© 2015. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2015) 8, 1013-1026 doi:10.1242/dmm.020529

REVIEW : how a common root can cause a tree of pathologies Nadia Danilova1,* and Hanna T. Gazda2,3,4,*

ABSTRACT DBA is a rare disease with an incidence of ∼5 cases per million Defects in biogenesis are associated with a group of diseases live births, but it has attracted substantial attention as a model called the ribosomopathies, of which Diamond-Blackfan anemia (DBA) disease for ribosomopathies, a group of pathologies associated with is the most studied. are composed of ribosomal defects in ribosome biogenesis (Armistead and Triggs-Raine, 2014; (RPs) and ribosomal RNA (rRNA). RPs and multiple other factors are James et al., 2014). Despite this common defect, phenotypes of necessary for the processing of pre-rRNA, the assembly of ribosomal ribosomopathies differ. A common feature among several subunits, their export to the and for the final assembly of ribosomopathies is p53 activation (Danilova et al., 2008b; subunits into a ribosome. Haploinsufficiency of certain RPs causes Elghetany and Alter, 2002; Jones et al., 2008), but the mechanisms DBA, whereas mutations in other factors cause various other involved have not been completely elucidated. A p53-independent ribosomopathies. Despite the general nature of their underlying response to RP deficiency has also been observed (Aspesi et al., 2014; defects, the clinical manifestations of ribosomopathies differ. In DBA, Danilova et al., 2008b; Singh et al., 2014; Torihara et al., 2011). The for example, red blood cell pathology is especially evident. In addition, pathways that lead from a particular defect in ribosome biogenesis to individuals with DBA often have malformations of limbs, the face and the phenotype of a are still not well understood. various organs, and also have an increased risk of cancer. Common Several insightful reviews about the mechanisms of ribosomopathies features shared among human DBA and animal models have emerged, have been published recently (Armistead and Triggs-Raine, 2014; such as small body size, eye defects, duplication or overgrowth of Ellis, 2014; Golomb et al., 2014; James et al., 2014; Ruggero and ectoderm-derived structures, and hematopoietic defects. Phenotypes Shimamura, 2014) with a focus on human data. However, cross- of ribosomopathies are mediated both by p53-dependent and species analysis might also provide additional clues as to the -independent pathways. The current challenge is to identify mechanisms of these diseases. differences in response to ribosomal stress that lead to specific Here, we review the phenotypes that are caused by RP deficiency in defects in various ribosomopathies. Here, we review recent findings in humans as well as in mouse, fly and zebrafish models, with an this field, with a particular focus on animal models, and discuss how, in emphasis on their common features. We also discuss the consequences some cases, the different phenotypes of ribosomopathies might arise of ribosomal stress at the molecular level, with a particular emphasis from differences in the spatiotemporal expression of the affected . on p53 activation, metabolic changes, the origin of erythroid defects, and congenital malformations. We also discuss potential new KEY WORDS: Ribosome biogenesis, Ribosomal , directions for DBA treatment that arise from recent findings. Ribosomopathy, Diamond-Blackfan anemia, p53, ΔNp63 Phenotypes caused by defects in ribosome biogenesis Introduction All ribosomopathies originate from defects in ribosome biogenesis, Diamond-Blackfan anemia (DBA) is a congenital syndrome yet every ribosomopathy has a unique phenotype with different associated with anemia, physical malformations and cancer tissues affected. In this section, we provide an overview of the (Halperin and Freedman, 1989; Vlachos et al., 2012). In the phenotypes caused by RP deficiency in human DBA and in its majority of individuals with DBA, mutations or deletions of a animal models, and compare them to phenotypes of other subset of ribosomal proteins (RPs; see Box 1) are found, with ribosomopathies. RPS19 mutations accounting for about 25% of all cases (Table 1). Although some mutations are dominant negative, the major RP deficiency mechanism of the disease is associated with haploinsufficiency of In humans, DBA is often diagnosed during the first year of life; an RP that disrupts the processing of pre-ribosomal RNA (pre- common clinical features include anemia, low reticulocyte count, rRNA), leading to abortive ribosome biogenesis (Choesmel et al., macrocytic erythrocytes (see Box 1 for a glossary of terms), increased 2007; Devlin et al., 2010; Farrar et al., 2014; Flygare et al., 2007; expression of fetal hemoglobin and elevated activity of adenosine Gazda et al., 2004; Leger-Silvestre et al., 2005; Panic et al., 2006). deaminase (ADA; Box 1) (Fargo et al., 2013; Halperin and Freedman, 1989). Approximately 40% of affected individuals have short stature 1Department of Molecular, Cell & Developmental Biology, University of California, and variable congenital malformations of craniofacial skeleton, eyes, Los Angeles, CA 90095, USA. 2Division of Genetics and Genomics, The Manton heart, visceral organs and limbs. Notably, duplication of some Center for Orphan Disease Research, Boston Children’s Hospital, Boston, 3 structures, including triphalangeal thumb, bifid thumb and extra ribs, MA 02115, USA. Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA. 4Broad Institute, Cambridge, MA 02142, USA. has been reported (Halperin and Freedman, 1989). The clinical symptoms of DBA rarely correlate with the type of causative mutation. *Authors for correspondence ([email protected]; hanna.gazda@childrens. harvard.edu) However, individuals with mutations in RPL5 often have a cleft palate, whereas this malformation was not observed in individuals This is an Open Access article distributed under the terms of the Creative Commons Attribution carrying RPS19 mutations (Gazda et al., 2008). The acquired License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. haploinsufficiency of RPS14 has been shown to underlie the Disease Models & Mechanisms

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brain apoptosis, pericardial edema, reduced pigmentation and Box 1. Glossary hematopoietic defects (Amsterdam et al., 2004a; Danilova et al., 2011; Taylor et al., 2012; Zhang et al., 2014). Similar phenotypes 5′ and 3′ external transcribed spacers (ETSs): non-functional RNA sequences of the pre-rRNA transcript that have structural roles and are were observed in zebrafish embryos in which rps19 or rpl11 were excised during pre-rRNA processing. knocked down using morpholinos; in addition, some morphants had 5q-myelodysplastic syndrome (5q-MDS): a form of MDS that is missing eyes, abnormal positioning of the heart and pancreas, and caused by loss of a part of the q arm of 5. increased fin size (Chakraborty et al., 2009; Danilova et al., 2008b; Adenosine deaminase (ADA): an enzyme involved in the metabolism Uechi et al., 2008). Adult zebrafish heterozygous for RP mutations of adenosine. have a high frequency of malignant peripheral nerve sheath tumors Anemia: a condition associated with an insufficient number of red blood (Amsterdam et al., 2004b). cells in blood. Asplenia: absence of spleen or very small spleen. In mice, mutations in Rps19 and Rps20 cause dark skin, reduced Biliary cirrhosis: cirrhosis caused by damage to the bile ducts in the liver. body size and a reduced erythrocyte count (McGowan et al., 2008). Epiboly: growth of a cell layer to envelope the yolk during gastrulation. RPS6-deficient mice also have hematopoietic defects (Keel et al., Haploinsufficiency: when a single copy of the gene is insufficient to 2012). A transgenic mouse carrying an Rps19 R62W mutation, which maintain normal function. is common in DBA, has been recently developed (Devlin et al., Internal ribosome entrysite (IRES): a sequence inside mRNA that allows 2010). The constitutive expression of the transgene bearing this for initiation of cap-independent in the middle of mRNA. Internally transcribed spacers (ITSs): spacers between 18S, 5.8S and mutation is developmentally lethal. Its conditional expression results 28S rRNA in the pre-rRNA transcript that play structural roles and, like in growth retardation and anemia. Another RPS19-deficient mouse ETSs, are excised during pre-rRNA processing. created by transgenic RNA interference also develops symptoms Jaundice: yellow color of the skin and whites of the eyes caused by similar to those found in individuals with DBA (Jaako et al., 2011). excess bilirubin in the blood. Mutation of the mouse Rpl24 leads to the Belly Spot and Tail Macrocytic erythrocytes: abnormally large red blood cells. (Bst) phenotype characterized by small size, eye defects, a Mechanistic target of rapamycin (mTOR): a serine/threonine kinase that is a central regulator of cellular metabolism. It forms mTORC1 and white ventral spot, white hind feet and various skeletal mTORC2 complexes, which mediate cellular responses to stresses such abnormalities including duplicated digits and phalanges as DNA damage and nutrient deprivation. (Fig. 1), which is similar to the anomaly noted in individuals Myelodysplastic syndrome (MDS): a syndrome caused by mutations with DBA (Oliver et al., 2004). Mutations in Rps7 also lead to in several genes, most often encoding splicing factors. It is associated skeletal malformations, ventral white spotting and eye defects with ineffective production of blood, which often leads to leukemia. (Watkins-Chow et al., 2013). Reticulocyte: immature erythrocyte. Thus, several cross-species features caused by RP deficiency RNA polymerase I and III (PolI/PolIII): enzymes involved in the of non-coding . have emerged, which include a smaller size, eye defects, congenital Ribosomal proteins (RPs): proteins that together with ribosomal RNA malformations often associated with the duplication or overgrowth (rRNA) make up the ribosome. They are called RPS (RP from small of structures derived from the epidermal ectoderm, and varying ribosomal subunit) or RPL (RP from large ribosomal subunit) depending degrees of anemia. on whether they associate with the small or large subunit of the ribosome. Small nucleolar RNA (snoRNA): a class of small RNAs that guide Examples of phenotypes of other ribosomopathies chemical modifications such as methylation and pseudouridylation of other RNAs. Treacher Collins syndrome (TCS) is caused by mutations in genes involved in rRNA transcription, such as the Treacher Collins- Franceschetti syndrome 1 (TCOF1) and POLR1D and POLR1C genes, which encode subunits of RNA polymerase I and III erythroid defect in 5q-myelodysplastic syndrome (MDS) (Ebert et al., (PolI/III; Box 1) (Dauwerse et al., 2011; Valdez et al., 2004). TCS is 2008; Pellagatti et al., 2008). associated with craniofacial deformities such as absent cheekbones Individuals with DBA have an increased frequency of both solid (Dixon et al., 2006). cancers and leukemia (Vlachos et al., 2012), and acquired mutations Shwachman Diamond syndrome (SDS) is caused by mutations in in RPL5 and RPL10 are found in T-cell acute lymphoblastic the Shwachman-Bodian-Diamond syndrome (SBDS) gene, which leukemia (De Keersmaecker et al., 2013). functions in ribosomal subunit joining (Boocock et al., 2003). A strikingly different phenotype is associated with the RPSA SDS is characterized by decreased production of white blood protein, an RP that is involved in the maturation of the 40S subunit cells, exocrine pancreatic insufficiency, and problems with bone and also serves as a receptor. Mutations in this RP lead to formation and growth. familial isolated congenital asplenia (see Box 1 for a glossary of Dyskeratosis congenita (DC) is caused by mutations in genes that terms) (Bolze et al., 2013). However, this phenotype is likely code for the components of small nucleolar ribonucleoprotein associated with the function of RPSA as a laminin receptor because complexes, which function in rRNA processing (Ruggero et al., laminin deficiency has been shown to lead to asplenia (Miner and 2003). Individuals with DC have mucocutaneous defects and bone Li, 2000). marrow failure. In the fruit fly, Drosophila, the haploinsufficiency of many North American Indian childhood cirrhosis (NAIC) is caused by RPs leads to a Minute phenotype, which is characterized by mutation in CIRH1A [cirrhosis, autosomal recessive 1A (cirhin)], developmental delay, small size, small bristles, small rough eyes, which encodes a protein that functions in rRNA processing and recessive lethality at late embryonic/early larval stages (Chagnon et al., 2002). The phenotype is restricted to neonatal (Kongsuwan et al., 1985; Marygold et al., 2005). Notably, jaundice that progresses to biliary cirrhosis (Box 1) that at some heterozygous Rpl38 and Rpl5 Drosophila mutants have abnormally point requires hepatic transplantation. large wings (Marygold et al., 2005). These examples illustrate the variability of phenotypes of Zebrafish embryos homozygous for RP mutations also exhibit ribosomopathies. A comprehensive description of these and other developmental delay and small size as well as a small head and eyes, ribosomopathies can be found in recent reviews (Armistead and Disease Models & Mechanisms

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Table 1. Genes mutated in DBA % of affected Gene individuals Type of mutation References RPS19 ∼25 Single-nucleotide variant (SNV) (missense, Draptchinskaia et al., 1999; Farrar et al., nonsense, splice site mutations), 2011; Gazda et al., 2004 microinsertions, microdeletions, single copy deletions RPL5 ∼7 SNV (missense, nonsense, splice site Cmejla et al., 2009; Gazda et al., 2008; mutations), microinsertions, Quarello et al., 2012 microdeletions RPS26 ∼6.5 SNV (missense and splice site mutations), Doherty et al., 2010; Farrar et al., 2011 microinsertions, single copy deletions RPL11 ∼5 SNV (missense, nonsense, splice site Cmejla et al., 2009; Gazda et al., 2008; mutations), microinsertions, Quarello et al., 2012 microdeletions RPL35A ∼3 SNV (missense, nonsense, splice site Farrar et al., 2008; Farrar et al., 2011 mutations), microdeletion, single copy deletions RPS10 ∼2.5 SNV (missense and nonsense), Doherty et al., 2010 microinsertion RPS24 ∼2 SNV (nonsense), microinsertion/ Gazda et al., 2006b; Landowski et al., 2013 microdeletion, large deletion RPS17 ∼1 SNV (missense), microinsertion, single copy Cmejla et al., 2007; Farrar et al., 2011; deletions Gazda et al., 2008 RPS7 <1 SNV (splice site mutation) Gazda et al., 2008 RPL26 <1 Microdeletion Gazda et al., 2012 GATA1 <1 SNV (missense, splice site mutations) Sankaran et al., 2012 RPL15 <1 Large deletion Landowski et al., 2013 RPS29 <1 SNV (missense mutations) Mirabello et al., 2014 RPS28 <1 SNV (missense mutations) Gripp et al., 2014 TSR2 <1 SNV (missense mutation) Gripp et al., 2014 RPL31 <1 Single copy deletion Farrar et al., 2014 RPS27 <1 Microdeletion Wang et al., 2015 RPL27 <1 SNV (splice site mutation) Wang et al., 2015 Genes ordered according to the percentage of DBA-affected individuals who carry a certain mutation. RPL, RP from large ribosomal subunit; RPS, RP from small ribosomal subunit.

Triggs-Raine, 2014; James et al., 2014). How a general defect can Abortive ribosome biogenesis and p53 activation lead to the malfunction of a specific tissue remains a focus of Ribosome biogenesis: stages and the role of RPs many ongoing studies, which uncovered that some phenotypic The is composed of a small (40S) and a large consequences of ribosomal stress are caused by p53 activation, as (60S) subunit (Kressler et al., 2010). The small subunit includes 18S discussed in more detail in the next section. rRNA and 33 RPs; the large subunit includes 5S rRNA, 28S rRNA, 5.8S rRNA and 46 RPs. The genes encoding rRNA (rDNA) are found in multiple copies organized into tandem repeats. 18S, 5.8S and 28S rRNAs are transcribed as a single pre-rRNA transcript by RNA polymerase I in a substructure of the nucleus called the nucleolus. In yeast, the rDNA repeat also encodes the 5S rRNA, which is transcribed in the reverse direction (Henras et al., 2015). In human cells, the 5S rRNA precursor is transcribed from multiple genes in the nucleoplasm by RNA polymerase III (Henras et al., 2015). Then, 5S rRNA migrates to the nucleolus for further processing and incorporation into the pre-60S subunit. The nascent pre-rRNA assembles co-transcriptionally with a subset of RPs and with multiple other factors that facilitate the folding, modification and cleavage of pre-rRNA and the formation of ribosomal subunits (Fig. 2). Pre-rRNA processing can take place co-transcriptionally as well as post-transcriptionally. In yeast, the pre-40S subunit most often is released by cleavage within the internal transcribed spacer 1 (ITS1) before the transcription of the 3′ end of the pre-rRNA is finished. Details of pre-rRNA processing can be found in recent reviews (Henras et al., 2015; Kressler et al., 2010). Most RPs are strictly required for the pre-rRNA processing. It is Rpl24 Fig. 1. Duplicated digits and phalanges in mice heterozygous for . thought that their role is to assist the proper folding of the pre-rRNA Skeletal stain of newborn forelimbs (upper) and hindlimbs (lower). Mice heterozygous for a mutation in Rpl24 (Bst/+ phenotype) show preaxial (Henras et al., 2015). RPs act in a hierarchical order. Approximately polydactyly (0) and triphalangy of the first digit (1). Figure reproduced with half of RPs from the 40S ribosomal subunit, including RPS24 and permission (Oliver et al., 2004). RPS7, associate early with the 5′ end of the nascent pre-rRNA and Disease Models & Mechanisms

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Nucleolus Nucleus

Transcription

RPS24 Processing or RPS7 deficiency

20S

Processing RPS19 Maturation or RPS17 deficiency Export

Cytoplasm Maturation

RPSs

Ribosome formation

Fig. 2. A simplified schematic of ribosome biogenesis in human cells. (A) 18S, 5.8S and 28S rRNAs are transcribed by Pol1 in the nucleolus as segments of a long precursor pre-rRNA, which also includes two externally transcribed spacers 5′ETS and 3′ETS and two internally transcribed spacers, ITS1 and ITS2 (B; Box 1). 5S rRNA is transcribed independently by PolIII in the nucleus. (B) Concomitant with transcription, the pre-rRNA assembles with accessory factors and a subset of ribosomal proteins (RPs: RPSs and RPLs). This facilitates the formation of a secondary structure necessary for the correct folding, modification andcleavageof pre-rRNA. (C) After removal of the 5′ETS and cleavage in the ITS1 site, pre-40S (which contains the 20S precursor of 18S rRNA) and pre-60S subunits are formed and continue to mature. 5S rRNA incorporates into pre-60S subunit. Subunits are then exported to the cytoplasm. (D) Once in the cytoplasm, small and large subunits undergo final maturation, which involves the removal of remaining accessory factors and incorporation of missing RPs. (E) A functional ribosome forms after transcribed mRNA bindsto the 40S subunit, which triggers association of the 60S subunit with this complex. More than 200 accessory factors, which include helicases, nucleases, small nucleolar RNAs (snoRNAs; Box 1), chaperones and transporters, temporally associate with the maturing ribosomal subunits at various steps. In human cells, pre-rRNA processing is differentially affected by deficiency of various RPs. For example, deficiency of RPS24 or RPS7 prevents formation of the 20S precursor of 18S rRNA, whereas deficiency of RPS19 or RPS17 prevents conversion of the 20S precursor to a mature 18S rRNA. are required for the initiation of cleavages at the 5′ external not required for 5′ETS removal but is necessary for ITS1 cleavage. transcribed spacer (5′ETS; Box 1) and ITS1 (Fig. 2). The second set Correspondingly, depletion of RPS24 or RPS7 in yeast and human of RPs, which includes RPS19 and RPS17 along with other RPs, is cells results in the failure of maturation of the 5′ end of 18S rRNA, Disease Models & Mechanisms

1016 REVIEW Disease Models & Mechanisms (2015) 8, 1013-1026 doi:10.1242/dmm.020529 whereas, in the absence of RPS19 or RPS17, the 3′ end of the 18S raises the question of how cells respond to a deficiency of RPL5 or rRNA cannot mature (Choesmel et al., 2007, 2008; Flygare et al., RPL11. In primary human lung fibroblasts and in murine embryonic 2007; Leger-Silvestre et al., 2005; Robledo et al., 2008). In all cases, stem cell lines, their depletion reportedly does not induce the p53 pre-40S particles that contain non-cleaved pre-rRNA cannot be pathway (Teng et al., 2013; Singh et al., 2014). However, Rpl11- exported and accumulate in the nucleus. Similarly, deficiency of deficient zebrafish do have an upregulated p53 pathway most other RPs that are mutated in DBA, both from the small and the (Chakraborty et al., 2009; Danilova et al., 2011). These large ribosomal subunits, affects pre-rRNA processing in a unique contradictory results flag the need for further investigation into way, leading to the accumulation of different rRNA precursors and the mode of action of these RPs in p53 regulation. disruption of ribosome biogenesis at different steps (Robledo et al., Besides the RPs, Mdm2 has over a hundred binding partners and 2008). some of them might be involved in response to ribosomal stress (James et al., 2014). One of them, p14ARF, an alternate reading RP deficiency and p53 activation frame protein product of cyclin-dependent kinase inhibitor 2A Ribosomal biogenesis is a highly energy-consuming process that (CDKN2A), is an Mdm2 inhibitor and is upregulated in response to is coupled to cell growth and requires tight regulation. It is oncogenic signaling. An increase in such signaling has been controlled by the TOR pathway, which regulates the synthesis of reported in hematopoietic progenitors derived from DBA patients ribosomal components in response to growth factors and nutrient (Gazda et al., 2006a) and in Rpl11-deficient zebrafish (Danilova availability (see Box 1 for a glossary of terms) (Kressler et al., et al., 2011) (Fig. 3). 2010). Ribosome biosynthesis requires coordinated activity of three Some data suggest that ataxia telangiectasia and Rad3 related RNA polymerases, PolI, PolII and PolIII. Cellular stress such as (ATR) and ataxia telangiectasia mutated (ATM) kinases, which hypoxia or DNA damage decreases ribosome biogenesis mostly are responsible for the replication stress and DNA-damage by inhibition of PolI transcription of rRNA through several checkpoints, might contribute to p53 upregulation in situations mechanisms (Boulon et al., 2010). Shutting down ribosome where rRNA synthesis is compromised (Fig. 3). The ATR-Chk1 biogenesis during stress preserves cellular homeostasis and axis was implicated in cell cycle arrest induced by inhibition of ensures that enough cellular resources can be relocated to a rRNA synthesis using actinomycin D (Ma and Pederson, 2013). response to stress. Another study reported upregulation of the ATR-ATM-Chk1-p53 One of the first indications that p53 is involved in controlling the pathway in RPS19-deficient human cells and in zebrafish models fidelity of ribosome biogenesis came from the finding that a of DBA (Danilova et al., 2014). The exact mechanism of ATR mutation in Bop1, which is involved in maturation of rRNAs, leads induction in these studies was not explored. However, some to p53-dependent cell cycle arrest in mouse cells (Pestov et al., researchers have hypothesized that the obstruction of pre-rRNA 2001) and that RPS6-gene haploinsufficiency activates p53 in processing might interfere with transcription and, ultimately, mouse embryos and T cells (Panic et al., 2006; Sulic et al., 2005). with DNA replication (Bermejo et al., 2012). This hypothesis is Subsequent studies have established p53 activation as a general based on the fact that pre-rRNA processing starts before its response to RP deficiency (Barlow et al., 2010; Danilova et al., transcription is finished (Osheim et al., 2004; Schneider et al., 2008b; Dutt et al., 2011; Fumagalli and Thomas, 2011; McGowan 2007). Pre-mRNA splicing also happens co-transcriptionally and et al., 2008; Taylor et al., 2012). Acquired loss of RPS14 in splicing defects increase the formation of DNA double-strand 5q-myelodysplastic syndrome (5q-MDS; Box 1) is also associated breaks (Li and Manley, 2005). with p53 upregulation (Dutt et al., 2011; Pellagatti et al., 2008). An additional source of p53 activation in RP-deficient cells might Several hypotheses to explain how p53 is activated in DBA have arise from the altered nucleotide metabolism (Fig. 3). In RP- been proposed. The nucleolus has been suggested to be a universal deficient zebrafish, the level of adenosine triphosphate (ATP), stress sensor that is responsible for the maintenance of a low level of which is a source of energy, is decreased, whereas that of p53 in the cell; the nucleolus disrupts and arrests the cell cycle in deoxythymidine triphosphate (dTTP), which is a building block response to various stresses, including DNA damage, hypoxia, heat of DNA, is increased (Danilova et al., 2014). Decreased ATP leads shock, nucleoside triphosphate (NTP) depletion, and others (Rubbi to activation of the energy sensor AMP-activated protein kinase and Milner, 2003). However, RP deficiency does not always lead to (AMPK), which, in turn, activates p53 (Hardie et al., 2012). The nucleolar disruption (Fumagalli et al., 2009), suggesting that other disproportional increase or decrease in one of deoxyribonucleotide mechanisms sense ribosomal stress as well. triphosphates can interfere with DNA synthesis and result in p53 One way in which altered ribosome biogenesis might affect p53 is upregulation (Austin et al., 2012; Sanchez et al., 2012). through RPs that are not incorporated into ribosomes (Fig. 3). RP deficiency in a fetal environment, in addition to p53 protein Several RPs have been shown to bind Mdm2 (mouse double minute stabilization, leads to the transcriptional upregulation of p53 2 homolog, which is a negative regulator of p53) and to inhibit its (Chakraborty et al., 2009; Danilova et al., 2008b) possibly binding to p53, leading to p53 stabilization and to cell cycle arrest because of the upregulation of certain growth factors such as Myc (Zhang and Lu, 2009). Attaining the right balance between the (Danilova et al., 2011). synthesis of rRNA and RPs seems to be important; when rRNA Reactive oxygen species (ROS) can also contribute to p53 synthesis is decreased, RPs are no longer used for ribosome building stabilization in RP-deficient cells (Heijnen et al., 2014). The and can stabilize p53 (Donati et al., 2011). Thus, p53 stabilization insufficient production of hemoglobin and the accumulation of the by RPs might be a general mechanism involved in the response to excess of heme might contribute to oxidative stress in erythroid various stresses. progenitors (Ellis, 2014). RPL5 and RPL11 have been proposed to be the essential players Various hypotheses of p53 activation are not mutually exclusive. in the regulation of p53 by RPs (Macias et al., 2010). Together with In fact, several mechanisms might function in parallel to induce p53 5S rRNA they form the Mdm2 regulatory complex (Sloan et al., to guard ribosome biogenesis. The contribution of each mechanism 2013). They are protected from the proteasomal degradation that could differ between species, tissues, and with the particular RP other RPs undergo when PolI is inhibited (Bursac et al., 2012). This involved. Disease Models & Mechanisms

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Nucleolus

- -

Nucleolar disruption

p53-independent pathways

Cell cycle

Nucleus

Decreased Low PIM number of ribosomes Low cyclins Low ATP

Cytoplasm Altered translational profile

Fig. 3. Defects in ribosomal biogenesis activate p53 and other stress-response mechanisms. A schematic showing pre-rRNA transcription, and assembly of accessory factors and RPs on the nascent pre-rRNA. (A) Recent studies have suggested that problems with pre-rRNA processing can affect DNA transcription, leading to the activation of ATR-ATM-Chk1/2 signaling (which is responsible for the replication-stress and DNA-damage checkpoints) and p53 upregulation. In addition, deoxynucleoside triphosphate (dNTP) imbalance caused by RP deficiency might interfere with transcription and replication and contribute to ATR-ATM activation. (B) Problems with pre-RNA processing compromise ribosome biogenesis and lead to nucleolar disruption. The nucleolus is involved in maintaining low p53 levels by exporting it for degradation. Various stressors disrupt nucleolar organization, compromising p53 export and leading to p53 accumulation in the nucleus. Nucleolar disruption might also lead to the release of factors that activate p53 or cause cell cycle arrest by p53-independent mechanisms. (C) An alternative pathway of p53 activation is through free RPs that, in complex with 5S RNA, bind the p53 negative regulator MDM2, releasing p53 from its control. (D) Upregulation of MYC and RAS pro-survival factors in DBA patients and in animal models suggests that they might activate p14ARF, which, in complex with 5S RNA, also negatively regulates MDM2. Hypothetically, additional not-yet-identified nucleolar factors might also negatively interact with MDM2 and contribute to p53 upregulation. (E) p53 might also be activated by secondary changes in RP-deficient cells, such as increased levels of ROS or decreased levels of ATP, which activates AMPK, which, in turn, activates p53. p53 then translocates to the nucleus. (F) p53 activation leads to cell cycle arrest and to the induction of downstream pathways ranging from cellular repair to apoptotic mechanisms. (G) A p53-independent response might also originate from the cytoplasm owing to a decreased number and altered activity of ribosomes, which also can lead to cell cycle arrest. For example, decreased levels of cyclins or PIM1 caused by RP deficiency might inhibit cell cycle progression. Abbreviations: AMPK, AMP-activated protein kinase; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3 related; Chk1/2, checkpoint kinase 1/2; MDM2, MDM2 oncogene, E3 ubiquitin protein ligase; MYC, avian myelocytomatosis viral oncogene homolog; p14ARF, alternate protein product of the CDKN2A, cyclin-dependent kinase inhibitor 2A; PIM, pim-1 oncogene; PolI, RNA polymerase I; RAS, rat sarcoma viral oncogene homolog; ROS, reactive oxygen species; 5S, rRNA. See Fig. 2 for a key. p53 activation in other ribosomopathies deficiency (Danilova et al., 2008b; Torihara et al., 2011). Yeast, an p53 activation occurs in other ribosomopathies as well. The organism that lacks the p53-Mdm2 pathway, responds to the craniofacial defects in TCS are mediated by p53 upregulation inhibition of ribosome biogenesis by cell cycle arrest, which (Jones et al., 2008). p53 protein overexpression has been found in involves a yeast functional equivalent of the human tumor bone marrow biopsies of patients with SDS (Elghetany and Alter, suppressor pRb (protein retinoblastoma) (Bernstein et al., 2007). 2002). In zebrafish and mouse models of DC, p53 is upregulated Some p53-independent responses to ribosomal stress are outlined (Pereboom et al., 2011; Zhang et al., 2012; Gu et al., 2008). below. Knockdown of the zebrafish homolog of the gene responsible for NAIC results in p53 activation (Wilkins et al., 2013). Reduced ribosome number or activity Reduced ribosome activity might contribute to DBA phenotypes. p53-independent responses to ribosomal stress Slowed proliferation of human RPL11- or RPL5-deficient lung Recent studies of transcriptional responses to RP deficiency induced fibroblasts was attributed to reduced ribosome content and in p53-negative human cell lines revealed changes in the expression translational capacity that suppressed the accumulation of cyclins of genes involved in metabolism, proliferation, apoptosis and cell and thus cell cycle progression (Teng et al., 2013). Although redox homeostasis (Aspesi et al., 2014). Studies of murine reduced ribosome number and activity might still be sufficient for embryonic stem cells haploinsufficient for Rps19 and Rpl5 survival, they might be not adequate to translate some mRNAs that indicate the presence of p53-independent cell-cycle and erythroid- have stringent conditions for initiation of translation (Ludwig et al., differentiation defects (Singh et al., 2014). Zebrafish studies also 2014). Moreover, ribosomal stress affects translation of internal point to both p53-dependent and -independent responses to RP ribosome entry site (IRES)-containing RNAs (Armistead and Disease Models & Mechanisms

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Triggs-Raine, 2014; Horos et al., 2012) (Box 1). Thus, ribosomal also respond to RP deficiency by the inhibition of proliferation and stress might lead to changes in the spectrum of translated mRNAs, by the upregulation of pro-apoptotic genes (Aspesi et al., 2014). which could differ between tissues. Metabolism Ribosomes as a platform for kinase signaling Rpl11-deficient zebrafish and RPS19-deficient mouse fetal liver Ribosomes were suggested to be a platform for signaling molecules cells downregulate genes that encode glycolytic enzymes and after the discovery that RAC1, a receptor for protein kinase C, is upregulate genes involved in aerobic respiration (Danilova et al., a constituent of the ribosome (Nilsson et al., 2004). The PIM1 2011). This is consistent with a known effect of p53 activation proto-oncogene serine/threonine kinase is also associated with (Matoba et al., 2006). Glycolysis provides not only ATP but also ribosomes (Chiocchetti et al., 2005). In human erythroid cell lines, intermediates for the biosynthesis of carbohydrates, proteins, lipids RP deficiency and other types of ribosomal stress such as inhibition of and nucleic acids, and is increased in normal cells during rRNA production at various steps with actinomycin, camptothecin or proliferation and in tumors, which is known as the Warburg effect cisplatinum cause a decrease of PIM1 levels, leading to inhibition of (Lunt and Vander Heiden, 2011). Suppressed glycolysis in RP- the cell cycle (Iadevaia et al., 2010). A pathway conserved from yeast deficient cells means that lower levels of intermediates are available to mammals involves the regulation of growth-related processes via for the biosynthesis of organic macromolecules. Moreover, in the interaction between ribosomes and TORC2 (see Box 1 for a Rpl11-deficient zebrafish, the expression of genes encoding glossary of terms), and ribosomal defects can inhibit TORC2 enzymes that are involved in the biosynthesis of lipids and (Zinzalla et al., 2011). These mechanisms might contribute to the proteins is downregulated, whereas expression of genes involved phenotypes of RP-deficient cells. in catabolism is upregulated (Danilova et al., 2011). Changes in the expression of genes involved in metabolism have also been found in p53-negative RP-deficient cells (Aspesi et al., 2014). RPs as regulators Autophagy is a mechanism used by normal cells, but especially In addition to their structural role in the ribosome, RPs are involved by stressed cells, to replenish their energy and biosynthetic in other cellular processes. Several RPs modulate the activity of intermediates. Consistently, disrupted ribosome biogenesis leads regulatory proteins such as NF-κB, p53, p21, Myc and nuclear to the activation of autophagy both in human cells and in zebrafish receptors, whereas others regulate translation by binding to embryos (Heijnen et al., 2014; Boglev et al., 2013). untranslated regions of mRNAs (Warner and McIntosh, 2009). Expression of genes encoding enzymes involved in ROS Some RPs act as a regulatory component of the ribosome to confer detoxification, such as superoxide dismutase 2, is decreased both in transcript-specific translational control. For example, translation of Rpl11 zebrafish mutants and in p53-negative RPS19-deficient human vesicular stomatitis virus mRNAs depends on RPL40 (Lee et al., cell lines (Aspesi et al., 2014; Danilova et al., 2011). These results 2013). This RP is also required for translation of some cellular indicate that RP-depleted cells are predisposed to oxidative stress. mRNAs, including those involved in the stress response (Lee et al., A common metabolic change in DBA is the upregulation of ADA 2013). RPS25 facilitates interactions of the ribosome with viral (Fargo et al., 2013). Overexpression of ADA causes ATP depletion IRES elements (Landry et al., 2009). In Rpl38-mutant mouse (Chen and Mitchell, 1994), which was also found in RP-deficient embryos, global translation is unchanged but the translation of a zebrafish (Danilova et al., 2014). ATP shortage leads to the subset of homeobox mRNAs is perturbed (Kondrashov et al., 2011). activation of AMPK, which inhibits biosynthesis and translation An imbalance in RP regulatory functions might contribute to the (Hardie et al., 2012). Translation is decreased in RP-deficient cells pathophysiology of DBA. For example, increased frequency of (Cmejlova et al., 2006; Gazda et al., 2006a; Signer et al., 2014) physical malformations in individuals with mutations in RPL5 and despite the overstimulation of mTOR and S6 kinase that is found in RPL11 (Gazda et al., 2008) might be related to impaired regulatory these cells (Heijnen et al., 2014; Payne et al., 2012). functions of these RPs. Another important metabolic change in RP-deficient cells is the dysregulation of the insulin pathway. Recent data suggest that RP Pathological changes in RP-deficient cells deficiency leads to the inhibition of this pathway through a Both p53-positive and p53-negative animal cells respond to RP mechanism that is reminiscent of insulin resistance (Heijnen et al., deficiency by global homeostatic changes that serve to stall 2014). Increased glucose levels and the upregulation of pre- proliferation and to create conditions for cellular repair, senescence proinsulin are also observed in zebrafish heterozygous for the rpl11 or apoptosis depending on the cell type and the intensity of the stress mutation (Danilova et al., 2011). Known processes contributing to signal. Here, we outline the most significant changes that have been the development of insulin resistance include p53 activation, observed in RP-deficient cells, both p53-dependent or -independent. increased ROS and increased production of proinflammatory A better understanding of these changes might help to find new cytokines (Minamino et al., 2009). treatments for DBA and other ribosomopathies. Structural changes Cell cycle arrest and apoptosis Multiple changes in the expression of genes that encode structural An increase in the expression of genes responsible for cell cycle proteins have been found in an rpl11 zebrafish mutant, such as the arrest has been reported in various DBA models (Barkic et al., 2009; downregulation of and the membrane components of red Danilova et al., 2008b; Miyake et al., 2008). Slower cell blood cells (Danilova et al., 2011). In DBA patients, the composition proliferation might promote repair and improve survival (Barkic of the membranes of red blood cells also changes, with the et al., 2009). However, when repair is not possible, cells activate accumulation of non-red blood cell proteins (Pesciotta et al., 2014). apoptosis. Increased apoptosis has been reported in animal and cellular models of DBA, as well as in cells from DBA patients Activation of the innate immune system (Avondo et al., 2009; Danilova et al., 2011, 2008b; Gazda et al., The upregulation of genes involved in interferon and TNFα (tumor

2006a; Miyake et al., 2008; Perdahl et al., 1994). p53-negative cells necrosis factor alpha) signaling has been reported in red blood cell Disease Models & Mechanisms

1019 REVIEW Disease Models & Mechanisms (2015) 8, 1013-1026 doi:10.1242/dmm.020529 progenitors and in fibroblasts obtained from DBA patients (Avondo defects of erythroid cells in DBA (Ludwig et al., 2014). In support et al., 2009; Gazda et al., 2006a), as well as in Rpl11-deficient of this, when GATA1 protein levels were increased in bone marrow zebrafish (Danilova et al., 2011). TNFα signaling has been found to mononuclear cells from DBA patients or in primary human contribute to the hematopoietic failure observed in RPS19-deficient hematopoietic cells with reduced levels of RPL11 or RPL5, red human hematopoietic progenitors and in Rps19-deficient zebrafish blood cell production and cell differentiation was improved embryos (Bibikova et al., 2014). In RP-deficient zebrafish, the (Ludwig et al., 2014). The mechanism of GATA1 suppression in upregulation of several components of the complement system and DBA might involve the highly structured 5′ end of GATA1 mRNA the downregulation of complement inhibitors have been reported that requires stringent conditions for translation initiation (Ludwig (Danilova et al., 2011; Jia et al., 2013). Notably, monocytes from et al., 2014). These findings show that GATA1, although not the the bone marrow of DBA, DC and SDS patients have an altered only target of translation dysregulation in DBA, is an important response to bacterial antigens (Matsui et al., 2013). factor in mediating the erythroid-specific defect observed in this condition. Cancer The increased incidence of cancer in an RP-deficient environment Increased proliferation causes increased demand for ribosomes might be attributed to upregulation both of factors that induce It has been suggested that, because the chromatin of erythroid cells apoptosis and factors that promote proliferation, as was reported becomes condensed and transcriptionally inactive prior to for zebrafish rpl11 mutants (Danilova et al., 2011). Also, in enucleation, the rapidly proliferating immature erythroid cells hematopoietic progenitors from DBA patients, some members of require very high ribosome synthesis rates in order to produce the RAS family are upregulated, whereas the tumor suppressors enough ribosomes to last for the duration of the mature erythrocyte breast cancer 2, early onset (BRCA2) and retinoblastoma 1 (RB1) life cycle (Sieff et al., 2010). This hypothesis was confirmed in fetal are downregulated (Gazda et al., 2006a). The RP-deficient liver cells of RP-deficient mice, in which cell numbers increased condition therefore resembles an after-irradiation environment in three to fourfold while RNA content increased sixfold, suggesting which both pro-apoptotic and survival factors are upregulated; an accumulation of an excess of ribosomes during early such an environment confers a dramatic selective advantage to erythropoiesis (Sieff et al., 2010). p53-deficient cells that avoid cell cycle arrest and apoptosis In addition, rapid proliferation itself might make cells vulnerable (Marusyk et al., 2010). Selection for p53-negative cells takes place to stressful conditions. It is known that irradiation does the most at the level of progenitor cells, which are the most sensitive to damage to hematopoietic and epithelial tissues, including the stress. Interestingly, in RP-deficient zebrafish tumors, the tp53 thymus, spleen and small intestine, where cell turnover is the fastest; gene is wild type and transcribed, but the p53 protein is not this sensitivity correlates with the increased induction of p53 in synthesized (MacInnes et al., 2008). The mechanism of the these tissues (Gottlieb et al., 1997; Komarova et al., 1997). selective suppression of p53 translation in these tumors is currently unknown. Erythroid-specific alteration in transcription, splicing or translation Some data suggest that erythroid progenitors respond to stress by Failure of erythropoiesis in DBA selective changes in transcription, splicing or in the translation of a Erythroid failure is the most distinctive feature of DBA, with specific set of genes. The decreased transcription of a crucial decreased proliferation of erythroid progenitors (Ebert et al., 2005; erythropoietic factor, MYB (v-myb avian myeloblastosis viral Flygare et al., 2005) and their increased sensitivity to apoptosis oncogene homolog), has been reported in erythroid progenitors (Perdahl et al., 1994). In RP-deficient erythroid progenitors, p53 is from DBA patients (Gazda et al., 2006a) and in Rpl11-deficient selectively activated (Dutt et al., 2011). Hematopoietic defects zebrafish (Danilova et al., 2011). Myb protein levels are also appear already at the stage of hematopoietic stem cells (HSCs). reduced in RP-deficient mouse fetal liver cells (Sieff et al., 2010). Their number in RP-deficient zebrafish is decreased (Danilova Splicing is a process that can be selectively affected under stress et al., 2011) and induced pluripotent stem cells derived from DBA (Dutertre et al., 2011). Immature erythroid cells from DBA patients patients exhibit impaired differentiation (Garcon et al., 2013). In this express alternatively spliced, non-functional isoforms of FLVCR1 section, we discuss factors that might contribute to erythroid defects (feline leukemia virus subgroup C cellular receptor 1), a heme in DBA. exporter required for erythropoiesis (Rey et al., 2008). These results suggest that FLVCR1 insufficiency contributes to the erythropoietic Decreased GATA1 levels defects seen in individuals with DBA. Mutations in the erythroid-specific gene GATA1 (GATA binding There are indications of increased translational suppression of protein 1) leads to a phenotype that is currently classified as DBA erythroid-specific genes in RP-deficient organisms in comparison (Sankaran et al., 2012). Although individuals with GATA1 to other genes. In addition to the selective suppression of GATA1 mutations do not have congenital malformations, their erythroid translation discussed above, selective suppression of globin phenotype is very similar to that of DBA-affected individuals with translation has been reported in RP-deficient zebrafish (Zhang RP mutations. Recent studies demonstrated that RP knockdown in et al., 2014). Decreased protein levels of some common factors that primary human hematopoietic progenitors or in an erythroid cell are normally highly expressed in differentiating erythroid cells, line leads to decreased GATA1 translation (Bibikova et al., 2014; such as BCL2-associated athanogene (BAG1), which encodes a Ludwig et al., 2014). A decreased level of Gata1 protein was also HSP70 co-chaperone, and cold shock domain containing E1, found in zebrafish mutant for rpl11 (Bibikova et al., 2014). RNA-binding (CSDE1), have been reported in mouse RP-deficient Moreover, a global gene expression profiling of erythroid cells from erythroblasts and erythroblasts cultured from DBA patients (Horos DBA patients showed that the expression of GATA1 transcriptional et al., 2012). target genes is downregulated in these cells (Ludwig et al., 2014), Additional suppression of GATA1 protein levels might be which is consistent with decreased GATA1 activity. These data mediated through the upregulation of TNFα in RP-deficient cells suggest that a decreased level of GATA1 protein underlies many (Bibikova et al., 2014). Disease Models & Mechanisms

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Metabolic and structural vulnerability of erythroid cells to the importance of a patient’s overall genetic background. Below In addition, red blood cells have a distinct physiology that might we discuss the origin of specific defects and factors that might cause make them selectively vulnerable to alterations in several common phenotypic variability in ribosomopathies. pathways. An example is the shift from glycolysis to aerobic respiration, which takes place in all cells after p53 upregulation Role of ΔNp63 in DBA congenital malformations (Matoba et al., 2006), including in RP-deficient cells (Danilova Extra digits or phalanges are among the notable congenital defects et al., 2011). Although this shift occurs in all cells of RP-deficient observed in individuals with DBA (Halperin and Freedman, 1989) organisms, only erythrocytes rely almost exclusively on glycolysis andintheRpl24 mouse mutant (Oliver et al., 2004) (Fig. 1). As and they are, therefore, selectively affected by this change. discussed above in the section devoted to the phenotypes of DBA Many membrane and cytoskeleton changes seen in DBA animal patients and animal models, eye defects are also a common models are not specific to erythroid cells but occur in all tissues feature. The combination of these defects suggests that they might (Danilova et al., 2011). However, defects originating from these originate during development from a misbalance between neural changes might particularly affect erythrocytes because of the and non-neural ectoderm in the early embryo. Development of especially high requirements for a strong and deformable cell non-neural ectoderm is controlled by a member of the p53 protein structure to withstand circulation in narrow blood capillaries family, ΔNp63. Besides its role during development, ΔNp63 is a (Steiner and Gallagher, 2007). p53 target gene and is upregulated in response to p53 activation Furthermore, erythroid cells mature in association with (Bourdon, 2007). In the gastrulating zebrafish embryos, ΔNp63 macrophages; alterations in membranes of RP-deficient erythroid expression is localized to the ventral side of the embryo and marks cells might lead to their increased elimination by macrophages. non-neural ectoderm; its overexpression leads to the expansion of non-neural ectoderm and to the suppression of neural structures Tissue specificity and variability of defects in (Bakkers et al., 2002). Later in development ΔNp63 controls the ribosomopathies development of limbs and its overactivity can lead to the DBA is characterized by broad phenotypic variability; moreover, duplication of limb structures (Mills et al., 1999). In Rps19- some first-degree relatives of DBA-affected individuals with RPS19 deficient zebrafish embryos, the area of ΔNp63 expression mutations carry an identical mutation but display no observable expands to the dorsal side (Danilova et al., 2008b) (Fig. 4A). phenotype (Willig et al., 1999). Congenital malformations are found This corresponds to the expansion of non-neural ectoderm into the in ∼40% of DBA patients and they differ between affected neural field, as confirmed by hybridization with gata2, a marker of individuals. No correlation between the type of mutation and the non-neural ectoderm (Fig. 4A). Shrinkage of the neural field phenotype has been found among individuals with DBA with affects mostly the forebrain and eyes, as illustrated by the RPS19 mutations (Willig et al., 1999). Similar to DBA, the expression of pax2, which has a key role in the development of the phenotype of TCS is highly variable and some mutation carriers, CNS, eyes, urogenital tract and kidneys (Krauss et al., 1991) parents of patients, are only mildly affected (Dixon and Dixon, (Fig. 4B,C). 2004; Teber et al., 2004). These findings point to a role for genetic It would be interesting to know whether ΔNp63 upregulation modifiers in the penetrance and severity of both DBA and TSC, and contributes to eye defects and to overgrown wings in Drosophila

Fig. 4. Origin of developmental defects in RP-deficient zebrafish embryos. (A) ΔNp63 expression (black arrow, upper panels) in early zebrafish embryos defines the non-neural ectoderm field and overlaps with a marker of non-neural ectoderm, gata2 (black arrow, lower panels). In Rps19-deficient zebrafish [in which rps19 expression has been knocked down with a morpholino oligonucleotide (MO)], this field is expanded (right upper and lower panels). Staining with a probe for goosecoid (gsc), necessary for the formation of the dorsoventral axis of the embryo, marks the dorsal side (red arrow). Arrowheads point to the neural field. This is an in situ hybridization image at gastrulation, 80% epiboly (Box 1). Dorsal is to the right. wt, wild type. (B) Expression of pax2, which has a key role in the development of the CNS, eyes, urogenital tract and kidneys, is altered in Rps19-deficient zebrafish embryos. Arrows and arrowheads point, respectively, to forebrain and eye fields, which are contracted in Rps19-deficient embryos. This is an in situ hybridization image at 16 hpf. (C) Schematics showing how expansion of non-neural ectoderm in early zebrafish embryos leads to the contraction of the neural field, especially the area of the forebrain and eye. This research was © originally published in Blood (Danilova et al., 2008b). American Society of Hematology. Disease Models & Mechanisms

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Minute mutants, or to the eye defects and the duplication of fingers the development of T cells, whereas knockdown of rpl22l1 impairs and phalanges in mice and humans with RP deficiency. the development of HSCs. Mice also have Rpl22 and Rpl22L1 ΔNp63 is a part of the p53 protein family network, which paralogs; Rpl22L1 is expressed at a low level in all tissues except involves hundreds of proteins that are differentially regulated in pancreas and its expression is regulated by Rpl22 (O’Leary et al., various tissues during development (Danilova et al., 2008a). 2013). A recent study found that other RPs and their paralogs are Because both environmental and genetic factors can modulate this differentially expressed in mouse embryos; the more recently network, ΔNp63 and other members of this network might evolved RP paralogs showed a much greater level of tissue-specific contribute to phenotypic variation of ribosomopathies. A expression (Wong et al., 2014). multitude of other modifiers might theoretically affect cellular Not only RPs but other genes involved in ribosome biogenesis response to ribosomal stress, as was recently discussed (Farrar and also show tissue-specific expression. Cirhin, mutations in which Dahl, 2011). cause childhood cirrhosis (NAIC), is expressed in embryonic mice at the highest levels in the liver, with much weaker expression in Mechanisms causing dissimilar phenotypes in DBA and TCS other tissues (Chagnon et al., 2002). In zebrafish, cirhin expression p53 activation contributes to the clinical features of both DBA and is also high in the developing liver (Wilkins et al., 2013). TCS. However, these two diseases have completely different There might be other ways of creating a different ribosome. phenotypes. In contrast to the bone marrow failure and various For example, RPs, rRNAs and various accessory factors involved in tissue defects seen in DBA, TSC defects are restricted to the pre-rRNA processing are modified post-transcriptionally and, craniofacial tissues that originate from the neural crest. Most cells in hypothetically, variations of these processes could also create the embryo seem to survive TCOF1 or PolI/III insufficiencies altered ribosomes. A comprehensive review of these and other despite their crucial role in ribosome biogenesis. To explain the mechanisms of the formation of specialized ribosomes have been paradox, it was hypothesized that different progenitor cell recently presented (Xue and Barna, 2012). populations at defined points during embryonic development have If ribosomes differ among tissues then mutations in some genes diverse ribosome requirements (James et al., 2014). involved in ribosome biogenesis could selectively affect only a Another explanation for this paradox might reside in the subset of ribosomes. spatiotemporal expression patterns of the affected genes. In zebrafish embryos, tcof1 is expressed at a high level at the one- Current and potential therapeutic approaches to DBA cell stage, which suggests the maternal origin of its mRNA (Weiner In spite of recent progress in understanding the pathophysiology et al., 2012). Moreover, tcof1 has the highest level of expression at of DBA, its treatment is still based on corticosteroids, red blood this stage. Therefore, enough protein can be produced in the embryo cell transfusions and HSC transplantation (Vlachos et al., 2014). from the maternal mRNA to pass through the early stages of DBA is a complex disease; it is rather a tree of pathologies. The development. Neural crest cells in zebrafish develop and start to only way to tackle its cause is with gene therapy and this migrate relatively late in development, at ∼15 hours post- possibility is being investigated. Replacement of the defective fertilization (hpf) through 24 hpf, and form the craniofacial RPs in experimental systems has produced positive results skeleton, the most affected structure in TCS, even later, at (Flygare et al., 2008; Garcon et al., 2013). This approach holds ∼72 hpf (Schilling and Kimmel, 1994). At this stage, all zebrafish promise for the future. Until then, the individual consequences of tissues and organs, including HSCs, are already formed and, RP deficiency can be targeted. therefore, p53 activation can affect only craniofacial tissues. polr1d, The most damaging effect of RP deficiency is the upregulation of the zebrafish ortholog of another gene mutated in TCS, is also p53. Direct suppression of p53 in DBA is often considered expressed maternally in zebrafish embryos [Thisse, B., Thisse, C. unacceptable owing to an increased risk of cancer. However, (2004) Fast Release Clones: A High Throughput Expression this approach might still be feasible, as illustrated by the effects Analysis. ZFIN Direct Data Submission (http://zfin.org)]. of cenersen in 5q-MDS. Cenersen is a 20-mer antisense In contrast to the genes mutated in TCS, only a trace expression of oligonucleotide complementary to TP53 exon 10; it suppresses rpl11 mRNA can be detected at the one-cell stage in zebrafish p53 expression and restores erythropoiesis in 5q-MDS (Caceres embryos; it increases from approximately 6 hpf, pointing to the et al., 2013). Zebrafish data suggest that corticosteroids might act, beginning of Rpl11 production (Danilova et al., 2011). p53 in part, by decreasing the expression of p53 (Danilova et al., 2011). upregulation in RP-deficient zebrafish can also be detected from An indirect way of suppressing p53 activation is suggested by ∼6 hpf (Danilova et al., 2008b), pointing to the onset of problems zebrafish studies. Treatment of RP-deficient zebrafish with a with ribosome biogenesis at this early time point. Early onset of p53 mixture of nucleosides decreased the levels of p53, improved upregulation in RP-deficient embryos would affect the formation of hematopoiesis and diminished developmental defects (Danilova all tissues and organs. The difference in timing of p53 activation et al., 2014). The efficiency of nucleoside treatment can be might therefore account for the multitude of defects in DBA versus attributed to the fact that stress changes metabolism. Non-stressed the relatively restricted phenotype of TCS. cells use de novo nucleotide synthesis to make their DNA; therefore, very few nucleosides from food end up in the DNA of healthy Specialized ribosomes as drivers of tissue-specific defects people. Because stressed cells start to use salvage pathways (Austin Recent studies suggest that composition of ribosomes can vary et al., 2012), nucleoside supplementation improves outcomes in between tissues. In yeast, many RPs are duplicated and paralogs physiologically stressed patients (Hess and Greenberg, 2012). have different functional roles (Komili et al., 2007). Based on these Nucleoside treatment in general decreases replication stress and data, Komili et al. proposed the existence of many different forms of DNA damage (Bester et al., 2011). Nucleosides in infant formulas functionally distinct ribosomes. Recent studies revealed that some have a positive effect on development (Singhal et al., 2010). animal RPs also have paralogs with specifically tailored functions. Nucleoside mixtures are now available as over-the-counter Zebrafish Rpl22 and Rpl22L1 play different roles during supplements. Their application might benefit DBA patients during hematopoiesis (Zhang et al., 2013). Knockdown of rpl22 blocks the acute phase of the disease. Disease Models & Mechanisms

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Inflammation is another damaging result of RP deficiency. Anti- Funding inflammatory properties of lenalidomide, an immunomodulatory This work was supported by National Institutes of Health grants R01 HL107558 and agent used to treat several types of cancer, might be responsible for K02 HL111156 to H.T.G. its positive effects in RP-deficient models (Keel et al., 2012; Narla References et al., 2011). The TNFα inhibitor etanercept also improves the Ak, P. and Levine, A. J. (2010). P53 and NF-kB: different strategies for responding condition of RP-deficient zebrafish (Bibikova et al., 2014). The to stress lead to a functional antagonism. FASEB J. 24, 3643-3652. upregulation of the complement system in zebrafish DBA models Amsterdam, A., Nissen, R. M., Sun, Z., Swindell, E. C., Farrington, S. and Hopkins, N. (2004a). Identification of 315 genes essential for early zebrafish (Danilova et al., 2011; Jia et al., 2013) suggests that complement development. Proc. Natl. Acad. Sci. USA 101, 12792-12797. inhibitors might also merit investigation as a potential treatment for Amsterdam, A., Sadler, K. C., Lai, K., Farrington, S., Bronson, R. T., Lees, J. A. DBA. The careful consideration of the timing of treatments that and Hopkins, N. (2004b). Many genes are cancer genes in zebrafish. PLoS Biol. 2, e139. target p53 or inflammation will still be necessary because these Armistead, J. and Triggs-Raine, B. (2014). Diverse diseases from a ubiquitous pathways usually antagonize each other (Ak and Levine, 2010). process: the ribosomopathy paradox. FEBS Lett. 588, 1491-1500. An important metabolic change in RP-deficient cells that Aspesi, A., Pavesi, E., Robotti, E., Crescitelli, R., Boria, I., Avondo, F., Moniz, H., deserves extra attention is the dysregulation of the insulin Da Costa, L., Mohandas, N., Roncaglia, P. et al. (2014). Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for pathway. Individuals with DBA might have a condition similar Diamond-Blackfan anemia. Gene 545, 282-289. to that of pre-diabetes, which could be targeted by the Austin, W. R., Armijo, A. L., Campbell, D. O., Singh, A. S., Hsieh, T., Nathanson, corresponding drugs. D., Herschman, H. R., Phelps, M. E., Witte, O. N., Czernin, J. et al. (2012). Nucleoside salvage pathway kinases regulate hematopoiesis by linking Another important metabolic change, the decrease in translation, nucleotide metabolism with replication stress. J. Exp. Med. 209, 2215-2228. has been recently targeted by L-leucine treatment in a DBA patient Avondo, F., Roncaglia, P., Crescenzio, N., Krmac, H., Garelli, E., Armiraglio, M., (Pospisilova et al., 2007). The treatment resulted in an increase in Castagnoli, C., Campagnoli, M. F., Ramenghi, U., Gustincich, S. et al. (2009). reticulocyte count and hemoglobin levels. Leucine treatment Fibroblasts from patients with Diamond-Blackfan anaemia show abnormal expression of genes involved in protein synthesis, metabolism and stimulates mTOR and improves anemia in a mouse model of cancer. BMC Genomics 10, 442. DBA (Jaako et al., 2012). It also decreases anemia and Bakkers, J., Hild, M., Kramer, C., Furutani-Seiki, M. and Hammerschmidt, M. developmental defects in RP-deficient zebrafish embryos and (2002). Zebrafish DeltaNp63 is a direct target of Bmp signaling and encodes a transcriptional repressor blocking neural specification in the ventral ectoderm. increases proliferation of Rps19- and Rps14-deficient erythroid Dev. Cell 2, 617-627. progenitors (Payne et al., 2012). It is not known, however, what the Barkic, M., Crnomarkovic, S., Grabusic, K., Bogetic, I., Panic, L., Tamarut, S., effect of mTOR overactivation, especially for prolonged periods of Cokaric, M., Jeric, I., Vidak, S. and Volarevic, S. (2009). The p53 tumor time, might be. Several ongoing clinical trials will determine the suppressor causes congenital malformations in Rpl24-deficient mice and promotes their survival. Mol. Cell. Biol. 29, 2489-2504. safety of this treatment in individuals with DBA. Barlow, J. L., Drynan, L. F., Hewett, D. R., Holmes, L. R., Lorenzo-Abalde, S., Conflicting signaling seems to be a characteristic of RP-deficient Lane, A. L., Jolin, H. E., Pannell, R., Middleton, A. J., Wong, S. H. et al. (2010). cells, such as the activation of p53 and inflammation. With many A p53-dependent mechanism underlies macrocytic anemia in a mouse model of human 5q-syndrome. Nat. Med. 16, 59-66. modifiers involved, pathways leading from RP deficiency to Bermejo, R., Lai, M. S. and Foiani, M. (2012). Preventing replication stress to erythroid defects in one patient might not be exactly the same as maintain genome stability: resolving conflicts between replication and in another patient. Moreover, these pathways might differ in the transcription. Mol. Cell 45, 710-718. same patient between the acute and remission phases of the disease. Bernstein, K. A., Bleichert, F., Bean, J. M., Cross, F. R. and Baserga, S. J. (2007). Ribosome biogenesis is sensed at the start cell cycle checkpoint. Mol. Therefore, individualized therapy is likely to be necessary for the Biol. Cell 18, 953-964. successful treatment of DBA. Bester, A. C., Roniger, M., Oren, Y. S., Im, M. M., Sarni, D., Chaoat, M., Bensimon, A., Zamir, G., Shewach, D. S. and Kerem, B. (2011). Nucleotide Conclusion deficiency promotes genomic instability in early stages of cancer development. Cell 145, 435-446. A tremendous amount of knowledge about DBA has been Bibikova, E., Youn, M.-Y., Danilova, N., Ono-Uruga, Y., Konto-Ghiorghi, Y., accumulated since the first discovery of RP mutation in DBA. Ochoa, R., Narla, A., Glader, B., Lin, S. and Sakamoto, K. M. (2014). TNF- DBA studies have stimulated the field of ribosome biogenesis, mediated inflammation represses GATA1 and activates p38 map kinase in RPS19-deficient hematopoietic progenitors. Blood 124, 3791-3798. resulting in a better understanding of the mechanisms of pre-rRNA Boglev, Y., Badrock, A. P., Trotter, A. J., Du, Q., Richardson, E. J., Parslow, processing and of ribosome subunit formation. The roles of the p53 A. C., Markmiller, S. J., Hall, N. E., de Jong-Curtain, T. A., Ng, A. Y. et al. and p53-independent pathways in DBA have also become better (2013). Autophagy induction is a TOR- and p53-independent cell survival defined. Multiple diseases have been found to originate from defects response in a zebrafish model of disrupted ribosome biogenesis. PLoS Genet. 9, e1003279. in ribosome biogenesis. However, treatment options lag behind this Bolze, A., Mahlaoui, N., Byun, M., Turner, B., Trede, N., Ellis, S. R., Abhyankar, newfound knowledge. 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