Mol Biol Rep (2014) 41:6481–6491 DOI 10.1007/s11033-014-3531-9

Alternative splicing and immune response of Crassostrea gigas tumor necrosis factor receptor-associated factor 3 Baoyu Huang • Linlin Zhang • Yishuai Du • Li Li • Tao Qu • Jie Meng • Guofan Zhang

Received: 7 April 2013 / Accepted: 19 June 2014 / Published online: 11 July 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Diverse alternative splicing isoforms play an important role in immune diversity and specificity. Their role in molluscan host-defense is however poorly understood. We characterized two alternative isoforms of tumor necrosis factor receptor-associated factor 3 (TRAF3) in the Pacific oyster, Crassostrea gigas, which were named CgTRAF3-S and CgTRAF3-L. An intron was retained in CgTRAF3-L, introducing a premature termination codon. Comparison and phylogenetic analysis revealed that CgTRAF3 shared a higher identity with other species, suggesting the conservation of the two gene transcripts. Quantitative real-time PCR was performed and the expression levels of CgTRAF3 isoforms were found to be significantly changed after Vibrio anguillarum and ostreid herpesvirus 1 challenges. These two isoforms represented contrary trends, indicating that CgTRAF3-L might function as a negative regulator of CgTRAF3-S. We also investigated the expression level of the transcripts of the two CgTRAF3 isoforms, following the silence of C. gigas mitochondrial anti-viral signaling protein like gene (CgMAVS-like). We concluded that CgTRAF3 might be involved in a MAVS-mediated immune signaling Electronic supplementary material The online version of this article (doi:10.1007/s11033-014-3531-9) contains supplementary material, which is available to authorized users. B. Huang  L. Zhang  Y. Du  L. Li (&)  T. Qu  J. Meng  G. Zhang (&) Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China e-mail: [email protected] G. Zhang e-mail: [email protected]; [email protected] B. Huang University of Chinese Academy of Sciences, Beijing 100049, China

pathway. This study suggests that CgTRAF3 may be a response to bacterial and viral stimulation and that the two isoforms may be involved in immune response pathways. It is also possible that the two alternative splicing isoforms could be inter-coordinated and may promote survival of these oysters under immune stress conditions. Keywords Crassostrea gigas  TRAF3  Alternative splicing  Immune response

Introduction The Pacific oyster, Crassostrea gigas, is a bivalve mollusca that has been introduced into many countries in the world. Crassostrea gigas is native to East Asia and distribute in the coastal waters of Japan, Korea and North China [1]. Oysters survive in the intertidal zone where the environment is extremely stressful, so these animals have evolved an advanced tolerance to such conditions, which makes the oyster a suitable model for the study of intertidal ecology and stress adaptation. The association of pathogens (such as Vibrio spp. and OsHV-1) with occasional high mortality levels among economically-important shellfish species means that there is a need to focus more attention on the study of the oyster immune system [2]. Previous studies have tried a lot on the immune response of commercially important species like abalone [3] and oyster in order to find the possible antiviral substances in invertebrates [4] or seek the explanation of the mortality [5, 6]. Nevertheless, there is relatively little known about the underlying genes that regulate the immune system in molluscs. As a result, the characterization of the genes that function in the immune system would benefit the understanding of the molecular mechanisms of the molluscan immunity. Nowadays the

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availability of the oyster genome sequence [7] would facilitate the study of the molecule research of the C. gigas. Animal immunity comprises adaptive immunity and innate immunity and it is widely accepted that only jawed vertebrates possess a well-developed adaptive immunity [8]. Invertebrates lacking in adaptive immunity must therefore evolve an elaborate innate immune system in order to survive possible pathogens challenges. Such a system will inevitably rely on several mechanisms of gene regulation to achieve the required diversity and flexibility of function [9]. Alternative splicing is one of the most important mechanisms for generating protein diversity and it can generate a large number of mRNA and protein isoforms from the relatively low number of gene. Alternative splicing can change the structure of transcripts and their encoded proteins thus it can modulate the function of proteins and the isoforms generated by alternative splicing may even have totally opposite function [10]. The innate immunity is well studied in the Deuterostomes, which are well represented by model species (sea urchin, human and so on). The immunity information is also deeply investigated in Drosophlia and Caenorhabditis, the representative Ecdysozoa. However, the immune systems of the Lophotrochozoa, which can be well represented by C. gigas, have not received much research attention. Indepth research and the acquisition of more information on the immune system of the Pacific oyster would therefore make a major contribution to our understanding of the evolution of the innate immune system. TRAF proteins are a family of crucial adaptor proteins that function downstream of members of the tumor necrosis factor receptors (TNFR) family [11], the toll-like receptor (TLR) family [12], the interleukin-1 receptor (IL-1R) family [13], and the RIG-I-like receptor (RLR) family [14]. To date, seven members of the TRAF family (TRAF1 through to TRAF7) have been identified in mammals and they share a common TRAF domain at the C terminal, with the exception of TRAF7. TRAF1 and TRAF2 were the first isolated and molecularly cloned TRAF molecules using biochemical purification and the yeast two-hybrid system, as they interact with the intracellular domain of TNFR2. The analysis also indicated that TRAF1 and TRAF2 were associated with the cytoplasmic domain of TNF-R2 in a heterodimeric complex in which TRAF2 contacted the receptor directly [15]. Among all known TRAFs, TRAF3 is a highly versatile regulator. Recent work has revealed that TRAF3 functions as a negative regulator of alternative nuclear factor-kB (NF-kB) signaling and, separately, as a positive regulator of type I interferon (IFN) production [16]. In Drosophila melanogaster, DmTRAF1 specifically activates the c-Jun N-Terminal Kinase (JNK) signaling pathway, whereas DmTRAF2 (in the TRAF domain which is most closely related to that of mammalian TRAF6), activates the NF-kB pathway and stimulates the antimicrobial immune

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functions [17]. Furthermore, in Caenorhabditis elegans, there are only weak homologs of TRAF and they may not function in nematode pathogen responses [18]. In molluscs, several other TRAFs—such as TRAF6 in Chlamys farreri [19], TRAF7 in Crassostrea hongkongensis [20] and TRAF3 in Pinctada fucata [21]—have been identified. The results of these studies have shed light on the diversity of TRAF function. Additional research on TRAF homologs in other invertebrates is however required in order to find out more about the details of TRAF function. In this paper, we report the identification and characterization of the full-length of a TRAF3 gene, termed CgTRAF3-S, in the Pacific oyster C. gigas. We also describe a novel isoform of CgTRAF3-S and named CgTRAF3-L. To better understand the function of TRAF3 in oysters, we investigated the expression profile of CgTRAF3 variant transcripts in multiple tissues, as well as following bacterial and viral challenge in hemocytes. To obtain further details on immune pathways of C. gigas, the expression patterns of CgTRAF3 transcripts were examined followed silence of CgMAVS-like.

Materials and methods Oysters, bacterial challenge and sample collection Adult oysters, C. gigas, with an average shell height of 60 mm, were collected from a farm of Qingdao, Shandong Province, China. Specimens for the experiment were acclimatized in aerated and filtered seawater at 15 °C for 10 days prior to the start of the experiment. Sixty oysters were employed for the Vibrio anguillarum (strain no. MVM425)challenge-experiment. These oysters were randomly divided into two groups of 30 individuals. Each of the oysters from one group then received a muscle injection of 100 lL phosphate buffered saline (PBS buffer, 0.14 mol L-1 sodium chloride, 3 mmol L-1 potassium chloride, 8 mmol L-1 disodium hydrogen phosphate dodecahydrate, 1.5 mmol L-1 potassium phosphate monobasic; pH 7.4). Each oyster from the other group received an injection, into the adductor muscle, of 100 lL live V. anguillarum at a concentration of 2 9 108 CFU mL-1, suspended in PBS. At 0, 6, 12, 24 and 48 h after the injection, three individuals from each group were randomly sampled and the hemolymph was collected and immediately centrifuged at 1,0009g for 10 min at 4 °C, to harvest the hemocytes for RNA preparation. Other tissue was also dissected from three oysters in each group. Total RNA extraction and cDNA synthesis Total RNA was isolated from the hemocytes of oysters using TRIzol Reagent (Invitrogen, USA), after which RQ1

Mol Biol Rep (2014) 41:6481–6491 Table 1 Sequences of primers used in this study

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Primer

Sequence

Application

CgTRAF3-F1

GGGACCTTCTAGCGGGGAATC

Cloning primer

CgTRAF3-R1

CCACGGCAACAGGGCATCA

Cloning primer

dTAP

GGCCACGCGTCGACTAGTACT16

30 RACE adaptor primer

dGAP

GGCCACGCGTCGACTAGTACG10

50 RACE adaptor primer

AP

GGCCACGCGTCGACTAGTAC

Adaptor primer

5CgTRAF3-1

CGCTCCTCCCCTGCTTCTC

50 RACE PCR

5CgTRAF3-2

CCCCGCTAGAAGGTCCCTGA

50 RACE PCR

5CgTRAF3-3

ACGGCCTCACGGAGAACCCC

50 RACE PCR

3CgTRAF3-1

CGGGATAACCACGCCTACCTG

30 RACE PCR

3CgTRAF3-2

AGAGAAGCAGGGGAGGAGCGG

30 RACE PCR

CgTRAF3-np-F1

GAGAAGCAGGGGAGGAGC

Cloning primer

CgTRAF3-np-R1

CCACGGCAACAGGGCATCA

Cloning primer

CgTRAF3-np-F2

CAGGGGAGGAGCGTGATG

Cloning primer

CgTRAF3-np-R2

GCGTCTTGCTTTCTTTGGGTGTA

Cloning primer

CgTRAF3-S-qRT-F1 CgTRAF3-S-qRT-R1

AGGGCGGAGCTTGTCGGCCTCG GCCTTTGCGTCTTGCTTTCTTTGGG

qRT-PCR qRT-PCR

CgTRAF3-L-qRT-F1

ACTTTCGTATCATCTTCTGATGTCT

qRT-PCR

CgTRAF3-L-qRT-R1

TCCGCAAATCTAGCATCCA

qRT-PCR

EF-qRT-F

AGTCACCAAGGCTGCACAGAAAG

qRT-PCR

EF-qRT-R

TCCGACGTATTTCTTTGCGATGT

qRT-PCR

Oligo(dT)-adaptor

GGCCACGCGTCGACTAGTACT16VN

cDNA synthesis

RNase-Free Dnase was used to eliminate the genomic DNA. After purification, total RNA was reverse transcripted with the assistance of Promega M-MLV RT Usage information, using adaptor primer oligo(dT)-adaptor (Table 1) as a primer. The reaction mixtures were performed at 42 °C for 1 h, and terminated by heating at 95 °C for 5 min. Cloning the full-length of CgTRAF3 cDNA Taking advantage of C. gigas genome [7], CgTRAF3-F1 and CgTRAF3-R1 were designed for the PCR amplification of the CgTRAF3 fragment. Once the fragment was obtained, gene specific primers for 30 and 50 ends, rapid amplification of cDNA ends (RACE) were designed. The 30 end of CgTRAF3 was cloned, using the primer 3CgTRAF3-1 and anchor primer AP. The PCR was conducted using the following program: 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min followed by an extension at 72 °C for 10 min. The 50 end of CgTRAF3 was obtained using primers 5CgTRAF3-1, 5CgTRAF3-2, 5CgTRAF3-3, and adaptor primer dGAP. In the 50 RACE cloning, two rounds of nested PCR were performed with the touchdown program: 10 cycles at 94 °C for 30 s, 60 °C (-1.3 °C per cycle) for 40 s, and 72 °C for 2 min, then 25 cycles at 94 °C for 30 s, 47 °C for 40 s, and 72 °C for 2 min followed by an extension at 72 °C for 10 min. For the first round, the cDNA tailed with dCTP at the 50 end by

terminal transferase TdT. According to the Invitrogen manual, this was used as the PCR template, with 5CgTRAF3-1 and dGTP as the forward and reverse primers. Then 5CgTRAF3-2/AP, or 5CgTRAF3-3/AP, was employed as the second-round PCR primer. One thousand bp of 50 RACE PCR product and 1,200 bp of 30 RACE PCR product were purifed using the E.Z.N.A Gel Extraction Kit (OMEGA, USA). Then the purified PCR product was cloned into pMD18-T vector (TaKaRa, Japan). The recombinant vector was transformed into Trans1-T1 competent cell (Transgen, China) and sequenced by ABI 3130 sequencer. Sequence analysis Software package DNAman (Version 5.2.2) was used to analyze cDNA and deduce amino acid. The sequences of TRAF3 from different species were compared using the BLAST program at the National Center for Biotechnology Information (NCBI). SMART (Simple modular architecture research tool (http://www.smart.emblheidelberg.de)) was utilized to predict domains of the deduced amino acid sequences of CgTRAF3. The sequences of TRAF3 from various species were downloaded from the NCBI database and compared, using the NCBI BLAST search program. A multiple sequence alignment was performed using ClustalW (http://www.ebi.ac.uk/clustalw/) and a phylogenetic tree of TRAF3 was constructed by the MEGA program

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(Version 5.05), using the neighbor-joining algorithm. Reliability of the branching was tested using bootstrap resampling (1,000 pseudo-replicates) (http://www.mega software.net). Quantitative real-time PCR analysis of TRAF3 mRNA expression Quantitative real-time RT-PCR was performed with the SYBR Green 29 Master mix (Takara, Japan), using an ABI 7500 fast Real-Time Thermal Cycler, according to the manual’s instructions (Applied Biosystems, USA), to investigate the expression of CgTRAF3-S and CgTRAF3L. Primers of this assay are shown in Table 1. As mentioned above, total RNA was extracted using TRIzol Reagent (Invitrogen, USA). Reverse transcription was performed with the Oligo(dT) primer. For CgTRAF3, CgTRAF3-qRT-F1 and CgTRAF3-qRT-R1 (Table 1) the forward and reverse primers and the elongation factor (EF) (GenBank accession number AB122066) were used as the internal control with the forward and reverse primers EFqRT-F and EF-qRT-R, respectively [22, 23]. The PCR parameters were 94 °C for 2 min, then 40 cycles of 94 °C for 15 s, 52 °C for 15 s and 72 °C for 30 s. SYBR green I (TaKaRa, Japan) was used as a fluorescence dye and the dissociation curve analysis was performed to confirm PCR amplification specificity at the end of each PCR reaction. The relative mRNA expression level of CgTRAF3 variant transcripts was calculated using the comparative Ct method (2-DDCt method) [24]. The statistical data in Fig. 3 comparing expression patterns in different tissues was tested using one factor ANOVA (analysis of variance). The data in Figs. 4, 5 and 6 was tested using a 2 factor ANOVA with post hoc tests to examine the differences between the infected and PBS control groups over time. If P \ 0.05, the differences were considered statistically significant. The statistical test was performed using SPSS 16.0 software. OsHV-1challenge of oyster Naturally infected oysters collected on the field where the mortality happened during the summer 2012 were used for the suspension preparation. A small piece of mantle (about 20 mg) was sampled from each oyster for DNA extraction and then qRT-PCR was employed for the OsHV-1 detection [25]. Oysters with positive virus detection were used for the preparation of tissue homogenates. Briefly, the total tissues of these animals were cut up, pooled together in a 50 mL sterile tube and weighted. Then ten volumes of 0.22 lm filtered artificial seawater were added in the tube (9 mL of seawater per g of tissues). After that, tissues were crushed on ice and centrifuged (1,0009g, 5 min, 4 °C). Supernatant was shifted to a new tube and diluted by addition of four volumes of

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filtered artificial seawater. Finally, the tissue homogenate was filtered consecutively in sterile conditions using syringe filters at 5, 2, 0.45 and 0.22 lm pore sizes [26]. 200 lL of filtered tissue supernatant was taken out for DNA extraction and quantification of OsHV-1 DNA. The left was stored at -80 °C until use. Two hundred apparently-healthy oysters were employed in this assay. These animals were randomly divided into two groups, each with one hundred oysters. Oysters from one group each received a muscle injection of 100 lL PBS. Those from the other group received injections, into the adductor muscle, of 100 lL filtered tissue supernatant (1.8 9 105 viral DNA copies/lL), using a 1 mL micro-syringe. At 0, 3, 6, 12, 24, 48, 60, 72, 96 and 120 h after injection, three individuals were randomly sampled from each group and the hemolymph was collected and immediately centrifuged at 1,0009g for 10 min at 4 °C in order to harvest the hemocytes for RNA preparation. The total RNA was then extracted, the template for the qRT-PCR was prepared, and qRT-PCR was performed to analyze the mRNA expression level of CgTRAF3 isoforms after the challenge of OsHV-1. Knockdown of CgMAVS-like gene in vivo via dsRNAmediated RNA interference A CgMAVS-like cDNA fragment from cDNA of C. gigas was amplified using primers incorporated with T7 promoter after which the PCR products were used as templates to synthesize dsRNA. According to the method described previously [27], the dsRNAs were generated by means of in vitro transcription. The integrity and the concentration of dsRNAs were tested by electrophoresis and absorbance at 260 nm, after which the concentration of dsRNAs was adjusted to a final concentration of 1 mg mL-1. One hundred oysters were then randomly divided into two groups of 50 individuals. In the first group, each oyster received an injection of 100 lg dsRNA, while oysters in the other group were injected with 100 lg PBS. At 0, 1, 2, 3, 4, 5, 6 and 7 days after injection, three individuals from each group were randomly chosen for collection of hemolymph. These samples were immediately centrifuged at 1,0009g for 10 min at 4 °C to harvest the hemocytes from which RNA was extracted. The template for the qRTPCR was prepared and qRT-PCR was performed to determine the mRNA expression level of CgTRAF3 isoforms, following the knockdown of CgMAVS-like.

Results cDNA and the deduced amino acid of CgTRAF3-S A full-length cDNA of 2,218 bp was isolated from a Pacific oyster cDNA library and designated CgTRAF3-S

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Fig. 1 a Schematic representation of CgTRAF3 gene structure. Exons and introns are represented by boxes and lines. b Multiple sequence alignment at alternative splicing site. RING really

interesting new gene; zf-TRAF TRAF domains with zinc finger domains; MATH meprin and TRAF homology domain

with an open reading frame (ORF) of 1,686 bp encoding a putative protein of 561 amino acids. The complete sequence of CgTRAF3-S cDNA consists of a 50 terminal untranslated region (UTR) of 34 bp, and 30 UTR of 498 bp (Supplementary File 1). The deduced CgTRAF3-S contains a RING finger domain, two TRAF domains with zinc finger domains, and a conserved C-terminal meprin and TRAF homology (MATH) domain, similar to those of other members of TRAF family (Fig. 1a).

amino acids that were encoded by nucleotides around the splicing sites were variable in both Homo sapiens and P. fucata (Fig. 1b).

Identification of a novel CgTRAF3 cDNA isoform A novel isoform of CgTRAF3-S was obtained and designated as CgTRAF3-L (the full-length cDNA of CgTRAF3L is 3,177 bp (Supplementary File 1)). Alignment of CgTRAF3 cDNA with the genomic sequence revealed that CgTRAF3 consisted of nine exons and eight introns spanning over 11 kb. CgTRAF3-L was found to be 959 bp longer than CgTRAF3-S, and retained the eighth intron of CgTRAF3-S. As the insertion contains a new translation stop site, the ORF of CgTRAF3-L is only 1,155 bp, encoding a 384 amino acid polypeptide. As a result, CgTRAF3-L is lacking the MATH domain (Supplementary File 2). Multiple sequences alignment indicated that the

Multiple sequences alignment and phylogenetic analysis A comparison of the deduced amino acid sequences of Pacific oyster TRAF3 to the corresponding sequences of other species is shown in Supplementary File 2. The putative protein sequence of CgTRAF3-S shared 45 % identity to that of PfTRAF-3, 41 % identity to BbTRAF-3, 37 % identity to XtTRAF-3, and 39 % identity to GgTRAF-3 and HsTRAF-3. The identity of the TRAF domain between oyster and other species is about 75 %, suggesting the conserved TRAF domains in different species. A phylogenetic tree was constructed using a neighborjoining method with 1,000 bootstrap test, based on the multiple alignments of CgTRAF3-S, CgTRAF3-L and TRAF3 from other species (Fig. 2). In the phylogenetic trees, CgTRAF3-S and CgTRAF3-L, were firstly clustered together, and then converged into a clade with PfTRAF-3. It was also noted that TRAF3s of invertebrates formed a cluster which is divergent from that of chordate TRAF3s.

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Fig. 2 Neighbor-joining phylogenetic tree of TRAF3s. The phylogenetic tree was constructed using MEGA software 5.05 with 1,000 replications of bootstrap. The scale bar indicated a branch length of 0.2. CgTRAF3s were marked with triangle. HsTRAF-3: Homo sapiens TRAF-3; MmTRAF-3: Mus musculus TRAF-3; BtTRAF-3: Bos taurus TRAF-3; GgTRAF-3: Gallus gallus TRAF-3; XtTRAF-3: Xenopus tropicali TRAF-3; DrTRAF-3: Danio rerio TRAF-3;

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CcTRAF-3: Cyprinus carpio TRAF-3; BbTRAF-3: Branchiostoma belcheri TRAF-3; CiTRAF-3: Ciona intestinalis TRAF-3; CgTRAF3: Crassostrea gigas TRAF3; PfTRAF-3: Pinctada fucata TRAF-3; SkTRAF-3: Saccoglossus kowalevskii TRAF-3; SjTRAF-3: Schistosoma japonicum TRAF-3; DyTRAF-3: Drosophila yakuba TRAF-3; BmTRAF-3: Bombyx mori TRAF-3

Fig. 3 Expression profile of CgTRAF3 isoforms in different tissues by qRT-PCR. GI gill; HM hemolymph; MA mantle; MU muscle; GO gonad. Elongation factor (EF) gene expression was used as internal control and gill was used as reference sample. Vertical bars represent the mean ± SD (N = 3)

CgTRAF3 gene expression in different tissues and response to V. anguillarum The qRT-PCR analysis was employed to study the tissuespecific expression of CgTRAF3-S and CgTRAF3-L. As shown in Fig. 3, the expression of CgTRAF3-S and CgTRAF3-L were found in all tested tissues of C. gigas and, similarly, both CgTRAF3-S and CgTRAF3-L were expressed at the highest level in the hemolymph. The

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expression level of CgTRAF3-S in the hemolymph was about 18-fold compared to that of the gill and mantle, whereas the expression level of CgTRAF3-L in the hemolymph was only about 2.8-fold compared to that of the gill and mantle. To examine the temporal expression profiles of the CgTRAF3 response to V. anguillarum, we performed a qRT-PCR of two oyster groups injected with PBS and V. anguillarum during the post challenge period (0, 6, 12, 24

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Fig. 4 CgTRAF3 isoforms mRNA expression in oyster hemolymph after V. anguillarum challenge. Elongation factor (EF) gene expression was used as internal control and time 0 h was used as reference samples. Vertical bars represent the mean ± SD (N = 3). Blue indicates injected with PBS (negative control) and red V. anguillarum. Results that are significantly different (P \ 0.05) between challenged and control at the same time point are showed with an asterisk. (Color figure online)

more than 14-fold compared to that of the original level, after which it declined (Fig. 4b). Expression pattern of CgTRAF3 following OsHV-1 stimulation

Fig. 5 CgTRAF3 isoforms mRNA expression in oyster hemolymph following ostreid herpesvirus 1 (OsHV-1) stimulation. Elongation factor (EF) gene expression was used as internal control and time 0 h was used as reference samples. Vertical bars represent the mean ± SD (N = 3). Blue indicates injected with PBS (negative control) and red OsHV-1. Results that are significantly different (P \ 0.05) between challenged and control at the same time point are showed with an asterisk. (Color figure online)

and 48 h). In the V. anguillarum group, it was noted that the expression level of CgTRAF3-S was gradually downregulated during the first 12 h of the post challenge period and the minimal expression level of CgTRAF3-S was less than half of that of the original level at 12 h post-challenge. It then gradually rose to the original level at 48 h postchallenge (Fig. 4a). Compared with the expression pattern of CgTRAF3-S after the challenge, CgTRAF3-L had nearly opposite expression style. The expression level of CgTRAF3-L was up-regulated until 24 h post-challenge and, at 24 h after injection, the CgTRAF3-L expressed

As shown in Fig. 5a, the expression of CgTRAF3-S fluctuated up and down as time passed. CgTRAF3-S was significantly up-regulated soon after injection, and at 6 h postinjection the expression level reached a level of about eight-fold that of the PBS control. During the following 6 h however, the expression level declined and then rose sharply, reaching a peak at 24 h. Thereafter, the expression level of CgTRAF3-S declined. The mRNA quantity of CgTRAF3-S was up-regulated again at 96 h post-injection. The expression pattern of CgTRAF3-L, shown in Fig. 5b, also fluctuated but in a different pattern to that of CgTRAF3-S. It seemed that the expression of CgTRAF3-L was generally up-regulated when the expression of CgTRAF3-S was down-regulated, with some exceptions at certain time points. The expression of CgTRAF3 isoforms during the silencing of CgMAVS-like To further understand the function of CgTRAF3 in the immune pathways, RNAi was performed to inhibit the expression of CgMAVS-like, which was considered an upstream molecular of TRAF3 in the RLR pathway [28]. As shown in Fig. 6a, during the gene silence of CgMAVSlike, the expression level of CgTRAF3-S was changing with the passage of time. In the first 2 days, the expression of CgTRAF3-S was slightly up-regulated while the expression of CgTRAF3-L was sharply decreased in the second day. But in the third day of RNAi, the mRNA expression level CgTRAF3-S decreased sharply, to below

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Fig. 6 CgTRAF3 isoforms mRNA expression in oyster hemolymph after CgMAVS-like was silenced. Elongation factor (EF) gene expression was used as internal control and time 0 h was used as reference samples. Vertical bars represent the mean ± SD (N = 3). Blue indicates injected with PBS (negative control) and red dsRNA. Results that are significantly different (P \ 0.05) between challenged and control at the same time point are showed with an asterisk. (Color figure online)

0.1-fold of the PBS control. In the remaining days, mRNA of CgTRAF3-S was up-regulated gradually and finally reached a peak on the seventh day. Compared to the CgTRAF3-S, CgTRAF3-L had an unequal expression pattern (Fig. 6b). The expression level of CgTRAF3-L was down-regulated as time progressed and on the fourth day it reached the minimum, about 0.1-fold of the PBS control, after which it increased gradually and reached the peak.

Discussion In the present study, we cloned the full-length of a new TRAF3 gene and its alterative isoform from Pacific oyster C. gigas for the first time, which were named CgTRAF3-S and CgTRAF3-L, respectively. We also analyzed the expression patterns of the two CgTRAF3 isoforms in different tissues and under the challenge of V. anguillarum and OsHV-1. Our results revealed the important role of CgTRAF3 in the immune response of the oyster. Moreover, the expression profile of CgTRAF3 isoforms after the silence of CgMAVS-like suggested that CgTRAF3 might participate and play a vital role in the immune response. To our knowledge, this is the first reported study elaborating that the alternative splicing of TRAF3 may play a crucial role in immune regulation.

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TRAF proteins play vital roles in the signaling pathways activated by TNFR, TLR and RLR family members. Acting alone or in combination, they control many biological processes, including cytokine production and cell survival. Among TRAF proteins, TRAF3 is a versatile molecule. TRAF3 was first identified as a CD40-binding protein and has been extensively studied in mammals because of its importance in such immune pathways above. The information on TRAF3 in invertebrates is however still insufficient and much research on TRAF3 is needed, to broaden and deepen our understanding of this versatile molecule. In this paper, a TRAF3 homology gene, CgTRAF3, was isolated from the Pacific oyster C. gigas. As is the case for other members of the TRAF family, the deduced CgTRAF3 protein contains a RING finger domain, two TRAF domains with zinc finger domains, and a conserved C-terminal MATH domain. As shown in our results, oyster TRAF3 was highly conserved, from oyster to human, which may indicate that TRAF3 plays a critical rule in multiple signaling pathways. Based on the phylogenetic tree that we determined, CgTRAF3 has a close evolutionary relationship with TRAF3 from the pearl oyster. A functional immune system needs to have a high degree of diversity and the ability to function at the level of individual cells. Such a system should rely on several mechanisms of gene regulation, to achieve the required diversity and flexibility of function. Alternative splicing is one of the most important mechanisms for generating a large amount of mRNA and protein isoforms from a relatively low number of genes. For example, alternative splicing of the Dscam gene of Drosophila can generate more than 38,000 distinct mRNA isoforms [29], which is more than two times the number of predicted genes in the entire genome [30]. Much research attention has been recently focused on alternative splicing in mammals. Unfortunately, only a few genes from mollusks have been reported to be generated by alternative splicing. To some extent, these studies shed light on the functioning of these genes. The alternative splicing of TRAF3 has not however been previously reported in mollusks. In the present study, a novel CgTRAF3 isoform, CgTRAF3-L generated from alternative splicing, was characterized for the first time from C. gigas. The retention of the eighth intron resulted in the introduction of a premature termination codon. The stop codon was present in more than 50–55 nucleotides upstream of the 30 -most exon–exon junction. This means that—according to the general rule for specifying whether a transcript would be targeted by the nonsense mediated mRNA decay (NMD) pathway [31, 32] (a surveillance mechanism that selectively degrades nonsense mRNAs)— the premature stop codon may be recognized and the mRNA may be degraded.

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Unveiling the mRNA distribution pattern of CgTRAF3 isoforms in different tissues will be useful for determining the potential functions of such isoforms. As shown in Fig. 3, CgTRAF3-S and CgTRAF3-L are predominantly expressed in hemolymph, although they are ubiquitous in all of the organs of the oyster. Given the pivotal role of hemocytes in host defense, hemolymph seems an ideal candidate tissue for the study of environmental challenges [33]. So naturally in our research, the hemolymph was sampled for the analyzation of gene expression after vibrio and virus stimulation and even for the analyzation of RNAi effect. Then the abundance of CgTRAF3 in hemolymph suggests a crucial role of CgTRAF3 in C. gigas immune response taking the importance of hemocytes in the oyster defense into account. The effects of alternative splicing range from subtle effects that are difficult to detect, to a complete loss of function [10]. Some isoforms generated by alternative splicing may even have markedly different activities. For example, human Bcl-x, Bcl-x (L) is an antiapoptotic factor, whereas Bcl-x (S) can induce apoptosis [34]. For the purpose of finding out more detail about the function of isoforms of CgTRAF3, a typical pathogen in mollusks, V. anguillarum [35] was used to challenge the oysters and then the mRNA transcripts of CgTRAF3-S and CgTRAF3L were measured using real-time PCR. The results showed that, in response to V. anguillarum, CgTRAF3-S was significantly down-regulated at 6 h post-stimulation, reaching a minimum level at 12 h in hemolymph, and then gradually rising to a normal level. This consequence was similar to the TRAF3 expression pattern in P. fucata after V. alginolyticus stimulation [21]. These data may help us to know more about TRAF3 function in the NF-kB signaling pathway. The mRNA transcript of CgTRAF3-L was also measured and it had a totally different expression pattern after bacteria stimulation. CgTRAF3-L was first up-regulated and then recovered. Since NMD may take part in regulating the quantity of CgTRAF3-L, the expression pattern of CgTRAF3 isoforms suggests that the biological activity of CgTRAF3 may be dictated by the relative abundance of the CgTRAF3-L and the CgTRAF3-S transcripts. The abundance of CgTRAF3-L mRNA transcripts might therefore result in a relative decrease of CgTRAF3S, which may act as a negative regulator of the NF-kB signaling pathway. An increase in CgTRAF3-L thus contributes to the oyster defense against V. anguillarum. Recent mass mortalities of oysters during summer months have been reported in most countries, such as France [36], Japan [37], and USA [38], where Pacific oysters are produced. Some research has supported the view that OsHV-1 infections are closely associated with significant mortality levels in the Pacific oyster [39–42]. To understand the immune response of CgTRAF3 to the virus

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stimulation, a OsHV-1 challenge assay was conducted, based on standard methods [26]. The expression analysis of CgTRAF3 after OsHV-1 infection was performed by qRTPCR. The results showed that CgTRAF3-S was up-regulated after OsHV-1 stimulation and its expression reached peaks at 6, 24 and 96 h. This led to the conclusion that the increase of CgTRAF3-S at 6 h might be related to a stress response to the invasion of foreign tissue. Then the immune system might be activated, following a replication of the virus, reaching peak activity at 24 h, which might resulte in the elimination of most virus particles. The up-regulation of CgTRAF3-S at 96 h, may have been a response to the proliferation of the surviving virus, which would have led to restarting the immune pathway in which CgTRAF3-S participates. The expression profile of CgTRAF3-L was different to that of CgTRAF3-S. To a certain extent, it seemed that the expression pattern of CgTRAF3-L was opposite to that of CgTRAF3-S. Further study of this phenomenon may deepen our understanding of the role that CgTRAF3-L plays in the immune pathways. Compared with CgTRAF3-L, CgTRAF3-S has a complete MATH domain and it is more likely to directly participate in the immune response. Because of the lack of the MATH domain, it is possible that CgTRAF3-L is not involved in the immune pathways, but it may influence the immune system indirectly, by regulating the quantity of CgTRAF3-S. The retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) pathway is a well-known immune pathway, which is sensitive to replicating viruses in the cytoplasm, particularly during the early phases of viral infection [43]. This results in the activation of innate immune responses, which leads to the induction of type I and III interferon and inflammatory cytokines. RIG-I recognizes viral RNA through its helicase domain and associates with MAVS whose activation results in the recruitment of downstream signaling molecules, one of which is TRAF3 [44]. In the present study, we investigated the expression profile of CgTRAF3 after CgMAVS-like was silenced via RNAi. The results showed that, when the expression of CgMAVS was suppressed via RNAi, the mRNA expression level of CgTRAF3-S and CgTRAF3-L decreased. According to the view of Segal et al. [45], the key genes involved in the same pathway always exhibit a similar gene expression pattern. Thus, the results of our experiment may support the view that CgMAVS-like and CgTRAF3 function in the same pathway. Our results also indicate that CgTRAF3-L acts more swiftly than CgTRAF3-S, in response to stimulation. This phenomenon may indicate that CgTRAF3-S is the functional molecule. CgTRAF3-L acted first to upregulate or down-regulate the amount of CgTRAF3-S, which can participate in the immune response. More research is needed to ascertain whether the RIG-I pathway

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exists in C. gigas and to find out whether CgTRAF3 plays an important role in this immune pathway. In conclusion, we demonstrated the presence of the TRAF3 homolog CgTRAF3-S in C. gigas and also identified CgTRAF3-L as a novel alternative isoform. Our analysis revealed that CgTRAF3 shared a high identity with other species. We also investigated the expression profile of CgTRAF3 in different tissues and under the stresses of V. anguillarum and OsHV-1. The results suggested that the two CgTRAF3 isoforms both took part in the immune response of the oyster. Moreover, the expression pattern of CgTRAF3 isoforms, after the silence of CgMAVS-like, was also researched. The results from this research could provide basal resources for further investigations into the immune pathways of C. gigas. Acknowledgments We thank everyone of laboratory for technical assistance and good suggestions. This research was supported by National Basic Research Program of China (973 Program, No. 2010CB126401), the National Natural Science Foundation of China (No. 40730845), National High Technology Research and Development Program (863 program, No. 2012AA10A405), Shandong Provincial Natural Science Foundation (SDNSF, ZR2010DQ024 & ZR2013DQ010), Mollusc Research and Development Center, CARS, Taishan Scholars Climbing Program of Shandong and Oversea Taishan Scholar Program of Shandong.

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Alternative splicing and immune response of Crassostrea gigas tumor necrosis factor receptor-associated factor 3.

Diverse alternative splicing isoforms play an important role in immune diversity and specificity. Their role in molluscan host-defense is however poor...
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