The bicistronic microRNA (miRNA) locus miR-144/451 is highly expressed during erythrocyte development, although its physiological roles are poorly understood. We show that miR-144/451 ablation in mice causes mild erythrocyte instability and increased susceptibility to damage after exposure to oxidant drugs. This phenotype is deeply conserved, as miR-451 depletion synergizes with oxidant stress to cause profound anemia in zebrafish embryos. At least some protective activities of miR-451 stem from its ability to directly suppress production of 14-3-3ζ, a phospho-serine/threonine-binding protein that inhibits nuclear accumulation of transcription factor FoxO3, a positive regulator of erythroid anti-oxidant genes. Thus, in miR-144/451−/− erythroblasts, 14-3-3ζ accumulates, causing partial relocalization of FoxO3 from nucleus to cytoplasm with dampening of its transcriptional program, including anti-oxidant-encoding genes Cat and Gpx1. Supporting this mechanism, overexpression of 14-3-3ζ in erythroid cells and fibroblasts inhibits nuclear localization and activity of FoxO3. Moreover, shRNA suppression of 14-3-3ζ protects miR-144/451−/− erythrocytes against peroxide-induced destruction, and restores catalase activity. Our findings define a novel miRNA-regulated pathway that protects erythrocytes against oxidant stress, and, more generally, illustrate how a miRNA can influence gene expression by altering the activity of a key transcription factor.

Cells remove unstable polypeptides through protein quality-control (PQC) pathways such as ubiquitin-mediated proteolysis and autophagy. In the present study, we investigated how these pathways are used in ␤-thalassemia, a common hemoglobinopathy in which ␤-globin gene mutations cause the accumulation and precipitation of cytotoxic ␣-globin subunits. In ␤-thalassemic erythrocyte precursors, free ␣-globin was polyubiquitinated and degraded by the proteasome. These cells exhibited enhanced proteasome activity, and transcriptional profiling revealed coordinated induction of most proteasome subunits that was mediated by the stress-response transcription factor Nrf1. In isolated thalassemic cells, short-term proteasome inhibition blocked the degradation of free ␣-globin. In contrast, prolonged in vivo treatment of ␤-thalassemic mice with the proteasome inhibitor bortezomib did not enhance the accumulation of free ␣-globin. Rather, systemic proteasome inhibition activated compensatory proteotoxic stress-response mechanisms, including autophagy, which cooperated with ubiquitin-mediated proteolysis to degrade free ␣-globin in erythroid cells. Our findings show that multiple interregulated PQC responses degrade excess ␣-globin. Therefore, ␤-thalassemia fits into the broader framework of protein-aggregation disorders that use PQC pathways as cellprotective mechanisms. (Blood. 2012; 119(22):5265-5275) IntroductionThe production of functional hemoglobin A (HbA) tetramers (␣ 2 ␤ 2 ) requires the coordinated synthesis and assembly of ␣-and ␤-globin protein chains and iron-containing heme groups. Individually, all HbA components are toxic to RBCs and their precursors, as illustrated by ␤-thalassemias, a common hemoglobinopathy in which ␤-globin gene (HBB) mutations cause the buildup of free ␣-globin. 1 These unpaired ␣ chains initiate an oxidative damage cascade and form damaging precipitates that contribute largely to the clinical problems associated with ␤-thalassemia.The pathophysiology of ␤-thalassemia bears similarities to a diverse group of protein-aggregation diseases affecting multiple organs (for review, see Khandros and Weiss 2 ). These disorders, which include Parkinson disease, Alzheimer disease, Huntington disease, amyotrophic lateral sclerosis, and ␣ 1 -antitrypsin deficiency, are caused by the accumulation of unstable, relatively insoluble proteins. It is believed that the affected cells can detoxify and remove these damaging proteins via multiple interacting biochemical pathways called protein quality-control (PQC) pathways, but that disease ensues when such compensatory mechanisms are overwhelmed (for review, see Ciechanover and Brundin,3 Ding and Yin, 4 and Jaeger and Wyss-Coray 5 ). Cellular PQC systems include molecular chaperones, ubiquitin-mediated proteolysis, and autophagy. Several lines of evidence suggest that ␤-thalassemic erythroid cells use PQC pathways to detoxify free ␣-globin (for review, see Khandros and Weiss 2 ): (1) the clinical severity of ␤-thalassemia is proportional to the degree of ␣-glo...

MicroRNAs are small noncoding RNAs that regulate cellular development by interfering with mRNA stability and translation. We examined global microRNA expression during the differentiation of murine hematopoietic progenitors into megakaryocytes. Of 435 miRNAs analyzed, 13 were up-regulated and 81 were down-regulated. Many of these changes are consistent with miRNA profiling studies of human megakaryocytes and platelets, although new patterns also emerged. Among 7 conserved miRNAs that were up-regulated most strongly in murine megakaryocytes, 6 were also induced in the related erythroid lineage. MiR-146a was strongly up-regulated during mouse and human megakaryopoiesis but not erythropoiesis. However, overexpression of miR-146a in mouse bone marrow hematopoietic progenitor populations produced no detectable alterations in megakaryocyte development or platelet production in vivo or in colony assays. Our findings extend the repertoire of differentially regulated miRNAs during murine megakaryopoiesis and provide a useful new dataset for hematopoiesis research. In addition, we show that enforced hematopoietic expression of miR146a has minimal effects on megakaryopoiesis. These results are compatible with prior studies indicating that miR-146a inhibits megakaryocyte production indirectly by suppressing inflammatory cytokine production from innate immune cells, but cast doubt on a different study, which suggests that this miRNA inhibits megakaryopoiesis cell-autonomously. (Blood. 2010;116(23):e128-e138)

Background:The ␣-hemoglobin-stabilizing protein (AHSP) facilitates hemoglobin assembly. Results: AHSP mutations that enhance binding affinity for ␣-globin or slow its rate of autooxidation in vitro do not affect normal or stressed erythropoiesis in mice. Conclusion: AHSP exhibits robust molecular chaperone activity in vivo even when its biochemical interactions with reduced ␣-globin are perturbed. Significance: Our findings support new models for AHSP activities in vivo.

Whereas these studies cannot exclude the role of genetic variation in the lamin a/c gene in isolated cases of PCOS, we can conclude that common variation in the lamin a/c gene does not contribute to the etiology of PCOS in women of European ancestry.