Recovery of cDNAs encoding ribosomal proteins S9 and L26 from Aedes albopictus mosquito cells and identification of their homologs in the malaria vector Anopheles gambiae.код для вставкиСкачать
ARCH 05005 44 Li and Fallon Archives of Insect Biochemistry and Physiology 60:44–53 (2005) Recovery of cDNAs Encoding Ribosomal Proteins S9 and L26 From Aedes albopictus Mosquito Cells and Identification of Their Homologs in the Malaria Vector, Anopheles gambiae Lei Li and A.M. Fallon* We used PCR-based approaches to obtain the full-length cDNA sequences encoding ribosomal protein (Rp) S9 and L26 from a mosquito (Aedes albopictus) C7-10 cell line. The deduced mosquito RpS9 protein has a mass of 22,826 Da and a pI of 11.41, while RpL26 had a mass of 17,442 Da and a pI of 11.52. Both cDNAs initiated with the 5′-polypyrimidine motif characteristic of ribosomal protein transcripts. Using the Aedes protein and nucleic acid sequences, we identified rpS9 and rpL26 as single copy genes in the Anopheles gambiae genome. In An. gambiae, the RpS9 coding region was distributed over 3 exons, spanning 2.6 kb, but the Anopheles rpL26 protein coding region lacked introns. The Aedes and Anopheles RpS9 and RpL26 proteins shared 96 and 92% identity, respectively. Despite low numbers of parsimony-informative amino acid substitutions, phylogenies based on the ribosomal protein sequences accurately group the Aedes and Anopheles proteins with high bootstrap values. Arch. Insect Biochem. Physiol. 60:44–53, 2005. © 2005 Wiley-Liss, Inc. KEYWORDS: Aedes albopictus; Anopheles gambiae; cell line; genome; mosquito; protein synthesis; ribosomal protein; RpS9; RpL26 INTRODUCTION In adult female mosquitoes, the blood meal initiates a cycle of ribosome biosynthesis, and in the fat body, the accumulated ribosomes provide protein synthetic machinery that supports synthesis of the egg yolk proteins, or vitellogenins. The approximately 80 individual ribosomal proteins (Rp) that are assembled into the small and large ribosomal subunits play diverse roles in maintaining ribosome structure and participating in specific aspects of protein synthesis. Some of the ribosomal proteins also have important extra-ribosomal functions, including roles in DNA replication and repair and in RNA processing (Wool, 1996). Our interest in ribosomal proteins relates to their potential manipulation in transgenic mosquitoes to disrupt vector-parasite interactions. Qian et al. (1988) showed that transgenic disruption of the synthesis of a single ribosomal protein caused sterility in female Drosophila melanogaster. With the exception of an unusual C-terminal extension on mosquito RpS6 (Hernandez et al., 2003), the amino acid sequences of homologous ribosomal proteins that have been described from Aedes and Anopheles mosquitoes are well conserved. In the cases where genomic DNA has been sequenced, exon-intron organization is also conserved. In general, Exon 1 in mosquito ribosomal protein genes is short, and may not be translated. Department of Entomology, University of Minnesota, St. Paul, Minnesota Contract grant sponsor: National Institutes of Health; Contract grant number: AI20385; Contract grant sponsor: University of Minnesota 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 22 January 2005; Accepted 18 April 2005 © 2005 Wiley-Liss, Inc. DOI: 10.1002/arch.20083 Published online in Wiley InterScience (www.interscience.wiley.com) Archives of Insect Biochemistry and Physiology Mosquito Ribosomal Proteins S9 and L26 The AUG start codon is typically located near the end of Exon 1 or close to the 5′-end of Exon 2. Aedes albopictus rpS6 (Hernandez et al., 2003), rpL8 (Lan and Fallon, 1992), and rpL34 (Niu and Fallon, 1999) genes each contain a long Exon 3, which encodes the remainder of the protein. The 5′-end of the known mosquito Rp mRNAs is C/Trich (Niu and Fallon, 1999), and the 3′-end is polyadenylated (Durbin et al., 1988). Because their sequence and structure are relatively well conserved, it will be of interest to compare phylogenies based on ribosomal proteins with results derived from rRNA genes, which have been used extensively as molecular data to reconstruct relationships among insect taxa (Pawlowski et al., 1996). In bacteria, phylogenies based on the fused sequences of the 53 ribosomal proteins have shown good agreement with rRNA-based phylogenies (Matte-Tailliez et al., 2002). Because mRNA encoding ribosomal proteins is relatively abundant (Durbin et al., 1988), our unanticipated recovery of mosquito RpS9 and RpL26 sequences among PCR products obtained with degenerate primers was not surprising. In the case of these two proteins, the availability of a full-length sequence from both Drosophila melanogaster and the silk moth, Bombyx mori, and our identification of homologs in the Anopheles gambiae database, allowed us to construct phylogenies based on all known insect RpS9 and RpL26 proteins, using the rat homolog as the outgroup. Despite relatively few parsimony-informative characters, individual phylogenies based on RpS9 and RpL26 group the Aedes and Anopheles proteins with high bootstrap values, but only RpS9 reliably includes Drosophila with the mosquitoes. Analyses with RpL26 consistently yield two trees, in which B. mori, or alternatively D. melanogaster, is the sister group to the mosquitoes. MATERIALS AND METHODS Cell Line and Culture Conditions Ae. albopictus C7-10 mosquito cells (Fallon, 1997) were maintained as monolayers at 28°C in Eagle’s minimal medium supplemented with nonSeptember 2005 45 essential amino acids, glutamine, and 5% heat-inactivated fetal bovine serum (Shih et al., 1998). RNA Isolation and PCR-Based Cloning Total RNA was obtained from C7-10 cells using Qiagen’s RNeasy kit (Valencia, CA). A PCR product encoding the 5′-end of rpS9 was recovered by 5′RACE, using the GeneRacer kit from Invitrogen (Carlsbad, CA) and a degenerate reverse primer designed to recognize a mosquito homolog of the tumor suppressor protein, p53. The remainder of the rpS9 cDNA sequence was obtained using reverse transcriptase (RT)-PCR with a specific forward primer, S9-1 (5′TCG AAT CTT GAC AAA GCA ACA G), from the 5′-untranslated region (Fig. 1A), and oligo(dT) as the reverse primer. PCR was done using 35 cycles of 30 sec denaturation at 94°C, 45 sec annealing at 58°C, and 1 min extension at 72°C. PCR was terminated with a 2-min extension at 72°C. The band was cloned as described below. A PCR product encoding the C-terminal end of RpL26 was likewise obtained fortuitously using a heterologous primer. Based on the sequence of the initial product, specific reverse primers (Fig. 1B) were designed to obtain the complete cDNA sequence using the 5′-primer supplied in the GeneRacer kit. Using L26-1, we obtained a mixture of PCR products using a touchdown PCR protocol (denaturation at 94°C for 2 min, followed by denaturation at 94°C for 30 sec and annealing at 72°C for 45 sec (5 cycles); denaturation at 94°C for 30 sec with annealing at 70°C for 45 sec (5 cycles), followed by 25 cycles of denaturation at 94°C for 30 sec, annealing at 62°C for 30 sec, and extension at 72°C for 45 sec. The PCR product was re-amplified with the internal primer L262 and the GeneRacer 5′-nested primer using 1 cycle at 94°C for 5 min, 25 cycles of denaturation at 94°C for 30 sec, annealing at 62°C for 30 sec, and extension at 72°C for 30 sec, followed by one cycle at 72°C for 3 min. The resulting, discrete band was cloned into pGEM®-T Easy vector (Promega, Madison, WI) and introduced into competent DH5α® Escherichia coli (Invitrogen, La Jolla, CA) using standard procedures. 46 Li and Fallon Fig. 1. Mosquito RpS9 (A) and RpL26 (B) cDNAs. The ATG translation start codons and TAA stop codons are boxed, and the polypyrimidine tracts at the 5′-end of each transcript are underlined. The position and orientation of horizontal arrows designate primers used to obtain the sequence. In A, inverted triangles indicate positions of introns within the coding sequence, based on a comparison with the An. gambiae genome. In B, a consensus polyadenylation signal is doubly underlined. RESULTS Aedes albopictus rpS9 and rpL26 cDNAs The complete nucleotide sequences of Ae. albopictus RpS9 cDNA (GenBank Accession no. AY847002) and Ae. albopictus RpL26 cDNA (GenBank Accession no. AY885229) are shown in Figure 1A and B. The 5′-ends of the RpS9 and RpL26 mRNAs extended 78 and 79 nucleotides, respec- tively, upstream of the AUG translation initiation codon. Both cDNAs contained the polypyrimidine motif characteristic of Rp transcripts at their 5′ends. In Figure 1, the initiation and termination codons are boxed, and the specific primers S9-1, L26-1, and L26-2 used to obtain the complete sequences are designated by arrows showing the direction of extension. In the rpL26 cDNA, a putative polyadenylation signal is doubly underlined. AlArchives of Insect Biochemistry and Physiology Mosquito Ribosomal Proteins S9 and L26 47 Fig. 1 (continued) though an exact consensus signal is absent from the 3′-untranslated region in the rpS9 cDNA, there are several A/T-rich motifs that may serve as polyadenylation signals. RpS9 and RpL26 Homologs in Anopheles gambiae When the protein sequences deduced from these cDNAs were compared to the An. gambiae genome using the program BLAST at the National Center for Biotechnology Information (NCBI) website (http: //www.ncbi.nlm.nih.gov/), we found the homologous rpS9 gene on An. gambiae chromosome 2L, encoding the conceptual protein identified by XP_313936), which was reported to be incomplete on the N-terminal end. Alignment with the Ae. albopictus sequence showed that the Anopheles protein initiated at the methionine represented by the third codon in XP_313936. An. gambiae rpL26 mapped to chromosome 2R, corresponding to protein XP_312471. This RpL26 entry was also reported September 2005 as incomplete at the N-terminus, but direct comparison with the Aedes sequence indicates that translation of Anopheles RpL26 initiates at an internal methionine (residue 25) in XP_312471. Both genes occurred as a single-copy in the An. gambiae genome. Translation of the An. gambiae genomic DNA sequence and alignment with the Ae. albopictus coding sequence indicated that in An. gambiae, the rpS9 coding region is distributed over 3 exons, spanning 2.6 kb. Within the coding sequence, intron boundaries were verified by the presence of gt..ag consensus nucleotides, and their relative positions are indicated by inverted open triangles in Figure 1A. Lengths of the upstream and downstream introns in the An. gambiae gene are 222 and 1,754 nucleotides, respectively. By way of contrast, the An. gambiae RpL26 coding sequence is not interrupted by introns, as is shown by the alignment of Anopheles genomic DNA with the Ae. albopictus cDNA (Fig. 2). In D. melanogaster, the rpL26 cod- 48 Li and Fallon Fig. 2. Alignment of An. gambiae RpL26 genomic DNA sequence with Aedes albopictus RpL26 cDNA. The alignment shows the Anopheles sequence at top (lowercase letters) and the Aedes sequence at bottom (uppercase letters). Protein initiation and termination codons are boxed. Identities are represented by vertical lines, and gaps (indicated by dots) were introduced to maximize the alignment. Archives of Insect Biochemistry and Physiology Mosquito Ribosomal Proteins S9 and L26 ing region of gene CG6846 also lacks introns (FlyBase Consortium, 2003). DNA Upstream of the Coding Region Using 5′RACE, obtaining the 5′-end of eukaryotic mRNAs is relatively straightforward, and the cDNAs shown in Figure 1 contain 5′-untranslated regions (UTR) upstream of the translational start codon. The cDNA sequence does not, however, include introns that may interrupt the 5′-UTR. Thus, comparison of the Ae.albopictus cDNA sequences with An. gambiae genomic DNA facilitates identification of introns present within the coding sequence by simple comparison of the An. gambiae open reading frame with the deduced translation product from the Aedes cDNA. Introns within the 5′-UTR are more difficult to detect because they lie outside the open reading frame defined by the cDNA. For example, with the introduction of an eightnucleotide gap in the Ae. albopictus RpL26 cDNA sequence, the 5′-ends of the cDNA and the genomic DNA upstream of the AUG start codon in An. gambiae were well conserved (Fig. 2), suggesting that in An. gambiae, the rpL26 gene lacks an intron in the 5′-UTR. In the Drosophila homolog, however, an intron is indicated in the DNA upstream of the RpL26 (CG6846) coding region Fig. 3. Analysis of the An. gambiae nucleotide sequence immediately upstream of the RpS9 coding region. In the top sequence, the 5′-end of the experimentally-determined Ae. albopictus RpS9 sequence (uppercase letters) is boxed. September 2005 49 (FlyBase Consortium, 2003), which would be represented by a gap corresponding to the length of the intron in the cDNA sequence, relative to genomic DNA. Without additional experimental data from Anopheles gambiae, however, we cannot exclude the possibility that the high level of nucleotide homology is simply fortuitous. In the case of rpS9, the alignment suggests that an intron is present upstream of the coding region. Here again, in silico comparisons between Aedes and Anopheles sequences are not entirely unambiguous because the Anopheles sequence contains a stretch of 20 unidentified residues (represented by a series of n’s in Fig. 3) upstream of the coding region. Using the GCG (Genetics Computer Group, Madison, WI) program Bestfit, we noted relatively weak homology between the Ae. albopictus 5′-UTR and the putative upstream region in the An. gambiae gene. Nevertheless, a polypyrimidine tract that resembles the 5′-end of a ribosomal protein mRNA is found in the Anopheles sequence immediately upstream of the coding region. RpS9 and RpL26 Proteins The Ae. albopictus RpS9 contained 195 amino acid residues, with a calculated mass of 22,826 and pI of 11.41. RpS9 from Ae. albopictus and An. In the An. gambiae sequence (lowercase), a potential polypyrimidine tract [poly(PY)] is boxed. The box surrounding both sequences corresponds to nucleotides encoding the N-terminus of the homologous RpS9 proteins. 50 Li and Fallon Phylogenetic Analysis gambiae shared 99% similarity, 96% identity. Features of mosquito RpS9 shared with homologs from other organisms include the ~45 residue putative RNA binding domain (boxed in Fig. 4) and a series of five to six C-terminal acidic amino acids, which show a single conservative E/D substitution between the two mosquito sequences. Some, but not all, of the tripeptide repeats described in rat RpS9 (Chan et al., 1993) are also well conserved in insect RpS9. The Ae. albopictus RpL26 protein (Fig. 5) contained 151 residues, a deduced molecular mass of 17,442 and a pI of 11.52. Relative to the An. gambiae sequence, identity was 92%, and similarity was 95%. Of three putative 9-residue repeats with the consensus sequence VQVXRXKYK described for rat RpL26 (Paz et al., 1989; see the bars at the top of the alignment shown in Fig. 5), the repeat closest to the C-terminus was poorly conserved between the rat and insect proteins. Although the deduced amino acid sequences of relatively few insect ribosomal proteins are available in the databases, we tested the construction of phylogenies using the protein sequences of Aedes, Anopheles, and Drosophila flies, and the moth, Bombyx mori. The alignments shown in Figures 4 and 5 were analyzed based on parsimony using the default parameters of PAUP* (Swofford, 2000) to obtain the phylograms shown in Figure 6, in which the rat sequence was designated as the outgroup. For RpS9, bootstrap values (Fig. 6, circled values) were based on 1,000 replications, while for RpL26, 10,000 replications were used. Note that for both proteins, An. gambiae was most closely related to Ae. albopictus. However, for RpL26, bootstrap support for inclusion of Drosophila with the mosquitoes was low. Fig. 4. Alignment of RpS9 proteins from mosquitoes with homologs from D. melanogaster, B. mori, and the rat. The alignment was produced using ClustalX, version 1.83 (Thompson et al., 1997) with default settings. A single gap in the B. mori sequence is indicated by a dash, and consensus residues are indicated by asterisks below the align- ment. Double dots (:) and single dots (.) designate highly conserved, and less well-conserved amino acid replacements, respectively. The boxed region designates the RNA binding domain, and bars above the alignment indicate triplet peptides identified in the rat sequence by Chan et al. (1993). Archives of Insect Biochemistry and Physiology Mosquito Ribosomal Proteins S9 and L26 51 Fig. 5. Alignment of RpL26 proteins. The alignment was produced using ClustalX, version 1.83, as described in the legend to Figure 4. Bars at the top of the alignment desig- nate putative 9-residue repeats in the rat sequence described by Paz et al. (1989). DISCUSSION quences are deposited in the databases, it will be of interest to revisit their potential value in phylogenetic comparisons. Eukaryotic RpS9 is a component of the small ribosomal subunit homologous to Escherichia coli RpS4 (Chan et al., 1993). The observation that bacterial RpS4 mutants have high translational error rates suggested that the wild type RpS4 participates in ribosome assembly (Davies and Nomura, 1992). Nowotny and Nierhaus (1988) showed that E. coli RpS4 and RpS7 nucleate an assembly domain for 16S rRNA. In the fungus, Podospora anserina, mutations in the gene encoding RpS9 confer resistance to paromomycin (Dequard-Chablat, 1985), which suggests that RpS9 plays a similar role in translational accuracy in eukaryotic cells. The crystal structure of RpS4 from Bacillus stearothermophilus suggests that the RNA binding domain is predominantly on one side of the protein, is highly positively charged, and contains two conserved arginine residues (Davies et al., 1998; Markus et al., 1998). Aside from mosquitoes and D. melanogaster, partial RpS9 sequences have been reported from the flies Drosophila yakuba and Sarcophaga crassipalpis. Insofar as sequence is available, the two Drosophila RpS9 proteins are 100% identical to each other, and to the partial sequence from S. crassipalpis. In the evolution of the ribosome, selection acts at the level of the amino acid sequence of the protein (see Wittmann-Liebold et al., 1990), which determines how the protein interacts with the rRNA scaffold and with other proteins. The degree of conservation between ribosomal proteins varies, with identities between rat and yeast homologs ranging from 40 to 80% (Wool et al., 1990). In the phylogenetic analysis presented here (Fig. 6) the three dipteran RpS9 sequences grouped together with high confidence, while for RpL26, the positions of D. melanogaster and B. mori were reversed roughly 50% of the time. Thus, for RpL26, a tree based on protein sequence correctly shows the two mosquitoes as most closely related to one another, but does not reliably distinguish between a member of the higher Diptera (D. melanogaster) and a moth. Given these differences, it is of interest to note that for RpS9, there were 195 characters (amino acids), of which 154 were constant, 32 variable, but parsimony uninformative, and only 9 parsimony informative characters. For RpL26, there were 151 characters, of which 92 were constant, 41 variable but uninformative, and 18 were parsimony informative. As additional insect ribosomal protein seSeptember 2005 52 Li and Fallon Fig. 6. Phylograms based on the alignments shown in Figures 4 and 5. A parsimony analysis was done with PAUP* (Swofford, 2000) with default settings, and the trees were rooted by designating the rat sequence as the outgroup. Branch lengths are displayed as integers. Bootstrap values are circled and based on 1,000 replicates for RpS9 and 10,000 replicates for RpL26. RNAi directed towards Drosophila RpS9 has been reported to affect cell growth and viability (FlyBase Consortium, 2003), and in rat neuronal cells, RpS9 has been associated with protection from oxidative damage (Kim et al., 2003). RpL26 is located on the large ribosomal subunit. In contrast to RpS9, RpL26 has been little studied, but like rpS9, sequences are available for B. mori and D. melanogaster. Eukaryotic RpL26 is most closely related to bacterial RpL24, and in yeast, Villarreal and Lee (1998) have shown that the protein is located at the interface between the small and large ribosomal subunits. Larede et al. (2001) identified RpL26 from a marine snail, and in this species, RpL26 is up-regulated during anoxia. Because larvae of mosquitoes and other aquatic insects may experience anoxic conditions during development, it will be of interest to examine this potential function in mosquitoes. ACKNOWLEDGMENTS We thank Dr. G. Jayachandran for participation in early stages of this work. LITERATURE CITED Chan YL, Paz V, Olvera J, Wool IG. 1993. The primary structure of rat ribosomal protein S9. Biochem Biophys Research Commun 193:106–112. Davies C, Gerstner RB, Draper DE, Ramakrishnan V, White SW. 1998. The crystal structure of ribosomal protein S4 reveals a two-domain molecule with an extensive RNAbinding surface: one domain shows structural homology to the ETS DNA-binding motif. EMBO J 17:4545–4558. Davies J, Nomura M. 1992. The genetics of bacterial ribosomes. Annu Rev Genet 6:203–234. Dequard-Chablat M. 1985. 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