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Control of mRNA translation in erythroid cells

Paolini, N.A.

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

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

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Download date:01 Oct 2021 Chapter 2

Diamond-Blackfan Anemia: From to Phenotype

eLife Sciences (2016)

Nahuel A Paolini1, Alyson W MacInnes1,2, Marieke von Lindern1

1 Department of Hematopoiesis at Sanquin Research, and Landsteiner laboratory AMC/UvA, Amsterdam, The Netherlands. 2 Laboratory Genetic Metabolic Diseases AMC-UvA, Amsterdam, The Netherlands. Chapter 2

Abstract

Diamond Blackfan Anemia (DBA) is a congenital disorder that presents in the first year of life as severe anemia, and in several patients is coupled with developmental defects. DBA is a ribosomopathy because almost all known mutations or deletions occur in encoding ribosomal (RP) that impair biogenesis. However, atypical examples of patients carry mutations in the erythroid specific transcription factor GATA1. DBA is a rare disease that displays a high level of heterogeneity with respect to the affected RP and disease penetrance. The reduced availability of affects several cellular processes, including stabilisation of the tumour suppressor, and impaired mRNA translation. Registration of the genetic and phenotypic characteristics of DBA patients worldwide is neededto understand the relation between mutations, patient symptoms, and cellular processes that underlie 2 this pathophysiology. Introduction

Diamond Blackfan Anemia (DBA) is diagnosed when patients under 1 year of age present with severe red cell aplasia and all other blood cell lineages appear normal. Macrocytic anemia and reticulocytopenia imply that proliferation of precursors is impaired. Approximately 40% of the DBA patients display congenital anomalies like short stature, craniofacial aberrations, thumb abnormalities and cleft palate. More complete diagnostic criteria of DBA, and subsequent treatment have been reviewed,1 and are summarized on the website of the DBA foundation (dbafoundation.org). It was initially difficult to conceive that a specific disease such as DBA could be caused by haploinsufficiency of Ribosomal S19 (RPS19), a protein that is required in every cell.2 Subsequently, it became clear that the majority of DBA patients carries a mutation in one of several (RP) encoding genes.1 Concurrently, big advances were made in understanding the structure and function of the ribosome. In 2009 the Nobel Prize in Chemistry was awarded to Venkatraman Ramakrishnan, Thomas Steitz and Ada Yonath for their studies of the structure and function of the ribosome, which boosted further research on the role of ribosomes in normal physiology and in pathological conditions such as DBA. In this review, we will highlight the heterogeneity of the genotype and phenotype of DBA, and link the genotypic and phenotypic variation to the disease mechanisms.

Genetics of DBA

Mutations in RPS19 occur in 25% of DBA patients. Screens in other RP genes uncovered recurrent mutations or large deletions in at least 14 different RP genes. Two thirds of patients carry mutations or deletions resulting in haploinsufficiency of proteins that are part of the small ribosomal subunit (RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPS27, RPS29) or of the large ribosomal subunit (RPL5, RPL11, RPL15, RPL26, RPL27, RPL35A).3; 4 In one third of the patients, however, the underlying mutation is still unidentified, while many genes encoding RPs have been screened. The chase for mutations in this latter group raised the question whether each case of DBA is a ribosomopathy. Initially it was expected that DBA patients without mutations in RP genes may carry mutations in genes encoding proteins or snoRNAs involved in rRNA maturation (Fig. 1). These, however, have not yet been reported, except for rare nonsense mutations inTSR2 (20S rRNA accumulation). The TSR2 transport protein delivers RPS26 to the nucleosome.5; 6 Of note, inactivating mutations in TSR2 and RPS26 were found in patients that suffer from symptoms of both DBA and Treacher-Collins (Fig. 2). Interestingly, mutations were also found inGATA1. 7; 8 GATA1 is translated from two start codons, resulting in a long and short isoform. The mutations change the splice donor site of exon 2, or the translation start codon of the full length GATA1 open reading frame, which reduces expression of GATA1 protein.7; 9 GATA1 is a crucial erythroid-specific transcription factor, and decreased GATA1 expression impairs erythropoiesis at the BFU-E to CFU-E stage, similar to impaired RPS19 expression. It is conceivable that mutations in other erythroid specific genes may be found in the 20-30% of DBA patients without molecular diagnosis.

26 Diamond-Blackfan Anemia: from Genotype to Phenotype

The question is whether mutations in GATA1 represent DBA as a ribosomopathy. It seems more likely that mutations in GATA1 only cause the specific red cell aplasia that characterises DBA. However, GATA1 was found to occupy the region upstream of the promoter of RP genes, including RPS19 during erythropoiesis,10 which suggests a potential role for GATA1 in ribosome function or biogenesis in erythroblasts. Yet, we suggest to discriminate between DBA with a clear ribosomal origin (DBA type I) and DBA with mutations in GATA1 and possibly other erythroid specific genes (DBA type II), similar to type I-IV of Congenital Dyserythropoietic Anemia (CDA).11

DBA phenotype in model systems

The phenotype of DBA patients includes anemia at a young age, and developmental aberrations affecting thumb, radius, and craniofacial aspects.1 Model systems, however, are needed to test the 2 contribution of specific genes to the disease process. Human cell models: Reduced expression of RPS19 in haematopoietic stem and progenitor cells (HSPC) left lineage commitment and differentiation largely unaffected, except for erythroid differentiation from the stage of the BFU-E (burst forming unit-erythroid).12; 13 Reduced expression of RPs from the small or large subunit yields largely overlapping phenotypes in reduced proliferation and differentiation capacity of erythroid progenitors, although there are specific differences as 14well. Recently, induced pluripotent stem cells (iPSC) were generated from patient fibroblasts with mutated RPS19 or RPL5. Differentiation of these iPSCs towards the erythroid lineage was impaired, but also towards the megakaryocyte and myeloid lineage.15 Mouse models: Mice carrying a heterozygous nonsense mutation in Rps19 did not display a phenotype, and complete abrogation of Rps19 expression is lethal.16 Expression of a doxycycline inducible Rps19 shRNA as a single or double allele, however, enabled knockdown of Rps19 to various levels. These mice developed anemia two weeks after Rps19 knockdown.17 A mouse model in which Rpl11 was deleted by inducible Cre expression also phenocopied the haematological defects seen in DBA. Deletion of a single Rpl11 allele in adult mice rapidly, and specifically impaired erythroblast expansion and maturation.18 Haploinsufficiency during embryonal development was lethal, suggesting severe developmental defects. The major advantage of mouse models is that they resemble the physiological state of the disease, and allow to study the effect of reducedRP expression in vivo. Zebrafish models: Mutant zebrafish lines carrying RP gene haploinsufficiency display characteristic developmental defects and develop .19 The heterozygous RP mutant fish exhibit a slight growth defect, however they do not have a hematopoietic phenotype.20 Injection of morpholinos reduces RP expression to a larger extent, similar to the bi-allelic expression of shRNAs in the Rps19 shRNA mouse model. Morpholinos specifically impaired erythropoiesis in addition to developmental defects.21-23 Zebrafish provide a model system in which the physiological effects of reduced gene expression can rapidly be tested, and it is amenable to large scale drug testing.

DBA phenotype results from underlying molecular mechanisms

Activation of p53.The tumour suppressor p53 has an important role in monitoring . When the synthesis of rRNA and RPs is out of balance, an excess of positively charged RPs sequester HDM2 (Mdm2) through its acidic domain. This reduces ubiquitinylation of p53, increases its stability, and results in cell cycle arrest and .24 All RPs can bind HDM2/Mdm2, but RPL5 and RPL11 appear to play a central role in the stabilisation of p53.25 This is difficult to unite with the observation that DBA patients that have a mutation in the RPL5 or RPL11 gene have a significantly higher incidence of congenital anomalies when compared to other DBA patients.26; 27 Possibly, it is not RPL5 and RPL11 that stabilise p53, but an excess of the 5S rRNA that is transported to the 60S pre-ribosome by these RPs.28 Deletion ofTP53 in human cell models, or expression of a Mdm2 mutant protein in the DBA mouse model restored erythropoiesis.14; 29; 30 This suggested that the DBA phenotype is caused by ribosomal stress and activation of the HDM2-P53 pathway. In contrast, reduced expression of Rps19 and Rpl11

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2

Figure 1. Ribosome biogenesis. Duplicated rRNA genes are transcribed by RNA polymerase-I (Pol-I) in the nucleoli to produce the 47S pre-rRNA, Pol-III transcribes 5S rRNA in the nucleus, and Pol-II is required to transcribe RP genes. RP mRNA is translated in the cytoplasm and subsequently transported to the where ribosomes are synthesised. RPs (blue and red), the pre-RNA and accessory proteins (green) first form the 90S pre-ribosome. Part of the RPs cooperates with enzymes and small nucleolar RNAs (snoRNA) in the processing of the pre-rRNA. Cleavage of the 47S pre- rRNA into a 21S and a 32S pre-rRNA generates the pre-40S and pre-60S ribosomal subunits. Inclusion of 5S rRNA into the large ribosomal subunit is mediated by RPL5 and RPL11. In several steps the pre-rRNAs are trimmed to produce the 18S rRNA of the small (40S) ribosomal subunit and the 5.8S plus 28S rRNA of the large (60S) ribosomal subunit. Immature ribosomes in the nucleolus associate with specific export complexes (including eIF6 for the 60S subunit) that translocate the subunits through the nucleus where they undergo further modifications and finally are exported to the cytoplasm where the final maturation steps take place (double line represents nuclear membrane). Liberated from the export complexes, the ribosomal subunits can join into a 80S ribosome during mRNA translation (for review see66 ).

28 Diamond-Blackfan Anemia: from Genotype to Phenotype

2

Figure 2. Ribosomopathies. Several congenital diseases are associated with defects in ribosome biosynthesis. Treacher-Collins (Treacher Collins) is associated with reduced transcription of rRNA genes; whereas DBA is associated with loss of RPs that are required for rRNA processing. Both DBA and Treacher Collins reduce ribosome biogenesis, resulting in an excess of RPs that sequester HDM2-Mdm2 such that p53 is no longer ubiquitinated. Stabilisation increases p53 expression. (DC) is characterised by impaired rRNA uridinylation, and Shwachman-Diamond Syndrome (SDS) is characterised by reduced release of the eIF6-containing protein complex that exports the 60S ribosomal subunit to the cytoplasm. The distinct diseases impair the availability and/or function of ribosomes, which impacts differently on the transcriptome. Cell specific effects may be caused by selective sensitivity of a cell’s transcriptome to the specific translation defect.

29 Chapter 2

in p53-deficient mouse erythroblasts still impaired erythroid proliferation and differentiation.31 In zebrafish models loss of Tp53 abrogated the developmental defects, but the erythroid phenotype was at most partially rescued21; 23. Moreover, homozygous RP mutations degrade p53 in zebrafish models.32 This loss of p53 protein is also observed in tumour cells that arise in adult RP+/- zebrafish.33 Therefore, the stabilisation of p53 does not appear to be linked to the loss of blood cells inRP mutant fish. Severe anemia in DBA has been explained by an extreme sensitivity of erythroblasts for P53 stabilisation.30 However, Treacher Collins is also characterised by an excess of RPs in the nucleolus, due to reduced rRNA synthesis (Fig. 2). In a Treacher Collins mouse model, genetic ablation ofTp53 prevents apoptosis of neural crest cells and rescues the craniofacial abnormalities typical for Treacher Collins.34 Thus, the Treacher Collins phenotype is also due to activation of the HDM2-p53 pathway. However, patients affected by Treacher Collins display strong facial abnormalities, but no haematological 2 abnormalities. Finally, p53 stabilisation is also supposed to cause neutropenia, failure of theHSPC compartment to maintain , and pancreas failure in the ribosomopathy Shwachmann- Bodium-Diamond Syndrome (SDS), caused by the failure to release the proteins involved in nuclear export from the 60S ribosomal subunit (Fig. 2).35; 36 These examples demonstrate that stabilisation of p53 causes part of the disease features such as cleft palate, triphalangial thumbs, and an absent radius. The phenotypic variability in developmental defects may be due to individual variations in the control of the p53 pathways. The disease specific features of the ribosomopathies, however, must have a basis that is linked to a pathway that is selectively affected by the disease specific mutation. Stabilisation of p53 may contribute to red cell aplasia in DBA, but it cannot be the mechanism that drives the DBA specific erythroid defect.

Autophagy. Autophagy is best known in the context of nutrient deprivation, when cells recycle their own cytoplasmic constituents in autophagosomes to generate new biomolecules. The same process is induced during erythroblast maturation to eliminate membrane structures, including mitochondria.37 DBA-linked mutations inRP genes induce autophagy in both patient-derived and zebrafish erythroblasts.38 Autophagy was, however, also induced in cells derived from a patient with SDS and may be a hallmark of ribosomopathies in general.38 Nevertheless, abnormal induction of autophagy may be part of the mechanism underlying erythrocyte loss in DBA patients.

Specific translation defects. Specific mRNA translation defects likely contribute to the disease specific phenotype of ribosomopathies. The effect of a specific translation defect depends on the transcriptome present in distinct cell types, and could result in the cell specific DBA phenotype. Translation of GATA1:Mutations in the gene encoding erythroid specific transcription factor GATA1 altered translation of GATA1 mRNA. This prompted analysis of GATA1 translation in DBA-derived erythroblasts with a mutation in RP genes.8 Both the short and long isoform of Gata1 (Gata1s and Gata1l) are poorly synthesised in erythroid progenitors of DBA patients with haploinsufficiency of RPs.9 Gata1s is not expressed in mouse cells, because the start codon of the short isoform is not conserved. Moreover, Gata1 translation was not specifically affected in mouse models, as it is in human cells.9; 31 This may explain why anemia is less pronounced in mouse models, but also implies that the erythroid defect in mouse and zebrafish models is independent of Gata1 regulation. Transcriptome analysis in a p53-deficient DBA model in human erythroid progenitors deficient in Rps19 or Rpl11 indicated that expression of GATA1 target genes (EpoR; Tal1) is increased.39 Because GATA1s retains the DNA binding domain, but not the GATA1 transactivation domain, it has a dominant negative effect on GATA1 target genes.40 Increased expression of GATA1 target genes does not support the hypothesis that reduced expression of GATA1 unites DBA with mutations in RP genes and DBA with GATA1 mutations. More research is needed to fully understand the role of GATA1 in DBA. IRES mediated translation: Profiling of actively translated, polyribosomal mRNA in p53-deficient mouse erythroblasts indicated that reduced expression of Rps19 or Rpl11 predominantly impairs IRES- mediated translation.31 Examples are Bag1 and Csde1, transcripts that are expressed at much higher levels in differentiating erythroblasts compared to other hematopoietic cells. Expression of Bag1 and Csde1 protein was also reduced in erythroblasts of DBA patients. Of note, both Bag1 and Csde1 are

30 Diamond-Blackfan Anemia: from Genotype to Phenotype required for erythropoiesis. Bag1-deficient mouse embryos die at embryonic day E13.5 with a lack of definitive erythrocytes.31; 41 Csde1 deficiency is lethal at the implantation stage. Reduced expression of Csde1 in erythroblasts impairs their proliferation and differentiation.31; 42 Predominant effects on IRES- dependent translation when ribosomes are limiting may occur because IRES-dependent translation is less competitive for available ribosomes compared to cap-dependent translation initiation. Leucine enhances mTOR-dependent translation: All DBA models display overall reduced protein synthesis that can be improved by addition of high levels of the essential amino acid leucine.43 Leucine improved in vitro expansion and differentiation of hematopoietic cells, and enhanced erythropoiesis in mouse and zebrafish models.22; 23; 43; 44 Leucine can activate mTORC1, and enhances phosphorylation of the mTORC1 targets eIF4E-Binding Protein (4EBP) and p70 S6-kinase (S6K).22 There is, however no indication that reduced expression of RPs impairs mTORC1 activity. In contrast, enhanced S6K phosphorylation is involved in induction of autophagy,38 and translation of transcripts that are 2 hypersensitive to mTORC1 activity is enhanced in erythroblasts.31 Of note, translation of branched- chain aminotransferase-1 (BCAT1) is reduced in lymphoid cell lines derived from DBA patients, which decreases leucine catabolism.45

Cell cycle regulation.Reduced expression of RPs in mammalian cell models inhibits cell cycle progression. Cell cycle arrest seems more prone to knock down of Rpl5 or Rpl11 compared to knock down of Rps19, but results are not always consistent and may depend on the remaining RP expression level.12; 14; 46 Impaired cell cycle progression may be the result of p53 activation,30 but could also be a direct effect of reduced RP expression. Among the IRES-mediated transcripts that are poorly translated in DBA is Cdc25b, encoding a dual specificity phosphatase that activates cyclin-dependent kinases 31 (CDKs). Cdc25b protein was reduced in erythroblasts derived from DBA patients Horos ( and von Lindern, unpublished). Of note, glucocorticoids are a first choice of treatment in DBA. Glucocorticoids support erythroblast renewal divisions in vitro, and are required for expansion of the erythroblast compartment in the spleen of mice during stress erythropoiesis. Glucocorticoids cooperate with SCF to maintain a long cell cycle G1 phase,47 and activation of the GR induces expression of cell cycle inhibitors that are in part the same inhibitors described for the anti-proliferative effect of glucocorticoids in lymphocytes. Glucocorticoids may be effective in DBA because their inhibition of S-phase entry provides more time to synthesize all required proteins. This would imply that slow cell cycle progression may not be harmful during expansion of erythroblasts. Impaired cell cycle progression during the final differentiation divisions, however, is the likely cause for macrocytosis associated with DBA.

Iron metabolism. DBA patients are extremely sensitive to iron overload.48 This has been ascribed to ineffective erythropoiesis and reduced use of iron. To prevent toxic effects, iron is always bound to carrier proteins and cellular uptake is regulated by a set of transporters in the outer cell membrane and mitochondrial membranes. A major heme exporter in erythroblasts is FLVCR1 (Feline Leukaemia Virus subgroup C Cellular Receptor 1). FLVCR1 deficiency in mice mainly affects erythropoiesis and causes a DBA-like phenotype.49 Notably, alternative splicing of FLVCR1, reducing its expression and function occurs in DBA-derived erythroblasts.50 The oxidative stress due to an increase in free iron is cytotoxic and contributes to the phenotype of DBA.51

Disease heterogeneity

The above shows that several distinct mechanisms contribute to the DBA phenotype. In addition, DBA is caused by mutation or deletion of several genes. As a result, the DBA phenotype can be very heterogeneous, which complicates its physical diagnosis. Molecular diagnostics is also complex because mutations have been found in 14 RP genes, in TSR2 and GATA1, whereas the genetic defect remained unknown in one third of DBA patients. High throughput analysis of the genome of DBA patients, to identify mutations in the latter group, confronts us with the huge challenge to discriminate disease- causing mutations from the immense natural variation in genomes of human individuals. These novel advances therefore prompt the questions: Do deficiencies in RPs always cause DBA?

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Mutation of RPs does not always cause DBA

Molecular diagnostics indicated that DBA patients may have parents that carry the mutation but do not display the disease.52 These parents can be classified as silent DBA. It is often suggested that mutations in RP genes are always an indication of (silent) DBA. We oppose this view. The relation between mutant RP genes and cellular biology is much more complex. For instance loss of Rpl38 in a mouse model results in skeletal defects,53 loss of RPSA leads to asplenia,54 loss of Rpl22 results in a B cell defect,55 and Rpl13a dissociates from ribosomes to bind a RNA element in specific transcripts during interferon responses.56 Erythropoiesis is unaffected by mutations in these RP genes. It needs to be said, however, that haploinsufficiency of RPs involved in DBA also fails to induce overt anemia in mouse models.17 It is important to distinguish mutations that cause haploid insufficiency from missense 2 mutations that may alter the functional properties of the RP. For instance congenital missense mutations in RPL10 were found in patients suffering from autism,57; 58 whereas acquired mutations in RPL10 and RPL5 were observed in patients with T-ALL,59 again suggesting that some mutations, or mutations in specific cell types may enhance function or expansion of that cellular compartment, rather than induce a cytopenia. Finally, deletions on human 5q that encompass RPS14 are acquired in (5q- MDS).60; 61 This deletion drives expansion of HSPC with a strong erythroid deficiency. RPS14 haploinsufficiency phenocopies DBA associated anemia, whereas deletion of a tumour suppressor gene or miRNA on 5q may be responsible for clonal expansion.62 As long as the specific translatome that is affected by each mutation is unknown, the assumption that mutations inRP genes all contribute to DBA is an oversimplification.

Closely related diseases that need to be excluded by molecular diagnosis. Finally, deletions in mitochondrial DNA (mtDNA) were found in 8 out of 176 patients that were clinically diagnosed with DBA, but in which no known mutation was found.63 These deletions are characteristic for Pearson marrow pancreas syndrome (PS), a syndrome with overlapping features including severe anemia during the first year of life. The rareness of the disease and the overlap in phenotype warrant molecular diagnosis for PS in all DBA cases that do not carry any of the so far known disease initiating mutations. Similarly, small deletions of chromosome 5q were found in two young patients of which one was diagnosed as Myelodysplastic Syndrome (MDS), and one as DBA. In both cases haematopoiesis recovered, and the 5q- clone disappeared upon treatment with lenolidomide.64 This too warrants careful molecular analysis of the RPS14 containing region of chromosome 5, because acquired deletion ofRPS14 needs to be discriminated from congenital defects in ribosome biogenesis.

Genetic factors predicting pathophysiology

The huge disease heterogeneity could be triggered by environmental factors, but is likely to have a genetic cause. Such genetic predisposition effects are, so far, unexplored. The exception is the response to glucocorticoids, the major treatment to alleviate anemia in DBA.1 The glucocorticoid receptor (GR) is highly polymorphic. Alternative splicing produces a dominant negative GRβ isoform that is stabilised by the A3669G (rs6198) SNP. The incidence of this SNP is increased in DBA, and particularly in transfusion- dependent DBA.65

Conclusion

The genetic basis of DBA indicates that DBA is a true ribosomopathy in most patients. Individual studies are mostly restricted to a specific phenotypic aspect to explain the pathophysiology of DBA. However, the central role of ribosomes in protein synthesis implies that the phenotype of DBA is likely to be more complex. In fact, the ribosomopathy Diamond Blackfan Anemia should be renamed to Diamond Blackfan Syndrome to emphasise that it is a systemic disease of which severe anemia at young age is the most common, but not the exclusive symptom. Alleviation of one aspect may largely improve a patient’s condition, just as p53 deficiency diminishes the developmental defects in DBA mouse and zebrafish

32 Diamond-Blackfan Anemia: from Genotype to Phenotype models. This does not necessarily mean that this feature is crucial to the disease phenotype. It may only imply that alleviation of one phenotypic aspect enables feedback mechanisms to compensate other aspects. To fully understand the genotype-phenotype relation in DBA and the effect of treatment, it will be important that clinicians, researchers and patient organisations collaborate in compiling a worldwide overview of patient mutations and associated disease symptoms to avoid a bias in conclusions due to small patient numbers and a high heterogeneity of the disease.

Acknowledgements Thanks go out to the Maria Daniela Arturi Foundation for stimulating the interaction between scien- tists working on DBA. And to DBAF and EuroDBA for organising meetings with scientists, clinicians and patients. We are grateful for support from the Landsteiner Foundation for Bloodtransfusion Research (#1239) to NAP and MvL, and ERA-Net for Research on Rare Diseases (ZonMW #113301205) to AWM. 2

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