Mosquito ribosomal protein S3 lacks a critical glutamine residue associated with DNA repair activity in homologous Drosophila proteins.код для вставкиСкачать
188 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 Drosophila Proteins 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 INTRODUCTION 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 b,d-elimination reaction. 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: firstname.lastname@example.org Received 18 January 2006; Accepted 10 September 2006 � 2006 Wiley-Liss, Inc. DOI: 10.1002/arch.20156 Published online in Wiley InterScience (www.interscience.wiley.com) Archives of Insect Biochemistry and Physiology December 2006 doi: 10.1002/arch. Role of RpS3 in DNA Repair in Mosquitoes 189 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 MATERIALS AND METHODS 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 atmosphere. have recently shown that mosquito cells express an internally-deleted variant of the ribonucleotide reductase R2 subunit, Aal DR2, RNA Isolation and PCR when exposed to ultraviolet light. The likelihood that Aal DR2 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 32 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. � � � 190 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. RESULTS (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 domain, these domains are highly conserved 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 translated to define exon-intron organization within the 244 amino acid protein coding region. An exon containing the initiating methionine plus 9 additional residues, ending with the KKRR 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 (http://www.ncbi.nlm.nih.gov), 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 191 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. 192 Li and Fallon Fig. 2. Alignment of RpS3 pro- teins from insects and representative vertebrates. The alignment was produced with ClustalX, 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 dashes represent gaps intro- 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, above the alignment. Within the KH and S3-C boxes, three residues that confer N-glycosylase and d-elimination ac- 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. 193 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 with DNA Ae. albopictus template. C: genomic Southern 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 � Drosophila 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. 194 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 vertebrates. boxes in Fig. 2). K134 is replaced by arginine (R) in the Lepidopteran species, but otherwise these DISCUSSION 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 195 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. LITERATURE CITED 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 lian p53R2 protein forms an active ribonucleotide reduc- 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 erating cells. J Biol Chem 276:40647�651. 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 Hegde V, Kelley MR, Xu Y, Mian IS, Deutsch WA. 2001. Conversion of the bifunctional 8-oxoguanine/ b-d apurinic/ apyrmidinic DNA repair activities of Drosophila ribosomal protein S3 into the human S3 monofunctional b-elimi- nation catalyst through a single amino acid change. J Biol Chem 276:27591�596. site (Hedge et al., 2001). Finally, genomic Southerns failed to show evidence of a second copy of Jayachandran G, Fallon, AM. 2004. The mosquito ribonucle- the RpS3 gene that might exist in addition to the otide reductase R2 gene: ultraviolet light induces expres- cDNA clones described here. In aggregate, these se- sion of a novel R2 variant with an internal amino acid quence data imply that mosquito RpS3 has only deletion. Insect Mol Biol 13:231�9. the modest repair capacity of the human protein. This difference between Drosophila and mosquito RpS3 led us to examine other insect RpS3 Koval TM. 1980. Relative responses of mammalian and insect cells. In: Myen RE, Withers RH, editors. Radiation Biology In Cancer Research. New York: Raven Press. p sequences that had been deposited in the data- 169�4. bases. We found that the available insect sequences generated a cladogram that accurately separated Lyamouri M, Enerly E, Lambertsson A. 2002. Organization, major orders, and within the Diptera, distinguished sequence and phylogenetic analysis of the ribosomal pro- between Drosophila and mosquitoes. RpS3 from tein S3 gene from Drosophila virilis. Gene 294:147�6. four Lepidoptera, and from a single beetle, uniformly lacked the critical Q59 residue. 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