HRI coordinates translation necessary for protein homeostasis and mitochondrial function in erythropoiesis

Abstract

Iron and heme play central roles in the production of red blood cells, but the underlying mechanisms remain incompletely understood. Heme-regulated eIF2α kinase (HRI) controls translation by phosphorylating eIF2α. Here, we investigate the global impact of iron, heme, and HRI on protein translation in vivo in murine primary erythroblasts using ribosome profiling. We validate the known role of HRI-mediated translational stimulation of integratedstressresponse mRNAs during iron deficiency in vivo. Moreover, we find that the translation of mRNAs encoding cytosolic and mitochondrial ribosomal proteins is substantially repressed by HRI during iron deficiency, causing a decrease in cytosolic and mitochondrial protein synthesis. The absence of HRI during iron deficiency elicits a prominent cytoplasmic unfolded protein response and impairs mitochondrial respiration. Importantly, ATF4 target genes are activated during iron deficiency to maintain mitochondrial function and to enable erythroid differentiation. We further identify GRB10 as a previously unappreciated regulator of terminal erythropoiesis.

ATF4 mediates fetal globin upregulation in response to reduced β-globin

“…Notably, ATF4ko-1 and ATF4ko-2 were unable to undergo in vitro differentiation, and instead exhibited extensive cell death after 5 days in differentiation media (Fig S3D). This is consistent with a known role of ATF4 in erythroid differentiation (Masuoka and Townes, 2002;Suragani et al, 2012;Zhang et al, 2019). Second, we explored N-terminal truncation mutants of ATF4 with separation-of-function properties (Steinmüller and Thiel, 2005).…”

Global translational repression induced by iron deficiency in yeast depends on the Gcn2/eIF2α pathway

“…In mammals, iron deficiency limits heme synthesis, which activates heme-regulated eIF2α kinase (HRI), one of the four mammalian eIF2α kinases that phosphorylate eIF2α. This causes a decrease in bulk translation, down-regulating the synthesis of globins while increasing the translation of specific transcripts, such as HbF (coding for fetal hemoglobin) in certain anemias [42][43][44][45] . The targeted deletion of HRI causes an improvement of global translation, similar to that observed for the deletion of yeast GCN2, which increases globin translation 42 .…”

Requirement of activating transcription factor 5 for murine fetal liver erythropoiesis

“…HRI-mediated ISR is necessary for effective erythropoiesis in iron deficiency (Zhang et al, 2018). The dependence of Atf5 expression on HRI supports the involvement of ATF5 in HRI-activated ISR under iron deficiency (Zhang et al, 2019). HRI-deficient mice develop ineffective erythropoiesis during iron deficiency (Han et al, 2001).…”

“…Five shRNA oligonucleotides targeting different regions of Atf5 mRNA were obtained from the Genetic Perturbation Platform of Broad Institute (Table SI). Preparations and infections of recombinant retroviruses were performed as previously described (Zhang et al , ). Briefly, erythroid progenitors (Lin − CD71 − Ter119 − ) were infected with five Atf5 shRNAs and a Green Fluorescent Protein (GFP) control recombinant retroviruses by centrifuging at 700 g for 1·5 h at room temperature in the presence of 8 μg/ml polybrene (Sigma‐Aldrich, Saint Louis, MO, USA).…”

“…Animals and progenitor enrichment C57BL/6J mice were maintained at the animal facility of Massachusetts Institute of Technology (MIT; Cambridge, MA), and all experiments using mice of 8-12 weeks old were carried out with protocols approved by the Committee on Animal Care (CAC). FLs from embryonic day 13Á5 (E13Á5) samples were used to enrich erythroid progenitors by magnetic sorting using EasySep Magnet (Stem Cell Technologies, Vancouver, Canada) as previously described (Thom et al, 2014;Zhang et al, 2019). Platform of Broad Institute (Table SI).…”