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Myelodysplastic syndromes with ring sideroblasts (MDS-RS) commonly originate from mutations in the splicing factorSF3B1(SF3B1mt).SF3B1mtcause RNA mis-splicing, mechanistically established as the major driver of RS development. However, little is known about RS fate and biology after their initial formation in the human bone marrow. We here achieve isolation of viable RS from patient samples, enabling the first complete investigation ofSF3B1mtdevelopment from stem cell to RS. We show that RS skew MACS-isolated CD34+data towards erythroid features not recapitulated in single-cell RNAseq. We demonstrate that RS divide, differentiate, enucleate and actively respond to mis-splicing/oxidative stress, decreasing wildtype stem cell fitness via GDF15 overproduction. We identify circulating RS as a uniform clinical feature associated with disease burden. Finally, we establish thatSF3B1mtmis-splicing intensifies during erythroid differentiation and demonstrate through combined transcriptomics/proteomics an uncoupling of RNA/protein biology in RS encompassing severe and dysfunctional mis-splicing of proapoptotic genes.Statement of significanceWe here combine a novel method for RS isolation with state-of-the-art multiomics to perform the first complete investigation ofSF3B1mtMDS-RS hematopoiesis from stem cell to RS. We identify the survival mechanisms underlyingSF3B1mterythropoiesis and establish an active role for erythroid differentiation and RS themselves inSF3B1mtMDS-RS pathogenesis.

Large scale high-throughput DNA sequencing studies have identified clonal hematopoiesis (CH) as a clinical phenomenon characterized by a disproportionately large clonal population in the hematopoietic system with a shared mutational background. CH originates through mutations in hematopoietic stem and progenitor cells (HSPCs) which provide a proliferative advantage over unmutated HSPCs and has been characterized as a risk factor for myeloid neoplasm (MN) development. Large population studies found that CH is an age-related event which is commonly found in association with milder phenotypes such as cytopenia, mild monocytosis, intravascular hemolysis, or chronic inflammation. More importantly, the vast majority of individuals with CH are asymptomatic and healthy people of advanced age, where the impact of CH is thus considered to be of indeterminate potential (CHIP). These conditions are sometimes referred to as benign to facilitate distinction from overt MN but, despite this definition, may still result in severe illness, reduced overall survival, and increased risk of hematologic neoplasms development and all-cause mortality. The purpose of this review is to describe clinical conditions associated with CH, the clinical significance of CH-related clinical phenotypes, and the determinants of progression from CH to overt MN following the paradigmatic example of SF3B1-driven CH.

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The process of maturation of reticulocytes into fully mature erythrocytes that occurs in the circulation is known to be characterized by a complex interplay between loss of cell surface area and volume, removal of remnant cell organelles and redundant proteins, and highly selective membrane and cytoskeletal remodeling. However, the mechanisms that underlie and drive these maturational processes in vivo are currently poorly understood and, at present, reticulocytes derived through in vitro culture fail to undergo the final transition to erythrocytes. Here, we used high-throughput proteomic methods to highlight differences between erythrocytes, cultured reticulocytes and endogenous reticulocytes. We identify a cytoskeletal protein, non-muscle myosin IIA (NMIIA) whose abundance and phosphorylation status differs between reticulocytes and erythrocytes and localized it in the proximity of autophagosomal vesicles. An ex vivo circulation system was developed to simulate the mechanical shear component of circulation and demonstrated that mechanical stimulus is necessary, but insufficient for reticulocyte maturation. Using this system in concurrence with non-muscle myosin II inhibition, we demonstrate the involvement of non-muscle myosin IIA in reticulocyte remodeling and propose a previously undescribed mechanism of shear stress-responsive vesicle clearance that is crucial for reticulocyte maturation.

The capacity to undergo substantial deformation is a defining characteristic of the red blood cell (RBC), facilitating transit through the splenic interendothelial slits and microvasculature. Establishment of this remarkable property occurs during a process of reticulocyte maturation that begins with egress through micron-wide pores in the bone marrow and is completed within the circulation. The requirement to undertake repeated cycles of deformation necessitates that both reticulocytes and erythrocytes regulate membrane-cytoskeletal protein interactions in order to maintain cellular stability. In the absence of transcriptional activity, modulation of these interactions in RBCs is likely to be achieved primarily through specific protein posttranslational modifications, which at present remain undefined. In this study, we use high-throughput methods to define the processes that underlie the response to deformation and shear stress in both reticulocytes and erythrocytes. Through combination of a bead-based microsphiltration assay with phosphoproteomics we describe posttranslational modification of RBC proteins associated with deformation. Using microsphiltration and microfluidic biochip-based assays, we explore the effect of inhibiting kinases identified using this dataset. We demonstrate roles for GSK3 and Lyn in capillary transit and maintenance of membrane stability following deformation and show that combined inhibition of these kinases significantly decreases reticulocyte capacity to undergo repeated deformation. Finally, we derive a comprehensive and integrative phosphoproteomic dataset that provides a valuable resource for further mechanistic dissection of the molecular pathways that underlie the RBC’s response to mechanical stimuli and for the study of reticulocyte maturation.

Investigating the role that host erythrocyte proteins play in malaria infection is hampered by the genetic intractability of this anucleate cell. Here we report that reticulocytes derived through in vitro differentiation of an enucleation-competent immortalized erythroblast cell line (BEL-A) support both successful invasion and intracellular development of the malaria parasite Plasmodium falciparum. Using CRISPR-mediated gene knockout and subsequent complementation, we validate an essential role for the erythrocyte receptor basigin in P. falciparum invasion and demonstrate rescue of invasive susceptibility by receptor re-expression. Successful invasion of reticulocytes complemented with a truncated mutant excludes a functional role for the basigin cytoplasmic domain during invasion. Contrastingly, knockout of cyclophilin B, reported to participate in invasion and interact with basigin, did not impact invasive susceptibility of reticulocytes. These data establish the use of reticulocytes derived from immortalized erythroblasts as a powerful model system to explore hypotheses regarding host receptor requirements for P. falciparum invasion., Here, the authors show that reticulocytes derived from immortalized erythroblasts support invasion and development of Plasmodium falciparum and use CRISPR-mediated gene knockout and complementation of an invasion receptor to demonstrate utility of this model system for research in malaria invasion.

Invasion of and intracellular development within the red blood cell by malaria parasites requires interaction with a multitude of host proteins expressed at the surface of or inside the cell. Perhaps the biggest obstacle to dissection of specific functions of host proteins during invasion is the inability to manipulate protein expression in the genetically intractable anucleate red blood cell. Whilst genetic manipulation and subsequent in vitro differentiation of nucleated erythroid precursors have facilitated progress in this area, such approaches are limited by the finite proliferative capacity of primary hematopoietic stem cells, and a failure of erythroleukemic cell lines to enucleate, respectively. Here, we report that reticulocytes derived through in vitro differentiation of an enucleation competent immortalized erythroblast cell line (BEL-A) support both successful invasion and growth by Plasmodium falciparum. Using CRISPR-mediated gene knockout and lentiviral expression of open reading frames, we validate an essential role for the erythrocyte receptor basigin in P. falciparum invasion and, for the first time, demonstrate that this can be rescued by re-expression of the receptor or of a mutant thereof. Specifically, using sustainable edited clones derived from this line, we exclude a functional role for the cytoplasmic domain of basigin during invasion, and challenge the reported requirement of its associated receptor cyclophilin B. These data establish the use of reticulocytes derived from immortalized erythroblasts as a crucial model system to explore specific hypotheses regarding host receptor requirements and involvement in P. falciparum invasion.