Stress Granules in Red Blood Cell Development - ABSTRACT Hematopoiesis involves stem and progenitor cell progression to the mature lineages. Unlike the steady production of new red cells in homeostasis, progenitors must rapidly regenerate many red blood cells under stress conditions. Throughout evolution, the erythroid program has adapted cell-specific processes for function. For example, translation regulation has also been adapted to ensure high levels of globin expression. However, the molecular mechanism facilitating the efficient translation of erythroid mRNAs has not been fully investigated, particularly during stress response. We found that stress granules (SGs) form in erythroid cells in multiple conditions, including in mice during phenylhydrazine-induced hemolytic anemia and in human CD34+ KO of the RNA demethylase, ALKBH5. ALKBH5 KO hCD34+ cells showed defective erythropoiesis measured by in vitro colony formation assays. Proteomic analysis of these cells showed significantly decreased ATXN2 levels. ATXN2 is a stress granule (SG) core component involved in translational regulation and RNA metabolism. ATXN2 overexpression (OE) in wild type erythroid progenitor cells led to accelerated erythropoiesis. STED microscopy of ALKBH5 KO K562 erythroid cells showed massive SGs containing SG structural proteins and m6A-modified RNAs. Restoring ATXN2 levels by lentiviral OE in the ALKBH5 KO cells dissolved SGs and rescued the blocked erythroid differentiation. We investigated the ATXN2 interactome in normal and stress erythroid cells by anti-ATXN2 pull-down mass-spectrometry and high-resolution microscopy. We discovered a novel ATXN2-SRSF RNA binding complex (primarily SRSF8) only in normal erythroid cells. SRSF proteins are involved in RNA metabolism and translation processes. In ALKBH5 mutant cells, ATXN2 bound SG proteins but not SRSF factors. hCD34+ cells with defective SRSF8 showed blocked erythroid differentiation, and the SRSF8 function is required for the ATXN2-OE rescue of erythropoiesis of ALKBH5-KO CD34+ cells. We propose that the ATXN2-SRSF8 complex is essential for the constant delivery of mRNAs to the ribosome in normal erythropoiesis. Under stress, the ATXN2-SRSF8 complex dissociates, and the complex-associated mRNAs are sequestered into SGs along with the ATXN2 protein. During the stress resolution phase, the ATXN2-SRSF8 complex reassociates to load SGs-stored mRNAs onto ribosomes. We will study how ATXN2 and SRSF8 work together at the molecular level to load erythroid-specific RNAs onto the ribosome for efficient translation and accelerate erythropoiesis following stress. We studied the erythropoietic phenotype of ATXN2 mutant mice. ATXN2 mutant mice have SGs, show abnormal red cell size and have defects in PHZ-induced recovery. We will investigate erythropoiesis in mice overexpressing ATXN2. We have made ATXN2 mutant zebrafish to probe SG formation and function in vivo. We plan to evaluate ATXN2 and SGs activity in erythroid cells derived from anemic patient CD34+ cells. Our functional analysis of ATXN2-SRSF8 complex and SGs in erythropoiesis will improve the understanding of normal- and stress-erythropoiesis and may lead to novel therapies for erythroid disorders.
