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HEMATOPOIESIS
The Shwachman-Diamond SBDS protein localizes to the nucleolus
Karyn M. Austin, Rebecca J. Leary, and Akiko Shimamura
Shwachman-Diamond syndrome (SDS) is
an autosomal recessively inherited disorder characterized by exocrine pancreatic
insufficiency and bone marrow failure.
The gene for this syndrome, SBDS, encodes a highly conserved novel protein.
We characterized Shwachman-BodianDiamond syndrome (SBDS) protein expression and intracellular localization in 7
patients with SDS and healthy controls.
As predicted by gene mutation, 4 patients
with SDS exhibited no detectable fulllength SBDS protein. Patient DF277, who
was homozygous for the IVS2 ⴙ 2 T>C
splice donor mutation, expressed scant
levels of SBDS protein. Patient SD101
expressed low levels of SBDS protein
harboring an R169C missense mutation.
Patient DF269, who carried no detectable
gene mutations, expressed wild-type levels of SBDS protein to add further support to the growing body of evidence for
additional gene(s) that might contribute
to the pathogenesis of the disease phenotype. The SBDS protein was detected in
both the nucleus and the cytoplasm of
normal control fibroblasts, but was particularly concentrated within the nucleolus. SBDS localization was cell-cycle dependent, with nucleolar localization
during G1 and G2 and diffuse nuclear
localization during S phase. SBDS nucleolar localization was intact in SD101 and
DF269. The intranucleolar localization of
SBDS provides further supportive evidence for its postulated role in rRNA
processing. (Blood. 2005;106:1253-1258)
© 2005 by The American Society of Hematology
Introduction
Shwachman-Diamond syndrome (SDS)1,2 is an autosomal recessive
disease3 characterized by exocrine pancreatic insufficiency, bone marrow failure, and leukemia predisposition (reviewed in Dror and Freedman4 and Smith5). Additional features commonly associated with this
syndrome include skeletal malformations,6 liver abnormalities, and
short stature.4,5 Neutropenia is the most common manifestation of bone
marrow failure in patients with SDS, although a subset of patients
develops aplastic anemia. Lymphocyte defects7 and bone marrow
stromal-cell dysfunction8 have also been described in patients with SDS.
Thus, the disease phenotype is not restricted to myeloid cells but affects
multiple hematopoietic lineages and multiple organ systems.
The Shwachman-Diamond gene, SBDS, was recently cloned.9 SBDS
is located on chromosome 7 near the centromere.10,11 An adjacent
pseudogene, SBDSP, shares a high degree of homology with SBDS but
contains deletions and nucleotide changes that prevent the generation of
a functional protein.9 The majority of patients (nearly 75%) with
Shwachman-Diamond syndrome were found to harbor SBDS mutations
arising from a gene conversion event with this pseudogene.9
The SBDS gene is predicted to encode a novel 250–amino acid
protein with no homology to other proteins of known function. The
SBDS gene is highly conserved throughout evolution, and orthologues
can be found in organisms ranging from plants, Archaea, and yeast to
vertebrate animals. The SBDS mRNA is widely expressed throughout
human tissues9; however, little is yet known about SBDS protein
expression patterns. Ubiquitous SBDS mRNA tissue expression, together with multiorgan involvement in patients with SDS, suggests that
the SBDS protein likely functions in a global cellular process.
To further investigate the molecular pathways affected in SDS,
we examined the SBDS protein in 7 patients with Shwachman-
Diamond. The SBDS gene was sequenced for all 7 patients. Using
an antibody generated against the carboxyl terminus of the SBDS
protein, we assessed SBDS protein expression in cell lines derived from
these 7 patients. We also examined the intracellular localization of the
SBDS protein in wild-type and SDS patient-derived cells.
Materials and methods
Cell culture
Dana-Farber Cancer Institute (DFCI) Institutional Review Board–approved
informed consent was obtained from participating patients.
Peripheral blood mononuclear cells from patients with ShwachmanDiamond syndrome followed at Children’s Hospital or from healthy volunteers
were isolated by centrifugation over a Histopaque 1077 (Sigma, St Louis, MO)
cushion for 30 minutes at 250g. Samples were stripped of identifying information
and assigned a code number to maintain patient confidentiality. Epstein-Barr
virus–immortalized lymphoblast lines were established and maintained in RPMI
1640 containing 15% heat-inactivated fetal bovine serum (FBS; Sigma), 2 mM
glutamine, 1% penicillin and streptomycin. Cell cultures were maintained in a
humidified incubator containing 5% carbon dioxide at 37°C.
Primary fibroblast cultures were established from bone marrow aspirates and
grown in Chang Medium D (Irvine Scientific, Santa Ana, CA).
SBDS gene sequencing
SBDS sequencing was performed on patient samples by GeneDx (GeneDx,
Gaithersburg, MD).
From the Department of Pediatric Hematology/Oncology, Children’s Hospital
Boston, Boston, MA; and the Department of Pediatric Oncology, Dana-Farber
Cancer Institute, Boston, MA.
An Inside Blood analysis of this article appears in the front of this issue.
Submitted February 28, 2005; accepted April 17, 2005. Prepublished online as
Blood First Edition Paper, April 28, 2005; DOI 10.1182/blood-2005-02-0807.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by the National Institutes of Health (grants 1 K23 HL68632-01 and 1
R01 HL079582-01) (A.S.) and the Gracie Fund.
BLOOD, 15 AUGUST 2005 䡠 VOLUME 106, NUMBER 4
Reprints: Akiko Shimamura, Karp Family Research Facilities, 300 Longwood
Ave, Boston, MA 02115; e-mail: [email protected].
© 2005 by The American Society of Hematology
1253
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1254
BLOOD, 15 AUGUST 2005 䡠 VOLUME 106, NUMBER 4
AUSTIN et al
SBDS antibody
A polyclonal antibody was generated against the terminal 18 amino acids of
the SBDS protein (Ac-SLEVLNLKDVEEGDEKFE-amide) (BioSource,
Hopkinton, MA). The peptide was injected into rabbits using standard
techniques. The antibody was affinity purified over a column bearing the
18–amino acid peptide.
Laboratories, Burlingame, CA). The cells were visualized with a Zeiss Axioplan
2 microscope (Thornwood, NY), and digital images were obtained using
OpenLab software (Improvision, Lexington, MA).
Confocal microscopy was performed with a Zeiss L5M510META NLO
in the MRRC (Mental Retardation Research Center) Imaging Core at
Children’s Hospital, Boston.
Retroviral infection
Fibroblasts or lymphoblasts were infected with a pBabe-puro retrovirus
containing the SBDS cDNA as previously described.12
Western blotting
protocol.13
The Western blot protocol was modified from a previous
Cells from
lymphoblasts or primary fibroblasts were washed twice in ice-cold phosphatebuffered saline (PBS) and lysed in lysis bufferA(50 mM Tris [tris(hydroxymethyl)
aminomethane] pH 6.8, 2% sodium dodecyl sulfate, and 86 mM 2-mercaptoethanol) or buffer B (NETN [50 mM Tris pH 7.4, 150 mM NaCl, 5 mM EGTA
(ethylene glycol-bis-(B-aminoethyl ether)-N-N-N⬘-N⬘-tetraacetic acid)], 1%
NP-40 (Nonidet P-40, [Octylphenoxy]polyethylene glycol ether), 2 ⫻ protease
inhibitor tablet (Roche, Indianapolis, IN), 1 ␮M pepstatin (Roche) for 15 minutes
on ice. If lysed in buffer B, the lysate was centrifuged at full speed in an
Eppendorf Microfuge centrifuge (Eppendorf, Westbury, NY) for 20 minutes at
4°C. Protein concentration was determined using a Bradford (BioRad, Hercules,
CA) or Lowry (BioRad) assay with a bovine serum albumen standard curve. For
Western blots, 20 to 30 ␮g protein were loaded per lane on a 10% sodium
dodecyl sulfate–polyacrylamide gel with a 5% stacking gel. The proteins were
transferred to a nitrocellulose membrane for 45 minutes at 0.8 amps, 4°C in
Tris-glycine buffer (25 mM Tris base, 200 mM glycine, 20% methanol). The
filter was washed in TBST (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1%
Tween 20), blocked for 1 hour in 5% nonfat dry milk in TBST at room
temperature, and then incubated in primary antibody for 1 hour at room
temperature or overnight at 4°C. The anti-SBDS antibody was incubated at a
1:10 000 dilution in TBST for the anti–C-terminal SBDS antibody. The
antitubulin antibody (Sigma) was incubated at a 1:800 dilution in TBST for 1
hour at room temperature. The filter was then washed 3 times (10 minutes per
wash) with TBST. The secondary antibody (anti–rabbit- horseradish peroxidase;
Amersham, Piscataway, NJ) was added at a 1:10 000 dilution in 1% milk/TBST
for 1 hour. The filter was washed 3 times (10 minutes per wash) in TBST
followed by chemiluminescence.
Immunofluorescence
Primary fibroblasts were plated onto sterile coverslips. The following day, the
cells were washed twice in PBS and fixed in 4% paraformaldehyde in PBS for 10
minutes. Following another 3 washes with PBS, the cells were permeabilized for
10 minutes with 0.3% Triton X-100 in PBS. The cells were washed 3 times in
PBS and incubated with blocking buffer (10% goat serum, 0.1% NP-40 in PBS)
for 1 hour. The anti-SBDS antibody (1:400 dilution in blocking buffer) and/or the
C-23 antinucleolin antibody (1:800; Santa Cruz Biotechnology, Santa Cruz, CA)
was added for 1 to 2 hours at room temperature. The cells were washed 5 times
with 0.1% NP-40 in PBS, 5 minutes per wash, then incubated for 1 hour with the
secondary fluorescein isothiocyanate (FITC)–labeled antirabbit antibody (1:1000
in blocking buffer) and/or Texas-red antimouse antibody (1:400) (Jackson
Immunoresearch, West Grove, PA) in the dark. After 5 washes in 0.1%
NP-40/PBS, the coverslips were mounted onto slides with Vectashield (Vector
Results
Characterization of SBDS-patient mutations
Seven patients fulfilled the diagnostic criteria for ShwachmanDiamond syndrome as outlined in a recent consensus statement
from an expert panel.14 All patients had evidence of exocrine
pancreatic insufficiency by fulfilling one of the following criteria:
(1) direct pancreatic enzyme measurement following pancreatic
stimulation, (2) low serum trypsinogen or isoamylase levels, or (3)
abnormal fecal fat balance study without intestinal pathology and a
pancreatic imaging study showing a small fatty pancreas. The
patients had peripheral blood cytopenias as noted in Table 1. All
patients had neutropenia, 2 patients had concomitant anemia,
and 1 patient underwent a bone marrow transplantation for
aplastic anemia with an isochromosome 7 clonal marrow
population in the absence of overt myelodysplastic syndrome or
acute myeloid leukemia.
The SBDS genes were sequenced for each patient (Table 1). The
broad distribution of these mutations within the gene is illustrated
in Figure 1A. Five of the patients exhibited mutations predicted to
lead to premature protein truncation. The intron 2 donor splice site
mutation is the most common mutation associated with SDS9 and
was seen in 6 of the patients in our cohort. Patient DF277 was
homozygous for this mutation, whereas the other 5 patients
harbored different mutations on the second allele. Of note, patient
SD101 harbored a 505C⬎T missense mutation, leading to an
amino acid substitution of a cysteine for an arginine at amino acid
position 169. Patient DF269 was particularly interesting. Although
this patient met the clinical diagnosis for SDS, he had no identified
mutations in the SBDS gene. Some types of mutations, such as
promotor mutations, gene inversions, or large gene deletion
mutations, would not be detected by the exon-directed gene
sequencing approach used here.
Patients with SDS exhibit variable levels of SBDS
protein expression
To assess the effects of these mutations on SBDS protein expression, a rabbit polyclonal antibody was generated against an SBDS
C-terminal peptide. The ability of the antibody to recognize SBDS
protein was first validated against epitope-tagged SBDS cDNAencoded proteins as well as against glutathione-S-transferase
(GST)–tagged recombinant SBDS expressed in bacteria (data not
shown). We next used this antibody to assess endogenous SBDS
Table 1. SBDS mutations
Patient
Age at diagnosis, mo
DF250
4
DF259
24
DF260
6
DF277
Hematologic abnormalities
SBDS mutations
Neutropenia ⫹ anemia
IVS2 ⫹ 2 T⬎C, IVS3-1 G⬎A
Neutropenia ⫹ anemia
IVS2 ⫹ 2 T⬎C, 183-184TA⬎CT(K62X)
Neutropenia ⫹ anemia ⫹ thrombocytopenia ⫹ i7 clone
IVS2 ⫹ 2 T⬎C, 119delG
24
Neutropenia
IVS2 ⫹ 2 T⬎C, IVS2 ⫹ 2 T⬎C
SD101
18
Neutropenia
IVS2 ⫹ 2 T⬎C, 505C⬎T (R169C)
DF269
19
Neutropenia
No mutations identified
DF307
8
Neutropenia
IVS2 ⫹ 2 T⬎C, 183-184TA⬎CT (K62X)
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BLOOD, 15 AUGUST 2005 䡠 VOLUME 106, NUMBER 4
Figure 1. Patients with SDS express variable levels of SBDS protein. (A)
Schematic diagram of SBDS mutations. The locations of the SBDS mutations
identified in our patient series are diagramed (not drawn to scale). The SBDS gene is
composed of 5 exons (depicted with boxes). The translated mRNA regions are
shaded gray. (B) SBDS expression in fibroblasts. Bone marrow fibroblast protein
lysates were analyzed by immunoblotting for SBDS protein (top). The SBDS
genotypes of each cell line are indicated in parentheses. The blot was also probed for
tubulin to ascertain equivalent protein loading (bottom). DF259 and DF307 harbored
SBDS mutations predicted to lead to premature translation termination. The wild-type
(WT) control (NMF-100) is a marrow fibroblast cell line derived from a healthy control
patient. (C) SBDS expression in lymphoblasts. Lymphoblast protein lysates were
analyzed by immunoblotting for SBDS protein (top) and tubulin (bottom). DF250,
DF259, and DF260 carried SBDS mutations predicted to lead to premature translation termination. SD101 carried a missense (R169C) mutation on 1 SBDS allele.
DF269 is derived from a patient with SDS who lacked any identified SBDS mutations.
(D) Retroviral transduction of the SBDS cDNA restores SBDS protein expression. A
lymphoblast (DF277.L) or fibroblast (DF259.F) SDS cell line was infected with a
pBabe retrovirus containing a wild-type SBDS cDNA (lanes 3 and 5) or empty vector
(lanes 2 and 4). Cell lysates were immunoblotted for SBDS protein. A normal
lymphoblast control cell line (DF213) was run in lane 1 (WT control). (E) SBDS protein
is expressed in a variety of tissues. Lysates from the indicated human cell lines were
immunoblotted for SBDS protein expression. Caco2 indicates intestinal epithelial cell
line; HepG2, hepatocellular carcinoma cell line; HL-60, myeloid leukemia cell line;
U2OS, osteosarcoma cell line; CAPAN-1, pancreatic adenocarcinoma cell line.
protein expression. Lysates from normal control fibroblasts were
analyzed by immunoblotting with an antibody directed against the
C-terminus of SBDS. As shown in Figure 1B, lane 1, a protein of
slighter more than 30 kDa was detected in normal control lysates.
This size was close to the predicted size of 28.8 kDa based on the
protein sequence and correlated with findings by Woloszynek et
al.15 SBDS protein expression was noted in all cell types examined,
including lymphocytes (Figure 1C, lane 1), myeloid cells (HL-60),
pancreatic cells (CAPAN-1), osteosarcoma cells (U2OS), liver
cells (HepG2), and intestinal epithelial cells (Caco2) (Figure 1E,
lanes 1-5).
Fibroblast cell lines derived from patients with SDS were then
analyzed for SBDS protein expression. As expected, SBDS protein
was not readily discernible in DF259 and DF307, who harbored
biallelic SBDS gene mutations predicted to lead to protein truncation (Figure 1B, lanes 2 and 3). Equivalent protein loading was
ascertained by both Ponceau stain and by probing the same blot
with an antibody against tubulin as an internal standard (Figure 1B,
bottom). Since the antibody is directed against the carboxyl
SHWACHMAN-DIAMOND SYNDROME AND SBDS PROTEIN
1255
terminus of SBDS, the production of smaller truncated SBDS
mutant proteins cannot be ruled out. Western blots of lymphocytes
from patients DF250, DF259, and DF 260 also lacked full-length
SBDS protein (Figure 1C, lanes 2-4). A small quantity of SBDS
protein expression was detectable in lysates from DF277 lymphoblasts (Figure 1D, lane 2). This patient was homozygous for
mutations at the IVS2 ⫹ 2 T⬎C intron 2 splice donor site. A trace
amount of SBDS protein could sometimes be discerned in some
SDS cell lines wherein one affected allele bore the IVS2 ⫹ 2 T⬎C
splice site mutation. This mutation could potentially produce a
small quantity of alternatively spliced SBDS mRNA encoding a
functional protein, and the additive production from 2 alleles in
DF277 likely facilitated the detection of this scant SBDS protein
product. A recent study of the yeast SBDS orthologue, YLR022C,
suggested that pathogenic SBDS mutations were likely to represent
the effects of hypomorphic alleles and that complete loss of SBDS
function was likely to be lethal.16 This observation provides a
possible rationale for the prevalence of this splice site mutation in
patients with SDS. In further support of the antibody specificity, the
introduction of a wild-type SBDS cDNA into the DF259 fibroblast
cell line or the DF277 lymphocyte cell line restored SBDS protein
expression (Figure 1D). DF314 is a cell line derived from a
6-year-old patient who had exocrine pancreatic insufficiency but no
significant marrow failure and was heterozygous for SBDS mutations (IVS2 ⫹ 2 T⬎C on one allele, no mutations detected on the
other allele). DF314 expressed wild-type levels of SBDS protein
(compare Figure 1B, lanes 1 and 4).
Patient SD101 harbored the arginine to cysteine point mutation
in the SBDS gene, and this patient expressed reduced levels of
SBDS protein (Figure 1C, lane 5). Interestingly, Patient DF269, for
whom no SBDS gene mutation was detected by sequencing,
expressed SBDS protein at levels comparable to that observed in
wild-type control cells (compare Figure 1C, lanes 1 and 6). The
presence of normal levels of full-length protein ruled out the
presence of other undetected gene mutations that might affect
protein expression. It was possible that this patient harbors a defect
in a different gene contributing to the SDS molecular pathway.
Another possibility was that there might be a separate second
molecular pathway that culminates in a phenotype similar to SDS.
SBDS protein is located in both the nucleus and the cytoplasm
but particularly concentrated within the nucleolus
We next investigated the intracellular localization of SBDS protein
by immunofluorescence. We first validated the antibody specificity
in this assay by probing DF259 cells, which lack detectable SBDS
protein expression by Western blot. No SBDS protein was detected
by immunofluorescence in either DF259.F fibroblasts infected with
empty vector retrovirus (Figure 2A, top) or uninfected DF259.F
cells (data not shown). SBDS expression was restored following
stable infection with a retrovirus containing a wild-type SBDS
cDNA (Figure 2A, bottom). SBDS protein was present throughout
the cell in both the cytoplasm and the nucleus, but it was most
prominent within subregions of the nucleus corresponding to the
nucleolus by DAPI stain in the majority of cells.
To confirm whether endogenous SBDS protein exhibited a
similar localization pattern, we probed normal control fibroblasts
(Figure 2C, top) or HeLa cells (Figure 2B, top) with the anti-SBDS
antibody. Again, endogenous SBDS was localized throughout the
cell but was concentrated within the nucleolus in the majority of
cells. To confirm the nucleolar localization of SBDS, HeLa cells
were simultaneously probed with an antibody against nucleolin
(reviewed in Ginisty et al17 and Srivastava and Pollard18) (Figure 2B,
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1256
AUSTIN et al
BLOOD, 15 AUGUST 2005 䡠 VOLUME 106, NUMBER 4
Figure 2. SBDS localizes to the nucleolus. (A) Retroviral SBDS
transduction restores SBDS expression by immunofluorescence.
DF259.F fibroblasts infected with retrovirus containing empty
vector (top) or SBDS cDNA (bottom) were fixed and probed with
the anti-SBDS antibody followed by a FITC-labeled secondary
antibody. The cells were counterstained with DAPI (4⬘, 6-diamino2-phenylindole) to visualize the nuclei. SBDS was located throughout the cell but was particularly prominent in the region corresponding to the nucleolus. (B) Endogenous SBDS localizes to the
nucleolus. HeLa cells were fixed and probed with both an
anti-SBDS polyclonal antibody and an antinucleolin monoclonal
antibody. SBDS was detected with an antirabbit FITC-labeled
secondary antibody, and nucleolin was detected with an antimouse Texas-Red (TR) secondary antibody. Cells were visualized
by standard fluorescence microscopy (top) or by confocal microscopy (bottom). (C) SBDS nucleolar localization is intact in SD101
fibroblasts. WT control fibroblasts (NMF-100) or SD101.F fibroblasts were analyzed by immunofluorescence for SBDS. Low
power, ⫻ 63 magnification; high power, ⫻ 100 magnification. (D)
SBDS localizes to the nucleolus in DF269 lymphoblasts and in
myeloid cells. The indicated lymphoblast cell lines or the myeloid
leukemia HL-60 cell lines were subjected to immunofluorescent
staining for SBDS protein and nucleolin. Nuclei were counterstained with DAPI.
top). Cells were also analyzed by confocal microscopy (Figure 2B,
bottom) to ascertain the colocalization of SBDS and nucleolin. Wildtype lymphocyte controls also exhibited nucleolar SBDS localization
(Figure 2D). This pattern of localization was not restricted to fibroblasts
and lymphocytes since a similar pattern of SBDS localization was also
observed in a myeloid cell line (Figure 2D).
We next assessed SBDS localization in patient SD101, who
carried a R169C SBDS missense mutation. By immunofluorescence, the R169C SBDS mutant protein was still able to localize to
the nucleolus (Figure 2C, bottom). Quantitation of the percentage
of cells with SBDS nucleolar localization failed to show any
significant difference between SD101 and a wild-type control cell
line (data not shown). We also examined DF269 cells, which were
derived from a patient with SDS lacking SBDS mutations. It was
possible that the defect in this patient might involve pathways
affecting SBDS localization; however, SBDS was observed in the
nucleolus in DF269 cells (Figure 2D). A slightly more prominent
cytoplasmic localization was generally noted in the DF269 cells;
however, quantitation of the percentage of DF269 cells exhibiting
nucleolar SBDS localization did not show any significant difference from that observed in the wild-type lymphocyte controls (data
not shown).
SBDS nucleolar localization is cell-cycle dependent
The observation that SBDS was localized to the nucleolus in only a
subset of cells (approximately 70%-80%) raised the intriguing
possibility that SBDS intracellular localization was dynamic, as is
the case for many nucleolar proteins.19 To investigate the regulation
of SBDS localization, we visualized SBDS in synchronized HeLa
cells following release from a double thymidine block. The
cell-cycle profile was analyzed by flow cytometry (Figure 3A).
SBDS protein was localized to the nucleolus when cells were
predominantly in the G1 or G2 phases of the cell cycle (Figure 3A, 0
hour and 7 hours). SBDS became diffusely localized throughout
the nucleus when cells were predominantly in S phase (Figure 3A,
4 hours). The nucleolus is disassembled during mitosis,20,21 and,
accordingly, SBDS nucleolar localization was also lost during that
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BLOOD, 15 AUGUST 2005 䡠 VOLUME 106, NUMBER 4
SHWACHMAN-DIAMOND SYNDROME AND SBDS PROTEIN
1257
Figure 3. SBDS nucleolar localization is cell-cycle dependent. (A) HeLa cells were synchronized with a double thymidine block, then released for the times indicated. Cells
were fixed and stained for SBDS. An aliquot of cells was also fixed for flow cytometry (right). Cells in late telophase are indicated with arrows. (B) SBDS protein levels remain
constant throughout the cell cycle. HeLa cells synchronized with a double thymidine block were released for the indicated times. Cell lysates were immunoblotted for Fanconi
anemia, complementation group D2 (FANCD2), SBDS, and tubulin.
time. A pair of cells in telophase can be seen on the right-hand side
of Figure 3A at 7 hours (arrows). SBDS is diffusely dispersed
throughout the mitotic cells, although some reaccumulation within
the nucleolus could be detected. The same cell-cycle–dependent
pattern of SBDS intracellular localization was observed following
synchronization with mimosine (data not shown).
We also assessed SBDS protein levels by Western blot at
different phases of the cell cycle (Figure 3B). The blot was also
probed for tubulin to confirm equivalent protein loading. FANCD2
was simultaneously immunoblotted as a marker for progression
through the cell cycle.22 FANCD2 is predominantly monoubiquitinated following a double thymidine block and is gradually
deubiquitinated as the cells are released to progress through the cell
cycle.22 As shown by Western blot analysis, SBDS protein expression remained constant throughout the cell cycle (Figure 3B).
Discussion
We characterized a rabbit polyclonal antibody raised against the
carboxyl terminus of SBDS. Using this antibody, we have identified
wild-type SBDS as an approximately 31-kDa protein located throughout the cell but primarily in the nucleus with accumulation in the
nucleolus in a cell-cycle–dependent pattern. Protein expression levels
varied between patients with SDS in a manner corresponding with that
predicted from the associated SBDS gene mutations.
The data from patient DF269 was provoking. This patient
fulfilled the current diagnostic criteria for SDS, yet carried no
SBDS gene mutations and expressed normal levels of SBDS
protein. Furthermore, SBDS nucleolar localization is intact in this
patient. It is possible that DF269 represents a different disease
process with phenotypic features that are similar to SDS. Another
possibility is that this patient could harbor a mutation in a second
gene involved in the pathogenesis of Shwachman-Diamond syndrome. The identification of a cellular phenotype or biochemical
pathway for Shwachman-Diamond syndrome would facilitate
further investigation into this question. Additional patients with
SDS lacking identifiable SBDS gene mutations have been previously described,9,15 and SBDS protein expression has been documented in some of these patients.15
Immunofluorescence studies showed that SBDS was localized
to both the nucleus and the cytoplasm but was particularly
concentrated within the nucleolus. The nucleolus is best known as a
site of ribosome biogenesis. RNA processing factors are localized
in the nucleolus, which is intriguing because the SBDS protein has
been postulated to play a role in RNA processing based on data
from SBDS orthologues.9 The yeast orthologue of SBDS clusters
with RNA processing genes in microarray expression profiles.23
Yeast microarray studies of noncoding RNAs revealed a potential
defect in rRNA processing in the yeast mutant strain lacking the
SBDS ortholog YLR022C.24 The Archaeal orthologue resides
within conserved operons encoding RNA processing genes.25 The
Arabidopsis orthologue has an extended C-terminus that contains
an RNA-binding motif.9 More direct evidence is provided by a
recent study in yeast, where the SBDS orthologue YLR022C
protein was shown to be associated with other proteins involved in
ribosome biosynthesis.26 Genetic interactions between the yeast
nonessential gene YHR087W, which shares structural similarities to
YLR022C, and other rRNA processing genes, have also been
reported.26 Our studies describing the intranucleolar accumulation
of SBDS in human cells provide further supportive evidence for a
role for SBDS in RNA processing.
The shuttling of SBDS protein in and out of the nucleolus raises
the intriguing possibility that its localization may play an important
role in SBDS protein function. To date, neither of the 2 patients
with SDS who produce SBDS protein manifested any gross defect
in SBDS nucleolar localization. Whether the regulation of SBDS
nucleolar localization is disrupted in these patients is currently
under investigation. Further systematic mutational analysis of the
SBDS protein will help clarify this question. The dynamic state of
the nucleolus was demonstrated in a recent study of nucleolar
proteomic analysis following different stressors.19 Recent studies
have implicated the nucleolus in cellular stress responses27 and
cell-cycle regulation.28 An intriguing discovery from proteomic
studies of the nucleolus29-31 was that approximately 30% of
nucleolar proteins constituted either novel or uncharacterized
proteins. Such data raise the possibility that the nucleolus may
harbor hitherto unappreciated molecular functions. The question of
whether SBDS might contribute to cellular processes in addition to
rRNA processing remains to be explored.
The nucleolus and RNA processing have been implicated in
other inherited marrow failure syndromes. A defect in the RNA
component of RNAse MRP is responsible for cartilage-hair hypoplasia,32 which shares clinical features of SDS, including metaphyseal
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BLOOD, 15 AUGUST 2005 䡠 VOLUME 106, NUMBER 4
AUSTIN et al
dysostosis, marrow failure, and short stature. Another inherited
marrow failure syndrome, dyskeratosis congenita,33 is associated
with mutations in DKC1 that encodes the nucleolar protein,
dyskerin.34 Dyskerin shares some homology with RNA pseudouridine synthases, which play a role in the isomerization of uridine
bases in ribosomal RNAs. The role of dyskerin in rRNA processing
is not clear, because dyskerin also associates with telomerase RNA
and affects telomerase function.35 Interestingly, telomerase has also
been observed in the nucleolus,36 and the telomeres in patients with
SDS are short.37 It is unknown whether telomere shortening
contributes to disease pathogenesis or merely represents a secondary effect of marrow failure because shortened telomeres are a
common feature of inherited and acquired marrow failure.38-42 One
of the genes responsible for the inherited marrow failure syndrome,
Diamond-Blackfan anemia (DBA), is the gene encoding the
ribosomal protein RSP19. RPS19 has recently been shown to be
localized to the nucleolus of normal cells, but it can be mislocalized
when it carries pathogenic mutations.43 Further investigation of
SBDS protein function may provide insights into the emerging
molecular themes underlying general pathogenic mechanisms of
the inherited marrow failure syndromes.
Acknowledgments
We thank David Nathan for critical discussions and advice
throughout this project. We thank Shannon MacKeigan for assistance with patient samples and Matthew Salanga for performing
the confocal microscopy in the Children’s Hospital Imaging Core.
We thank Jim Huang for helpful discussions. We thank Dr Richard
Grand and Dr Robert Montgomery for the Caco2 and HepG2 cells.
We are especially grateful for the support of the Gracie Fund.
References
1. Shwachman H, Diamond LK, Oski FA, Khaw KT.
The syndrome of pancreatic insufficiency and
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2005 106: 1253-1258
doi:10.1182/blood-2005-02-0807 originally published online
April 28, 2005
The Shwachman-Diamond SBDS protein localizes to the nucleolus
Karyn M. Austin, Rebecca J. Leary and Akiko Shimamura
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