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Review Series
BONE MARROW FAILURE
Marrow failure: a window into ribosome biology
Davide Ruggero1 and Akiko Shimamura2,3,4
1
Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA; 2Clinical Research Division, Fred
Hutchinson Cancer Research Center, Seattle, WA; 3Pediatric Hematology/Oncology, Seattle Children’s Hospital, Seattle, WA; and 4Department
of Pediatrics, University of Washington, Seattle, WA
Diamond-Blackfan anemia, ShwachmanDiamond syndrome, and dyskeratosis
congenita are inherited syndromes characterized by marrow failure, congenital
anomalies, and cancer predisposition.
Genetic and molecular studies have uncovered distinct abnormalities in ribosome
biogenesis underlying each of these 3
disorders. How defects in ribosomes, the
essential organelles required for protein
biosynthesis in all cells, cause tissuespecific abnormalities in human disease
remains a question of fundamental scientific and medical importance. Here we
review the overlapping and distinct clinical
features of these 3 syndromes and discuss
current knowledge regarding the ribosomal pathways disrupted in each of these
disorders. We also explore the increasing
complexity of ribosome biology and how
this informs our understanding of developmental biology and human disease.
(Blood. 2014;124(18):2784-2792)
Introduction
The study of inherited bone marrow failure syndromes has yielded
unique insights into global molecular pathways regulating hematopoiesis and clonal evolution. These clinically heterogeneous syndromes are
all characterized by marrow failure, frequent physical anomalies, and
cancer predisposition.1 Impairment of ribosome biogenesis is emerging
as a common molecular pathogenic mechanism underlying many
of these marrow failure syndromes. Ribosomes are ribonucleoprotein
complexes that catalyze protein synthesis by translating the mRNA
message into its cognate protein product. This basic cellular function is
required by all cells and is essential for life. Previously, ribosomes had
been largely relegated to the status of passive cellular drones in the
ranks of the protein translational corps. The unexpected revelation that
disruption of such an essential cellular function could preferentially
affect specific tissues in human disease stimulated a reexamination
of ribosome biology. The relevance of ribosome pathology for more
common diseases was highlighted by the identification of acquired
somatic mutations affecting ribosomal proteins in myelodysplastic
syndrome (MDS) and leukemias arising in the general population. The
question of how ribosomal abnormalities cause marrow failure and
cancer predisposition is of fundamental biological and clinical interest.
In this review, we discuss the clinical, genetic, and molecular
features of marrow failure ribosomopathies (Figure 1) and explore
the clinical implications of recent advances in our understanding
of ribosomal functions in cellular and developmental biology. There
are prior excellent reviews for information regarding other nonhematologic ribosomal diseases.
Overview of ribosome biogenesis and
protein synthesis
Ribosome biogenesis is a highly-regulated, complex process.2
Eukaryotic ribosomes are comprised of 2 subunits: the small 40S
Submitted March 31, 2014; accepted May 27, 2014. Prepublished online as
Blood First Edition paper, September 18, 2014; DOI 10.1182/blood-2014-04526301.
2784
subunit and the large 60S subunit. These subunits join together to
form the active 80S ribosome. Ribosomes contain 4 structural
ribosomal RNAs (rRNAs). The 40S subunit contains the 18S rRNA,
and the large 60S subunit contains the 28S, 5.8S, and 5S rRNAs.
These rRNAs are complexed with approximately 79 ribosomal
proteins in eukaryotic ribosomes. Ribosome assembly is a complex
and highly regulated process using a significant investment of
cellular biosynthetic energy. More than 200 assembly factors and
small nucleolar RNAs (snoRNAs) are required to synthesize
ribosomes.
In the nucleolus, ribosomal RNA is initially transcribed into a
long 47S pre-rRNA by RNA polymerase I. 5S rRNA is transcribed
separately by RNA polymerase III. During this process, ribosomal
proteins, nonribosomal assembly/processing factors, and snoRNAs
associate with the nascent pre-rRNA to form the 90S preribosomal
particle. The binding of ribosomal proteins to the maturing preribosomal RNA is required for rRNA processing. The 47S pre-rRNA
undergoes a series of carefully orchestrated cleavage and modification events, including methylation and pseudouridylation, to form
the pre-40S and pre-60S precursor particles. These subunits are
exported to the cytoplasm, where additional maturation steps take
place before they are assembled in the active translating ribosomes.
The most general mechanism of translation initiation, cap-dependent
translation, requires the assembly of a complex of proteins, known
as eukaryotic initiation factors (eIFs), to recruit the 40S ribosomal
subunit on the 59 end (59cap structure or m7GTP) of the mRNA. The
40S bound to the ternary complex (eIF2-GTP-initiator methionyl
tRNA [Met-tRNAiMet]) forms the 43S preinitiation complex and,
with the help of the eIFs, scans the 59 untranslated region (UTR) in
search of a start codon (AUG). After scanning, the AUG codon
base pairs with the anticodon of the initiator tRNA in a site of the
ribosomal subunit known as P-site. This complex is joined by the
60S subunit to form an 80S ribosome competent for translation
elongation. This is the mechanism engaged by the majority of mRNAs
© 2014 by The American Society of Hematology
BLOOD, 30 OCTOBER 2014 x VOLUME 124, NUMBER 18
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BLOOD, 30 OCTOBER 2014 x VOLUME 124, NUMBER 18
MARROW FAILURE AND RIBOSOMOPATHIES
2785
anemia in DBA remains unclear. The subset of DBA patients who do
not respond to corticosteroids or who require high doses with
unacceptable toxicities receive chronic red cell transfusions. Transfusional iron overload remains a major cause of morbidity and
mortality in DBA. Bone marrow transplant is the only curative
treatment of marrow failure in DBA and is generally reserved for
patients for whom corticosteroids or red cell transfusions are not
viable options. A spontaneous remission rate of ;20% by age 25
has been reported.3 The molecular pathophysiology of remission
from anemia in DBA remains unknown, but such patients remain
at risk for malignancy.
Diamond-Blackfan anemia: genetics
Figure 1. Overlapping and distinct clinical features of inherited marrow failure
ribosomopathies. DBA, SDS, and DC are all characterized by marrow failure, predisposition to MDS/AML, and congenital anomalies. The primary feature of marrow
failure in DBA is red-cell aplasia, although other hematologic lineages may also
be variably affected. Although neutropenia is the most common feature of marrow
failure in SDS, all 3 lineages may be depressed. Cellular and humoral immunologic
abnormalities have been reported in DC and SDS. The spectrum of physical
anomalies in these 3 syndromes shares both overlapping and distinct features. 1
Exocrine pancreatic lipomatosis is characteristic of SDS, whereas pulmonary
fibrosis is a common characteristic of DC. The risk of soft tissue sarcomas is increased in DBA, and the risk of squamous cell carcinomas of the oropharynx and
gastrointestinal tract is elevated in DC. Data are insufficient to determine whether
solid tumor risk is elevated in SDS. More detailed descriptions of clinical phenotypes
have been reviewed.1
to initiate translation. Internal ribosome entry site (IRES)-dependent
translation is an alternative mode of translation initiation and follows
an RNA-based mechanism whereby the 40S is recruited directly
by the IRES element independent of some, or even all, of the eIFs
involved in the cap-dependent translation. The mechanism by which
translation control is perturbed in ribosomopathies still remains an
outstanding question.
Diamond-Blackfan anemia: clinical features
Diamond-Blackfan anemia (DBA) classically presents with redcell aplasia in the first year of life, with a median age of presentation
of 2 months, though, rarely, presentation may be delayed into
adulthood.3 It remains unclear why DBA patients generally do not
have development of severe anemia in utero during fetal development.4,5 The normochromic anemia is typically macrocytic with
reticulocytopenia. The bone marrow is classically normocellular,
with a paucity of erythroid precursors. The erythroid burst-forming
units (BFU-E) and erythroid colony-forming units (CFU-E) in vitro
are severely reduced, with relative sparing of the granulocyte-monocyte
colony-forming units (CFU-GM).6 The erythrocyte adenosine deaminase levels are often elevated.7,8 Approximately 50% of DBA
patients have physical anomalies such as craniofacial abnormalities,
including clefting of the lip or palate, thumb abnormalities, cardiac
malformations, and short stature.3 The risk of solid tumors, MDS,
or leukemia is elevated in DBA. The cumulative incidence of
malignancy was approximately 20% by age 46 years.9
Severe or symptomatic anemia is currently treated with corticosteroids or chronic red cell transfusions.10 Corticosteroids exert a
cell-autonomous effect on hematopoietic stem cells to improve
erythropoiesis.11 The mechanism whereby corticosteroids ameliorate
Heterozygous mutations resulting in haploinsufficiency have been
identified for 11 genes encoding ribosomal proteins RPS19, RPL5,
RPS10, RPL11, RPL35A, RPS26, RPS24, RPS17, RPS7, RPL26,
and RPL15 (Figure 2).12 Mutations in ribosomal genes account for
60% to 70% of DBA cases. Of these ribosomal gene mutations,
around 20% involve large deletions that require analysis of copy
number variation for detection.13 Ribosomal gene mutations may
be inherited in an autosomal dominant pattern or may arise spontaneously. X-linked mutations in the transcription factor GATA1,
which is essential for erythropoiesis, have also been associated with
DBA.14 Overall, it is estimated that approximately 35% of DBA
patients remain as yet genetically undefined.
Diamond-Blackfan anemia: molecular
pathophysiology
In general, the heterozygous DBA mutations result in loss of function
in a single copy of a ribosomal protein gene. A dominant-negative
mechanism has also been demonstrated in a murine model of DBA.15
The large number of ribosomal proteins mutated in DBA fail to
cluster in any specific region of the ribosome. Haploinsufficiency
or reduced expression of a ribosomal protein results in decreased
levels of the cognate 40S or 60S subunit and a defect in processing
of the ribosomal RNA precursor.16-19 The effect of decreased
ribosomal protein activity in vivo and in a tissue-specific manner
is still poorly understood. Interestingly, induced pluripotent stem
cells derived from DBA patients recapitulate the aberrant rRNA
processing, decreased ribosomal subunit levels, and impaired
hematopoiesis that characterize DBA.20 Insights from and
limitations of animal models of DBA have been recently
reviewed.21
P53 activation
P53 activation has been observed in bone marrows from DBA
patients, after depletion of ribosomal proteins in human erythroid
progenitor cells, and in murine models.22,23 P53 is a stress response
gene whose activation results in apoptosis or cell cycle arrest
and appears to contribute to the pathogenesis of marrow failure
in DBA. Because ribosomal protein haploinsufficiency results in
reduced ribosomal protein gene expression in multiple different
tissues in DBA, it remains to be demonstrated why erythroid precursors are particularly sensitive to p53 induction by ribosomal
stress.24 What is the mechanism underlying the activation of P53
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2786
RUGGERO and SHIMAMURA
BLOOD, 30 OCTOBER 2014 x VOLUME 124, NUMBER 18
Figure 2. Mutations of key ribosome components
underlying ribosomopathies. The DKC1 gene encoding dyskerin (rRNA pseudouridine synthase) is frequently
mutated in X-DC. The SBDS gene encodes the SDBS
protein and is found to be mutated in SDS. RP genes
encoding ribosomal proteins belonging to both the
large and small ribosomal subunits are found to be
mutated in DBA.
in DBA? The association of distinct ribosomal proteins with a
critical regulator of P53 stability provides one working model.
Specifically, several different ribosomal proteins, including RPS7,
RPS14, RPL5, RPL11, RPL23, and RPL26, have been shown to
interact with HDM2/MDM2.25,26 HDM2/MDM2 is a ubiquitin
ligase that associates with p53 and targets p53 for degradation by
proteosomes. Interaction of any of these ribosomal proteins with
HDM2/MDM2 inhibits the ability of HDM2/MDM2 to target p53
for degradation with consequent stabilization and increased levels
of p53, which in turn promotes apoptosis.25 Mice harboring an
Mdm2C305F mutation that impairs Mdm2 binding to Rpl5/Rpl11
lost the p53 response to actinomycin D, an inducer of ribosome
stress, while retaining a normal p53 response to DNA damage.27
The Mdm2C305F accelerated Myc-induced lymphomagenesis,27
suggesting that abrogation of the p53 response to ribosome stress
provides a potential mechanism contributing to cancer predisposition.
Advances in our understanding of ribosomal disorders in cancer have
been recently reviewed.28
Clinical, genetic, and biochemical evidence points to a unique
role for the ribosomal proteins RPL5 and RPL11. RPL5 and RPL11
appear to be central regulators of the MDM2/HDM2-p53 pathway.
RPL5, RPL11, and the 5S ribosomal RNA (rRNA) form a nascent
pre-ribosomal complex that coordinately inhibits HDM2 to upregulate
p53.29 The formation of such a complex may explain the codependence of RPL5 and RPL11 on the activation of p53.30,31 DBA patients
with RPL5 or RPL11 mutations are distinguished clinically by the
higher incidence of congenital anomalies such as cleft palate and
thumb abnormalities compared with other DBA patients.32 However, the observation that haploinsufficiency of RPL5 or RPL11
causes DBA indicates that additional pathways other than ribosomal
stress activation of the HDM2-p53 pathway likely also contribute to
DBA. Intriguingly, depletion of RPL5 or RPL11 impairs cell-cycle
progression in a p53-independent manner.33,34
Mouse21 and zebrafish35 animal models also provide evidence
for both p53-dependent and p53-independent pathways in DBA.
Indeed, p53 activation appears to be a common downstream stress
response pathway activated in response to a variety of different
mechanisms underlying nonribosomal marrow failure syndromes
such as anemia.36,37
Protein synthesis
The role of ribosomal proteins in gene expression at the translation
level is still a key question in normal physiology and disease
pathogenesis. In some DBA patients, global translation was reduced
by 48% to 73% in lymphocytes.38 Furthermore, altered translation of
59TOP mRNAs has been observed in response to deficiencies of 40S
ribosomal subunit proteins, suggesting that translation of specific
subsets of mRNAs might be preferentially altered by ribosomal
abnormalities.39 Similarly, knockdown of Rps19 or Rpl11 by about
50% in fetal liver erythroblasts resulted in alterations in the
polysome-associated mRNA pool, particularly for mRNAs
containing an internal ribosome entry site (IRES) in their
59UTR. Translational profiling of Epstein-Barr virus–immortalized
lymphoblasts from DBA patients harboring mutations in RPS19 or
RPL11 also showed differential polysome loading of a subset of
transcripts.40 Differential translational control of specific mRNA
subsets is an attractive hypothesis that provides a potential mechanism
for tissue-specific phenotypes resulting from ribosomal protein gene
haploinsufficiency.
mTOR signaling
The importance of translation control in DBA pathogenesis is also
highlighted by recent evidence that the addition of the essential
branched chain amino acid L-leucine to the lymphocytes of a subset
of DBA patients increased global translation in vitro.38 A DBA
patient with severe transfusion-dependent anemia received a trial
of leucine with subsequent improvement in her hemoglobin levels
and reticulocyte count free of transfusion support.41 Leucine also
ameliorated the anemia and reduced p53 levels in a murine Rps19
model of DBA.42 Leucine treatment improved erythroid differentiation in zebrafish rps19 and rps14 morpholino knockdown models43
and in human CD341 cells wherein RPS19 or RPS14 had been
knocked down.43 These data suggest that the beneficial effect of
L-leucine on DBA cells may be ascribed to its regulation of protein
metabolism through activation of a key kinase known as mammalian
target of rapacmycin (mTOR), a master regulator of cell growth and
protein synthesis43-45 and the resulting stimulation of translation.46
The potential effect of leucine on protein translation of specific
mRNAs in DBA has yet to be determined and clinical studies are
ongoing.45
Acquired mutations in ribosomal protein genes: 5q– MDS
An acquired MDS characterized by a 5q– cytogenetic clonal
abnormality shares clinical features with DBA.47 Clinically,
5q– syndrome presents with severe macrocytic anemia, thrombocytosis, and variable mild neutropenia. The bone marrow shows
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BLOOD, 30 OCTOBER 2014 x VOLUME 124, NUMBER 18
red cell aplasia and dysplastic hypolobulated small megakaryocytes. 5q– MDS typically follows a more indolent course, with
a lower rate of progression to acute myelogenous leukemia (AML)
compared with other MDS subtypes.
The common deleted region of 5q region contains the RPS14
gene, which encodes a protein subunit of the small 40S ribosome.
A screen using small interfering RNAs to target each gene located
within the common deleted region of 5q demonstrated that knockdown of RPS14 was sufficient to impair erythropoiesis, with relative
sparing of the megakaryocytes, thus phenocopying 5q– syndrome.48
Introduction of the RPS14 gene rescued erythroid differentiation in
primary patient bone marrow CD341 cells.48 Additional genes and
microRNAs contained within the 5q locus may additively contribute
to the disease phenotype.47
The molecular pathophysiology of this acquired myelodysplastic
syndrome involving haploinsufficiency of RPS14 shares overlap
with that of DBA.49 Indeed, a small somatic deletion of 5q including
RPS14 was identified in a 5-year-old girl who carried the clinical
diagnosis of nonclassical DBA.50 Knockdown of RPS14 resulted
in defective pre-18S rRNA processing and decreased levels of the
40S small ribosomal subunit.48 A mouse model haploinsufficient
for the Cd74 – Nid67 regions syntenic to the commonly deleted
region of 5q– resulted in a macrocytic anemia and dysplastic
megakaryocytes.51 Elevated levels of p53 were noted in the bone
marrows of the Cd74 – Nid67 haploinsufficient mice. Co-deletion
of p53 rescued erythropoiesis in vitro. Altogether, these findings
in 5q– MDS, wherein an acquired deletion of RPS14 causes erythroid
failure, support the notion that a ribosomal protein gene mutation
exerts an effect intrinsic to the hematopoietic stem cell.
Shwachman-Diamond syndrome:
clinical features
Shwachman-Diamond syndrome (SDS) is classically characterized
by marrow failure and exocrine pancreatic dysfunction.52,53 Patients
typically present early in life with neutropenia and steatorrhea;
however, a subset of patients have atypical or cryptic clinical presentations.54 Although neutropenia is the most common hematologic
abnormality, anemia and thrombocytopenia are also common. Severe
aplastic anemia may develop in a subset of patients. The bone
marrow is typically mildly dysplastic and hypocellular. Abnormalities of neutrophil chemotaxis have been reported,55,56 though
patients are able to form purulent abscesses and empyemas.57 Neutrophil
respiratory burst activity is normal.58 Immunologic abnormalities of
B- and T-cell numbers and function have been reported.59 SDS is a
multisystem disorder with variable abnormalities in other organs including the skeletal, neurocognitive, endocrine, and cardiac systems.53
A variety of congenital anomalies have been reported.54 SDS patients
are at increased risk for clonal cytogenetic abnormalities, MDS, and
AML.60,61 The prognosis of leukemia in SDS is generally poor.
Although early-onset solid tumors have been described in patients
carrying the diagnosis of SDS,62-65 data are insufficient to determine
whether SDS patients are at increased risk for solid tumors.
Hematologic complications such as severe or symptomatic
marrow failure, MDS, or AML are treated with hematopoietic stem
cell transplant. SDS patients are at increased risk for treatmentrelated complications, but outcomes with reduced-intensity conditioning regimens are promising.53 Supportive measures to
manage cytopenias include granulocyte colony-stimulating factor
for neutropenia and transfusion support of red cells or platelets.
MARROW FAILURE AND RIBOSOMOPATHIES
2787
Exocrine pancreatic insufficiency is treated with pancreatic enzyme
supplements.
Shwachman-Diamond syndrome: genetics
SDS is inherited in an autosomal recessive fashion. Approximately
90% of patients with the clinical features of SDS harbor biallelic
mutations in the SBDS gene.66 Most SBDS mutations correspond to
sequences found in an adjacent highly homologous pseudogene
and likely result from gene conversion. The pseudogene contains
mutations that disrupt protein production. The SBDS gene encodes
an evolutionarily conserved protein and is expressed across a broad
range of tissues. Although aplastic anemia has been described
in a few patients harboring only a single SBDS mutation, it is
currently unclear whether an increased risk of aplastic anemia is
seen in obligate SBDS heterozygous carriers such as parents or
grandparents.
Shwachman-Diamond syndrome:
molecular pathophysiology
Knockdown of SBDS in hematopoietic cells impairs proliferation
and hematopoietic colony formation.67-69 iPSCs derived from SDS
patients manifest deficits in exocrine pancreatic and hematopoietic
differentiation and enhanced apoptosis.70 Deletion of murine Sbds
resulted in early embryonic lethality.71 Targeted knockdown of
Sbds in mouse osteoprogenitor cells resulted in leukopenia and
lymphopenia and dysplasias of the neutrophils and megakaryocytes.72 Observations of in vitro assays for abnormalities of marrow
stroma from SDS patients have varied.73,74 A variety of cellular
phenotypes has been observed in SDS, including mitotic spindle
destabilization,75,76 Fas ligand–induced apoptosis,77 heightened
cellular stress responses78 and Rac2-mediated monocyte migration,79
decreased mitochondrial membrane potential and oxygen consumption, and increased the production of reactive oxygen species.80,81
SBDS promotes the release of EIF6 from the pre-60S ribosome,
which is required for the formation of a mature 80S functional
ribosome82-84 (Figure 2). Human SBDS associates with the 60S large
ribosomal subunit but not with mature polysomal ribosomes.85 Yeast
deficient for Sdo1, the ortholog of SBDS, results in a slow-growth
phenotype that is suppressed by Tif6 mutations that impair Tif6
binding to 60S ribosomes.82 Tif6, whose mammalian homolog is
EIF6, is implicated in the maturation and nuclear export of the 60S
subunit86,87 and sterically prevents premature joining of the 60S
subunit to the 40S subunit.88-90 Complete abrogation of Sbds expression in animal models produces polysome profiles with halfmers, a pattern that arises when 40S subunits have not associated
with the 60S subunit, and is consistent with a defect in ribosome
joining.83 Half-mers have not been observed in cells from SDS
patients, likely because of the retention of some scant SBDS
expression in SDS patients.91 SDS patient cells exhibit impaired
ribosome association in vitro.91 This observation was recapitulated with SBDS knockdown. The ribosome association defect
was rescued by the expression of wild-type but not mutant SBDS
cDNA. Knockdown of EIF6 improved ribosome association in
SDS patient cells but did not improve hematopoietic colony
formation of SBDS-deficient CD341 cells.91
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RUGGERO and SHIMAMURA
The release of eIF6 from pre-60S subunits was catalyzed in
vitro by the addition of SBDS, EFL1, and GTP.83 Introduction of
pathogenic SBDS mutations impaired EIF6 release. SBDS stimulated the GTPase activity of EFL1 in vitro. Kinetic studies of
EFL1 in the presence or absence of SBDS suggest that SBDS may
stabilize the binding of GTP to EFL1.92 Structural studies of the
SBDS protein revealed a conserved internal flexible hinge region
allowing rotation of the SBDS amino terminal domain.83 A direct
interaction between recombinant SBDS and EFL1 alters the conformation of the interacting domain of EFL1.93 These experiments
support a model wherein SBDS conformational changes couple
ELF1 GTPase activity to EIF6 release from the pre-60S subunit.
An important role in malignancy for ribosomal maturation
defects akin to those in SDS was highlighted by the demonstration
in pediatric T-cell acute lymphoblastic leukemia of recurrent
acquired mutations of arginine 98 or histidine 123 in RPL10.94 Expression of these RPL10 mutations resulted in reduced proliferation,
impaired ribosome biogenesis, and abnormal nuclear accumulation
of Nmd3 and Tif6 in yeast. These defects were ameliorated by
introducing a mutation in Nmd3 that weakened its binding to the
ribosome, suggesting that the RPL10 mutations affected the release
of Nmd3 and Tif6.
Knockdown of SBDS expression using shRNAs in HEK293
cells resulted in alterations in mRNA transcript levels and mRNA
polysome-loading, suggesting that disruption of ribosome biogenesis by SBDS may affect translation of specific mRNA subsets.95
Yeast models, wherein ribosomal joining is impaired by decreased
levels of 60S subunits, manifest altered translation of specific
mRNAs.96 Why distinct clinical phenotypes arise from mutations
in ribosomal protein genes vs genes affecting ribosome assembly
remains an intriguing question.
Dyskeratosis congenita: clinical features
Dyskeratosis congenita (DC) is an inherited marrow failure
syndrome whose diagnosis was originally based on the clinical
triad of dystrophic nails, mucosal leukoplakia, and a reticular or
mottled rash.97 The phenotype of DC is now recognized to be
broad, with many atypical or cryptic presentations. DC patients
may also affect additional organ systems including the pulmonary, gastrointestinal, skeletal, neurologic, immunologic, and
ophthalmologic systems.1,98 Congenital physical anomalies are also
common. Cytopenias may involve any or all of the hematologic
lineages. The bone marrow is typically hypocellular, with mild
dysplasias at baseline. Patients with DC are at increased risk for
MDS, AML, and solid tumors, namely adenocarcinomas of the
head and neck and gastrointestinal tract.99
Marrow failure may respond to androgens such as oxymetholone.
Androgens have been reported to stimulate telomerase.100-102 Premature telomere shortening is a key feature of DC.97 Hematopoietic
stem cell transplant is the only curative therapy for the hematologic
complications of DC, but it does not treat the life-threatening
complications of other organ systems such as pulmonary fibrosis.103
Dyskeratosis congenita: genetics
To date, 9 genes have been reported to cause DC: DKC1, TINF2, TERC,
TERT, WRAP53 (TCAB1), CTC1, RTEL1, NHP2, and NOP10.97 The
BLOOD, 30 OCTOBER 2014 x VOLUME 124, NUMBER 18
mode of inheritance may be autosomal dominant (TINF2, TERC,
TERT), autosomal recessive (TERT, WRAP53/TCAB1, CTC1,
RTEL1, NHP2, NOP10), or X-linked (DKC1). Mutations in these
9 genes account for approximately 50% of patients who fulfill
clinical criteria for DC.
Dyskeratosis congenita: molecular
pathophysiology
Although disruption of telomere maintenance is central to the
pathogenesis of DC, a subset of genes found mutated in DC plays
an important role in both ribosome and telomere activity.104-106
Interestingly, approximately half of the genes mutated in DC
(DKC1,NHP2,NOP10, and WRAP53/TCAB1) encode for proteins
associated with abundant regulatory noncoding RNAs (ncRNAs)
termed H/ACA small RNAs.107 H/ACA small RNAs lie at the
nexus of several important RNA-based cellular processes implicated
in telomere, ribosome, and splicing biology.107,108 One wellcharacterized example of an H/ACA small RNA found mutated
and deregulated in several forms of DC is the telomerase RNA
component (TERC) that plays an essential role in telomere
maintenance.105,109 An outcome of deregulated TERC is the agedependent shortening of telomeres, which is one common feature
of DC.110 Importantly, the evolutionary conserved RNA-binding
proteins encoded by DKC1,NHP2,NOP10, and WRAP53/TCAB1)
associate with hundreds of additional H/ACA small RNAs to form
H/ACA small ribonucleoprotein (RNP) complexes involved in
modifying ribosomal RNA (rRNA) and small nuclear RNA
(snRNA).107,111,112 Therefore, in addition to a role in telomere
maintenance mediated by the stabilization of TERC, a large subset of
genes mutated in DC exhibit additional RNA-based cellular
functions.
Importantly, the DKC1 gene, encoding the pseudouridine synthase
dyskerin, is mutated in the most common form of DC, X-linked
dyskeratosis congenita (X-DC), as well as in the clinically severe
variant of DC known as Hoyeraal-Hreidarsson (HH) syndrome.113,114
Notably, the most well-characterized role of dyskerin is the modification of pseudouridine residues on rRNA106,107,115-117 (Figure 2).
The largest subgroup of dyskerin-associated H/ACA small RNAs,
termed H/ACA small nucleolar RNAs (snoRNAs), is responsible
for guiding dyskerin to convert uridine to pseudouridine at select
nucleotides within the rRNA.118 Intriguingly, a recent study has
identified remarkable heterogeneity in the expression of specific
subsets of H/ACA snoRNAs in primary cells isolated from X-DC
patients, including CD341 hematopoietic progenitor cells.119 Specifically, these findings demonstrate that distinct DKC1 mutations lead to
a significant decrease in the levels and activity of several H/ACA
snoRNAs known to guide pseudouridine modifications on rRNA.
Indeed, X-DC patient cells harbor site-specific defects in rRNA
pseudouridine modifications at distinct residues, providing functional readouts of H/ACA snoRNA dysfunction in X-DC.119
Notably, it was also demonstrated that the pseudouridine synthase
activity of dyskerin in modifying RNA is important for hematopoietic differentiation because expression of a catalytically inactive
dyskerin mutant failed to rescue hematopoietic stem cell differentiation in X-DC patient cells when compared with catalytically active
dyskerin.119 These exciting new findings highlight an important, yet
perhaps unappreciated, role for impaired dyskerin pseudouridine
synthase activity, H/ACA snoRNAs deregulation, and ribosome
dysfunction in DC pathogenesis. For example, reduced levels of
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BLOOD, 30 OCTOBER 2014 x VOLUME 124, NUMBER 18
rRNA pseudouridylation appear to have no overall effect on global
rates of protein synthesis; however, a role for pseudouridine
modifications in maintaining translation fidelity has been established.120 In addition, it has also been demonstrated that rRNA
pseudouridine modifications have an evolutionarily conserved role
in the recruitment of structured RNA components, such as IRES
elements, to the ribosome.120 Ultimately, these studies illustrate that
pseudouridine modifications on rRNA guided by H/ACA snoRNAs
may play an important role in modulating the expression of specific
mRNAs such as those containing IRES elements. In agreement
with this hypothesis, impairments in the translation of distinct
mRNAs known to harbor IRES elements such as p53 and p27 were
identified in primary X-DC patient cells and also in a mouse model
of X-DC.119,121,122 Thus it seems likely that deregulated ribosome
function may contribute to DC disease pathogenesis by altering the
translation of specific mRNAs in DC patient cells. How ribosomal
perturbations might act in concert with impaired telomere maintenance to contribute to the DC phenotype remains to be explored.
These studies built the foundation for the hypothesis that alterations
in the translation of distinct mRNAs are key determinants for the
development of ribosomopathies.
Ribosomal complexity in cellular and
developmental biology
Most of the ribosomal proteins appear to be dispensable for the
ribosomal peptidyl transferase activity to catalyze peptide bone
formation, which is largely dependent on the ribosomal RNA.123
Eukaryotic ribosomes contain additional ribosomal proteins and
additional segments (expansion segments) within the rRNAs compared with prokaryotic ribosomes. Most of the additional ribosomal
proteins and rRNA expansion segment found in eukaryotes are
located over the surface of the ribosome apart from the conserved
core catalytic elements of the ribosome; thus the roles of these
additional proteins and RNA segments remain largely unclear.
Variations in the composition and posttranslational modifications
of ribosomal components raise the intriguing possibility of differential function of specialized ribosomes.124
Ribosomal proteins undergo a variety of posttranslational modifications, including phosphorylation, acetylation, methylation,
ubiquitination, and O-linked b-D-N-acetylglucosamine (O-GlcNAc)
modification.124 Ribosomal RNA is also subject to posttranscriptional modifications including pseudouridylation (see previous
discussion) and methylation. Such modified residues are found
within functionally important domains of the rRNA.125 Alterations
in ribosome-associated proteins and mRNA structures may also
contribute additional layers of translational regulation. Additional
extraribosomal functions of ribosomal proteins have also been
described.126 Nonredundant effects of disrupting specific ribosomal
paralogues suggest that ribosomal proteins may serve specific cellular
MARROW FAILURE AND RIBOSOMOPATHIES
2789
and developmental functions. For example, knockdown of rpl22 in
zebrafish embryos results in T-cell developmental arrest, whereas
knockdown of the paralogue rpl22l1 blocks the development of
hematopoietic stem cells independent of p53 levels.127 Importantly, in mice, mutations in Rpl38 cause selective effects on axial
skeletal patterning and the formation of the mammalian body plan.128
These tissue-specific phenotypes are mirrored by selective impairment
in the translation of Hox genes, key vertebrate developmental
regulators. These studies thereby link a key component of the
ribosome to fundamental control of vertebrate embryonic development. They also suggest a deeper level of ribosome-mediated
regulation in organogenesis that may be central to the understanding
of congenital birth defects associated with ribosomopathies.
Summary
The inherited marrow failure syndromes provided the initial insights
into the previously unexpected role of ribosomes in cellular and
developmental processes contributing to human disease. The cancer
predisposition of these inherited syndromes provides compelling
evidence for an initiating or driving role for the observed ribosomal
pathway mutations in cancers arising in the general population. The
study of inherited marrow failure syndromes is revealing ribosomal
functions in stress pathways and protein translational regulation
regulating hematopoiesis and clonal evolution, and is opening up
new avenues of investigation for targeted therapies.
Acknowledgments
The authors apologize to those whose work could not be included in
this review because of space limitations. The reader is referred to
additional primary literature within the cited reviews.
D.R. is a Leukemia & Lymphoma Society Scholar.
This work was supported by National Institutes of Health grants
from the National Heart, Lung, and Blood Institute 5 R01 HL079582-11
(A.S.), the National Institute of Diabetes and Digestive and Kidney
Diseases 5 P30 DK056465 (A.S.), and R01 DK098057 (D.R.), and
the Seattle Children’s Hospital Butterfly Guild (A.S.).
Authorship
Contribution: D.R. and A.S. wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing
financial interests.
Correspondence: Akiko Shimamura, Fred Hutchinson Cancer
Research Center, 1100 Fairview Ave N, D2-100, Seattle, WA 98109;
e-mail: [email protected].
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From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
2014 124: 2784-2792
doi:10.1182/blood-2014-04-526301 originally published
online September 18, 2014
Marrow failure: a window into ribosome biology
Davide Ruggero and Akiko Shimamura
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