1 Department of Human Genetics, University of Michigan Medical School, Ann
Arbor, MI 48109, USA
2 Department of Internal Medicine, University of Michigan Medical School, Ann
Arbor, MI 48109, USA
* Author for correspondence (e-mail: tglaser{at}umich.edu)
Accepted 12 May 2004
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SUMMARY |
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Key words: Mouse, Genetics, Minute, Ribosome, Cell cycle, Retina, Bst
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Introduction |
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Although riboprotein mutations have also been identified in Escherichia
coli and Saccharomyces cerevisiae
(Warner, 1999), little is
known about riboprotein mutations in mammals. The mammalian ribosome comprises
79 ribosomal proteins and four rRNAs, which combine in equimolar ratios to
form the small (40S) and large (60S) subunits. Each riboprotein is encoded by
a single gene. Despite the large number of riboprotein genes
(Uechi et al., 2001
), only one
is known to be mutated in mammals. RPS19, a component of the small
ribosomal subunit, is mutated in a quarter of humans with
DiamondBlackfan anemia (DBA, MIM205900), a congenital red blood cell
hypoplasia (Draptchinskaia et al.,
1999
). In addition to anemia, these patients commonly have low
birth weight, small stature, and craniofacial and skeletal defects. Aside from
reduced somatic growth, it is unclear how these tissue-specific phenotypes
result from mutations in a gene that is universally required for protein
synthesis.
Belly spot and tail (Bst) is a semidominant, homozygous lethal
mutation. Heterozygotes have decreased pigmentation and a kinked tail
(Southard and Eicher, 1977).
Bst/+ mice also have a significant retinal abnormality, characterized
by delayed closure of the choroid fissure, marked deficiency of ganglion cells
and subretinal neovascularization (Rice et
al., 1995
; Rice et al.,
1997
; Smith et al.,
2000
; Tang et al.,
1999
). Accordingly, Bst has been proposed as a model for
human optic nerve atrophy and age-related macular degeneration.
Here, we report that the riboprotein gene Rpl24 is mutated in Bst/+ mice. The mutation impairs Rpl24 mRNA splicing and L24 production, which in turn affects ribosome biogenesis, protein synthesis and the cell cycle. Bst/+ cells have a significant growth disadvantage in chimeras that is similar to the classical cell competition effect observed in Drosophila Minute somatic mosaics. These findings establish Bst as a mouse Minute.
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Materials and methods |
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Bst mapping
Mouse strains were obtained from The Jackson Laboratory (Bar Harbor, ME).
An intersubspecific backcross was performed between (C57BLKS Bst/+
x CAST/Ei) F1 and C57BLKS +/+ mice. N2 progeny were scored at weaning
for white spotting, white hind feet and tail kinks, and were genotyped using
MIT markers (Dietrich et al.,
1992) and four new microsatellites:
PCR products were separated using 6% polyacrylamide sequencing or 4% Metaphor agarose gels. To confirm Bst genotypes, critical recombinants and N2 mice with low expressivity were test-crossed to C57BLKS. Opa1 was genotyped using PCR primers 5'-GAGCAGAAGAGCCCTGGAC-3' and 5'-CAATGGAGGGTGGATGTTTC-3' and a BlpI polymorphism in the 3'UTR. To type Hes1, a genomic segment corresponding to the terminus of bacterial artificial chromosome (BAC) clone RP24-110I11 was amplified using primers 5'-TTATTTGTTTCTACGACCCAGCTT-3' and 5'-ACTGGTCTTGAACTCCTAGACTCC-3' and scored for a single-strand conformational polymorphism (SSCP) by MDE gel electrophoresis (FMC Bioproducts).
Rpl24 genotyping
The Bst allele was typed by PCR, using upstream primer
5'-TTTGCAGCGCACATACGAG-3' which overlaps the first intron
branchpoint deletion and downstream primer
5'-GCTGACTCACATTTTGCATTAAGA-3' within the third exon. The
wild-type allele was amplified using upstream primer
5'-CTCTTTGCAGCGCACATACTAAC-3', which overlaps the intact first
intron branchpoint and downstream primer
5'-GGAAAACCTGCAGTTAACAAATTC-3' within the second intron. PCR
parameters were 3 minutes at 95°C, followed by 40 cycles (30 seconds at
95°C, 1 minute at 56°C, 1 minute at 72°C) and 7 minutes extension
at 72°C.
Reverse transcriptase PCR
Total RNA was extracted using Trizol reagent (Invitrogen) from livers of
C57BLKS+/+, C57BLKS Bst/+ and SPRET/Ei adult mice; livers of P21
weanlings and embryonic day (E) 18.5 embryos derived from a (C57BLKS
Bst/+ x CAST/Ei) F1 x SPRET/Ei cross; and heads of
co-isogenic C57BLKS Bst/+ and +/+ E13.5 embryos. First strand cDNA
synthesis was primed using random hexamers. To evaluate Bst candidate
genes, 40 cycles of RT-PCR were performed on E13.5 head and body RNA using
gene-specific primers. To evaluate Bst effects on Rpl24
splicing, three RT-PCRs were performed (designated A, B and C in
Fig. 3D,E). These reactions
share a common downstream primer (5'-CTGTCTTCTTTGATGCCTGCTTAG-3'),
which was 32P or HEX end-labeled. The upstream primers were
5'-GCAGGCCGACATCTATCAC-3' (A),
5'-GGATGGCTCCTCTTTGCAG-3' (B) and
5'-ACATCTATCACCATGAAGGTCGAG-3' (C). The upstream primer in
reaction C spans the exon 1-2 junction and only amplifies from correctly
spliced Rpl24 mRNA. PCR parameters were 3 minutes at 95°C,
followed by 35-40 cycles (30 seconds at 95°C, 1 minute at 56°C, 1
minute at 72°C) and 7 minutes at 72°C. To eliminate heteroduplex
products in reaction C, a final 10-minute extension step was performed after
adding fresh PCR reagents. Samples were then digested overnight with
AluI at 37°C. 32P-labeled products were denatured at
95°C, separated on 6% polyacrylamide sequencing gels and analyzed using a
Phosphorimager (Amersham Biosciences). HEX-labeled products were separated on
an ABI sequencer.
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To confirm the presence of Rpl24 within each transgene, we crossed founders to SJL mice and compared the ratio of SJL and C57 alleles in offspring using a Sau3A1 RFLP within intron 4 or a microsatellite marker (D16Ero9) located 1 kb upstream of Rpl24. D16Ero9 was typed using primers 5'-CTGGCTAGCATAGCTTTTCTTTTT-3' and 5'-GGAAAGGTGAGCAGGTTAAGAATA-3'. To evaluate transgene correction, founders were crossed to C57BLKS Bst/+ mice. The resulting progeny were examined for Bst phenotypes and assessed using BAC-specific and Rpl24 allele-specific PCRs.
The human RPL24 cDNA was amplified by PCR from IMAGE clone 5609137 (Open Biosystems) using an upstream primer with an engineered BamHI site (5'-CGTGGATCCGTCGCCATGAAGGTCGAGCTGTG-3') and a downstream primer with an engineered EcoRI site (5'-TTTAGAATTCTAATCTGCCAGTTTAGCGTTTTCC-3'). Following digestion, the resulting 0.5 kb BamHI-EcoRI fragment was subcloned into BglII- and EcoRI-digested pBROAD3 (Invivogen). The final construct (R26-huL24) contains the entire 471 bp RPL24 coding sequence, is transcribed from the 1.9 kb murine ROSA26 promoter, and terminates in a ß-globin polyadenylation site (Fig. 4D). The 3.0 kb transgene was excised with PacI, gel purified and injected into pronuclei of coisogenic C57BLKS Bst/+ and +/+ eggs. Transgenic mice were identified by PCR using primers 5'-ACAGGTGTGAAACAGGAAGAGAAC-3' and 5'-GGGAACAAAGGAACCTTTAATAGA-3', which selectively amplify the human cDNA construct.
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Polysome profiles
Livers from sex-matched adult littermates were homogenized in ice-cold
hypotonic lysis buffer (1.5 mM KCl, 2.5 mM MgCl2, 5 mM Tris-HCl pH
7.4, 1% Na deoxycholate, 1% Triton X-100) using 1 ml buffer per 10 mg liver.
After centrifugation for 15 minutes at 2500 g, heparin (1
mg/ml) and cycloheximide (100 µg/ml) were added, and 700 µl of the
supernatants were applied to 13 ml 7-47% (w/v) sucrose gradients. Samples were
centrifuged in a Beckman SW-41 rotor at 288,000 g for 2 hours
at 4°C. Gradients were collected using a Biocomp fractionator with UV
photometer.
Murine embryonic fibroblasts
Murine embryonic fibroblast (MEF) cultures were generated at E13.5 from
co-isogenic C57BLKS Bst/+ and +/+ embryos and from (CAST/Ei x
C57BLKS Bst/+) F2 embryos as described
(Robertson, 1987) and tested
at equivalent early passages (<P8). Experiments were performed in
triplicate unless otherwise noted.
To evaluate protein synthesis, MEFs were plated at a density of 5x104 cells per 35 mm dish. After 24 hours, cells were incubated for 2 hours in 2 ml fresh media containing 10 µCi [3H]leucine, washed with ice-cold phosphate buffered saline (PBS), and treated with 5% trichloroacetic acid (TCA) for at least 2 hours at 4°C. [3H]leucine incorporation was measured after lysis in 0.5 N NaOH, 0.5% sodium dodecyl sulphate (SDS) and was normalized to mitochondrial dehydrogenase activity in parallel cultures, measured using a water soluble tetrazolium (WST) assay (Roche).
To measure cell-doubling time, parallel cultures were plated in complete media at a density of 5x104 cells per 35 mm dish. Starting 36 hours after plating, adherent cells were harvested at 12-hour intervals and counted using a Coulter Z2 counter. The growth rate was determined between 36 and 60 hours after plating.
Bivariate cell cycle analysis was performed on asynchronous cultures using
a BrdU Flow Kit (BD Pharmingen). Cultures were labeled with 10 µM BrdU for
75 minutes, stained with an FITC-conjugated anti-BrdU antibody (new DNA
synthesis) and 7-AAD (total DNA content), and sorted using a Coulter Elite ESP
Fluorescence Cell Sorter. The fraction of cells in G1, S and G2/M is directly
related to the duration of each phase in the cell cycle
(Gray et al., 1990). Flow
cytometry data were analyzed using WinMDI v.2.8 software.
To evaluate S-phase entry after serum starvation, parallel MEF cultures
were plated in complete media at 5x104 cells per 35 mm dish.
After plating, cells were washed twice with serum-free media and refed
Dulbecco's modified Eagle's medium (DMEM) containing 0.5% fetal bovine serum
(FBS) for 48 hours. Cultures were released from serum starvation
(Herrera et al., 1996) by the
addition of complete DMEM containing 15% FBS. DNA synthesis was measured
periodically by adding 0.8 µCi [3H]thymidine per dish for 1
hour. These cultures were washed twice with ice-cold PBS and treated with 5%
trichloroacetic acid (TCA) for at least 3 hours at 4°C. The cells were
washed, lysed in 0.5 N NaOH, 0.5% SDS and counted for [3H]thymidine
incorporation. This experiment was repeated using MEFs derived from three
separate litters.
Chimera analysis
E3.5 blastocysts were harvested as described
(Robertson, 1987) from 24- to
30-day-old C57BLKS +/+ females that had been superovulated and mated to
C57BLKS Bst/+ males. Blastocysts were microinjected with exactly five
or ten ROSA26 embryonic stem (ES) cells, which were between passage 15 and 18
(kindly provided by Liz Robertson and Phil Soriano). After reimplantation into
pseudopregnant (C57BL/6 x DBA) F2 females, chimeras were analyzed at
E12.5 or after birth.
The percent agouti coat color was estimated independently by our lab and the University of Michigan transgenic core at the time of weaning. Rpl24 genotypes were determined afterward from tail DNA by allele-specific PCR. To further evaluate the extent of chimerism, adult tissues were fixed at 4°C for 2-3 hours in 2% paraformaldehyde, 0.2% glutaraldehyde 0.1 M NaPO4 (pH 7.3), 0.02% NP40, 0.02% Na deoxycholate; cryopreserved through 10-30% sucrose in PBS with 5 mM MgCl2; and embedded in optimal cutting temperature (OCT) medium (TissueTek). Cryosections (10 µm) were stained for ß-galactosidase in 100 mM NaPO4 (pH 7.3), 3 mM K3Fe(CN)6, 3 mM K4Fe(CN)6, 1.3 mM MgCl2, 0.1% X-gal for 7 hours at 37°C, counterstained with neutral red, dehydrated and mounted in Permount.
Genomic DNA was extracted from E12.5 embryos, amplified by PCR, and tested
using an XbaI polymorphism in the ßh1 locus
that distinguishes 129/SvEv (ROSA26) and C57BLKS alleles
(Fiering et al., 1995). The
relative abundance of alleles was determined using a Phosphorimager.
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Results |
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Molecular identification of Bst
Bst arose spontaneously in the inbred C57BLKS strain
(Southard and Eicher, 1977)
and was mapped to a 10 cM interval on mouse chromosome 16
(Rice et al., 1995
). To
further localize Bst, we performed an intersubspecific backcross
between (C57BLKS Bst/+ x CAST/Ei) F1 and C57BLKS+/+ mice. N2
progeny were scored for Bst pigmentation and tail phenotypes and
genotyped with informative microsatellite DNA markers. Among 828 N2 progeny,
386 were Bst/+. This segregation ratio (386:442) is less than 1:1
(P=0.092), consistent with decreased viability of Bst/+
mice. Bst maps within a 0.5 cM interval, delimited by
D16Mit199 and D16Ero1
(Fig. 2A). Our data formally
exclude Opa1, the mouse homolog of the human optic nerve atrophy gene
(MIM605290), and the Hes1 basic helix-loop-helix transcription
factor, which has roles in retinal and somite development
(Saga and Takeda, 2001
;
Tomita et al., 1996
). Both
genes lie more than 5cM centromeric to Bst
(Fig. 2A).
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Rpl24 encodes a protein component of the large (60S) ribosomal
subunit. L24 is a highly basic protein of 157 amino acids and is conserved
among eukaryotes and archaebacteria. Its position near the polypeptide exit
and elongation factor binding sites (Ban et
al., 2000), its contacts with the 40S subunit
(Spahn et al., 2001
) and the
properties of L24 mutant yeast (Dresios et
al., 2000
) suggest that L24 helps catalyze the peptidyl
transferase reaction. Rpl24 spans six exons, extends over 5.2 kb and
contains a characteristic 5' terminal oligopyrimidine (TOP) initiator
element (Hariharan et al.,
1989
). More than 20 processed Rpl24 pseudogenes are
interspersed throughout the genome (data not shown), similar to other
riboprotein genes (Zhang et al.,
2002
). However, only one of these loci, within the Bst
critical interval, contains introns and is therefore the bona fide
Rpl24 gene.
Rpl24 branchpoint mutation alters mRNA splicing
Bst destroys the first intron splice branchpoint of
Rpl24, which matches the consensus sequence (UACUAAC) exactly. The
deletion removes four nucleotides, including the critical adenosine
(underlined) that reacts to form the lariat intermediate
(Fig. 3A,B). No other obvious
branchpoints are present within this 108 bp intron. Using an allele-specific
PCR assay, we determined that the deletion in Rpl24 is unique to the
Bst mice (Fig. 3C).
This branchpoint is intact in the parental C57BLKS strain, highly related C57
strains, different Mus species (M. spretus, M. castaneus and
M. molossinus) and rat Rpl24 (accession no. X78443).
To determine how Bst affects Rpl24 splicing, we performed RT-PCR experiments (Fig. 3D). Using primers in exons 1 and 5, we amplified a novel 500 bp product from Bst/+ RNA, which reflects inclusion of intron 1. No other products were detected, including a 320 bp product that would result if exon 2 were skipped. This result was confirmed by direct PCR sequencing and a second RT-PCR using primers in intron 1 and exon 5 (Fig. 3D). Rpl24 translation begins in the first exon (Fig. 3A). Retention of intron 1 would cause premature termination of the L24 polypeptide after the first two codons. The first intron contains two other stop codons and the next AUG codon (M91) is in the fourth exon.
Activation of a cryptic branchpoint could restore proper splicing
(Ruskin et al., 1985). Because
Bst homozygotes are not available, we evaluated Rpl24
splicing in interspecies hybrid mice, derived from a (C57BLKS Bst/+
x CAST/Ei) F1 x SPRET/Ei cross, by a PCR strategy, using a
polymorphic AluI restriction site in exon 4 and an upstream primer
spanning the exon 1-2 junction. This allowed us to resolve and compare the
amount of correctly spliced Rpl24 transcripts from wild-type and
Bst alleles (Fig.
3E-G). These results show that 20-25% of Bst transcripts
are correctly spliced. Bst is therefore hypomorphic. Inbred C57BLKS
Bst heterozygotes and homozygotes thus have approximately 60% and 20%
of normal Rpl24 transcript levels, respectively.
Rpl24 BAC and cDNA transgenes correct Bst
Like the Drosophila Minutes, Bst has general effects on somatic
growth and specific effects on different tissues
(Fig. 1). To confirm that the
Rpl24 mutation is solely responsible for the pleiotropic Bst
phenotypes, we created transgenic mouse lines expressing wild-type
Rpl24 from a 181 kb mouse BAC (RP23-10L10) or a human cDNA expression
cassette (Fig. 4A,D). Four BAC
transgenic lines were bred to C57BLKS Bst/+ mice
(Fig. 4C). Founders Tg321 and
Tg326 have intact transgenes that completely correct the Bst
phenotypes (Fig. 4B,C).
Founders Tg898 and Tg901 have partial BAC transgenes lacking Rpl24
and these fail to correct the Bst phenotype. We generated cDNA
transgenics using human RPL24 and the ubiquitously active murine
ROSA26 promoter (Zambrowicz et al.,
1997). Human and mouse L24 proteins are identical. We injected
co-isogenic C57BLKS Bst/+ and +/+ eggs with the R26-huL24 construct
(Fig. 4D) and recovered seven
transgenic founders. Three of the four Bst/+ founders were
indistinguishable from wild-type littermates (data not shown), proving that
RPL24 alone is sufficient to correct the Bst phenotype and
that Rpl24 is Bst. Taken together, our data show
Bst acts via a partial loss-of-function mechanism and should be
considered a mouse Minute.
Bst impairs ribosome biogenesis
Ribosome biogenesis requires highly coordinated synthesis of ribosomal
proteins and rRNAs (Warner,
1999). These are assembled in a tightly ordered process involving
more than 200 accessory proteins and small nucleolar ribonucleoproteins
(Fatica and Tollervey, 2002
).
Decreased availability of individual components can impair these events
(Saveanu et al., 2001
;
Volarevic et al., 2000
;
Zhao et al., 2003
).
Although large and small subunits assemble independently, transcription and
processing of the 45S rRNA precursor into 18S and 28S rRNAs couples formation
of both subunits (Fatica and Tollervey,
2002). As such, rRNA processing is a sensitive indicator of
ribosome biogenesis. We therefore examined rRNA synthesis in a pulse labeling
experiment. Sex-matched co-isogenic C57BLKS Bst/+ and wild-type
littermates were fasted for 48 hours, injected with
32P-orthophosphate and re-fed for 2 hours. Profiles of unlabeled
(steady state) rRNA were similar after re-feeding
(Fig. 5A, Methylene Blue), but
the amount of newly processed rRNA in the Bst/+ mouse was
dramatically decreased (Fig.
5A, autoradiogram). The 28S rRNA was reduced to a greater extent
than the 18S rRNA, consistent with a defect in large subunit assembly
(Fig. 5A, densitometry). In
yeast, mutations specific to the 60S subunit can affect 18S rRNA processing
indirectly, most probably through the exosome
(Zanchin et al., 1997
). This
nuclease complex degrades improperly spliced mRNAs, excess rRNA precursors and
unused rRNA intermediates, and is required for rRNA processing
(Allmang et al., 2000
;
Butler, 2002
;
Mitrovich and Anderson, 2000
).
Likewise, ribosome subunits are stable once assembled and most unincorporated
riboproteins are rapidly eliminated
(Warner, 1977
). Bst
is therefore unlikely to affect the abundance of L24 at steady state. The
labeling of the 34S and 45S pre-rRNAs was similar in both mice
(Fig. 5A, autoradiogram),
suggesting that the decrease in 28S rRNA is due to increased turnover rather
than a specific bottleneck in rRNA processing. This cellular response is
consistent with a dynamic shortage of L24 polypeptide.
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Bst/+ fibroblasts have altered properties
In spite of its apparent tissue specificity, L24 deficiency should affect
every cell in the Bst/+ mouse, in the same way that cell growth is
universally slower in Minute flies
(Simpson and Morata, 1981). To
test this hypothesis, we examined cell size, protein synthesis, proliferation
rates and cycle kinetics in exponentially growing mouse embryonic fibroblast
(MEF) cultures. We found no difference in size between Bst/+ and
wild-type MEFs by flow cytometry (forward light scatter, data not shown). This
is consistent with recent studies in Drosophila showing no size
difference between Minute and wild-type imaginal disc cells
(Montagne et al., 1999
). We
examined overall protein synthesis in MEFs by [3H]leucine
incorporation, normalized to cell viability
(Fig. 6A). Bst/+
cultures showed a 31.2±5.8% reduction in the rate of protein synthesis,
similar to the 30% reduction described for M(3)i55/+
Drosophila Minute embryos (Boring
et al., 1989
).
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To further investigate the prolongation of G1 by Bst, we compared
the time required for Bst/+ MEF cultures to enter S phase after serum
stimulation. Parallel cultures were arrested in G1 by serum starvation,
released by the addition of serum-rich media, and assayed periodically for DNA
synthesis by [3H]thymidine incorporation. We observed a
2.4±0.5 hour increase in the time required to reach half-maximal
incorporation in Bst/+ cultures compared with wild type
(Fig. 6D). These results
confirm the increased length of G1 in Bst/+ MEFs and are consistent
with classical studies in which the protein synthesis inhibitor cycloheximide
was shown to specifically lengthen G1 phase following serum stimulation
(Brooks, 1977).
Wild-type cells outcompete Bst/+ cells during development
Among the most striking features of Drosophila Minutes is the
competition that occurs between M/+ and wild-type cells in somatic
mosaics (Garcia-Bellido et al.,
1973; Morata and Ripoll,
1975
; Simpson,
1979
). The increased doubling time of Bst/+ MEFs suggests
that a similar competition effect may occur between mutant and wild-type cells
in developing mice. To test this prediction, we generated chimeras by
injecting exactly five or ten wild-type ROSA26 (R26) embryonic stem (ES) cells
into a/a (non-agouti) C57BLKS Bst/+ and +/+ E3.5
blastocysts (Fig. 7A). The R26
ES cells are derived from the A/A (agouti) 129/SvEv strain
and contain a ubiquitously expressed lacZ enhancer trap
(Zambrowicz et al., 1997
). In
every experiment, we found that a greater fraction of mice derived from
Bst/+ blastocysts were detectably chimeric (
5% agouti
coloration). These chimeras had a greater agouti coat content
(Fig. 7B,C) and more extensive
lacZ staining throughout the body
(Fig. 7D). Some tissues, such
as brain, have a greater R26 contribution than others, such as liver,
regardless of the recipient blastocyst genotype. This is most likely due to
strain differences between C57 and the 129 ES cells. A similar strain effect
was described in neural tissues of interspecific aggregation chimeras
(Goldowitz, 1989
).
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Discussion |
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If wild-type cells outcompete Bst/+ cells in the germline as
expected, our results may have practical implications for generating high
level ES cell chimeras, as a useful alternative to tetraploid recombination
methods (Nagy et al., 1993;
Wang et al., 1997
).
Bst/+ blastocysts may represent ideal hosts, as the introduced ES
cells should readily populate the embryo.
Paucity of mammalian riboprotein mutations
Riboprotein mutations appear to be rare in mammals, in contrast to
bacteria, yeast and Drosophila. The paucity of mutations can be
explained if heterozygous phenotypes are too mild, too severe, heterogeneous
or otherwise poorly ascertained. The fact that Bst is hypomorphic
suggests that a threshold level of gene expression might be required for
survival. Null mutations may be lethal or subvital. Likewise, ribosomes may
tolerate mutations in few of their components. L24 does not exist in
eubacteria and is one of seven dispensable riboproteins in yeast
(Baronas-Lowell and Warner,
1990). Yeast lacking L24 have an increased doubling time and a
reduced peptidyl transferase rate
(Baronas-Lowell and Warner,
1990
; Dresios et al.,
2000
). These observations suggest that L24 enhances translation
efficiency but does not have a unique structural role. Indeed, L24 is added to
the 60S subunit relatively late, most probably during export through the
nuclear pore or in perinuclear cytoplasm
(Harnpicharnchai et al., 2001
;
Saveanu et al., 2001
). A
special case of redundancy might also exist for L24. An evolutionarily
conserved paralog, Rlp24 (ribosomal-like protein 24), associates with pre-60S
complexes (Saveanu et al.,
2001
). Mouse Rlp24 and L24 are 31% identical and equally similar
to the archaean L24 homolog. It has been proposed that Rlp24 plays an
essential role in ribosome biogenesis and that Rlp24 and L24 sequentially
occupy the same docking site on the 60S subunit, in the nucleus and cytoplasm,
respectively (Saveanu et al.,
2001
). If Rlp24 can reach the cytoplasm, it might in principle
partially substitute for L24 in the mature ribosome. Alternatively, a large
number of unrecognized riboprotein mutations may exist in mice as they do in
Drosophila, with semidominant spotting, tail defects and small size
as core phenotypes, and homozygous lethality
(Hrabe de Angelis and Balling,
1998
; Morgan,
1950
; Tease and Fisher,
1993
).
Bst phenotypes
Since Rpl24 is essential for protein translation, Bst
homozygotes are likely to die at or before the blastocyst stage, when the pool
of maternal ribosomes is exhausted (Copp,
1995), or slightly later, owing to low-level Rpl24
expression from the Bst allele
(Fig. 4E-G). In a normal mouse
blastocyst,
20% of the 2.5x108 ribosomes originate from
the oocyte (Piko and Clegg,
1982
) and ribosomal proteins account for 8% of total protein
synthesis (LaMarca and Wassarman,
1979
). By contrast, 0-nu Xenopus mutants lacking rRNA
genes can survive to the tadpole stage using maternal ribosomes only
(Brown and Gurdon, 1964
).
The specific defects in Bst/+ mice
(Fig. 1) are difficult to
reconcile with a global reduction in protein synthesis. Developing retinas,
somites and melanocytes may be hypersensitive because mRNAs critical for these
tissues are translated at or near a required threshold. Indeed, different
mRNAs are translated with widely varying rates of initiation and elongation
(Dever, 2002;
Lodish, 1976
). However,
Bst phenotypes may be more easily explained by tissue differences in
(1) the extent of coupling between cell division and organogenesis, (2) levels
of riboprotein gene expression or (3) extraribosomal functions.
The cell cycle alterations in Bst/+ mice
(Fig. 6) provide the basis for
the first model. In Drosophila, mutations in the promitotic
dmyc gene are phenotypically similar to Minutes
(Johnston et al., 1999). Mice
with graded c-myc alleles are likewise small, have short tails and
prolonged cell cycles (Trumpp et al.,
2001
). Bst phenotypes could arise in tissues in which the
timecourse of organogenesis is relatively uncoupled from cell proliferation.
During the life cycle of Minute flies, delayed eclosion allows for
complete development. In mice, however, gestation cannot be prolonged;
Bst/+ pups are born at the same time as wild-type littermates.
Organogenesis must proceed within the time allotted to ensure viability and
must be relatively uncoupled from cell proliferation. In principle, defects
could result from situations in which development outpaces cell proliferation.
Such a dyschronic mechanism may explain the phenotypes in the retina
and vertebral column, where timing of developmental events is particularly
critical.
In the case of the developing retina, a single progenitor population gives
rise to seven major cell types in a characteristic order
(Turner et al., 1990). The
progenitors are thought to proceed through a sequence of competency states,
each of which favors the differentiation of one or a few particular cell types
(Livesey and Cepko, 2001
).
RGCs are the first-born retinal neurons in all vertebrate species
(Altshuler et al., 1991
).
Although histogenesis can occur when cell division is blocked
(Harris and Hartenstein,
1991
), the timing of cell cycle exit greatly influences retinal
development and progenitor cell fate (Dyer
and Cepko, 2001
). Furthermore, cell cycle length is significantly
modulated during the normal course of retinal histogenesis
(Alexiades and Cepko, 1996
;
Li et al., 2000
). In the
Bst/+ retina, there is no change in the fraction of cells undergoing
proliferation or apoptosis, but the total number of neuroblasts is reduced by
roughly 60% (E10.5) to 20% (E13.5), and the onset of terminal mitoses is
delayed from E10.5 to E12.5 (Tang et al.,
1999
). These findings can now be considered in light of the
general increase in Bst/+ cell-cycle length
(Fig. 6). Assuming our MEF
results apply in vivo, Bst is likely to have a disproportionately
greater effect during the early stages of retinal histogenesis, when the cycle
time is shorter (Alexiades and Cepko,
1996
), protein synthetic demands are greatest, and RGCs are born
(Livesey and Cepko, 2001
). The
subretinal neovascularization (Smith et
al., 2000
) is probably a secondary consequence of RGC deficiency,
similar to that observed in Math5 mutant mice and humans with optic
nerve aplasia (Brown et al.,
2001
; Lee et al.,
1996
; Brzezinski et al.,
2003
).
Timing is similarly critical during somite development. Somites are added
iteratively to the caudal end of the neural tube, in response to periodic
waves of gene expression (Saga and Takeda,
2001). This process is controlled by an intrinsic clock that is
sensitive to cycloheximide (Hirata et al.,
2002
; Palmeirim et al.,
1997
). The vertebral defects in Bst/+ mice are more
severe posteriorly, consistent with a progressive deterioration in periodicity
and phase coherence of the intrinsic somite clock
(Saga and Takeda, 2001
). This
pattern could occur if high levels of the protein synthesis were needed to
propagate successive cycles of somitogenesis.
In the second model, pleiotropic Bst phenotypes may reflect
tissue-specific differences in Rpl24 expression. To achieve unimolar
stoichiometry, dispersed riboprotein genes are coordinately expressed via
5'TOP elements, which define transcriptional start sites and control
translation of riboprotein mRNAs
(Hariharan et al., 1989;
Jefferies et al., 1997
). In
spite of this convergently evolved feature, 5'TOP sequences may vary in
potency across tissues. Differential regulation of riboprotein mRNAs has been
described in pluripotent embryonal carcinoma cells, various cancer cell lines
and in RNA profiling studies (Blackshaw et
al., 2001
; Panda et al.,
2002
). If the developing retina, somites and melanoblasts express
relatively low levels of Rpl24 mRNA, they may be more sensitive to
quantitative reduction caused by the Bst mutation.
In the third model, Bst phenotypes may occur because L24 has
unique extraribosomal functions, apart from protein translation
(Wool, 1996). For example, the
small subunit riboprotein S3 functions as an endonuclease during DNA repair,
while S0 acts on the cell surface, as the primary laminin receptor
(Ardini et al., 1998
). This
explanation would be supported if mice with mutations in other riboprotein
genes have phenotypes unlike Bst. The high incidence of
RPS19 mutations among DBA patients
(Draptchinskaia et al., 1999
)
suggests that S19 has extraribosomal functions in erythropoiesis. Recent
studies show that L24 from a variety of species interacts directly with the
transactivator (TAV) protein of cauliflower mosaic virus to promote
translation re-initiation on polycistronic mRNAs
(Park et al., 2001
). In
principle, the unique Bst phenotypes could reflect tissue specificity
of cellular mRNAs that utilize internal ribosome entry
(Hinnebusch, 1997
;
Johannes et al., 1999
;
Morris and Geballe, 2000
).
Implications for human disease
Human RPS19 and mouse Rpl24 mutations are both associated
with decreased growth and skeletal defects. Curiously, triphalangeal first
digits are frequent in Bst/+ mice and are the single most common
extramedullary findings in DBA patients. This suggests a shared mechanism in
which limb malformations arise through impairment of ribosome biogenesis.
However, unlike DBA patients, Bst/+ mice appear hematologically
normal in the absence of erythroid stress (data not shown). Conversely,
RPS19 mutations do not cause optic nerve disease or obvious
hypopigmentation. These findings might reflect differences between humans and
mice, or specific roles for S19 and L24 in hematopoiesis and retinogenesis,
respectively. Accordingly, if the retinal phenotype is unique to
Bst/+ mice, then RPL24 is a good candidate for dominant
human optic nerve disorders associated with small stature, including colobomas
of the posterior segment (Onwochei et al.,
2000) and septo-optic dysplasia (De Morsier syndrome, MIM182230).
Affected children have small optic nerves and growth retardation, similar to
Bst/+ mice. A few patients have mutations in the HESX1
homeobox gene (Dattani et al.,
1998
), but in most cases the cause is unknown. Growth hormone
levels are often decreased, but pituitary anatomy and endocrine profiles are
usually normal.
Riboprotein gene mutations may also contribute more broadly to common human
malformations, such as the CHARGE (MIM214800) and VACTERL (MIM192350)
syndromes, which involve a constellation of developmental defects but as yet
have no clear genetic basis (Lalani et
al., 2003). Eighty percent of CHARGE syndrome patients have
retinal colobomas, anatomically equivalent to those in Bst/+ mice
(Fig. 1B,C), and growth
retardation as core diagnostic features
(Russell-Eggitt et al., 1990
;
Tellier et al., 1998
).
Conversely, 11% of patients with ocular colobomas have diagnostic features of
CHARGE syndrome (Chestler and France,
1988
).
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ACKNOWLEDGMENTS |
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