Intron retention in a salmon gonadotropin releasing-hormone in three species of Chirostoma genus.

Abstract

Event Abstract Back to Event Intron retention in a salmon gonadotropin releasing-hormone in three species of Chirostoma genus. Monica Chavez1, Jorge E. Campos2, Antonio Campos3, RODOLFO CARDENAS1* and Carlos A. Martinez-Palacios4 1 UNIVERSIDAD NACIONAL AUTONOMA DE MEXICO, FES IZTACALA, UNIDAD DE MORFOLOGIA Y FUNCION, Mexico 2 UNIVERSIDAD NACIONAL AUTONOMA DE MEXICO, FES IZTACALA, UNIDAD DE BIOTECNOLOGIA Y PROTOTIPOS, Mexico 3 UNIVERSIDAD MICHOACANA DE SAN NICOLAS HIDALGO, FACULTAD DE BIOLOGIA, Mexico 4 UNIVERSIDAD MICHOACANA DE SAN NICOLAS HIDALGO, INSTITUTO DE INVESTIGACIONES AGROPECUARIAS Y FORESTALES, ACUACULTURA, Mexico The Atherinomorphae family includes several genera in Mexico. One of the most representative is the genus Chirostoma, which, in turn, includes several species (Dyer and Chernof, 1996). A recent report, in another species of this family, indicated that the brain of that species has three different isoforms of gonadotropin releasing-hormone (GnRH) (Guilgur et al., 2007), this condition is characteristic of the main group of teleost fish (Kah et al., 2007, Okubo and Nagahama, 2008). These three isoforms of GnRH are located in discrete areas of the brain. Those present in neurons in the preoptic area are named GnRH-1 system, usually specific for an isoform of each species. Other neurons in the mesencephalon are the GnRH-2 system, expressing the isoform chicken II GnRH (cGnRH-II) in all cases, and a third group of neurons are in the telencephalon and are called GnRH-3 system, which corresponds always to the salmon GnRH (sGnRH). It is accepted that GnRH-1 has hypophysiotrophic functions. In general, systems 2 and 3 have been postulated to play a neuromodulation/neurotransmission role, especially in reproductive behaviour (Lethimonier et al., 2004; Guilgur et al., 2006, Kah et al., 2007, Okubo and Nagahama, 2008). The GnRH gene is highly conserved in vertebrates, consisting of four exons (1, 2, 3, and 4) separated by three introns (A, B, and C). The 5’UTR is contained in exon 1. Exon 2 contains the sequences of the signal peptide, GnRH decapeptide, processing site, and several nucleotides of the GnRH associated peptide (GAP), which is interrupted by intron B. Exons 3 and 4 contained GAP sequences and the 3’UTR, interrupted by intron C. The mature mRNA is translated into a prepropeptide sequence that starts in exon 2 and contains the signal peptide, hormone peptide, the processing site, and the GAP (Okubo and Nagahama, 2008; Tsai and Zhang, 2008). In Chirostoma humboldtianum an unusual transcript of the sGnRH isoform retaining intron B was found. The present study was aimed at assessing whether intron B is retaining in the sGnRH transcript in the brain for these three important species of the genus in Mexico, Ch. estor, Ch. Promelas, and Ch. humboldtanum. Materials and Methods Animals Chirostoma estor and Ch. promelas fishes came from the experimental unit of the Instituto de Investigaciones Agropecuarias y Forestales of the Universidad Michoacana de San Nicolás Hidalgo, Michoacán, Mexico. Ch. humboldtianum fish were caught in the Zacapu lagoon, Michoacan, Mexico, located between 19º40’40” and 19º49’26” N, and 101º46’45” and 101º47’25” W.Fish were anesthetized with 0.05% tricaine methanesulfonate (MS 222, Sigma Co. St. Louis, MO) before tissue collection. The brain was removed and collected in microcentrifuge tubes previously treated with diethyl pyrocarbonate (DEPC), and stored in dry ice until their arrival to the laboratory. The samples were stored at -40 °C. On the next day, brains were processed for total RNA extraction. Procedures Isolation Total RNA was extracted from brains of Chirosotoma humboldtianum, Ch. estor, and Ch. promelas with Trizol-based phenol-chloroform- guanidinium acid method (Chomczynski and Sacchi, 1987). cDNA was obtained, according to the first-strand kit instructions (Roche) from 1 g of total RNA. The first-strand was used as template for PCR. The primers were designed according to those reported to amplify the sGnRH coding region of the Argentine- Pejerrey (Guilgur et al., 2007), Set 1: FORWARD, 5’-CAG CAY TGG TCI TAY GGI TGG CT-3’ and REVERSE, 5’-TCT CTC TTG GAT TTG GGC ACT T-3’. PCR was conducted at an annealing temperature of 58°C and 35 cycles. The PCR products obtained were isolated in 1.5% agarose gels, purified and cleaned by means of a MiniElute (QIAGEN Inc. Francia ). The products were cloned in a pGEM-Teasy vector system (Promega) and the positive clones were selected and sequenced in an automated DNA sequencer (ABI 3100, Perkin Elmer). Sequencing analysis DNA sequences were analyzed with the NCBI-BLAST software, available at http://blast.ncbi.nlm.nih.gov/Blast.cgi. Results A 310 bp cDNA from Ch. humboldtianum (JN157840), Ch. estor (JN157841) and Ch. promelas (JN157842) was obtained. The sequences were identical for the three species, which corroborates the tight relationship between species. The sequences correspond to coding regions are from 1st to 69 nucleotide (the hormone peptide’s, processing site, the first part of GAP), the intron is from 70 to 267 nucleotide, and the another coding region is from268 to 310 nucleotide (the second aprt of GAP). When the sequence of intron B is removed, the partial GAP sequence is very similar to that reported for this isoform in other species, both nucleotides and amino acids. Discussion To the best of our knowledge this is the first time that intron B retention in sGnRH mRNA is reported for the brain of any teleost fish. The partial sequence of sGnRH after the intron is removed showed high similarity with other sGnRH sequences of teleosts, in particular with the most related species such as Odonthestes bonariensis and Epinephelus (Table 1). There are some reports on GnRH mRNAs longer than the classical sequence for these hormones. In the rainbow trout (Oncorhynchus mykiss), intron retention has been reported in the brain and gonads (Gray et al., 2002). However, some considerations have been made: 1) O. mykiss is a very well studied species, and like other teleost fish, rainbow trout is a tetraploid species (Ohno et al., 1968; Onho,1999; Hoegg et al, 2004), in consequence, there are reports of two sGnRH genes named sGnRH-1 and sGnRH-2, respectively (Ashihara et al., 1995), and the propeptide sequences from both genes are exactly the same, however, there are some differences in the 5’ untranslated region for promoters (Von Schalburg and Sherwood, 1999 ). Similar conditions have been reported for salmon (Higa et al., 1997); 2) In rainbow trout and many other salmonid species there are just two isoforms of GnRH in the brain and gonads, sGnRH and cGnRH-II, being sGnRH the variant that plays a double role like the GnRH-1system (hypophysiotrophic) and GnRH-3 system (possibly involved in reproductive behaviour) (Sherwood et al., 1983; Amano et al., 1997); 3) The longer sGnRH mRNA forms only the transcript of sGnRH-2 gene. This transcript was found four months just before the reproductive season, the retained intron was intron A (Gray et al., 2002). The same findings have been reported by Uzbekova et al. (2002) in gonads of the same species. Similar situation has been reported for Anguila japonica, in which, like in the mammalian GnRH isoform, intron A is retained in several organs, brain and gonads included (Okubo et al., 1999). The mGnRH plays the role of system-1 in this species. Also, in Sparus aurata (sea bream), evidence indicates that gonads have an intron retention in the sea bream GnRH isoform (sbGnRH)(Nabissi et al., 2000). Then intron retention seems to be a natural phenomena observed in several teleost species, specially for gonads. In mammals, in the brain of rats it was found that the first intron spliced is B, followed by intron C and A (Jakubowsky and Roberts, 1994). In vitro systems and using nuclear extract of GnRH neurons demonstrated that introns B and C are easily removed, being intron A the last one to be removed,. In that study, exonic splicing enhancer in exons 3 and 4 was found to splice intron A (Seong et al., 1999). The importance of splicing intron A was investigated by Son et al. (2003) their results showed that intron A is a splicing variant not a splicing intermediate, demonstrating that only intron A-retained transcripts can be transported to the cytoplasm, whereas RNAs that contain intron B or C are restricted to the nucleus. The splice variant of transcripts that retain intron A is untranslated and this is because intron A contains a tandem repeat of the ATG sequence within intron blocking the translation (Son et al., 2003). Splicing of intron A is a key regulatory step in GnRH prohormone biosynthesis (Son et al., 2005). In Chirostoma, the functional significance of intron B retention is not clear. The sequence is similar to that reported for intron B from Oryzias latipes (AB041335.1) and from Cynoscion (DQ179647.1). However, for these species, the intron sequence was taken from complete gene sequence and no intron retention of sGnRH is reported in either the brain or the gonads. In the three species of Chirostoma studied, the intron contains a stop codon, which could synthesize a shorter pre-propeptide, especially, in reference to GAP, which could regulate the translation and avoid the right folding propeptide to release the GnRH decapeptide, acting like a physiological mechanism of self-regulation. However, based on the primer design we cannot discard the presence of another RNA variant that contains intron A, C, or both besides intron B. Interestingly is to note that in other fish species, the isoform that depicts intron retention is always the one that plays the role of GnRH-1 (sGnRH, mGnRH, and sbGnRH). However, in Chirostoma, the GnRH-1 system is played by the peje GnRH (pjGnRH) isoform, and sGnRH is associated with GnRH-3. Another explanation is that total RNA extraction was from whole brain, and some neurons in other areas of the brain, outside the telencephalum and preoptic area, could have a different splicing rate (Son et al., 2005). Specific studies about the sGnRH distribution in whole brain of Chirostoma are necessary. On the other hand, RNA contamination with DNA seems unlikely because RNA extraction was performed at different times for each species, minimizing the possibility of having the same DNA fragment for each sample. 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Regulation and expression of gonadotropin-releasing hormone gene differs in brain and gonads in rainbow trout. Endocrinol. 140, 3012-3024. Keywords: Brain, Chirostoma, GnRH, Intron B, teleost Conference: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology, Ann Arbor, United States, 13 Jul - 16 Jul, 2011. Presentation Type: Poster Topic: Neuroendocrine control of reproduction Citation: Chavez M, Campos JE, Campos A, CARDENAS R and Martinez-Palacios CA (2011). Intron retention in a salmon gonadotropin releasing-hormone in three species of Chirostoma genus.. Front. Endocrinol. Conference Abstract: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology. doi: 10.3389/conf.fendo.2011.04.00115 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 02 Aug 2011; Published Online: 09 Aug 2011. * Correspondence: Dr. RODOLFO CARDENAS, UNIVERSIDAD NACIONAL AUTONOMA DE MEXICO, FES IZTACALA, UNIDAD DE MORFOLOGIA Y FUNCION, Tlalnepantla, Edo. de Mexico, 54090, Mexico, rodolf@unam.mx Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Monica Chavez Jorge E Campos Antonio Campos RODOLFO CARDENAS Carlos A Martinez-Palacios Google Monica Chavez Jorge E Campos Antonio Campos RODOLFO CARDENAS Carlos A Martinez-Palacios Google Scholar Monica Chavez Jorge E Campos Antonio Campos RODOLFO CARDENAS Carlos A Martinez-Palacios PubMed Monica Chavez Jorge E Campos Antonio Campos RODOLFO CARDENAS Carlos A Martinez-Palacios Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.