Download Genetic Analysis of RpL38 and RpL5, Two Minute Genes Located in

Copyright © 2005 by the Genetics Society of America
DOI: 10.1534/genetics.104.034124
Genetic Analysis of RpL38 and RpL5, Two Minute Genes Located in the Centric
Heterochromatin of Chromosome 2 of Drosophila melanogaster
Steven J. Marygold, Carmen M. A. Coelho and Sally J. Leevers1
Growth Regulation Laboratory, Cancer Research UK—London Research Institute, London WC2A 3PX, United Kingdom
Manuscript received July 29, 2004
Accepted for publication October 25, 2004
ABSTRACT
The Minute mutations of Drosophila melanogaster are thought to disrupt genes that encode ribosomal proteins (RPs) and thus impair ribosome function and protein synthesis. However, relatively few Minutes have
been tied to distinct RP genes and more Minute loci are likely to be discovered. We have identified point
mutations in RpL38 and RpL5 in a screen for factors limiting for growth of the D. melanogaster wing. Here,
we present the first genetic characterization of these loci. RpL38 is located in the centric heterochromatin
of chromosome arm 2R and is identical to a previously identified Minute, M(2)41A, and also l(2)41Af.
RpL5 is located in the 2L centric heterochromatin and defines a novel Minute gene. Both genes are haploinsufficient, as heterozygous mutations cause the classic Minute phenotypes of small bristles and delayed
development. Surprisingly, we find that RpL38 ⫺/⫹ and RpL5 ⫺/⫹ adult flies have abnormally large wings
as a result of increased cell size, emphasizing the importance of translational regulation in the control of
growth. Taken together, our data provide new molecular and genetic information on two previously
uncharacterized Minute/RP genes, the heterochromatic regions in which they reside, and the role of their
protein products in the control of organ growth.
F
IRST described in Drosophila melanogaster over 80 years
ago, the Minutes comprise at least 50 distinct genetic
loci that produce a similar set of phenotypes when mutated
(Bridges and Morgan 1923; Schultz 1929; Lindsley
and Zimm 1992; reviewed in Lambertsson 1998). All
Minute mutations are lethal when homozygous and are
associated with the dominant phenotypes of prolonged
development and short, slender bristles on the adult
body. Together, these three effects define the classic
Minute phenotype. In addition, many Minute heterozygotes have reduced viability and fertility, and several
show additional patterning and growth defects such as
roughened eyes, abnormal wings, defective abdominal
segmentation, and small body size. Finally, numerous
Minutes show dominant genetic interactions with other
mutations, especially with those that perturb wing development (Schultz 1929; Hart et al. 1993). All these
dominant phenotypes are the result of haplo-insufficiency; that is, having only one copy of a Minute gene
produces inadequate gene product for normal development.
Most, if not all, Minute phenotypes are a direct result
of suboptimal protein synthesis. For example, bristle
production and gametogenesis require maximal protein synthesis and are therefore particularly sensitive to
a reduction in the translational capacity of the cell.
1
Corresponding author: Growth Regulation Laboratory, Cancer
Research UK—London Research Institute, 44 Lincoln’s Inn Fields,
London WC2A 3PX, United Kingdom.
E-mail: [email protected]
Genetics 169: 683–695 (February 2005)
Indeed, it is now generally accepted that Minute mutations disrupt genes that encode cytosolic ribosomal proteins (RPs). There is both direct and indirect evidence
for this conclusion. First, the number of genes encoding
(cytosolic) RPs in the D. melanogaster genome (ⵑ90;
http://flybase.bio.indiana.edu; S. J. Marygold, unpublished results) compares well with the number of described Minutes (⬎50; Lindsley and Zimm 1992), considering that potentially separable Minute loci may well
have been grouped together in the past (see below).
Second, reduction in any single RP is expected to result
in the same Minute phenotype because ribosome function depends on an equimolar balance of all RPs (together with rRNAs; Warner 1999). Moreover, the vast
majority of D. melanogaster RPs are present in a single
copy in the genome (S. J. Marygold, unpublished results). Third, of the historically defined Minute loci,
several have since been unambiguously linked to RP
genes, and many others map to the same genomic region as cloned RP genes (Lambertsson 1998). More
recent work has generated new mutations in RP genes
and many of these have also been shown to cause the
Minute phenotype. Finally, to date, only mutations in
RP genes have been shown to display the full complement of Minute traits (Lambertsson 1998).
Of the ⵑ50 well-documented classical Minute loci, only
10–15 have been unequivocally assigned to one of the
ⵑ90 RP genes in the D. melanogaster genome (Lambertsson 1998; http://flybase.bio.indiana.edu; S. J. Marygold,
unpublished results). This relatively poor Minute-to-RP
gene correspondence is because most classical Minutes
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S. J. Marygold, C. M. A. Coelho and S. J. Leevers
have been discovered through the phenotype of a deficiency (i.e., deletion-bearing) heterozygote rather than
a point mutation heterozygote (Lindsley and Zimm
1992). Thus, many Minutes have only a crude cytogenetic
location on the chromosome that is hard to correlate
with a specific RP gene predicted from the physical,
sequence-based genome. Furthermore, as deletions often remove many genes, some Minute deficiencies may
uncover two or more RP genes located in close proximity, leading to an underestimation of the number of
distinct Minute loci in the genome. Two key questions
therefore remain: Do all currently described Minutes
disrupt RP genes? And does a 50% reduction in the
dosage of any RP gene generate the Minute phenotype?
It is evident that molecularly defined, nondeficiency
mutations of RP genes will be required to address these
questions. Such mutations will also permit specific genetic analyses of the function of individual RPs and
interactions between them. Much progress has been made
in this direction in recent years, mainly through P-elementmediated mutagenesis, and today discrete, molecularly
defined mutations are described for ⵑ30 of the ⵑ90 RP
genes in the D. melanogaster genome (Lambertsson 1998;
http://flybase.bio.indiana.edu ; S. J. Marygold, unpublished results).
All Minute/RP mutations described to date reduce
the overall rate of organismal growth in a dominant
manner, thereby resulting in retarded development
(Brehme 1939, 1941a; Lambertsson 1998). Like other
Minute traits, these growth defects are likely to be
caused by suboptimal cellular protein synthesis, leading
to a cell-autonomous lengthening of individual cell cycles (Morata and Ripoll 1975). Although many heterozygous Minutes attain a normal final body size, some
have been reported to be smaller than wild type (Brehme
1939; Lambertsson 1998; Montagne et al. 1999). Moreover, Brehme (1941a) reported that individual adult
wing cells in three different Minutes were abnormally
small and that at least in the case of M(3)95A1 (RpS31),
this reduction in cell size was sufficient to account for
an overall reduction in wing and body size (Brehme
1941a). It should also be noted that the rate of growth
and/or the final organ size attained is perturbed by
mutations in genes encoding other components or regulators of the translational machinery such as translation
initiation and elongation factors, S6 kinase and components of the insulin/phosphatidylinositol 3-kinase (PI3K)
pathway, and the cMyc transcription factor (Lehner
1999; Ruggero and Pandolfi 2003). However, unlike
RP mutants, the growth defects in these cases are not
associated with the dominant Minute phenotype.
Curiously, other work has linked a reduction in RP
gene dosage to hyperplasia and overgrowth. For example, mutation of D. melanogaster RpS21 or RpS6 causes
overgrowth of the imaginal discs and/or hematopoietic
organs (Watson et al. 1992; Stewart and Denell 1993;
Torok et al. 1999), while a recent mutagenesis screen
in zebrafish embryos revealed that many RP genes behave as tumor suppressors in this organism (Amsterdam et al. 2004). Furthermore, certain RPs have been
shown to be downregulated in some human cancers or
cancer syndromes (Kondoh et al. 1996; Loftus et al.
1997; Draptchinskaia et al. 1999). Recent studies in
D. melanogaster have also challenged earlier reports that
cells heterozygous for a Minute/RP mutation are small
(Brehme 1941a,b): RpS3 Plac92/⫹ cells in the developing
wing disc are wild type in size (Neufeld et al. 1998),
whereas RpS13 1/⫹ wing-disc cells are abnormally large
(Martin-Castellanos and Edgar 2002). It is unclear
at the present time whether these discrepancies reflect
different methodologies or gene/allele-specific differences. In summary, differential expression of RPs and
modification of the translational apparatus can impinge
on growth and size regulation in a number of ways.
Here, we report the isolation and analysis of point
mutations in two previously uncharacterized D. melanogaster RP genes located in the centric heterochromatin
of the second chromosome, namely RpL38 and RpL5.
We find that these mutants display the classic Minute
phenotypes of small adult bristles and delayed development. In addition, trans-heterozygous viable combinations of RpL38 mutant alleles generate flies with distinct
patterning defects. Compared to wild-type flies, flies
with a reduced dosage of either RpL38 or RpL5 have
larger wings that contain larger cells.
MATERIALS AND METHODS
D. melanogaster strains and crosses: The w 1118-iso strain was
used as a wild-type control in all crosses; it is isogenic for the
first, second, and third chromosomes and is available from
the Bloomington Stock Center at Indiana University. RpL38 2b1,
RpL38 2b2, RpL5 2d1, and RpL5 2d2 were generated in our laboratory. RpL38 NC21 (formerly l(2)41Af NC21) and l(2)NC204 NC204 were
provided by M. Peifer and are described in Myster et al.
(2004). RpL38 KPL1, RpL38 P3, and Df(2L)l t5 were provided by
P. Dimitri. Df(2L)lt31, Df(2L)lt64, and Df(2L)lt68 were provided
by B. Wakimoto; Df(2L)lt64 is described in Wakimoto and
Hearn (1990). RpL38 1 (formerly l(2)41Af 1), wg Sp-1 Bl 1 L rm Bc 1
Pu 2/CyO, Df(2R)M41A1 (formerly M(2)41A1), Df(2R)M41A2
(formerly M(2)41A2), M(2)39F 1, and all other deficiency strains
were obtained from the Bloomington Stock Center and are
described at http://flybase.bio.indiana.edu. All crosses were
performed in incubators with a 10-hr light/14-hr dark cycle
at 25⬚ except those to generate RpL38 NC21 trans-heterozygote
adults, which were performed at 18⬚. Strains were balanced
with a CyO or CyO, Kr-Gal4 UAS-GFP, or In(2LR)Gla balancer
to allow genotyping of progeny from crosses by virtue of the
Cy or GFP or Gla dominant markers.
Crosses to assess developmental delay, dominant effects on
wing size, and notal bristle phenotypes were set up in vials and
then transferred to egg-laying cages for 2-hr laying periods.
Embryos were then aged for ⵑ26 hr and 60 control or [30
Rp⫺/⫹ and 30 CyO, Kr-Gal4 UAS-GFP/⫹] first instar larvae
were transferred to fresh vials supplemented with wet yeast.
An appropriate control cross was set up at the same time and
kept under the same conditions as the test crosses for each
experiment. To measure the delay in eclosion, vials were
checked and the number of eclosed adults counted every 2
D. melanogaster RpL38 and RpL5
hr throughout the day, starting 8 days after egg deposition
and continuing until no more flies emerged.
Cuticle preparations: For wing preparations, adult flies were
collected 2–3 days after eclosion and stored in isopropanol.
Wings were subsequently dissected in isopropanol, mounted
in Euparal (Agar Scientific), and baked at 65⬚ overnight. Male
and female flies were dissected and their wings mounted separately. Only one wing from each fly was analyzed when assessing dominant phenotypes while both wings were taken from
RpL38 NC21 trans-heterozygous flies, owing to the small number
of escapers. For notal preparations, adult female flies were
boiled in 5 m KOH for 10 min to dissolve the soft tissues,
rinsed and dissected in H2O, mounted in Euparal (Agar Scientific), and baked at 65⬚ overnight. Cuticle preparations were
viewed on a Zeiss Axioplan 2 microscope, captured using an
AxioCam HRm digital camera and Axiovision 4.1 software,
and processed in Adobe Photoshop.
Bioinformatics and mapping strategy: The mapping strategy
and location of single nucleotide polymorphisms (SNPs) will
be described in detail elsewhere (our unpublished results).
The design of primer pairs and the determination of SNP
locations on the physical map were based on the D. melanogaster
Genome Release 3.1 and relied entirely on the BDGP GadFly
annotation database. The location of deficiency breakpoints
is based on the data in Myster et al. (2004) at http://flybase.
bio.indiana.edu and on our own studies. The CLUSTAL alignment in Figure 5C was achieved by first performing a BLAST
search with the D. melanogaster RpL5 protein sequence against
the RefSeqP data library; the highest-scoring hits from various
species were then aligned using CLUSTALW at Pôle BioInformatique Lyonnais (http://pbil.ibcp.fr) and subsequently were
produced using ESPript 2.1 (http://prodes.toulouse.inra.fr/
ESPript). Pfam 12.0 was used to identify protein motifs (http://
www.sanger.ac.uk/Software/Pfam).
PCR and sequencing: Genomic DNA was isolated from ⵑ10
heterozygous adult flies: the RpL38 2b1, RpL38 2b2, RpL5 2d1, and
RpL5 2d2 strains were each isogenized for the mutant and the
balancer chromosome. Flies were homogenized in 400 ␮l of
buffer (0.1 m Tris-HCl pH 9, 0.1 m EDTA, 1% SDS) and the
homogenate was incubated at 70⬚ for 30 min. Protein was
precipitated by adding 56 ␮l of 8 m potassium acetate and
incubating for 30 min on ice. Protein was then pelleted by
centrifugation at 4⬚ for 15 min and DNA precipitated from
the resulting supernatant by adding 0.6 volumes of isopropanol and incubating for 5 min at room temperature. DNA was
pelleted by centrifugation for 5 min, washed with 70% ethanol,
and finally resuspended in 200 ␮l distilled water.
Primers were designed using the Primer3 program (http://
frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi). The following primers were used to amplify the RpL38 gene: RpL38-FOR,
5⬘-CAAAGACAGCCCTCGAAAAG-3⬘; RpL38-REV, 5⬘-TTTTC
CGTACGCGTTAAAGG-3⬘. PCR conditions for RpL38 amplification were 95⬚ for 5 min, followed by 30 cycles of 95⬚ for
30 sec, 48⬚ for 1 min, and 72⬚ for 55 sec. RpL5 was amplified
in two steps using the following primers: RpL5-FOR1, 5⬘CGCTGTCTTGCCTTATATTGG-3⬘; RpL5-REV1, 5⬘-ATCGG
CTAAACTGCCTTTTG-3⬘; RpL5-FOR2, 5⬘-TATATTTGGCCA
GCACGTTG-3⬘; and RpL5-REV2, 5⬘-TTCTGTCACATTTCT
CGGCC-3⬘. RpL5 PCR conditions were the same as above except that an extension time of 1 min 15 sec was used. Primers
and PCR conditions for CG12775 are available upon request.
All PCRs were performed using 2 ␮l of genomic DNA as
template in a total volume of 25 ␮l using Taq PCR Master Mix
(QIAGEN, Chatsworth, CA).
PCR products were cleaned up using either the QIAquick
PCR purification kit or the QIAquick gel extraction kit (QIAGEN). Sequencing reactions were performed using the BigDye
Terminator v3.1 cycle sequencing kit (Applied Biosystems,
685
Foster City, CA) according to the manufacturer’s instructions.
Sequencing reactions were cleaned up using the DyeEx spin
kit (QIAGEN) and sequencing was performed on an ABI 3730
DNA analyzer (Applied Biosystems). Sequences were analyzed
using AutoAssembler (Applied Biosystems), Sequencher (Gene
Codes, Ann Arbor, MI), or SeqManII (Lasergene, DNASTAR)
software. Mutations were recognized as double peaks on sequence traces as DNA from both the mutant chromosome
and the balancer chromosome from heterozygous flies was
amplified and sequenced. All RpL38 mutations were verified
by sequencing DNA from heterozygote adults on both strands.
RpL5 mutations were confirmed by sequencing DNA from
hemizygous embryos. The genomic region containing the entire transcript ⫾100–200 bp was sequenced in each case and
found to be wild type except for the mutations reported in
the text.
Wing and cell density measurements: Whole-wing area was
calculated from a low-magnification image of the wing and measured using the magnetic Lasso tool and histogram function of
Adobe Photoshop. Cell density was calculated by drawing a 200 ⫻
200 pixel square (⫽ 40,000 pixel2) on a high-magnification image
of the area described in Table 2 and counting the number of
wing hair roots within it. Dimensions were converted from pixels
to micrometers and the cell area and total cell number were
calculated as described in Table 2.
RESULTS
E-2b and E-2d are Minute genes: To identify novel
growth regulators or growth regulatory mechanisms in
D. melanogaster, we performed a genetic interaction
screen (our unpublished results). We screened for modifiers of a small-wing phenotype induced by misexpression of a kinase-dead (KD) D. melanogaster PI3K (Dp110/
Pi3K92E) specifically in the wing (Leevers et al. 1996).
Briefly, EMS-mutagenized males were crossed to smallwing females and F1 progeny containing either a dominant suppressor or a dominant enhancer mutation were
retained and subsequently grouped into lethal complementation groups. The E-2b (for Enhancer on chromosome 2, complementation group b) and E-2d loci are
each represented by two independent mutations isolated in this screen: all four mutations dominantly enhance the KD-Dp110 small-wing phenotype (data not
shown).
Flies heterozygous for a mutation at either the E-2b
or the E-2d locus also show dominant phenotypes in a
wild-type genetic background: they have short, slender
bristles (Figure 1, A–C) and eclose with a delay of 12–24
hr compared to control flies (Figure 1D). In addition,
flies with the genotype E-2b1/E-2b 2 or E-2d 1/E-2d 2 are embryonic or larval lethal, respectively (data not shown).
Taken together, these phenotypes are characteristic of
the Minute class of mutants (Lambertsson 1998). As
Minute mutations are thought to disrupt RP genes, we
suspected that the E-2b and E-2d genes might also encode RPs.
Mapping of the E-2b and E-2d loci: We adapted the
method of Martin et al. (2001) to map and identify the
genes disrupted by the E-2b and E-2d mutations. This
strategy is based on standard meiotic recombination-
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S. J. Marygold, C. M. A. Coelho and S. J. Leevers
Figure 1.—E-2b and E-2d are Minute genes. (A–C) Notal cuticle preparations from control (A), E-2b2/⫹
(B), and E-2d1/⫹ (C) adult flies. The
notal bristles are abnormally small
and slender in B and C, as indicated
on the scutellum (arrows). Note that
the notal cleft seen in these preparations is an artifact of the mounting procedure. (D) Flies heterozygous for
E-2b1, E-2b2, E-2d 1, or E-2d 2 eclose later
than flies of the control genotype.
Time in days after egg deposition is
shown on the x-axis. The w 1118-iso strain
was used as the control genotype and
the other mutations were crossed into
this background.
based mapping using a combination of visible markers
and SNPs and will be described elsewhere (our unpublished results).
Mapping using visible markers showed that both E-2b
and E-2d loci are located close to the centromere of
the second chromosome (data not shown). SNP-based
mapping confirmed these initial findings and placed
physical limits on the chromosomal region that contains
these genes (Figure 2A). Unfortunately, we were unable
to achieve a high mapping resolution by SNP analysis
of recombinants, owing to lower rates of both recombination and SNP discovery close to the centromere, a phenomenon that has also been described by other researchers (Berger et al. 2001; Hoskins et al. 2001;
Martin et al. 2001). We therefore performed complementation tests with deficiency strains that lack genomic
sequence in the vicinity of the centric region of the
second chromosome (Figure 2B and data not shown).
Four overlapping deficiencies that failed to complement
E-2b mutations placed the E-2b gene within the centric
heterochromatin in band h46 on chromosome arm 2R
(ⵑ41C–E on the cytological map; Corradini et al.
2003). Similar complementation analyses localized E-2d
to a small region near the euchromatin-heterochromatin border on the 2L arm that corresponds to cytological
bands 40A–B and includes the distal part of heterochromatic band h35 (Hoskins et al. 2002).
E-2b corresponds to M(2)41A: At the time that we
completed the mapping described above, previous genetic analyses by other groups had identified seven
genes within the h46 interval (Coulthard et al. 2003;
Figure 3A). Of these, the Minute M(2)41A was a clear
candidate for being allelic to the E-2b gene. Consistent
with this idea, the only two extant M(2)41A mutant
strains, M(2)41A1 and M(2)41A2 (http://flybase.bio.
indiana.edu ), failed to complement the two E-2b mutations (data not shown). However, further work by us
and others has demonstrated that both these putative
M(2)41A alleles are in fact deficiencies (Myster et al.
2004; Figure 3B) and should therefore be referred to as
Df(2R)M41A1 and Df(2R)M41A2. Previous studies have
shown that all other listed alleles of M(2)41A are also
deficiencies (Lindsley and Zimm 1992). Thus, to date,
the M(2)41A locus has been defined by the Minute phenotype of several overlapping deficiency chromosomes
rather than by point mutation. Although this raises the
possibility that there is more than one Minute gene in
this chromosomal region, we consider E-2b and M(2)41A
to be identical loci (see discussion).
E-2b encodes RpL38: Although the M(2)41A locus has
not been previously associated with a particular RP, a
good candidate has emerged through recent efforts of
two laboratories. Dimitri and colleagues used FISH analysis on deficiency chromosomes to show that ⵑ20 computed genes (CGs) are located in the h46 band (Corradini et al. 2003). Peifer and colleagues confirmed and
extended these results and also defined several anchor
points between the genetic and physical maps in the h46
region to tile the genomic scaffolds correctly (Myster et
al. 2004; Figure 3B). Of the genes thus calculated to lie
D. melanogaster RpL38 and RpL5
687
Figure 2.—Mapping the E-2b
and E-2d loci. (A) Summary of the
meiotic mapping. The euchromatin (solid line) and centric heterochromatin (boxed area) of the
second chromosome are shown.
wingless Sp-1 (wg), Bristle 1 (Bl), and
Lobe rm (L ) were used as dominant
visible marker mutations in the initial round of mapping. The genomic regions containing the E-2b
and E-2d genes were ultimately
delimited to between the two SNP
markers shown (with their approximate physical locations on
Genome Release 3.1). (B) Summary of the deficiency-based mapping. The second chromosome
centric heterochromatin (boxed
area) is shown divided into its 12
cytological regions (h35–h46;
Dimitri 1991). The flanking euchromatin is labeled with respect
to its cytological banding patterns
(not drawn to scale). The distal
edges of heterochromatic bands
h35 and h46 are located approximately in polytene bands 40A and
41E, respectively (Hoskins et al.
2002; Corradini et al. 2003). A
subset of the deficiencies used to
map the E-2b (top) and E-2d (bottom) genes is shown; the horizontal line represents the approximate region removed by the deficiency (http://flybase.bio.indiana.edu; Wakimoto and Hearn
1990; B. Wakimoto, personal communication; our unpublished data). Note that Df(2R)nap14 does not extend as far proximally
as reported previously (http://flybase.bio.indiana.edu). Deficiencies that failed to complement E-2b or E-2d mutations are marked
with an asterisk. The vertical dotted lines delimit the regions that contain E-2b and E-2d. 2L , left-arm euchromatin; 2R, rightarm euchromatin; 2Lh, left-arm heterochromatin; 2Rh, right-arm heterochromatin; C, centromere.
within h46, only CG18001 (also known as CG40278) is
predicted to encode a RP. Conceptual translation of
the CG18001 coding sequence predicts a 70-amino-acid
protein that contains a Ribosomal_L38e (eukaryotic ribosomal protein 38 of the large 60S subunit) domain
that comprises almost all the protein (from amino acid
2 to 69). The size, sequence, and organization of the
CG18001 protein are typical of RpL38 orthologs from
other species. For example, human RpL38 is also 70
amino acids long and shares 73% identity with the D.
melanogaster version, and RpL38 from Arabidopsis thaliana comprises 69 residues and is 64% identical to
CG18001. Importantly, CG18001 is the only gene in the
D. melanogaster genome that is predicted to encode a
protein with a Ribosomal_L38e domain (S. J. Marygold, unpublished results) and has recently been renamed RpL38 in FlyBase (http://flybase.bio.indiana.edu)
to reflect this. The RpL38 protein is found only in eukaryotes, and although no specific function has been
ascribed to this RP, its strong evolutionary conservation
suggests that it is a critical component of the eukaryotic
ribosome (Espinosa et al. 1997).
DNA sequence analysis revealed independent point
mutations in the RpL38 gene in the E-2b 1 and E-2b 2
mutant strains (Figure 3, D and E, and Table 1). The
E-2b 1 mutation changes the initiating ATG codon to
AGG, so it is likely to be a null allele. The E-2b 2 mutation
alters the canonical CAG splice acceptor sequence in
the single intron immediately before the initiating ATG,
so it is predicted to disrupt proper splicing of the premRNA (Mount et al. 1992). We therefore conclude that
E-2b/M(2)41A corresponds to RpL38 on the physical
map and henceforth refer to this gene as RpL38 and to
the two mutant alleles isolated in our screen as RpL38 2b1
and RpL38 2b2 (Table 1).
RpL38 is identical to l(2)41Af: In addition to taking
a candidate gene approach to identifying E-2b, we conducted further complementation tests with deficiency
strains in the region generated by Myster et al. (2004).
This approach reduced the number of candidate genes
to only three: l(2)NC204, l(2)41Af, and RpL38 (Figure
3B). Further tests showed that a l(2)NC04 mutation but
not two independently derived l(2)41Af mutations complement our RpL38 alleles (Hilliker 1976; Myster et
al. 2004; data not shown). Moreover, flies heterozygous
for the l(2)41Af 1 mutation show a strong Minute bristle
phenotype and a delay in eclosion similar to RpL38 2b1
or RpL38 2b2 heterozygotes (Figure 4H and Table 1).
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S. J. Marygold, C. M. A. Coelho and S. J. Leevers
Figure 3.—E-2b is RpL38. (A)
Genetic map showing the genes in
h46 that were identified at the
start of this project. The correct
ordering of the l(2)41Ae, l(2)IR3,
and l(2)IR23 loci (bracketed) was
unknown at this time. (B) Map of
the h46 region adapted from Corradini et al. (2003) and Myster et
al. (2004). RpL38 (boldface type)
and genes to the right of RpL38
were positioned on the physical
map (not drawn to scale here)
whereas genes to the left of RpL38
were ordered on a genetic map.
The extent of the gap between the
genetic and physical maps was not
determined and the correct ordering of the l(2)41Af and l(2)NC204
loci (bracketed) at the distal end
of the genetic map was unknown.
Note that the l(2)41Ae mutation
shown in A is now thought to be
a deletion that removes l(2)IR23,
l(2)IR3, and five other more proximal genes (Myster et al. 2004).
Deficiencies are drawn and annotated as described in the legend to Figure 2. Here, deficiency strains were tested for their ability to complement the E-2b1 and
E-2b2 mutations. (C) Physical map (not to scale) of the correct gene order in the vicinity of the RpL38 gene (boldface type).
The centromere is to the left and the 2R euchromatin is to the right in A–C. (D) RpL38 gene structure showing the location
of mutations. Open boxes, UTRs; solid box, protein-coding region; thin lines, noncoding sequences. (E) DNA sequence surrounding the initiating ATG codon of the RpL38 gene in wild-type (WT) and mutant strains. Lowercase letters, intronic sequence;
uppercase letters, protein-coding sequence; solid line, initiating ATG codon; dotted line, splice-acceptor sequence; boxed letter,
point mutation.
Together, these data suggest that the l(2)41Af locus is
identical to RpL38. This was confirmed by sequencing
the RpL38 gene region in the two l(2)41Af mutant strains
(Figure 3, D and E, and Table 1). l(2)41Af 1 is a point
mutation in the splice acceptor sequence, immediately
adjacent to the mutation found in the RpL38 2b2 allele.
l(2)41Af NC21 is a C-to-T point mutation 7 bases upstream
of the predicted transcription start site and therefore
probably disrupts a regulatory element of the RpL38
gene that results in reduced mRNA levels. We conclude
TABLE 1
Defining the RpL38 allelic series
Dominant
phenotype a
RpL38 allele Former name
RpL38 2b1
RpL38 1
RpL38 2b2
RpL38 P3
RpL38 KPL1
RpL38 NC21
a
E-2b 1
l(2)41Af 1
E-2b 2
l(2)41Af P3
l(2)41Af KPL1
l(2)41Af NC21
Delay b
ⵑ1 day
ⵑ1 day
ⵑ0.5 day
ND f
ND f
None
Bristles c
Phenotype in trans
with RpL38 NC21
Viability d
Bristles c
Medium
2/98 (2%)
Strong
Strong
8/144 (5%) Very strong
Medium
6/88 (6%)
Strong
Medium 20/95 (17%) Strong
Very weak 67/147 (31%) Weak
Very weak
NAg
NAg
Molecular description
Point mutation
Point mutation
Point mutation
P insertion
P insertion
Point mutation
Ranke
in first codon: null? Strong
in splice acceptor
Strong
in splice acceptor
Strong
Medium
Weak
upstream of 5⬘ UTR Weak
All RpL38 mutations were crossed into the w 1118-iso background to assess dominant phenotypes.
Approximate delay of eclosion compared to the w 1118-iso control.
c
Nota were assessed for the Minute small-bristle phenotype.
d
Number of nonbalancer progeny/balancer progeny (% of nonbalancer progeny compared to total progeny) from a cross
of RpL38 NC21/CyO ⫻ RpL38 ⫺/CyO flies.
e
Relative strength of mutation taking all phenotypes into account.
f
Not determined.
g
Not applicable: adult RpL38 NC21 homozygotes were not recovered, perhaps because of additional lethal mutations on the
chromosome.
b
D. melanogaster RpL38 and RpL5
689
Figure 4.—Flies with reduced
RpL38 dosage show patterning defects and a severe Minute phenotype. (A) RpL38 NC21/⫹ wings have
no obvious defects (compare with
Figure 6A). (B–D) Wings that are
trans-heterozygous for RpL38 NC21
and a second RpL38 mutation
have extra venation and notches
in the wing margin. The images
shown are typical of each genotype. (E and F) High-magnification images of part of the region
bounded by vein IV, vein V, the
posterior cross-vein, and the wing
margin, oriented as in A–D. (E)
Wing hairs from a wild-type wing
point toward the distal edge of the
wing margin to form an organized
pattern. (F) Wing hair polarity in
wings trans-heterozygous for RpL38
mutations is often disorganized. A
severe example is shown here.
(G–J) Notal cuticle preparations
from adult flies. (G) RpL38 NC21/⫹ nota show a very weak Minute bristle phenotype. Compare to Figure 1A. (H) RpL38 1/⫹ nota show
a strong Minute bristle phenotype, stronger than RpL38 2 b1/⫹ or RpL38 2 b2/⫹. Compare with Figure 1B. (I) RpL38 NC21/RpL38 2 b2
flies have a stronger Minute phenotype than either heterozygote. Compare to G and Figure 1B. ( J) RpL38 NC21/RpL38 1 flies have
a very strong phenotype that is stronger than either heterozygote. Compare to G and H. Notal bristles are frequently absent,
either because they are not formed or because they are fragile and break off easily. Flies were reared at 18⬚ in this experiment
to increase the number of trans-heterozygous escapers.
that the l(2)41Af 1 and l(2)41Af N C21 mutations disrupt the
RpL38 gene and therefore refer to them as RpL38 1 and
RpL38 N C21, respectively (Table 1).
Our discovery that l(2)41Af is allelic to RpL38 allows
two improvements to the recent map of the region produced by Myster et al. (2004; Figure 3B). First, the two
most distal genes within the genetic map, l(2)41Af and
l(2)NC04, can now be ordered with respect to each other
(compare Figure 3, B and C). Second, the fact that
l(2)41Af on the genetic map is identical to RpL38 on
the physical map effectively eliminates the gap between
the end of the physical and genetic maps (compare
Figure 3, B and C).
Defining the RpL38 allelic series: In addition to the
four RpL38 point mutations described so far, we acquired two additional P insertion alleles: RpL38 P3 and
RpL38 KPL1 (N. Corradini, K. Gazzetti and P. Dimitri,
unpublished results). The different RpL38 mutants vary
in the strength of their Minute phenotype and their
behavior in trans with RpL38 NC21, and this allowed us to
order them into an allelic series that fits well with their
molecular descriptions. By these criteria, RpL38 NC21 and
RpL38 KPL1 are weak alleles, RpL38 P3 is a medium strength
allele, and RpL38 1, RpL38 2 b1, and RpL38 2b2 are strong
alleles (Figure 4 and Table 1). Note that the RpL38 1
allele shows a remarkably strong bristle phenotype relative to its other characteristics, perhaps because of additional mutations on this chromosome. Together, the
molecular description and genetic characterization of
six RpL38 mutant alleles make RpL38 one of the most
genetically tractable Minute/RP genes in the D. melanogaster genome.
In addition to the strong Minute bristle phenotype
seen on the notum of RpL38 NC21 trans-heterozygotes (Table 1 and Figure 4, G–J), these flies also show patterning
defects such as large rough eyes, ectopic wing venation,
aberrant wing-blade hair polarity, and notches at the
wing margin (Figure 4, A–F, and data not shown). The
wing notching and venation phenotypes are also seen
in RpL38 ⫺/⫹ flies at low penetrance (data not shown).
Similar wing phenotypes have been reported for a number of other Minute/RP mutations either in a wild-type
genetic background or in the context of a second mutation. For example, lowering RpL14 levels at the wing
margin results in a severe notching phenotype (Enerly
et al. 2003) and several Minutes enhance the venation
defects of plexus, net, and Delta mutants (Lambertsson
1998). Wing-notching effects were also seen when flies
were made doubly heterozygous for the RpL38 1 mutation and any of several mutations in genes required
for wing development, such as Notch, vestigial, and cut
(Rollins et al. 1999). One explanation that can account
for many of these effects on wing morphology is that the
Notch pathway is exquisitely sensitive to the impaired rate
of protein synthesis caused by a reduction in RP expression
(Hart et al. 1993).
E-2d encodes RpL5: Deficiency-based mapping placed
the E-2d gene in a region containing cytological bands
40A–B and h35 of the 2L centric heterochromatin (Figure 2B). Previous genetic studies have mapped two Mi-
690
S. J. Marygold, C. M. A. Coelho and S. J. Leevers
Figure 5.—E-2d is RpL5. (A) Physical map (not to scale) of genes surrounding the euchromatin-centric heterochromatin
boundary region of 2L (adapted from Hoskins et al. 2002 and Yasuhara et al. 2003). Note the presence of two RP genes; RpL5
is shown in boldface type. The break represents a BAC contig gap of ⵑ100 kb (Yasuhara et al. 2003). The centromere is to the
right. (B) The RpL5-PA/PB protein showing the location of the E-2d mutations. Shaded region is the Ribosomal_L5e domain.
(C) CLUSTAL alignment of D. melanogaster RpL5-PA/PB and RpL5 sequences from a variety of eukaryotes. Identical residues
have a black background while similar residues have a shaded background. The dotted line represents the Ribosomal_L5e
domain as defined in D. melanogaster RpL5 and the position of the mutations shown in B are indicated by vertical arrows. Amino
acid numbering is for the D. melanogaster RpL5 sequence.
nute loci to this region: M(2)39F at 39F1–40A4 (http://
flybase.bio.indiana.edu ) and a second Minute at 40B–F
(Howe et al. 1995). As M(2)39F 1 complements both
E-2d mutations (data not shown), it is likely that E-2d
corresponds to the more proximal Minute.
To identify the physical gene disrupted by E-2d mutations, we searched for RP genes in the 40A–B/h35 region using the D. melanogaster Genome Annotation Database (http://flybase.bio.indiana.edu). This search revealed
two potential candidates for E-2d: CG12775, which encodes RpL21, and yip6/CG17489, which encodes RpL5
(Figure 5A). Both genes were sequenced in the two
E-2d mutant strains and point mutations were discovered only in the yip6 coding sequence (Figure 5, B and
C). yip6 is located in the h35 band and encodes the
single RpL5 protein in the D. melanogaster genome (Hoskins et al. 2002; Yasuhara et al. 2003; S. J. Marygold,
unpublished results). We therefore refer to this gene
as RpL5 and to the mutant alleles as RpL5 2d1 and RpL5 2d2.
EST evidence suggests the existence of many alternative
RpL5 transcripts that could theoretically encode three different protein products: RpL5-PA/PB, -PC, and -PD/PE
(http://flybase.bio.indiana.edu). RpL5-PA/PB is the longest version at 299 amino acids in length and contains
a Ribosomal_L5e (eukaryotic ribosomal protein 5 of the
large subunit) domain from residues 26 to 173 (Figure
5, B and C). RpL5-PC consists of just the carboxy-terminal half of PA/PB and entirely lacks the Ribosomal_L5e
domain, while RpL5-PD/PE comprises the amino-terminal two-thirds of PA/PB and so retains the RibosomalL5e motif but lacks the carboxy-terminal third. Of these
three, RpL5-PA/PB is supported by the vast majority of
EST evidence and is validated by a full-length cDNA
clone. A CLUSTAL alignment of RpL5 protein sequences
from diverse eukaryotes confirms that PA/PB is the most
likely RpL5 protein product (Figure 5C). All of these
orthologs are similar in length to the D. melanogaster
RpL5-PA/PB and share a high degree of identity with
it. For example, both human and Saccharomyces cerevisiae
RpL5 are 297 amino acids in length and share 67 and
55% identity with D. melanogaster RpL5, respectively. Furthermore, the RpL5 2d1 and RpL5 2d2 mutations are predicted to have deleterious effects only within the RpL5PA/PB protein (see below). We therefore conclude that
D. melanogaster RpL38 and RpL5
691
Figure 6.—Wings heterozygous
for RpL38 or RpL5 mutations are increased in final size because of larger
cell size. (A) Wild-type control wing.
A small amount of ectopic vein tissue near the distal tip of vein II is
often seen in both the w 1118-iso and
Oregon-R strains when reared at 25⬚
under uncrowded conditions. Note
that flies reared at 25⬚ grow to smaller
final sizes than those reared at 18⬚.
For this reason the ⫹/⫹ (25⬚) wing
shown here is smaller than the
RpL38 NC21/⫹ (18⬚) wing in Figure 4A;
flies of these genotypes have wings of
the same size when reared at the same
temperature (data not shown). (B
and C) Wings heterozygous for
RpL38 2b1 (B) or RpL5 2d2 (C) are larger than control wings. A silhouette of the control wing shown in A is overlaid to allow direct
comparison. (A⬘–C⬘) High-magnification views of part of the wing region bounded by vein IV, vein V, the posterior cross-vein,
and the wing margin from flies of the respective genotypes in A–C. The increased spacing between wing hairs in B⬘ and C⬘
compared to the control (A⬘) reflects the larger cell size in these wings as each wing-blade cell protrudes a single hair.
RpL5-PA/PB is likely to be the major protein produced
from the RpL5 gene in vivo and refer to it henceforth
as RpL5.
The RpL5 2d1 mutation creates a premature termination codon just after the Ribosomal_L5e domain, so it
is predicted to produce a truncated protein missing the
carboxy-terminal third of the protein (Figure 5, B and
C). This carboxy-terminal region contains motifs involved in both 5S rRNA binding and nuclear/nucleolar
localization (Michael and Dreyfuss 1996; Rosorius
et al. 2000; see below). RpL5 2d2 is a missense mutation
that changes an alanine to a threonine within the
Ribsomomal_L5e domain (Figure 5, B and C). This
alanine residue is conserved within higher eukaryotes
and is therefore expected to be important for normal
RpL5 folding/function. Although the dominant Minute
phenotypes associated with each mutation are similar,
RpL5 2d1 hemizygous larvae die earlier and at a smaller
size than RpL5 2d2 hemizygotes (data not shown), suggesting that RpL5 2d1 is the stronger mutant allele.
Eukaryotic RpL5 is homologous to prokaryotic RpL18,
suggesting that this protein is of ancient origin and has
a key function in the ribosome. Indeed, RpL5 has been
shown to specifically bind 5S rRNA and transport it from
the nucleoplasm to the nucleolus for assembly into the
60S ribosomal subunit (Steitz et al. 1988; Deshmukh
et al. 1993, 1995; Michael and Dreyfuss 1996; Rosorius
et al. 2000). Furthermore, RpL5 has a role in anchoring
peptidyl-tRNAs to the P-site of the ribosome to prevent
frameshifting (Meskauskas and Dinman 2001).
Reducing RpL38 or RpL5 gene dosage increases final
wing size: Mutations in RpL38 and RpL5 were originally
identified as dominant enhancers of a small-wing phenotype generated by inhibiting PI3K signaling in the
fly wing (our unpublished results). Rather than representing a genuine genetic interaction, this observation
may have resulted from purely additive effects as some
Minutes show dominant reductions in wing and/or body
size (Brehme 1939, 1941a). To address this point, we
examined the size of adult wings heterozygous for a RpL38
or RpL5 mutation but otherwise wild type. Surprisingly,
these wings are 5–11% larger than wild-type control wings
(Figure 6, A–C, and Table 2). In RpL38 2b1/⫹ wings, these
increases in wing area are associated with proportional
increases in cell size without any detectable effect on
the total number of cells in the wing (Figure 6, A⬘
and B⬘; Table 2). Although RpL38 2b2/⫹, RpL5 2d1/⫹, and
RpL5 2d2/⫹ wings also comprise large cells, these wings
contain fewer cells than wild-type wings and therefore
show relatively smaller increases in total wing area compared to RpL38 2b1/⫹ wings (Figure 6 and Table 2). It
is not immediately apparent why cell number is reduced
in wings heterozygous for the RpL38 2b2, RpL5 2d1, or
RpL5 2d2 mutations but not for the RpL38 2b1 mutation.
However, we note that RpL38 2b1 is the only null mutation
and the consequent severe impairment of cellular protein synthesis in RpL38 2b1/⫹ wings may have quantitatively different effects on cell proliferation (see discussion). In summary, mutation of one copy of either
RpL38 or RpL5 increases overall wing and individual
cell size and either does not affect or reduces wing cell
number. The role of RpL38, RpL5, and other RPs in
the control of cell and organ growth is discussed below.
DISCUSSION
This article describes the first genetic analyses of D.
melanogaster RpL38 and RpL5. We find that RpL38 is
identical to the previously identified l(2)41Af and
M(2)41A loci and that RpL5 is a novel Minute gene that
has previously been called yip6. These data are of interest from several perspectives: first, analysis of the Minutes
692
S. J. Marygold, C. M. A. Coelho and S. J. Leevers
TABLE 2
RpL38
Genotype
Control e
RpL38 2b1/⫹
RpL38 2b2/⫹
RpL5 2d1/⫹
RpL5 2d2/⫹
⫺/⫹
and
RpL5 ⫺/⫹
wings are abnormally large because of increased cell size
Mean wing area a
Wing area
Mean cell density b
Cell area c
Cell area
Total no. of
Cell no.
(␮m2 ⫾ SD ⫻ 106) (% of control) (cells/␮m2 ⫾ SD ⫻ 10⫺3) (␮m2) (% of control) cells d (⫻ 103) (% of control)
1.69
1.87
1.77
1.76
1.78
⫾
⫾
⫾
⫾
⫾
0.0321
0.0607**
0.0743**
0.0313**
0.0433**
NA
111
105
105
106
6.35
5.77
5.82
5.58
5.56
⫾
⫾
⫾
⫾
⫾
0.188
0.213**
0.364**
0.249**
0.309**
158
174
172
179
180
NA
110
109
114
114
10.7
10.8
10.3*
9.86**
9.89**
NA
101
96.4
92.2
92.5
Nineteen female wings from the control genotype and 10 female wings from the mutant genotypes were analyzed; all figures
are reported to three significant figures. P-values were calculated using a two-tailed Student’s t -test assuming equal variances:
*P ⬍ 0.05, **P ⬍ 0.01.
a
The area of the whole wing exclusive of the alula and costal cell was measured.
b
Each wing-blade cell protrudes a single hair; wing hairs were counted in an area of 40,000 pixel2 (ⵑ15,000 ␮m2) from the
middle of the region flanked by vein IV, vein V, the posterior cross-vein, and the wing margin.
c
Reciprocal of the mean cell density.
d
Estimated by multiplying the mean wing area by the mean cell density.
e
Control genotype was the w 1118-iso strain; all other mutants were crossed into this background.
and their connection to RPs; second, annotation of the
centric heterochromatin of chromosome 2; and third,
the role of RPs and protein synthesis in growth regulation.
Linking RPs to Minute loci: Approximately 50 welldocumented Minute loci are identified in the D. melanogaster genome (Lambertsson 1998). Although it is
generally agreed that these loci correspond to RP genes,
this has been proven molecularly in relatively few cases.
Here, we show that the M(2)41A locus corresponds to
the RpL38 gene on the basis of three separate observations. First, comparison of the physical and genetic
maps in the h46 region shows that the physical location
of RpL38 corresponds well with the genetically defined
M(2)41A locus (Figure 3, A and B). Second, several deficiency strains that were used to define M(2)41A all fail to
complement point mutations in the RpL38 gene (Figure
3B). Third, RpL38 point mutation heterozygotes have a
similar Minute bristle phenotype to flies heterozygous for
one of the original M(2)41A alleles, Df(2R)M41A2 (data
not shown). Hilliker and colleagues have speculated
previously that M(2)41A is a repetitive or duplicated
locus (Coulthard et al. 2003). Although our data suggest otherwise, we note that flies heterozygous for the
relatively large Df(2R)M41A1 deletion show a more severe Minute phenotype than flies heterozygous for either the small Df(2R)M41A2 deletion or the RpL38 2b1
null allele (data not shown). Thus, additional genes in
the region, perhaps in the unannotated heterochromatin proximal to RpL38, may contribute to the Minute
phenotype defined by the larger Df(2R)M41A1 deletion.
M(2)39F, which maps to cytological bands 39F1-40A4,
is the only previously defined Minute locus lying in the
40A–B region that contains the RpL5 gene. However,
M(2)39F and RpL5 are not allelic to one another, and
M(2)39F probably corresponds to the RpL21 gene that
is located just distal to RpL5 on the physical map (Yasu-
hara et al. 2003; Figure 5A). Howe et al. (1995) refer
to a second Minute locus proximal to M(2)39F that
maps to 40B–F and it is probable that this unnamed
Minute corresponds to RpL5. The existence of two separable Minute loci in the vicinity of the 40A band would
have been overlooked by many deficiency-based studies
in the past because of the close proximity of the RpL21
and RpL5 genes. The same is likely to apply to other
regions of the genome and, in part, explains why there
are almost twice as many RP genes as well-documented
Minutes (see Introduction).
Annotation of the centric heterochromatin of chromosome 2: Heterochromatin is characteristically rich
in repetitive sequence elements and transposons and
has a lower gene density than euchromatin (Hoskins
et al. 2002). Nevertheless, the heterochromatin portion
of the D. melanogaster genome is substantial, comprising
ⵑ60 megabases (Mb) of the ⵑ175-Mb genome of a
female fly, and is predicted to include at least 450 genes
(Hoskins et al. 2002). Although the repetitive nature
of heterochromatic DNA has hampered its sequencing
and annotation, a number of recent studies have made
significant advances to rectifying this situation (Hoskins
et al. 2002; Corradini et al. 2003; Yasuhara et al. 2003;
Myster et al. 2004; http://www.dhgp.org).
RpL38 is the most proximal gene within the heterochromatic scaffold AABU01002769 (formerly the “Release 3 whole-genome shotgun centromere extension
sequence”) on chromosome arm 2R (Celniker et al.
2002; Corradini et al. 2003; Myster et al. 2004; http://
flybase.bio.indiana.edu). By demonstrating that RpL38
is allelic to l(2)41Af, we have been able to correctly order
the genes proximal to RpL38 on a genetic map (Myster
et al. 2004; Figure 3C). In doing so, we have also created
an overlap between the genetic and physical maps described by Myster et al. (2004), thus correlating the
two maps at this key region (Figure 3C). RpL38 should
D. melanogaster RpL38 and RpL5
therefore be a useful “anchor point” for extending the
annotated map of the 2R centric heterochromatin toward the centromere.
RpL5 may be one of the most distal genes in the 2L
centric heterochromatin, lying near the transition zone
between the heterochromatin and euchromatin (Hoskins et al. 2002; Yasuhara et al. 2003). Again, the molecular and genetic data provided here should aid sequence
assembly and analysis in this chromosomal region.
We were intrigued to find that RpL38 and RpL5 are
located in heterochromatin as this region of the genome
is generally associated with transcriptional silencing,
whereas RPs are required at high levels in the cell and
are known to be genetically haplo-insufficient (Lambertsson 1998; Warner 1999; Elgin and Grewal 2003). However, several other vital genes are located in the heterochromatin of D. melanogaster (Dimitri et al. 2003), some
of which also encode ribosomal components, such as Qm
(⫽ RpL10; 3h), RpL15 (3h), and the bobbed locus (Xh)
that harbors rRNA genes (Ritossa 1976; Corradini et al.
2003; http://flybase.bio.indiana.edu). All essential heterochromatic genes must therefore lie within transcriptionally active domains to be expressed at levels appropriate for their efficient biological function.
Ribosomal proteins and growth regulation: One explanation for identifying RpL38 and RpL5 mutations as
enhancers of the small-wing phenotype in our original
screen is that RPs are a direct and critical target of
the PI3K pathway in promoting growth (Lehner 1999;
Thomas 2000). Stimulation of PI3K signaling activates
S6 kinase, which in turn leads to the phosphorylation of
RpS6 and the selective increase in translation of mRNAs
containing an oligopyrimidine tract at their 5⬘ end (5⬘
TOPs; Thomas 2000). 5⬘ TOPS are principally found
in mRNAs that encode components of the translation
machinery, including RPs (Meyuhas 2000). Indeed, the
major transcripts of the RpL38 and RpL5 genes contain
a 5⬘ TOP: CTTTCCTTCT and CTTTTT, respectively
(http://flybase.bio.indiana.edu; J. Yasuhara, personal
communication). However, as we find that a small-wing
phenotype generated by inhibiting epidermal growth
factor receptor signaling is also enhanced by mutation
of either RpL38 or RpL5 (data not shown), we favor the
idea that optimal RP production and protein synthesis
are more generally required to support wing growth
rather than being required specifically for PI3K-driven
growth.
Most of the data from D. melanogaster and other species suggests that reducing RP expression slows growth
rates and, in some cases, leads to smaller cell, organ, or
body size (see Introduction). Consistent with this idea,
we find that reducing the dosage of RpL38 or RpL5
slows the organismal growth rate and, in most cases,
reduces cell number in the adult wing (Figure 1D and
Table 2). However, RpL38⫺/⫹ and RpL5⫺/⫹ adult wings
are significantly larger than wild-type controls as a result
693
of increased cell size (Figure 6 and Table 2). This latter
finding provokes new questions regarding the role of
RPs in growth regulation. First, how might a reduction
in RP gene dosage, and therefore ribosome biogenesis
and cellular protein synthesis, lead to increased cell
size? Second, why should mutations in RpL38 and RpL5
dominantly enhance, rather than suppress, the PI3Ksensitized small-wing phenotype?
Similar to adult wing cells that are heterozygous for
either a RpL38 or a RpL5 mutation, RpS131/⫹ cells in the
D. melanogaster larval wing disc are enlarged compared
to wild-type cells (Martin-Castellanos and Edgar
2002). Likewise, RPL3 deficiency increases cell size in
tobacco plants (Popescu and Tumer 2004). Furthermore, overexpression of the fly brain tumor gene, which
inhibits rRNA synthesis and therefore ribosome production, also increases the size of wing imaginal-disc cells
(Frank et al. 2002). Perhaps the simplest explanation
of this hypertrophic growth is that a reduction in the
protein synthetic capacity of the cell slows the cell division cycle to a greater degree than it impairs the cellular
growth rate. Thus, reduction in RP expression results
in large, slowly dividing cells. Alternatively, the extended
larval period of Rp⫺/⫹ animals may simply allow more
time for cell growth to occur in a given cell cycle or
for more food to be eaten and/or assimilated. Such
mechanisms have been proposed to account for the
increased growth seen when flies are raised at a low
temperature (French et al. 1998) and we note that the
growth effects of reduced RP gene dosage and decreased rearing temperature are strikingly similar
(French et al. 1998; Azevedo et al. 2002). In a third
model, impaired ribosome function may lead to a reduction in the levels of a critical growth-inhibitory protein.
Future work will investigate how mutations in RpL38,
RpL5, and other RP genes may alter cell size, cell proliferation, and cell death at earlier developmental stages
to modify tissue growth and final body size.
The finding that mutations in RpL38 or RpL5 dominantly increase wing size in a wild-type genetic background suggests that they should cause dominant suppression, rather than the observed enhancement, of the
original small-wing phenotype. This paradox may be
explained by considering the different mechanisms by
which reduced PI3K activity (by overexpression of the
KD-Dp110 transgene) and mutation of RP genes affect
wing growth. Wings overexpressing KD-Dp110 are small
because they contain fewer cells of smaller size (Leevers
et al. 1996). Although RpL38⫺/⫹ and RpL5⫺/⫹ wings
can also contain fewer cells, they are increased in size
overall because they contain larger cells (Table 2).
When these two genetic manipulations are combined,
it is likely that Dp110-KD overexpression prevents the
increase in cell size normally caused by lowering RP
gene dosage. In this way, the small-wing phenotype may
be enhanced by heterozygosity for RP mutations as a
result of a further reduction in cell number. Further
694
S. J. Marygold, C. M. A. Coelho and S. J. Leevers
research will be necessary to elucidate the true mechanism by which the combined perturbation of both PI3K
signaling and protein translation impinges on cell
growth and division.
We thank Nic Tapon, Patrizio Dimitri, and Helen McNeill for helpful comments on the manuscript. We also thank Patrizio Dimitri,
Mark Peifer, Barbara Wakimoto, and the Bloomington Stock Center
for fly stocks. We are grateful to Irene Lavagi for assistance with
mapping, Jiro Yasuhara and Patrizio Dimitri for sharing unpublished
data, and Patrizio Dimitri for many useful discussions. We acknowledge the Cancer Research-UK (CR-UK) Equipment Park for DNA
sequencing services and the CR-UK Oligonucleotide Synthesis service.
This work was funded by the Medical Research Council (UK) and
Cancer Research UK.
Note added in proof : It has been brought to our attention that the
RpL381 allele was induced on a Pin-bearing mutant chromosome (D.
Sinclair, personal communication; A. J. Hilliker, 1976, Genetic
analysis of the centromeric heterochromatin of chromosome 2 of
Drosophila melanogaster : deficiency mapping of EMS-induced lethal
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RpL381 mutant flies have such a strong bristle phenotype because Pin
mutations are dominant mutations that result in shortened thoracic
bristles.
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Communicating editor: T. C. Kaufman