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Prepublished online as a Blood First Edition Paper on August 1, 2002; DOI 10.1182/blood-2002-04-1131.
RED CELLS
From the New York Blood Center and Mount
Sinaï Medical Center, NY; the Lawrence Berkeley
National Laboratory, Life Sciences Division, Berkeley, CA; The Jackson
Laboratory, Bar Harbor, ME; and the Laboratoire d'Hématologie,
AP-HP, Faculté de Médecine Paris XI, INSERM U473,
Hôpital de Bicêtre, Le Kremlin Bicêtre,
France.
The gene encoding ribosomal protein S19 (RPS19) has been shown to
be mutated in 25% of the patients affected by Diamond-Blackfan anemia
(DBA), a congenital erythroblastopenia. As the role of RPS19 in
erythropoiesis is still to be defined, we performed studies on RPS19
expression during terminal erythroid differentiation. Comparative
analysis of the genomic sequences of human and mouse RPS19
genes enabled the identification of 4 conserved sequence elements
in the 5' region. Characterization of transcriptional elements allowed
the identification of the promoter in the human RPS19 gene
and the localization of a strong regulatory element in the third
conserved sequence element. By Northern blot and Western blot analyses
of murine splenic erythroblasts infected with the anemia-inducing
strain Friend virus (FAV cells), RPS19 mRNA and protein expression were
shown to decrease during terminal erythroid differentiation. We
anticipate that these findings will contribute to further development
of our understanding of the contribution of RPS19 to erythropoiesis.
(Blood. 2003;101:318-324) The mammalian ribosome is composed of 4 RNA species
and 80 different ribosomal proteins.1-5 One of these
proteins, the ribosomal protein S19 (RPS19) is localized at the beak of
the small ribosomal subunit 40S6 and mutations in the gene
encoding RPS19 have been identified in 25% of the patients affected by
Diamond-Blackfan anemia (DBA), a rare congenital
erythroblastopenia.7,8 However, the mechanistic
understanding of the relation between RPS19 and erythropoiesis and,
specifically, the impact of RPS19 mutations in DBA remains to be defined.
At the transcriptional level, all ribosomal protein genes must be
coordinated to allow for efficient and balanced protein synthesis.
Although mammalian ribosomal protein genes are not clustered9 but rather dispersed throughout the
genome,2 they are transcribed at very similar rates due to
the equivalent strength of their promoters.10 Such
coordinated activity of the promoters of these numerous ribosomal
protein genes is regulated transcriptionally through the binding of
transcription factors to specific promoter sequence
elements.11 The cis-acting transcriptional regulatory elements and the trans-acting factors, which bind
to these elements, have been characterized in the region located upstream of the translation initiation codon, in a few
human12,13 and mouse ribosomal protein
genes.10,11,14-25 However, transcriptional control of most
of these genes, including the RPS19 gene, remains to be defined.
In the present study, we provide information on the transcriptional
regulatory elements in the 5' region upstream of the translation initiation codon in the human RPS19 gene. Alignment of the
human and mouse genomic sequences from this region allowed us to
delineate 4 consensus regions present in both sequences. The Neural
Network Promoter Prediction software (LBNL, Berkeley,
CA)14-16 identified a human RPS19 gene promoter,
which was confirmed to have promoter activity. Furthermore, we found a
strong transcriptional regulatory element in the third consensus
region, upstream of the translation initiation site but downstream of
the promoter element. A putative transcription factor, which is likely
a member of the nuclear factor- Mouse RPS19 gene characterization
Alignment of human and mouse genomic sequences
DNA cloning
Cell culture
Cell transfection 293T cells were transfected with 3 µg DNA per 300 000 cells with Lipofectamine 2000 (Life Technologies). At 24 hours after transfection, cells were assayed for luciferase activity using a luminometer (Dynex, Chantilly, VA). Transfection efficiency was normalized by cotransfection with a second plasmid encoding renilla luciferase.Production and purification of chicken antibody against mouse RPS19 protein We produced an antibody raised in chicken-against-mouse His-tagged RPS19 protein with the assistance of Washington Biotechnology (Baltimore, MD). Mouse RPS19 cDNA was cloned, NsiI-XhoI, into pET31b(+) vector (Novagen, Madison, WI) and transformed in BL21 bacteria (Stratagene, La Jolla, CA). Of particular note, this cloning strategy results in removal of the insoluble protein originally encoded by the empty pET31b(+) vector. This vector was chosen because it allows C-terminal expression of the protein of interest with only 8 additional amino acids including the 6xHis tag. Mouse RPS19 protein was expressed as a His-tagged recombinant RPS19 protein after induction of BL21 bacteria with 1 mM isopropyl -D thiogalactopyranoside (ITPG) (Sigma, St Louis, MO). After induction, bacteria were spun down and lysed by freeze-thaw. Lysed bacteria were
resuspended in binding buffer (Novagen) supplemented with 0.1 mM
diisopropyl fluorophosphate (DFP) as a protease inhibitor (Sigma) and 6 M urea in order to solubilize the RPS19 protein. Bacteria suspension
was then sonicated for 60 seconds at 4°C and centrifuged at 17 000
rpm for 10 minutes at 4°C. Mouse recombinant His-tagged RPS19 protein
was purified on a nickel column according to the manufacturer's
instructions (Histidine Binding Buffer Kit; Novagen) with some
modifications. RPS19 protein solubilized in binding buffer containing 6 M urea was bound to the column. The column was subsequently washed with
wash buffer containing decreasing concentrations of urea in order to
renature the protein as much as possible. Recombinant RPS19 protein was
then eluted with elution buffer and dialyzed in phosphate-buffered
saline (PBS). Two hens were immunized with purified mouse His-tagged
recombinant RPS19. Prior to immunization, nonspecific IgYs and after
immunization, specific IgYs (corresponding to chicken IgGs) contained
in egg yolk were subject to delipidation. Then, nonspecific IgYs were just precipitated using Eggcellent IgY precipitation reagent according to the manufacturer's instructions (Pierce, Rockford, IL) whereas the
specific IgYs were affinity-purified using an Affi-Gel 10 column
(Bio-rad, Hercules, CA) previously coupled with mouse recombinant RPS19
His-tagged protein. Specific IgYs bound to mouse-affigel column RPS19
were eluted with 0.2 M glycine-HCl, pH 2.2, neutralized with 1 M Tris,
pH 8.5, extensively dialyzed against PBS, and quantified by absorbance
at 280 nm. This affinity-purified antibody was used in immunoblotting
and immunofluorescence experiments.
Northern blotting Total RNA (10 µg) extracted from FAV cells was separated on 1% agarose genetic technology grade (GTG) gel and transferred by capillarity to a nylon membrane (Amersham, Piscataway, NJ) overnight at room temperature using NorthernMax one-hour transfer buffer (Ambion, Austin, TX). The membrane was prehybridized for 6 hours at 65°C, then hybridized overnight at 65°C with a 361-bp -[32P]
deoxycytosine triphosphate (dCTP)-labeled cDNA probe amplified from RPS19 cDNA and with a 2-kb -[32P] dCTP-labeled
cDNA probe amplified from human -actin, as a control. The membrane
was then washed and autoradiographed for 8 hours at  80°C.
Immunoblotting Proteins were subject to 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred onto a polyvinylidenefluoride (PVDF) membrane (Millipore, Portsmouth, NH) for one hour at a constant current using a semidry electroblotter according to the manufacturer's instructions (Integrated separation system; Enprotech, Natick, MA). Protein transfer was assessed after staining PVDF membrane with red Ponceau. The membrane was blocked for one hour at room temperature in Blotto solution (10 mM Tris, pH 7.5, 140 mM NaCl, 1% bovine serum albumin [BSA], 4% nonfat milk, 1% donkey serum [anti-RPS19 blotting] or 1% goat serum [antiactin and anti-4.1R blotting], 1% Tween-20, 0.02% sodium azide). The membrane was then incubated overnight at 4°C with various affinity-purified primary antibodies, either an antibody against His-tagged recombinant mouse RPS19 raised in chicken, or an IgG1 kappa light chain antibody against chicken actin raised in mouse (clone C4) (ICN Biomedicals, Aurora, OH), or an antibody against a 16-kDa His-tagged recombinant protein encoding mouse 4.1R exon 13, raised in rabbit. The 4.1R antibody was a kind gift from Drs Loren Walensky and Solomon Snyder (The Johns Hopkins University School of Medicine, Baltimore, MD). These antibodies were diluted in Blotto solution at 0.05 µg/mL to 0.3 µg/mL for the anti-RPS19 antibody, at the dilution 1:20 000 for the antiactin antibody, and at 0.1 µg/mL for the anti-4.1R antibody. After 2 rinses, 3 washes of 10 minutes each, and a short blocking in Blotto solution without serum for 15 minutes, the membrane was incubated for 1 hour at room temperature with secondary antibodies coupled to the horseradish peroxidase, donkey anti-chicken (RDI, Flanders, NJ) for RPS19 detection, goat anti-mouse (Jackson ImmnoResearch Laboratories, West Grove, PA) for actin detection, and goat anti-rabbit (Sigma) for 4.1R detection, diluted 1:5000, 1:20 000, and 1:200 000, respectively, in Blotto solution without serum. After extensive washing as described above, the membranes were probed with enhanced chemiluminescence reagent R (ECL; Nen Life Science Products, Boston, MA).
Mouse RPS19 gene Subcloning of an 18-kb fragment from BAC clones into a bluescript vector enabled us to sequence the entire mouse RPS19 gene (5.1 kb), a 2.5 kb region upstream of the 5' UTR, and a 5.8 kb region downstream of the 3' UTR (GenBank accession no. AF216207). It is composed of 5 coding exons and 4 introns. The structure of the murine gene is very similar to that of the human gene. RPS19 was nonrecombinant with Lipe, D7Mit20, and D7Ertd462e, placing RPS19 5.5 cM distal to the centromere on mouse chromosome 7, a region that shows conserved synteny with human 19q13.1-q13.2, where the human RPS19 gene segregates.1 Our data have been added to Mouse Genome Database under accession no. J: 58250 and can be accessed through the World Wide Web (http://www.jax.org). No obvious potential candidate mouse mutations map to the region containing RPS19 in chromosome 7 (1997 Chromosome Committee data). Linkage data were confirmed by FISH analysis of BAC DNA containing the RPS19 gene localized to the proximal end of chromosome 7 (data not shown).Genomic sequence analysis Alignment of 5' sequences upstream of the translation initiation site in mouse and human RPS19 genes identified 4 distinct regions with significant identity. These 4 regions in human and mouse sequences respectively were: (1) region 1 from position1219 to 1054, and 1160 to 998 (77% identity); (2) region 2 from position 992 to 934, and 933 to 874 (83% identity); (3) region 3 from position 631 to 474, and 609 to 455 (86%
identity); (4) region 4 from position 398 to 263, and 381 to
249 (80% identity) (Figure 1). Interestingly, consensus region 3 spanned the exon 1 of the RPS19 gene, which corresponded to
the 2 previously identified 5' UTRs of the gene, from 557 to 488
for the longer 5' UTR described by Strausberg et al50
(GenBank entry accession no. BC000023) and from position 509
to 488 for the shorter one described by Kondoh et al25
(GenBank accession no. NM_001022) (Figure 1).
The computer software, NNPP,14-16 identified only one
potential promoter region in the 1314-bp long region upstream of the translation initiation start codon from Promoter analysis of human RPS19 gene The presence of a functional promoter in the region upstream of the translation initiation start codon in the human RPS19 gene was confirmed by the fact that this promoter was able to drive transcription of the luciferase reporter gene (Figure 2B). In the promoter assay system used, the luciferase activity generated by the human wild-type RPS19 construct was at least 40-fold higher than that measured by transfection with empty vector.No alteration in luciferase activity was observed when we introduced
into the region upstream of the translational initiation start codon
the 2 mutations found in patients with DBA (Figure 2A): the missense
mutation G>T at RPS19 gene and protein expression We investigated by Northern blot analysis the level of RPS19 mRNA in FAV cells, kindly provided by Mark Koury and colleagues.17,24 Over the time course of 0 to 44 hours in suspension culture, the proerythroblasts at 0 hours matured into enucleating erythroblasts by 44 hours. RPS19 mRNA expression decreased markedly compared with actin mRNA expression during terminal erythroid differentiation (Figure 3).
To monitor the level of RPS19 protein expression during erythroid
differentiation, we produced an antibody raised in chicken against
mouse recombinant His-tagged RPS19. After affinity purification, anti-RPS19 antibody used in a range of concentrations of 0.02 µg/mL
to 0.1 µg/mL was able to detect 100 ng of human recombinant RPS19
protein (Figure 4A). Over the time course
of 0 to 44 hours of the terminal differentiation of FAV cells, there
was a progressive decrease in RPS19 protein expression with no
detectable level of protein at 44 hours in enucleating erythroblasts
(Figure 4B). This result was reproducible in 3 independent experiments.
Importantly, specificity of the decrease in RPS19 expression during
terminal erythroid differentiation was further supported by the finding that the expression of cytoskeletal protein 4.1R, a protein known to
accumulate late in erythroid differentiation, showed increased expression (data not shown), whereas the expression of actin did not
change during erythroid differentiation over the same time course
(Figure 4B).
The present study highlights several interesting features of the
RPS19 gene expression. The comparison of human and mouse sequences upstream of the translation initiation start site enabled us
to identify 4 consensus regions containing putative binding motifs for
5 transcription factors. These include SP1, GATA1, CACCCBf in the first
consensus region, c-Rel and Rel-A in the third consensus
region, and N-Myc transcription factor in the fourth consensus
region. The strong homology between human and mouse
RPS19 genes in the promoter region already reported for the
RPL13A and RPS1113 genes
suggested to us that some conserved regions and transcription
binding motifs could also be functionally involved in the regulation of
RPS19 promoter activity. The location of the human
RPS19 gene promoter predicted by the computer program NNPP14-16 was confirmed at position In order to further investigate the role of consensus region 3 in
promoter function, we deleted binding sites for putative transcription
factors within that region. Surprisingly, we found that deletion of the
putative binding site for c-Rel and Rel-A subunits of NF- These results were in accordance with the general features of other mammalian ribosomal protein gene promoters, as previously described for the mouse RPL30,10,11,26-28 RPL32,10,27-35 and RPS16 10,34,36-38 genes. These features included (1) location of regulatory transcription elements in a short region of 200 nt immediately upstream of the transcriptional start site; (2) existence of regulatory elements located downstream of the transcription cap; and (3) regulatory elements close to the cap site which play a major role in the promoter function. In human RPS6,12 and in mouse RPS16 and RPL32, the farther-upstream elements contributed to but were not essential for promoter activity.11,31,32,36 The only exception described to date is the mouse RPL30 gene promoter in which the major determinant elements of transcriptional activity were located farther from the cap site.11,26,27 The RPS19 gene, like other housekeeping genes and ribosomal protein genes,12,13,36,37 contains several SP1 binding sites, often located very far upstream of the transcriptional start site. At least 6 potential SP1 binding motifs are present in the human RPS19 gene (all located in GC-rich regions), but they do not appear essential for promoter activity. Furthermore, in accordance with the features of mammalian ribosomal protein gene promoters,10,12,29,34,37 the human RPS19 gene promoter lacked the canonical TATA-box and exhibited a transcriptional start at cytidine residues embedded in a polypyrimidine tract, a short 5' UTR of 22 nt for the shortest 5' UTR described by Kondoh (GenBank accession no. NM_001022).25 In contrast to the predicted promoter region and the third consensus
region, neither of the 2 mutations found in DBA in the 5' region
upstream of the translation initiation start site altered promoter
activity. The missense mutation G>T at The NF- Since mutations in the RPS19 gene have been identified in 25% of the patients affected by DBA, we performed detailed Northern blot and Western blot analyses of the levels of transcription and translation of RPS19 during terminal erythroid differentiation. We chose the FAV cell system17,24 for this study since it faithfully recapitulates terminal erythroid differentiation. In this system, the pure population of proerythroblasts, put into suspension cultures, undergoes terminal differentiation and after 44 hours produces enucleating erythroblasts and reticulocytes. We observed a decrease in both RPS19 mRNA and protein expression during terminal erythroid differentiation. Furthermore, the finding of high levels of RPS19 expression in proerythroblasts and decreasing levels during terminal erythroid differentiation is consistent with the finding of maturation arrest at early stages of erythroid differentiation in DBA. Based on the present findings with regard to the expression pattern of RPS19 during erythroid differentiation, we are in the process of investigating the role of RPS19 in DBA pathogenesis. Identification of erythroid-specific binding partners for RPS19 will be important for elucidating the contribution of RPS19 defects to the DBA phenotype.
We gratefully acknowledge Dr Robert Lersch and Dr Uli Weier (Lawrence Berkeley National Laboratory) for performing FISH analysis, Dr Mark Koury for providing us with FAV cells, Dr Loren Walensky and Dr Solomon Snyder for giving us the antibody against 4.1R, Dr Victor Hou for Western blot analysis of protein 4.1R in FAV cells, Dr Philippe Gascard for his helpful advice, and Michael Patterson for his help in DNA sequencing.
Submitted April 15, 2002; accepted July 25, 2002.
Prepublished online as Blood First Edition Paper, August 1, 2002; DOI 10.1182/blood-2002-04-1131.
Supported by National Institutes of Health Grant DK26263 (N.M.), HL64885 (L.L.P.), the Daniella Maria Arturi Foundation, the Diamond Blackfan Anemia Foundation, la Direction de la Recherche Clinique Assistance Publique-Hopitaux de Paris (CRC95183 G.T.), and contrats INSERM/AFM (RD: 4MR09F et ARC 5636).
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.
Reprints: Lydie Da Costa, Red Cell Physiology Laboratory, New York Blood Center, 310 East 67th St, New York, NY 10021; e-mail: ldacosta{at}lbl.gov, or lydie.dacosta{at}bct.ap-hop-paris.fr.
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Top
Abstract
Introduction
B (NF-
) between the mouse and human
5' sequence upstream of the initiation start ATG. The human genomic
sequence upstream of the translational initiation start site is
represented as the black line at the top of the panel. The predicted
promoter found in the human sequence between the second and third
consensus regions is also indicated. The consensus binding motifs for
putative transcription factors in the consensus regions 1, 3, and 4 are
noted with arrowheads. We cloned the full-length human 5' end
(wild-type RPS19 gene at the top of the panel and
mutants below) in a promoterless pGL3 vector, which contained a
luciferase reporter gene. The 2 mutations found in patients affected by
DBA in the 5' sequence upstream of the translational initiation start
site were also cloned into pGL3 vector. Their positions are indicated.
We deleted each of the consensus regions, the predicted promoter, both
in a same construct the predicted promoter and the consensus region 3, and finally the putative 
