suggests a role for ADAR1 in a cell-intrinsic DNA-sensing mechanism that involves the putative Z-DNA binding protein DAI, a sensor for cytosolic DNA39
. Consistent with a possible role for ADAR1 in intracellular DNA sensing pathways, DAI transcript abundance was increased 15-fold in Adar−/−
HSCs as compared with control HSCs (not shown). Finally, another recent study suggests that NF-κB-mediated transcriptional activation of the human IFNB
gene upon virus infection involves the use of NF-κB binding sites embedded in an Alu repeat40
. Interestingly, widespread A-to-I editing of Alu repeat-containing mRNAs has been described in humans2-4
. Thus, ADAR1 may regulate the expression of interferon-inducible genes by targeting regulatory elements embedded in Alu or Alu-like repeats.
Future work will be required to elucidate whether ADAR1 plays a direct role in the regulation of interferon signaling, for example, by editing critical transcripts of protein-coding or regulatory RNA genes, or if activation of the interferon pathway by ADAR1 deficiency represents a cellular response to immunoreactive nucleic acid that may result from failure of an unknown ADAR1-dependent mechanism. Independent of the underlying mechanism, our study identifies ADAR1 as essential in vivo suppressor of interferon-induced gene expression.
Our finding that ADAR1 was essential during the activated state of a rare cell population comprising < 0.05 % of total bone marrow may explain, in part, why intensive efforts over the past years have failed to identify critical targets of ADAR1. Without substantial enrichment of specific, rare cells, the search for low abundance or edited transcripts may prove unsuccessful. Identifying the targets of ADAR1, which may include protein and/or non-protein coding transcripts, or possibly interacting proteins36
, will be critical in a fuller understanding of the unanticipated requirements for ADAR1 in the maintenance of hematopoiesis and suppression of interferon signaling.