Prediction and Expression Analysis of G Protein-Coupled Receptors in the Laboratory Stick Insect, Carausius Morosus
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Turkish Journal of Biology Turk J Biol (2019) 43: 77-88 http://journals.tubitak.gov.tr/biology/ © TÜBİTAK Research Article doi:10.3906/biy-1809-27 Prediction and expression analysis of G protein-coupled receptors in the laboratory stick insect, Carausius morosus 1 1,2, Burçin DUAN ŞAHBAZ , Necla BİRGÜL İYİSON * 1 Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Boğaziçi University, İstanbul, Turkey 2 Center for Life Sciences and Technologies, Boğaziçi University, İstanbul, Turkey Received: 16.09.2018 Accepted/Published Online: 18.12.2018 Final Version: 07.02.2019 Abstract: G protein-coupled receptors (GPCRs) are 7-transmembrane proteins that transduce various extracellular signals into intracellular pathways. They are the major target of neuropeptides, which regulate the development, feeding behavior, mating behavior, circadian rhythm, and many other physiological functions of insects. In the present study, we performed RNA sequencing and de novo transcriptome assembly to uncover the GPCRs expressed in the stick insect Carausius morosus. The transcript assemblies were predicted for the presence of 7-transmembrane GPCR domains. As a result, 430 putative GPCR transcripts were obtained and 43 of these revealed full-length sequences with highly significant similarity to known GPCR sequences in the databases. Thirteen different GPCRs were chosen for tissue expression analysis. Some of these receptors, such as calcitonin, inotocin, and tyramine receptors, showed specific expression in some of the tissues. Additionally, GPCR prediction yielded a novel uncharacterized GPCR sequence, which was specifically expressed in the central nervous system and ganglia. Previously, the only information about the anatomy of the stick insect was on its gastrointestinal system. This study provides complete anatomical information about the adult insect. Key words: GPCR, stick insect, RNAseq 1. Introduction The laboratory stick insect is an organism defined as G protein-coupled receptors (GPCRs) constitute the an agricultural pest in some countries but fed as a pet major targets of small regulatory peptides or peptide in others. Its locomotion behavior is widely studied and hormones. They transduce these extracellular stimuli to modeled (Bläsing and Cruse, 2004; Gruhn et al., 2016; intracellular molecular responses and become the key Dallmann et al., 2017). Additionally, other peptidergic regulators of endocrine signaling. They contain conserved mechanisms, such as ecdysis behavior, circadian rhythm, 7-transmembrane (7TM) helices, a variable extracellular and heartbeat frequency, are as well studied (Wadsworth et N-terminus, and an intracellular C-terminus. They al., 2014; Marco et al., 2018). Although the most researched interact with the trimeric G-proteins from the intracellular mechanisms of the stick insect rely on peptidergic site. GPCRs are the largest group of proteins in eukaryotes pathways, there is limited information on its neuropeptides and are clustered in separate classes, namely rhodopsin- and GPCRs. Even though its neuropeptidome was revealed like Class A, secretin-like Class B1, adhesion GPCRs Class recently (Liessem et al., 2018), we need to uncover the B2, glutamate receptors Class C, Frizzled receptors Class GPCRs they target and activate in order to understand F, and Taste-2 receptors. Within these receptors, peptides the mechanism of action of the neuropeptides. There are interact with the GPCRs of Class A and Class B1. Our well-fitted approaches and tools for GPCR prediction previous work identified a C type of Allatostatin receptor from RNA sequencing (RNAseq) data. For instance, the (AlstR) from Class A in the laboratory stick insect, RNAseq approach has been recently used to predict the Carausius morosus (Duan Sahbaz et al., 2017). All types GPCR profiles of different arthropods (Buckley et al., of AlstRs (A, B, and C) have a common function, which is 2016; Guerrero et al., 2016). These studies utilized the open inhibition of juvenile hormone (JH) secretion. However, reading frame and 7TM domain prediction tools to predict their expression profiles differ in a stage-specific, tissue- GPCR candidates. Then they filtered the reliable GPCRs specific, and species-specific manner. In addition, their with the help of the GPCRPred tool, which annotates and inhibitory effect varies between species. predicts GPCRs from other proteins with an accuracy of * Correspondence: [email protected] 77 This work is licensed under a Creative Commons Attribution 4.0 International License. DUAN ŞAHBAZ and BİRGÜL İYİSON / Turk J Biol 99.5% (Bhasin and Raghava, 2004). Therefore, we utilized annotations were performed via blastx against NCBI B. the transcriptome data for identification of the GPCRome germanica sequences. The data were submitted to Sequence of C. morosus including all AlstRs and predicted the Read Archives (SRA7949781). functions of some of these GPCRs in comparison to their 2.4. GPCRome prediction expression profiles and neuropeptide ligands. In order to predict open reading frames (ORFs), the sequences were submitted to ORFPREDICTOR (Min et 2. Materials and methods al., 2005). All six ORFs were analyzed for the presence 2.1. Animals of TM helices in TMHMM (Krogh et al., 2001). TM- The stick insects (Carausius morosus) were obtained from containing ORFs were separated into different files. The the University of Cologne, Germany. They were kept in ORFs, which included more than 2 TM regions, were cages at room temperature (RT) and fed ad libitum in a aligned with the NCBI BLASTp tool. Results of the top 12-h light/dark cycle. Sampling was performed in daylight ten hits were taken and filtered according to the presence when the animals were least active. The adult females, of GPCR domains. In order to check for the functional which were ready to lay eggs, were sacrificed via CO2 and units and patterns of these sequences ExPASy (Gasteiger cooled down in PBS at –20 °C. Dissection was performed et al., 2003), BLASTp, and SMART (Schultz et al., 1998) in the presence of cold PBS. The organs were immediately were used. Structural GPCR domains were determined put into TRIzol reagent and stored at –80 °C. For RNA in GPCRHMM (Wistrand et al., 2006). Finally, putative sequencing, the animals were flash-frozen in liquid GPCR classification was performed in 6 groups according nitrogen, disrupted by a mortar and pestle, and stored at to the nomenclature generated by GPCRdb (Pándy- –80 °C. Szekeres et al., 2018). 2.2. RNA isolation 2.5. cDNA synthesis Frozen total animal tissues were mixed with TRIzol and The amount of RNA of different tissues was adjusted to the mixture was homogenized in a MagNA Lyser for 1 min. 1 µg. First strand cDNA synthesis was performed as The homogenates were put in clean tubes and incubated recommended in the protocol of the SensiFAST cDNA at RT for 5 min. Centrifugation was performed at 12,000 Synthesis Kit (BIO-65054, BIOLINE, London, UK). × g for 10 min at 4 °C. The clear lysate was transferred 2.6. qPCR into a clean microcentrifuge tube and chloroform was The primers were designed from the putative GPCR added in 1:5 ratio. The tube was mixed vigorously for 20 transcripts via the BlastPrimer tool. Quantitative s and incubated at RT for 2–3 min. Centrifugation was PCR reactions were prepared according to the performed at 10,000 × g for 18 min. The aqueous part was recommendations of the SensiFAST SYBR No-ROX Kit transferred into a clean microcentrifuge tube and 1 volume (SF581-B054620, BIOLINE). Each sample was prepared of 100% EtOH was added. The tube was inverted 6 times. in technical duplicates. The PCR efficiency of each primer Next, 700 µL of this sample was loaded into a NucleoSpin set was calculated by the standard curve method taking RNA column. Centrifugation was performed at 11,000 × g the curves with R2 ≥ 0.99. Fold changes in expression for 30 s and the flowthrough was discarded. The rest of the levels were calculated via the REST method (including the procedure was performed as recommended in the protocol PCR efficiencies) with regard to GAPDH as the reference of NucleoSpin RNA (740955.50, MN, Germany). The RNA gene and ovary tissue as the calibrator. The other tissues samples were evaluated in MOPS gel electrophoresis in included in the analysis were the brain together with the denaturing conditions. For RNA sequencing one animal endocrine glands corpora allata (CA) and corpora cardiaca sample was prepared and dried in RNAstable (Biomatrica) (CC), ganglia, Malpighian tubules, crop and foregut, for shipping. For expression analyses, at least three adult gastric cecum, postposterior midgut with the hindgut, fat animals were utilized in one replica, according to the mass body, and the aorta. Each qPCR reaction was performed of the organ. in biological triplicates, each containing at least three 2.3. Sequencing and de novo RNA assembly animals. The specificity and consistency of the reactions Paired-end sequencing (2 × 100 bp) was generated by were determined via RT-PCR. GENEWIZ Inc. Assembly was performed by Epigenetiks Ltd. Co. using Trinity (Grabherr et al., 2013). Sequence- 3. Results read files were retrieved in FASTQ format. Quality control 3.1. Novel GPCR sequences were predicted from the was performed on FastQC (Andrews, no date). Blatella transcriptome of the adult stick insect germanica and Zootermopsis nevadensis were chosen as The total body of adult female C. morosus specimens was the genomes closest to C. morosus. The assembly file was used for total RNA sequencing