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First published online 4 July 2007
doi: 10.1242/dev.003517
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1 Department of Cell and Developmental Biology, University of Pennsylvania
School of Medicine, Philadelphia, PA 19104-6048, USA.
2 Section of Cell and Developmental Biology, University of California, San
Diego, La Jolla 92093, USA.
* Author for correspondence (e-mail: sdinardo{at}mail.med.upenn.edu)
Accepted 2 June 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Spermatogenesis, Meiosis, Cell cycle, Differentiation, Translation initiation, eIF4G, Drosophila
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Ten `spermatocyte arrest' genes are required for both meiosis and
differentiation and are sorted into two classes according to their molecular
targets and specific role in promoting transcription
(Ayyar et al., 2003;
Hiller et al., 2004;
Hiller et al., 2001;
Jiang and White-Cooper, 2003;
Lin et al., 1996;
Perezgasga et al., 2004). The
always early (aly) class affects the transcription of meiotic genes such
boule, twine and cyclin B, as well as that of
differentiation genes such as fuzzy onions (fzo) and don
juan (White-Cooper et al.,
1998). Notably, these mutations do not effect transcription of
other spermatocyte genes, such as pelota, cyclin A and
roughex. The Aly class proteins are thought to alter chromatin
structure to permit the high levels of transcription necessary in
spermatocytes (Ayyar et al.,
2003; Jiang and White-Cooper,
2003; Perezgasga et al.,
2004; White-Cooper et al.,
2000). The cannonball (can) class affects boule and
twine expression post-transcriptionally only and has no effect on
cyclin B. The post-transcriptional effects must be indirect, because
all can class loci encode testis-specific components of the general
transcriptional machinery (Hiller et al.,
2004; Hiller et al.,
2001). Together, the spermatocyte arrest genes reveal how a
diverse set of genes is selectively transcribed in spermatocytes.
The transcriptional regulatory pathway does not address the timing of
meiotic entry and differentiation, however. Although transcripts necessary for
these processes accumulate in early spermatocytes, the corresponding proteins
do not appear until much later
(White-Cooper et al., 1998).
Because there is little, if any, transcription after the G2-M
transition in flies (Olivieri and
Olivieri, 1965), spermatocytes must delay meiotic division until
all the necessary transcripts have accumulated. A similar dilemma exists
during the mitotic cycle in yeast. For cells to maintain the same average size
over several divisions, control points act during the gap phases and allow
cell cycle progression only when the cell has reached a threshold size, with
G1 predominating in budding yeast and G2 in fission
yeast (Rupes, 2002). Cell
growth rates also feed back on mitotic cell cycle progression in
Drosophila cells (Stocker and
Hafen, 2000). Less is known about how growth might affect the
specialized meiotic cell cycle.
Our identification and characterization of off-schedule
(ofs) provides evidence that cell growth is linked to the
coordination of meiosis and differentiation. Spermatocytes in ofs
mutant males failed to execute the G2-M transition of meiosis or
substantive post-meiotic differentiation and had a significant cell size
defect. The Off-schedule protein was found to resemble the eukaryotic
initiation factor 4G (eIF4G), which is a member of the eIF4F translation
initiation complex and bridges mature mRNAs and the ribosome
(Prevot et al., 2003). The
eIF4G activity of Ofs was apparent in its ability to associate with mRNA caps
and to functionally replace canonical eIF4G (also known as eIF-4G-FlyBase) in
cell culture. Because translation is primarily regulated at initiation, eIF4G
is instrumental in determining the translational capacity of a cell and thus
its ability to accumulate mass. Thus, the ofs mutant phenotype
suggests that sufficient cell mass must accumulate before spermatocytes
execute meiosis and differentiation.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). rux9
(roughex9) is a presumed null
(Gönczy et al., 1994).
Mosaic analysis with repressible cell marker (MARCM) clones were generated in
y w hsFLP Tub-GAL4 UAS-GFP:myc-nls; +; FRT82B
ofsZ3-3283/FRT82BTub-GAL80 males [MARCM stocks from Ken Irvine
(Rutgers, Piscataway, NJ) and Gary Struhl (Columbia University, New York, NY)]
(Lee and Luo, 1999). Clones
were induced in wandering third instar larvae, and flies were aged 1-3 days at
25°C and an additional 6 days at 29°C. For lacZ-marked
clones, we used FRT82B ms(3)1011 (an insertion at effete, which
results in lacZ expression throughout the male germline)
(Castrillon et al., 1993).
These clones were induced in pupae or newly eclosed adult males; flies were
aged at 25°C. twine-lacZ has been described previously
(White-Cooper et al.,
1998).
Microscopy
For phase microscopy of unfixed samples, testes were dissected in
Drosophila ringers solution, placed in 15 µl on a slide and
overlaid with a coverslip. BrdU pulse labeling was for 20-30 minutes at room
temperature, as was previously described
(Wallenfang et al., 2006).
Frozen split squashes were as described in Li et al.
(Li et al., 2004).
ß-galactosidase-activity stains on testes from newly eclosed males
homozygous for the twine-lacZ insertion were as in Gönczy et al.
(Gönczy et al., 1992).
Immunofluorescence on whole mounts was as described in Terry et al.
(Terry et al., 2006). Primary
antibody incubations were overnight at 4°C unless noted: 1:50-100 mouse
-Cyclin A-A19, 1:1000 rabbit -Boule (R383)
(Cheng et al., 1998), 1:4000
rabbit -Aly (White-Cooper et al.,
1998), 1:700 rabbit -Fzo
(Hales and Fuller, 1997), 1:20
mouse -Cyclin B-F2, 1:4000 rabbit -lamin-Dm0
(Smith and Fisher, 1989),
1:1000 rabbit -GFP (1 hour, room temperature; Molecular Probes), 1:1000
rabbit -Rux (Avedisov et al.,
2000), 1:500 rabbit -eIF4G (p180)
(Zapata et al., 1994) and 1:10
mouse -BrdU (90 minutes, 25°C; Becton-Dickinson). Images were
captured using a Zeiss microscope with Apotome illumination.
In situ hybridizations
Probes were synthesized from cDNAs AT30049 (ofs;
Fig. 5A) and RE34257
(eIF4G) using the Dig RNA labeling kit (Roche Boehringer Mannheim),
treated with DNase I, hydrolyzed in carbonate buffer and precipitated. Testes
were fixed in 4% formaldehyde in PBS for 15 minutes on ice, then in 4%
formaldehyde in PBS with 0.1% Tween-20 and 0.1% Na deoxycholate for 15 minutes
at room temperature, treated with Proteinase K and fixed a third time in 4%
formaldehyde in PBS with 0.1% Tween-20 for 30 minutes at room temperature.
Hybridization was overnight at 65°C in 50% formamide, 5xSSC, 100
µg/ml salmon sperm DNA, 50 µg/ml heparin and 0.1% Tween-20, adjusted to
pH 4.5 with citric acid. An -Digoxygenin-AP secondary antibody (Roche)
was used at 1:2000, either overnight at 4°C or for 1 hour at room
temperature, and reactions were developed with NBT/BCIP.
Cloning
The 3572D-175ca deletion was generated by male recombination using
insertion EP(3)3572 (Preston and Engels,
1996). Complementation between 3572D-175ca and other deletions
that we generated, including 3572D-197e (which complemented 3572D-175ca),
defined a roughly 2 kb relevant region. This comprised the 5' UTR for
two nested genes, CG10192 and CG33111, and included 45 bases of the CG10192
coding region (Fig. 5A).
CG10192 and CG33111 coding regions were then sequenced from
ofsZ3-3283 and two isogenic controls (Z3-0105 and
Z3-5364). Only one base change was found: a C to T change (Gln798>Stop) in
CG10192.
cDNA mapping and 5' and 3' RACE showed that CG10192 and CG33111
share an alternatively spliced 5' UTR
(Fig. 5A, transcripts A-D) but
have distinct ORFs and 3' UTRs. A 117 bp region encoding no recognizable
protein domains is alternatively spliced out of the first exon of CG10192
(Fig. 5A,B). Reverse
transcriptase (RT)-PCR analysis from testis RNA verified that this was the
only alternative splicing in the CG10192 coding region. Details of transcript
mapping are available upon request. The rescue construct contained 328 bp of
5' UTR common to all splice variants, the full-length coding region and
298 bp of the 3' UTR, assembled by RT-PCR and cloned into an hs83
promoter P element vector (Horabin and
Schedl, 1993).
Cell culture
We plated 4.5 million S2R+ cells in six-well plates in Insectagro
(Mediatech) supplemented with 7.5% fetal bovine serum and
penicillin/streptomycin. Transfections were carried out the following day
(Effectene; Qiagen). For the cap-association experiments, cells were harvested
2 days post-transfection and lysed in 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1%
Tween-20, 0.3% Igepal with a protease inhibitor cocktail (Sigma), 1 mM NaF, 1
mM activated sodium orthovanadate and 1 mM PMSF on ice for 15 minutes. GTP
(100 mM) and 1 mM DTT were added for 10 minutes on ice, and then lysates were
incubated with pre-blocked 7-methyl-GTP Sepharose 4B or Glutathione Sepharose
4B (GE, Amersham) overnight at 4°C. Beads were washed twice in lysis
buffer then in PBS and resuspended in 1xSDS-PAGE sample buffer, boiled
and analyzed by western blot.
dsRNA target regions were: DSRed, first 
600 bp of the pDSRed2 coding
region (Clontech); eIF4G, first 650 bp of the coding region
(Herold et al., 2001);
ofs, HFA14207 (GenomeRNAi Database,
www.dkfz.de/signaling2/rnai/);
and eIF4A (eIF-4A), HFA03526. Target regions were amplified
from cDNA using primers with 5' T7 promoter sequences appended, and
dsRNAs were synthesized (MEGAscript T7 kit. Ambion). dsRNA knockdown was
performed as described (Clemens et al.,
2000). Double knockdowns used twice as much total dsRNA.
Growth and rescue experiment overview: day 1, plated
4.5x106 cells per well and dsRNA treated; day 2, transfected
as above; days 4 and 7, split cells 1:3; days 2, 3, 4, 6, 8 and 10, counted
cells (hemacytometer). On day 8, cells were harvested, washed, fixed overnight
in cold 70% ethanol and stained with propidium iodide solution. They were then
sorted on a Becton-Dickinson FACSort machine running CellQuest Pro software.
Cell cycle profiles were determined on FlowJo software using the Watson
algorithm. All constructs used the Actin5C promoter, with eGFP kindly provided
by Sara Cherry (University of Pennsylvania, Philadelphia, PA), and
GFP-ofs and GFP-eIF4G both generated using the
Drosophila Gateway system and the pENTR/D-TOPO kit (Invitrogen)
(Huynh and Zieler, 1999).
Western blotting
Each 1 ml of S2R+ cells from the growth experiment was lysed in 50 µl of
50 mM Tris (pH 7.5), 150 mM NaCl, 1% NP40, 10% glycerol, 1.5 mM EGTA on ice,
combined with 2xSDS-PAGE sample buffer and boiled. Approximately 1-5
µl of lysate was loaded, adjusting for differences in cell numbers. Blots
were blocked in 5% milk in TBS +0.1% Triton X-100, then cut to probe the same
filter with experimental and loading control antibodies: 1:5000 mouse
-GFP (Clontech), 1:100,000 rabbit -eIF4G (p180)
(Zapata et al., 1994),
1:10,000 mouse -alpha-tubulin (B512; Sigma), 1:5000 rabbit
-eIF4E (eIF-4E), 1:5000 mouse -FLAG (F3165; Sigma), diluted in
2% milk (GFP antibody) or 5% milk (all others). HRP-coupled secondary
antibodies (1:2000; Vector) were used for ECL detection (Amersham).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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)
uncovered a second, stronger allele, Z3-3283, mutations in which produced no
sperm tails (Fig. 1B). Z3-3283
testes were instead filled with cells resembling primary spermatocytes, having
large nuclei and prominent nucleoli (Fig.
1D,E, red nuclei). In wild type, spermatid differentiation was
first evident with the appearance of a spherical, phase-dark mitochondrial
derivative (Fig. 1D, purple).
In the mutants, mitochondria instead formed a dissociated mass next to the
nucleus (Fig. 1E, blue). These
observations suggested that mutant spermatocytes did not execute the meiotic
G2-M transition or significant spermatid differentiation. The
phenotype was recessive (Fig.
1A), and no stronger with Z3-3283 placed over a deficiency
(Fig. 1C). Thus, the Z3-3283
allele was probably a strong hypomorph or null for the gene we tentatively
called off-schedule (ofs), and was used for all subsequent
analyses. Mutant spermatocytes appeared smaller than wild type (Fig. 1D,E, compare red nuclei). To confirm this, we induced both homozygous-mutant and homozygous-wild-type clones in heterozygous testes, and compared the cross-sectional area of young spermatocytes of each genotype. Mutant cells were identified by the loss of a lacZ-expressing chromosome (lacZ-), whereas homozygous wild-type cells were identified by higher lacZ expression (roughly 2x higher) compared with heterozygotes (homozygous wild-type cells were termed 2xlacZ+). We outlined the cell periphery, revealed by anti-phospho-Tyrosine, and found that the cross-sectional area of lacZ- ofs cells was significantly smaller than age-matched 2xlacZ+ wild-type cells induced in the same testis. For example, the mean±s.e.m. was 192±5.2 µm2 versus 291±6.1 µm2 (n=5 cells of each genotype; Student's t-test for matched pairs, P<0.001). Similar results were obtained in comparisons in two other testes. To rule out any contribution from second-site mutations, we also compared spermatocyte size in ofs/Df(3R)mbc-R1 testes versus heterozygous siblings. Young ofs/Df spermatocytes, defined as those first accumulating Boule protein (see below), were smaller than heterozygous sibling cells (mean±s.e.m.=314±15 µm2 versus 448±14 µm2, respectively; n=25 cells, five testes for each genotype; P=3.3x10-8; see Fig. S1 in the supplementary material). Because decreased size could reflect a difference in ploidy, we assayed for pre-meiotic S phase in ofs mutants by BrdU incorporation. All germ cells within a cyst cycle synchronously, allowing us to distinguish pre-meiotic S phase from the mitotic S phases by counting the number of cells in each BrdU-positive (BrdU+) cyst. The fraction of cysts per testis that were undergoing pre-meiotic S phase was similar in ofs compared to heterozygotes (0.4 versus 0.6, n=21 and 15 testes, respectively; P=0.42). Thus, the ofs spermatocytes progressed through pre-meiotic S phase normally, and were indeed in G2 of their meiotic cell cycle. The smaller size of ofs spermatocytes must therefore reflect a defect in mass accumulation.
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).
ofs did not control Aly accumulation, which had the same spermatocyte
nuclear localization in ofs mutants as in wild type
(Fig. 2A,B, red). Furthermore,
Cyclin A accumulation in ofs mutants revealed a phenotype distinct
from that of the spermatocyte-arrest class. Normally, Cyclin A accumulated in
the cytoplasm of G2 spermatocytes
(Fig. 2A, green). As
spermatocytes near the G2-M transition, Cyclin A translocates into
the nucleus, where it is visible only briefly before it is degradation at
metaphase (Eberhart et al.,
1996; Lin et al.,
1996). In spermatocyte-arrest mutants, Cyclin A accumulates
normally, but then persists (Lin et al.,
1996). By contrast, in ofs mutants, Cyclin A shifted to
the nuclei of spermatocytes and was degraded
(Fig. 2B, green, arrows; also
see Fig. 3B, gold arrow). In
addition, the timing of both nuclear translocation and eventual degradation
dramatically differed from wild type. First, nuclear accumulation appeared
more proximal to the testis tip than would normally occur
(Fig. 2B). To establish whether
this implied precocious movement of Cyclin A to the nucleus in mutant cells,
we analyzed ofs mutant clones using cell position along the testis as
a measure of developmental time. Indeed, Cyclin A was visible in the nucleus
of mutant cells (Fig. 3A,
arrows) that were much more proximally located relative to heterozygous cells,
in which the translocation only occurred at meiosis I
(Fig. 3A, arrowhead). Second,
degradation of Cyclin A appeared delayed, because each ofs mutant
testis had several cysts with nuclear Cyclin A, whereas there was, on average,
less than one cyst in wild type (Fig.
2, green) (Lin et al.,
1996). Mosaic analysis confirmed this, because Cyclin A persisted
much longer in ofs mutant cells than in heterozygous control cysts
(Fig. 3B, white arrows versus
outline), although, eventually, it was degraded in mutant cells
(Fig. 3B, gold arrow).
Whereas Cyclin A translocation was precocious, Boule accumulation was
delayed. Boule is one target of the spermatocyte-arrest pathway and
accumulates late in G2 (Cheng et
al., 1998; White-Cooper et
al., 1998). In ofs/+ heterozygotes, germ cells
approaching meiotic entry expressed both proteins
(Fig. 3C, arrow). In
ofs mutants, however, Cyclin A was degraded before Boule was first
detected (Fig. 3D, line).
Because Cyclin A persisted longer in ofs mutants, the fact that
Cyclin A and Boule did not overlap suggested that the onset of Boule
accumulation was significantly delayed. This was confirmed by scoring for
Boule accumulation in homozygous mutant (lacZ-
ofs) and homozygous wild-type (2xlacZ+)
clones at 2, 3, 6 and 8 days after clone induction. Whereas wild-type
2xlacZ+ clones accumulated Boule by day 3, we did
not detect Boule in ofs clones until day 6
(Table 1).
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). The delay in Boule accumulation should therefore cause
a delay in Twine accumulation. Indeed, in ofs mutant testes,
accumulation of a Twine translational reporter
(White-Cooper et al., 1998)
was observed in most mutant testes (15 out of 25), but at low levels and only
in cells towards the base of the testis
(Fig. 3F). Taken together, our
data show that several key meiotic regulators exhibited advanced or delayed
patterns in ofs mutants; hence the name off-schedule.
off-schedule spermatocytes overexpress Roughex, which causes aberrant Cyclin A localization
The early cytoplasmic-to-nuclear shift of Cyclin A in ofs
spermatocytes either reflected precocious execution of a normal event
associated with the G2-M transition or a different mechanism
entirely. Normally, Cyclin A translocation is coincident with chromosomal
condensation from a crescent-shaped to being more compact
(Lin et al., 1996)
(Fig. 3A, inset ii,
lower cells). However, ofs mutant cells with nuclear Cyclin A
exhibited chromosomal organization that more closely resembled early-stage
heterozygous spermatocytes (Fig.
3A, insets). Therefore, the nuclear import of Cyclin A in
ofs mutants was dissociated from other aspects of the G2-M
transition.
Similar precocious nuclear import had been observed upon overexpression of
Roughex (Rux), a cyclin-dependent kinase inhibitor
(Avedisov et al., 2000;
Gönczy et al., 1994;
Thomas et al., 1997). Indeed,
we found that ofs mutant spermatocytes exhibited abnormal
accumulation of Rux protein, and that Rux colocalized with nuclear Cyclin A
(Fig. 4B, arrow). Furthermore,
removal of rux activity prevented Cyclin A from accumulating in the
nuclei of mutant spermatocytes (Fig.
4D, arrow). Thus, in ofs mutants, excess Rux accumulated,
leading to aberrant, early nuclear Cyclin A localization
(Fig. 4A). In contrast to this
aberrant program, the normal cytoplasmic-to-nuclear transition of Cyclin A in
wild-type cells was not associated with Rux accumulation, and still occurred
when rux was absent (Fig.
4C). In summary, the changes in Cyclin A subcellular distribution
observed in ofs mutants do not reflect precocious timing for the
normal mechanism, but rather the engagement of a different mechanism
entirely.
Examining the block to meiosis in off-schedule spermatocytes
We considered whether the changes in protein accumulation that we observed
for Cyclin A, Twine and Rux were sufficient to explain the block the
G2-M transition in ofs mutants. Because Twine accumulation
was delayed in ofs mutants, we drove Twine accumulation in early
spermatocytes using the transcriptional and translational regulatory sequences
of ß2-tubulin to try to rescue meiotic entry
(Maines and Wasserman, 1999).
However, the ofs mutant phenotype was not noticeably suppressed, even
after also removing rux inhibition of Cyclin A/cyclin-dependent
kinase (Cdk) activity (data not shown). This suggests that other effectors of
the G2-M transition are also affected in ofs mutants
(perhaps Cyclin B, see Discussion).
off-schedule encodes the predominant eIF4G-like protein in spermatocytes
To clone ofs, we defined the portion of the
ofs3572D-175ca deletion crucial for its effects on
spermatogenesis and identified a nonsense mutation in
ofsZ3-3283 (Fig.
5A; see Materials and methods). Both affected the CG10192 locus.
We conducted transcript mapping, and assembled a full-length cDNA for
transgenic rescue, using a promoter that expresses well in spermatocytes (see
Materials and methods). ofs homozygotes inheriting the transgene were
fully rescued to fertility (data not shown).
ofs encodes a 2072-residue protein with striking homology in the C
terminus to the eukaryotic initiation factor 4 gamma (eIF4G). The N terminus
did not exhibit any significant homology to other proteins. The bona fide
eIF4G ortholog in D. melanogaster is CG10811 (also known as eIF-4G or
eIF4G) (Hernandez et al.,
1998; Zapata et al.,
1994), which is 1666 amino acids in length and has higher overall
similarity to eIF4G proteins from other species than does Ofs
(Fig. 5B). Ofs and the
Drosophila eIF4G protein have strong conservation in their C termini.
The premature stop in ofsZ3-3283 is upstream of the
conserved regions, consistent with its strong loss-of-function phenotype.
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Off-schedule is pulled down by 7-methyl-GTP Sepharose from S2R+ cell extracts
In cap-dependent translation, the eIF4F complex, made up of eIF4G, eIF4A
and eIF4E, acts as a bridge between the mRNA cap and the 40S ribosomal
subunit. eIF4E binds the modified cap structure of mature mRNAs, whereas eIF4A
is an RNA helicase that unwinds secondary structure
(Prevot et al., 2003). To
establish whether Ofs could associate with mRNA caps, we incubated extracts of
S2R+ cells expressing epitope-tagged versions of Ofs or eIF4G with
7-methyl-GTP Sepharose and identified bound proteins by SDS-PAGE western
analysis. Both Ofs and eIF4G associated with 7-methyl-GTP Sepharose but not
with control Sepharose beads (Fig.
6). Thus, by inference, Ofs can associate with mature mRNAs in
cultured cells.
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To examine what effect depletion of these proteins had on the cell cycle,
we analyzed DNA content by FACS on day 8. A larger proportion of cells in the
eIF4G alone and eIF4G+Ofs depletion conditions had G1 DNA content,
indicating that the G1-S transition was delayed
(Fig. 7D). This is consistent
with the fact that the G1-S transition is thought to be the major
control point for growth sensing in mitotic cells of the fly
(Stocker and Hafen, 2000). If
a G1 delay was the only effect, we would have expected to see a
similar, compensatory decrease in both S and G2. Instead, most of
the decrease occurred in S phase. Thus, G2 had also slowed, but to
a lesser degree than G1. Similar results have been reported
previously (Bjorklund et al.,
2006). As with the growth curves, the Ofs single depletion had no
effect on the cell cycle profile.
We next assessed the ability of GFP-Ofs to rescue the growth defect. Cells were treated with dsRNA on day 1, transfected with a rescue construct on day 2, and GFP expressers (transfected) and non-expressers (untransfected) were counted for each well from days 3 through to 10. Whereas GFP alone could not rescue, cells expressing either GFP-Ofs or GFP-eIF4G now exhibited growth curves and cell cycle profiles indistinguishable from controls (Fig. 7C,E, respectively). Either protein rescued eIF4G deficiency with equal effectiveness. Because dsRNA treatment probably reduced the effectiveness of the cognate GFP-tagged protein (Fig. 7B), rescue was likely to be underestimated.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Of 12 crucial residues, ten were identical in Ofs, one was
a conservative (L>I) change, and the twelfth diverged in
Drosophila eIF4G as well (data not shown). With regard to eIF4E
binding, the putative binding site in Ofs has an arginine substituted for the
usual hydrophobic residue (Mader et al.,
1995). However, a similar substitution is tolerated in
Drosophila eIF4E binding protein 1
(Miron et al., 2001), and
Baker and Fuller present evidence for interaction with Drosophila
eIF4E1 (Baker and Fuller,
2007). Taken together, it is quite likely that Ofs participates in
cap-dependent translation initiation.
eIF4G (CG10811) and Ofs (CG10192) appear to be the only two eIF4G proteins
encoded in the fly genome. One other candidate, l(2)01424, is more related to
the proposed translational inhibitor, NAT1/p97 (Rpn1)/DAP5, than to eIF4G
proteins (Takahashi et al.,
2005). Although the novel N-terminus of Ofs raised the possibility
that it would play a role distinct from eIF4G, our data suggest that Ofs can
act as the only eIF4G in cultured cells. Whether these two proteins always act
redundantly in vivo cannot be assessed without mutations in eIF4G.
Nevertheless, eIF4G, at its endogenous level, cannot substitute for Ofs in
spermatocytes. Perhaps this is simply due to a relatively lower level of eIF4G
compared with Ofs. Alternatively, Ofs might uniquely aid in the translation of
a special class of mRNAs, specific to spermatocyte development. Perhaps
sequences in its novel N-terminus assist in such a role. Although further
experiments are needed to distinguish between these possibilities, one reason
for a distinction between spermatocytes and other cells might be in their
respective mode of growth control. In cultured eIF4G-deficient mitotic cells,
the cell cycle effect we observed was on G1, whereas the defect in
spermatocyte progression was in G2. Although the G1-S
transition is the major control point for growth sensing in mitotic cells of
the fly (Stocker and Hafen,
2000), G2 might make more sense as the control point
for meiosis, because it is during this phase of the cycle that spermatocytes
need to prepare not just for division, but for differentiation. Furthermore,
spermatocytes might commit to the meiotic cycle, versus returning to the
mitotic cycle, during G2, as is the case for the yeast
Saccharomyces cerevisiae (Simchen
et al., 1972). Perhaps expressing a unique eIF4G (Ofs) in
spermatocytes helps serve this role. Given the functional role for
ofs as presented here and in the accompanying paper
(Baker and Fuller, 2007), we
propose that ofs henceforth be known as eIF4G2 (also known
as eIF-4G2-FlyBase).
|
).
Indeed, one of the earliest phenotypes in eIF4G2 spermatocytes was
their small size. Yet, we also found that Aly accumulation appeared normal and
that Rux protein appeared to accumulate to an excess degree in early
spermatocytes. These data demonstrate that some mRNAs are not affected by the
translational deficit, and raise an alternative scenario wherein spermatocytes
actively monitor their size. If they do not achieve proper growth, a
checkpoint is induced to prohibit meiosis and differentiation. Because meiosis
involves two cell divisions with little intervening interphase, size
monitoring would be especially important before these cells commit to
divide.
Circumstantial support for a growth checkpoint includes the accumulation of
the Cdk inhibitor Rux, which leads to aberrant behavior of Cyclin A. In this
model, the postulated checkpoint causes the striking delay in the accumulation
of Boule, which, in turn, explains the delay in Twine accumulation.
Eventually, Boule does accumulate to reasonable levels, perhaps as cells leak
through the checkpoint, just as eventually occurs in mitotic checkpoints
(Hartwell and Weinert, 1989).
However, by then, Cyclin A has been degraded, and without it, the eventual
accumulation of Twine cannot trigger meiosis, so the checkpoint has
succeeded.
To establish that a checkpoint exists, one would need to identify the
sensor, which detects the problem, and effectors, which execute inhibitory
functions until the cell resolves the problem. We do not have a candidate for
the sensor that detects growth at this time, nor for effectors controlling
differentiation. However, we can speculate that Rux is one effector regulating
the meiosis branch, where it could serve to inhibit Cyclin A-driven Cdc2
kinase activity (Avedisov et al.,
2000). Rux is not the only effector regulating meiosis, however.
Previous work showed that directly increasing the level of Rux only blocked
entry into the second meiotic division
(Gönczy et al., 1994).
Consequently, the accumulation of Rux that we observe in eIF4G2
mutants cannot fully explain the absence of the first meiotic division or the
defect in differentiation. As would be typical for cell cycle regulation,
several effectors must be activated at once to completely block the
G2-M transition.
The existence of other effectors could explain why forcing early Twine
accumulation failed to restore meiotic entry to eIF4G2 mutants in a
rux background. Alternatively, there might be additional positive
factors necessary for G2-M transition that have not accumulated in
eIF4G2 spermatocytes. Consistent with this, prior work driving
expression of another Cdc25, string (stg), in early
spermatocytes directed a normal rather than a precocious G2-M
transition (Sigrist et al.,
1995). Thus, advancing Cdc25 activity is insufficient to trigger a
precocious G2-M even in the absence of a growth defect. Perhaps
early spermatocytes have not had enough time to accumulate an essential
component, such as Cyclin B, for the meiotic divisions. We found that
eIF4G2 mutant clones exhibited Cyclin B levels comparable to
neighboring heterozygous cells (data not shown). However, there is a peak in
Cyclin B accumulation just prior to meiosis I
(White-Cooper et al., 1998),
and Baker and Fuller describe a deficit of this Cyclin B peak in
eIF4G2 mutants (Baker and Fuller,
2007). Thus, Cyclin B remains a candidate factor.
|
).
Similarly, during G2 in the fission yeast, Schizosaccharomyces
pombe, accumulation of CDC25 is disproportionately affected by defects in
translation (Daga and Jimenez,
1999). Perhaps the translation of Boule, along with a few other
meiotic cell cycle regulators, is disproportionately affected when translation
is compromised in spermatocytes. Although this should be investigated, this
simpler model does not explain the aberrant accumulation of Rux and the
nuclear sequestration of Cyclin A that we observed.
The role of growth control in spermatid differentiation
The defects in differentiation in eIF4G2 mutants are not secondary
to the meiotic block, because several cell cycle mutants fail to divide but
still undergo substantial post-meiotic differentiation
(Alphey et al., 1992;
Eberhart et al., 1996;
Eberhart and Wasserman, 1995;
Sigrist et al., 1995). Several
spermatid differentiation genes, such as don juan and fuzzy
onions, are transcribed in primary spermatocytes under the control of
spermatocyte arrest genes (White-Cooper et
al., 1998). Translational control delays the accumulation of their
protein products. This delay is functionally relevant, because precocious
don juan accumulation leads to sterility
(Hempel et al., 2006). In
principle, then, the lack of significant differentiation in eIF4G2
mutants could simply be due to a more pronounced translational delay for key
differentiation genes. Alternatively, the block in differentiation might
reflect a direct effect of the proposed growth checkpoint. Consistent with
either model, the accumulation of the mitochondrial fusion protein Fuzzy
onions is delayed, although we did not time this precisely (data not shown).
We expect that other differentiation targets will also be abnormally delayed
in eIF4G2 mutants.
Possible conservation of coupling translation initiation with meiotic progression
There are striking parallels to the role of eIF4G2 during spermatogenesis
in other organisms. For instance, there are also two major isoforms of eIF4G
in Caenorhabditis elegans, encoded by ifg-1. When the
longest isoform was depleted from the germ line, oocytes arrested in meiosis I
(B. D. Keiper, personal communication). The requirement for ifg-1 in
spermatogenesis has not yet been examined. However, one of the five isoforms
of eIF4E in the worm, IFE-1, is clearly essential for spermatogenesis. RNA
interference against ife-1 results in delayed meiotic progression,
and in defective sperm, in both hermaphrodites and males
(Amiri et al., 2001).
Furthermore, mouse testes carrying the Y chromosome deletion Spy
(also known as Eif2s3y-Mouse Genome Informatics) have a meiotic
arrest phenotype due to a lack of EIF2 (also known as EIF2S2-Mouse Genome
Informatics) function (Mazeyrat et al.,
2001). Taken together, these examples suggest that translational
control, and therefore possibly growth control, is a common theme for meiotic
cycle cells.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/15/2851/DC1
|
ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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