Adenosine-to-inosine (A-to-I) editing is a highly prevalent posttranscriptional modification of RNA, mediated by ADAR (adenosine deaminase acting on RNA) enzymes. In addition to RNA editing, additional functions have been proposed for ADAR1. To determine the specific role of RNA editing by ADAR1, we generated mice with an editing-deficient knock-in mutation (Adar1E861A, where E861A denotes Glu861→Ala861). Adar1E861A/E861A embryos died at ~E13.5 (embryonic day 13.5), with activated interferon and double-stranded RNA (dsRNA)–sensing pathways. Genome-wide analysis of the in vivo substrates of ADAR1 identified clustered hyperediting within long dsRNA stem loops within 3′ untranslated regions of endogenous transcripts. Finally, embryonic death and phenotypes of Adar1E861A/E861A were rescued by concurrent deletion of the cytosolic sensor of dsRNA, MDA5. A-to-I editing of endogenous dsRNA is the essential function of ADAR1, preventing the activation of the cytosolic dsRNA response by endogenous transcripts.

The small molecules thalidomide, lenalidomide, and pomalidomide induce the ubiquitination and proteasomal degradation of the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) by recruiting a Cys2-His2 (C2H2) zinc finger domain to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase. Here we screened the human C2H2 zinc finger proteome for degradation in the presence of thalidomide analogs, identifying 11 zinc finger degrons. Structural and functional characterization of the C2H2 zinc finger degrons demonstrates how diverse zinc finger domains bind the permissive drug-CRBN interface. Computational zinc finger docking and biochemical analysis predict that more than 150 zinc fingers bind the drug-CRBN complex in vitro, a larger number than previously anticipated, and we show that selective zinc finger degradation can be achieved through compound modifications. Our results provide a rationale for therapeutically targeting transcription factors that were previously considered undruggable.

Erythropoietin (Epo) has been used in the treatment of anemia resulting from numerous etiologies, including renal disease and cancer. However, its effects are controversial and the expression pattern of the Epo receptor (Epo-R) is debated. Using in vivo lineage tracing, we document that within the hematopoietic and mesenchymal lineage, expression of Epo-R is essentially restricted to erythroid lineage cells. As expected, adult mice treated with a clinically relevant dose of Epo had expanded erythropoiesis because of amplification of committed erythroid precursors. Surprisingly, we also found that Epo induced a rapid 26% loss of the trabecular bone volume and impaired Blymphopoiesis within the bone marrow microenvironment. Despite the loss of trabecular bone, hematopoietic stem cell populations were unaffected. Inhibition of the osteoclast activity with bisphosphonate therapy blocked the Epo-induced bone loss. Intriguingly, bisphosphonate treatment also reduced the magnitude of the erythroid response to Epo. These data demonstrate a previously unrecognized in vivo regulatory network coordinating erythropoiesis, B-lymphopoiesis, and skeletal homeostasis. Importantly, these findings may be relevant to the clinical application of Epo. (Blood. 2011; 117(21):5631-5642) IntroductionAnemia can result from benign conditions, such as acute/chronic bleeding, dietary deficiency, renal disease or hemoglobinopathies, and malignant diseases, such as myelodysplastic syndrome. 1,2 In response, the body produces erythropoietin (Epo). 3 Epo acts via its receptor (Epo-R) to stimulate and support increased erythropoiesis. 4,5 It has been widely used clinically for treating anemia of numerous causes, including infection, inflammation, gastrointestinal diseases, and cancer. 2,6 However, the use of Epo in cancer treatment is controversial because of adverse effects on survival, which have been reported in some clinical trials. [7][8][9] Within the erythroid lineage, Epo is essential for the survival, proliferation, and differentiation of definitive progenitors. Whereas erythroid colony-forming-units (CFU-Es) are highly Epo-dependent, Epo is dispensable for later stages of erythroid development. 10 Lack of Epo signaling in the mouse results in reduced primitive erythropoiesis and death at approximately embryonic day 13 (E13) because of failure of definitive erythropoiesis. 11,12 Targeted disruption of Epo or its receptor Epo-R generates identical phenotypes, suggesting that there are no alternate physiologic ligands or receptors. 11 In addition to its erythropoietic activity, Epo has also been proposed to act as a tissue-protective factor in tissues, such as the heart, blood vessels, and brain. 13,14 However, the expression of Epo-R in nonerythroid tissues is contentious. Recently described antibody studies suggest that Epo-R is restricted to the erythroid lineage. [15][16][17][18] This raises several questions regarding the effects of Epo in nonhematopoietic cell types.We have treated wild-type mice with a clinically relevant dose of E...

BackgroundAdenosine-to-inosine (A-to-I) editing of dsRNA by ADAR proteins is a pervasive epitranscriptome feature. Tens of thousands of A-to-I editing events are defined in the mouse, yet the functional impact of most is unknown. Editing causing protein recoding is the essential function of ADAR2, but an essential role for recoding by ADAR1 has not been demonstrated. ADAR1 has been proposed to have editing-dependent and editing-independent functions. The relative contribution of these in vivo has not been clearly defined. A critical function of ADAR1 is editing of endogenous RNA to prevent activation of the dsRNA sensor MDA5 (Ifih1). Outside of this, how ADAR1 editing contributes to normal development and homeostasis is uncertain.ResultsWe describe the consequences of ADAR1 editing deficiency on murine homeostasis. Adar1 E861A/E861A Ifih1 -/- mice are strikingly normal, including their lifespan. There is a mild, non-pathogenic innate immune activation signature in the Adar1 E861A/E861A Ifih1 -/- mice. Assessing A-to-I editing across adult tissues demonstrates that outside of the brain, ADAR1 performs the majority of editing and that ADAR2 cannot compensate in its absence. Direct comparison of the Adar1 -/- and Adar1 E861A/E861A alleles demonstrates a high degree of concordance on both Ifih1 +/+ and Ifih1 -/- backgrounds, suggesting no substantial contribution from ADAR1 editing-independent functions.ConclusionsThese analyses demonstrate that the lifetime absence of ADAR1-editing is well tolerated in the absence of MDA5. We conclude that protein recoding arising from ADAR1-mediated editing is not essential for organismal homeostasis. Additionally, the phenotypes associated with loss of ADAR1 are the result of RNA editing and MDA5-dependent functions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1301-4) contains supplementary material, which is available to authorized users.

Thalidomide and its derivatives, lenalidomide and pomalidomide, are clinically effective treatments for multiple myeloma and myelodysplastic syndrome with del(5q). These molecules lack activity in murine models, limiting investigation of their therapeutic activity or toxicity in vivo. Here, we report the development of a mouse model that is sensitive to thalidomide derivatives because of a single amino acid change in the direct target of thalidomide derivatives, cereblon (Crbn). In human cells, thalidomide and its analogs bind CRBN and recruit protein targets to the CRL4 E3 ubiquitin ligase, resulting in their ubiquitination and subsequent degradation by the proteasome. We show that mice with a single I391V amino acid change in Crbn exhibit thalidomide-induced degradation of drug targets previously identified in human cells, including Ikaros (Ikzf1), Aiolos (Ikzf3), Zfp91, and casein kinase 1a1 (Ck1α), both in vitro and in vivo. We use the model to demonstrate that the in vivo therapeutic activity of lenalidomide in del(5q) myelodysplastic syndrome can be explained by heterozygous expression of Ck1α in del(5q) cells. We found that lenalidomide acts on hematopoietic stem cells with heterozygous expression of Ck1α and inactivation of causes lenalidomide resistance. We further demonstrate that is sufficient to confer thalidomide-induced fetal loss in mice, capturing a major toxicity of this class of drugs. Further study of the model will provide valuable insights into the in vivo efficacy and toxicity of this class of drugs.

Adenosine deaminases that act on RNA (ADARs) convert adenosine residues to inosine in double-stranded RNA. In vivo, ADAR1 is essential for the maintenance of hematopoietic stem/progenitors. Whether other hematopoietic cell types also require ADAR1 has not been assessed. Using erythroid- and myeloid-restricted deletion of Adar1, we demonstrate that ADAR1 is dispensable for myelopoiesis but is essential for normal erythropoiesis. Adar1-deficient erythroid cells display a profound activation of innate immune signaling and high levels of cell death. No changes in microRNA levels were found in ADAR1-deficient erythroid cells. Using an editing-deficient allele, we demonstrate that RNA editing is the essential function of ADAR1 during erythropoiesis. Mapping of adenosine-to-inosine editing in purified erythroid cells identified clusters of hyperedited adenosines located in long 3’-untranslated regions of erythroid-specific transcripts and these are ADAR1-specific editing events. ADAR1-mediated RNA editing is essential for normal erythropoiesis.

The conversion of genomically encoded adenosine to inosine in dsRNA is termed as A-to-I RNA editing. This process is catalyzed by two of the three mammalian ADAR proteins (ADAR1 and ADAR2) both of which have essential functions for normal organismal homeostasis. The phenotype of ADAR2 deficiency can be primarily ascribed to a lack of site-selective editing of a single transcript in the brain. In contrast, the biology and substrates responsible for the Adar1(-/-) phenotype have remained more elusive. Several recent studies have identified that a feature of absence or reductions of ADAR1 activity, conserved across human and mouse models, is a profound activation of interferon-stimulated gene signatures and innate immune responses. Further analysis of this observation has lead to the conclusion that editing by ADAR1 is required to prevent activation of the cytosolic innate immune system, primarily focused on the dsRNA sensor MDA5 and leading to downstream signaling via MAVS. The delineation of this mechanism places ADAR1 at the interface between the cells ability to differentiate self- from non-self dsRNA. Based on MDA5 dsRNA recognition requisites, the mechanism indicates that the type of dsRNA must fulfil a particular structural characteristic, rather than a sequence-specific requirement. While additional studies are required to molecularly verify the genetic model, the observations to date collectively identify A-to-I editing by ADAR1 as a key modifier of the cellular response to endogenous dsRNA.

Pharmacologic agents that modulate ubiquitin ligase activity to induce protein degradation are a major new class of therapeutic agents, active in a number of hematologic malignancies. However, we currently have a limited understanding of the determinants of activity of these agents and how resistance develops. We developed and used a novel quantitative, targeted mass spectrometry (MS) assay to determine the relative activities, kinetics, and cell-type specificity of thalidomide and 4 analogs, all but 1 of which are in clinical use or clinical trials for hematologic malignancies. Thalidomide analogs bind the CRL4CRBN ubiquitin ligase and induce degradation of particular proteins, but each of the molecules studied has distinct patterns of substrate specificity that likely underlie the clinical activity and toxicities of each drug. Our results demonstrate that the activity of molecules that induce protein degradation depends on the strength of ligase-substrate interaction in the presence of drug, the levels of the ubiquitin ligase, and the expression level of competing substrates. These findings highlight a novel mechanism of resistance to this class of drugs mediated by competition between substrates for access to a limiting pool of the ubiquitin ligase. We demonstrate that increased expression of a nonessential substrate can lead to decreased degradation of other substrates that are critical for antineoplastic activity of the drug, resulting in drug resistance. These studies provide general rules that govern drug-dependent substrate degradation and key differences between thalidomide analog activity in vitro and in vivo.