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The gene encoding ribosomal protein S19 is mutated in Diamond-Blackfan anaemia

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Diamond-Blackfan anaemia (DBA) is a constitutional erythroblastopenia characterized by absent or decreased erythroid precursors. The disease, previously mapped to human chromosome 19q13, is frequently associated with a variety of malformations. To identify the gene involved in DBA, we cloned the chromosome 19q13 breakpoint in a patient with a reciprocal X;19 chromosome translocation. The breakpoint occurred in the gene encoding ribosomal protein S19. Furthermore, we identified mutations in RPS19 in 10 of 40 unrelated DBA patients, including nonsense, frameshift, splice site and missense mutations, as well as two intragenic deletions. These mutations are associated with clinical features that suggest a function for RPS19 in erythropoiesis and embryogenesis.
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nature genetics • volume 21 • february 1999 169
The gene encoding ribosomal protein S19 is mutated in
Diamond-Blackfan anaemia
Natalia Draptchinskaia
1*
, Peter Gustavsson
1*
, Björn Andersson
2
, Monica Pettersson
3
, Thiébaut-Noël Willig
4,5
,
Irma Dianzani
6
, Sarah Ball
7
, Gil Tchernia
4,5
, Joakim Klar
1
, Hans Matsson
1
, Dimitri Tentler
1
,
Narla Mohandas
4
, Birgit Carlsson
1
& Niklas Dahl
1
*These authors contributed equally to this work.
Diamond-Blackfan anaemia (DBA) is a constitutional erythroblastopenia characterized by absent or decreased ery-
throid precursors. The disease, previously mapped to human chromosome 19q13, is frequently associated with a
variety of malformations. To identify the gene involved in DBA, we cloned the chromosome 19q13 breakpoint in a
patient with a reciprocal X;19 chromosome translocation. The breakpoint occurred in the gene encoding ribosomal
protein S19. Furthermore, we identified mutations in RPS19 in 10 of 40 unrelated DBA patients, including non-
sense, frameshift, splice site and missense mutations, as well as two intragenic deletions. These mutations are
associated with clinical features that suggest a function for RPS19 in erythropoiesis and embryogenesis.
1
Unit of Clinical Genetics,
2
Unit of Medical Genetics and
3
Unit of Pathology, Department of Genetics and Pathology, Uppsala University, 751 85 Uppsala,
Sweden.
4
Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
5
Department of Hematology, Hôpital Bicêtre, AP-HP, Kremlin-Bicêtre
and Faculté Paris-Sud, Bicêtre, France.
6
Department of Pediatrics, University of Turin, 10126 Turin, Italy.
7
Department of Haematology, St. George’s Hospital
Medical School, London SW17 0RE, UK. Correspondence should be addressed to N.D. (e-mail: niklas.dahl@klingen.uu.se).
Introduction
Diamond-Blackfan anaemia (DBA; McKusick 205900) is charac-
terized by a chronic constitutional aregenerative anaemia with
absent or decreased erythroid precursors in bone marrow but
otherwise normal cellularity
1,2
. Most patients present with
anaemia in the neonatal period or in infancy. Approximately
30% of affected children present with a variety of associated
physical anomalies. Thumb and upper limb malformations as
well as craniofacial abnormalities are common. Other defects fre-
quently observed include atrial or ventricular septal defects, uro-
genital anomalies and prenatal or postnatal growth
retardation
1–5
. A moderately increased risk of developing
haematological malignancies also exists
5
. Most cases with DBA
are sporadic, with an equal sex ratio, but at least 10% have a posi-
tive family history for the disorder
6,7
. The pathophysiology and
the basic molecular defect(s) of DBA remain unclear. Normal
erythropoiesis can be restored by bone marrow allografting
8
,
ascribing the anaemia to an intrinsic defect of haematopoietic
progenitor cells. Different in vitro assays as well as molecular
studies have failed to demonstrate a direct role of growth factors
or their receptors in the pathogenesis of DBA (refs 912).
Our previous genetic mapping studies localized a gene respon-
sible for DBA to a 1-Mb region on 19q13 on the basis of: (i) the
identification of a balanced translocation breakpoint in a patient
with the disease
13
; (ii) linkage analysis in multiplex DBA
families
6
; and (iii) de novo microdeletions associated with the
disease
7
. Furthermore, we recently found evidence for genetic
heterogeneity in DBA (ref. 7), but without correlation between
the 19q13 locus and clinical expression of the disease.
The cloning of translocation breakpoints from rare patients
with a specific phenotype has proven to be a powerful method
for the positional identification of disease genes
14
. Using this
strategy, we identified transcribed sequences adjacent to the
translocation breakpoint on chromosome 19q13 associated with
DBA. We report here the cloning of the chromosome 19 translo-
cation breakpoint, which interrupts the gene encoding riboso-
mal protein S19. Our subsequent findings of mutations in
RPS19 in several unrelated DBA patients provide genetic evi-
dence for the primary role of this gene in the pathogenesis of
DBA in a subset of affected individuals. Our results suggest an
involvement of RPS19 in erythroid differentiation and prolifera-
tion as well as in the normal development of other tissues fre-
quently affected in DBA.
Results
Cosmid spanning the 19q13 translocation breakpoint
We recently reported a female patient with a de novo balanced
translocation 46,XX,t(X;19)(p21;q13) (ref. 13) who presented
with constitutional erythroblastopenia as well as short stature
(–3 SD) and left kidney hypoplasia. To map the chromosome 19
breakpoint, we hybridized cosmids from a chromosome 19
map
15
to metaphase chromosomes from the patient. The 19q13
breakpoint was found to be located in a 400-kb region flanked by
cosmids 14353 and 24450 (Lawrence Livermore National Labo-
ratory). Of three overlapping cosmids in this region, cosmid
27589 showed hybridization signals on both chromosome 19
derivatives from the patient with a t(X;19) (Fig. 1). This finding
implies that the cosmid spans the breakpoint. Different restric-
tion fragments of the cosmids were isolated and hybridized with
genomic DNA from the patient with the translocation. One 0.9-
kb BamHI subfragment from the centromeric part of the cosmid
identified a rearranged band when hybridized with HindIII-
digested genomic DNA of the patient. These results confirmed
that cosmid 27589 spans the 19q13 breakpoint. We also analysed
three patients with DBA and microdeletions in the 19q13 region
7
with cosmid 27589. FISH analysis to metaphase chromosomes
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170 nature genetics • volume 21 • february 1999
revealed hybridization signals on only one chromosome 19q in
each of the three patients. Our results showed that cosmid 27589
is included in the three microdeletions.
Sequence analysis and characterization of RPS19
Partial genomic sequences were generated from the overlapping
cosmids 27589, 14688 and 27678 (LLNL). The cosmids were
sheared and fragments were cloned in M13 and randomly
sequenced. Resulting sequences (a total stretch of 70 kb) were
aligned and analysed for the presence of genes using both
BLASTN and BLASTX to search public databases. Genes known
to encode RPS19 (ref. 16), IGA (CD79a; ref. 17) and Rho-GEF
(ref. 18) were identified. Corresponding cDNAs were used for
Southern-blot analysis on EcoRI-digested genomic DNA from
the female with a t(X;19). Hybridization with RPS19 cDNA
identified two aberrant bands of 7 kb and 13 kb, which sug-
gested that this gene was interrupted by the breakpoint. Com-
parison between the published RPS19 cDNA sequence and the
sequence derived from cosmid 27589 enabled us to determine
the genomic structure and the intron/exon boundaries of
RPS19 (Table 1). The gene is 11 kb with 6 exons (Fig. 2a). The
first exon is untranslated and the ATG, which corresponds with
the start codon (AUG) in the cDNA, is located at the beginning
of exon 2. No TATA or CAAT boxes were identified, but
genomic sequence analysis of 1,260 bp of sequence upstream of
the translational start site revealed several sequence motifs
which match human promoter elements. The RPS19 coding
sequence predicted from the sequenced cosmid is identical to
the published cDNA sequence. Further comparision of genomic
sequence with restriction fragments identified by the RPS19
cDNA revealed that the 19q13 breakpoint was located in the
third intron (Fig. 2b).
Expression of RPS19
Sequence of the published human RPS19 cDNA contains a 435-bp
ORF and encodes a 145-aa protein
16
. Several homologous ESTs
were identified, two of which contained sequences from exon 1 and
exon 2. These ESTs enabled us to extend the 5´ UTR to 160 bp
upstream of the start site; this sequence is continuous on genomic
DNA. The 5´ UTR contains an oligopyrimidine tract of 13 nt,
including the core CTTTCC motif observed in several other
mRNAs encoding ribosomal proteins
19
. The 3´ UTR spans 40 nt;
thus, the predicted size of the RPS19 transcript is 635 bp.
The expression pattern of RPS19 was examined by northern-
blot analysis. Human adult tissues including bone marrow,
peripheral blood, spleen, liver and non-haematopoietic tissues
showed ubiquitous expression of a single 0.60.7-kb transcript
(Fig. 3). The same pattern was seen using a probe corresponding
to either the 5´ UTR region only or exons 26 of RPS19 cDNA.
The same filters were subsequently hybridized with a cDNA
probe for ribosomal protein S29 (RPS29; ref. 20), which revealed
a similar hybridization pattern when compared with that of the
RPS19 probe (Fig. 3). These results confirm that RPS19 is tran-
scribed in haematopoietic and non-haematopoietic tissues.
Fig. 1 Identification of the chromosome 19 translocation break-
point in the patient with t(X;19). FISH analysis to metaphase chro-
mosomes shows that cosmid 27589 (LLNL) hybridizes to both
chromosome 19 derivatives (arrows) and normal chromosome 19.
Table 1 • Intron-exon boundaries of RPS19
Exon Exon size 3´ splice site 5´ splice site
number (bp)
1 159 AGGCCGCACGgtaagcgggg
2 71 tctccctcagATGCCTGGAG TCCTCAAAAAgtgagtttgg
3 101 ttggtcttagGTCCGGGAAG ACGCGAGCTGgtgaggaact
4 184 ctacccccagCTTCCACAGC ACCAAGATGGgtaagcaggg
5 56 ctccccacagCGGCCGCAAA CGCCGGACAGgtaaggcctg
6 64 tttcccacagGTGGCAGCTG
Fig. 2 Map of the human RPS19 region. a, A
schematic representation of RPS19 structure.
Boxes, exons; filled boxes, coding sequences.
Exon 1 encodes the 5´ UTR. The AUG is
located at the beginning of exon 2. b, South-
ern-blot analysis with hybridization of
probes for exon 3 and exon 4 from RPS19 to
EcoRI digests of DNA from the female with a
t(X;19) and two control individuals. Arrows
indicate rearranged bands in DNA from the
t(X;19) female. The top arrow indicates the
13-kb band detected by the exon 3 probe,
the bottom arrow indicates the 7-kb band
detected by the exon 4 probe.
a
b
1 kb
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nature genetics • volume 21 • february 1999 171
Identification of mutations in unrelated patients with DBA
Molecular analysis of the X;19 translocation associated with DBA
allowed us to infer RPS19 as a candidate. Therefore, we screened
for mutations using direct sequencing of genomic DNA of 40
probands with DBA: 21 with a family history of the disease and
19 sporadic cases. Nine different mutations were found in RPS19
in ten probands (Table 2). Six of the patients with mutations had
a family history of the disease. No mutations were found in the 5´
UTR sequence or in the sequence encoding the five translated
exons in 30 other probands. An intragenic deletion of 295 nt that
spans exon 5 was identified on genomic DNA of two affected
members of family F13. The mutation predicts the loss of nt 357
411 in the mRNA with a frameshift after codon 120. The muta-
tion was confirmed by direct sequencing of RT-PCR products
from lymphoblastoid-derived mRNA from one affected family
member, which revealed a truncated transcript missing 55 nt
from exon 5 (Fig. 4a). A CT transition causing an Arg94Stop
was identified in two sisters and their mother (family F01;
Fig. 4b). The sisters were discordant for associated malforma-
tions: one of them presented with thumb malformations and
duplicated ureter, whereas the other had congenital glaucoma.
The mother had normal haemoglobin levels and no malforma-
tions. An AG transition in the start codon was found in family
F18 (Fig. 4c). The three affected family members presented with
similar degrees of hypoplastic anaemia. The transition was veri-
fied in RT-PCR products from lymphoblastoid-derived mRNA of
the proband. Different single nucleotide insertions were identi-
fied in two sporadic cases. Insertion of an A in exon 2 (13insA;
family S14) predicted a frameshift after codon 4 (Fig. 4d).
Another insertion of an A was found in exon 3 (104insA; family
S11), resulting in a frameshift after codon 34. A GT transver-
sion that alters the acceptor splice site of exon 2 was found in a
proband and his affected father (family F38; Fig. 5a). The muta-
tion predicts a truncated transcript missing exon 2. A 4-bp dele-
tion was found in the donor splice site of intron 2
(AAgtgagtttAAgttt) in the mother and four children of family
F03, which indicates dominant segregation. A disruption of the
consensus donor splice site was predicted when the sequence was
analysed with a human splice-site prediction program
21
.
A CT transition at position 184 was identified in two non-
related patients of Swedish and Italian origin (F04 and S05). The
patients do not share a flanking haplotype, suggesting recurrent
mutation events. This substitution results in the replacement of
arginine with tryptophan (Arg62Trp). The mutation was found
to segregate with the two affected individuals of the Swedish fam-
ily, whereas the mutation in the Italian family occurred de novo in
a sporadic case (Fig. 5b,c). The presence of the transition was
confirmed by restriction digestion due to the loss of a BstUI site.
Finally, a TC transition resulting in a change from a trypto-
phan to an arginine (Trp52Arg) was found in a sporadic case
(S08). The mutation destroys a BsrI site and was confirmed on
genomic DNA. The two different missense mutations were not
present on any of 100 control chromosomes analysed. Moreover,
the missense mutations predicted amino acid substitutions at
positions that were conserved in eukaryotes as well as in the
archaebacterium Methanococcus jannaschii. The mutations iden-
tified in the six multiplex families were found to cosegregate with
affected family members. All patients with mutations were het-
erozygous for the alterations and no additional sequence varia-
tions in the protein-coding region of the gene were found.
Ribosomal protein S19
Ribosomal protein S19 consists of 145 aa with a predicted molec-
ular weight of 16 kD and an isoelectric point of 10.3. The protein
lacks cysteine residues and the hydropathy profile predicts the
presence of hydrophobic domains. In agreement with the
BLASTN homology searches, at the peptide level, BLASTX,
BLASTP and BEAUTY searches revealed highly significant
homologies of RPS19 with proteins from diverse organisms. The
high degree of conservation of RPS19 with full-length RPS19 of
Rattus norvegicus (99% identity, 99% similarity), Mus musculus
(98% identity, 98% similarity), Drosophila melanogaster (65%
identity, 77% similarity), ribosomal protein 55 of Saccharomyces
cerevisiae (52% identity, 68% similarity) and M. jannaschii (42%
identity, 59% similarity) is exemplified in the CLUSTAL align-
ment (Fig. 6). In addition, homologies of RPS19 with peptides
from Archaeologlobus fulgidus and Haloarcula marismortui were
also observed.
Fig. 3 Expression analysis of RPS19. Multiple-tissue northern blots (Clontech)
were hybridized with the RPS19 cDNA probe PN1 (top bands, 0.60.7 kb). The
same filters were subsequently hybridized with the RPS29 cDNA probe MP
(bottom bands) as a reference.
Table 2 • Mutations in RPS19
N Pedigree Exon/intron Nucleotide change Predicted effect
1 F38 intron 1 1(1)agATGatATG acceptor splice site defect
2 F18 exon 2 1AG mutation in the start codon
3 S14 exon 2 13 ins A frameshift at codon 5;
stop at codon 50
4 F03 intron 2 71(+3+6) deletion of 4 bp donor splice site defect
AAgtgagtttggg AAgtttggg
5 S11 exon 3 104 ins A frameshift at codon 35;
stop at codon 50
6 S08 exon 3 154TC Trp52Arg
7 F01 exon 4 280CT Arg 94Stop
8 F04 exon 4 184CT Arg62Trp
9 S05 ″″
10 F13 intron 4intron 5 deletion of 295 bp spanning deletion of exon 5;
exon 5 (nt 357411 of the cDNA) frameshift at codon 120
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172 nature genetics • volume 21 • february 1999
Discussion
Diamond-Blackfan anaemia is a model for a specific differentia-
tion arrest of the erythroid lineage. The specific reduction of
erythroid precursor cells in the bone marrow suggests that the
deficient protein has a key regulatory role in erythropoiesis.
From the cloning and characterization of the 19q13 breakpoint
region associated with DBA we identified three candidate genes.
One candidate, encoding an S19 ribosomal protein associated
with the ribosomal 40S subunit
16
, was found to be interrupted
by the breakpoint.
To determine the possible involvement of RPS19 in DBA, we
screened DNA from affected individuals for mutations in this
gene. We identified mutations in 10 of 40 DBA patients (25%)
analysed. Three probands carry missense mutations and seven
patients have mutations that predict either an altered translation
of RPS19 or a truncated RPS19. The six RPS19 mutations identi-
fied in multiplex families were found to cosegregate with the
DBA phenotype and the observation of mutations in consecutive
generations shows an autosomal dominant mode of transmis-
sion. We saw a variable expression for RPS19 mutations in several
families and incomplete penetrance in one family.
S19 is one of 79 ribosomal proteins. They constitute a major
component of cellular proteins, but their functions, apart from
being part of the ribosome, are unknown
19
. As the ribosome is
Fig. 4 Identification of RPS19 mutations in Diamond-Blackfan anaemia patients. Sequence chromograms are shown for control (top) and affected (bottom)
individuals. The sequence depicted over the trace data is that of the sense strand. a, The deletion of exon 5 in the cDNA of the proband of family F13.
b,ACT transition resulting in a premature stop codon in the proband of family F01. c, An AG substitution in the start codon in family F18 from the
sequencing of lymphoblastoid-derived cDNA. d, Insertion of an A at position 13 of RPS19 cDNA in a patient of family S14. The sequence of F01 and S14 is gen-
erated from genomic DNA.
Fig. 5 Segregation of RPS19 mutations in families. a, Family F38 with a GT transversion in the acceptor splice site of intron 1. The mutation results in loss of an
SfaNI site, which is detected as the top band after the separation of digested PCR products from genomic DNA using primers flanking exons 2–3. b, Family F04,
segregating for a missense mutation (C184T). The mutation results in loss of an BstUI site, which is detected as the upper band after the separation of digested
PCR products from genomic DNA using primers flanking exons 4–5. c, The missense mutation (C184T) that has occurred de novo in a sporadic case of family S05.
The upper band in the proband indicates the mutant allele. The mutation was not found in the parents or in the two healthy siblings who share the proband’s
genotype for markers D19S197, PG1 (ref. 7) and D19S408. RPS19 is located between markers D19S197 and PG1.
a
b
c
d
a
b
c
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nature genetics • volume 21 • february 1999 173
known to be mandatory for cellular growth, one may expect
haploinsufficiency for a ribosomal protein to result in protein
synthesis rate limitation in most tissues with a high prolifera-
tive activity. The potential role of ribosomal proteins in human
disease has received considerable attention recently
22,23
. It has
been suggested that RPS4X and RPS4Y, which are homologous
on the X and Y chromosomes, contribute to the Turner syn-
drome phenotype
24,25
; however, much of the evidence is still
circumstantial. Dyskeratosis congenita was recently shown to
be caused by mutations in a gene encoding NAP57, a protein
essential for the processing of pre-rRNA (ref. 26). It has been
suggested that NAP57 may act as a chaperone in the assembly
of ribosomes. Consequently, the disease may be caused
by an impaired ribosomal function in either a quantitative or
qualitative fashion.
Our findings of a ribosomal protein as a cause of DBA is
unexpected in view of the fact that clinical symptoms in the
majority of patients are confined to erythropoiesis, and in
some cases to organs during embryogenesis. Although a
human ribosomal protein gene mutation is expected to have a
generalized effect, there is now accumulating evidence that
some ribosomal proteins have a second and extra-ribosomal
function
23
. The nematode Ascaris lumbricoides has two
homologous genes encoding RPS19, one of which is elimi-
nated (S19G) in presomatic cells during early development
27
.
It has been suggested that the S19G copy, which encodes a pro-
tein that differs at 24 aa from that encoded by the somatic S19
copy, has a function in early embryogenesis apart from the
ribosome
23
. This is supported by the observed preferential
transcription of S19G in germline cells. In Drosophila, evi-
dence for extra-ribosomal functions of ribosomal proteins has
emerged from the identification of mutations in the genes
encoding ribosomal proteins S2, S6 and L19. Mutations in
RPS2 (string-of-pearls) result in an arrest of oogenesis at stage
5 of development
28
, mutations in RPS6 results in hypertro-
phied haemopoietic organs
29
and RPL19 mutants display
abnormal wing blade development
30
. Analogous to the
Drosophila RPS2, RPS6 and RPL19 mutants, the clinical fea-
tures of DBA could suggest extra-ribosomal and tissue-spe-
cific functions for RPS19. Drosophila mutants for RPS2, RPS6
and RPL19 also share some features with a set of about 50
other Drosophila mutations designated Minute
31
. Flies with
the Minute phenotype have delayed larval development,
diminished viability, reduced body size, decreased fertility and
thin bristles. Several Minute genes have been identified that all
encode ribosomal proteins. In view of the Drosophila Minute
phenotype, it is tempting to speculate that the unspecific fea-
tures frequently associated with DBA, for example, short
stature, reflect a general dysfunction of the translational appa-
ratus or a second function for this ribosomal protein.
The expression of rRNA and ribosomal proteins is coordi-
nated and correlates with the requirements for protein synthe-
sis at different rates of growth
23,32
. In vertebrates, the control
of ribosomal protein gene expression at the translational level
is the most prevalent regulatory mechanism. The regulatory
cis-acting element consists of a 5´ oligopyrimidine tract (5´
TOP) located in the 5´ UTR of the mRNAs (ref. 32). This 5´
TOP, also shared by RPS19, is critical for the translational con-
trol of ribosomal proteins
32
. Recent results, however, suggest
that the TOP-containing mRNAs of elongation factor 2 (EF2)
and RPS16 are translationally regulated in a growth-dependent
manner in cells of haematopoietic origin but not in other
cell types
33
.
By sequencing RPS19, we identified mutations in 25% of
DBA patients, with similar numbers for sporadic and familiar
cases. The unexpectedly low proportion of RPS19 mutations
found in multiplex families in our study may have several
explanations. One possibility is that mutations in regulatory
elements or in other non-coding regions of RPS19 are
involved. Furthermore, small deletions involving one or both
primers used to generate sequencing templates from genomic
DNA may have escaped identification. Another possibility is
that the DBA phenotype in some of the smaller families
included in previous linkage analysis segregates with 19q13 by
chance. The results from recent genetic linkage analysis suggest
that at least 10% of multiplex families, possibly a larger pro-
portion, contain mutations in genes unlinked to 19q13 (ref. 7).
In conclusion, our data provide evidence that mutations in
RPS19 cause DBA in a subset of patients. We hypothesize that
the phenotype associated with DBA may result from haploin-
suffiency of a ribosomal function and/or altered extra-riboso-
mal functions. Whether a partial DBA phenotype or the entire
complex of clinical features are caused by either one or both of
these mechanisms remains to be clarified.
Fig. 6 Alignment of RPS19 from
human and other species. The high
degree of conservation is illus-
trated by the CLUSTAL/BOXSHADE
alignment. White letters on a black
background indicate identical
amino acids; black letters on a
white background indicate differ-
ent and non-conserved amino
acids, black letters on a grey back-
ground depict different but con-
served amino acids. Asterisks above
the sequence indicate positions of
two missense mutations (Trp52Arg
and Arg62Trp).
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174 nature genetics • volume 21 • february 1999
Methods
Patients. Most patients were included in a previous study in which periph-
eral blood samples were obtained from unselected DBA patients and their
families
6,7
. All patients had been diagnosed in pediatric haematology/
oncology departments in their country of origin. The diagnostic criteria
included normochromic anaemia in infancy (<2 years), low reticulocyte
counts and absent or decreased bone marrow red cell precursors
34
(<5%
of nucleated cells). Additional features included presence of malforma-
tions, macrocytosis, elevated fetal haemoglobin, elevated erythrocyte
ADA levels and a normal chromosome fragility test (mitomycin C or
diepoxybutane). Lymphoblastoid cell lines were established from periph-
eral blood of a few patients, by transformation with the Epstein-Barr
virus, for further genetic analysis.
FISH analysis. Flourescent in situ hybridization to chromosome prepara-
tions from the patient with t(X;19) was performed as described
13
. Cos-
mid DNA was labelled with biotin or digoxigenin using nick translation.
The labelled probes were visualized with FITC-avidin (Vector) or rho-
damine-labelled anti-digoxigenin (Boehringer) and the chromosomes
were counterstained with DAPI (Serva). Hybridizations were analysed
with a Zeiss Axioskop epifluorescence microscope and images were cap-
tured using a CCD camera (Photometrics) and the Quips SmartCapture
FISH software (Vysis).
Subcloning of cosmids into M13. DNA from each of the three cosmids
(LLNL cosmids 27589, 14688, 27678) was purified using a Qiagen midi-
prep kit and sheared by nebulization to an average size of approximately 2
kb. The random fragments were cloned into a modified M13 vector using
the ‘double adaptor’ method
35
. M13 clones were grown in a 96-well format
and high-quality DNA templates were prepared using a 96-well glass-fiber
filter protocol
36
.
Sequencing of M13 clones and PCR products. Fluorescent automated
DNA sequencing was performed using Applied Biosystems 377 DNA
sequencers (Perkin Elmer) and sequencing reagents from Perkin Elmer and
Amersham, using automated fluorescent methods
37
. The sequence reads
were assembled and the contiguous sequences edited using the Staden
package
38
and the PHRED-PHRAP package (courtesy of P. Green). Gap
closure and finishing was carried out using a mapped-gap strategy
39
and
walking using specific oligonucleotide primers.
Gene identification and characterization. Expressed sequences were iden-
tified by database searches using BLAST (ref. 40) and by the program
GRAIL (ref. 41). Prediction of promoter elements was performed with the
TSSW promoter recognition program (http://dot.imgen.bcm.tcm.edu:
9331/gene-finder/Help/tssw.html).
Mutation detection and confirmation. Genomic DNA from DBA patients
and normal controls was amplified by PCR. Four distinct genomic frag-
ments were amplified spanning 722 bp (containing exon 1 and the 5´
UTR), 642 bp (containing exons 2 and 3), 850 bp (containing exons 4 and
5) and 354 bp (containing exon 6), respectively (primer sequences available
on request). Amplification was carried out with template DNA (200 ng) in
reactions (50 µl) using Taq polymerase (Perkin Elmer Cetus). Amplifica-
tion bands were excised from 1% agarose gels, purified using QIAquick
spin columns (Qiagen) and sequenced using ABI dye-terminator sequenc-
ing kits. Mutations were sequenced on both strands derived from two inde-
pendent PCR reactions for verification. The presence of missense muta-
tions was confirmed by restriction digestion due to the loss of a BstUI site
(pedigrees F04, S05) and a BsrI site (pedigree S08). The mutations were
analysed by genomic amplification across the recognition sites followed by
appropriate restriction digestion. The 4-bp deletion in family F03 was
detected after the amplification of genomic DNA with primers flanking the
deletion. Splice-site mutations were analysed by the human splice-site pre-
diction software HSPL (service@bioinformatics.weizmann.ac.il) and
Neural Networks (http://www-hgclbl.gov/inf/).
RNA isolation, RT-PCR and cDNA synthesis. Total RNA was extracted
42
from EBV-transformed lymphoblastoid cell lines (probands of families
F13, S05, S08 and F18). First strand cDNA was synthesized using a primer
designed from nt 448469 of RPS19 cDNA. The cDNA was amplified using
the first strand synthesis primer and a forward primer from nt 76 to 57.
PCR was performed with an initial cycle of 94 °C for 3 min, then 34 cycles
of 94 °C for 40 s, 62 °C for 40 s, 72 °C for 60 s, followed by 72 °C for 10 min.
Control reactions for the RT-PCR without reverse transcriptase were run
in all experiments to exclude contaminations of genomic DNA containing
RPS19 homologous sequences.
Expression studies and Southern-blot analysis. Hybridization probes for
northern- and Southern-blot analyses were labelled by random priming
with
32
P-(dCTP). Northern blots 7760-1 and 7768-1 (Clontech) were
hybridized as described
43
and Southern blots containing DNA from leuko-
cytes were hybridized for 16 h at 42 °C in 50% formamide, 5×SSC, 1×Den-
hardt’s solution, phosphate buffer (20 mM, pH 7.6), 1% SDS and salmon
sperm DNA (100 µl/ml). The filters were then washed twice in 2×SSC and
0.5% SDS at 60 °C. Kodak XAR-5 film was exposed at 70 °C with Dupont
Cronex Lightning Plus intensifying screens.
Probes. Hybridization probes were gel-purified DNA fragments amplified
from a human thymus cDNA library (Clontech HL5010b) or from genom-
ic DNA. A 450-bp probe (PN1) for the human RPS19 cDNA (exons 26)
was amplified using primers designed from nt 7 to 13 and nt 432453.
Genomic probes for exon 3 and exon 4, respectively, of the human RPS19
cDNA were amplified with intron-derived primers. Three overlapping
cDNA probes for Rho-GEF were amplified, spanning nt 881,600, nt
1,2472,371 and nt 2,2673,053. The cDNA probe for IGA (CD79a) was
amplified with primers spanning nt 39748 and the human RPS29 probe
of 290 bp (MP) was amplified from a human thymus cDNA library. A 450-
bp 5´ UTR probe for RPS19 was amplified from genomic DNA (primer
sequences available upon request).
GenBank accession numbers. Human RPS19 cDNA, M81757; human ESTs
with homology to RPS19, D28389, AA170846, R68032; human genomic
RPS19 sequences, AF092906, AF092907; human RPS29 cDNA, U14973;
mouse Rps19 cDNA, W54527; rat Rps19 cDNA, X51707; D. melanogaster,
P39018; S. cerevisiae, P07280; M. jannaschii, P54057.
Acknowledgements
We are grateful to the patients and their families for their participation in this
study. We thank O. Nygård and H. Johansson for discussions; L. Gordon for
cosmids in the 19q13 region; and the European DBA consortium of the
European Society for Pediatric Hematology and Immunology and the
Resource Center of the German Human Genome Project, Berlin. This work
was supported by grants from the Childrens Cancer Foundation of Sweden,
the Swedish Medical Research Council, the DBA Foundation Inc., T. and R.
Söderbergs Fund, The Swedish Cancer Society, The Beijer Foundation, the
Borgström Foundation, Ronald McDonalds fund, Lundbergs Foundation,
Wera Ekströms fund, Uppsala University, Association Française Contre les
Myopathies, Généthon and DRC (CRC 950183) AP-HP, Telethon Italia
(grant E.619), NIH grants DK 32094 and DK 26263 and Max Reinhart
Charitable Trust.
Received 20 October; accepted 22 December 1998.
© 1999 Nature America Inc. • http://genetics.nature.com
© 1999 Nature America Inc. • http://genetics.nature.com
article
nature genetics • volume 21 • february 1999 175
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© 1999 Nature America Inc. • http://genetics.nature.com
... Given the centrality of translation, it was long believed that mutations in components of the translational apparatus would be incompatible with successful completion of highly coordinated and complex vertebrate developmental programs. This view was challenged with the discovery that Diamond-Blackfan Anemia is caused by mutations in a ribosomal protein (6). With the advent of improved sequencing technologies, the list of "ribosomopathies" has continued to expand: the current list of mutations responsible for this class of disorders includes genes encoding ribosomal proteins, ribosome biogenesis factors, and posttranscriptional rRNA-modification complexes (7). ...
... Accordingly, in tandem with the increased availability of patient genome sequencing, a growing list of genes either directly involved in or adjacent to translation have been implicated in genetic disorders. These include mutations in tRNA processing (6,7) and tRNA modification proteins (11)(12)(13)(14), aminoacyl-tRNA synthetases (15,16), and in the translation elongation factors (17)(18)(19)(20)(21)(22)(23). These "translationopathies" tend to share common clinical features including developmental abnormalities and neurological defects and, on a molecular level, loss of translational fidelity (24). ...
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Thesis
Le ribosome, qui traduit le code génétique en protéines, est une machinerie moléculaire essentielle au fonctionnement optimal des cellules. La formation, ou biogenèse, de ces ribosomes est un processus très complexe, qui doit être finement contrôlé et régulé pour produire rapidement des sous-unités ribosomiques matures et fonctionnelles. Des mutations dans des composants de cet appareil cellulaire essentiel sont à l'origine de diverses maladies, comme des anémies, des retards de croissance mais aussi certains cancers. Dans ce contexte, la caractérisation précise de l'ensemble des mécanismes d'assemblage et de fonctionnement des ribosomes en conditions normales et pathologiques devient un enjeu primordial. Ces questions scientifiques ont récemment connu des avancées majeures, notamment grâce à l'utilisation de la cryo-microscopie électronique (cryo-EM). En effet, couplée à des analyses fonctionnelles, la résolution de différents états structuraux du ribosome a permis la description de nombreux mécanismes moléculaires responsables de la biogenèse des ribosomes, ainsi que de différentes étapes de la traduction. Mon projet de thèse s'inscrit pleinement dans ces analyses structure/fonction de l'assemblage et du fonctionnement des ribosomes. Mon premier projet de thèse s'est focalisé sur les étapes finales de la maturation de la petite sous-unité ribosomique humaine, aussi appelée sous-unité 40S. Pour cela, en me basant sur des analyses structurales par cryo-EM, j'ai mené des études fonctionnelles sur des particules pré-40S purifiées à une étape tardive de leur maturation, grâce à la forme catalytiquement inactive de l'ATPase RIO1. Ce travail m'a permis de mettre en évidence les rôles combinés de RIO1 et de la protéine ribosomique RPS26 dans le déclenchement du clivage du pré-ARNr 18S-E par l'endonucléase NOB1, entrainant la maturation finale de l'ARNr 18S. Mon second projet de thèse visait à comprendre les effets d'une mutation ponctuelle de la protéine ribosomique RPS15 (mutation RPS15-P131S) sur les mécanismes de traduction. Des mutations ponctuelles dans le domaine C-terminal de cette protéine de la petite sous-unité ribosomique sont en effet retrouvées dans de nombreux cas de leucémies et de tumeurs solides. Pour cela, j'ai entamé une analyse structure/fonction de ces ribosomes mutants. J'ai purifié et déterminé par cryo-EM les structures 3D de ribosomes en cours de traduction. J'ai comparé les structures de ribosomes " sauvages " à leurs homologues mutants, portant la mutation RPS15-P131S. Mes résultats montrent que la mutation provoque la déstabilisation structurale du domaine C-terminal de RPS15, ce qui entraînerait un ralentissement de l'étape d'élongation de la traduction. Une hypothèse pour expliquer ce phénomène serait que la déstructuration du domaine C-ter de RPS15 empêche l'accommodation correcte des ARNt amino-acylés dans le site A du ribosome.
... Alterations in the ribosome imply deviations in the translation of different mRNAs [2-5, 9, 10]. Mutations and deletions in ribosomal proteins or ribosome biogenesis factors were first linked to rare congenital diseases, the ribosomopathies, which are accompanied by a hyperproliferative phase with an increased risk of developing cancer [11]. Lately, changes in ribosomal composition have been directly related to cancer. ...
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Thesis
La traduction est le processus qui assure la synthèse protéique indispensable à la croissance et la prolifération cellulaire. Le ribosome est l’acteur principal de ce processus. Les ribosomopathies telles que l’anémie de Blackfan-Diamond et le syndrome myélodysplasique 5q- sont associées à une mutation ou une délétion d’un gène codant une protéine ribosomique. Ces deux pathologies aboutissent à une anémie hypoplasique. Pour comprendre le tropisme érythroïde des ribosomopathies, nous avons étudié les régulations de la traduction en cas d’altération de la biogenèse des ribosomes et mis en évidence l’importance de certaines caractéristiques des transcrits au cours des ribosomopathies et au cours de l’érythropoïèse humaine normale. Nous avons montré qu’il existe une diminution d’expression de la protéine GATA1 par coloration immunohistochimique de biopsie ostéo-médullaire de patients 5q-. Le transcrit GATA1 n’étant pas diminué nous avons identifié une régulation post-transcriptionnelle de GATA1. Deux modèles mimant le syndrome 5q- ont été développés dans les lignées cellulaires UT-7/EPO et K562. La diminution d’expression du gène codant la protéine ribosomique RPS14 par stratégie shARN conduit à une réduction de moitié́ du contenu cellulaire en ribosomes. Ces nouvelles conditions cellulaires entraînent une traduction sélective au détriment du facteur de transcription érythroïde majeur GATA1. Nous avons réalisé une analyse « omique » de la traduction afin de déterminer d’éventuelles caractéristiques de séquence ou de structure présentes au niveau des régions 5’UTR, CDS ou 3’UTR impliquées dans les dérégulations observées. Ainsi, nous montrons que la longueur des séquences, la composition en codons dit optimaux, la structuration des séquences en 3’UTR mais aussi de manière moindre en 5’UTR sont les caractéristiques majeures pour expliquer les modifications traductionnelles observées en condition shRPS14. Plus généralement nous avons étendu ces analyses à des données issues de modèles mimant l’anémie de Blackfan-Diamond via des shRPS19 et shRPL5. Ces analyses confirment les différentes caractéristiques impliquées dans la régulation de la traduction en condition de concentration cellulaire limitée en ribosome. Nous avons ensuite validé par approche fonctionnelle l’impact sur la traduction des différentes caractéristiques en 5’UTR, 3’UTR et au niveau de la séquence codante. De plus, une analyse intégrée du transcriptome et du protéome des cellules UT-7/EPO shRPS14 confirme que les régulations post-transcriptionnelles sont directement liées à ces critères de sélectivité de traduction. Par ailleurs, nous avons étudié le caractère dynamique de la traduction au cours de l’érythropoïèse normale. Les caractéristiques des transcrits dont la traduction est limitée en condition shRPS14, shRPS19 ou encore shRPL5 sont les mêmes que celles des transcrits dont la traduction augmente au cours de l’érythropoïèse normale. Nous montrons que les protéines les plus abondantes dans les stades érythroïdes matures sont issues de transcrits court, dont la séquence comporte un taux élevé de codons optimaux. Ces résultats indiquent que les transcrits codant pour les protéines qui s’accumulent au cours de l’érythropoïèse possèdent des caractéristiques optimales pour la traduction en conditions physiologiques, mais qui s’avèrent délétères en condition de disponibilité limitée en ribosomes. Ces observations nous ont permis d’émettre plusieurs hypothèses mécanistiques. Ces dernières pourraient impliquer le système de recyclage-réinitiation du ribosome, des voies de signalisation impliquées dans la réponse intégrée au stress, des systèmes de régulation non canonique de la traduction ou encore l’altération de la régulation traductionnelle par les miARN. Ces mécanismes ne sont pas mutuellement exclusifs.
Chapter
Eukaryotic ribosomes are extraordinarily complex cellular machines that catalyze the peptide bond synthesis necessary for producing new proteins. Ribosomes are composed of protein and RNA components, but the catalytic activity is found within the RNA itself. Each ribosome contains four ribosomal RNAs (rRNAs) initially synthesized from two precursor genes. These precursors undergo a laborious maturation process to release the mature rRNAs. In this article, we will discuss the mechanistic details governing pre-rRNA processing, focusing on those processes in human and yeast cells.
Chapter
Ribosomal proteins are fundamental building blocks for ribosome that is made through ribosomal biogenesis, a finely coordinated multi-step process engaging hundreds of molecules, including ribosomal proteins, RNA polymerases, ribosomal RNAs, and other auxiliary proteins, and essential for protein translation. However, numerous studies over the past decades have unveiled extra-ribosome functions of many ribosomal proteins. These functions are implemented independently of the translational machinery and often triggered by ribosomal or nucleolar stress. This article offers a review on the extra-ribosome functions of the ribosomal proteins in ribosomal stress signaling, normal development, tumorigenesis, and genetic diseases, and a prospective on their clinical significance as potential anti-disease therapeutic targets.
Thesis
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