UvA-DARE (Digital Academic Repository)

The spiders at the center of the web Csde1 and strap control translation in erythropoiesis Moore, K.S.

Publication date 2018 Document Version Other version License Other Link to publication

Citation for published version (APA): Moore, K. S. (2018). The spiders at the center of the web: Csde1 and strap control translation in erythropoiesis.

General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).

Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible.

UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)

Download date:05 Oct 2021 Chapter 1

General Introduction

An adapted version of this chapter has been published as:

RNA-binding and regulation of mRNA translation in erythropoiesis

Kat S Moore, Marieke von Lindern

Frontiers in Physiology 9 (2018): 910

Introduction

In the human body, the production of red blood cells (erythropoiesis) is a delicate balance between supply and demand. The body must maintain a population of eryth- 1 rocytes that is large enough to adequately oxygenate tissue, while avoiding elevated risk of hypertension, clotting and stroke when the erythrocyte population is too large. This chapter presents an overview of the molecular mechanisms which control expression during erythropoiesis, with particular emphasis on the translation of mRNA transcripts to , and the consequences when translational control is disrupted.

1.1 Hematopoiesis & erythropoiesis

Hematopoiesis is the process by which new blood cells (red and white) are formed. The process is governed by the maintenance of hematopoietic stem cells (HSCs) in the bone marrow, which undergo asymmetric division into one daughter HSC and a lineage specific progenitor [1]. HSCs and their immediate daughter cells are character- ized by the presence of cell surface markers Sca1 and c-kit, and lack of markers associ- ated with cells committed to a specific branch of differentiation [2]. Hence, these cells at the top of the hematopoietic hierarchy are referred to as the LSK (lineage-/Sca1+/c- kit+) compartment [3]. Long-term hematopoietic stem cells (LT-HSCs) are mostly quiescent and can sustain lifelong hematopoieis, whereas short-term hematopoietic stem cells (ST-HSCs) divide more frequently but have a limited lifespan [4–6]. LT-HSCs are negative for cell surface markers CD34 and Flt3, while ST-HSCs are Flt3-positive. A third member of the LSK compartment are multipotent progenitors (MPPs), which are positive for both CD34 and Flt3 and form the proliferating compartment that actively replenishes all the blood cell lineages with the required number of progenitors. The hematopoietic tree branches at this point into the lymphoid and myeloid common progenitors (CLP and CMP, respectively). CLPs differentiate into B, T, and NK immune cells, whereas CMPs split further into granulocyte/macrophage progenitors (GMPs) and megakaryocyte/erythrocyte progenitors (MEPs) [3]. MEPs subsequently differen- tiate into megakaryocytic and erythroid precursors (Figure 1A). From CMP to mature erythrocyte, the erythropoietic lineage is characterized by morphology and colony-forming capacity (Figure 1B). The CMP is described as colo- ny-forming unit- granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) [7,8]. A CFU-GEMM gives rise to a burst-forming unit-erythroid (BFU-E) containing multiple thousands of cells expressing the erythropoietin receptor (EpoR) at low levels [9]. EpoR expression is increased during the subsequent stage of differentiation, dur- ing which a smaller, hemoglobin-containing colony-forming unit-erythroid (CFU-E) is formed. Further differentiation towards the mature erythrocyte occurs in multiple morphologically recognizable stages [10]. The proerythroblast contains a large nucleus

13 Chapter 1 with multiple observable nucleoli. The basophilic erythroblast is smaller in diameter with a proportionally smaller nucleus : cytoplasm ratio and granular cytoplasm. The polychromatophilic erythroblast displays initial condensation of the nucleus without visible nucleoli and a regionally acidophilic cytoplasm. The orthochromatic erythro- blast contains a darker nucleus with a further reduction in nuclear volume relative to the cytoplasm and a visible reddish tint due to increased hemoglobin concentration. By the reticulocytic stage, the cell has lost its nucleus. Upon subsequent loss of the ribosomes and mitochondria, the erythrocyte has fully matured.

Figure 1. Schematic overview of the hematopoiesis and erythropoiesis. (A) Self-renewing cells in the LSK compartment differentiate via the CLP (common lymphoid progenitor) into lymphocytes (B: B-cell, T: T-cell, NK: natural killer cell) or the CMP (common myeloid progenitor) and the GMP (gran- ulocyte-macrophage progenitor) into granulocytes (G) and macrophages (M). The MEP (megakaryo- cyte-erythroid progenitor) yields megakaryocytes (MK) or progress down the erythroid lineage. (B) Erythroid-committed cells display characteristic morphology. Maturation progresses through BFU-E (burst-forming unit-erythroid), CFU-E (colony-forming unit-erythroid), Pro-EB (pro-erythroblast), Baso-EB (basophilic erythroblast), PC-EB (polychromatophilic erythroblast), Ortho-EB (orthochromatic erythroblast). The cell loses the nucleus to become the reticulocyte and finally, loss of the mitochondria and ribosomes mark the mature erythrocyte.

14 Introduction

1.1.1 factors controlling erythropoiesis Erythrocytes circulate through human peripheral blood for approximately 120 days. 1 Every day, the human body produces 1011 new erythrocytes, which requires a tight balance between progenitor proliferation and maturation. An excess of erythrocytes poses a severe risk of thrombosis, whereas a paucity of erythrocytes causes anemia with a risk of ischemic damage in the tissues. Maintenance of this balance requires tightly controlled regulation of erythropoeitic homeostasis, enacted by a cascade of transcrip- tion factors and signaling pathways that control erythropoiesis. Several transcription factors are essential for determining the erythroid program of gene expression. Gata1 functions as the master regulator of erythropoiesis, inducing the expression of EpoR [11–13] and commitment to the erythroid lineage. Together with cofactor Fog1, it pro- motes the transcription of β-globin [14]. Nfe2 controls globin synthesis via chromatin remodeling and by binding directly to the promoters of both α- and β-globin as well as ferrochelatase, a heme biosynthetic enzyme [15]. Also of importance in terminal erythroid differentiation is Klf1 (Kruppel-like factor 1), a transcription factor essential for the synthesis of β-globin [16]. Haploinsufficiency in Klf1 causes hereditary persis- tence of fetal hemoglobin (HPFH) [17]. Furthermore, Klf1 is involved in many cellular changes required for the maturation of erythroblasts to erythrocytes [18].

1.1.2 Signaling in erythropoiesis Signaling cascades allow for the adaption of erythropoeitic expansion and differ- entiation upon demand. For instance, during hypoxic stress, Epo production in the kidneys is dramatically increased, allowing the body to counteract tissue hypoxia via the expansion of erythrocyte production [16]. The binding of Epo to EpoR triggers a conformational change that activates the cytoplasmic tyrosine kinase Jak2 [10]. In turn, Jak2 triggers a downstream signaling cascade via the Stat5, Akt and PI3 kinase pathways to support the proliferation and differentiation of erythropoeitic progenitors [19–21]. Stat5 induces the expression of anti-apoptotic Bcl-xL to maintain viability during terminal maturation [11]. In the absence of Bcl-xL, erythropoiesis is negatively regulated primarily via caspase-mediated apoptosis. The activation of caspases 8 and 9 activates downstream effector caspases (such as caspase 3), resulting in the cleav- age of Gata1 [10]. This molecular safety mechanism prevents the overproduction of erythrocytes, which can lead to hyperviscosity and ischemic stroke. In summary, low levels of Epo cause cell death by apoptosis, intermediate levels prevent differentiation, and high levels of Epo allow the production of mature erythrocytes [10]. Epo therefore functions as a dosage-dependent regulator of erythropoiesis in response to physi- ological demand. Stem cell factor (Scf), the ligand for the c-Kit receptor, acts in conjunction with Epo to repress differentiation and maintain the population of erythroid progenitors [22]. Scf

15 Chapter 1 signaling is enacted via the phosphoinositide-3-kinase (Pi3k) pathway, resulting in the activation of protein kinase B (Pkb) [16]. Pi3k/Pkb signaling has two major effector path- ways in erythroblasts. On the one hand, it prevents nuclear localization of the Foxo3a transcription factor that otherwise induces erythroid differentiation [23]. Concurrently, it activates mammalian target of rapamycin (mTOR), which interacts with translation initiation factors to promote the translation of a specific set of transcripts [24]. Increasingly, it is understood that erythropoietic signaling pathways enact not only transcriptional, but also translational control of gene expression. In particular, tight regulation of the initiation of translation is critical for the balance between prolifera- tion and differentiation. The mechanisms which govern the initiation of translation in the context of erythropoiesis is discussed in the next section. This includes an overview of cap-dependent translation, which is enhanced by Epo/Scf signaling and inhibited by heme deficiency or oxidative stress, as well as features within the 5’UTR which determine transcript sensitivity to regulatory pathways.

1.2 Cap-dependent translation initiation

According to the central dogma of molecular biology, genetic information encoded in DNA is transcribed into mRNA in the nucleus, which is exported into the cytoplasm and translated into proteins. Studies on gene expression have historically focused on transcription as the primary regulatory mechanism. However, there is increasing evidence to suggest that translation is another major determinant of protein abun- dance in the cell [25]. In other words, transcription determines whether a given gene is expressed, but translation determines the level of expression. Control of translation is mediated by a complex web of overlapping mechanisms, including, but not limited to, regulation of translation initiation, the length of the poly(A) tail and the presence of short upstream open reading frames (uORFs). Below, a brief overview of these topics as pertains to erythropoiesis is presented. Canonically, translation in higher eukaryotes is initiated via an interaction between the 5’ cap structure of the mRNA and a family of translation initiation factors, which cooperate to recruit the ribosome [26]. Briefly summarized, a ternary complex (TC) consisting of Met-tRNA, eIF2 and GTP associates with the 40S ribosomal subunit and with eIFs 1, 1A, 3 and 5 to form the 43S pre-initiation complex (PIC). At the same time, cap-binding factor eIF4E associates with scaffolding protein eIF4G and the RNA heli- case eIF4A to form the eIF4F complex [27]. eIF4F interacts with poly-A binding protein (Pabp), forming a closed-loop structure between the 5’ and 3’ end of the mRNA. The recruitment complex is subsequently formed via an interaction between eIF4G in the closed-loop structure and eIF3 in the PIC. Upon recruitment, the complex is referred

16 Introduction to as the 48S initiation complex. Th e preinitiation scanning complex scans the bound mRNA until encountering an initiation codon, at which point recognition of the initia- 1 tion 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 begin- ning of elongation (Figure 2). Availability of GTP-loaded eIF2 is the limiting factor of translation initiation [28]. Once translation initiation has occurred, hydrolysis of GTP to GDP ensures that eIF2 must be reloaded before a new round of translational initiation can begin. Th is is ac- complished via GDP-GTP exchange factor eIF2B, allowing the pre-initiation complex containing eIF2-GTP and Met-tRNA to reform. Th is mechanism is subject to control by phosphorylation (for example, by HRI, see section 1.2.2: eIF2 phosphorylation in erythropoiesis) of the α subunit of eIF2, which prevents the GDP-GTP exchange, resulting in the inhibition of cap-dependent translation (Figure 3a).

Figure 2. Cap-dependent translation initiation. Cap-dependent translation begins when the ternary complex (consisting of Met-tRNA, eIF2 (2) and GTP) associates with the 40S ribosomal subunit and eukaryotic initiation factors (eIFs) 1, 1A, 3 and 5 (labelled numerically in the fi gure). Th is assembly is referred to as that 43S pre-initiation complex (PIC). Th e PIC interacts with mRNA in a closed-loop for- mation, enacted by the binding of eIF4E (4E) to the 5’ cap and binding of PABP to the poly(A) tail, with scaff olding protein eIF4G (4G) bridging the two. RNA helicase eIF4A (4A) promotes a confi guration con- ducive to ribosomal landing. Th e heterotrimer consisting of eIF4A, eiF4E and eiF4G is referred to as the eIF4F complex. After docking with the ribosome, the 48S pre-initiation complex (PIC) 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 transla- tional elongation begins. Figure adapted from Kong and Lasko, 2012 (with permission).

17 Chapter 1

1.2.1 Epo and Scf control translational initiation in erythroblasts Stem cell factor (Scf) cooperates with erythropoietin (Epo) to expand erythroblast numbers in vivo and in vitro, whereas erythroblasts mature to erythrocytes in pres- ence of Epo only. The crucial pathway activated by Scf is mTOR-dependent release of the cap binding factor eIF4E from its binding protein, 4EBP [29] (Figure 3B). Overexpression of eIF4E inhibits erythroid differentiation. Upon release from 4EBP, eIF4E can once again bind the scaffold protein eIF4G, allowing the formation of the pre-initiation scanning complex [27,30]. Epo signaling overlaps with Scf signaling on the mTOR pathway. The result of Epo signaling is a partial phosphorylation of 4EBP, which requires synergy with Scf stimulation for the phosphorylation of all 4EBP phosphorylation sites to sustain erythroid proliferation [29]. The primary antagonist of mTOR-mediated 4EBP phosphorylation is protein phosphatase 2A (Pp2a), a ubiq- uitously expressed phosphatase that changes target specificity based upon complex formation with regulatory proteins [31]. Although the availability of eIF4E controls overall translation initiation, transcripts with a TOP (terminal oligopyrimidine tract) or secondary structures are hypersensitive to the available eIF4E concentration [32,33]. One such transcript is immunoglobulin binding protein 1 (Igbp1), the mRNA of which is constitutively expressed in erythro- blasts, but selectively translated only upon Scf-induced eIF4E release [24]. Igbp1 is more suitably known as the alpha4 subunit of Pp2a. As a regulatory subunit, it inhibits the catalytic activity of Pp2a on 4EBP. Expression of Igbp1/alpha4 thereby 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 mul- tiple eIF4E-sensitive mRNAs, resulting in the attenuation of erythroid differentiation.

1.2.2 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. This huge iron reservoir has a large oxidative, damaging potential. 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 [34]. Therefore, in- and export of iron in erythroblasts, and the synthesis of heme and globin needs to be tightly coupled. This process is controlled by several mRNA translation mechanisms [35]. Heme acts as a signaling molecule which binds to eIF2 associated kinase (eIF2ak1), also called HRI (Heme Regulated Inhibitor) [36]. During heme deficiency, HRI phosphorylates eIF2 alpha, preventing the exchange of eIF2-bound GDP for GTP by eIF2B and therefore also preventing the reassociation of the preinitiation scanning complex (Figure 3A) [37,38]. This results in a global inhibition of protein synthesis [39]. This mechanism

18 Introduction

1

Figure 3. molecular regulation of cap-dependent translation in erythropoiesis. (a) eIF2 phosphoryla- tion inhibits cap-dependent translation under stress conditions. After a round of translation initiation, eIF2 must be reloaded with new 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 misfolded proteins), or GCN (General control nonderepressible, during amino acid starvation), preventing eIF2B from exchanging eIF2-bound GDP for GTP. Th e lack of unphosphorylated eIF2 leads to translational arrest. (b) 4E-BP phosphorylation regulates cap-dependent translation. Scf sig- naling, and, to a lesser extent, Epo signaling, activates the mTOR pathway, which leads to the phosphory- lation of 4E-BP and the release of cap-binding protein eIF4E. Increased availability of eIF4E stimulates translation globally, but has a pronounced eff ect on transcripts containing structured 5’ UTRs, including Igbp1. Ibp1 inhibits the dephosphorylation of 4E-BP by Pp2a, strengthening the eff ect of mTOR activation.

19 Chapter 1 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 starva- tion via eIF2ak4 (GCN2) [40]. In addition to heme deficiency, HRI is activated both by oxidative stress and by denatured cytoplasmic proteins [35].

1.2.3 Upstream open reading frames Sensitivity to eIF2 phosphorylation is predicted by the presence of upstream open reading frames (uORFs) in the 5’ UTR of the transcript [27,41]. uORFs render mRNA transcripts hypersensitive to translation initiation factors because their translation met requires reassociation of eIF2:GTP and tRNAi with the scanning complex (Figure 2). uORFs may also inhibit the translation of select transcripts when they overlap with the coding sequence (CDS), in which case, the start codon is mostly in a less favor- able Kozak consensus sequence. Multiple uORFs in the 5´UTR can impose a complex regulation of translation of both the uORFs and the CDS to suppress protein expres- sion. The thrombopoietin (TPO) transcript contains seven uORFs which negatively in- hibit translation, the seventh of which overlaps with the protein-coding open-reading frame [42]. Although translation is generally inhibited upon eIF2 phosphorylation, the translation of specific transcripts may be enhanced under these conditions. One such transcript is activating transcriptional factor 4 (Atf4), which is essential for erythroid differentiation and for reduction of oxidative stress during the basophilic erythroblast stage [43]. Atf4 contains two uORFs, the second of which overlaps with the CDS [44]. Translation of the second uORF inhibits Atf4 protein expression by overwriting the CDS [45]. Decreased abundancy of unphosphorylated eIF2 due to HRI activity allows for a sufficient delay in ribosomal reassociation to select for the CDS instead of the inhibi- tory uORF, providing a mechanism for Atf4 expression under stress conditions. Other uORF-containing transcripts may, instead, be hypersensitive to eIF2 phosphorylation and translation may be impaired more than average upon eIF2 phosphorylation. These transcripts include Pabpc1 and Csde1 [46], the latter of which will be discussed in detail in section 1.6.

1.2.4 Translational control of iron homeostasis Iron homeostasis is achieved via a balanced regulation of iron import via transferrin receptor 1 (Tfr1), storage in ferritin and export via ferroportin [47]. Post-transcription- al 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 5’UTR [48,49]. Binding of the IRP to the iron responsive element (IRE) prevents the association of the 43S preinitiation complex to the mRNA transcript [50]. 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 [51].

20 Introduction

Interestingly, a splice variant of ferroportin lacking the IRE is expressed in duodenum and erythroid cells permits the escape of IRP-mediated translational control [52]. By 1 contrast, the Tfr1 transcript possesses 5 IREs in the 3’UTR rather than the 5’UTR [53,54]. Binding of IRPs to Tfr1 confers increased mRNA stability to the transcript, resulting in an inverse relationship between Tfr1 protein expression and iron abundancy [55,56]. 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. It possesses the capacity to act as an aconitase in the catalyzation of citrate to isocitrate [57]. The catalytic activity is dependent upon the assimilation of an ad- ditional 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 [58–60]. Because the formation of the iron-sulphur cluster in the active site requires an oxygen- free environment [61], the RNA-binding form is preferentially induced in the presence of the vasodilating agent nitrous oxide [62]. Although Irp2 is 57% homologous with Irp1 in humans, it does not function as an aconitase under iron-rich conditions [63]. Unlike Irp1, Irp2 is rapidly degraded by the proteasome when iron and oxygen levels are high [63,64]. Degradation of Irp2 is prevented by low oxygen pressure [65]. Taken together, Irp1 and Irp2 are capable of controlling iron homeostasis under both low and high iron and oxygen supply, allow- ing a proportional response to environmental stimuli [47].

1.3 IRES-dependent translation in hematopoiesis

In addition to cap-dependent translation, ribosomes can associate on a subset of transcripts carrying an internal ribosomal entry site (IRES) [66]. While there is no consensus sequence or universal structural motifs for IRESs, they typically contain complex structural elements which include stem loops and pseudoknots [67,68]. The majority of IRESs are found within the 5’UTR directly upstream of the initiation codon, though they can also exist within the coding region, causing synthesis of a truncated protein [69,70]. IRES-mediated translation is preferred under stress conditions, which are characterized by decreased availability of cap-dependent translation [71,72], due, for example, to eIF2 phosphorylation [73,74] or cleavage of scaffolding protein eIF4G [67]. This includes, but is not limited to, viral infection, hypoxia, nutrient starvation, ER stress and cell differentiation [67,75]. Translation via an IRES requires the binding of IRES transactivating factors (ITAFs) such as Polypyrimidine Tract Binding protein (Ptb) [76,77] and Csde1 [78–80]. IRES- mediated translation initiation is less competitive in ribosome recruitment. It is prob- ably for that reason that primarily IRES-dependent translation initiation is suppressed

21 Chapter 1 when fewer ribosomes are present, which is a hallmark of Diamond Blackfan Anemia (DBA, see section 1.5.1 Diamond-Blackfan Anemia) [81]. The induction of severe anemia due to loss of ribosomes in this latter disease indicates that IRES-mediated translation is of particular importance in erythropoiesis. Several involved in hematopoiesis are subject to IRES-mediated translation, among which are Bag1 and Runx1. Bag1, an Hsp70 cochaperone, is required for terminal differentiation of erythroblasts [81]. All three Bag1 isoforms are produced from a single transcript, dependent upon the involvement of either cap-dependent or IRES-mediated translation [82]. Two ITAFs are involved in this process: Pcbp1, which remodels the RNA to allow ribosomal entry, and Ptb, which is necessary for the recruitment of the ribosome to the Bag1 transcript [83]. In hematopoiesis, Bag1 defi- ciency is lethal at day E13.5 in mice with pronounced defects in brain and liver tissue [84]. shRNA-mediated knockdown of Bag1 in erythroblasts results in the production of fewer hemoglobinated daughter cells from erythroblasts cultured under differentia- tion conditions. Bag1 is implicated in DBA, wherein haploinsufficiency of ribosomal proteins results in the loss of Bag1 and other transcripts from polysomes [81]. In a DBA model, reduced expression of Rps19/Rpl11 resulted in diminished IRES-driven translation of Bag1 in a bicistronic reporter construct, indicating a possible deregula- tion of IRES-mediated translation in DBA. Another example of IRES-mediated translation is Runx1 (also called Aml1), a tran- scription factor essential for fetal liver hematopoiesis [85], which contains an IRES proximally upstream to one of its two promoters [86,87]. The absence of the IRES in Runx1 causes embryonic fatality due to pericyte development and disordered prolif- eration and differentiation of hematopoietic cells in the fetal liver [88].

1.4 The role of the poly(A) tail in mRNA stability and translation

The mRNA poly(A) tail protects the transcript from degradation, but the length of the poly(A) tail also affects translation initiation. The length of the poly(A) tail is determined by the recruitment of polyadenylation proteins. In the nucleus, there are four sequence elements and their binding proteins involved in selecting the site of cleavage and polyadenylation [89]. Upon cleavage site recognition, a nuclear poly(A) polymerase is recruited to synthesize the poly(A) tail. Typically this is carried out by poly(A) polymerase alpha, but depending on the cell type and specific transcript, another non-canonical polymerase may be involved [89]. Upon export to the cytoplasm, interaction with the poly(A) tail may enhance or repress translation of the mRNA transcript. A broad array of RNA-binding proteins

22 Introduction governs this process. Here, an overview of some of the larger families of poly(A)- interacting proteins and their influence on hematopoiesis will be presented. 1

1.4.1 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 (Pabp) to the poly(A) tail. PABPs directly interact with the eIF4G scaffold protein of the eIF4F cap-binding complex. This brings the mRNA tail close to the cap, and forms a circular mRNA conformation that is believed to optimize recycling of translation initiation and elongating factors [90]. The interaction of Pabp with eIF4G stabilizes pre-initiation scanning complexes on the 5’UTR [91]. A longer poly(A) tail increases Pabp affinity for target transcripts, which enhances the stabilization of multiple pre- initiation scanning complexes and thereby enhances translation initiation efficiency. Additionally, the binding of Pabp to the poly(A) tail protects the transcript from dead- enylation [92], a process which is discussed in more detail below. The most common Pabp is Pabpc1. In erythroid cells, however, there is a prominent role for Pabpc4 [93]. Pabpc4 binds to a specific subset of transcripts with short poly(A) tails containing an AU-rich motif, including α-globin, and protects them from further degradation. Depletion of Pabpc4 blocks induced terminal differentiation of mouse erythroblast leukemia (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 is not redundant with Pabpc1. Given that downregulation of c-Kit is essential for cell cycle arrest prior to terminal differentiation [16,94], the rescue of c-Kit expression is likely responsible for the inability of Pabpc4-depleted MEL cells to form mature erythrocytes [93]. Also induced were c-Myb, c-Myc, CD44, and Stat5a, all well-studied genes which promote erythroblast maintenance at the expense of differentiation [95].

1.4.2 CPEBs control poly(A) tail length In the cytoplasm, the length of the poly(A) tail may undergo additional processing to control translation of target transcripts. Critical to this process are the Cytoplas- mic Polyadenylation Element Binding proteins (CPEBs). CPEBs recruit cytoplasmic poly(A) polymerases to promote translation, the most well-characterized of which is Gld-2 (Germ line development 2) [96]. Depending on the cell type, a cytoplasmic vari- ant of the classic nuclear poly(A) polymerase may be recruited instead [97–99]. Cpeb1 is known to increase mRNA stability by binding to Pabpc1 and Pabpc1L (also called Epab) [100,101]. The process is further regulated by the involvement of additional RNA- binding proteins, such as Pumilio, which may stabilize the binding of CPEBs but also may function as a deadenylation factor and translational repressor [89]. CPEBs can

23 Chapter 1 also recruit the deadenylating CCR4/NOT complex, which cooperates with Pumilio to deadenylate target transcripts. The CCR4/NOT complex is composed of several Cnot subunits, which individually have varying roles in a myriad of physiological processes [102]. In particular, Cnot9 was identified as an erythropoietin-responsive gene, indi- cating a role for the complex in erythropoiesis [103]. Deadenylation initially represses translation, because less Pabp can bind and connect to eIF4G proteins in preinitiation complexes. Ultimately, deadenylation results in silencing of mRNA via 3’-5’ degrada- tion by the exosome, or 5’-3’ degradation by Xrn1 after decapping by Dcp1-Dcp2 [102]. Of the CPEB family members, Cpeb4 is specifically induced during erythroid dif- ferentiation [104], via the transcription factors Gata1 and Tal1 [95,105]. Cpeb4 not only promotes deadenylation, but also represses translation via binding to the eIF3 complex [104]. Cpeb4 is additionally capable of binding to and repressing its own mRNA, forming a feedback loop that maintains Cpeb4 levels within a range required for terminal erythropoiesis.

1.4.3 Musashi-mediated translational control in hematopoiesis In addition to CPEBs and PABPs, cytoplasmic polyadenylation is regulated by Musashi-1 and -2 (Msi1, Msi2), which interact with mRNA via the MSI-binding ele- ment (MBE) [89]. The MBE is known to confer cytoplasmic polyadenylation in the absence of CPEB activity, and knockdown of Msi1 prevents polyadenylation of Mos kinase, a regulator of meiosis during oocyte maturation [106]. Strikingly, however, no interactions between MSI proteins and the polyadenylation machinery have been described [89]. Although it is unclear how Msi proteins influence polyadenylation, there is a signifi- cant body of research detailing their function as RNA-binding proteins. Msi proteins are oppositely regulated from the Auf1/HnrpD family of AUBPs (see section 1.4.4: AUBPs in hematopoiesis), indicating their role as global translational regulators [107]. 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 [108]. Transcripts silenced by Msi1 include cell cycle regulators such as Numb, an in- hibitor of the Notch pathway and p21, and cyclin-dependent kinase inhibitor [109,110]. RNA binding domains between Msi1 and Msi2 are largely homologous (85-95%), but Msi2 has no Pabp-binding domain [111]. However, there is evidence to suggest that Msi2 alters Notch localization and upregulates Hes1, a Notch reporter protein [112], 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 [112–114]. The expression of Msi2 is subse- quently downregulated during differentiation. Downregulation of Msi2 alters the

24 Introduction balance between self-renewal and differentiation of HSCs via Notch pathway interac- tion [112,114]. This effect is achieved without influencing apoptotic rates or homing 1 behavior. A mouse line [Msi2Gt/Gt] expressing a truncated Msi2 gene results in a marked decrease in short term hematopoietic stem cells (ST-HSCs) and multipotent pro- genitors (MPPs) while the effect on long term hematopoietic stem cells (LT-HSCs) was nominal [113]. In contrast to RNA interference experiments, Msi2-defective LSKs from the mouse model display impaired proliferation. A non-competitive bone marrow transplantation experiment showed a decrease in the LT-HSC population [112]. This is in contrast with findings under steady state conditions. In addition, a doxycycline- inducible Msi2 transgenic mouse model observed an increase in ST-HSC/MPP popu- lations and a decrease in LT-HSC, whereas Msi2 overexpression increased LT-HSC self-renewal in transplanted mice. Furthermore, Msi2 was found to be significantly downregulated in erythroblasts derived from the CD34+ hematopoietic stem cells of β-thalassemia patients relative to those derived from healthy controls [115]. Taken together, these findings suggest that Msi2 is of particular importance during stress hematopoiesis as well as self-renewal and stem cell homeostasis. Studies on Msi2 in hematopoieis 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 [114], Ras, MAPK, cyclin D1, and Myc [112,113]. The role of Msi2 in hematopoiesis has been extensively reviewed in de Andrés-Aguayo et al., 2012.

1.4.4 AUBPs in hematopoiesis AU-rich elements (AREs) are sequence motifs (typically AUUUA) in the 3’UTR which recruit a large family of AU-binding proteins (AUBPs) that regulate translation. Trans- lational regulation via AUBPs can occur via a number of mechanisms, namely via deadenylation, but also via transcript sequestration to P-bodies and stress granules [116]. AUBPs key to erythropoiesis include the Tristetraprolin (TTP) family members (Zfp36, Zfp36l1, Zfp36l2) and HuR/Elav1. TTP family members interact with Not1 to promote the rapid deadenylation by the CCR4/NOT complex [117]. Interestingly, Zfp36l1 and Zfp36l2 demonstrate opposite regulation in erythropoiesis. Zfp36l1 downregulates Stat5b expression, reducing the formation of erythroid colonies [118]. By contrast, Zfp36l2 is required for burst-form- ing unit-erythrocyte (BFU-E) renewal [119]. Therefore, maintenance of erythrocyte homeostasis requires simultaneous downregulation of Zfp36l1 and upregulation of Zfp36l2. Elav1 is a ubiquitously expressed AUBP with thousands of direct and functional targets [120,121]. Relevant hematopoietic roles for ELAV1 in humans include the

25 Chapter 1 stabilization of mRNAs for BCL2, MCL1, cyclin A, cyclin B1, cyclin D1, lymphotoxin-α, GM-CSF, IL4, VEGF, CD3, CD95L, GATA-3, XIAP, and survivin, and destabilization of mRNAs for AML1/RUNX1, CD2, VAV1, NFκBIE, CD3ε, TNFα, and STAT3 (reviewed in [116]). ELAV1 additionally enhances the translation of mRNAs encoding p53, cyto- chrome c, XIAP, and BCL2 while suppressing translation of p27, MYC, and WNT5 (re- viewed in [116]). Interestingly, ZFP36l1 is a functional target of ELAV1 in human cells exposed to oxidative stress, wherein ionizing radiation decreases ZFP36L1 transcript binding by ELAV1, resulting in a decreased recruitment of ZFP36L1 to polysomes [122]. This suggests that Elav1 may synergize with Zfp36l1 in regulating erythropoiesis under some conditions.

1.5 Aberrant translation and hematopoietic disease

Disruption of translation has serious physiological consequences. Specifically, muta- tions in ribosomal proteins can cause a family of diseases known as ribosomopathies that result in abnormal hematopoiesis. Among these are Diamond-Blackfan Anemia (DBA), Shwachman-Diamond Syndrome (SDS), and Dyskeratosis Congenita (DC). It is important to note that all of these hematopoietic ribosomopathies present with clinically diverse symptoms, suggesting that dysregulation of ribosomal protein expression results in complex defects with organ-specific effects. It remains unclear why ribosomal mutations result in diverse clinical outcomes, instead of translational deregulation in an organism-wide, embryonically lethal manner.

1.5.1 Diamond-Blackfan Anemia DBA is an inherited anemia that presents with severe red cell aplasia in infant patients (<1 year of age) while all other blood lineages remain normal [123]. The primary feature of DBA is a reduction of erythroid precursors in the bone marrow [124], but the DBA phenotype can vary widely in severity, with approximately half of patients afflicted with skeletal and growth abnormalities, such as craniofacial deformities, di- minished stature, abnormalities of the thumb and the presence of a cleft palate [125]. Genetically, DBA is characterized by a haploinsufficiency of ribosomal proteins [126]. Loss of ribosome functionality can be the result of one or more mutated genes, most prominently RPS19 (25% of patients) [127], but mutations in both the small ribosomal subunit (RPS7, RPS10, RPS17, RPS24, RPS26, RPS29) and the large ribosomal subunit (RPL5, RPL11, RPL15, RPL26, RPL27, RPL35A) have been observed [126,128,129]. An imbalance of ribosome synthesis inhibits cell proliferation via p53 activation [130]. However, in one third of patients, the underlying mutation is yet to be identified, though there are many ribosomal proteins that remain unscreened [123].

26 Introduction

In atypical cases, DBA is associated with mutations of erythroid transcription factor GATA1 [131,132]. GATA1 mutations resulting in DBA may alter the splice donor site 1 of exon 2 or the start codon of the GATA1 open reading frame, resulting in reduced expression of GATA1 [132,133]. In this case, it is possible that GATA1 causes the red cell aplasia of DBA via a signaling defect during erythropoiesis rather than behaving as a ribosomopathy [123]. However, GATA1 binds to the region upstream of the promoter for several ribosomal proteins, including RPS19, suggesting that it may also play a role in the ribosome abnormalities found in DBA patients [134].

1.5.2 Shwachman-Diamond Syndrome SDS is an autosomal recessive syndrome characterized by bone marrow failure, neu- tropenia, decreased red cell/platelet counts, deficient pancreatic functioning, and an increased risk of leukemia (reviewed in [135]). SDS is caused by homozygous mutations in the Shwachman Bodian Diamond syndrome (SBDS) gene [136]. SBDS is involved in the regulation of RNA metabolism and ribosome functioning [137,138]. The ortholog of SBDS in S. cerevisiae, Sdo1p, regulates assembly of the 60S ribosomal subunit via as- sembly protein TIF6P and also regulates the interaction of the 60S subunit with the 40S subunit, playing a critical role in the formation of the complete ribosome [139]. Cells derived from SDS patients are unusually sensitive to rRNA transcription inhibition [135,140,141]. Comparisons between yeast models for DBA (RPL33A) and SDS (SDO1) show decreased formation of the 60S ribosomal subunit and an increase in polysomes containing stalled 48S initiation complexes [142]. However, in the SDS model, these incompletely assembled ribosomal precursors accumulate in the nucleus, whereas in the DBA model, the 60S precursors are rapidly degraded. This suggests that the defect in ribosome assembly observed in SDS is distal to the ribosomal defects observed in DBA [135].

1.5.3 Dyskeratosis Congenita DC is both clinically and genetically heterogenous (reviewed in [143]). Physiologically, it is characterized by mucocutaneous aberrations, bone marrow failure, anemia, and an increased vulnerability to carcinogenesis [144]. There are three recognized genetic subtypes of DC. The X-linked recessive version is caused by mutations in dyskerin [145], a component of small nucleolar RNA ribonucleoproteins (snoRNPs) involved in splicing that is also a telomerase component. The autosomal dominant version of DC has heterozygous mutations in either the RNA component (TERC) [146] or the enzymatic component (TERT) [147,148] of the telomerase complex. There is also an autosomal recessive version of DC, wherein the genetic cause remains unclear. The pathology of DC when caused by dyskerin, TERT or TERC mutations is believed to be a consequence of instability due to defective telomerase activity [143]. Pa-

27 Chapter 1 tients with dyskerin mutations display additional defects in rRNA synthesis, ribosome biogenesis, and mRNA splicing, suggesting that translational control is paramount to causing the DC phenotype in this genetic subtype [149–151]. In this chapter, the importance of both cap-dependent and cap-independent trans- lation in determining gene expression programming during erythropoiesis has been demonstrated. Ribosomopathies indicate that mutations in the broader translational machinery may, surprisingly, have tissue-specific consequences. At the intersection of these concepts is a protein known as Cold Shock Domain-containing E1 (Csde1), an ITAF implicated in DBA [81]. The next section summarizes the known functions of Csde1 in translational regulation and erythropoiesis.

1.6 Csde1 is at the crossroads of translational regulation

Csde1, originally called Unr (upstream of N-ras) is an RNA-binding protein with five cold-shock domains [152]. The first evidence for Csde1’s biological function involved the silencing of Msl (male sex lethal) during sex determination of Drosophila mela- nogaster via binding to the 3’UTR [153–158]. Repression of Msl-2 occurs via a direct interaction between Csde1 and 3’UTR-bound Pabp, resulting in the prevention of Pabp-mediated recruitment of the 43S PIC [159]. Paradoxically, the Csde1-Pabp complex has also been reported to stimulate translation by stabilizing the interaction between Pabp and eIF4G [160].

1.6.1 Csde1 and IRES-mediated translation Csde1 is implicated in both endogenous and viral IRES-mediated translation in mam- malian cells. The presence of viral double-stranded RNA causes eIF2 phosphorylation via PKR, resulting in a preferential advantage for cap-independent translation via IR- ESs. Both rhinovirus and poliovirus contain IRES elements that require an interaction between Csde1 and Ptb [79,161]. Early studies indicated that all five of Csde1’s cold shock domains were necessary for maintenance of Csde1’s affinity for rhinovirus -IR ESs, whereas cold shock domains 1 and 2 had the most impact on Csde1’s binding to Msl-2 [156,162], and cold shock domains 2 and 4 were the only required elements for stimulation of translation via Pabp [160]. The cooperation between Csde1 and Ptb in regulating IRES activity extends to cellular transcripts. The Csde1 transcript contains an IRES within the 5’UTR which, when bound by Ptb and Csde1 itself, downregulates IRES-dependent translation of Csde1 [78,163,164]. Pathways such as mitosis and apoptosis specifically promote IRES-mediated translation at the expense of cap-dependent translation via, among other mechanisms, phosphorylation of eIF2 [67,73]. Csde1 IRES activity is strongly

28 Introduction upregulated during mitosis due to increased binding of hnRNP C1/C2 proteins with simultaneous release of Csde1 and Ptb [163]. The resultant increase in Csde1 expres- 1 sion during the G2/M phase of the cell cycle facilitates the IRES-mediated translation of cyclin-dependent PITSLRE kinases, which are essential for centrosome maturation and mitotic spindle formation [73,165]. During apoptosis, Csde1 and Ptb upregulate Apaf-1 (apoptotic protease-activating factor 1) via binding to the IRES within the 5’UTR [166]. Binding of Csde1 and Ptb changes the conformation of the IRES of Apaf-1 to a single-stranded region, granting access for ribosomal recruitment and thereby permitting the translation of the transcript [80].

1.6.2 Csde1 and the poly(A) tail Csde1 is also implicated as a regulator of mRNA stability via poly(A) deadenylation. Csde1 binds to c-fos major protein-coding determinant of instability (mCRD) motifs in conjunction with Pabp [167]. In contrast to the stabilizing influence of Pabp when interacting with CPEBs, 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., (2004) propose a model in which the Csde1-Pabp complex initially protects the poly(A) tail from deadenyl- ation 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 decreased expression as a consequence of transcript deadenylation and subsequent degradation.

1.6.3 Csde1 and 4E-T Yet another role for Csde1 in the regulation of translation is apparent in its interaction 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 asso- ciation with scaffolding protein eIF4G [168] while simultaneously reducing ribosomal access to the 5’ cap via interaction with RNA-binding proteins at the 3’UTR [169,170]. Furthermore, 4E-T is a component of the CPEB translation repressor complex [171], the CCR4-NOT complex [172], and enhances decay of transcripts containing AU-rich elements [173–175]. Finally, 4E-T is involved in shuttling between the cytoplasm and the mRNA triage and degradation centers known as Processing Bodies (P-bodies) [176,177]. 4E-T is directly bound by Csde1 and indirectly bound by Strap (also known as Unrip, Unr-interacting protein) through Csde1 [178]. Kamenska et al., (2016) shows that Csde1 binds to Cnot4 in manner mutually exclusive with binding of Cnot4 to 4E- T, theoretically abrogating 4E-Ts role as a bridge between Cnot1 and Cnot4 complex subunits. 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

29 Chapter 1 authors suggest that Csde1 and Ddx6, an RNA helicase component of the CPEB repres- sor 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, disrup- tion of which unravels a network of interactions necessary for translational repression.

1.6.4 Csde1 in DBA CSDE1 is implicated in Diamond-Blackfan Anemia (DBA). A study by Horos et al., (2012) employed the knockdown of Rps19 and Rpl11 to simulate the DBA phenotype in Trp53 -/- mouse erythroblasts. Polysomal and subpolysomal mRNA was isolated via a sucrose gradient and hybridized separately to microarrays to identify which transcripts were susceptible to diminished polysome recruitment in the DBA model. Among the transcripts lost was Csde1. Protein expression levels were correspondingly decreased after Rps19/Rpl11 knockdown, whereas mRNA expression was unaltered. These results were reproducible in erythroblast derived directly from DBA patients and in p53 competent cells, making the negative inhibition of CSDE1 translation p53- independent. Expression of Csde1 steadily increases during progression through the hematopoietic lineage, with levels 250x higher in erythroblasts versus LSKs, CMPs, and GMPs. Additionally, knockdown of Csde1 results in impairment of erythroblast proliferation and differentiation. The known functions of Csde1 are summarized in Figure 4.

1.7 Scope of the thesis

Erythropoiesis needs to be tightly regulated to maintain the number of erythrocytes in peripheral blood between narrow limits. At the molecular and cellular level, this re- quires a transcriptional program that is fine-tuned at the level of mRNA translation and protein stability. Given that Csde1 is an essential protein that is abundantly expressed in erythroid cells, it stands to reason that Csde1 is a critical regulator of translation during erythropoiesis. We aimed to unravel the mechanism of Csde1-dependent translational regulation in erythropoiesis, via identification of not just Csde1’s target transcripts, but also its protein binding partners, and whether protein complex forma- tion alters Csde1 function. We examined the phenotypic differences in erythroblasts after reduction of Csde1 expression and after removal of a select RNA binding domain (Chapter 2). We subsequently investigated the role of Strap, a Csde1-binding partner, on the translation of Csde1-bound transcripts and identified a possible role for Strap during hypoxic stress (Chapter 3). More globally, we quantified the effect of eIF2 phos-

30 Introduction

1

Figure 4. Hierarchical overview of Csde1-mediated translational regulation. Csde1 has been empiri- cally demonstrated to regulate both cap-dependent and IRES-mediated translation, as well as directly af- fecting the stability of select mRNA transcripts. It may either promote or repress cap-dependent transla- tion, as its interaction with PABP can either facilitate or inhibit ribosomal recruitment. Th e role of Csde1 in IRES-mediated translation is generally stimulatory, as seen in the increased IRES activity of Apaf-1 under apoptotic conditions. IRES-driven translation of PITSLRE kinase and of Csde1’s own transcript is enhanced during the cell-cycle. Th e same holds true for viral IRESs. Csde1 may additionally both pro- mote or inhibit mRNA degradation via transient interaction with the CCR4-NOT complex. Interaction with PABP contributes to the protection of the poly(A) tail from CCR4-NOT. Finally, Csde1 interacts with 4-ET, which shuttles transcripts between the cytoplasm and P-bodies, where they are sequestered and degraded. As 4-ET also functions as part of the CPEB repressor complex and the CCR4-NOT complex, Csde1 likely cooperates with 4-ET to infl uence translational stability as part of a highly complex and overlapping group of regulatory pathways. phorylation on ribosome occupancy as a mechanism for assaying which transcripts are diff erentially translated under stress conditions (Chapter 4).

31 Chapter 1

References

1. Ogawa M. Differentiation and proliferation of hematopoietic stem cells. Blood. 1993;81. 2. Spangrude G, Heimfeld S, Weissman I. Purification and characterization of mouse hematopoi- etic stem cells. Science (80- ). 1988;241. 3. Blank U, Karlsson G, Karlsson S. Signaling pathways governing stem-cell fate. Blood. 2008;111. 4. Adolfsson J, Borge OJ, Bryder D, Theilgaard-Mönch K, Astrand-Grundström I, Sitnicka E, et al. Upregulation of Flt3 expression within the bone marrow Lin(-)Sca1(+)c-kit(+) stem cell compartment is accompanied by loss of self-renewal capacity. Immunity. 2001;15: 659–69. 5. Yang L, Bryder D, Adolfsson J, Nygren J, Månsson R, Sigvardsson M, et al. Identification of Lin– Sca1+kit+CD34+Flt3– short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients. Blood. 2005;105. 6. Morrison SJ, Weissman IL. The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype. Immunity. 1994;1: 661–73. 7. Carow CE, Hangoc G, Broxmeyer HE. Human multipotential progenitor cells (CFU-GEMM) have extensive replating capacity for secondary CFU-GEMM: an effect enhanced by cord blood plasma. Blood. 1993;81: 942–9. 8. Roodman GD, LeMaistre CF, Clark GM, Page CP, Newcomb TF, Knight WA. CFU-GEMM corre- late with neutrophil and platelet recovery in patients receiving autologous marrow transplanta- tion after high-dose melphalan chemotherapy. Bone Marrow Transplant. 1987;2: 165–73. 9. Wu H, Liu X, Jaenisch R, Lodish HF. Generation of committed erythroid BFU-E and CFU-E pro- genitors does not require erythropoietin or the erythropoietin receptor. Cell. 1995;83: 59–67. 10. Sposi NM. Interaction between Erythropoiesis and Iron Metabolism in Human β-thalassemia - Recent Advances and New Therapeutic Approaches. Inherited Hemoglobin Disorders. InTech; 2015. doi:10.5772/61716 11. Gregory T, Yu C, Ma A, Orkin SH, Blobel GA, Weiss MJ. GATA-1 and erythropoietin cooperate to promote erythroid cell survival by regulating bcl-xL expression. Blood. 1999;94: 87–96. 12. Kim S-I, Bresnick EH. Transcriptional control of erythropoiesis: emerging mechanisms and principles. Oncogene. 2007;26: 6777–94. doi:10.1038/sj.onc.1210761 13. Ribeil J-A, Arlet J-B, Dussiot M, Cruz Moura I, Courtois G, Hermine O. Ineffective Erythropoi- esis in Beta-Thalassemia. Sci World J. 2013;2013: 1–11. doi:10.1155/2013/394295 14. Ferreira R, Ohneda K, Yamamoto M, Philipsen S. GATA1 function, a paradigm for transcription factors in hematopoiesis. Mol Cell Biol. American Society for Microbiology (ASM); 2005;25: 1215–27. doi:10.1128/MCB.25.4.1215-1227.2005 15. Andrews NC. The NF-E2 transcription factor. Int J Biochem Cell Biol. 1998;30: 429–32. 16. Dzierzak E, Philipsen S. Erythropoiesis: development and differentiation. Cold Spring Harb Perspect Med. Cold Spring Harbor Laboratory Press; 2013;3: a011601. doi:10.1101/cshperspect. a011601 17. Borg J, Papadopoulos P, Georgitsi M, Gutiérrez L, Grech G, Fanis P, et al. Haploinsufficiency for the erythroid transcription factor KLF1 causes hereditary persistence of fetal hemoglobin. Nat Genet. 2010;42: 801–805. doi:10.1038/ng.630 18. Perkins A, Xu X, Higgs DR, Patrinos GP, Arnaud L, Bieker JJ, et al. Krüppeling erythropoiesis: an unexpected broad spectrum of human red blood cell disorders due to KLF1 variants. Blood. 2016;127: 1856–62. doi:10.1182/blood-2016-01-694331

32 Introduction

19. Fang J, Menon M, Kapelle W, Bogacheva O, Bogachev O, Houde E, et al. EPO modulation of cell-cycle regulatory genes, and cell division, in primary bone marrow erythroblasts. Blood. American Society of Hematology; 2007;110: 2361–70. doi:10.1182/blood-2006-12-063503 1 20. Socolovsky M, Murrell M, Liu Y, Pop R, Porpiglia E, Levchenko A. Negative autoregulation by FAS mediates robust fetal erythropoiesis. PLoS Biol. Public Library of Science; 2007;5: e252. doi:10.1371/journal.pbio.0050252 21. Menon MP, Karur V, Bogacheva O, Bogachev O, Cuetara B, Wojchowski DM. Signals for stress erythropoiesis are integrated via an erythropoietin receptor-phosphotyrosine-343-Stat5 axis. J Clin Invest. American Society for Clinical Investigation; 2006;116: 683–94. doi:10.1172/ JCI25227 22. Broudy VC, Lin NL, Priestley G V, Nocka K, Wolf NS. Interaction of stem cell factor and its recep- tor c-kit mediates lodgment and acute expansion of hematopoietic cells in the murine spleen. Blood. 1996;88: 75–81. 23. Bakker WJ, Blázquez-Domingo M, Kolbus A, Besooyen J, Steinlein P, Beug H, et al. FoxO3a regulates erythroid differentiation and induces BTG1, an activator of protein arginine methyl transferase 1. J Cell Biol. 2004;164: 175–84. doi:10.1083/jcb.200307056 24. Grech G, Blázquez-Domingo M, Kolbus A, Bakker WJ, Müllner EW, Beug H, et al. Igbp1 is part of a positive feedback loop in stem cell factor–dependent, selective mRNA translation initiation inhibiting erythroid differentiation. Blood. 2008;112. 25. Schwanhäusser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, et al. Global quantification of mammalian gene expression control. Nature. 2011;473: 337–342. doi:10.1038/nature10098 26. Kong J, Lasko P. Translational control in cellular and developmental processes. Nat Rev Genet. 2012;13: 383–394. doi:10.1038/nrg3184 27. Hinnebusch AG. The scanning mechanism of eukaryotic translation initiation. Annu Rev Bio- chem. 2014;83: 779–812. doi:10.1146/annurev-biochem-060713-035802 28. Proud CG. eIF2 and the control of cell physiology. Semin Cell Dev Biol. 2005;16: 3–12. doi:10.1016/j.semcdb.2004.11.004 29. Blázquez-Domingo M. Translation initiation factor 4E inhibits differentiation of erythroid progenitors. Mol Cell …. 2005; 30. Siddiqui N, Sonenberg N. Signalling to eIF4E in cancer. Biochem Soc Trans. Portland Press Ltd; 2015;43: 763–72. doi:10.1042/BST20150126 31. Grech G, von Lindern M. The role of translation initiation regulation in haematopoiesis. Comp Funct Genomics. 2012;2012: 576540. doi:10.1155/2012/576540 32. Graff JR, Zimmer SG. Translational control and metastatic progression: enhanced activity of the mRNA cap-binding protein eIF-4E selectively enhances translation of metastasis-related mRNAs. Clin Exp Metastasis. 2003;20: 265–73. 33. Modelska A, Turro E, Russell R, Beaton J, Sbarrato T, Spriggs K, et al. The malignant phenotype in breast cancer is driven by eIF4A1-mediated changes in the translational landscape. Cell Death Dis. Nature Publishing Group; 2015;6: e1603. doi:10.1038/cddis.2014.542 34. Jacob H, Winterhalter K. Unstable hemoglobins: the role of heme loss in Heinz body formation. Proc Natl Acad Sci U S A. 1970;65: 697–701. 35. Chen J-J. Translational control by heme-regulated eIF2$α$ kinase during erythropoiesis. Curr Opin Hematol. 2014;21: 172–178. doi:10.1097/MOH.0000000000000030 36. Han A-P, Fleming MD, Chen J-J. Heme-regulated eIF2alpha kinase modifies the phenotypic se- verity of murine models of erythropoietic protoporphyria and beta-thalassemia. J Clin Invest. American Society for Clinical Investigation; 2005;115: 1562–70. doi:10.1172/JCI24141

33 Chapter 1

37. Chung J, Chen C, Paw BH. Heme metabolism and erythropoiesis. Curr Opin Hematol. 2012;19: 156–162. doi:10.1097/MOH.0b013e328351c48b 38. Chen J-J. Regulation of protein synthesis by the heme-regulated eIF2alpha kinase: relevance to anemias. Blood. 2007;109: 2693–2699. doi:10.1182/blood-2006-08-041830 39. Liu S, Bhattacharya S, Han A, Suragani RNVS, Zhao W, Fry RC, et al. Haem-regulated eIF2alpha kinase is necessary for adaptive gene expression in erythroid precursors under the stress of iron deficiency. Br J Haematol. NIH Public Access; 2008;143: 129–37. doi:10.1111/j.1365- 2141.2008.07293.x 40. Wek RC, Jiang H-Y, Anthony TG. Coping with stress: eIF2 kinases and translational control. Biochem Soc Trans. 2006;34: 7–11. doi:10.1042/BST20060007 41. Young SK, Willy JA, Wu C, Sachs MS, Wek RC. Ribosome Reinitiation Directs Gene-specific Translation and Regulates the Integrated Stress Response. J Biol Chem. American Society for Biochemistry and Molecular Biology; 2015;290: 28257–71. doi:10.1074/jbc.M115.693184 42. Stockklausner C, Breit S, Neu-Yilik G, Echner N, Hentze MW, Kulozik AE, et al. The uORF- containing thrombopoietin mRNA escapes nonsense-mediated decay (NMD). Nucleic Acids Res. Oxford University Press; 2006;34: 2355–63. doi:10.1093/nar/gkl277 43. Suragani RNVS, Zachariah RS, Velazquez JG, Liu S, Sun C-W, Townes TM, et al. Heme-regulated eIF2$α$ kinase activated Atf4 signaling pathway in oxidative stress and erythropoiesis. Blood. 2012;119: 5276–5284. doi:10.1182/blood-2011-10-388132 44. Vattem KM, Wek RC. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. PNAS. 2004;101: 11269–11274. 45. Lu PD, Harding HP, Ron D. Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response. J Cell Biol. 2004;167: 27–33. doi:10.1083/ jcb.200408003 46. Andreev DE, O’Connor PB, Fahey C, Kenny EM, Terenin IM, Dmitriev SE, et al. Translation of 5’ leaders is pervasive in genes resistant to eIF2 repression. Elife. 2015;4: 1–21. doi:10.7554/ eLife.03971 47. Kühn LC, Samuelsson T, Bottke W, Laftah AH, Takeuchi K, Halliday N, et al. Iron regula- tory proteins and their role in controlling iron metabolism. Metallomics. The Royal Society of Chemistry; 2015;7: 232–243. doi:10.1039/C4MT00164H 48. Aziz N, Munro HN. Both subunits of rat liver ferritin are regulated at a translational level by iron induction. Nucleic Acids Res. 1986;14: 915–27. 49. Hentze MW, Caughman SW, Rouault TA, Barriocanal JG, Dancis A, Harford JB, et al. Identifica- tion of the iron-responsive element for the translational regulation of human ferritin mRNA. Science. 1987;238: 1570–3. 50. Gray NK, Hentze MW. Iron regulatory protein prevents binding of the 43S translation pre- initiation complex to ferritin and eALAS mRNAs. EMBO J. 1994;13: 3882–91. 51. Abboud S, Haile DJ. A Novel Mammalian Iron-regulated Protein Involved in Intracellular Iron Metabolism. J Biol Chem. 2000;275: 19906–19912. doi:10.1074/jbc.M000713200 52. Cianetti L, Segnalini P, Calzolari A, Morsilli O, Felicetti F, Ramoni C, et al. Expression of al- ternative transcripts of ferroportin-1 during human erythroid differentiation. Haematologica. 2005;90: 1595–606. 53. Müllner EW, Neupert B, Kühn LC. A specific mRNA binding factor regulates the iron-dependent stability of cytoplasmic transferrin receptor mRNA. Cell. 1989;58: 373–82.

34 Introduction

54. Koeller DM, Casey JL, Hentze MW, Gerhardt EM, Chan LN, Klausner RD, et al. A cytosolic pro- tein binds to structural elements within the iron regulatory region of the transferrin receptor mRNA. Proc Natl Acad Sci U S A. 1989;86: 3574–8. 1 55. Owen D, Kühn LC. Noncoding 3’ sequences of the transferrin receptor gene are required for mRNA regulation by iron. EMBO J. 1987;6: 1287–93. 56. Müllner EW, Kühn LC. A stem-loop in the 3’ untranslated region mediates iron-dependent regulation of transferrin receptor mRNA stability in the cytoplasm. Cell. 1988;53: 815–25. 57. Kennedy MC, Emptage MH, Dreyer JL, Beinert H. The role of iron in the activation-inactivation of aconitase. J Biol Chem. 1983;258: 11098–105. 58. Haile DJ, Rouault TA, Tang CK, Chin J, Harford JB, Klausner RD. Reciprocal control of RNA- binding and aconitase activity in the regulation of the iron-responsive element binding protein: role of the iron-sulfur cluster. Proc Natl Acad Sci U S A. National Academy of Sciences; 1992;89: 7536–40. 59. Emery-Goodman A, Hirling H, Scarpellino L, Henderson B, Kühn LC. Iron regulatory factor expressed from recombinant baculovirus: conversion between the RNA-binding apoprotein and Fe-S cluster containing aconitase. Nucleic Acids Res. 1993;21: 1457–61. 60. Hirling H, Henderson BR, Kühn LC. Mutational analysis of the [4Fe-4S]-cluster converting iron regulatory factor from its RNA-binding form to cytoplasmic aconitase. EMBO J. European Molecular Biology Organization; 1994;13: 453–61. 61. Beinert H, Kennedy MC. Aconitase, a two-faced protein: enzyme and iron regulatory factor. FASEB J. 1993;7: 1442–9. 62. Drapier JC, Hirling H, Wietzerbin J, Kaldy P, Kühn LC. Biosynthesis of nitric oxide activates iron regulatory factor in macrophages. EMBO J. 1993;12: 3643–9. 63. Guo B, Yu Y, Leibold EA. Iron regulates cytoplasmic levels of a novel iron-responsive element- binding protein without aconitase activity. J Biol Chem. 1994;269: 24252–60. 64. Guo B, Phillips JD, Yu Y, Leibold EA. Iron regulates the intracellular degradation of iron regula- tory protein 2 by the proteasome. J Biol Chem. 1995;270: 21645–51. 65. Hanson ES, Rawlins ML, Leibold EA. Oxygen and Iron Regulation of Iron Regulatory Protein 2. J Biol Chem. 2003;278: 40337–40342. doi:10.1074/jbc.M302798200 66. Komar AA, Hatzoglou M. Cellular IRES-mediated translation: the war of ITAFs in pathophysi- ological states. Cell Cycle. Taylor & Francis; 2011;10: 229–40. doi:10.4161/cc.10.2.14472 67. Komar AA, Hatzoglou M. Internal Ribosome Entry Sites in Cellular mRNAs: Mystery of Their Existence. J Biol Chem. 2005;280: 23425–23428. doi:10.1074/jbc.R400041200 68. Baird SD, Lewis SM, Turcotte M, Holcik M. A search for structurally similar cellular internal ribosome entry sites. Nucleic Acids Res. 2007;35: 4664–4677. doi:10.1093/nar/gkm483 69. Komar AA, Lesnik T, Cullin C, Merrick WC, Trachsel H, Altmann M. Internal initiation drives the synthesis of Ure2 protein lacking the prion domain and affects [URE3] propagation in yeast cells. EMBO J. 2003;22: 1199–1209. doi:10.1093/emboj/cdg103 70. Grover R, Candeias MM, Fähraeus R, Das S. p53 and little brother p53/47: linking IRES activities with protein functions. Oncogene. 2009;28: 2766–2772. doi:10.1038/onc.2009.138 71. Spriggs KA, Stoneley M, Bushell M, Willis AE. Re-programming of translation following cell stress allows IRES-mediated translation to predominate. Biol Cell. 2008;100: 27–38. doi:10.1042/ BC20070098 72. Lewis SM, Holcik M. For IRES trans-acting factors, it is all about location. Oncogene. 2008;27: 1033–1035. doi:10.1038/sj.onc.1210777

35 Chapter 1

73. Tinton SA, Schepens B, Bruynooghe Y, Beyaert R, Cornelis S. Regulation of the cell-cycle-de- pendent internal ribosome entry site of the PITSLRE protein kinase: roles of Unr (upstream of N-ras) protein and phosphorylated translation initiation factor eIF-2alpha. Biochem J. Portland Press Ltd; 2005;385: 155–63. doi:10.1042/BJ20040963 74. Gerlitz G, Jagus R, Elroy-Stein O. Phosphorylation of initiation factor-2 alpha is required for activation of internal translation initiation during cell differentiation. Eur J Biochem. 2002;269: 2810–9. 75. Graber TE, Holcik M. Cap-independent regulation of gene expression in apoptosis. Mol Biosyst. 2007;3: 825. doi:10.1039/b708867a 76. Bushell M, Stoneley M, Kong YW, Hamilton TL, Spriggs KA, Dobbyn HC, et al. Polypyrimidine Tract Binding Protein Regulates IRES-Mediated Gene Expression during Apoptosis. Mol Cell. 2006;23: 401–412. doi:10.1016/j.molcel.2006.06.012 77. Cobbold LC, Wilson LA, Sawicka K, King HA, Kondrashov A V, Spriggs KA, et al. Upregulated c-myc expression in multiple myeloma by internal ribosome entry results from increased inter- actions with and expression of PTB-1 and YB-1. Oncogene. Nature Publishing Group; 2010;29: 2884–2891. doi:10.1038/onc.2010.31 78. Cornelis S, Tinton SA, Schepens B, Bruynooghe Y, Beyaert R. UNR translation can be driven by an IRES element that is negatively regulated by polypyrimidine tract binding protein. Nucleic Acids Res. Oxford University Press; 2005;33: 3095–108. doi:10.1093/nar/gki611 79. Hunt SL, Hsuan JJ, Totty N, Jackson RJ. unr, a cellular cytoplasmic RNA-binding protein with five cold-shock domains, is required for internal initiation of translation of human rhinovirus RNA. Genes Dev. Cold Spring Harbor Laboratory Press; 1999;13: 437–48. 80. Mitchell SA, Spriggs KA, Coldwell MJ, Jackson RJ, Willis AE. The Apaf-1 internal ribosome entry segment attains the correct structural conformation for function via interactions with PTB and unr. Mol Cell. 2003;11: 757–71. 81. Horos R, Ijspeert H, Pospisilova D, Sendtner R, Andrieu-Soler C, Taskesen E, et al. Ribosomal deficiencies in Diamond-Blackfan anemia impair translation of transcripts essential for dif- ferentiation of murine and human erythroblasts. Blood. American Society of Hematology; 2012;119: 262–72. doi:10.1182/blood-2011-06-358200 82. Coldwell MJ, deSchoolmeester ML, Fraser GA, Pickering BM, Packham G, Willis AE. The p36 isoform of BAG-1 is translated by internal ribosome entry following heat shock. Oncogene. 2001;20: 4095–4100. doi:10.1038/sj.onc.1204547 83. Pickering BM, Mitchell SA, Spriggs KA, Willis AE, Stoneley M. Bag-1 Internal Ribosome Entry Segment Activity Is Promoted by Structural Changes Mediated by Poly ( rC ) Binding Protein 1 and Recruitment of Polypyrimidine Tract Binding Protein 1 Bag-1 Internal Ribosome Entry Segment Activity Is Promoted by Structural C. 2004; doi:10.1128/MCB.24.12.5595 84. Götz R, Wiese S, Takayama S, Camarero GC, Rossoll W, Schweizer U, et al. Bag1 is essential for differentiation and survival of hematopoietic and neuronal cells. Nat Neurosci. NIH Public Access; 2005;8: 1169–78. doi:10.1038/nn1524 85. Okuda T, van Deursen J, Hiebert SW, Grosveld G, Downing JR. AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoi- esis. Cell. 1996;84: 321–30. 86. Pozner A, Goldenberg D, Negreanu V, Le SY, Elroy-Stein O, Levanon D, et al. Transcription- coupled translation control of AML1/RUNX1 is mediated by cap- and internal ribosome entry site-dependent mechanisms. Mol Cell Biol. American Society for Microbiology (ASM); 2000;20: 2297–307.

36 Introduction

87. Levanon D, Groner Y. Structure and regulated expression of mammalian RUNX genes. Onco- gene. 2004;23: 4211–9. doi:10.1038/sj.onc.1207670 88. Nagamachi A, Htun PW, Ma F, Miyazaki K, Yamasaki N, Kanno M, et al. A 5’ untranslated region 1 containing the IRES element in the Runx1 gene is required for angiogenesis, hematopoiesis and leukemogenesis in a knock-in mouse model. Dev Biol. 2010;345: 226–36. doi:10.1016/j. ydbio.2010.07.015 89. Charlesworth A, Meijer HA, de Moor CH. Specificity factors in cytoplasmic polyadenylation. Wiley Interdiscip Rev RNA. Wiley-Blackwell; 2013;4: 437–61. doi:10.1002/wrna.1171 90. Wakiyama M, Imataka H, Sonenberg N, Fukunaga R, Hunter T, Sonenberg N. Interaction of eIF4G with poly(A)-binding protein stimulates translation and is critical for Xenopus oocyte maturation. Curr Biol. Elsevier; 2000;10: 1147–50. doi:10.1016/S0960-9822(00)00701-6 91. Kahvejian A, Svitkin Y V, Sukarieh R, M’Boutchou M-N, Sonenberg N. Mammalian poly(A)- binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms. Genes Dev. Cold Spring Harbor Laboratory Press; 2005;19: 104–13. doi:10.1101/gad.1262905 92. Norbury CJ. Cytoplasmic RNA: a case of the tail wagging the dog. Nat Rev Mol Cell Biol. 2013;14: 643–53. doi:10.1038/nrm3645 93. Kini HK, Kong J, Liebhaber SA. Cytoplasmic poly(A) binding protein C4 serves a critical role in erythroid differentiation. Mol Cell Biol. American Society for Microbiology (ASM); 2014;34: 1300–9. doi:10.1128/MCB.01683-13 94. Munugalavadla V, Dore LC, Tan BL, Hong L, Vishnu M, Weiss MJ, et al. Repression of c-kit and its downstream substrates by GATA-1 inhibits cell proliferation during erythroid maturation. Mol Cell Biol. 2005;25: 6747–59. doi:10.1128/MCB.25.15.6747-6759.2005 95. Hattangadi SM, Wong P, Zhang L, Flygare J, Lodish HF. From stem cell to red cell: regulation of erythropoiesis at multiple levels by multiple proteins, RNAs, and chromatin modifications. Blood. American Society of Hematology; 2011;118: 6258–68. doi:10.1182/blood-2011-07-356006 96. Barnard DC, Ryan K, Manley JL, Richter JD. Symplekin and xGLD-2 Are Required for CPEB- Mediated Cytoplasmic Polyadenylation. Cell. 2004;119: 641–651. doi:10.1016/j.cell.2004.10.029 97. Bilger A, Fox CA, Wahle E, Wickens M. Nuclear polyadenylation factors recognize cytoplasmic polyadenylation elements. Genes Dev. 1994;8: 1106–16. 98. Gebauer F, Richter JD. Cloning and characterization of a Xenopus poly(A) polymerase. Mol Cell Biol. American Society for Microbiology (ASM); 1995;15: 1422–30. 99. Benoit P, Papin C, Kwak JE, Wickens M, Simonelig M. PAP- and GLD-2-type poly(A) polymer- ases are required sequentially in cytoplasmic polyadenylation and oogenesis in Drosophila. Development. 2008;135. 100. Seli E, Lalioti MD, Flaherty SM, Sakkas D, Terzi N, Steitz JA. An embryonic poly(A)-binding protein (ePAB) is expressed in mouse oocytes and early preimplantation embryos. Proc Natl Acad Sci U S A. National Academy of Sciences; 2005;102: 367–72. doi:10.1073/pnas.0408378102 101. Guzeloglu-Kayisli O, Pauli S, Demir H, Lalioti MD, Sakkas D, Seli E. Identification and charac- terization of human embryonic poly(A) binding protein (EPAB). MHR Basic Sci Reprod Med. Oxford University Press; 2008;14: 581–588. doi:10.1093/molehr/gan047 102. Shirai Y-T, Suzuki T, Morita M, Takahashi A, Yamamoto T. Multifunctional roles of the mam- malian CCR4-NOT complex in physiological phenomena. Front Genet. Frontiers Media SA; 2014;5: 286. doi:10.3389/fgene.2014.00286 103. Gregory RC, Lord KA, Panek LB, Gaines P, Dillon SB, Wojchowski DM. SUBTRACTION CLON- ING AND INITIAL CHARACTERIZATION OF NOVEL EPO-IMMEDIATE RESPONSE GENES. Cytokine. 2000;12: 845–857. doi:10.1006/cyto.2000.0686

37 Chapter 1

104. Hu W, Yuan B, Lodish HF. Cpeb4-mediated translational regulatory circuitry controls terminal erythroid differentiation. Dev Cell. 2014;30: 660–72. doi:10.1016/j.devcel.2014.07.008 105. Kerenyi MA, Orkin SH. Networking erythropoiesis. J Exp Med. 2010;207: 2537–41. doi:10.1084/ jem.20102260 106. Charlesworth A, Wilczynska A, Thampi P, Cox LL, MacNicol AM. Musashi regulates the temporal order of mRNA translation during Xenopus oocyte maturation. EMBO J. European Molecular Biology Organization; 2006;25: 2792–801. doi:10.1038/sj.emboj.7601159 107. Sakakibara S, Nakamura Y, Yoshida T, Shibata S, Koike M, Takano H, et al. RNA-binding protein Musashi family: roles for CNS stem cells and a subpopulation of ependymal cells revealed by targeted disruption and antisense ablation. Proc Natl Acad Sci U S A. National Academy of Sciences; 2002;99: 15194–9. doi:10.1073/pnas.232087499 108. Kerwitz Y, Kühn U, Lilie H, Knoth A, Scheuermann T, Friedrich H, et al. Stimulation of poly(A) polymerase through a direct interaction with the nuclear poly(A) binding protein allosteri- cally regulated by RNA. EMBO J. European Molecular Biology Organization; 2003;22: 3705–14. doi:10.1093/emboj/cdg347 109. Bardwell VJ, Zarkower D, Edmonds M, Wickens M. The enzyme that adds poly(A) to mRNAs is a classical poly(A) polymerase. Mol Cell Biol. American Society for Microbiology (ASM); 1990;10: 846–9. 110. Topalian SL, Kaneko S, Gonzales MI, Bond GL, Ward Y, Manley JL. Identification and func- tional characterization of neo-poly(A) polymerase, an RNA processing enzyme overexpressed in human tumors. Mol Cell Biol. American Society for Microbiology (ASM); 2001;21: 5614–23. doi:10.1128/MCB.21.16.5614-5623.2001 111. de Andrés-Aguayo L, Varas F, Graf T. Musashi 2 in hematopoiesis. Curr Opin Hematol. 2012;19: 268–272. doi:10.1097/MOH.0b013e328353c778 112. Kharas MG, Lengner CJ, Al-Shahrour F, Bullinger L, Ball B, Zaidi S, et al. Musashi-2 regulates normal hematopoiesis and promotes aggressive myeloid leukemia. Nat Med. NIH Public Ac- cess; 2010;16: 903–8. doi:10.1038/nm.2187 113. de Andrés-Aguayo L, Varas F, Kallin EM, Infante JF, Wurst W, Floss T, et al. Musashi 2 is a regu- lator of the HSC compartment identified by a retroviral insertion screen and knockout mice. Blood. 2011;118. 114. Hope KJ, Sauvageau G. Roles for MSI2 and PROX1 in hematopoietic stem cell activity. Curr Opin Hematol. 2011;18: 203–207. doi:10.1097/MOH.0b013e328347888a 115. Forster L, McCooke J, Bellgard M, Joske D, Finlayson J, Ghassemifar R. Differential gene ex- pression analysis in early and late erythroid progenitor cells in β-thalassaemia. Br J Haematol. 2015;170: 257–267. doi:10.1111/bjh.13432 116. Baou M, Norton JD, Murphy JJ. AU-rich RNA binding proteins in hematopoiesis and leukemo- genesis. Blood. 2011;118. 117. Sandler H, Kreth J, Timmers HTM, Stoecklin G. Not1 mediates recruitment of the deadenylase Caf1 to mRNAs targeted for degradation by tristetraprolin. Nucleic Acids Res. Oxford University Press; 2011;39: 4373–86. doi:10.1093/nar/gkr011 118. Vignudelli T, Selmi T, Martello A, Parenti S, Grande A, Gemelli C, et al. ZFP36L1 negatively regulates erythroid differentiation of CD34+ hematopoietic stem cells by interfering with the Stat5b pathway. Mol Biol Cell. American Society for Cell Biology; 2010;21: 3340–51. doi:10.1091/ mbc.E10-01-0040

38 Introduction

119. Zhang L, Prak L, Rayon-Estrada V, Thiru P, Flygare J, Lim B, et al. ZFP36L2 is required for self- renewal of early burst-forming unit erythroid progenitors. Nature. NIH Public Access; 2013;499: 92–6. doi:10.1038/nature12215 1 120. Lebedeva S, Jens M, Theil K, Schwanhäusser B, Selbach M, Landthaler M, et al. Transcriptome- wide Analysis of Regulatory Interactions of the RNA-Binding Protein HuR. Mol Cell. 2011;43: 340–352. doi:10.1016/j.molcel.2011.06.008 121. Mukherjee N, Corcoran DL, Nusbaum JD, Reid DW, Georgiev S, Hafner M, et al. Integrative Regulatory Mapping Indicates that the RNA-Binding Protein HuR Couples Pre-mRNA Process- ing and mRNA Stability. Mol Cell. 2011;43: 327–339. doi:10.1016/j.molcel.2011.06.007 122. Mazan-Mamczarz K, Hagner PR, Zhang Y, Dai B, Lehrmann E, Becker KG, et al. ATM regulates a DNA damage response posttranscriptional RNA operon in lymphocytes. Blood. 2011;117. 123. Paolini NA, MacInnes AW, von Lindern M, Paolini NA, MacInnes AW, Lindern M. Diamond- Blackfan Anaemia: From Genotype to Phenotype. eLS. Chichester, UK: John Wiley & Sons, Ltd; 2016. pp. 1–8. doi:10.1002/9780470015902.a0024471 124. Vlachos A, Ball S, Dahl N, Alter BP, Sheth S, Ramenghi U, et al. Diagnosing and treating Diamond Blackfan anaemia: results of an international clinical consensus conference. Br J Haematol. 2008;142: 859–876. doi:10.1111/j.1365-2141.2008.07269.x 125. Ellis SR, Gleizes P-E. Diamond Blackfan Anemia: Ribosomal Proteins Going Rogue. Semin Hematol. 2011;48: 89–96. doi:10.1053/j.seminhematol.2011.02.005 126. Boria I, Garelli E, Gazda HT, Aspesi A, Quarello P, Pavesi E, et al. The ribosomal basis of diamond-blackfan anemia: mutation and database update. Hum Mutat. 2010;31: 1269–1279. doi:10.1002/humu.21383 127. Dahl N, Draptchinskaia N, Gustavsson P, Andersson B, Pettersson M, Willig T-N, et al. The gene encoding ribosomal protein S19 is mutated in Diamond-Blackfan anaemia. Nat Genet. 1999;21: 169–175. doi:10.1038/5951 128. Danilova N, Gazda HT. Ribosomopathies: how a common root can cause a tree of pathologies. Dis Model Mech. 2015;8: 1013–26. doi:10.1242/dmm.020529 129. Farrar JE, Dahl N. Untangling the phenotypic heterogeneity of Diamond Blackfan anemia. Semin Hematol. 2011;48: 124–35. doi:10.1053/j.seminhematol.2011.02.003 130. Deisenroth C, Zhang Y. Ribosome biogenesis surveillance: probing the ribosomal protein- Mdm2-p53 pathway. Oncogene. 2010;29: 4253–4260. doi:10.1038/onc.2010.189 131. Sankaran VG, Ghazvinian R, Do R, Thiru P, Vergilio J-A, Beggs AH, et al. Exome sequencing identifies GATA1 mutations resulting in Diamond-Blackfan anemia. J Clin Invest. American Society for Clinical Investigation; 2012;122: 2439–43. doi:10.1172/JCI63597 132. Parrella S, Aspesi A, Quarello P, Garelli E, Pavesi E, Carando A, et al. Loss of GATA-1 full length as a cause of Diamond-Blackfan anemia phenotype. Pediatr Blood Cancer. Europe PMC Funders; 2014;61: 1319–21. doi:10.1002/pbc.24944 133. Ludwig LS, Gazda HT, Eng JC, Eichhorn SW, Thiru P, Ghazvinian R, et al. Altered translation of GATA1 in Diamond-Blackfan anemia. Nat Med. Nature Publishing Group; 2014; doi:10.1038/ nm.3557 134. Amanatiadou EP, Papadopoulos GL, Strouboulis J, Vizirianakis IS. GATA1 and PU.1 Bind to Ri- bosomal Protein Genes in Erythroid Cells: Implications for Ribosomopathies. Simos G, editor. PLoS One. 2015;10: e0140077. doi:10.1371/journal.pone.0140077 135. Huang JN, Shimamura A. Clinical spectrum and molecular pathophysiology of Shwachman- Diamond syndrome. Curr Opin Hematol. NIH Public Access; 2011;18: 30–5. doi:10.1097/ MOH.0b013e32834114a5

39 Chapter 1

136. Boocock GRB, Morrison JA, Popovic M, Richards N, Ellis L, Durie PR, et al. Mutations in SBDS are associated with Shwachman–Diamond syndrome. Nat Genet. 2002;33: 97–101. doi:10.1038/ ng1062 137. Boocock GRB, Marit MR, Rommens JM. Phylogeny, sequence conservation, and functional complementation of the SBDS protein family. Genomics. 2006;87: 758–771. doi:10.1016/j. ygeno.2006.01.010 138. Savchenko A, Krogan N, Cort JR, Evdokimova E, Lew JM, Yee AA, et al. The Shwachman-Bodian- Diamond Syndrome Protein Family Is Involved in RNA Metabolism. J Biol Chem. 2005;280: 19213–19220. doi:10.1074/jbc.M414421200 139. Menne TF, Goyenechea B, Sánchez-Puig N, Wong CC, Tonkin LM, Ancliff PJ, et al. The Shwa- chman-Bodian-Diamond syndrome protein mediates translational activation of ribosomes in yeast. Nat Genet. 2007;39: 486–495. doi:10.1038/ng1994 140. Austin KM, Leary RJ, Shimamura A. The Shwachman-Diamond SBDS protein localizes to the nucleolus. Blood. 2005;106: 1253–1258. doi:10.1182/blood-2005-02-0807 141. Ganapathi KA, Austin KM, Lee C-S, Dias A, Malsch MM, Reed R, et al. The human Shwach- man-Diamond syndrome protein, SBDS, associates with ribosomal RNA. Blood. 2007;110: 1458–1465. doi:10.1182/blood-2007-02-075184 142. Moore JB, Farrar JE, Arceci RJ, Liu JM, Ellis SR. Distinct ribosome maturation defects in yeast models of Diamond-Blackfan anemia and Shwachman-Diamond syndrome. Haematologica. 2010;95: 57–64. doi:10.3324/haematol.2009.012450 143. Kirwan M, Dokal I. Dyskeratosis congenita: a genetic disorder of many faces. Clin Genet. 2007;73: 103–112. doi:10.1111/j.1399-0004.2007.00923.x 144. Drachtman RA, Alter BP. Dyskeratosis congenita: clinical and genetic heterogeneity. Report of a new case and review of the literature. Am J Pediatr Hematol Oncol. 1992;14: 297–304. 145. Heiss NS, Knight SW, Vulliamy TJ, Klauck SM, Wiemann S, Mason PJ, et al. X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions. Nat Genet. 1998;19: 32–8. doi:10.1038/ng0598-32 146. Vulliamy T, Marrone A, Goldman F, Dearlove A, Bessler M, Mason PJ, et al. The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita. Nature. 2001;413: 432–435. doi:10.1038/35096585 147. Armanios M, Chen J-L, Chang Y-PC, Brodsky RA, Hawkins A, Griffin CA, et al. Haploinsuf- ficiency of telomerase reverse transcriptase leads to anticipation in autosomal dominant dys- keratosis congenita. Proc Natl Acad Sci. 2005;102: 15960–15964. doi:10.1073/pnas.0508124102 148. Vulliamy TJ, Walne A, Baskaradas A, Mason PJ, Marrone A, Dokal I. Mutations in the reverse transcriptase component of telomerase (TERT) in patients with bone marrow failure. Blood Cells, Mol Dis. 2005;34: 257–263. doi:10.1016/j.bcmd.2004.12.008 149. Luzzatto L, Karadimitris A. Dyskeratosis and ribosomal rebellion. Nat Genet. 1998;19: 6–7. doi:10.1038/ng0598-6 150. Ruggero D, Grisendi S, Piazza F, Rego E, Mari F, Rao PH, et al. Dyskeratosis congenita and can- cer in mice deficient in ribosomal RNA modification. Science. 2003;299: 259–62. doi:10.1126/ science.1079447 151. Mochizuki Y, He J, Kulkarni S, Bessler M, Mason PJ. Mouse dyskerin mutations affect accumu- lation of telomerase RNA and small nucleolar RNA, telomerase activity, and ribosomal RNA processing. Proc Natl Acad Sci U S A. 2004;101: 10756–61. doi:10.1073/pnas.0402560101

40 Introduction

152. Triqueneaux G, Velten M, Franzon P, Dautry F, Jacquemin-Sablon H. RNA binding specificity of Unr, a protein with five cold shock domains. Nucleic Acids Res. Oxford University Press; 1999;27: 1926–34. 1 153. Jacquemin-Sablon H, Triqueneaux G, Deschamps S, le Maire M, Doniger J, Dautry F. Nucleic acid binding and intracellular localization of unr, a protein with five cold shock domains. Nucleic Acids Res. Oxford University Press; 1994;22: 2643–50. 154. Gebauer F, Corona DF, Preiss T, Becker PB, Hentze MW. Translational control of dosage com- pensation in Drosophila by Sex-lethal: cooperative silencing via the 5’ and 3’ UTRs of msl-2 mRNA is independent of the poly(A) tail. EMBO J. European Molecular Biology Organization; 1999;18: 6146–54. doi:10.1093/emboj/18.21.6146 155. Abaza I, Coll O, Patalano S, Gebauer F. Drosophila UNR is required for translational repression of male-specific lethal 2 mRNA during regulation of X-chromosome dosage compensation. Genes Dev. Cold Spring Harbor Laboratory Press; 2006;20: 380–9. doi:10.1101/gad.371906 156. Abaza I, Gebauer F. Functional domains of Drosophila UNR in translational control. RNA. Cold Spring Harbor Laboratory Press; 2008;14: 482–90. doi:10.1261/rna.802908 157. Duncan K, Grskovic M, Strein C, Beckmann K, Niggeweg R, Abaza I, et al. Sex-lethal imparts a sex-specific function to UNR by recruiting it to the msl-2 mRNA 3’ UTR: translational repression for dosage compensation. Genes Dev. Cold Spring Harbor Laboratory Press; 2006;20: 368–79. doi:10.1101/gad.371406 158. Grskovic M, Hentze MW, Gebauer F. A co-repressor assembly nucleated by Sex-lethal in the 3’UTR mediates translational control of Drosophila msl-2 mRNA. EMBO J. European Molecu- lar Biology Organization; 2003;22: 5571–81. doi:10.1093/emboj/cdg539 159. Duncan KE, Strein C, Hentze MW. The SXL-UNR Corepressor Complex Uses a PABP-Mediated Mechanism to Inhibit Ribosome Recruitment to msl-2 mRNA. Mol Cell. 2009;36: 571–582. doi:10.1016/j.molcel.2009.09.042 160. Ray S, Anderson EC. Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction. Sci Rep. Nature Publishing Group; 2016;6: 22461. doi:10.1038/srep22461 161. Boussadia O, Niepmann M, Créancier L, Prats A-C, Dautry F, Jacquemin-Sablon H. Unr is re- quired in vivo for efficient initiation of translation from the internal ribosome entry sites of both rhinovirus and poliovirus. J Virol. American Society for Microbiology (ASM); 2003;77: 3353–9. doi:10.1128/jvi.77.6.3353-3359.2003 162. Brown EC, Jackson RJ. All five cold-shock domains of unr (upstream of N-ras) are required for stimulation of human rhinovirus RNA translation. J Gen Virol. Microbiology Society; 2004;85: 2279–2287. doi:10.1099/vir.0.80045-0 163. Schepens B, Tinton SA, Bruynooghe Y, Parthoens E, Haegman M, Beyaert R, et al. A role for hnRNP C1/C2 and Unr in internal initiation of translation during mitosis. EMBO J. European Molecular Biology Organization; 2007;26: 158–69. doi:10.1038/sj.emboj.7601468 164. Dormoy-Raclet V, Markovits J, Jacquemin-Sablon A, Jacquemin-Sablon H. Regulation of Unr expression by 5’- and 3’-untranslated regions of its mRNA through modulation of stability and IRES mediated translation. RNA Biol. 2005;2: e27-35. doi:10.4161/rna.2.3.2203 165. Petretti C, Savoian M, Montembault E, Glover DM, Prigent C, Giet R. The PITSLRE/CDK11p58 protein kinase promotes centrosome maturation and bipolar spindle formation. EMBO Rep. 2006;7: 418–24. doi:10.1038/sj.embor.7400639

41 Chapter 1

166. Mitchell SA, Brown EC, Coldwell MJ, Jackson RJ, Willis AE. Protein Factor Requirements of the Apaf-1 Internal Ribosome Entry Segment: Roles of Polypyrimidine Tract Binding Protein and upstream of N-ras. Mol Cell Biol. 2001;21: 3364–3374. doi:10.1128/MCB.21.10.3364-3374.2001 167. Chang TC, Yamashita A, Chen CYA, Yamashita Y, Zhu W, Durdan S, et al. UNR, a new partner of poly(A)-binding protein, plays a key role in translationally coupled mRNA turnover mediated by the c-fos major coding-region determinant. Genes Dev. Cold Spring Harbor Laboratory Press; 2004;18: 2010–2023. doi:10.1101/gad.1219104 168. Sonenberg N, Hinnebusch AG. Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell. 2009;136: 731–45. doi:10.1016/j.cell.2009.01.042 169. Rhoads RE. eIF4E: new family members, new binding partners, new roles. J Biol Chem. 2009;284: 16711–5. doi:10.1074/jbc.R900002200 170. Kamenska A, Simpson C, Standart N. eIF4E-binding proteins: new factors, new locations, new roles. Biochem Soc Trans. 2014;42: 1238–45. doi:10.1042/BST20140063 171. Minshall N, Reiter MH, Weil D, Standart N. CPEB interacts with an ovary-specific eIF4E and 4E-T in early Xenopus oocytes. J Biol Chem. 2007;282: 37389–401. doi:10.1074/jbc.M704629200 172. Kamenska A, Lu W-T, Kubacka D, Broomhead H, Minshall N, Bushell M, et al. Human 4E-T represses translation of bound mRNAs and enhances microRNA-mediated silencing. Nucleic Acids Res. 2014;42: 3298–313. doi:10.1093/nar/gkt1265 173. Ferraiuolo MA, Basak S, Dostie J, Murray EL, Schoenberg DR, Sonenberg N. A role for the eIF4E-binding protein 4E-T in P-body formation and mRNA decay. J Cell Biol. 2005;170: 913–24. doi:10.1083/jcb.200504039 174. Chang S-H, Elemento O, Zhang J, Zhuang ZW, Simons M, Hla T. ELAVL1 regulates alternative splicing of eIF4E transporter to promote postnatal angiogenesis. Proc Natl Acad Sci U S A. National Academy of Sciences; 2014;111: 18309–14. doi:10.1073/pnas.1412172111 175. Nishimura T, Padamsi Z, Fakim H, Milette S, Dunham WH, Gingras A-C, et al. The eIF4E- Binding Protein 4E-T Is a Component of the mRNA Decay Machinery that Bridges the 5’ and 3’ Termini of Target mRNAs. Cell Rep. 2015;11: 1425–36. doi:10.1016/j.celrep.2015.04.065 176. Eulalio A, Behm-Ansmant I, Izaurralde E. P bodies: at the crossroads of post-transcriptional pathways. Nat Rev Mol Cell Biol. 2007;8: 9–22. doi:10.1038/nrm2080 177. Parker R, Sheth U. P bodies and the control of mRNA translation and degradation. Mol Cell. 2007;25: 635–46. doi:10.1016/j.molcel.2007.02.011 178. Kamenska A, Simpson C, Vindry C, Broomhead H, Bénard M, Ernoult-Lange M, et al. The DDX6-4E-T interaction mediates translational repression and P-body assembly. Nucleic Acids Res. Oxford University Press; 2016;44: 6318–34. doi:10.1093/nar/gkw565

42