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Control of mRNA translation in erythroid cells

Paolini, N.A.

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Citation for published version (APA):Paolini, N. A. (2018). Control of mRNA translation in erythroid cells.

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Download date:23 Jul 2021

General Introduction and Scope of this Thesis

Chapter 1

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Erythropoiesis

Erythropoiesis is needed to replenish erythroid cells. Hematopoiesis takes place in the bone marrow and is the process in which new blood cell lineages are formed from Hematopoietic Stem Cells (HSCs). Several models of hematopoiesis have been proposed, but the common view is that adult mammalian HSCs give rise to multipotent progenitors that are able to expand and to differentiate into the various blood cell lineages to replenish peripheral blood with mature, functional blood cells.1–4 Erythropoiesis is the branch of hematopoiesis, in which erythrocytes are produced.5,6 Erythrocytes are red blood cells that transport oxygen to tissues in the body and remove carbon dioxide from these tissues through hemoglobin.7 Hemoglobin consists of 4 globin chains each bound to an Fe2+-containing heme molecule. Two globin chains are encoded by the α-globin locus, two from the β-globin locus. The human β-globin locus contains ε-, γ- and β-globin genes that are subsequently expressed during embryonic development.8,9

The earliest cell with restricted erythroid potential is the megakaryocyte-erythroid progenitor (MEP). MEPs commit towards the erythroid lineage and generate erythroid progenitors that gradually mature while expanding. The most immature erythroid progenitors have BFU-E potential (erythroid burst-forming units), as cells mature they form CFU-Es (erythroid colony-forming unit). The pro-erythroblast is the first stage in which an erythroblast can be morphologically discerned. Pro-erythroblasts differentiate into basophilic erythroblasts, polychromatic and orthochromatic erythroblasts and ultimately into mature erythrocytes.5,6 Erythroid progenitors produce on average 1011 erythrocytes per day. Maintaining a tight balance between expansion and differentiation is important to prevent anemia (too few) or increased risk for stroke (too many). Canonically, the cytokine erythropoietin (EPO) stimulates erythroid expansion and maturation in the bone marrow. However, other growth factors like Stem Cell Factor (SCF) and glucocorticoids are needed for expansion of the erythroid compartment, whereas differentiation is induced by EPO.10,11 During terminal erythropoiesis, the cells produce hemoglobin, that yields the characteristic red color, and they expel their nucleus.12 Enucleation yields a larger surface for optimal expression of hemoglobin molecules, and increases cell flexibility required to pass through tissue capillaries. Hematopoietic lineage commitment is canonically governed by transcription factors, that bind DNA to regulate a lineage specific gene expression profile.5,6

Transcription factors are the canonical drivers of erythropoiesis. Master transcriptional regulators of erythropoiesis include the transcription factors Pu.1, Gata1 and Klf1. Elevated levels of Pu.1 increase the expression of myeloid specific receptors and cytokines, which pushes multipotent progenitors towards the myeloid lineage.13 Gata1 and Klf1 are needed at later stages to commit towards erythropoiesis.5,14

Gata1 expression increases from the MEP stage towards the proerythroblast stage.15 Loss of Gata1 in embryonic stem cells in mice causes death between embryonal day E10.5 and E11.5, due to a lack of embryonic mature erythrocytes.16 The loss of Gata1 also impairs differentiation of in vitro cultured hematopoietic cells, as these cells are not able to differentiate beyond the proerythroblast stage.17 These results may be explained by the pro-survival role of Gata1 in erythropoiesis. A well-known transcriptional target of Gata1 is the EPO receptor (EpoR).18 EPO signaling is needed for survival of erythroid cells, as erythroid precursors deprived of EPO in vitro go into apoptosis.19 Gata1 and EpoR signaling result in the upregulation of the anti-apoptotic Bcl-xL, that enables red blood cell survival during terminal differentiation.20 During erythropoiesis, Gata1 induces the expression of α- and β-globins,21 which is needed to produce hemoglobin.

Next to Gata1, Klf1 is also essential during terminal erythropoiesis by regulating β-globin gene expression.22 Disruption of the Klf1 gene in mice results in small and pale fetal livers at day E12.5 of embryonic development, which leads to their death at day E14. Klf1 heterozygous fetal livers contained normal levels of all erythroid stages.23 Fetal hemoglobin combines 2 α-globin chains with 2 γ-globin chains expressed from the β-globin locus. In humans, KLF-1 is involved in the regulation of fetal to adult hemoglobin switching by directly binding to the β-globin locus and activating the transcription of the β-globin gene.24,25 In addition, KLF-1 suppresses γ-globin expression by directly activating transcription of BCL11A .26,27 BCL11A was characterized as a major repressor of the γ-globin genes, shifting the enhancer

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at the locus control region to the β-globin genes.28 Individuals with a haploinsufficient mutation in the KLF-1 gene, that impairs DNA-binding, have increased levels of fetal hemoglobin and have deregulated expression of known KLF-1 target genes.26 Additionally, in mature erythroid cells, KLF-1 regulates the expression of genes involved in heme synthesis and membrane stability.29

Although transcriptional regulators are considered as the canonical drivers of erythropoiesis, additional evidence has shown that mRNA translational control also plays a role in the regulation of erythropoiesis, particularly in response to environmental factors. First, growth factors control translation of mRNAs with long structured 5’UTRs in erythroid cells. Second, the loss of ribosomes particularly affects the erythroid compartment. Third, the balance of heme and globin needs to be tightly balanced. Iron deficiency disrupts this balance, which leads to translation of specific mRNAs that protect erythroid cells. These topics will be discussed in this thesis.

Control of mRNA translation

mRNA translation imposes another level of gene expression control. The central dogma of biology is the transcription of RNA from DNA, and the translation of messenger RNA (mRNA) into a peptide sequence.30 Translation is carried out by ribosomes that scan and translate mRNA triplet codons into amino acids. mRNAs consist of three regions: a 5’ untranslated region (UTR), a coding sequence (CDS) that codes for a protein, and a 3’UTR. Translation can be divided in initiation, elongation and termination. Translation initiation control involves multiple translation initiation factors (eIFs), and facilitates the recognition of start codons (discussed below). Once a start codon is encountered, the ribosomes translate mRNAs and elongate the peptide sequence. Elongation control is modulated through phosphorylation of elongation factor eEF2, which leads to inactivation of its function and suppression of translation during mitosis.31,32 Termination occurs upon encountering a stop codon, where termination factors control the dissociation of ribosomes from mRNAs.33 Translational control at the 3’ end involves microRNAs (miRNAs) or other RNA binding proteins.34 miRNAs are small non-coding RNA molecules (~22 nt) that can bind complementary seed sequences located commonly on the 3’UTR of mRNAs.35 miRNA-bound mRNAs are then targeted for degradation at RNA-induced silencing complexes or their translation is impaired. Therefore, miRNAs negatively affect gene expression.36,37 In addition, some RNA-binding proteins can bind to regions on the 3’UTR. For example, some RNA-binding proteins can bind AU-rich elements on the 3’ region of specific cytokine mRNAs to regulate transcript stability in T-cells and thereby modulate the immune response.38 In addition, protein kinase C (PKC) signaling is also activated during T-cell activation, and PKC can enhance mTOR (mammalian target of rapamycin) signaling and thereby cap-dependent translation.39,40 The mTOR signaling pathway is discussed further below.

Cap-dependent mRNA translation. Translational control also occurs at the 5’ end, which involves ribosome recruitment in a cap-dependent and cap-independent mechanism, and which may involve RNA structures recognized by RNA binding proteins and upstream open reading frames (uORFs).41 In this thesis, we will focus on translational control from the 5’ end of mRNAs, which is currently considered to be the most complex form of translation control. Translation initiation is the rate limiting phase and starts with binding of eIF4E at the cap structure of mRNAs present at the 5’ end. The cap is a structure that consists of a 7-methylguanosine bound to the 5’ mRNA end by a triphosphate bridge. The 5’ cap is, besides the initiation of translation, also involved in mRNA stability, nuclear export and 5’ proximal intron excision.42 Translation initiation factors and ribosomal subunits are involved in mRNA start codon scanning and subsequent translation initiation and ensure start codon translation fidelity.

In this process, the initiation complex eIF4F binds at the cap of the 5’UTR. eIF4F is a heterotrimeric protein consisting of eIF4E, eIF4G and eIF4A.43–45 eIF4F binds the cap structure through eIF4E, eIF4A is the helicase that unwinds RNA secondary structure and eIF4G facilitates recruitment of the 40S small

Appendix chapter. We investigated how mRNA stability, transcription and translation efficiency in CD8+ cytotoxic T-cells contributed to the production of the cytokines TNF-α, IFN- γ and IL-2. We did this by investigating relative polysome recruitment and RNA stability of these cytokines in resting and activated T-cells.

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subunit of the ribosome through binding eIF3. The 40S ribosomal subunit, in turn, associates with the GTPase eIF2 that is charged with GTP and carries a methionine-loaded initiator tRNA (tRNAi

met). This complex is also known as the 43S preinitiation complex (PIC) that scans mRNAs for start codons (AUGs) in an optimal Kozak sequence context.46 The PIC is assembled with the help of eIFs 1, 1A, 3 and 5.47 Recognition of AUGs in an optimal Kozak sequence is thought to be mediated by control of eIF2 GTPase activity through eIF1 and eIF5. Start codon stringency is dependent on their levels; high levels of eIF1 results in more stringent AUG recognition from optimal Kozak sequences, whereas low levels of eIF1 or high levels of eIF5 leads to recognition of start codons in a poor Kozak context as well.48,49 The recognition of a start codon by the 43S PIC will lead to dislocation of eIF1 from the complex and the loss of a phosphate from eIF2, and subsequent release of eIF2-GDP and an empty tRNAi from the scanning complex.50 In addition, start codon recognition strengthens the interaction between eIF1A and eIF5 in the 43S PIC, which may be needed for 43S conformational change from an open scanning state to a closed translation starting state and for ensuring start codon recognition fidelity.51

Recognition of a start codon also leads to recruitment of the 60S large subunit of the ribosome, through eIF5B. The formation of an 80S ribosome is then mediated by eIF5 after eIF2 GTP hydrolysis.52 Which is followed by unloading of the methionine to start formation of the peptide sequence. After completion of translation initiation, the 80S ribosome will engage in translation elongation to complete mRNA translation until encountering a stop codon, which will lead to translation termination.43 Reinitiation is facilitated by the interaction of polyA binding protein (PABP) with eIF4F, which enables a closed mRNA loop by bringing the 3’ mRNA end towards the 5’ end.53

eIF2 controls translation of mRNAs with upstream open reading frames. Free eIF2-GDP is inactive and cannot be reloaded into a 40S scanning complex. Therefore, eIF2-GDP is recycled to eIF2-GTP by the guanine nucleotide exchange factor eIF2B, to generate active eIF2. eIF2-GTP reassociates with a tRNAi

met and is loaded into the 40S subunit to start a new round of translation. Translation is only initiated when eIF2, bound to tRNAi

met, is present in the 43S preinitiation complex. eIF2 is composed of three subunits: an α, β and γ subunit. GTP and tRNAi

met are bound by the γ subunit, whereas the α and β subunits stabilize strong binding to the tRNAi

met.54 The serine at position 51 of the α subunit of eIF2 can be phosphorylated by eIF2α kinases PERK,

PKR, HRI and GCN2, that are activated during endoplasmic reticulum (ER) stress, viral infection, heme deficiency or amino acid deprivation, respectively (Fig. 1).55 This phosphorylation prevents binding of eIF2B to eIF2 and hence eIF2 will remain inactive and cannot be reloaded into the scanning complex.56 The result is a block in protein synthesis to prevent proteotoxicity. Proteotoxic stress is a result of build-up of misfolded proteins, accumulation of viral proteins or aggregation of globin protein precipitates. These aggregated proteins can damage the cell and therefore the cell stops its protein synthesis to conserve nutrients, prevent toxicity of misfolded or aggregated proteins, and goes into survival mode.55

Although the 5’ end of mRNAs have been defined as an untranslated region, a large number of mRNAs contain start codons on their 5’UTR that can be recognized by the scanning complex and translated by 80S ribosomes.57 Translation of such uORFs may inhibit translation of the downstream CDS. Therefore, these uORFs impose another layer of translational control. Inhibition of downstream CDSs may occur due to uORFs overlapping the CDS AUG, leaky scanning of uORFs in a suboptimal Kozak sequence, that allows bypass of ribosomes during stress and ribosome stalling or dissociation.58

The best studied example of uORF-mediated translational control is Atf4, in which uORFs impair translation of the CDS. The Atf4 mRNA contains two uORFs on its 5’UTR that allow selective translation of the CDS during proteotoxic stress.59 The first uORF is 3 codons long and is always recognized and translated both in normal and stress conditions. The second uORF is ~60 codons long and is positioned ~90 nucleotides downstream of uORF1. The Atf4 uORF2 overlaps the Atf4 CDS and inhibits translation of the Atf4 ORF; in non-stressed cells Atf4 protein will therefore not be produced due to translation of the inhibitory second uORF. During stress however, eIF2 is phosphorylated and hence less available, which leads to less active eIF2 molecules present in the cell. It will take longer for the scanning complex to be assembled. The distance between both uORFs is such that eIF2-tRNAi

met will not be loaded in time into the scanning complex before reaching the start codon of uORF2 but upon reaching the Atf4 start codon.

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This process results in skipping of the second uORF and recognition of the Atf4 CDS and production of the Atf4 protein (Fig. 2).59 Cells have evolved this mechanism to rapidly adapt to proteotoxic stress, in which the mRNA is already present and ribosomes switch translation to a different part of the mRNA. This mechanism enables the bypass of gene transcription, which can be more time consuming, to enable translation of stress responsive transcripts.

Figure 1. Induction of the integrated stress response. Endoplasmic reticulum stress, ROS and heme deficiency, nutrient starvation and viral infection are sensed by the eIF2 kinases PERK, HRI, GCN2 and PKR, respectively. These kinases phosphorylate eIF2, which leads to a reduction in global protein synthesis, but also to specific translation of, for example, Atf4 that regulates the transcription of downstream targets like Chop and Gadd34 to adapt to proteotoxic stress. Ddit3 induces apoptosis when the damage persists. Gadd34 and Ppp1r15b recruit phosphorylated eIF2 to the phosphatase PPase1, that then dephosphorylates eIF2, to alleviate the ISR.

An integrated stress response protects cells from proteotoxic stress. Atf4 is a transcription factor that is part of an integrated stress response (ISR) involved in proteotoxic stress adaptation. The ISR consists of two major events, the upregulation of the CCAAT-enhancer-binding protein homologous protein (CHOP, also called Ddit3 or Gadd153) which induces apoptosis, and the upregulation of Gadd34 (also called Ppp1r15a) that alleviates stress (Fig. 1).58,60 On the one hand, prolonged expression of Ddit3 activates apoptosis, at least in part, by downregulation of the anti-apoptotic protein Bcl-2.61,62 Additionally, Ddit3 induces cell death by inducing the expression of the pro-apoptotic transcription factor Atf5.63 On the other hand, Ddit3 is also associated with increased expression of Gadd34, which makes Ddit3 also a pro-survival factor.64 In line with this, there is evidence that both Ddit3 and Atf4 positively regulate the expression of genes involved in autophagy that are able to protect cells during proteotoxic stress.65 Gadd34 is involved in the dephosphorylation of eIF2 to induce cell recovery after stress.66 Gadd34 and Ppp1r15b are subunits of the eIF2 phosphatase PPase1. The subunits are needed for recruitment of phosphorylated eIF2 to PPase1, that then dephosphorylates eIF2 to enable new binding with the tRNAi

met to start new rounds of translation (Fig. 1).66,67 Therefore, it is thought that cells go into apoptosis or survive depending on the severity and persistence of the stress.58,60

Ddit3 is a direct target of Atf4 and is transcriptionally upregulated during proteotoxic stress. Gadd34 is also transcriptionally activated during proteotoxic stress. In addition, there is also evidence that Ddit3 and Gadd34 are translationally controlled by uORFs during stress Whereas Atf4 has two uORFs of which uORF2 overlaps the Atf4 CDS, Ddit3 has one conserved ~105 nt long uORF, ~26 nt upstream of the start codon. The start codon of the uORF is in a poor initiation context, whereas the Ddit3 start codon has an optimal Kozak sequence. Therefore, in the absence of stress, ribosomes recognize and translate the Ddit3 uORF, whereas reduced eIF2 availability during stress enables the scanning complex to bypass the uORF and initiate Ddit3 CDS translation (Fig. 3A).68 Translational control of Gadd34 is more complex and involves two overlapping uORFs. uORF1 lays in a poor Kozak context and is bypassed both in non-stressed and stressed cells. The inhibitory uORF2 is translated during high eIF2 availability, and ribosomes are released from the mRNA at the uORF2 stop codon, which prevents translation of the downstream Gadd34 ORF. Because uORF2 also has a poor Kozak sequence, this uORF is bypassed during stress, which leads to Gadd34 protein production (Fig. 3B).58,69,70

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Ribosomes may bypass some parts of the 5’UTR. The 5’UTR of some mRNAs can contain secondary structures that are unwound by the scanning ribosome. However, some highly structured stem loops contain uORFs that can inhibit translation of the downstream CDS. These stem loops are often preceded by a short uORF that terminates a couple of codons from the base of the stem loop. The 3’ end of the stem loop base contains a pyrimidine rich region. Ribosomes are able to bypass these secondary structures, this mechanism is called ribosome shunting, which is still poorly understood.71 Ribosome shunting has been described for the 35S RNA of the Cauliflower mosaic virus (CaMV) and Rice tungro bacilliform virus (RTBV),72,73 but also for the mammalian mRNA cIAP2, whose 5’ secondary structure is bypassed by ribosomes during moderate levels of ROS.74

IRES dependent mRNA translation. Not all mRNAs depend on cap-dependent translation and eIFs and the formation of closed loop mRNA. Some mRNAs bypass translational block during eIF2 phosphorylation due to complex secondary structures on their 5’UTR that can serve as internal ribosome entry sites (IRESs).75 IRES-mediated translation is found in viral RNA. This mechanism serves the compact viral genome because multiple cistrons can be translated from a single mRNA. Because viruses shut down cap-dependent translation by cleavage of eIF4G, the IRES is used by viruses to bypass the translational block imposed by host cells.76,77 However, IRES structures are also present in some mammalian mRNAs that play a role in stress adaptation, cell death, or growth and differentiation.78–80 For instance, IRES dependent translation of pro-apoptotic transcripts still enables virus infected cells to enter apoptosis to stop the viral infection. IRES-trans activating factors (ITAFs) are thought to play a role in recruiting ribosomes to IRES structures. Some of these ITAFs include Poly-pyrimidine Tract Binding Protein (PTB) or Cold Shock Domain Containing E1 (Csde1).79,81–83

Translational control of erythropoiesis

Growth factors activate the mTOR pathway to release eIF4E. Expansion of erythroblasts in the mouse spleen and human bone marrow under conditions that induce erythropoiesis and expansion of the erythroid progenitor compartment in vitro, are both dependent on the activation of the SCF receptor cKit by SCF.10,84 Activation of the cKit tyrosine kinase induces the PI3K/mTOR signaling cascade, which leads to recruitment of PI3K, that produces PIP3.85 PDK1 and PKB home to PIP3 upon which PKB is phosphorylated by PDK1. Activated PKB then phosphorylates Tsc1 and Tsc2, that leads to the release of Rheb, a GTPase that activates the kinase mTOR.86–88 Next, activated mTOR phosphorylates S6 kinase (S6K) and 4E-Binding Protein 1 (4E-BP1), that sequesters and inactivates eIF4E.89 Activated S6 kinase then phosphorylates ribosomal protein S6, and hyperphosphorylated 4E-BP1 releases eIF4E to initiate cap-dependent mRNA translation and increased translation mRNAs with long structured 5’UTRs.86,89,90

Overexpression of eIF4E in mouse erythroblasts in vitro impaired their differentiation and promoted their expansion in the absence of SCF, showing that indeed SCF controls erythroid expansion through the release of eIF4E.85 In vitro stimulation of mouse erythroblasts with SCF plus EPO revealed selective

Figure 2. Mechanism of translation of Atf4. Atf4 has two uORFs that are translated when eIF2 is available. Ribosomes will skip the Atf4 start codon, because uORF2 overlaps the Atf4 CDS. During stress, uORF1 is recognized and translated, but uORF2 is skipped due to low eIF2 availability. Instead the Atf4 CDS will be translated, which will lead to production of Atf4 protein.

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Figure 3. Mechanism of translation of Ddit3, Gadd34 and Ppp1r15b. (A) Ddit3 has one uORF that is translated when eIF2 is available. During low eIF2 availability the scanning complex will bypass this uORF due to its poor Kozak context and instead the Ddit3 CDS will be translated. (B) Gadd34 has two overlapping uORFs. The scanning complex bypasses uORF1 both during stress and no stress. When eIF2 is available, uORF2 is translated, which will lead to release of ribosomes upstream of the CDS. During stress, also uORF2 is bypassed by the scanning complex and Gadd34 CDS is translated. (C) Ppp1r15b has two in-frame uORFs; uORF1 is thought to be bypassed both during stress and steady state. uORF2 is translated when eIF2 is available, whereas it is bypassed during eIF2 phosphorylation to enable translation of the CDS.

recruitment of 111 mRNAs towards polysomes, among which Igbp1, whereas total RNA remained unchanged. Igbp1 (also called α4) plays a role in the mTOR pathway by sequestering the serine/threonine phosphatase PP2A, which prevents dephosphorylation and inactivation of pS6K and 4E-BP1, thereby enhancing translation initiation. Therefore, Igbp1 is involved in a positive feedback loop in which SCF mediated activation of the mTOR pathway enhances Igbp1 protein synthesis, which further enhances eIF4E dependent translation.91 Overexpression of Igbp1 inhibited differentiation of murine erythroid progenitors and increased phosphorylation of S6K and 4E-BP1, further establishing the role of the mTOR pathway in controlling erythroid cell expansion.91

Iron regulates mRNA stability and translation in erythroid cells. Erythroid cells are dependent on iron homeostasis. Iron is essential for the production of heme, which is the oxygen-binding component of hemoglobin and is needed for hemoglobin synthesis. Epithelial cells of the duodenum take up dietary iron through the divalent metal transporter 1 (DMT1).92 Next, iron is transported into the

Chapter 3. To increase our understanding of how control of mRNA translation regulates erythropoiesis, we further investigated control of Igbp1 translation. We investigated how structural elements in the 5’UTR of Igbp1 were involved in translation of its mRNA.

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bloodstream through the iron exporter ferroportin and binds to soluble transferrin, which can be taken up by erythroid cells, through the transferrin receptor (Tfr1).93,94 Tfr1 is highly expressed in erythroid progenitors95. Deletion of TfR1 in mice led to anemia due to increased apoptosis in erythroid cells. TfR1 deficient mice contained lower iron in liver and spleen compared to wildtype mice.96 Once taken up by erythroid cells, iron will be bound by protoporfyrin IX to form heme. Protoporfyrin is synthesized through a series of enzymatic reactions, whereby erythroid-specific Alas2 (5’-Aminolevulinate Synthase 2) is highly regulated, and a rate limiting enzyme.93 Iron is also deposited in the cell in ferritin stores.97

Iron availability is monitored by iron regulatory proteins IRP1 and IRP2 that can sense intracellular iron. IRP1 and IRP2 are RNA-binding proteins that can bind iron-response elements (IREs). IREs are hairpin structures present on the 3’ and 5’ UTRs of some mRNAs.98 When cellular iron stores are low, IRP1 and IRP2 bind IREs in the 3’ UTR of TfR1 mRNAs to increase their stability, which leads to an increase in iron uptake.99 On the other hand ferritin, ferroportin and Alas2 protein synthesis is inhibited to reduce iron storage, efflux and heme synthesis.100,101 Ferritin and Alas2 contain IREs on their 5’UTR, which prevents interaction with the 43S PIC and subsequent start codon scanning, upon IRP binding.101 Interestingly, also EPO stimulation leads to an increase in Alas2 transcriptional and translational control in erythroid progenitors, by disrupting the interaction of IRP 1 and 2 with Alas2 mRNA.102 In iron-replete cells, IRP1 is inactive upon binding to an iron-sulfur molecule, whereas IRP2 is ubiquitinated and degraded in the proteasome.93

The integrated stress response is essential for normal erythropoiesis. Next to IRP1 and IRP2, another way that erythroid cells monitor heme levels, is through Heme-regulated Inhibitor (HRI), that is activated when heme levels drop.103 Heme, together with the globin protein chains, forms hemoglobin. It is important that cells carefully monitor the heme:globin ratio. A reduction in heme distorts the ratio of heme and globins and leads to the accumulation of globin protein aggregates that can damage the cell.103 To reduce globin production, HRI then phosphorylates eIF2 to block global protein synthesis and to induce the ISR (Fig. 1). On the other hand, excess iron can be oxidized and can lead to the generation of reactive oxygen species (ROS).104 It was found that ROS can also lead to activation of HRI and phosphorylation of eIF2.105 Therefore, also increased uptake of iron during erythropoiesis may activate the ISR. Indeed, components of the ISR like HRI, Atf4 and Ddit3 were upregulated in erythroid cells that were differentiated in vitro.106–108 A low iron diet led to ineffective erythropoiesis in HRI knockout mice and increased reactive oxygen species (ROS) were observed in fetal liver erythroblasts of HRI knockout mice, treated with arsenite in vitro.106 Therefore, the ISR is important in immature and more mature erythroblasts.

Several knockout mouse models have shown the importance of the feedback control of the ISR in erythropoiesis. Adult Atf4-/- mice are mildly anemic with pale hypoplastic fetal livers, and bone marrow-derived CFU-E and BFU-E colonies are small due to a reduction in erythroid cell numbers.109 Mice lacking the Atf4 target gene Gadd34 also displayed ineffective erythropoiesis with a reduction in mature erythrocytes and an increase in immature erythroid cells. In addition, red blood cells of these mice displayed an abnormal morphology, contained less hemoglobin and erythroid numbers recovered poorly during iron deficiency.110 Gadd34 and Ppp1r15b enhance the phosphatase activity of PPase1. Ppp1r15b-/- mice die perinatally with low hematocrit count, low red blood cell count, deformed erythrocyte shape and reduced number of late erythroid cells in fetal liver.111 Gadd34-/- mice have morphologically abnormal erythrocytes with decreased volume and reduced hemoglobin content and large spleens with increased number of erythroid progenitors.110 Rescue of Ppp1r15b-/- by mutating eIF2 Ser51 to Ala51, that cannot be phosphorylated during stress, restored red blood cell count. All these studies combined show the importance of the feedback loop of the ISR in controlling immature and mature erythropoiesis. Despite that the ISR and erythropoiesis are tightly linked, it is not known if there are erythroid-specific transcripts with uORFs that are responsive to proteotoxic stress. Interestingly, treating erythroid cells with Salubrinal, that prevents dephosphorylation of eIF2 by PPase1, led to an increase of fetal globin mRNA translation, which can be important for protection against oxidative stress.112 However, there are no annotated uORFs on the fetal globin mRNA, and therefore this transcript may be regulated by other factors than eIF2.

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Genome wide investigation of mRNA translation

A powerful tool to study mRNA translation. The enhanced expression of the Atf4, Gadd34, and Ddit3 upon eIF2 phosphorylation is in part controlled by uORFs in the 5’UTR of the transcripts encoding these proteins. Polysome profiling, the comparison between polysome-bound mRNA to free RNA, gives an indication of which mRNAs are preferentially translated in a particular cell.113 However, mRNAs with uORFs are always present in the polysome fraction. During stress, ribosomes are “shifted” from the inhibitory 5’ UTR of Atf4 mRNA to the CDS. Polysome profiling makes it difficult to discriminate whether a different part of an mRNA is translated. Therefore, an mRNA like Atf4 will always appear in the polysomal compartment, even though in unstressed cells this does not lead to Atf4 protein production. Recently, ribosome profiling (also called Ribo-seq or ribosome footprinting) was developed to overcome this challenge.

Ribosome profiling is a genome wide deep sequencing technique that allows for quantification of mRNA translation.114,115 To do this, translating ribosomes are stalled on the mRNA of living cells with translation-blocking drugs, like cycloheximide (CHX) and/or harringtonine (Ht). CHX stalls elongating ribosomes by binding to the E-site of the 60S subunit and can be used to study ribosome density along the whole mRNA strand.116 On the other hand, Ht stalls initiating ribosomes by binding to the A-site of free 60S subunits and inhibiting aminoacyl-tRNA binding to the ribosome, while elongating ribosomes run off the mRNA.117,118 With Ht treatment, 80S initiating ribosomes are formed but are stalled at start codons. Therefore, Ht can be used to identify translation initiation sites (TISs), while CHX is used to analyse elongating ribosomes. This treatment is followed by RNAse1 digestion, which breaks down single stranded RNA. Treating cell lysates with this enzyme results in digestion of polysomes into monosomes. Monosomes are then isolated, with for example, sucrose gradient separation. Ribosome footprints (RFPs) are the parts of mRNAs that are protected by the ribosome during this treatment, i.e. the parts of mRNAs that ribosomes were translating. These RFPs are then isolated, sequenced and aligned to the genome. Total RNA sequencing (RNA-seq) is performed in parallel to quantify total transcript abundance (Fig. 4).119

Figure 4. Schematic representation of the ribosome profiling protocol. For ribosome profiling, ribosomes are stalled on the mRNA and cell lysate is treated with RNAse1 to digest single stranded RNA. Resulting monosomes are isolated and ribosome footprints (RFPs) are isolated from denaturing gel. RFPs are then sequenced and aligned to the genome. In parallel, whole RNA or polyA-enriched mRNA is fragmented, sequenced and aligned on the genome. Together, both techniques measure how many ribosomes are present on an amount of mRNA (ribosome density).

Bioinformatics analysis of ribo reads and RNA reads yields several characteristic parameters of translation. First, an mRNA has three reading frames, a triplet of mRNA nucleotides codes for a particular amino acid, which causes ribosomes to translate mRNAs each three nucleotides (codons). Therefore triplet periodicity analysis allows for investigation of which frame is translated and whether ribosomal frame-shifting occurs on specific mRNAs.120,121 Second, efficiency of codon usage can be analyzed by the novel differential ribosome codon reading (diricore), by investigating ribosome build-up at a particular codon. Diricore was found to be important for detection of which amino acids are limiting for cancer

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cell growth. Accumulation of ribosomes at particular codons suggests that these amino acids are scarce in some cells and that ribosomes are stalled until the amino acid is available.122 Third, Ht can be used to investigate which start codons are used on specific mRNAs to identify alternative start codon usage or protein isoforms. Canonically, ribosomes recognize AUGs, but ribosome profiling has shown that CUG, UUG or GUG codons are also used widespread as TISs,115 consistent with results of previous studies that reported non-canonical start codon translation initiation of specific transcripts.123,124 Fourth, by comparing ribosome reads to RNA reads, it is possible to investigate which parts of mRNAs are being translated and it is possible to investigate translation efficiency or ribosome density, i.e. how many ribosomes were present on an amount of mRNA strands.115 Fifth, translation elongation rate can be calculated by treating cells with Ht and assessing ribosome run-off rate.115

Therefore, in theory, a transcript like Atf4 will have increased ribosome density in its CDS during proteotoxic stress with a reduction of ribosomes reads on the second uORF at least in the 5’UTR region. However, ribosome profiling studies have shown that some translation of the second uORF still occurs during eIF2 phosphorylation.125 In agreement with this, also expression of peptides produced from both Atf4 uORFs was observed during proteotoxic stress.126 Therefore, ribosome profiling is a powerful tool to investigate translation at the nucleotide level. To date, one study has been published on ribosome profiling of erythroid cells during terminal differentiation, where it was found that uORF translation is widespread and is used to dampen gene expression during erythropoiesis.127

Ribosome profiling reveals novel players in the integrated stress response. Ribosome profiling performed on HEK 293 cells treated with arsenite, that causes oxidative stress, showed preferential translation of novel mRNAs containing uORFs.125 These mRNAs included SLC35A4, C19orf48, HOXB2, IFRD1 (Tis7), PTP4A1, PCNXL4, PPP1R15B and UCP2 and can be involved in the ISR. During stress, global translation was reduced, however the CDS of these novel transcripts had an increased or unaffected ribosome density during stress, whereas the 5’UTR exhibited a reduction in ribosome density compared to their CDS.125 Interestingly, another group found similar targets having upregulated or unchanged ribosome density during Tm treatment of HEK cells.128

The mechanism of translational control of these novel transcripts was different than the Atf4 translational control. Whereas Atf4 has two uORFs, of which the second uORF overlaps the Atf4 CDS, the composition and the amount of uORFs of these novel transcripts was different. For example, the 5’UTR of SLC35A4 contains 11 uAUGs with one of them being conserved, however it is not yet known how these uORFs regulate translation of the SLC35A4 CDS. In contrast, the 5’UTR of Tis7 has a single 53 codons-long uORF 43 nucleotides upstream of the CDS, which is needed for translation reinitiation at the CDS and for mRNA stabilization during eIF2 phosphorylation.125,129 The uAUG is in a suboptimal sequence context and can be bypassed during stress, which is a similar mechanism to Ddit3 translational control (Fig. 3A). PPP1R15B has 2 in frame uORFs that are separated by 21 nucleotides, the second uORF is inhibitory and needed for reinitiation at the CDS during eIF2 inactivation (Fig. 3C).125 This study has shown that stress responsive transcripts can have different numbers, organization and sizes of uORFs, but all these uORFs lead to selective translation of the CDS during eIF2 phosphorylation. It is unknown why these transcripts have evolved different types of 5’UTRs. Elucidating what the exact roles of the uORFs are on these novel stress resistant mRNAs are, will be the topic of further research.

The biological function of these novel transcripts in proteotoxic stress adaptation is not known. SLC35A4 is a member of the SLC35 family of nucleotide sugar transporters and it is speculated that upregulation of this protein increases nutrient uptake during stress adaptation.58,130 Tis7 was linked before to being upregulated during hypoxic stress and muscle injury and is a transcription factor that upregulates the transcription of muscle specific genes.131,132 However, its role in the ISR is still unexplored. Therefore, further research is needed to investigate the biological roles of these transcripts

Chapter 4. To establish if and how translation of uORFs contributes to control of mRNA translation following eIF2 phosphorylation in erythroid progenitors, we used ribosome profiling and we analyzed both initiating and elongating ribosomes to investigate used translation initiation sites and ribosome density, respectively.

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during reduced eIF2 availability.

Loss of ribosomes particularly affects the red blood cell compartment. Translational control in erythroid cells also plays a role in Diamond-Blackfan anemia (DBA). DBA is caused by mutations in genes encoding ribosomal proteins (RPs), which leads to impaired ribosome biogenesis and a reduction of ribosome numbers.133,134 DBA is termed a ribosomopathy, a disease caused by lack of ribosomes, which leads to anemia.135 DBA patients also experience physical malformations, like abnormal thumbs, low nasal bridge, cleft lip/palate, or small skull, among others.136 It is still unknown why mostly red blood cells suffer from a mutation in ribosomal proteins, that are ubiquitously expressed in all cell types. Several mechanisms have been proposed: (i) The loss of ribosomes can lead to the activation of P53, that induces erythroid cell death;137 (ii) Selective reduction in polysome recruitment of specific transcripts like BAG1 and CSDE1, that are highly expressed in erythroid cells. The loss of these 2 proteins during DBA can also lead to impaired erythropoiesis;138 (iii) A reduction in globin synthesis may induce cell death due to build-up of toxic heme in erythroid cells;139 (iv) Ludwig et al reported reduced translation of GATA1 and subsequent disruption of GATA1 target genes, that prevents maturation of red blood cells.140 (v) O’Brien et al performed microarray analysis of RNA from erythroid cells from RP and from GATA1 mutant DBA patients, which showed that heme synthesis genes were upregulated and ribosomal machinery genes were downregulated, respectively.141 O’Brien et al showed that the expression of GATA1 genes were normal in DBA patients, opposed to Ludwig et al. This discrepancy may be due to Ludwig et al investigating immature cells, whereas O’Brien et al investigated more mature cells, where GATA1 is expressed.140,141 However, re-analysis of the microarray data from O’Brien et al, showed that RP and GATA1 mutant cells were composed a heterogeneous population and not a pure erythroblast population, which may have caused confounding. This re-analysis showed that expression of heme synthesis and translation machinery genes were not different when normalized for differentiation stage. However, these analyses also showed that GATA1 target genes were not different in DBA patients and controls, which contradicts previous study that showed downregulation of GATA1 target genes in DBA patients with haploinsufficient RP mutations.140,142

While we hardly understand why ribosomal haploinsufficiency causes DBA, it also becomes clear that not all mutations in ribosomal proteins cause DBA. Ribosomal protein mutations are also found in cancer or in patients with intellectual disability.143,144 For a more extensive discussion on ribosomopathies and DBA in particular, see chapter 2.

Scope of this thesis

Translation initiation control poses additional levels to regulate gene expression. First, the release of translation initiation factor (eIF) eIF4E mediated by growth factor signaling enables the translation of cap-bound mRNAs with long 5’ untranslated regions (5’UTR). Second, ribosomal proteins (RPs) are involved in ribosome biogenesis and play a role in modulation of ribosome function. Third, phosphorylation of eIF2, during proteotoxic stress, controls selective translation of mRNAs harboring upstream open reading frames (uORFs) on their 5’UTR. Whereas transcription factors have been recognized as the main drivers of erythropoiesis, translational control also plays a role in erythroid homeostasis. First, EPO and SCF signaling leads to release of eIF4E and subsequent recruitment of Igbp1 to polysomes. Second, mutations in genes coding for RPs can cause the ribosomopathy Diamond-Blackfan anemia (DBA) in

Chapter 5. Because the ISR and erythropoiesis are linked, we investigated the role of IFRD1/Tis7 in Tm-challenged erythroblasts. We did this by knocking down Tis7 in erythroblasts and investigating the phenotype upon knockdown. To investigate which targets were regulated by Tis7 we performed RNA sequencing.

Chapter 6. We investigated whether mutations in RPS23, observed in two individuals with developmental aberrations, affected the pathways involved in DBA. We investigated how this mutation impaired ribosome function.

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which erythroblasts exhibit increased apoptosis and altered mRNA translation. Third, eIF2 is inhibited during heme deficiency which leads to selective translation of transcripts involved in the integrated stress response (ISR). In this thesis we investigated the role of translational control in erythropoiesis and mRNA translation.

In chapter 3 we explored how structural elements in the 5’UTR of Igbp1 mRNA control Igbp1 CDS translation. We investigated which RNA-binding protein(s) interacts with these elements and if ribosomes are able to bypass certain parts of the Igbp1 5’UTR to reinitiate at the CDS. In chapter 4 we have investigated if there are other transcripts that are translationally controlled during eIF2 phosphorylation in erythroid cells and that are involved in erythroid cell survival. To do this we performed ribosome profiling in tunicamycin (Tm) treated erythroblasts. Tm inhibits N-glycosylation of proteins, which leads to ER stress and activation of PERK and subsequent eIF2 phosphorylation. In chapter 5 we show that Tis7 is essential for survival at the expansion phase and shed further light on which target genes may be regulated by Tis7 during erythroid expansion and proteotoxic stress. In chapter 6 we present a case of an 11-year-old Dutch patient with DBA-like dysmorphic features, like cleft-palate, small skull, abnormal thumbs, but also with a mild form of intellectual disorder. Exome sequencing revealed that the child had a novel mutation in the gene encoding RPS23. We investigated whether this mutation was pathogenic and whether it caused alterations in translation fidelity that gave further insight into the abnormal occurrence of this patient. However, the patient did not have impaired erythropoiesis, which further supports the notion that not all mutations in RP genes cause DBA, as is reviewed in chapter 2. Finally, in Appendix chapter we investigated how PKC orchestrates mRNA transcription, stability and translation of specific cytokines.

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