Genomic structure and expression analysis of the RNase j family ortholog gene in the insect Ceratitis capitata Theodoros N. Rampias*, Emmanuel G. Fragoulis and Diamantis C. Sideris Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Athens, Greece Ribonucleases have been extensively studied in terms of structure, function and enzymatic properties in terms of their specific RNA degradation in a variety of organisms. In addition to the well known ribonucleases of pancreatic type [1,2], there are numerous intracellu- lar ribonucleases whose biological roles have not yet been established. Additionally, the burst of determina- tion of genome sequences in recent years has led to identification of homologs of many ribonucleases in a variety of diverse organisms. Many close homologs of bacterial or yeast ribonucleases have also been detected in insect genome sequences [3,4]. In Drosophila melanogaster, the JhI-1 gene encodes both forms of the tRNA 3¢ endonuclease RNase Z, which participates in endonucleolytic tRNA 3¢ end processing [5]. Additionally, JhI-1 is a juvenile hor- mone-regulated gene with several important functions in insects, such as moulting regulation during pre-adult Keywords alternative polyadenylation; AUUUA motifs; Cc RNase; RNase j; specific ribonuclease Correspondence D. C. Sideris, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Athens, Panepistimioupolis, 15701 Athens, Greece Fax: +30 210 7274158 Tel: +30 210 7274515 E-mail: dsideris@biol.uoa.gr *Present address Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA Database The nucleotide sequences of Ceratitis capi- tata RNase j have been submitted to the DDBJ ⁄ EMBL ⁄ GenBank databases under the accession numbers AJ874689 (cDNA) and AJ874690 (genomic DNA) (Received 28 July 2008, revised 9 September 2008, accepted 16 October 2008) doi:10.1111/j.1742-4658.2008.06746.x Cc RNase is the founding member of the recently identified RNase j family, which is represented by a single ortholog in a wide range of animal taxonomic groups. Although the precise biological role of this protein is still unknown, it has been shown that the recombinant proteins isolated so far from the insect Ceratitis capitata and from human exhibit ribonucleo- lytic activity. In this work, we report the genomic organization and molec- ular evolution of the RNase j gene from various animal species, as well as expression analysis of the ortholog gene in C. capitata. The high degree of amino acid sequence similarity, in combination with the fact that exon sizes and intronic positions are extremely conserved among RNase j orthologs in 15 diverse genomes from sea anemone to human, imply a very significant biological function for this enzyme. In C. capitata, two forms of RNase j mRNA (0.9 and 1.5 kb) with various lengths of 3¢ UTR were identified as alternative products of a single gene, resulting from the use of different polyadenylation signals. Both transcripts are expressed in all insect tissues and developmental stages. Sequence analysis of the extended region of the longer transcript revealed the existence of three mRNA instability motifs (AUUUA) and five poly(U) tracts, whose functional importance in RNase j mRNA decay remains to be explored. Abbreviations: ARE, AU-rich element; miRNA, micro RNA. FEBS Journal 275 (2008) 6217–6227 ª 2008 The Authors Journal compilation ª 2008 FEBS 6217 development, and control of reproductive maturation and sexual behaviour in adults [6]. Drosophila RNase H 1 is one of the best-studied insect ribonucleases [7], and its expression is essential for development but not for proliferation [8]. The two RNase III proteins, Dro- sha and Dicer, collaborate in the stepwise processing of micro RNAs (miRNAs), and play key roles in miRNA-mediated gene regulation of processes such as development and differentiation [9,10]. In Bombyx mori, a homolog of the RNase L inhibi- tor has been detected, cloned and characterized [11]. This finding implies the existence of RNase L, which plays an important role in the response of cells to dsRNA during events such as virus infection in insects. In Ceratitis capitata, three RNA-degrading enzymes have been detected. Two of these have been purified and characterized as alkaline [12] and acidic RNases [13], and a third poly(U)- and poly(C)-specific ribo- nuclease has also been isolated and characterized [14,15]. In order to further characterize this poly(U)- and poly(C)-specific ribonuclease, designated as Cc RNase, the full-length cDNA encoding this protein was cloned and characterized [16,17]. Cc RNase belongs to the recently identified RNase j family represented by a single ortholog in a wide spectrum of metazoans from anthozoans to humans. All family members are highly conserved, indicating a significant biological role for these molecules. Although the members of the RNase j family do not show any significant similarities to other known ribonucleases, the recombinant proteins isolated from two representatives (human and the insect C. capitata) exhibited ribonucleolytic activity on different RNA substrates. Additionally, the EST sequences deposited in NCBI databank for the human protein show that human RNase j is widely expressed in a large number of normal fetal, juvenile and adult tissues, a fact that strongly indicates a basic housekeep- ing cellular role for this ribonuclease [18]. This paper reports the identification, isolation and characterization of a novel Cc RNase transcript in the insect C. capitata. Moreover, we describe the expression profile of the RNase j gene at various developmental stages and in several tissues of the insect C. capitata. We have also determined the general genomic organization of this gene and analyzed its structural evolution. Results Expression analysis of Cc RNase In our previous work, we reported the cloning and overexpression of a cDNA sequence of 864 bp (Cc RNase cDNA) that encodes a specific RNase of the insect C. capitata [17]. In order to investigate the expression pattern of this novel RNase, a northern blot analysis was performed using RNA isolated from various developmental insect stages, as well as from male and female adults (Fig. 1). Our results show that the Cc RNase gene is expressed at all stages of the medfly’s life cycle, with higher levels of expression evi- dent in 6-day-old larvae. In addition to a Cc RNase mRNA band at 0.9 kb, a second unexpected band at 1.5 kb was also detected. Densitometric analysis of northern blot data revealed that the 1.5 kb transcript was more abundant than the 0.9 kb transcript at all the developmental stages studied, with ratios ranging from 1.1 (6-day-old larvae) to 2.2 (female adults). In order to investigate the tissue-specific distribution of the Cc RNase transcripts, total RNA isolated from the 0 2 4 6 8 10 12 14 16 18 20 EL3L6WPP3P4M F Stages Intensity of signal (arbitrary densitometric units) 1.5 kb 0.9 kb E L 3 L WP P 3 P 4 M F 1.5 Kb 0.9 Kb L 6 M F 1.5 Kb 0.9 Kb C 28S rRNA A B Fig. 1. Developmental northern blot analysis of Cc RNase. (A) Approximately 15 lg of total RNA isolated from various develop- mental stages of the insect C. capitata (E, eggs; L, larvae; WP, white pupae; P, pupae; M, adult male; F, adult female; the num- bers indicate the age in days) were loaded per lane, separated by electrophoresis in a 1.2% agarose–formaldehyde gel and trans- ferred to GeneScreen membranes. The membrane was hybridized with a 32 P-labeled probe corresponding to nucleotides 69–767 of the previously isolated Cc RNase cDNA (accession number AJ441124). Sizes were estimated by comparison with RNA size markers. (B) 28S rRNA was used as a control for the amounts of RNA loaded. (C) Densitometric analysis of the northern blot data, normalized to 28S rRNA, was performed using NIH IMAGEJ software. The vertical black and grey bars represent the 0.9 and 1.5 kb tran- scripts, respectively. Error bars denote the standard error of the mean for three independent measurements. Genomic structure and expression analysis of the Cc RNase T. N. Rampias et al. 6218 FEBS Journal 275 (2008) 6217–6227 ª 2008 The Authors Journal compilation ª 2008 FEBS epidermis and intestine of 6-day-old larvae and adult heads and ovaries, was blotted and hybridized using the Cc RNase cDNA as a probe. As shown in Fig. 2, both transcripts were expressed in all tissues examined. The signal for the longer transcript was found to be 2.5 times more intense than the signal for the shorter transcript in adult heads. Isolation and characterization of the Cc RNase 02 cDNA Given that the shorter transcript corresponds to the previously reported Cc RNase cDNA, a modified version of the hybrid selection technique was used in order to isolate and clone the longer transcript. Poly(A) + RNA from 6-day-old larvae was reverse- transcribed and the single-stranded cDNA molecules were hybridized with a 5¢-biotinylated DNA probe cor- responding to the main part of the Cc RNase cDNA. The selected hybrids were amplified by PCR and cloned into the pCR 2.1 vector. Twenty-four cDNA clones were selected and verified by restriction endonuclease mapping and DNA sequence analysis. The sequencing results revealed that the majority of the clones matched the Cc RNase cDNA, but seven clones corresponded to a longer cDNA molecule of 1.5 kb, referred to as Cc RNase cDNA 02 (EMBL database accession number AJ874689). This novel cDNA clone has a precise length of 1466 bp and contains a 5¢ UTR of 138 bp, an ORF of 288 nucleotides and a long 3¢ UTR of 1040 bp. The Cc RNase 02 cDNA encodes a 95 amino acid polypep- tide whose amino acid sequence is identical to that of the protein encoded by the previously isolated and characterized Cc RNase cDNA clone. Nucleotide sequence alignment between these cDNAs (Fig. 3) showed that Cc RNase cDNA 02 contains a 626 bp DNA sequence extension in the 3¢ UTR. The extended region is extremely AU-rich (71% A and U residues) and contains three mRNA instability motifs (AUUUA) and five poly(U) tracts (one U 16 stretch, one U 10 stretch, one U 9 stretch, one U 7 stretch and one U 9 G stretch). Three potential poly(A) signals were also found in the 3¢ UTR, one of them just 46 bp upstream of the poly(A) tail. The remaining two poly(A) signals are upstream of the region that contains the AUUUA motifs and the poly(U) tracts, so that if one of them were used as the poly(A) signal, a transcript encoding the same protein but without any instability motif in the 3¢ UTR would be produced. To confirm that the cloned Cc RNase cDNA 02 cor- responds to the 1.5 kb transcript seen in the northern blot experiments, the membranes were re-probed using a specific cDNA fragment (nucleotides 825–1314) from the extended 3¢ UTR sequence. In this case, only the 1.5 kb mRNA hybridized (data not shown), which strongly suggests that the cloned Cc RNase cDNA 02 corresponds to the longer Cc RNase transcript. Addi- tionally, the recombinant protein expressed in Escheri- chia coli by Cc RNase 02 ORF exhibits ribonucleolytic activity, as verified by direct visualization of the puri- fied recombinant protein on an SDS–PAGE gel into which poly(U) had been incorporated (Fig. 4). The fact that the recombinant proteins expressed by both Cc RNase cDNAs exhibits similar mobility and enzymatic activity confirms experimentally that the translation product of the two different cDNAs is the same. Epidermis Gut Head Ovary 1.5 kb 0.9 kb A B 28S rRNA 0 2 4 6 8 10 12 Epidermis Gut Head Ovary Tissues Intensity of signal (arbitrary densitometric units) 1.5 kb 0.9 kb C Fig. 2. Tissue-specific northern blot analysis of Cc RNase. (A) Approximately 15 lg of total RNA isolated from various tissues of the insect C. capitata (epidermis, guts, heads and ovaries) were loaded per lane, separated by electrophoresis in a 1.2% agarose– formaldehyde gel and transferred to GeneScreen membrane. The membrane was hybridized with a 32 P-labeled probe corresponding to nucleotides 69–767 of the previously isolated Cc RNase cDNA (accession number AJ441124). Sizes were estimated by compari- son with RNA size markers (Pharmacia). (B) 28S rRNA was used as a control for the amounts of RNA loaded. (C) Densitometric analy- sis of the northern blot data, normalized to 28S rRNA, was per- formed using NIH IMAGEJ software. The vertical black and grey bars represent the 0.9 and 1.5 kb transcripts, respectively. Error bars denote the standard error of the mean for three independent mea- surements. T. N. Rampias et al. Genomic structure and expression analysis of the Cc RNase FEBS Journal 275 (2008) 6217–6227 ª 2008 The Authors Journal compilation ª 2008 FEBS 6219 Cc RNase is present as a single-copy gene in the C. capitata genome Southern blot analysis was performed on genomic DNA of C. capitata digested with HindIII, SalI and EcoRI and hybridized with probe A (nucleotides 58– 784), which corresponds to the common sequence of the two cDNA clones, at high stringency (Fig. 5A). The autoradiograph shows a single band in lanes con- taining genomic DNA digested with HindIII or SalI, Fig. 3. Nucleotide and deduced amino acid sequences of the Cc RNase cDNAs. The cDNA 01 (top line, accession number AJ441124), cDNA 02 (middle line, accession number AJ874689) and amino acid sequences (bottom line) are numbered on the right. Nucleotides of the cDNA 02 that are identical those of the cDNA 01 are replaced by asterisks, and alignment gaps are indicated by dashes. The three potential poly(A) signals are boxed with a white background, and the poly(T) tracts and ATTTA motifs in the 3¢ UTR are boxed with a gray background. Genomic structure and expression analysis of the Cc RNase T. N. Rampias et al. 6220 FEBS Journal 275 (2008) 6217–6227 ª 2008 The Authors Journal compilation ª 2008 FEBS and two bands (approximately 0.9 and 1.5 kb) when the genomic DNA was digested with EcoRI. These data show that the Cc RNase gene occurs as single copy in the C. capitata diploid genome. Additionally, the fact that only the 0.9 kb EcoRI fragment disap- pears from the hybridization pattern when probe B (nucleotides 825–1314) specific for the 3¢ UTR of Cc RNase 02 cDNA was used (Fig. 5B) indicates that both Cc RNase mRNAs derive from the same gene. The Cc RNase gene contains two introns and three exons To determine the structure of the Cc RNase gene, a PCR reaction was performed using a primer pair designed to anneal to the 5¢ and 3¢ UTR terminal sequences of Cc RNase 02 cDNA, with genomic DNA of C. capitata as a template. The amplified product was found to be 1642 bp long upon cloning and sequencing in both directions (EMBL database accession number AJ874690). Multiple alignment of cDNAs and genomic nucleotide sequences revealed that the Cc RNase gene consists of three exons (218, 120 and 1150) interrupted by two introns (180 and 214 bp). All intron ⁄ exon boundaries follow the GT ⁄ AG rule, with GT being the splice donor and AG the splice acceptor [19]. Alternative polyadenylation of the Cc RNase gene generates two mRNA isoforms The finding that no additional intronic sequence is present in the 3¢ UTR of the Cc RNase gene indicates that the derived mRNAs 01 and 02 cannot derive from a primary transcript by alternative splicing. An exten- sive analysis of the Cc RNase cDNA 02 sequence revealed that two putative polyadenylation signals are found in the 3¢ UTR, located at nucleotide positions 788–793 (AAUAUA) and 1386–1391 (AAUAAA). The existence of these polyadenylation signals in the same exon of the gene encoding Cc RNase, in combination with the fact that the first signal (AAUAUA) is located just 19 bp upstream of the poly(A) tail of cDNA 01 and the second one is located 46 bp upstream of the poly(A) tail of cDNA 02, suggest that the two Cc RNase mRNA isoforms probably derive from a primary transcript by alternative polyadenyla- tion (Fig. 6). Intron/exon structure comparison of RNase j family genes from various taxa In addition to the characterized Cc RNase gene reported above, a search against all the available -94 -67 -43 -30 -20.1 -14.4 C 1 2 1 2 -94 -67 -43 -30 -20.1 -14.4 AB Fig. 4. SDS–PAGE and activity staining of the purified Cc RNase. One microgram of the purified recombinant protein expressed in E. coli from the ORF of the previously isolated cDNA (lane 1) and from the Cc RNase cDNA 02 described here (lane 2) were analysed on an 11–15% polyacrylamide gel (A) or on the same gel containing 0.25 mgÆmL )1 poly(U) (B). After electrophoresis, the gel in (A) was stained with Coomassie blue and the gel in (B) was stained for RNase activity as described in Experimental procedures. A set of marker proteins of known molecular weights (94, 67, 43, 30, 20.1 and 14.4 kDa, top to bottom) (C) was run in parallel and the pro- teins were stained with Coomassie blue. Hind III Sal I EcoR I Hind III Sal I EcoR I 10 kb 8 kb 5 kb 2.5 kb 1.5 kb 1 kb A B Fig. 5. Southern blot analysis of Cc RNase. Approximately 50 lgof chromosomal DNA were loaded per lane after complete digestion with HindIII, SalIorEcoRI, separated by electrophoresis in a 0.8% agarose gel and transferred to GeneScreen membrane. The mem- brane was then hybridized with (A) probe A (nucleotides 58–784) or (B) probe B (nucleotides 825–1314) of the alternative Cc RNase cDNA 02 sequence. Positions of the DNA markers are given on the right. T. N. Rampias et al. Genomic structure and expression analysis of the Cc RNase FEBS Journal 275 (2008) 6217–6227 ª 2008 The Authors Journal compilation ª 2008 FEBS 6221 genome nucleotide databases was performed in order to identify other RNase j homolog genes. This search resulted in the retrieval of 14 genomic sequences from various animal species, including mammals, fish, insects, echinoderms and anthozoans. Alignment of the genomic sequences with the corresponding ESTs led to identification of their intron ⁄ exon structure. In all cases, only one functional gene was detected in each genome. The intron ⁄ exon organization of the ORF region of the RNase j family genes is shown in Fig. 7A. In all organisms examined, the region coding for RNase j is interrupted by two introns, with the exception of the sea urchin Strongulocentrotus purpura- tus and the hymenopteran insects Nasonia vitripennis and Apis mellifera, in which only one intron is present. The intronic regions among these 15 species demon- strate an expectedly higher degree of variability than the exons, although remarkable similarities exist among several species. For example, the higher-verte- brate lineages represented by dog, cow, rat, mouse and human exhibit relatively few differences in the size of their respective introns. Furthermore, there is a strong conservation of exon organization. The exon size and the intronic positions are identical within mammals and only slightly different compared to species from other taxa. The intronic architecture of the selected RNase j genes was mapped onto a multiple protein sequence alignment (Fig. 7B). In addition to the high degree of amino acid sequence similarity, there is a considerable preservation of both the position and size of exons among RNase j genes in several taxa. For example, there is an absolute conservation of the loca- tion, size and phase of exon 1 from primitive animals to humans. However, the amino acid sequence from the C-terminus of exon 2 to the N-terminus of exon 3 is more divergent between the vertebrates and the other taxa analyzed. Discussion In the insect C. capitata, an RNase j mRNA of 864 bp has previously been cloned and characterized [17]. Northern blot analysis of expression of the RNase j revealed that, in addition to the band at 0.9 kb, a second band at 1.5 kb was also detected at all developmental stages and in all tissues examined. Using a modified version of the hybrid selection tech- nique, we cloned the full-length longer transcript. Nucleotide sequence alignment between the two alter- native transcripts revealed that the longer transcript is identical to the previously isolated Cc RNase mRNA in coding sequence, and differs only in the length of the 3¢ UTR, which contains a 626 bp extension. Addi- tionally, the recombinant protein expressed by the longer transcript exhibits the same molecular weight and the same enzymatic activity as the recombinant protein expressed by the shorter transcript. This result was expected due to the fact that the ORF is totally conserved between the two mRNA isoforms. The first question is whether the longer transcript is produced from the same RNase j gene by alternative RNA processing. The data obtained by Southern blot analysis revealed that there is a single copy of RNase j in the C. capitata genome. The genomic DNA frag- ATG TAA Poly A Poly A AAUAAA AAUAUA Gene mRNA 01 PA S1 PA S2 mRNA 02 100 bp Fig. 6. Schematic organization of the Cc RNase gene and its derived mRNA isoforms. The rectangles represent the exons and the horizon- tal bold lines the introns. The black parts of the rectangles show the protein-coding region, the white parts represent the non-coding regions and the gray parts show the poly(A) tail. The positions of the ATG, TAA and two putative polyadenylation signals PA S1 and PA S2 are indicated by vertical lines. The non-horizontal broken lines represent the alternative polyadenylation signal selection. Fig. 7. Gene structure and deduced amino acid alignment of RNase j among various species. (A) A schematic representation of the exon (rect- angles) and intron (horizontal lines) structure of the ORF region of the RNase j genes from Homo sapiens (NC_000017:6856522–6858575), Mus musculus (NC_000077:c70053348–70051628), Rattus norvegicus (NC_0051109), Bos taurus (NC_007317:c57078994–57077154), Canis familiaris (NC_006587:35037037–35038710), Danio rerio (NC_007118:c20053898-20052649), Nasonia vitripennis (NW_001820416 c1450413–1449631), Apis mellifera (AmeLGUn_WGA1021_4:8811–9751), Drosophila melanogaster (NT_033778:c75661–74991), Ceratitis cap- itata (reported here), Anopheles gambiae (NT_078265:c31050091–31048592), Culex pipiens (NW_001886701:1836614–1837565), Triboli- um castaneus (TcaLG7_WGA100_1: c3103336–3102941), Strongulocentrotus purpuratus (SpuUn_WGA56223_2:75333–76952) and Nematostella vectensis (NW_001834409:c1450413–1449631). The numbers above the boxes indicate the size of the exons, and those below the lines indicate the size of the introns. (B) Multiple amino acid sequence alignment of the RNase j family orthologs. Amino acid sequences are numbered on the right. Exon 1 is shown in dark gray and exon 2 in light gray. Where an intron does not fall at phase 0, the corresponding amino acid residue is highlighted. Dashes indicate alignment gaps. Genomic structure and expression analysis of the Cc RNase T. N. Rampias et al. 6222 FEBS Journal 275 (2008) 6217–6227 ª 2008 The Authors Journal compilation ª 2008 FEBS A B T. N. Rampias et al. Genomic structure and expression analysis of the Cc RNase FEBS Journal 275 (2008) 6217–6227 ª 2008 The Authors Journal compilation ª 2008 FEBS 6223 ment with the terminal sequences of the longer tran- script was amplified by PCR and the RNase j gene structure was determined. The C. capitata RNase j gene consists of three exons interrupted by two introns. Multiple alignment between the cDNA and genomic nucleotide sequences did not reveal any intronic sequences that could be involved in any alter- native splicing processes. We have therefore excluded this possibility for generation of the two alternative transcripts, and present evidence that C. capitata RNase j pre-mRNA undergoes processing at two polyadenylation sites. Sequence analysis of the cDNA corresponding to the longer transcript revealed the presence of two puta- tive polyadenylation signals in the 3¢ UTR, located at nucleotide positions 788–793 (AAUAUA) and 1386– 1391 (AAUAAA). The first signal (AAUAUA) is located just 19 bp upstream of the poly(A) tail of the shorter transcript and the second one is located 46 bp upstream of the poly(A) tail of the alternative transcript. The hexanucleotide AAUAAA is the pre- dominant cleavage-directing and polyadenylation sequence among eukaryotic pre-mRNAs; however, the AAUAUA sequence is also present at a lower frequency in Diptera transcripts [20,21]. These two polyadenylation signals were found in the C. capitata RNase j gene sequence. Alternative use of these polyadenylation signals in RNase j pre-mRNA is likely to generate the two mRNA isoforms. As the putative polyadenylation signal for the shorter tran- script (AAUAUA) is slightly different from the con- sensus signal that is used for formation of the longer transcript, it is possible that these transcripts are expressed at different levels due to a lower efficiency of polyadenylation driven by the AAUAUA hexamer. Northern blot analysis data demonstrated that the levels of the longer transcript are higher than the levels of the shorter transcript. A tblastn search of available EST sequences from Drosophila melanogaster revealed the existence of a single RNase j transcript containing the AAUAAA polyadenylation signal 15 nucleotides upstream of the polyA tail. This difference in the poly- adenylation process of the RNase j transcript between D. melanogaster and C. capitata could be explained by accelerated gene evolution in the group of dipterans [22,23]. As the first functional polyadenylation signal used to produce the smaller transcript (0.9 kb) is also pres- ent in the longer transcript (1.5 kb), a question con- cerning the functional significance of the 3¢ UTR extension was raised. It is well known that the presence of a long 3¢ UTR is frequently inductive of post-tran- scriptional regulation of gene expression via modula- tion of mRNA stability [24,25]. For this reason, we further investigated the existence of regulatory ele- ments in the 3¢ extended UTR of the longer transcript. An extensive analysis revealed that this particular region is extremely AU-rich, containing three copies of the AUUUA motif and five poly(U) tracts. Repeats of the pentamer AUUUA in the 3¢ UTRs of mRNAs promote de-adenylation and subsequent degradation in a wide variety of mRNAs [26,27]. Additionally, in sev- eral AU-rich elements (AREs), U stretches combined with AUUUA motifs have a destabilizing effect, lead- ing to rapid mRNA decay [28]. The complexity of ARE-mediated pathways is manifested by the presence of multiple ARE-binding proteins, including stabilizing proteins [29,30] or proteins that promote degradation of mRNAs that contain AREs [31]. Recent findings reveal that the RNase L expression is regulated by binding of the stabilizing HuR protein to ARE sequences located in the 3¢ UTR [32]. Alternative polyadenylation is also associated with miRNA targeting, considering that individual miRNA target sites may be included in or excluded from vari- ous mRNA isoforms [33,34]. Using the regulatory RNA motifs and elements finder [35], a search for putative miRNA binding sites in the extended 3¢ UTR sequence of the longer RNase j mRNA was per- formed. This search resulted in retrieval of a putative site for hsa-miR-206 miRNA (located at position 818– 840), whose homolog in Drosophila (dme-miR-1) was found to regulate the expression of a large number of genes [36]. These results generate an attractive hypoth- esis that alternative polyadenylation is one of the mechanisms that regulates RNase j expression in C. capitata. Another focus of this work was the study of the exon ⁄ intron structural evolution of the RNase j gene, by means of integration of blast-based analysis data obtained from all available EST and genome sequences from various taxa. No RNase j gene sequences were detected in the genomes of fungi, plants, bacteria or protists, a finding that clearly indi- cates that the RNase j family is limited to metazoans. It should be noted that, in all metazoan genomes ana- lyzed, only one copy of RNase j was identified, con- firming the conclusion that RNase j is an orthologous protein family. In early animal evolution, there appear to have been two intronic insertion events, leading to the extremely conserved three-exon structure of the RNase j gene that is observed from the old eumetazo- an phylum, the Cnidaria (Nematostella vectensis), to humans. As a result of this conservation, there is considerable preservation of the position and size of exons. Genomic structure and expression analysis of the Cc RNase T. N. Rampias et al. 6224 FEBS Journal 275 (2008) 6217–6227 ª 2008 The Authors Journal compilation ª 2008 FEBS An intronic deletion event appears to have occurred within the phylum Echinodermata, leading to the two-exon configuration identified in S. purpuratus. Prior to divergence of hymenoptera from diptera (> 290 mil- lion years ago) [37], an additional independent intronic deletion may have taken place and led to the two-exon structure seen in the insects N. vitripennis and A. mellif- era. As a result of these events, the sum of the sizes exons 2 and 3 detected in all other analyzed species is very similar to the size of exon 2 in S. purpuratus, N. vit- ripennis and A. mellifera. Additionally, there is absolute conservation of the location, size and phase of exon 1 among all RNase j genes in the available species. The above results imply that there has been a highly conservative selective pressure imposed on the RNase j gene structure during evolution, a fact that further supports the conclusion that the encoded protein plays a central role in RNA metabolism. Experimental procedures Animals The insect C. capitata was grown under controlled condi- tions of temperature and humidity as described previously [14]. Six-day-old larvae were collected and stored in liquid nitrogen prior to use. Materials Oligonucleotides were custom-synthesized by MWG Bio- tech (Ebersberg, Germany). Restriction enzymes and T 4 DNA ligase were purchased from New England Biolabs (Hitchin, UK). DyNAZymeÔ II DNA polymerase was obtained from Finnzymes Oy (Espoo, Finland). Plasmid preparation kits were obtained from Qiagen Inc. (Hilden, Germany). All expression vectors, bacterial strains and the His-Bind resin were from Novagen (Darmastadt, Ger- many). Hepes and poly(U) were purchased from Serva (Heidelberg, Germany). Marker proteins for SDS–PAGE molecular weight estimations and RNA markers were obtained from Pharmacia (Uppsala, Sweden). All other reagents used were of analytical grade and obtained from Merck (Darmstadt, Germany). Isolation of the Cc RNase cDNA 02 clone Total RNA was prepared from 6-day-old larvae of the insect C. capitata (Bouhin et al. [38]) and poly (A) + RNA was isolated using DynabeadsÒ oligo(dT) 25 (Dynal, Oslo, Norway). Poly(A) + RNA (1 lg) was reverse-transcribed using the SMART PCR cDNA synthesis kit (Clontech Laboratories, Palo Alto, CA, USA) as described by the manufacturer. In order to isolate the alternative Cc RNase cDNA, a modified version of the hybrid selection technique [39] was employed. A 5¢-biotinylated DNA probe was syn- thesized by PCR using the complete previously character- ized cDNA clone as template [17]. The biotinylated strand of the PCR product was attached to DynabeadsÒ M-280 streptavidin (Dynal), and hybridized overnight to the single-stranded cDNAs at 63 ° Cin3· SSC, 0.1% SDS, 1.25· Denhardt’s. Following hybridization, the beads were selected and washed three times in 2· SSC, 0.1% SDS at 60 °C for 10 min each, twice in TE buffer (10mm Tris-HCl, pH 7.4, 1 mm EDTA) at room temperature for 5 min each, and finally resuspended in distilled H 2 O. The selected hybrids were then amplified by PCR using the LD primer (5¢-AAGCAGTGGTAACAACGCAGAGT-3¢), which anneals at the 5¢ and 3¢ ends of the cDNA strands. The PCR prod- ucts were cloned into the pCR 2.1 cloning vector (Invitro- gen, Paisley, UK), and the isolated clones were sequenced in both directions. Amplification and cloning of the Cc RNase gene The Cc RNase gene was amplified by PCR using genomic DNA isolated from the insect C. capitata as described previously [40] as template and the DS74 (5¢-TTG TGGAAAATCATACGAGA-3¢) and R1286 (5¢-CAAAC ACACATCGAGGAGC-3¢) oligonucleotides corresponding to the 5¢ and 3¢ terminal regions of Cc RNase cDNA 02, respectively, as primers. PCR amplification was performed using a Perkin-Elmer (Waltham, MA, USA) 9600 thermal cycler, and the cycle conditions were as follows: one cycle at 95 °C for 2 min, followed by 35 cycles at 95 °C for 1 min, 56 °C for 1 min and 72 °C for 2 min, with a 10 min final extension at 72 °C. The PCR product was analyzed by agarose gel electrophoresis, and the extracted DNA was cloned into the pCR 2.1 cloning vector. Three clones were selected, and their authenticity was verified initially by restriction endonuclease mapping and then by DNA sequencing in both directions. Northern and Southern blot analysis Total RNA (15 lg) extracted from various developmental stages and tissues of the insect C. capitata were resolved by electrophoresis in 1% agarose containing 1.25 m formalde- hyde and transferred to a GeneScreen (DuPont-NEN, Bos- ton, MA, USA) membrane. For Southern blotting, 50 lg of genomic DNA were digested overnight using 30 units of the restriction endonucleases. Hybridization was performed at 68 °C as described previously [41], using two 32 P-labeled probes corresponding to nucleotides 58–784 (probe A) and 825–1314 (probe B) of the alternative Cc RNase cDNA 02 sequence. After hybridization, the membranes were washed and exposed to X-ray film at )80 °C for 2 days. Densito- metric analysis of Northern blot data was performed using nih imagej software (http://rsbweb.nih.gov/ij/). T. N. Rampias et al. Genomic structure and expression analysis of the Cc RNase FEBS Journal 275 (2008) 6217–6227 ª 2008 The Authors Journal compilation ª 2008 FEBS 6225 Expression and purification of Cc RNase recombinant protein The ORF of the Cc RNase cDNA 02 sequence was ampli- fied by PCR and then sub-cloned into the pSCREEN- 1b(+) expression vector (Novagen) producing the PF2 con- struct. This construct was initially transformed into the Nova blue E. coli host strain for characterization, and then into BL21(DE3) pLysS E. coli cells for protein expression. Purification of the recombinant protein was performed as previously described [17], and the ribonucleolytic activity towards poly(U) was tested by analyzing the purified enzyme in an activity staining gel [42]. 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Genomic structure and expression analysis of the RNase j family ortholog gene in the insect Ceratitis capitata Theodoros N. Rampias*,. with the terminal sequences of the longer tran- script was amplified by PCR and the RNase j gene structure was determined. The C. capitata RNase j gene