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Mosquito ribosomal protein S3 lacks a critical glutamine residue associated with DNA repair activity in homologous Drosophila proteins.

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Li and Fallon
Archives of Insect Biochemistry and Physiology 63:188�6 (2006)
Mosquito Ribosomal Protein S3 Lacks a Critical
Glutamine Residue Associated With DNA Repair
Activity in Homologous
Lei Li and A. M. Fallon*
In Drosophila melanogaster, ribosomal protein RpS3 has extra-ribosomal activities including apurinic/apyrimidinic lyase activity and N-glycosylase activity that participate in DNA repair. It has been suggested that these activities couple DNA repair to
the translational machinery. To establish a basis for participation of RpS3 in DNA repair in mosquitoes, we cloned RpS3 cDNAs
from Aedes aegypti and Aedes albopictus mosquito cell lines. The sequence data were used to reconstruct the homologous
gene from the Anopheles gambiae database. Mosquito RpS3 is a single copy gene, which in Aedes albopictus, lacks introns in
the amino acid coding region. Although RpS3 proteins are well-conserved among eukaryotes, a critical glutamine residue,
Q59, essential to robust DNA repair activity in the Drosophila protein, is replaced by an asparagine (N) in all three mosquito
RpS3 proteins. In this respect, the mosquito protein resembles human RpS3, which has relatively modest DNA repair activity.
None of the insect RpS3 proteins available in the database, other than those from Drosophila, contain glutamine at position
59. However, in the Lepidoptera, N59 is consistently replaced by serine (S), and the putative interactive site at position 134 is
replaced by arginine (R). These data suggest that in the case of RpS3, the Drosophila protein may be uniquely unusual in
having robust DNA repair activities that are unlikely to be common to RpS3 from other insects. Arch. Insect Biochem. Physiol.
63:188�6, 2006.
� 2006 Wiley-Liss, Inc.
KEYWORDS : mosquito; ribosomal protein; RpS3; DNA repair; N-glycosylase; phylogeny
that Drosophila RpS3 can remove sugar-phosphate
products via a deoxyribophosphodiesterase activ-
Among ribosomal proteins, RpS3 is unusual in
ity. Drosophila RpS3 also has N-glycosylase activity
that it has multiple functions, including an apu-
capable of removing the oxidized base, 7,8-dihydo-
rinic/aprymidinic (AP) lyase activity that partici-
8-oxoguanine, from damaged DNA (Deutsch et al.,
pates in repair of DNA damage. Wilson et al.
1997). These activities repair DNA damage caused
(1994) used a cDNA encoding rat RpS3 to obtain
by oxidizing agents and ionizing radiation.
the Drosophila melanogaster homolog, and showed
Relative to these repair activities associated with
that the Drosophila protein cleaved phosphodiester
Drosophila RpS3, human RpS3 lacks N-glycosylase
activity, has reduced AP-lyase activity, and is less
More recently, Sandigursky et al. (1997) showed
effective in DNA repair. Hedge et al. (2001) used a
bonds at AP sites via a
Department of Entomology, University of Minnesota, St. Paul
Contract grant sponsor: US National Institutes of Health; Contract grant number: AI20385; Contract grant sponsor: University of Minnesota Agricultural Experiment
Station, St. Paul, MN.
*Correspondence to: Ann M. Fallon, Department of Entomology, University of Minnesota, 1980 Folwell Ave., St. Paul, MN 55108. E-mail:
Received 18 January 2006; Accepted 10 September 2006
� 2006 Wiley-Liss, Inc.
DOI: 10.1002/arch.20156
Published online in Wiley InterScience (
Archives of Insect Biochemistry and Physiology
December 2006
doi: 10.1002/arch.
Role of RpS3 in DNA Repair in Mosquitoes
hidden Markov model analysis to show that the
These comparisons suggest that the robust DNA
absence of N-glycosylase and
repair activities of Drosophila RpS3 are unusual, and
d-elimination activi-
ties is due to replacement of a glutamine at amino
are not universally shared among the Diptera, or
acid 59 (Q59) in the Drosophila protein by an as-
by other insect species.
paragine (N) residue in the human homolog. Likewise, these activities were lost from the Drosophila
protein when site-directed mutagenesis was used
Cell Lines and Culture Conditions
to replace Q59 with an alanine residue. Thus, a
single residue change reduced DNA repair activity
of Drosophila RpS3 to a level similar to that of the
human protein (Hedge et al., 2001).
Aedes albopictus C7-10 cells and Aedes aegypti
Aag-2 cells were maintained in Eagle抯 minimal
medium supplemented with glutamine, vitamins,
Insects are known to have increased resistance
to various agents that damage DNA (for a review,
see Koval, 1980), but the molecular mechanisms
underlying this resistance remain unknown. We
nonessential amino acids, and 5% fetal bovine serum as described previously (Shih et al., 1998).
Cultures were maintained at 28 C, in a 5% CO2
have recently shown that mosquito cells express
an internally-deleted variant of the ribonucleotide
reductase R2 subunit, Aal
RNA Isolation and PCR
when exposed to
ultraviolet light. The likelihood that Aal
is a
Total RNA was extracted using Qiagen抯 (Valen-
functional participant in DNA metabolism was sug-
cia, CA) miniRNeasy kit according to the manu-
gested by enhancement of DNA repair in an in
facturer抯 instructions, and purified RNA was stored
vitro system (Jayachandran and Fallon, 2004), by
at � C. An initial cDNA, unexpectedly obtained
the presence of a similar gene (rnr4) in yeast (Wang
using degenerate primers and RACE-PCR, con-
et al., 1997), and by the presence of a novel ho-
tained the 5 -end of the RpS3 sequence. This ini-
molog called p53R2 in mouse and human cells
tial sequence was used to design specific forward
(Guittet et al., 2001).
primer S1, and reverse primers S2 and S10, which
In a screen for other genes related to DNA re-
were used to obtain the complete RpS3 cDNA se-
pair, we recovered a partial cDNA encoding mos-
quence (see Fig. 1). PCR products were visualized
quito RpS3. Because D. melanogaster and Drosophila
by electrophoresis on 0.9% agarose gels, cloned
virilis RpS3 proteins both contain Q59, while se-
into pGEM-T Easy vector (Promega, Madison, WI),
quences from representative vertebrates, nema-
and transformed into Escherichia coli XL-1 Blue
todes, yeast, and plants contained N (or D) at this
competent cells (Stratagene, La Jolla, CA). Three
site (Lyamouri et al., 2001), it was of interest to
independent clones were sequenced in both direc-
determine whether Q59 in Drosophila was common
tions using SP6 and T7 primers. Sequencing was
to RpS3 from other insects. Here we used PCR-
done at the Advanced Genetic Analysis Center at
based methods to obtain RpS3 cDNAs from Aedes
the University of Minnesota. Southern blots were
albopictus and Aedes aegypti mosquito cells, and
probed with
P-labeled full-length cDNA.
identified the Anopheles gambiae homolog from the
RpS3 gene organization was determined using
genomic database. Ae. albopictus RpS3 is a single
two sets of PCR primers designed to detect introns,
copy gene that lacks introns in the protein coding
with genomic DNA from Ae. albopictus C7-10 cells
sequence, while the An. gambiae coding sequence
as the template. Primer P5 corresponded to the 5 -
is interrupted by two introns. Despite high levels
end of the cDNA, upstream of the AUG initiation
of overall amino acid identity, all three mosquito
codon. P5 was used with reverse primer R3 (see
proteins, as well as four Lepidopteran and one Co-
Fig. 1). P3 extended from the 3 -UTR across the
leopteran RpS3 protein available in the NCBI da-
stop codon, and into the 3 -end of the coding se-
tabase, fail to conserve the Drosophila Q59 residue.
quence and was used with the internal forward
Archives of Insect Biochemistry and Physiology
December 2006
doi: 10.1002/arch.
Li and Fallon
primer F3. The PCR conditions included 1 cycle at
EAA01737.3, but lacked the first 10 N-terminal resi-
95癈 for 3 min, followed by 35 cycles of denatur-
dues and differed from the translation product de-
ation at 95癈 for 60 sec, annealing at 56癈 (P5/
duced from genomic DNA, shown in Figure 2, at
R3) or at 66癈 (F3/P3) for 45 sec, and extension
the two C-terminal residues. In addition, the exon-
at 72癈 for 60 sec, with a final extension at 72癈
intron organization (CDS) designated under EAA
for 5 min.
01737.3 appears to contain minor errors.
(Fig. 2). The nuclear localization signal (NLS) con-
The RpS3 protein had three conserved domains
Mosquito rpS3 Coding Sequences
Using PCR-based methods, we obtained RpS3
cDNA sequences from Aedes albopictus C7-10 cells
(Genbank DQ111982), and from Aedes aegypti Aag2 cells (DQ111983; Fig. 1). The full-length RpS3
cDNAs from both mosquitoes had identical 46 nucleotide, 5�-UTRs, beginning with a polypyrimidine-rich tract (CTTTTCT; see the filled bar in Fig.
1) typical of mosquito ribosomal protein cDNAs.
The 3�-UTR was less well-conserved between Ae.
albopictus and Ae. aegypti, but shared an identical
A/T-rich region that plausibly serves as a polyadenylation signal (represented by an open bar in
Fig. 1). In addition, a perfect polyadenlyation signal occurs 14 nucleotides downstream in the Ae.
sists of 4 consecutive basic residues at the N-terminal end of the protein. The K homology (KH
domain; below the filled bar) is an ~50 residue RNA
binding motif, which was first described in the human heterogeneous ribonucleoprotein (hnRNP) K,
and has since been identified in diverse nucleic
acid-binding proteins. Finally, the ~90 residue S3C domain (open bar) represents the RpS3 minimal carboxyl terminal domain. With the exception
of a stretch of four to seven residues in the KH
among insects and vertebrates. In contrast, residues
downstream of the S3-C domain varied considerably, even between Aedes and Anopheles mosquitoes. Overall, the An. gambiae protein differed in
17 residues, relative to that of Ae. aegypti.
aegypti sequence, but the initial A is replaced by a
G in Ae. albopictus. Between the two Aedes species,
RpS3 Copy Number and Gene Structure
nucleotide identity of the protein coding region
was 95%. The deduced Ae. albopictus RpS3 protein
had a mass of 26,941 Da and a pI of 10.41; in Ae.
aegypti, the deduced mass was 26,909 Da and the
pI was 10.41. The deduced protein sequences differed at only two sites near the C-terminal end of
the protein (Fig. 2).
The An. gambiae genomic DNA sequence was
within the 244 amino acid protein coding region.
An exon containing the initiating methionine plus
nuclear localization signal, was followed by a 508
nucleotide intron, a downstream exon encoding
66 residues, an 88 nucleotide intron, and a final
RpS3 From An. gambiae
exon encoded the remaining 168 amino acid residues (Fig. 3A). The Drosophila melanogaster gene
When the Ae. albopictus RpS3 cDNA sequence
was used to query the EST_others database on the
NCBI website (, we
recovered accession BM622280 (710 nucleotides)
corresponding to an An.
(CG6779) contained a single intron, which interrupted the coding sequence after the 12th amino
acid residue.
We used PCR with a genomic DNA template to
gambiae cDNA clone,
determine the size of intron(s) that might be
which in turn mapped to a 1,293-nucleotide region
present in the Ae. albopictus RpS3 coding sequence.
on An. gambiae chromosome 2R (map element
Primers P5 and P3 corresponded to the 5�- and 3�-
AAAB01008987). The deduced protein sequence
ends of the cDNA, respectively (Fig. 3A). A second
encoded by this region was similar to accession
set of primers, F3 and R3, was based on sequences
Archives of Insect Biochemistry and Physiology
December 2006
doi: 10.1002/arch.
Role of RpS3 in DNA Repair in Mosquitoes
Alignment of Aedes
Fig. 1.
top) and Aedes
bottom) RpS3 cDNAs.
albopictus (
aegypti (
Vertical lines designate nucleotide identities. The polypyrimidine motif at the 5�-end
of the cDNA is underlined
with a filled bar, and putative
polyadenylation signals downstream of the stop codon are
underlined with open bars.
The ATG start and TAA stop
codons are boxed. Arrows
with solid heads designate
primers used to obtain the
full-length cDNA sequences;
arrows with open heads designate primer pairs shown
schematically in Figure 3 and
used to examine the exon-intron organization of Ae. albo-
pictus RpS3.
Archives of Insect Biochemistry and Physiology
December 2006
doi: 10.1002/arch.
Li and Fallon
Fig. 2.
Alignment of RpS3 pro-
teins from insects and representative vertebrates. The alignment
version 1.83 (Thompson et al.,
1997). Pairwise alignments used
a gap opening penalty of 35.00,
and a gap extension penalty of
0.75. Multiple alignments used
a gap opening penalty of 15,
and a gap extension penalty of
0.30. In the alignment, identities to the Ae. aegypti sequence
were replaced with dots, and
duced to maximize alignment.
The consensus is designated below the alignment. An open box
designates the nuclear localization signal (NLS); the KH and
S3-C domains are designated by
filled and open bars, respectively,
Within the KH and S3-C boxes,
three residues that confer N-glycosylase and
tivity in Drosophila melanogaster
RpS3 are outlined as single columns of amino acids. Accession
numbers were: gi|54781367|,
Homo sapiens; gi|57164151|, Rattus
norvegicus ; gi|37595356|,
Danio rerio; gi|27769401|, Xenopus laevis; gi|527680|, Manduca
sexta ; gi|16566719|, Spodoptera
frugiperda; gi|54609285|, Bombyx
mori; gi|50284382|, Papilio dardanus ; gi|50344456|, Carabus
granulatus; gi|54637431, Drosophila pseudoobscura; gi|548856|,
Drosophila melanogaster; gi|1849
6083|, Drosophila virilis.
Archives of Insect Biochemistry and Physiology
December 2006
doi: 10.1002/arch.
Role of RpS3 in DNA Repair in Mosquitoes
Fig. 3.
Structure of RpS3 genes.
A: The exon-intron organization
of RpS3 genes in D. melanogaster
and in An. gambiae. Exons (E1,
E2, E3) are represented by filled
boxes, and introns are designated by lines. Note the relatively long intron at the 5�-end
of these genes. The Ae. albopictus
cDNA downstream of the 5 � UTR and Exon 1 is shown as an
open box. PCR primers P5 and
P3 corresponded to the extreme
5�- and 3�-ends of the Ae. albo-
pictus cDNA, respectively (see
Fig. 1). Primers F3 and R3 were
based on sequence within Exon
3 in the An. gambiae sequence.
B: Sizes of PCR bands obtained
blot. Ae. albopictus genomic DNA
was digested with EcoRI (lane 1),
XhoI (lane 2), and MboII (lane
3). The blot was probed with
full-length cDNA. An autoradiogram is shown, and bands are
labeled by asterisks.
within Exon 3 of the An. gambiae gene. The P5/R3
cated that RpS3 is a single copy gene in both An.
pair produced a fragment that corresponded in size
gambiae and D. melanogaster.
to the cDNA sequence (Fig. 3B), indicating that
the Ae. albopictus RpS3 gene lacks the relatively long
Absence of Q59 in Insects
intron, present in both D. melanogaster and An.
Other Than
gambiae, near the 5 -end of the coding sequence.
Likewise, the pair F3 and P3 verified that the C-
The most unexpected aspect of mosquito RpS3
terminal end of RpS3 in Ae. albopictus, like that in
proteins is the absence of the crucial Q59 residue
other Dipterans, lacked an intron downstream of
associated with N-glycosylase and
R3. Finally, we verified that Ae. albopictus RpS3 is a
tivities that have been characterized biochemically
single copy gene using Southern blots containing
in Drosophila RpS3. Three sequences are available
d-elimination ac-
genomic DNA digested with EcoRI and XhoI, which
from the genus Drosophila, and Q59 is conserved
do not cut within the cDNA, and generate only a
in all three species. Mosquitoes represent the only
single band on autoradiograms (Fig. 3C, lanes 1
other group of Diptera in which RpS3 has been
and 2; see the bands identified by stars; two spuri-
examined, and in both Anopheles and Aedes, Q59
ous artifacts appear in lane 2). As expected, the
is replaced by N, as is the case in humans, rats,
single MboII site in the cDNA generated two bands
fish, frogs, and the single sequence available from
(Fig. 3C, lane 3). Likewise, genome analysis indi-
a beetle (Fig. 2). In four representatives from the
Archives of Insect Biochemistry and Physiology
December 2006
doi: 10.1002/arch.
Li and Fallon
order Lepidoptera (Manduca, Spodoptera, Bombyx,
of highly conserved sequence. When the alignment
Papilio), Q59 is replaced by serine (S). In view of
was used to create a cladogram, the individual
the relatively high level of sequence conservation
members of the three insect orders clustered ac-
in RpS3, this difference was surprising.
cording to accepted phylogenies with high boot-
In a three-dimensional structure, Q59 interacts
strap support (see the circled values in Fig. 4), as
with lysine (K) and aspartic acid (D) at positions
would be expected for the sequence of an ancient
134 and 156 in the Drosophila sequence (Hedge et
protein that predates the divergence of insects and
al., 2001; Lyamouri et al., 2002; see the narrow
boxes in Fig. 2). K134 is replaced by arginine (R)
in the Lepidopteran species, but otherwise these
positions are invariant across species. Casual inspection of amino acid differences in Figure 2
Coordination of metabolic processes in cells
shows striking commonalities among closely re-
and organisms is a highly regulated process that
lated groups of organisms against a background
remains poorly understood. Increasing evidence
Fig. 4.
Rectangular cladogram showing relationships
vertical lines, and bootstrap values that exceed 50%, based
based on RpS3 protein sequences. The tree was constructed
on 1000 replicates, are shown in circles. The four verte-
in PAUP* (Swofford, 2000) from the alignment in Figure
brate species were designated as the outgroup.
2 by Neighbor Joining. Branch lengths are shown along
Archives of Insect Biochemistry and Physiology
December 2006
doi: 10.1002/arch.
Role of RpS3 in DNA Repair in Mosquitoes
that some ribosomal proteins have additional, non-
in the extent to which RpS3 participates in DNA
ribosomal functions and the observation that RpS3
repair. Further analysis of this interesting protein,
in Drosophila has robust DNA repair activity (Hedge
including biochemical analyses of the insect pro-
et al., 2001) suggest a coupling between transla-
teins, may contribute insights into the evolution
tion and DNA repair. However, the DNA repair ac-
of DNA repair activities and their coordination
tivity of human RpS3 is modest, relative to that of
with other metabolic processes.
Drosophila, and cannot remove a common product of oxidative DNA damage, 8-oxoguanine.
In general, insects are thought to be relatively
resistant to agents that damage DNA (Koval, 1980),
Deutsch WA, Yacoub A, Jaruga P, Zastawny TH, Dizdaroglu
but the enzymatic activities that contribute to re-
M. 1997. Characterization and mechanism of action of
sistance have received little attention. We anticipated that RpS3 in mosquitoes would have the
robust DNA repair activity that had been described
in Drosophila, but analysis of mosquito RpS3 pro-
Drosophila ribosomal protein S3 DNA glycosylase activity
for the removal of oxidatively damaged DNA bases. J Biol
Chem 272:32857�860.
Guittet O, Hakansson P, Voevodskaya N, Fridd S, Graslund
tein sequences deduced from cDNA clones for Ae.
A, Arakawa H, Nakamura Y, Thelander L. 2001. Mamma-
albopictus and Ae. aegypti, and from a conceptual
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translation of An. gambiae genomic DNA showed
tase in vitro with the R1 protein, which is expressed both
that despite overall high levels of sequence con-
in resting cells in response to DNA damage and in prolif-
servation in the KH and S3-C domains, the critical
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Q59 residue in the Drosophila protein is replaced
by N in all three mosquitoes. Inspection of the
alignment further showed that mosquitoes lack
compensatory changes in two additional sites,
K134 and D156 (Lyamouri et al., 2002) that are
thought to participate in formation of the active
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Jayachandran G, Fallon, AM. 2004. The mosquito ribonucle-
the RpS3 gene that might exist in addition to the
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Archives of Insect Biochemistry and Physiology
December 2006
doi: 10.1002/arch.
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