# THE RED CELL LIFE-CYCLE FROM ERYTHROPOIESIS TO CLEARANCE

EDITED BY : Lars Kaestner and Anna Bogdanova PUBLISHED IN : Frontiers in Physiology

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# THE RED CELL LIFE-CYCLE FROM ERYTHROPOIESIS TO CLEARANCE

Topic Editors: Lars Kaestner, Saarland University, Germany Anna Bogdanova, University of Zürich, Switzerland

Human red blood cell recorded with a Scanning Ion Conductance Microscope, by Greta Simionato Cover image: High level specialist/Shutterstock.com

The eBook 'The red cell life-cycle from erythropoiesis to clearance' continues the discussion of questions like: What are the changes associated with red blood cell maturation, adulthood and senescence? What are the determinants of red blood cell life span and clearance? What are the mechanisms in control of red blood cell mass in healthy humans and patients with various forms of anaemia? Can red blood cells be 'trained' to provide the body with more oxygen during endurance exercises? What are the markers of circulating red blood cell senescence and in cells during storage and transfusion? And what can be learned from various species that developed advanced adaptations to maintain oxygen delivery under stress conditions such as exercising to the limit, diving or living in anaerobic aquatic habitats or at high altitude?

Within the approximately 120 days (or 40 in a mouse, or 150-170 in a horse) life span of 'healthy' red blood cells, many cellular properties change leading to aged mixed cell populations in the circulation. Red blood cells seem to be genetically terminated by the time they become red blood cells and the contributions of this eBook increase the understanding of this process. There are surprisingly versatile remodeling processes happening during the red blood cell life span. Numerous disorders are associated with the premature onset of the 'ageing process' of red blood cells. Furthermore, in vitro ageing and/or modifications as well as the slowing down of the modifications is an important issue in transfusion medicine. Many of the molecular mechanisms behind such effects are elucidated in this eBook.

Citation: Kaestner, L., Bogdanova, A., eds. (2019). The Red Cell Life-Cycle From Erythropoiesis to Clearance. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-702-1

# Table of Contents

*06 Editorial: The Red Cell Life-Cycle From Erythropoiesis to Clearance* Lars Kaestner and Anna Bogdanova

### I. ERYTHROPOIESIS AND MATURATION OF RETICULOCYTES

*09 RNA Binding Proteins and Regulation of mRNA Translation in Erythropoiesis*

Kat S. Moore and Marieke von Lindern

*26 From Erythroblasts to Mature Red Blood Cells: Organelle Clearance in Mammals*

Martina Moras, Sophie D. Lefevre and Mariano A. Ostuni

### II. RED BLOOD CELL AGEING, SENESCENCE AND CLEARANCE

Pedro J. Romero and Concepción Hernández-Chinea

### III. RED BLOOD CELL ION CHANNELS AND MEMBRANE TRANSPORT

### *129 Red Blood Cell Passage of Small Capillaries is Associated With Transient Ca2+-mediated Adaptations*

Jens G. Danielczok, Emmanuel Terriac, Laura Hertz, Polina Petkova-Kirova, Franziska Lautenschläger, Matthias W. Laschke and Lars Kaestner

### IV. DIAGNOSTICS AND PATHOPHYSIOLOGY

*140 Influence of Standard Laboratory Procedures on Measures of Erythrocyte Damage*

Lena Wiegmann, Diane A. de Zélicourt, Oliver Speer, Alissa Muller, Jeroen S. Goede, Burkhardt Seifert and Vartan Kurtcuoglu

*151 Use of Laser Assisted Optical Rotational Cell Analyzer (LoRRca MaxSis) in the Diagnosis of RBC Membrane Disorders, Enzyme Defects, and Congenital Dyserythropoietic Anemias: A Monocentric Study on 202 Patients*

Anna Zaninoni, Elisa Fermo, Cristina Vercellati, Dario Consonni, Anna P. Marcello, Alberto Zanella, Agostino Cortelezzi, Wilma Barcellini and Paola Bianchi

Joanna F. Flatt and Lesley J. Bruce

Luanne L. Peters and Lionel Blanc

*194 Increased Reactive Oxygen Species and Cell Cycle Defects Contribute to Anemia in the RASA3 Mutant Mouse Model* scat Emily S. Hartman, Elena C. Brindley, Julien Papoin, Steven L. Ciciotte, Yue Zhao,

### V. RED BLOOD CELL STORAGE AND TRANSFUSION

Michel Prudent, Julien Delobel, Aurélie Hübner, Corinne Benay, Niels Lion and Jean-Daniel Tissot

# Editorial: The Red Cell Life-Cycle From Erythropoiesis to Clearance

Lars Kaestner 1 \* and Anna Bogdanova2 \*

1 Theoretical Medicine and Biosciences and Experimental Physics, Saarland University, Saarbrücken, Germany, 2 Red Blood Cell Research Group, Institute of Veterinary Physiology Vetsuisse Faculty and the Center for Integrative Human Physiology, University of Zürich, Zurich, Switzerland

Keywords: red blood cell, Erythropoiesis, calcium, turnover, stored blood

**Editorial on the Research Topic**

#### **The Red Cell Life-Cycle From Erythropoiesis to Clearance**

Most of the articles in this research topic focus on processes associated with the plasma membrane of reticulocytes and mature erythrocytes. Membranes of red blood cells (RBCs) do not only serve as a barrier between the cytosol and the plasma compartments. They are actively involved in volume regulation, metabolic control, sensing of environmental stimuli, and control of life span of the circulating RBCs. Dynamic changes occurring at the membrane level play a key role in transformation of reticulocytes to mature RBCs and in selection of RBCs for clearance. Receptors initiate the signaling cascades that control gene expression in erythroid precursor cells undergoing differentiation. Fine-tuning of mRNA translation in differentiating erythroblasts is further controlled by RNA binding proteins that respond to the availability of growth factors, nutrients, and iron (Moore and von Lindern). Interference with signaling processes in control of proliferation and apoptosis, e.g. by way of introducing mutations into Ras GTPase RASA3 results in bone marrow failure syndrome and severe anemia. Using mouse model of the disease oxidative stress and membrane fragmentation during the terminal stages erythroid differentiation were revealed as a cause of disease (Hartman et al.). Enucleation and maturation of reticulocytes is associated with major changes in RBC membrane lipid and protein composition and massive re-arrangements of the membrane cytoskeleton (Moras et al.; Minetti et al.; Ovchynnikova et al.). These changes result from complex sorting processes in which some proteins are actively removed from the membrane surface whereas others are retained and even enriched if normalized to the membrane surface (Minetti et al.). Understanding of the mechanisms underlying reticulocytes production, release, and their maturation is a key to successful production of RBCs from stem cells in a bioreactor in the future (Ovchynnikova et al.).

Processes of maturation and aging of circulating RBCs are covered by several contributions to this volume. In nucleated RBCs of rainbow trout aging and senescence is associated with massive changes in DNA transcription along with loss of membrane surface and increase in density (Götting and Nikinmaa). Of importance, most of the age-dependent changes in transcriptome were reported for the genes involved in sensing stress (e.g., beta adrenoreceptors), ion transport and volume regulation (e.g., Na+/H+ exchanger) and in genes coding for cytoskeletal proteins (Götting and Nikinmaa). In human RBCs maturation and senescence is associated with decrease in deformability (Huisjes et al.). The spleen performs a selection for the least deformable cells that are not able to pass through the splenic sinuses, removing them from the circulation defining thereby RBC lifespan. Mechanical stress, the cells are exposed to when squeezing through the narrow capillaries, is sensed by mechano-sensitive ion channels that open upon mechanic stimulation and allow a short bout of Ca2+ entry (Danielczok et al.) that is followed by activation

#### Edited by:

Mario Diaz, Universidad de La Laguna, Spain

#### \*Correspondence:

Lars Kaestner<EMAIL_ADDRESS>Anna Bogdanova<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Membrane Physiology and Membrane Biophysics, a section of the journal Frontiers in Physiology

> Received: 26 September 2018 Accepted: 15 October 2018 Published: 08 November 2018

#### Citation:

Kaestner L and Bogdanova A (2018) Editorial: The Red Cell Life-Cycle From Erythropoiesis to Clearance. Front. Physiol. 9:1537. doi: 10.3389/fphys.2018.01537

**6**

of Ca2+ pumps and rapid restoration of gradients (Kaestner et al.). Extreme sensitivity of RBCs to mechanical stress is almost never considered when designing experiments with these seemingly robust cells. In their study Wiegemann et al. show that simple pipetting or centrifugation may result in damage and loss of most unstable RBCs selecting more stable ones for further experiments.

Reduced oxygen availability (hypoxia) triggers adaptive responses in all cells and tissues including RBCs. In RBCs acute decrease in oxygenation is associated with a switch in metabolism favoring pentosophosphate pathway over glycolysis, increase in deformability and ATP release from RBCs of healthy humans (Grygorczyk and Orlov). Longer exposures of lowlanders to hypoxia triggers increase in RBC mass and the consequent upregulation in blood viscosity. RBCs newly produced at high altitude (neocytes) are most likely the first to be destroyed within the first days after descent from the mountains, the effect known as neocytolysis. The present state of research, the controversies and discussions of the neocytolysis concept are summarized in a review of Mairbäurl.

Extracoporeal circulation during open heart surgery represents one more stress condition causing RBC damage and hemolysis due to extensive mechanical abuse. Improvement of RBC membrane deformability and stability as well as reduction in oxidative stress by means of low-level light treatment supports RBCs and reduces hemolytic activity (Walski et al.). Stored RBCs also undergo premature removal that is driven by reversible and irreversible lesion formation. Whereas, membrane loss is associated with irreversible damage, metabolic depletion is a major contributor to the reversible lesion formation (Barshtein et al.). Membrane loss and the underlying alterations in cytoskeletal structure occurring during storage are making the cells less deformable. Mechanisms of microvesicle generation and their protein composition are intensively explored as they may provide insights into the such co-morbidities observed in patients with hereditary hemolytic anemias as thrombosis and autoimmune reaction (Leal et al.). Protein composition of vesicles produced during storage has been studied by Prudent et al. and provides further evidence that shedding of vesicle enriched with flotillin-2—band 3 complexes as well as α-adducin occurs during RBC storage. Loss of membrane or dehydration and the resulting decrease in deformability has multiple consequences. This important parameter may now be quantified and used as an indicator of severity of hereditary hemolytic anemias (Huisjes et al.). Recently introduced Laser Optical Rotational Red Cell Analyzer is becoming more and more abundant as a tool to assess deformability. Diagnostic power of this method for diagnosing rare hereditary anemias is discussed by Zaninoni et al. two processes converging and causing decrease in RBC deformability in senescent RBCs of healthy subjects include membrane loss and redistribution of inorganic cations and water. Loss of transmembrane gradients of Na+, K+, and Ca2+ in RBCs is known to be a common feature of several hereditary hemolytic anemias, often described as diseases of RBC volume regulation (Gallagher, 2013). Basic understanding of the molecular players in this dysregulation could result in new symptomatic therapies for this group of patients. In their review Flatt and Bruce give a detailed update on defects of cation permeability of RBC membranes in patients with hereditary stomatocytosis.

Free pseudo-steady state Ca2+ is maintained in the RBCs' cytosol at the level of approximately 60 nM (Tiffert et al., 2003) whereas plasma levels are up to 1.5 mM building up a gradient of more than four orders of magnitude. This gradient is supported by a low permeability of the plasma membrane of RBCs to Ca2+ and effective extrusion by the plasma membrane Ca2+ ATPases. Transient bouts of Ca2+ uptake via several types of Ca2+-permeable ion channels (Kaestner et al., accepted) sensitive to mechanical and chemical stimulation and changes in transmembrane potential are used for an extensive array of Ca2+-driven signaling in RBC (Danielczok et al.; Hertz et al.; Kaestner et al.). The role Ca2+ ions play in regulation of RBC life-span in vivo is discussed in several reviews in this volume (Lew and Tiffert; Romero and Hernandez-Chinea; Barshtein et al.).

Although, this research topic provides a significant contribution to our understanding of the RBC life-cycle, it is only one milestone on the way to go. We are delighted that in the frame of 'Frontiers in Red Cell Physiology, new, more specialized research topics are initiated. We believe "Membrane Processes in Erythroid Development and Red Cell Life Time," "Pathophysiology of Rare Hemolytic Anemias" and "Time Domains of Hypoxia Adaptation: Evolutionary Insights and Applications" and further research topics to come will facilitate RBC research and keep us updated.

### AUTHOR CONTRIBUTIONS

Both authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

### ACKNOWLEDGMENTS

We acknowledge the contributors to this research topic and funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement No 602121 (CoMMiTMenT) and from the European Union's Horizon 2020 research and innovation programme under grant agreement No 675115 (RELEVANCE).

### REFERENCES

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Kaestner and Bogdanova. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# RNA Binding Proteins and Regulation of mRNA Translation in Erythropoiesis

#### Kat S. Moore and Marieke von Lindern\*

Department of Hematopoiesis, Sanquin Research, and Landsteiner Laboratory, Amsterdam UMC, Amsterdam, Netherlands

Control of gene expression in erythropoiesis has to respond to signals that may emerge from intracellular processes or environmental factors. Control of mRNA translation allows for relatively rapid modulation of protein synthesis from the existing transcriptome. For instance, the protein synthesis rate needs to be reduced when reactive oxygen species or unfolded proteins accumulate in the cells, but also when iron supply is low or when growth factors are lacking in the environment. In addition, regulation of mRNA translation can be important as an additional layer of control on top of gene transcription, in which RNA binding proteins (RBPs) can modify translation of a set of transcripts to the cell's actual protein requirement. The 50 and 30 untranslated regions of mRNA (50UTR, 30UTR) contain binding sites for general and sequence specific translation factors. They also contain secondary structures that may hamper scanning of the 50UTR by translation complexes or may help to recruit translation factors. In addition, the term 50UTR is not fully correct because many transcripts contain small open reading frames in their 50UTR that are translated and contribute to regulation of mRNA translation. It is becoming increasingly clear that the transcriptome only partly predicts the proteome. The aim of this review is (i) to summarize how the availability of general translation initiation factors can selectively regulate transcripts because the 50UTR contains secondary structures or short translated sequences, (ii) to discuss mechanisms that control the length of the mRNA poly(A) tail in relation to mRNA translation, and (iii) to give examples of sequence specific RBPs and their targets. We focused on transcripts and RBPs required for erythropoiesis. Whereas differentiation of erythroblasts to erythrocytes is orchestrated by erythroid transcription factors, the production of erythrocytes needs to respond to the availability of growth factors and nutrients, particularly the availability of iron.

Keywords: erythropoiesis, mRNA translation, translation initiation factors, eIF4E, eIF2, CSDE1, polyadenylation

### GENE EXPRESSION AND SIGNAL TRANSDUCTION SET THE STAGE FOR ERYTHROPOIESIS

Erythrocytes circulate through human peripheral blood for approximately 120 days. Every day, the human body produces 1011 new erythrocytes to replenish the cells that are taken out of the circulation by macrophages. This requires a tight balance between progenitor proliferation and maturation, in conjunction with erythropoietin (Epo) dependent cell survival. Erythroid homeostasis is controlled by a cascade of transcription factors and by signaling pathways triggered by environmental factors.

#### Edited by:

Anna Bogdanova, Universität Zürich, Switzerland

#### Reviewed by:

Eitan Fibach, Hadassah Medical Center, Israel Andrew Charles Perkins, Monash University, Australia Theodosia A. Kalfa, Cincinnati Children's Hospital Medical Center, United States

#### \*Correspondence:

Marieke von Lindern<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Red Blood Cell Physiology, a section of the journal Frontiers in Physiology

Received: 28 February 2018 Accepted: 21 June 2018 Published: 24 July 2018

#### Citation:

Moore KS and von Lindern M (2018) RNA Binding Proteins and Regulation of mRNA Translation in Erythropoiesis. Front. Physiol. 9:910. doi: 10.3389/fphys.2018.00910

**9**

Several transcription factors that are specifically expressed in erythroid progenitors, are essential for determining the erythroid program of gene expression (**Figure 1**). For instance, GATA-1 (GATA-binding factor 1) functions as one of the master regulators of erythropoiesis, inducing for instance (i) the expression of the Epo receptor (EpoR), (ii) commitment to the erythroid lineage, and (iii) transcription of the β-globin locus (Gregory et al., 1999; Ferreira et al., 2005; Kim and Bresnick, 2007; Ribeil et al., 2013). GATA1 protein synthesis is reduced in Diamond-Blackfan anemia (DBA), a congenital red cell aplasia caused by reduced biosynthesis of ribosomes, and mutations in GATA1 can cause a DBA-like phenotype (Ludwig et al., 2014; Vlachos et al., 2014; Paolini et al., 2016). Also of importance, particularly during terminal erythroid differentiation, is KLF1 (Kruppel-like factor 1, or E-KLF: erythroid KLF), a transcription factor involved in many cellular changes required for the maturation of erythroblasts to erythrocytes, including expression of β-globin (Dzierzak and Philipsen, 2013; Perkins et al., 2016). Haploinsufficiency in KLF1 causes hereditary persistence of fetal hemoglobin (HPFH) (Borg et al., 2010), and a specific mutation in the DNA-binding domain of KLF1 causes type IV congenital dyserythropoietic anemia (CDA) (Iolascon et al., 2013).

Signaling cascades allow for the adaption of the gene expression program laid down by transcription factors such as GATA1 and KLF1 during erythropoietic expansion and differentiation upon demand (**Figure 1**). For instance, during hypoxic stress, Epo production in the kidneys is dramatically increased (Sposi, 2015). The binding of Epo to its cognate receptor (EpoR) activates the cytoplasmic tyrosine kinase JAK2 (Witthuhn et al., 1993), which triggers a downstream signaling cascade via the signal transducer and activator of transcription 5 (STAT5), phosphoinositide-3-kinase (PI3K), and protein kinase B (PKB) pathways to support the proliferation and differentiation of erythropoietic progenitors (Sui et al., 2000; Menon et al., 2006; Fang et al., 2007; Socolovsky et al., 2007). STAT5 induces the expression of, for instance, anti-apoptotic B-cell lymphomaextra large (BCL-XL) to maintain viability during terminal maturation (Gregory et al., 1999). Recently, many erythroid specific STAT5 targets were identified (Gillinder et al., 2017). Stem cell factor (SCF), the ligand for the SCF receptor KIT, acts in conjunction with Epo to repress differentiation and promote expansion of the population of erythroid progenitors (Broudy et al., 1996). SCF signaling is enacted via the PI3K pathway, resulting in the activation of PKB (Sui et al., 2000; van Dijk et al., 2000). PI3K/PKB signaling has two major effector pathways in erythroblasts. On the one hand, it prevents nuclear localization of the Forkhead box O3 (FOXO3) transcription factor that otherwise induces erythroid differentiation (Bakker et al., 2004). Concurrently, the PI3K/PKB signaling pathway activates mammalian target of rapamycin (mTOR), which interacts with translation initiation factors to promote the translation of a specific set of transcripts (Gingras et al., 1999; Grech et al., 2008).

Studies on gene expression have historically focused on transcription as the primary regulatory mechanism. However, it has become increasingly clear that protein levels correlate poorly with transcript expression, and that translation is a major determinant of protein abundance in the cell (Schwanhäusser et al., 2011; Brar and Weissman, 2015). In other words, DNA transcription determines whether a given gene is expressed and determines the fundamentals of the gene expression program. Subsequently, mRNA translation has this program at

regulation (left hand arrows). In addition, signal transduction (Epo and SCF in erythroid progenitors) can modify signaling molecules to activate transcription and translation. For instance, phosphorylation of STAT5 causes dimerization and activates the transcription activity, ERK phosphorylation cause nuclear location and phosphorylation of transcription factors. PI3K activates mTOR, a critical factor in protein synthesis.

its disposal for control of protein synthesis. Finally, protein modification and degradation also control protein expression (**Figure 1**). This review focusses on specificity in control of translation, which is generated by the combination of nucleotide sequences in the 50 and 30 untranslated regions (50UTR, 3 0UTR), together with expression levels and posttranslational control of constitutive translation factors and RNA binding proteins (RBPs). Control of mRNA translation is mediated by a complex web of overlapping mechanisms, including, but not limited to, regulation of translation initiation and the length of the poly(A) tail, by the presence of short upstream open reading frames (uORFs), and by sequence-specific association with RBPs. In this review, a brief overview of these topics of selective mRNA translation as pertains to erythropoiesis will be presented.

### TRANSLATION INITIATION FACTORS IN CAP-DEPENDENT PROTEIN SYNTHESIS

Canonically, translation is initiated via an interaction between the 50 7-methylguanylate (7mG) cap structure of the mRNA and the cap-binding protein eukaryotic initiation factor 4E (eIF4E) (Kong and Lasko, 2012). Interaction of eIF4E with the cap is the major rate limiting step, which is followed by binding of scaffolding protein eIF4G, and the RNA helicase eIF4A to form the eIF4F complex. The scaffolding protein eIF4G in this complex interacts with poly-A binding protein (PABP) to form a closed-loop structure between the 50 and 30 end of the mRNA (**Figure 2**). Concurrently, the ternary complex (TC), consisting of the methionine loaded initiator tRNA (tRNAi met) and the GTP-loaded GTPase eIF2 associates with the 40S ribosomal subunit and with translation factors eIF1, -1A, -3, and -5 to form the 43S pre-initiation complex (**Figure 2**; Hinnebusch, 2014). The preinitiation scanning complex, also referred to as the 48S initiation complex, is subsequently formed via an interaction between eIF4G and eIF3. The preinitiation scanning complex scans the bound mRNA until encountering an initiation codon, at which point recognition of the initiation codon triggers the hydrolysis of eIF2-bound GTP by eIF5 and the recruitment of the 60S large ribosomal subunit, marking the end of initiation phase and the beginning of elongation (**Figure 2**).

Next to eIF4E, the availability of GTP-loaded eIF2 is an important rate limiting factor of translation initiation (Proud, 2005). Once translation initiation starts, the TC dissociates upon hydrolysis of GTP to GDP. To reassociate with the TC and rejoin the scanning complex, eIF2 must be reloaded before a new round of translational initiation can begin. This is accomplished via GDP-GTP exchange factor eIF2B, allowing the pre-initiation complex containing eIF2-GTP and tRNAi met to reform. This mechanism is subject to control by phosphorylation of the α subunit of eIF2, which prevents the GDP-GTP exchange, resulting in the inhibition of cap-dependent translation (**Figure 2**).

The control of rate-limiting factors eIF4E and eIF2 in erythropoiesis and the corresponding signaling pathways are discussed in the next two paragraphs.

FIGURE 2 | Mechanism and regulation of cap-dependent translation initiation. (A) Growth factor signaling activates phosphorylation of 4EBP by mTOR, which releases the cap-binding factor eIF4E. Upon binding to the mRNA cap, eIF4E associates with helicase eIF4A and scaffold protein eIF4G to form the eIF4F complex. The interaction between the poly(A)-tail binding protein PABP with eIF4G forms a closed loop between cap and tail. Concurrently, the ternary complex, existing of eIF2:GTP and tRNAi met, associates with the 40S ribosomal subunit and eIF1, -1A, -3, and -5 (labeled numerically in the figure). This assembly is referred to as that 43S pre-initiation complex. (B) eIF3 interacts with eIF4G in the eIF4F complex. After docking with the ribosome, the 48S pre-initiation scanning complex is formed, and the transcript is scanned until encountering a start codon, upon which eIF2-bound GTP is hydrolyzed and the 60S ribosomal subunit is recruited. At this stage, initiation of translation is complete, and translational elongation begins. (C) Cap-dependent translation is regulated by the phosphorylation of eIF2. After a round of translation initiation, eIF2 must be recharged with GTP by eIF2B in order for the ternary complex to be reformed and for the 60S ribosomal subunit to be recruited in subsequent cycles of translational initiation. Under stress conditions, eIF2 is phosphorylated by, for example, HRI (heme regulated inhibitor, during heme scarcity), PKR (Protein kinase R, upon recognition of viral dsRNA), PERK (PRK-like endoplasmic reticulum kinase, upon aggregation of malfolded proteins), or GCN (General control non-derepressible, during amino acid starvation), preventing eIF2B from exchanging eIF2-bound GDP for GTP. The lack of unphosphorylated eIF2 leads to translational arrest.

## Epo and SCF Controlled Translational Initiation in Erythroblasts

Stem cell factor (SCF) cooperates with Epo to expand erythroblast numbers in vivo and in vitro, whereas erythroblasts mature to erythrocytes in presence of Epo only (Wessely et al., 1999).

The crucial pathway activated by SCF is the mTOR-dependent release of the cap binding factor eIF4E from its binding protein 4EBP (Blázquez-Domingo et al., 2005). Overexpression of eIF4E inhibits erythroid differentiation (Blázquez-Domingo et al., 2005; **Figure 3**). This is in part due to SCF-independent proliferation of erythroblasts, but also due to a block in erythroid differentiation (Chung et al., 2015). Upon release from 4EBP, eIF4E binds the mRNA cap-structure, allowing the formation of the pre-initiation scanning complex (Hinnebusch, 2014; Siddiqui and Sonenberg, 2015). In addition to release of eIF4E, PI3K dependent phosphorylation of eIF4B increases its association with the helicase eIF4A and enhances eIF4A activation, which facilitates scanning of the 50UTR (Shahbazian et al., 2010). Thus, PI3K activation increases formation of the preinitiation complex at the cap, but also increases the capacity of the complex to unwind structures in the 50UTR. The increased scanning capacity controls overall translation initiation, but transcripts with a TOP (terminal oligopyrimidine tract) or secondary structures are hypersensitive to PI3K activity and eIF4E availability (Graff and Zimmer, 2003; Modelska et al., 2015).

One such transcript is immunoglobulin binding protein (IGBP1), which is constitutively expressed in erythroblasts, but selectively translated only upon SCF-induced eIF4E release (Grech et al., 2008). IGBP1 is more suitably known as the alpha4 subunit of protein phosphatase 2A (PP2A) (Nanahoshi et al., 1998). As a regulatory subunit, it inhibits the catalytic activity of PP2A on 4EBP (**Figure 3**). Therefore, expression of IGBP1/Alpha4 enhances the effect of low levels mTOR activation on mRNA translation. SCF-induced expression of IGBP1 acts as a positive feedback mechanism in the polysome recruitment of multiple eIF4E-sensitive mRNAs, resulting in SCF-independent proliferation of erythroblasts and attenuation of erythroid differentiation (Grech et al., 2008). Another transcript subject to the mTOR/eIF4E pathway and crucial for erythroid differentiation encodes USE1 (Unusual SNARE protein in the ER 1), which controls retrograde vesicle transport between the Golgi apparatus and the endoplasmic

plus SCF induces full phosphorylation of 4EBP and release ofeIF4E, which enables asembly of the preinitiation scanning complex at the mRNA cap, and translation of IGBP1 mRNA to produce IGBP1 also known as the alpha4 inhibitory subunit of PP2A. (C) Overexpression of eIF4E out-titrates 4EBP and enables translation of IGBP1 in absence of mTOR activation. (D) Constitutive expression of IGBP1/Alpha4 inhibits dephosphorylation of 4EBP and enables weak PI3K/mTOR activation to result in full phosphorylation of 4EBP, release of eIF4E and translation of IGBP1/Alpha4. (B–D) IGBP1 is an example representing many other transcripts with highly structured 50UTRs. Cytospins indicate that translation of IGBP1 is associated with proliferation of erythroid progenitors in absence of differentiation.

reticulum (Grech et al., 2008; Nieradka et al., 2014). Finally, also mitochondrial biogenesis is under control of the mTOR/eIF4E pathway in erythroblasts (Liu et al., 2017).

### eIF2 Phosphorylation in Erythropoiesis

Regulation of translation initiation is of particular importance in hemoglobin synthesis. Erythrocytes carry approximately 250 million hemoglobin molecules, each consisting of 4 globin peptides and 4 iron-containing heme molecules. Iron deficiency reduces heme availability and presents a risk of cell damage from the accumulation of free α- and β-globins that form toxic precipitates known as Heinz bodies (Jacob and Winterhalter, 1970). Therefore, in- and export of iron in erythroblasts, and the synthesis of heme and globin needs to be tightly coupled (Chen, 2014). Heme acts as a signaling molecule which binds to eIF2 associated kinase (eIF2ak1), also called HRI (Heme Regulated Inhibitor) (Han et al., 2005). During heme deficiency, HRI phosphorylates the α-chain of trimeric eIF2, preventing the exchange of eIF2-bound GDP for GTP by eIF2B and therefore also preventing the reassociation of the TC and the preinitiation scanning complex (**Figure 2**; Chen, 2007; Chung et al., 2012). This results in a global inhibition of protein synthesis (Liu et al., 2008). This mechanism is similar to eIF2 alpha phosphorylation resulting from detection of (viral) double-stranded RNA via eIF2ak2 (PKR), ER-stress via eIF2ak3 (PERK), and amino acid starvation via eIF2ak4 (GCN2) (Wek et al., 2006). Control of eIF2 phosphorylation is crucial during erythroid differentiation. Mice carrying alleles in which control of eIF2 phosphorylation is disturbed suffer from anemia. Phosphorylation of eIF2 induces translation of ATF4 (Activating transcriptional factor 4), and expression of its target PPR15a (Protein phosphatase 1, regulatory subunit 15A, also known as GADD34). GADD34 enhances the enzyme activity of the catalytic PP1 subunit PPR15b (Harding et al., 2009). Mice that lack ATF4 suffer from mild compensated anemia, whereas mice lacking HRI or GADD34 are anemic with inefficient erythropoiesis (Masuoka, 2002; Patterson et al., 2006; Zhang et al., 2018).

### Upstream Open Reading Frames

Increased sensitivity to translation factors, and particularly to eIF2, is predicted by the presence of uORFs in the 50UTR of the transcript. The uORFs render mRNA translation hypersensitive to translation initiation factors at the level of (i) start codon recognition, (ii) interaction of the peptide or the uORF sequence with the ribosome, and (iii) reinitiation of the TC (eIF2:GTP and tRNAi met) with the scanning complex (Hinnebusch, 2014; Young et al., 2015). Translation of the uORF first requires recognition of the start codon. The better a start codon is embedded in a Kozak consensus sequence, the longer the scanning complex pauses, and the higher the chance that the GTPase activity of eIF2 is activated and translation is initiated (Kozak, 1986). Not only the local nucleotide sequence, also a downstream secondary structure may delay the scanning while the eIF4A helicase activity unfolds the transient obstacle. Finally, the activity of the eIF2 GTPase activity is controlled

by eIF1 and eIF5 that regulate eIF2 activity and specificity. Interestingly, expression of eIF1 and −5 is controlled by SCF and during erythroid differentiation (Grech et al., 2008). Translation of the uORF causes eIF2 to dissociate from the complex, and requires reassociation of a new TC with the scanning complex before the scanning complex reaches the start codon of the next ORF. Fast reassociation depends on eIF2 expression and phosphorylation (**Figure 2C**). In cases where the uORFs overlaps with the protein-coding ORF (indicated as CDS, coding sequence), start codon recognition is mostly in a less favorable Kozak consensus sequence, whereas the presence of multiple uORFs in the 50UTR typically renders translation dependent on the distance between uORFs and between the uORF and the CDS.

Whereas translation is generally inhibited upon eIF2 phosphorylation, the translation of specific transcripts may be enhanced under these conditions. One such transcript is ATF4, which is essential for erythroid differentiation and for reduction of oxidative stress during the basophilic erythroblast stage (Suragani et al., 2012). ATF4 contains two uORFs, the second of which overlaps with the CDS (Vattem and Wek, 2004). Translation of the second uORF inhibits ATF4 protein expression by overwriting the CDS (Lu et al., 2004). Phosphorylation of eIF2 decreases translation of the second uORF and increases translation of the ATF4 CDS ORF. Whether uORFs are translated, has to be determined experimentally. Ribosome footprint analysis is a novel breakthrough in this field, because it provides the position of ribosomes on a transcript, and can identify which uORFs are actually translated (Ingolia, 2014). Treatment of erythroblasts with tunicamycin to induce eIF2 phosphorylation via PERK reduces the overall density of ribosomes on the transcriptome (Paolini et al., 2018). The ribosome density increases, however, on at least 140 transcripts among which ATF4, but also PNRC2 (proline rich nuclear receptor coactivator 2, involved in RNA stability) and IFDR1 (interferon-related developmental regulator 1, involved in cellular stress resistance) (Paolini et al., 2018). Other uORF-containing transcripts, instead, are hypersensitive to eIF2 phosphorylation and translation decreases more than average upon eIF2 phosphorylation in erythroblasts, among which CSDE1 (Cold shock domain protein e1, see below), PABPC1 (PolyA binding protein c1), and PAPOLA, [poly(A) polymerase alpha], which suggests that eIF2 phosphorylation may result in shortening of mRNA polyA-tails. The response to eIF2 phosphorylation is cell type specific, as indicated by a comparison between ribosome footprints in erythroblasts and HEK293 cells (Andreev et al., 2015; Paolini et al., 2018).

The analysis of uORF translation in erythroblasts indicates that uORFs are involved in eIF2-dependent translational regulation, but they do not predict eIF2-dependent translational regulation. This characterizes the advances of the field. We increasingly understand how general translation factors act together to control cap-dependent translation in commonly used cell models. For specific transcripts we understand the interplay of general translation initiation factors and specific sequences or structural elements in the 50UTR of the transcript. However, when this information is used to make general predictions, the

predictions are often wrong. For instance, Mfold is a well-known program for making structural predictions (Zuker, 2003). This is a useful tool, but chaperones and RBPs present in specific cell types may be more important determinants of structures and the energy required to unfold these structures. Therefore, we need to increase our understanding on the role of sequences and structures in the 50UTR that have often been neglected when promoter studies were performed to understand gene expression and only the protein coding ORF of a transcript was used to study the role of the encoded protein.

### Alternative 50UTR Through Erythroid Specific Promoters or Alternative Splicing

The use of erythroid specific promoters and alternative splicing of the 50UTR is common and has largely been ignored when it does not affect protein identity. It has been estimated that up to one third of all genes expressed in erythroid progenitors may be expressed from an alternative promoter (Tan et al., 2006; Cheng et al., 2014). The presence of an alternative 50UTR can include or exclude structural elements (stem-loop structures or uORFs), with major consequence for translation initiation. RBP involved in alternative splicing of the 50UTR are also modifiers of translation. Notably, erythroid differentiation is associated with extensive alternative splicing (Pimentel et al., 2014), with a prominent role for the RBP Muscleblind (Cheng et al., 2014). Alternative splicing mainly involves coding sequences (Cheng et al., 2014), but may specifically alter translation regulation in the 50UTR. For instance, translation and stability of Heme Oxygenase-1 (HO1) is regulated by hemin as part of feedback control. Alternative splicing generates a hemin resistant alternative transcript (Kramer et al., 2013). Similarly, transcript variants of Ferroportin are expressed during erythropoiesis that vary in their 50UTR to enable iron responsive and non-responsive translation (Cianetti et al., 2005). In case of the SNARE-complex protein USE1, a predicted G-quadruplex in an alternatively spliced 50UTR results in USE1 protein expression mainly from the small ratio of transcripts with intron retention (Nieradka et al., 2014). Alternative splicing can also alter the translation initiation codon that is used. Detection of alternative translation start sites can be established by ribosome footprinting or by mass spectrometry, in which case novel predicted N-termini must first be added to the reference library (Van Damme et al., 2014; Floor and Doudna, 2016).

### IRES TRANS-ACTIVATING FACTORS

In addition to cap-dependent translation, ribosomes can associate on a subset of transcripts that carries an internal ribosomal entry site (IRES) (Komar and Hatzoglou, 2011). While there is no consensus sequence or universal structural motifs for IRESs, they typically contain complex structural elements which include stem loops and pseudoknots (Komar and Hatzoglou, 2005; Baird et al., 2007). The majority of IRESs are found within the 50UTR directly upstream of the initiation codon, though they can also exist within the coding region, causing synthesis of a truncated protein (Komar et al., 2003; Grover et al., 2009). IRESmediated translation is preferred under conditions of decreased availability of cap-dependent translation (Lewis and Holcik, 2008; Spriggs et al., 2008), due, for example, to eIF2 phosphorylation (Gerlitz et al., 2002; Tinton et al., 2005) or cleavage of scaffolding protein eIF4G (Komar and Hatzoglou, 2005). This includes, but is not limited to, viral infection, hypoxia, nutrient starvation, ER stress, and cell differentiation (Komar and Hatzoglou, 2005; Graber and Holcik, 2007).

Translation via an IRES requires the binding of IRES transactivating factors (ITAFs) such as Polypyrimidine Tract Binding protein (PTB) (Bushell et al., 2006; Cobbold et al., 2010) and CSDE1 (Hunt et al., 1999; Mitchell et al., 2003; Cornelis et al., 2005). Some ITAFs are required to unfold the secondary structure, which subsequently enables other ITAFs to bind to specific sequence elements, and to recruit the ribosomal subunits. IRES-mediated translation initiation is less competitive in ribosome recruitment. Compared to the 7mG cap structure, IRES elements are less competitive to recruit ribosomes. It is probably for that reason that primarily IRES-dependent translation initiation is suppressed when less ribosomes are present, which is a hallmark of DBA (Horos et al., 2012). The induction of severe anemia due to loss of ribosomes indicates that IRES-mediated translation is of particular importance in erythropoiesis.

Several genes involved in hematopoiesis are subject to IRESmediated translation. BCL2-associated athanogen 1 (BAG1) and Heat Shock Protein 70 (HSP70) cochaperone are required for terminal differentiation of erythroblasts (Horos et al., 2012). All three BAG1 isoforms are produced from a single transcript dependent upon the involvement of either cap-dependent or IRES-mediated translation (Coldwell et al., 2001). Two ITAFs are involved in this process: PCBP1 [poly(rC) binding protein 1], which remodels the RNA to allow ribosomal entry, and PTB, which is necessary for the recruitment of the ribosome to the BAG1 transcript (Pickering et al., 2004). Bag1 deficiency is lethal at day E13.5 in mice because of a complete lack of definitive erythropoiesis (Götz et al., 2005). shRNA-mediated knockdown of BAG1 in erythroblasts results in the production of fewer hemoglobinated daughter cells under differentiation conditions.

Another example of IRES-mediated translation is transcription factor RUNX1, which is essential for fetal liver hematopoiesis (Okuda et al., 1996). The RUNX1 gene has two promoters that yield RUNX1 transcripts with two distinct 50UTRs, one of which contains an IRES (Pozner et al., 2000; Levanon and Groner, 2004). The absence of the IRES in Runx1 causes embryonic fatality due to disordered proliferation and differentiation of hematopoietic cells in the fetal liver (Nagamachi et al., 2010).

Internal ribosomal entry sites have a unique role in circular RNAs that are generated by backsplicing. Without head and tail, these circular RNAs are hardly degraded, and lack the potential of being translated by cap-dependent mRNA translation. Interestingly, some circular RNAs encode proteins because translation initiation of circular RNA can be initiated from an IRES (Legnini et al., 2017; Pamudurti et al., 2017; Begum et al., 2018).

### RBPs CONTROL mRNA STABILITY AND TRANSLATION THROUGH THE mRNA POLY(A) TAIL

Almost all protein coding mRNAs (except histones) are polyadenylated at their 30 end. The poly(A) tail protects from degradation, and the length of the poly(A) tail also affects translation initiation (**Figure 2**). The length of the poly(A) tail is determined by the recruitment of polyadenylating and deadenylating enzymes to the transcript. Polyadenylation initially occurs in the nucleus where mRNA cleavage and polyadenylation are coupled (Charlesworth et al., 2013). Upon cleavage site recognition, a nuclear poly(A) polymerase is recruited to synthesize the poly(A) tail. Typically this is carried out by PAPOLA, but depending on the cell type and specific transcript, another, non-canonical polymerase may be involved (Charlesworth et al., 2013). Upon export to the cytoplasm, RBP binding to the 30UTR may recruit polyadenylating or deadenylating proteins that may enhance or repress translation of the mRNA transcript. A broad array of RNA-binding proteins governs this process. Here, an overview of some of the larger families of poly(A)-interacting proteins and their influence on erythropoiesis will be presented (**Figure 4**).

### PABPs Promote Translational Initiation and Stabilize Transcripts via poly(A) Binding

Essential to the initiation of translation is the binding of poly(A) binding proteins (PABPs) to the poly(A) tail. PABPs directly interact with the eIF4G scaffold protein of the eIF4F cap-binding complex (Hinnebusch and Lorsch, 2012). This brings the mRNA tail close to the cap, and forms a mRNA loop conformation that is believed to optimize recycling of translation initiation and elongating factors (Wakiyama et al., 2000). The interaction of PABP with eIF4G stabilizes pre-initiation scanning complexes on the 50UTR (Kahvejian et al., 2005; **Figure 2**). A longer poly(A) tail increases PABP affinity for target transcripts, which enhances the stabilization of multiple preinitiation scanning complexes and thereby enhances translation initiation efficiency. Additionally, the binding of PABP to the poly(A) tail protects the transcript from deadenylation and degradation (Norbury, 2013), a process which is discussed in more detail below.

The most common PABP is PABPC1. In erythroid MEL (mouse erythroid leukemia) cells, however, there is a prominent role for Pabpc4 (Kini et al., 2014). Pabpc4 binds to a specific subset of transcripts with short poly(A) tails containing an AUrich motif, including α-globin, and protects them from further degradation. Depletion of Pabpc4 blocks induced terminal differentiation of MEL cells by altering the expression of five genes associated with erythroid maturation. Receptor tyrosine kinase c-Kit is strongly upregulated in Pabpc4-depleted MEL cells. This activity is specific to Pabpc4 and not redundant with Pabpc1. Given that downregulation of c-Kit is essential for terminal differentiation (Munugalavadla et al., 2005; Dzierzak and Philipsen, 2013), the rescue of c-Kit expression is likely responsible for the inability of Pabpc4-depleted MEL cells to mature (Kini et al., 2014). Also induced were c-Myb, c-Myc, CD44, and Stat5a, all well-studied genes which promote erythroblast maintenance at the expense of differentiation (Hattangadi et al., 2011).

## CPEBs Control poly(A) Tail Length

Among the proteins able to recruit cytoplasmic polyadenylation enzymes are the cytoplasmic polyadenylation element binding proteins (CPEBs). CPEBs, however, are promiscuous proteins

able to recruit either cytoplasmic poly(A) polymerases such as the Drosophila GLD2 protein (Germ Line Development 2; TENT2, terminal nucleotidyltransferase 2, in man; PAPD4, PAP associated domain containing 4 in mouse), which promotes mRNA stability and translation, or the deadenylating CCR4/NOT complex (Barnard et al., 2004; Ogami et al., 2014; **Figure 4**). Whether binding of CPEBs increases the length of the poly(A) tail or causes deadenylation depends on the interaction with additional RBP such as PUMILIO, or on phosphorylation of CPEB, for instance by Aurora kinase A (AURA) (Charlesworth et al., 2013). Deadenylation by CPEB depends on recruitment of the CCR4/NOT complex that is composed of several CNOT subunits, which individually have varying roles in a myriad of physiological processes. CAF1 and CCR4 are subunits with deadenylation activity (Shirai et al., 2014). In particular, CNOT9 was identified as an erythropoietin-responsive gene, indicating a role for the complex in erythropoiesis (Gregory et al., 2000). Deadenylation initially represses translation, because less PABP can bind and connect to eIF4G proteins in preinitiation complexes. Ultimately, deadenylation results in silencing of gene expression via mRNA degradation (Shirai et al., 2014).

Of the CPEB family members, CPEB4 is specifically induced during erythroid differentiation (Bakker et al., 2007; Hu et al., 2014). Erythroid expression of CPEB4 is regulated via the transcription factors GATA1 and TAL1 (Hu et al., 2014), its strong upregulation late in differentiation depends on FOXO3 (Bakker et al., 2007; Kerenyi and Orkin, 2010; Hattangadi et al., 2011). Because the recruitment of polyadenylating or deadenylating complexes depends on the context of the transcripts, i.e., on the complexes formed with RBP that bind to nearby elements in the 30UTR, CPEB4 enhances or decreases mRNA translation with transcript-specific regulation. In erythroblasts, CPEB4 also associates with the eIF3 complex and the major mechanism appears to be repression of translation via the interaction with eIF3 (Hu et al., 2014). Both over- and underexpression of CPEB4 impairs the terminal differentiation of erythroblasts to reticulocytes, indicating that the level of CPEB4 needs to be controlled. Interestingly, CPEB4 is capable of binding to and repressing its own mRNA, forming a feedback loop that maintains CPEB4 levels within a range required for terminal erythropoiesis (Hu et al., 2014).

Although CPEBs are generally considered as factors that recruit adenylating or deadenylating enzymes, interaction with other general translation factors may also contribute to regulation of translation. Whereas CPEB4 binds eIF3, CPEB1 is known to increase mRNA stability by binding to PABPC1 and PABPC1L (Seli et al., 2005; Guzeloglu-Kayisli et al., 2008).

### Musashi-Mediated Translational Control in Hematopoiesis

In addition to CPEB and PABP, cytoplasmic polyadenylation is regulated by MUSASHI-1 and -2 (MSI1 and MSI2). These proteins do not specifically control erythropoiesis, but they are very important for hematopoietic stem- and progenitor cells (HSPC) from which erythropoiesis develops. MSI1 and MSI2 interact with mRNA via the MSI-binding element (MBE) (Charlesworth et al., 2013). The MBE element is known to confer cytoplasmic polyadenylation in the absence of CPEB activity (Charlesworth et al., 2006). The poly(A) polymerase GLD2 interacts with MSI in Xenopus oocytes, but interactions between MSI proteins and the polyadenylation machinery have been described in mammalian cells (Charlesworth et al., 2013; Cragle and MacNicol, 2014).

The majority of studies done on MUSASHI-mediated translational repression have been done with MSI1. MSI1 represses the translation of target transcripts by competing with eIF4G for binding with PABP, preventing the formation of the 80S ribosome subunit (Kerwitz et al., 2003). Transcripts silenced by Msi1 include cell cycle regulators such as Numb, an inhibitor of the NOTCH pathway, and the cell cycle inhibitor p21 (Bardwell et al., 1990; Topalian et al., 2001). RNA binding domains between MSI1 and MSI2 are largely homologous (85–95%), but MSI2 has no PABP-binding domain (de Andrés-Aguayo et al., 2012). However, there is evidence to suggest that MSI2 alters NOTCH localization and upregulates HES1, a NOTCH reporter protein (Kharas et al., 2010), suggesting that MSI1 and MSI2 overlap in regulating common targets.

MSI2 is abundantly expressed in primitive LSK cells of the hematopoietic lineage, where MSI1 expression is nearly absent (Kharas et al., 2010; de Andrés-Aguayo et al., 2011; Hope and Sauvageau, 2011). The expression of MSI2 is subsequently downregulated during differentiation. Downregulation of MSI2 alters the balance between self-renewal and differentiation of HSCs via regulation of the NOTCH pathway (Kharas et al., 2010; Hope and Sauvageau, 2011). This effect is achieved without influencing apoptotic rates or homing behavior. A mouse line expressing a truncated MSI2 gene (Msi2Gt/Gt) results in a marked decrease in short term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MPPs) while the effect on long term hematopoietic stem cells (LT-HSCs) was nominal (de Andrés-Aguayo et al., 2011). MSI2-defective LSKs display impaired proliferation, and the LT-HSC population is decreased following non-competitive bone marrow transplantation (Kharas et al., 2010). In addition, a doxycycline-inducible MSI2 transgenic mouse model observed an increase in ST-HSC/MPP populations and a decrease in LT-HSC, whereas MSI2 overexpression increased LT-HSC self-renewal in transplanted mice. Taken together, these findings suggest that MSI2 is important for HSPC self-renewal and stem cell homeostasis particularly during stress hematopoiesis. Studies on MSI2 in hematopoiesis have been largely functional in focus and do not mechanistically investigate mRNA polyadenylation via MSI2. Although the direct molecular targets of MSI2 in hematopoiesis are unknown, gene expression profiling indicates a regulatory function for pathways involved in HSC proliferation, including MEIS1, HOXA9, HOXA10 (Hope and Sauvageau, 2011), RAS, MAPK, CyclinD1, and MYC (Kharas et al., 2010; de Andrés-Aguayo et al., 2011). The role of MSI2 in Hematopoiesis has been extensively reviewed in de Andrés-Aguayo et al. (2012).

### AUBPs in Hematopoiesis

The AAU1/HNRPD family of AU-rich element binding proteins (AUBPs) are oppositely regulated from the MSI proteins (Sakakibara et al., 2002). AU-rich elements (AREs) are sequence

motifs (typically AUUUA) in the 30UTR that recruit a large family of AU-binding proteins (AUBPs) that regulate translation (**Figure 4**). They were among the first sequence elements known to affect stability of mRNA (Shaw and Kamen, 1986). Translational regulation via AUBPs can occur via a number of mechanisms including deadenylation, and transcript sequestration to P-bodies and stress granules (Baou et al., 2011). AUBPs key to erythropoiesis include the tristetraprolin (TTP) family members (ZFP36, ZFP36L1, and ZFP36L2) and HuR/ELAV1.

TTP family members interact with NOT1 to promote the rapid deadenylation by the CCR4/NOT complex (Sandler et al., 2011; Fabian et al., 2013). Interestingly, ZFP36L1 and ZFP36L2 demonstrate opposite regulation in erythropoiesis. ZFP36L2 is expressed in erythroid progenitors in response to glucocorticoids and downregulated during terminal differentiation. ZFPL1, instead, is only expressed in more mature erythroblasts (Zhang et al., 2013; MvL unpublished data). Concordantly, ZFP36L2 is required for burst-forming unit-erythrocyte (BFU-E) renewal (Zhang et al., 2013), whereas ZFP36L1 downregulates STAT5B expression, reducing the formation of erythroid colonies (Vignudelli et al., 2010). Why these highly homologous factors have such opposite effects in erythropoiesis, and how they regulate the transcripts to which they are bound, is unclear. In keratinocytes, ZFP36L1 and ZFP36L2 also have non-redundant roles in the regulation of growth factor expression, but in T cells, both ZFP36L1 and ZFP36L2 inhibit cell proliferation by inhibiting the expression of cell cycle genes, particularly D Cyclins (Galloway et al., 2016; Prenzler et al., 2016).

ELAV1, also known as HuR, is a ubiquitously expressed AUBP with thousands of direct and functional targets (Lebedeva et al., 2011; Mukherjee et al., 2011). Relevant transcripts bound by ELAV1 encode proteins that control cell cycle progression, apoptosis, signal transduction, and lineage specific transcription factors (reviewed in Baou et al., 2011). Interestingly, ZFP36L1 is a functional target of ELAV1 in cells exposed to oxidative stress, wherein ionizing radiation decreases ZFP36L1 transcript binding by ELAV1, resulting in a decreased recruitment of ZFP36L1 to polysomes (Mazan-Mamczarz et al., 2011). This suggests that ZFP36L1 may synergize with ZFP36L1 in regulating erythropoiesis under some conditions.

### Alternative Polyadenylation Controls mRNA Stability and Translation

The examples mentioned above are only a brief and incomplete overview of how proteins binding to the 30UTR may control mRNA stability and translation through recruitment of polyadenylases or the deadenylating complex. It is long known that the nuclear cleavage and polyadenylation complex may recognize alternative sites for mRNA cleavage resulting in alternative length of the 30UTR. The exclusion of binding sites for RBPs can have major consequences for mRNA stability and translation (Mayr, 2016). The implications of single transcripts have been studied, but systematic analysis of alternative polyadenylation (APA) and their consequences for mRNA stability are scarce (Sun et al., 2012). Novel methods are available to determine APA sites, but it is difficult to combine for instance ribosome footprint analysis with APA because the output of ribosome footprinting exists of short ribosome protected fragments that cannot be attributes to distinct transcripts. To date only few examples of APA are known in erythropoiesis but nothing is known about the relation of APA and control of gene expression by RBPs (Kudo et al., 1994; Boehmelt et al., 1998).

### RBPs INVOLVED IN ADDITIONAL CONTROL OF TRANSLATION

In addition to general translation initiation factors and RBP that control the poly(A), mRNAs contain secondary structures and specific sequences that bind RBP to specifically control their translation. An important posttranscriptional mechanism of gene expression is mediated by microRNAs. Argonaut proteins are the miRNA-binding RBPs that associate with RNA. Mice deficient in Argonaut 2 (Ago2) suffer from severe anemia, which is caused by the failure of miR-144/451 to act on its targets (Rasmussen et al., 2010). However, Ago2 is the Argonaut protein with slicing activity and appears to control mRNA stability more than translation (Jee et al., 2018). We therefore focus our discussion on the role of a few selected RBP that are important for erythropoiesis, rather than on miRs.

### Translational Control of Iron Homeostasis

Iron homeostasis in erythroid cells is achieved via a balanced regulation of iron import via transferrin receptor 1 (TfR1), storage in ferritin and export via ferroportin (Kühn et al., 2015). Posttranscriptional control over these proteins is enacted by iron regulatory proteins (IRPs). IRPs are recruited to ferritin mRNA via a conserved sequence that forms a hairpin structure in the 50UTR (Aziz and Munro, 1986; Hentze et al., 1987). Binding of the IRP to the IRE (Iron Response Element) prevents the association of the 43S preinitiation complex to the mRNA transcript (Gray and Hentze, 1994). The presence of iron blocks the IRE-IRP interaction, allowing translation of the formerly repressed ferritin transcript. A similar mechanism governs the translation of ferroportin (Abboud and Haile, 2000). Interestingly, a splice variant of ferroportin lacking the IRE is expressed in duodenum and erythroid cells permits the escape of IRPmediated translational control (Cianetti et al., 2005). By contrast, the TfR1 transcript possesses 5 IREs in the 30UTR rather than the 50UTR (Koeller et al., 1989; Müllner et al., 1989). Binding of IRPs to TfR1 confers increased mRNA stability to the transcript, resulting in an inverse relationship between TfR1 protein expression and iron abundancy (Owen and Kühn, 1987; Müllner and Kühn, 1988).

Both IRPs cooperate to control iron homeostasis, yet they are regulated via different mechanisms. IRP1 is a bifunctional protein with both enzymatic and RNA-binding activity. Its possesses the capacity to act as an aconitase in the catalyzation of citrate to isocitrate (Kennedy et al., 1983). The catalytic activity is dependent upon the assimilation of an additional iron atom in the

active site, with the result that IRP1 functions as an enzyme when iron is abundant, and as an RNA-binding protein when iron is depleted (Haile et al., 1992; Emery-Goodman et al., 1993; Hirling et al., 1994). Because the formation of the iron-sulfur cluster in the active site requires an oxygen-free environment (Beinert and Kennedy, 1993), the RNA-binding form is preferentially induced in the presence of the vasodilating agent nitrous oxide (Drapier et al., 1993).

Although IRP2 is 57% homologous with IRP1 in humans, it does not function as an aconitase under iron-rich conditions (Guo et al., 1994). Unlike IRP1, IRP2 is rapidly degraded by the proteasome when iron and oxygen levels are high (Guo et al., 1994, 1995). Degradation of IRP2 is prevented by low oxygen pressure (Hanson et al., 2003). Taken together, IRP1 and IRP2 are capable of controlling iron homeostasis under both low and high iron and oxygen supply, allowing a proportional response to environmental stimuli (Kühn et al., 2015).

### The RBP Csde1 Is Essential for Erythropoiesis

CSDE1, originally called UNR (upstream of N-ras) is an RNAbinding protein with five cold-shock domains (Triqueneaux et al., 1999).  Repression of MSL2 occurs via a direct interaction between CSDE1 and 30UTR-bound PABP, resulting in the prevention of PABP-mediated recruitment of the 43S complex (Duncan et al., 2009). Paradoxically, the CSDE1-PABP complex has also been reported to stimulate translation by stabilizing the interaction between PABP and eIF4G (Ray and Anderson, 2016).

CSDE1 is implicated in both endogenous and viral IRESmediated translation in mammalian cells. Both rhinovirus and poliovirus contain IRES elements that require an interaction between CSDE1 and PTB (Hunt et al., 1999; Boussadia et al., 2003). Early studies indicated that all five of CSDE1's cold-shock domains were necessary for maintenance of CSDE1's affinity for rhinovirus IRESs, whereas cold-shock domains 1 and 2 had the most impact on CSDE1's binding to MSL-2 (Brown and Jackson, 2004; Abaza and Gebauer, 2008), and cold-shock domains 2 and 4 were the only required elements for stimulation of translation via PABP (Ray and Anderson, 2016).

CSDE1 and PTB also cooperate in IRES binding of the CSDE1 transcript itself, resulting in repression of translation of CSDE1 (Cornelis et al., 2005; Dormoy-Raclet et al., 2005; Schepens et al., 2007). IRES-mediated translation is promoted during mitosis and apoptosis at the expense of cap-dependent translation (Komar and Hatzoglou, 2005; Tinton et al., 2005). The CSDE1 IRES activity is strongly upregulated during mitosis due to increased binding of hnRNPC1/C2 proteins with simultaneous release of CSDE1 and PTB (Schepens et al., 2007). The resultant increase in Csde1 expression during the G2/M phase of the cell cycle facilitates the IRESmediated translation of cyclin-dependent PITSLRE kinases, which are essential for centrosome maturation and mitotic spindle formation (Tinton et al., 2005; Petretti et al., 2006). During apoptosis, CSDE1 and PTB upregulate APAF1 (apoptotic protease-activating factor 1) (Mitchell et al., 2001). Binding of CSDE1 and PTB changes the conformation of the APAF1 IRES, granting access for ribosomal recruitment and thereby permitting the translation of the transcript (Mitchell et al., 2003).

CSDE1 is also implicated as a regulator of mRNA stability via poly(A) deadenylation. CSDE1 binds to cFOS in conjunction with PABP (Chang et al., 2004). In contrast to the stabilizing influence of PABP when interacting with CPEB, the CSDE1- PABP complex promotes transcript degradation via recruitment of the deadenylase CCR4. Given that binding of PABP and CSDE1 to the poly(A) tail can also increase transcript stability, Chang et al. propose a model in which the CSDE1-PABP complex initially protects the poly(A) tail from deadenylation by CCR4 prior to initiation of translation. Upon ribosomal transit of the mCRD, a conformational change is triggered that forms a landing pad for CCR4, resulting in transcript repression by deadenylation.

CSDE1 also interacts with the 4E-Transporter (4E-T), itself a translational regulator with a wide breadth of functions. 4E-T competitively binds to cap-binding protein eIF4E, preventing its association with scaffolding protein eIF4G (Sonenberg and Hinnebusch, 2009; Kamenska et al., 2016), while simultaneously reducing ribosomal access to the 50 cap via interaction with RNA-binding proteins at the 30UTR (Rhoads, 2009; Kamenska et al., 2014b). Furthermore, 4E-T is a component of the CPEB translation repressor complex (Minshall et al., 2007), the CCR4/NOT complex (Kamenska et al., 2014a), and enhances decay of transcripts containing AREs (Ferraiuolo et al., 2005; Chang et al., 2014; Nishimura et al., 2015). 4E-T is directly bound by CSDE1 (Kamenska et al., 2016). 4E-T binding to CSDE1 and CNOT4 is mutually exclusive, which predicts that Csde1 abrogates 4E-Ts role as a bridge between CNOT1 and CNOT4 complex subunits (Kamenska et al., 2016). Due to the complexity of the overlapping pathways associated with 4E-T, it is unclear precisely how CSDE1 cooperates with 4E-T in the regulation of translation. The authors suggest that CSDE1 and DDX6, an RNA helicase component of the CPEB repressor complex, simultaneously bind 4E-T to either redundantly repress translation or to selectively affect specific translational stages. Another possibility is that CSDE1 acts as a competitive inhibitor of 4E-T binding to other, unknown cofactors/repressors, disruption of which unravels a network of interactions necessary for translational repression.

Notably, CSDE1 expression is decreased in erythroblasts with a haploinsufficiency in ribosomal proteins. Expression of CSDE1 is >100-fold upregulated in erythroblasts compared to early progenitors, and expression of Csde1 is required for erythroblast proliferation and differentiation in mouse erythroblasts (Horos et al., 2012). Csde1-bound transcripts and associated proteins that are identified using Csde1 pulldown, encode proteins involved in ribogenesis, mRNA translation and protein degradation, but also proteins associated with the mitochondrial respiratory chain and mitosis (Moore et al., 2018). RNA expression and/or protein expression of Csde1-bound

transcripts are deregulated in MEL clones in which the first coldshock domain of Csde1 is deleted by Crispr/Cas9. For instance, protein expression of Pabpc1 was enhanced while Pabpc1 mRNA expression was reduced indicating more efficient translation of Pabpc1 followed by negative feedback on mRNA stability (Moore et al., 2018). Under the conditions used for Csde1 pull down experiments, Csde1 associates most prominently with Strap (serine threonine kinase receptor associated protein). Strap does not affect Csde1/mRNA association, but it alters expression of some transcripts and/or proteins (Moore et al., 2017). In addition to Strap, also Pabpc1 and Pabpc4 associate with Csde1 in MEL cells.

### Heterogeneous Nuclear Ribonucleoproteins hnRNPK and GRSF1

Among the first RBP that were identified and characterized are members of the large superfamily of heterogeneous nuclear ribonucleoproteins (hnRNPs). Within this superfamily four different RNA binding domains and several auxiliary domains are discerned, on basis of which 16 independent families are identified by capital letters (Geuens et al., 2016). The hnRNP family members do not specifically control translation. They are involved in many levels of gene expression regulation including gene transcription, mRNA splicing, mRNA nuclear-cytoplasmic transport, mRNA stability, and translation.

The RBPs hnRNPE1 and hnRNPK are important for late erythroid maturation. Together they control translation of the reticulocyte enzyme 15-lipoxygenase (r15-LOX) (Ostareck et al., 2001). This enzyme is at the base of mitochondrial degradation through dioxygenation of phospholipids in mitochondrial membranes (Grüllich et al., 2001). The silencing of r15-LOX translation by hnRNPK involves association of r15-LOX with DDX6 in P-bodies, and is regulated by SRC kinase through an intricate feed-back control of hnRNPK and SRC (Naarmann et al., 2008). Translational regulation of mitochondrial degradation in late erythroblasts and reticulocytes is not limited to control of r15-LOX translation. Expression of Non-muscle myosin heavy chain IIA (NmhcIIA), involved in intracellular vesicle transport, is also regulated by hnRNPK (Naarmann-de Vries et al., 2016). Thus, the maturation of reticulocytes depends on hnRNPE1 and hnRNPK. These are the only two subtypes of the hnRNP superfamily that carry the unique RNA binding domain indicated as a K-homology domain (KH) (Geuens et al., 2016).

The subfamilies hnRNPF and hnRNPH share a high degree of homology and contain 3 QRRM domains (Quasi RNA recognition motif) (Geuens et al., 2016). Interestingly, hnRNPF/H members were prominently detected among G-quadruplex binding proteins (von Hacht et al., 2014). Similar to hnRNP proteins, the G-quadruplex structure that can occur in DNA and RNA is widely employed to control gene expression, mRNA splicing, transport, stability, and translation (Song et al., 2016). In translational control, they are mainly detected in transcripts expressed during mitosis, and particularly in transcripts that are well expressed in tumor cells (Wolfe et al., 2014). The Guanine-rich RNA sequence binding factor 1 (GRSF1) contains three RNA binding domains and is a member of the heterogeneous nuclear ribonucleoprotein F/H family (hnRNPF/H) (Ufer, 2012; Sofi et al., 2018). GRSF1 binds a G-rich element in Glutathione Peroxidase 4 (GPX4), and in the transcript encoding Unusual SNARE protein in the ER-1 (USE1) (Ufer et al., 2008; Nieradka et al., 2014). Translation of USE1 depends on the PI3K/mTOR pathway, and on the availability of eIF4E in erythroblasts. Constitutive expression of a truncated USE1 isoform inhibits erythroid differentiation, whereas clones expressing full length USE1 could not be established (Grech et al., 2008). This suggests that regulation of USE1 expression during erythropoiesis is critical. Differential splicing creates transcripts with a long and short 50UTR. Although most transcripts are spliced and contain a short 50UTR, the unspliced USE1 transcript is predominantly present in polyribosomes (Nieradka et al., 2014). This is due to the presence of a G-rich element that scores high in G-quadruplex prediction models. Both USE1 and GRSF1 are expressed in proliferating erythroblasts and are downregulated during differentiation. Suppression of USE1 and GRSF1 expression in erythroblasts decreases the capacity to undergo renewal divisions, and induces terminal erythroid differentiation instead (Nieradka et al., 2014). Thus, the RBP GRSF1 is crucial to erythropoiesis, either through regulation of USE1 translation or through regulation of other G-Quadruplex containing transcripts.

### More RBPs and Methods to Decipher Translation Efficiency

Transcripts that are subject to control of translation can be identified by density centrifugation to separate subpolysomal from polyribosomal transcripts. In erythroblasts, this resulted in the identification of numerous transcripts that are dependent on for instance growth factors Epo and SCF for their translation (Joosten et al., 2004; Grech et al., 2008). Detailed mRNA translation, at the nucleotide level, can be determined using ribosome footprinting technology (Ingolia et al., 2011). This technology uncovered changes in mRNA translation efficiency in response to tunicamycin-induced phosphorylation of eIF2 (paragraphs 2.2 and 2.3; Paolini et al., 2018). Ribosome footprinting during terminal differentiation of erythroblasts to reticulocytes revealed several classes of transcripts subject to regulated translation (Alvarez-Dominguez et al., 2017). RNA binding motif 38 (RBM38) is an RBP that is prominently expressed in erythroblast. Its expression steadily increases from basophilic erythroblasts to the reticulocyte stage. Interestingly, a polymorphism in RBM38 is associated with erythrocyte volume (van der Harst et al., 2012). RBM38 is known to be associated with erythroid differentiation through control of mRNA splicing (Heinicke et al., 2013; Ulirsch et al., 2016). Of interest, Rbm38 serves as a tumor suppressor gene, and mice that lack Rbm38 are prone to cancer development. In addition, however, they suffer from anemia (Zhang et al., 2014). RBM38 binds to a UGUGU element in the 30UTR of its target transcripts and interacts with the scaffold protein eIF4G in the eIF4F capbinding complex. Therefore, Alvarez-Dominguez et al. (2017) hypothesize that RBM38 interferes with the closed loop model

of mRNA translation and thereby decreases the efficiency of translation.

Finally, ribosome profiling also showed that ribosomes fail to be recovered in reticulocytes and therefore accumulate in the 30UTR (Mills et al., 2016). This is due to loss of ABCE1. This loss of ribosome recovery may be the start of total RNA degradation which characterizes the maturation of reticulocytes to erythrocytes during the first 2 days after release of reticulocytes in the peripheral circulation.

### CONCLUSION

This review presents a general overview of distinct mechanisms and major players that control mRNA translation in erythropoiesis. However, it is clear that there is still much we have yet to understand regarding mechanisms in mRNA translation. The advent of novel molecular and biochemical technologies has revolutionized the large-scale identification of RBPs and their transcripts. Technologies such as iCLIP (Individual-nucleotide resolution UV crosslinking and immunoprecipitation) and ribosome profiling have the exciting potential to rapidly advance the field.

### REFERENCES

After transcription, translation functions as an additional regulatory layer of gene expression. Precise translational control enables coordinated protein synthesis in response to environmental signals, or the differentiation stage of cells. It also enables the dissociation of gene transcription and protein synthesis in time, which is of particular importance once gene transcription has ceased in late erythroblasts and reticulocytes undergoing terminal erythropoiesis. We expect that the coming years will provide exciting new insights in selective translation of mRNA during erythropoiesis.

### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

### FUNDING

This work was supported by the Landsteiner Foundation for Blood Transfusion Research (LSBR) project 1140.

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conversion between the RNA-binding apoprotein and Fe-S cluster containing aconitase. Nucleic Acids Res. 21, 1457–1461. doi: 10.1093/nar/21.6.1457

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Moore and von Lindern. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# From Erythroblasts to Mature Red Blood Cells: Organelle Clearance in Mammals

#### Martina Moras, Sophie D. Lefevre and Mariano A. Ostuni\*

UMR-S1134 Integrated Biology of Red Blood Cell, INSERM, Université Paris Diderot, Sorbonne Paris Cité, Institut National de la Transfusion Sanguine, Laboratoire d'Excellence GR-Ex, Paris, France

Erythropoiesis occurs mostly in bone marrow and ends in blood stream. Mature red blood cells are generated from multipotent hematopoietic stem cells, through a complex maturation process involving several morphological changes to produce a highly functional specialized cells. In mammals, terminal steps involved expulsion of the nucleus from erythroblasts that leads to the formation of reticulocytes. In order to produce mature biconcave red blood cells, organelles and ribosomes are selectively eliminated from reticulocytes as well as the plasma membrane undergoes remodeling. The mechanisms involved in these last maturation steps are still under investigation. Enucleation involves dramatic chromatin condensation and establishment of the nuclear polarity, which is driven by a rearrangement of actin cytoskeleton and the clathrin-dependent generation of vacuoles at the nuclear-cytoplasmic junction. This process is favored by interaction between the erythroblasts and macrophages at the erythroblastic island. Mitochondria are eliminated by mitophagy. This is a macroautophagy pathway consisting in the engulfment of mitochondria into a double-membrane structure called autophagosome before degradation. Several mice knock-out models were developed to identify mitophagy-involved proteins during erythropoiesis, but whole mechanisms are not completely determined. Less is known concerning the clearance of other organelles, such as smooth and rough ER, Golgi apparatus and ribosomes. Understanding the modulators of organelles clearance in erythropoiesis may elucidate the pathogenesis of different dyserythropoietic diseases such as myelodysplastic syndrome, leukemia and anemia.

Keywords: erythropoiesis, mitophagy, organelle clearance, reticulocytes, enucleation, erythroblast maturation

### INTRODUCTION

Mature red blood cells (RBCs) result from a finely regulated process called erythropoiesis that produces 2 million RBCs every second in healthy human adults (Palis, 2014). The standard model of erythropoiesis starts with hematopoietic stem cells (HSCs) in the bone marrow (BM), giving rise to multipotent progenitors that go on to erythroid-committed precursors to mature RBC. This hierarchical relationship is, however, challenged, showing a greater plasticity for the cell's potential fates, with several studies in mice (Adolfsson et al., 2005) and recent new data in human (Notta et al., 2016).

#### Edited by:

Lars Kaestner, Saarland University, Germany

#### Reviewed by:

Pablo Martín-Vasallo, Universidad de La Laguna, Spain Rodrigo F. M. De Almeida, Universidade de Lisboa, Portugal

> \*Correspondence: Mariano A. Ostuni<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Membrane Physiology and Membrane Biophysics, a section of the journal Frontiers in Physiology

> Received: 11 October 2017 Accepted: 06 December 2017 Published: 19 December 2017

#### Citation:

Moras M, Lefevre SD and Ostuni MA (2017) From Erythroblasts to Mature Red Blood Cells: Organelle Clearance in Mammals. Front. Physiol. 8:1076. doi: 10.3389/fphys.2017.01076

**26**

Maturation from erythroid-committed precursors is called terminal erythropoiesis and occurs in the BM within erythroblastic islands, which consist of a central macrophage surrounded by erythroblasts, and ends in the blood stream where reticulocytes complete their maturation within 1–2 days. During this phase, proerythroblasts (Pro-E) undergo morphological changes, such as cell size reduction and chromatin condensation, produce specific proteins, such as hemoglobin, and exhibit a reduced proliferative capacity to give rise to basophilic (Baso-E), polychromatophilic (Poly-E) and orthochromatophilic (Ortho-E) erythroblasts, successively. Even though several growth factors are known to regulate erythropoiesis, Epo is the main regulator of erythropoiesis driving RBC precursor proliferation and differentiation, preventing erythroblast apoptosis (Koury and Bondurant, 1990; Ji et al., 2011). The macrophage-erythroblast interaction in the BM is essential since macrophages facilitate proliferation and differentiation and provide iron to the erythroblasts (de Back et al., 2014).

At the end of the terminal maturation, mammalian erythroblasts expel their nuclei and lose all their organelles, such as the Golgi apparatus, endoplasmic reticulum (ER), mitochondria and ribosomes. After expelling its nucleus, the reticulocyte maturation continues, losing 20–30% of the cell surface (Waugh et al., 1997; Da Costa et al., 2001) and eliminating any remaining membrane-bound cytosolic organelles through an autophagy/exosome-combined pathway (Blanc et al., 2005).

While extensive literature is done concerning the general mechanisms of erythropoiesis (Palis, 2014), this review focuses on the mechanisms and molecular actors involved during organelle clearance and membrane remodeling in order to produce fully functional biconcave mature RBCs. **Figure 1** summarizes the best characterized steps of organelle clearance throughout erythroblast terminal differentiation.

### ENUCLEATION

The most spectacular aspect of mammalian erythropoiesis is the generation of enucleated cells. Enucleation occurs in orthochromatic erythroblasts producing two kinds of cells, the reticulocyte and the pyrenocyte [the nucleus surrounded by a tiny layer of cytoplasm and the plasma membrane (PM)]. Pyrenocytes are rapidly eliminated by the macrophages of the erythroblastic island, where phosphatidylserine exposure acts as an "eat me" signal (Yoshida et al., 2005).

Among the changes occurring during terminal differentiation, cell cycle arrest, chromatin and nuclear condensation and nuclear polarization are important for enucleation. In addition, nucleus expulsion is believed to be dependent on adhesion protein reorganization across the PM and macrophage interactions (Lee et al., 2004; Soni et al., 2006). The transcription factor KFL1 is required for enucleation (Parkins et al., 1995; Magor et al., 2015), regulating the expression of cell cycle proteins, deacetylases, caspases, and nuclear membrane proteins (Gnanapragasam et al., 2016; Gnanapragasam and Bieker, 2017).

Nuclear and chromatin condensation is essential for enucleation (Popova et al., 2009; Ji et al., 2010) and is dependent on the acetylation status of histones H3 and H4 under the control of histone acetyl transferases (HATs) and histone deacetylases (HDACs). Accordingly, Gcn5, an HAT protein, is down-regulated, and H3K9 and H4K5 histone acetylation decreases during mouse fetal erythropoiesis. In addition, Gcn5 is up regulated by c-Myc, which is known to decrease during the late phase of the erythropoiesis (Jayapal et al., 2010). With the same model, the role of HDAC2 protein was shown to be essential not only for chromatin condensation but also for the formation of the contractile actin ring (CAR), which is involved in nuclear pyknosis (Ji et al., 2010). Moreover, it was recently shown that major histones are released through a nuclear opening that is induced by caspase 3 activity-dependent lamin B cleavage and chromatin condensation (Gnanapragasam and Bieker, 2017).

Many studies demonstrate the cell cycle dependence of enucleation (Gnanapragasam and Bieker, 2017). Interestingly, the cyclin-induced E2F-2 transcription factor, which is a direct target of KLF1 during terminal erythropoiesis, appears to play a role in enucleation by inducing the expression of CRIK (Citron Rho-interacting kinase). Away from its regular targets related to microtubule organization and cytokinesis, CRIK participates in nuclear condensation (Swartz et al., 2017).

Cytoskeletal elements play an important role in erythroblast enucleation, acting in a similar manner to cytokinesis but in an asymmetric way. Specifically, as observed by electron and immunofluorescence microscopy, actin filaments (F-actin) condensate behind the extruding nucleus to form the CAR. The use of cytochalasin D, an F-actin inhibitor, causes the complete blockage of enucleation (Koury et al., 1989). Furthermore, the formation of the CAR is dependent on Rac1 GTPase and on mDia2, a Rho GTPase downstream effector, since downregulating these two proteins disrupts the CAR formation and blocks erythroblast enucleation (Ji et al., 2008).

Regarding other cytoskeleton elements, the pharmacological inhibition of vimentin does not affect enucleation, which is in agreement with its decrease during human erythropoiesis (Dellagi et al., 1983). However, the deregulation of microtubules diminishes the enucleation rate. Microtubules form a basket around the nucleus (Koury et al., 1989), which is displaced near the PM at the late erythroblast stages, suggesting that this network must be essential for the polarization of the nucleus. Recently, the importance of the molecular motor dynein, which mediates unidirectional movement toward the minus end of the microtubules, was shown. Furthermore, PI3K activity is induced by microtubule polymers, improves the polarization efficiency and promotes nuclear movement. However, PI3K inhibition does not block, but only delays, mice enucleation (Wang et al., 2012).

In 2010, Crispino's group observed, by electron microscopy, the formation of vesicles close to the nuclear extrusion site in both primary murine and human erythroblasts, suggesting that another mechanism contributes to enucleation. Additionally, as shown by genetic invalidation, clathrin is needed for the vesicle formation (Keerthivasan et al., 2010). More recently, it was shown that survivin is required for erythroblast enucleation, but instead of acting on cytokinesis via the chromosome passenger complex, survivin contributes to enucleation through

an interaction with EPS15 and clathrin (Keerthivasan et al., 2012).

Clearly, we are still at the beginning of unraveling the molecular players involved in the enucleation process. Moreover, as shown in **Table 1**, most of the molecular players were identified in mice, and we are still lacking a demonstration that these players are also involved in human erythropoiesis.

### Mitochondrial Clearance

The main mechanism for mitochondrial clearance is mitophagy, a selective type of autophagy that allows the degradation of damaged mitochondria. The importance of this process is highlighted by knowing that an impairment in mitochondrial function triggers an increase in reactive oxygen species production, which can in turn cause damage to cellular components (proteins, nucleic acid, and lipids) and trigger cell death (Lee et al., 2012).

During regular autophagy processes, stress or nutrient deprivation activates APM-activated protein kinase (AMPK), triggering two ubiquitin-dependent pathways (**Figure 1A**). One of these allows the assembly of the phagophore and involves several autophagy-related proteins (Atg), such as Atg5 and Atg7. The other aims to activate and lipidate LC3 (MAPLC3, microtubule-associated protein 1 light channel 3) by Atg4, a redox regulated protein. Atg4 and Atg7 cooperate to conjugate LC3 onto phosphatidylethanolamine in the lipid bilayer of the membrane originated from the ER-mitochondria contact site (Tooze and Yoshimori, 2010; Hamasaki et al., 2013). The elongated phagophore is then recruited to engulf targets via adaptor proteins, containing an LC3-interacting region (LIR) that forms a double-membrane autophagosome, which will fuse with a lysosome, initiating the degradation of the autophagosome components.

Upon mitochondria damage or depolarization, the mitochondrial membrane proteins are exposed and act as a beacon to recruit the phagophore membranes (Liu et al., 2014). An example is the PINK1 (P-TEN-induced kinase 1)-dependent recruitment of Parkin. Upon mitochondria depolarization, PINK1 accumulates at the OMM (outer mitochondrial membrane) and induces the mitochondrial translocation of Parkin, an RBR (ring-in-between)-type E3 ubiquitin ligase by direct phosphorylation (Kim et al., 2008; Narendra et al., 2010). The stabilization of Parkin at the OMM leads to the poly-ubiquitination of many proteins, inducing mitochondria fission and mobility stop and the phagophore recruitment by interacting with p62/SQSTM1, a LIR containing protein

TABLE 1 | Comparison between studies in human or mice erythroid cells or in other cell models.

(Geisler et al., 2010). Unlike regular mitophagy induction, targeted mitochondria, during erythroblast maturation, are fully functional. BNIP3L/NIX, a BH3-only integral OMM protein first identified in mouse reticulocytes, appears to be the major mitochondrial protein involved during terminal differentiation (Schweers et al., 2007; Sandoval et al., 2008). This protein is upregulated during erythropoiesis and induces mitochondrial membrane depolarization and membrane conjugated LC3 recruitment to the mitochondria (Aerbajinai et al., 2003; Novak et al., 2010). Nix action is not mediated by its BH3 domain but rather seems to be due to a cytoplasmic short linear motif, acting as a cellular signal to recruit other proteins (Zhang et al., 2012). However, whether Nix-induced mitochondrial depolarization activates the Parkin-dependent pathway is still unknown (Yuan et al., 2017).

Recently, other mitochondrial receptors were found to participate in mitophagy, such as FUNDC1, induced by MARCH5, an E3 ubiquitin ligase acting in hypoxic condition (Chen et al., 2017), Bcl2-L-13 (Murakawa et al., 2015), optineurin (Wong and Holzbaur, 2014), and Prohibitin 2 (Wei et al., 2017). It remains unknown whether they play a role in erythroid maturation.

Canonical Atg proteins also participate in terminal maturation. In human erythropoiesis, LC3 cleavage is under the control of the endopeptidase Atg4 and is needed for autophagosome maturation (Betin et al., 2013). In mice, Ulk1 (Atg1) expression correlates with terminal differentiation and participates in mitochondria and ribosome elimination (Chan et al., 2007; Kundu et al., 2008). The ubiquitination-dependent pathway also plays a role in reticulocyte maturation but is not essential. Indeed, in Atg7−/− reticulocytes, mitochondrial clearance is only partially affected (Zhang and Ney, 2009; Zhang et al., 2009). However, Nix and Ulk1 activation appears to be essential (Mortensen et al., 2010; Honda et al., 2014), suggesting the coexistence of both Atg5/Atg7-dependent and independent pathways during terminal differentiation.

Some studies suggest that the Atg5/7-independent degradation of mitochondria involves endosomal trafficking regulatory Rab proteins. Autophagosomes, formed in a Ulk1 dependent pathway, fuse with Golgi-derived vesicles and late endosomes in a Rab9a-dependent manner before they are targeted to the lysosomes (Wang et al., 2016). Interestingly, Rab proteins were also recently shown to be involved in mitochondria removal in a complete autophagy-independent pathway. Depolarized mitochondria appear to be engulfed in Rab5-positive endosomes that mature into Rab7-positive late endosomes and then fuse with lysosomes (Hammerling et al., 2017a,b). Unlike canonical autophagy, which involves the surrounding of a ubiquitin-decorated target by a double membrane structure, the entire mitochondria appears to be engulfed by an early endosome membrane invagination through the ESCRT machinery. Whether this might also occur in maturing erythroblasts is not known.

Mitophagy also appears to be transcriptionally regulated. Indeed, hemin-dependent differentiation of an erythroid cell line shows features of mitophagy (Fader et al., 2016). The NF-E2 transcription factor involved in globin gene expression also regulates mitophagy through the regulation of Nix and Ulk1 genes (Gothwal et al., 2016; Lupo et al., 2016). Another key regulator is the KRAB/KAP1-miRNA regulatory cascade, which acts as an indirect repressor of mitophagy genes in mice as well as in human cells, probably by the down and up regulation of a series of miRNAs, such as miR-351 that targets Nix (Barde et al., 2013).

In parallel to the autophagic pathway, cytosolic degradation seems to occur during reticulocyte maturation. 15-lipoxygenase (15-LOX), an enzyme that catalyzes the dioxygenation of polyunsaturated fatty acids, is translationally inhibited until the reticulocyte stage and acts to permeabilize organelle membranes, allowing proteasome access and degradation. Interestingly, only mitochondria elimination is affected, while ribosome clearance remains efficient when using a lipoxygenase inhibitor (Grüllich et al., 2001). This mechanism is still controversial, as 15-LOX might also act in the autophagy pathway as an OMM pH gradient disruptor that can induce mitophagy (Vijayvergiya et al., 2004), and on the oxidation of phospholipids conjugating with LC3 during the autophagosome formation; even so, these features, as shown in **Table 1** were not demonstrated in erythroid cells yet (Morgan et al., 2015).

### Ribosomes and Other Organelles

In general, autophagy plays an essential role in the elimination of other organelles, such as lysosomes, peroxisomes and ER. However, the literature presents only very few studies in erythroid cells (**Table 1**).

While Nix is required for mitochondria removal, Ulk1 is involved in ribosome and mitochondria degradation (Schweers et al., 2007; Kundu et al., 2008; Sandoval et al., 2008). Similarly, an efficient clearance of ribosomes and ER and the inhibition of mitophagy was observed in Atg7−/− mice (Mortensen et al., 2010). These data suggest that non-autophagic or Atg7 independent autophagic pathways might exist for the elimination of other organelles (**Figure 1A**).

In non-erythroid cells from mammals, it was proposed that peroxisomes are eliminated by three different pathways: macroautophagy (Iwata, 2006), 15-LOX mediated (Yokota et al., 2001) and the peroxisomal Lon proteases (Yokota et al., 2008). Furthermore, the autophagic degradation of lysosomes (lysophagy) was recently identified in HeLa cells where it is mediated by ubiquitination and involves p62 protein (Hung et al., 2013). The similarities between pexophagy/lysophagy and mitophagy in non-erythroid cells suggest that autophagy pathways might also be involved in erythroblast terminal maturation.

After enucleation, reticulocytes mature in the bone marrow (R1) and then exit in the blood stream (R2) to complete the process. While the degradation of organelles starts at the time of enucleation, the elimination of mRNA occurs in the blood stream and is mediated by ribonucleases, generating nucleotides that are degraded by the erythroid pyrimidine nucleotidase. This elimination is crucial, as the deficiency in this enzyme causes hemolytic anemia (Valentine et al., 1974). mRNAs in R2 reticulocytes mainly belong to three overlapping categories: transport, metabolic and signal transduction (Lee et al., 2014), and their presence is essential to reach the mature RBC stage. This supports the importance of the exosome pathway for the final maturation into RBCs with an active elimination of other subcellular components.

### Exocytosis and Membrane Remodeling

Exosomes are small vesicles that are secreted into the extracellular medium from various kind of cells. PM invaginations form early endosomes that engulf various targets forming multivesicular bodies (MVB, late endosomes) that eventually fuse with the PM and release exosomes. In reticulocytes, this pathway is thought to be involved in cell volume and membrane remodeling to reduce volume and remove unwanted membrane proteins. This was first discovered in sheep reticulocytes where transferrin receptor (TfR) is first internalized into small vesicles of 100–200 nm before being engulfed into the MVBs (Pan et al., 1985; Johnstone et al., 1989). The internalization step is clathrin-dependent, and the degradation is lysosome-independent and occurs by exocytosis after the fusion of the MVBs with the PM as shown in **Figure 1B** (Killisch et al., 1992). This process is required for the final elimination of other membrane proteins that are essential for the reticulocyte but are absent in the mature cell. Proteins such as aquaporin-1 (AQP1) (Blanc et al., 2009), α4β1 integrin (Rieu et al., 2000), glucose transporter and acetylcholinestarase (Johnstone et al., 1987) are found in glycophorine-A (GPA) positive endosomes while cytoskeletal proteins, such as actin or spectrin have never been found in these endosomes (Liu et al., 2010).

While plenty of evidence notes the role of autophagy in removing organelles during terminal maturation, the degradation step itself shows discrepancies with canonical proteolysis involving lysosomal proteins because of the disappearance of the lysosomal compartment during the maturation and removal of LAMP2 by exocytosis (Barres et al., 2010). Recently, GPA-positive endosomes were found to express LC3 at the endosome membrane, suggesting the cooperation of both autophagy and exocytosis in the removal of remnant organelles in R2 reticulocytes. These hybrid vesicles contain mitochondria, Golgi and lysosomes might be formed by the fusion of the outer-membrane of the autophagosome and the PM derived endosome (Griffiths et al., 2012). The exocytosis of this vesicle might be favored by the spleen, as splenectomized patients present large vacuoles inside reticulocytes (Holroyde and Gardner, 1970).

It should be pointed out the importance of lipids domain such as cholesterol and sphingomyelin-enriched domains in the PM remodeling, as they were find both in membrane vesiculation specific sites (Leonard et al., 2017).

### CONCLUSION

Even if all the animal models used to identify the molecular players involved during terminal differentiation exhibit maturation defects and anemia, links between organelle clearance and human hematological diseases are still mostly unknown. Erythroid disorders, such as β-thalassemia and myelodysplastic syndrome (MDS), are characterized by ineffective hematopoiesis, anemia, dissociation between proliferation and differentiation of progenitor cells and the inefficient elimination of aggregated protein (Arber et al., 2016; Taher et al., 2017). Indeed, defects in reticulocyte maturation and autophagy are identified in HbE/β-thalassemia patients (Lithanatudom et al., 2011; Khandros et al., 2012; Butthep et al., 2015), and enucleation defects are found in MDS patients (Garderet et al., 2010; Park et al., 2016). Impaired autophagy is involved in cytosolic toxic Lyn accumulation and mitochondria and lysosome degradation delay in chorea-acanthocytosis (Lupo et al., 2016). The use of autophagy modulators is beneficial in the case of SCD or β-thalassemia (Franco et al., 2014; Jagadeeswaran et al., 2017). Moreover, anemia in Pearson's syndrome was recently linked to incomplete mitochondrial clearance from reticulocytes (Palis, 2014) and an asynchronization of iron loading (Ahlqvist et al., 2015), while sickle cells patients showed an accumulation of proteins in their erythrocytes suggesting a defect in exosomal pathway (De Franceschi, 2009; Carayon et al., 2011).

Unraveling the molecular mechanisms and interplays ruling erythroblast terminal maturation would be priceless in hematological disease therapy. However, much of our knowledge regarding human erythropoiesis is based on animal models and/or ex vivo cultured human progenitor cells (**Table 1**). Great care should be applied when interpreting results, considering the important differences between mouse and human erythropoiesis as well as the in vivo and in vitro environments, as highlighted in the extensive transcriptome analysis across a terminal erythroid differentiation study (An et al., 2014).

### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

### FUNDING

This study was supported by grants from Laboratory of Excellence GR-Ex, reference ANR-11-LABX-0051. The labex GR-Ex is funded by the program "Investissements d'avenir" of the French National Research Agency, reference ANR-11-IDEX-0005-02.

### ACKNOWLEDGMENTS

MM is funded by the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 665850. We thank I. Marginedas-Freixa and C. Hattab for helpful discussions.

### REFERENCES

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by an ALS-linked mutation. Proc. Natl. Acad. Sci. U.S.A. 111, E4439–E4448. doi: 10.1073/pnas.1405752111

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2017 Moras, Lefevre and Ostuni. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Continuous Change in Membrane and Membrane-Skeleton Organization During Development From Proerythroblast to Senescent Red Blood Cell

#### Giampaolo Minetti 1 \*, Cesare Achilli 1 , Cesare Perotti 2 and Annarita Ciana1

1 Laboratori di Biochimica, Dipartimento di Biologia e Biotecnologie, Università degli Studi di Pavia, Pavia, Italy, 2 Servizio Immunoematologia e Medicina Trasfusionale, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy

Within the context of erythropoiesis and the possibility of producing artificial red blood cells (RBCs) in vitro, a most critical step is the final differentiation of enucleated erythroblasts, or reticulocytes, to a fully mature biconcave discocyte, the RBC. Reviewed here is the current knowledge about this fundamental maturational process. By combining literature data with our own experimental evidence we propose that the early phase in the maturation of reticulocytes to RBCs is driven by a membrane raft-based mechanism for the sorting of disposable membrane proteins, mostly the no longer needed transferrin receptor (TfR), to the multivesicular endosome (MVE) as cargo of intraluminal vesicles that are subsequently exocytosed as exosomes, consistently with the seminal and original observation of Johnstone and collaborators of more than 30 years ago (Pan BT, Johnstone RM. Cell. 1983;33:967-978). According to a strikingly selective sorting process, the TfR becomes cargo destined to exocytosis while other molecules, including the most abundant RBC transmembrane protein, band 3, are completely retained in the cell membrane. It is also proposed that while this process could be operating in the early maturational steps in the bone marrow, additional mechanism(s) must be at play for the final removal of the excess reticulocyte membrane that is observed to occur in the circulation. This processing will most likely require the intervention of the spleen, whose function is also necessary for the continuous remodeling of the RBC membrane all along this cell's circulatory life.

Keywords: artificial red blood cells, reticulocyte maturation, red blood cell ageing, multivesicular endosome, membrane rafts, fluid phase endocytosis, autophagy, spleen

"In fact, it may almost be true to say that the red cell continuously changes until its senescent properties are recognized and it is eliminated from the circulation."

(Ahn and Johnstone, 1991)

## INTRODUCTION

Recapitulation of erythroid differentiation and amplification in vitro is an actively investigated topic for the potentially revolutionary impact on regeneration and transfusion medicine, for all the conditions where reintegration of functional blood volume is required. Significant advancements in the field have made it possible to expand and differentiate in vitro erythroid precursors to

#### Edited by:

Guido Santos-Rosales, Universitätsklinikum Erlangen, Germany

#### Reviewed by:

Ashley Toye, University of Bristol, United Kingdom Pasquale Stano, University of Salento, Italy

> \*Correspondence: Giampaolo Minetti<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Membrane Physiology and Membrane Biophysics, a section of the journal Frontiers in Physiology

> Received: 15 January 2018 Accepted: 12 March 2018 Published: 26 March 2018

#### Citation:

Minetti G, Achilli C, Perotti C and Ciana A (2018) Continuous Change in Membrane and Membrane-Skeleton Organization During Development From Proerythroblast to Senescent Red Blood Cell. Front.

Paramount for the possibility of developing a simplified in vitro culture of RBCs was the discovery that the last stage in erythroblast development, the expulsion of the nucleus, is an intrinsic property of the erythroblast that does not require an erythroblastic island to occur. In fact, the central macrophage in the island appears to be actively involved, at this stage, only in the phagocytosis of the extruded nucleus (Koury et al., 1988; Qiu et al., 1995; Ji et al., 2010). Although it is possible that significant scale-up will require co-culture with bone marrow stromal cells (Giarratana et al., 2005; Mountford et al., 2010; Rousseau et al., 2014), to date differentiation to the reticulocyte (retic) stage can be obtained in liquid cultures using only soluble factors and it is reasonable that in a relatively short time the target concentration of 5 × 107 cells/ml could be reached (Rousseau et al., 2014). Yet, full development of the retic to a mature biconcave RBC under artificial conditions appears to still be a major challenge (Giarratana et al., 2005, 2011; Koury et al., 2005; Miharada et al., 2006). A number of reviews are available on the erythroblastic island and erythroid differentiation (Mountford et al., 2010; An and Mohandas, 2011; Anstee et al., 2012; Griffiths et al., 2012a; de Back et al., 2014; Satchwell et al., 2015a; Mankelow et al., 2016; Moras et al., 2017). The present article reviews the literature on retic maturation, one of the unsolved problems of erythropoiesis (Chasis et al., 1989; Koury et al., 1989; Blanc and Vidal, 2010; Ney, 2011), seen as a continuum of differential remodeling of the lipid bilayer and of the membrane-skeleton that leads to the mature circulating RBC first and then to the senescent RBC, and focusing on aspects of membrane topology and selectivity of protein sorting.

### SORTING OF PROTEINS DURING ENUCLEATION

In early seminal work, synthesis of RBC membrane proteins was described to occur asynchronously in avian and murine RBCs (Lazarides and Moon, 1984; Hanspal and Palek, 1987; Hanspal et al., 1992). In murine erythroblasts, band 3 synthesis, through a complex dynamics of trafficking from internal membranes to the plasma membrane of dimeric and tetrameric forms of the protein (Hanspal et al., 1998), was completed while the translation of spectrins was still in progress. More recent studies of human erythropoiesis have revealed that band 3 is synthesized early (Gautier et al., 2016) and assembled into multiprotein complexes, especially with protein 4.2, in the intracellular compartment (Satchwell et al., 2011). In a study (Chen et al., 2009) carried out with murine erythroblasts infected with the anemia-inducing strain of Friend erythroleukemia virus (FVA cells) (Koury et al., 1984), many RBC proteins were followed throughout maturation from proerythroblast to retic. Three patterns of protein turnover were observed for integral proteins. The levels of some proteins, including band 3 and glycophorin A (GPA), increased toward the late orthochromatic stage, those of others (CD44, Lu, ICAM-4, and β1 integrin) decreased throughout differentiation, and still others, among which the transferrin receptor (TfR or CD71), remained stable. Of the membrane-skeletal proteins all were shown to increase steadily except for actin, which decreased from proerythroblast to retic (Chen et al., 2009). Similar results were obtained with human erythroblasts (Hu et al., 2013). Comparative transcriptome and expression analysis of human and murine erythroid differentiation (An et al., 2014; Satchwell et al., 2015a; Gautier et al., 2016) is beginning to shed more light on this developmental program and its interspecies differences, which are responsible for generating the heterogeneous phenotypes seen in RBCs of different mammals (Ahn and Johnstone, 1991). Moreover, additional useful information would come from analysis of retic transcriptome, evaluated at different stages of retic maturation (Goh et al., 2007; Malleret et al., 2013).

Whatever the set of membrane proteins with which developing erythroblasts arrive at the orthochromatic stage, this composition will undergo a major redistribution with the enucleation event. This concept was already clear in early seminal studies carried out mostly in mice. Concerning transmembrane proteins, in enucleating mouse erythroblasts isolated from bone marrow, band 3 was shown to partition at a higher concentration in the membrane surrounding the nucleus, than in the membrane of the incipient retic. On the other hand, GPA, the second most abundant integral protein of the RBC membrane, was found to be equally distributed between these two domains of the membrane at enucleation (**Figure 1A**; Geiduschek and Singer, 1979). In erythroblasts isolated from rat bone marrow a similar partition pattern was described (Skutelsky and Farquhar, 1976).

More recently, GPA was found to partition almost exclusively to the retic during enucleation of FVA cells, possibly because of transiently increased connectivity, of unknown origin, of GPA to the spectrin skeleton (Lee et al., 2004). It should be observed that the cell model adopted may influence the results obtained. Many studies were carried out with FVA cells that may not exactly represent physiological erythroid differentiation. Nevertheless, this scenario was largely confirmed in a recent proteomic study of protein distribution during human erythroblast enucleation, although e.g., the restriction of band 3 to the retic membrane seemed to be less defined than what seen in the murine model (Bell et al., 2013).

Concerning membrane-skeletal proteins, they all partition to the retic by a remarkably selective sorting process whose mechanism remains unknown (Geiduschek and Singer, 1979; Zweig et al., 1981; Wickrema et al., 1994; Bell et al., 2013).

### SORTING OF PROTEINS DURING RETIC-TO-RBC MATURATION

A major remodeling of the reticulocyte in the maturation to RBC entails the loss of approximately 20% plasma membrane and the continued removal of residual intracellular components.

FIGURE 1 | (A) Schematic distribution of membrane proteins in the enucleating mouse erythroblast. Roman numerals I, II, and III correspond to the domains identified by the authors of the work (Geiduschek and Singer, 1979). Domain III corresponds to the incipient reticulocyte. The nucleus (N), not completely extruded, shows the characteristic constriction which separates domain I from domain II. As discussed in the text of the cited work, two classes of ConA receptors (ConA is a lectin that binds to band3, so ConA receptors are band 3 molecules) are assumed to be present in the erythroblast membrane, with ConA-Rn (but not ConA-Rr) present in domain I; both ConA-Rn and ConA-Rr in the membrane of domain II; and ConA-Rr (but not ConA-Rn) in domain III. Most strikingly, spectrin is confined to domains II and III. Re-drawn from Geiduschek and Singer (1979). (B) Rudimentary early depiction of a model for the remodeling of the cell membrane during retic maturation to RBC in vivo. Panel 1 depicts the invaginations of spectrin-free domains of the retic membrane occurring in the circulation, and their subsequent endocytosis. In panel 2, the endocytosed vesicles are pictured as associating with the membrane either to fuse with it or to be exocytosed. In panel 3, the exocytosis of vesicles in a larger, spectrin-containing body is pictured as occurring by mediation of the spleen. Re-drawn from Zweig et al. (1981).

Concerning membrane-skeletal proteins, they are thought to be largely retained in the maturing RBC through retic-to-RBC transition (Tokuyasu et al., 1979; Pan and Johnstone, 1983). A recent study on stress murine retics documents this in detail (Liu J. et al., 2010). Here all the main membrane-skeletal proteins (α-spectrin, β-spectrin, ankyrin R, protein 4.1R, membraneassociated actin, protein 4.2, p55, tropomodulin, protein 4.9) were found to be unchanged between retics and RBCs. Other cytoskeletal components declined (myosin, tropomyosin, adducin) and some of them were completely lost (tubulin, cytosolic actin). Concerning transmembrane proteins, a decline of Na+/K+ATPase, NHE1, GPA, CD47, Duffy, and Kell was observed, together with the complete loss of CD71 and of ICAM4. Interestingly and surprisingly, the following proteins were found to be increased in the membrane of the mature RBC: band 3, GPC, Rh, RhAG, XK, and GPA (for GPA, only the subset of molecules that act as receptor for the TER-119 antibody; Kina et al., 2000). This phenomenon was not commented further in the cited paper (Liu J. et al., 2010) (for a plausible explanation see below). A more recent proteomic analysis of human retics obtained by in vitro expansion and differentiation in in vitro culture of cord blood-derived CD34+ cells, largely confirmed a similar pattern of protein partitioning between cultured retics and the autologous mature RBCs recovered from cord blood (Wilson et al., 2016).

### MODELS OF RETICULOCYTE MATURATION

The loss of retic's excess membrane was originally proposed to occur through the mechanism depicted in **Figure 1B**, for which hints were already available since the 1960's (Kent et al., 1966; Holroyde and Gardner, 1970). Here the excess surface area first invaginates as endocytic spectrin-free vesicles, which are then hypothetically removed as the content of a larger spectrin-containing vesicle, possibly with the active intervention of the spleen. Later, Johnstone et al. (Pan and Johnstone, 1983; Johnstone, 2005) and Stahl and co-workers (Harding et al., 1984, 2013) almost at the same time discovered that in maturing retics a special endosomal compartment is formed, named multivesicular endosome or body (MVE, MVB), which contains intraluminal vesicles (ILVs) carrying the TfR and other selected transmembrane proteins, but lacking band 3 and GPA. Fusion of the MVE with the plasma membrane and exocytosis of the ILVs is the process though which maturing retics dispose of no longer needed membrane proteins. Although the discovery by Johnstone and co-workers was seminal for subsequent understanding of endocytic processes and vesicular trafficking and may explain the loss of most of the TfR, it does not account entirely for the loss of membrane surface area from retic to mature RBC (see below).

According to the current view of the vesicular trafficking around the complex endosomal compartment, several endocytic processes exist in eukaryotic cells, clathrin-dependent and -independent, for the purpose of captating extracellular components and for the internal needs of the cell of turning over plasma membrane constituents. Thus, the endocytic vesicles produced by clathrin-mediated endocytosis, caveolar endocytosis, flotillin-dependent endocytosis and Arf6-dependent endocytosis all fuse generating a common compartment, the early (sorting) endosome (Otto and Nichols, 2011; Bohdanowicz and Grinstein, 2013; Johannes et al., 2015). From the sorting endosome, a MVE can form that can take two different routes: one is fusion with the lysosome for complete hydrolytic degradation of the ILVs; the other is fusion with the plasma membrane for discharging the ILVs, that can now be defined as exosomes (Cocucci and Meldolesi, 2015), in the extracellular space.

Topologically, the orientation of transmembrane proteins in ILVs and exosomes is the same as in the plasma membrane (right-side-out) and as in ectosomes (intended as vesicles released extracellularly directly from the plasma membrane), whereas it is opposite in endocytic vesicles (inside-out).

There are therefore two steps at which proteins must be sorted along the above-described pathway. The first entails the selection at the plasma membrane for endocytosis. In the specific of retic maturation, endocytosis of TfR from the plasma membrane. The next sorting step occurs on the membrane of the sorting endosome where the TfR must be again selected among other membrane proteins to be inserted in ILVs. This process is mediated by a special molecular machinery based on ESCRT protein complexes (Gruenberg and Stenmark, 2004; Kowal et al., 2014) although an ESCRT-independent, ceramidebased mechanism has also been described (Trajkovic et al., 2008).

Concerning the sorting of plasma membrane proteins, as mentioned in the previous section, an increase in band 3 and GPC was observed in the maturation of mouse retics to RBCs by Western blotting of whole cell proteins, normalizing the quantity of sample by loading the same amount of cholesterol for RBCs and retics (Liu J. et al., 2010). Whereas, a decline of proteins during the retic-to-RBC transition can be easily accepted, their stability or increase in a membrane that is at the same time decreasing in size is less easily understood, especially for a complex multi-pass transmembrane protein like band 3. This is certainly not due to de novo synthesis in a cell that is progressively dismantling its internal translational apparatus and consuming the molecular machinery required for vesicular trafficking and where band 3 was synthesizes and already partially assembled in complexes in earlier erythroblastic stages (van den Akker et al., 2011). As the decrease in membrane surface area that occurs in the retic-to-RBC maturation is of the same order of magnitude as the observed "increase" in band 3 and GPC, the latter can only be explained by the increase in concentration that follows a selective loss of cholesterol (and other components different from band 3) by the retic in the process. Previous and independently obtained evidence exists on stable amounts of band 3 in retics and RBCs, despite the large difference in surface area between the two cell types (Foxwell and Tanner, 1981), pointing to a peculiar and as yet unknown mechanism for such selective retention of band 3 in the membrane of maturing retics that must be capable of discriminating between two almost equally abundant transmembrane proteins, band 3 with approximately 1.2 × 106 copies/cell and CD71 with approximately 8 × 105 copies/cell (Van Bockxmeer and Morgan, 1979) and completely get rid of the latter while largely saving the former.

To explain the selective retention in the reticulocyte membrane of such abundant transmembrane proteins as band 3, GPA, RhAG, GPC, CD47, a mechanism based on the formation of stable multiprotein complexes among the abovementioned species, with the association to components of the membrane-skeleton (Satchwell et al., 2015b, 2016) could be conceived. This mechanism would work by mechanically preventing incorporation of the bulky protein complexes into endovesicles. However, this is difficult to reconcile with the evidence that, for instance, some subpopulations of band 3 molecules or even band 3 complexes (with 4.2 and ankyrin), may still move around unanchored to the skeleton in retics (due to the extra membrane present at this stage, that cannot be supported by a membrane-skeleton of smaller area). Moreover, even in fully mature normal RBCs there is a significant 30% of band 3 molecules which is highly mobile, being not anchored to the skeleton. This population of band 3 molecules should be in principle free to leave the cell if size reduction was driven by a non-specific mechanism, but it is not: band 3 (as well as GPA and GPC) are not found in exosomes derived from ILVs in MVEs, as discovered already by Johnstone and co-workers.

There is actually one particular experimental model in which a peculiarly sharp segregation of different membrane proteins is observed, and that is during the isolation of membrane rafts as detergent resistant membrane (DRM). When DRMs are properly isolated from RBCs, they meet the canonical requirements for membrane rafts of being enriched in cholesterol, sphingolipids and specific classes of proteins like GPI-linked proteins, flotillins and stomatin, to name only the most relevant.

We infer from literature data and from our own experimental evidence that the step at which band 3, GPC, and GPA (Johnstone, 1992) are selected for being retained in the plasma membrane at the retic-to-RBC transition is in the trafficking of MVEs, and implies a mechanism based on differential partitioning of a given transmembrane protein into the membrane raft phase. To the best of our knowledge this is the first time that a membrane raft-based process for the sorting of membrane proteins during retic maturation is proposed.