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Transcriptome analysis provides insights into the regulatory function of alternative splicing in antiviral immunity in grass carp (Ctenopharyngodon idella).

Wan Q, Su J - Sci Rep (2015)

Bottom Line: Characterization of the transcriptomic response to infection is an effective approach to understanding the immune mechanisms.Furthermore, the splicing transcripts of IL-12p40 and IL-1R1 are firstly found to play diverse roles in the antiviral response of fishes.This study provides a complete transcriptome dataset of C. idella, which is valuable for the studies of immune complexity and, moreover, throws light on the regulatory role of AS in antiviral immunity.

View Article: PubMed Central - PubMed

Affiliation: 1] College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China [2] Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070, China.

ABSTRACT
Characterization of the transcriptomic response to infection is an effective approach to understanding the immune mechanisms. Herein we challenged grass carp (Ctenopharyngodon idella) with grass carp reovirus (GCRV) and sequenced four cDNA libraries obtained from head-kidney and spleen by using Illumina Miseq. As a result, we gained a total of 21.52 Gb clean data with 107.96 million reads, and de novo assembled 55,199 unigenes with an average length of 1,470 bp. Comparative transcriptome analysis reveals that 217 unigenes are differentially expressed (fold-change of at least 4) between resistant and susceptible fish in both head-kidney and spleen, and of which 36 unigenes were validated by RT-qPCR experiment. The expression profile of immune-related genes demonstrates that the immune response of spleen is more intense than that of head-kidney. Remarkably, 11,811 unigenes contain multiple transcripts, of which 322 unigenes possess notably differentially expressed transcripts between the four transcriptomic datasets. Furthermore, the splicing transcripts of IL-12p40 and IL-1R1 are firstly found to play diverse roles in the antiviral response of fishes. This study provides a complete transcriptome dataset of C. idella, which is valuable for the studies of immune complexity and, moreover, throws light on the regulatory role of AS in antiviral immunity.

No MeSH data available.


Related in: MedlinePlus

Splicing transcripts of IL-1R1 gene and their expressions in the head-kidney and spleen of C. idella.(a) Illustration of gene structures of the splice variants. Primers for RT-qPCR validation of the gene expression are showed on the corresponding structures. Primer ILF1666, ILF1703 and ILR1707 were designed at the corresponding junctions; (b) The FPKM values of the 12 splice transcripts in RNA-seq dataset, and the schematic diagram of suppositional proteins ‘encoded’ by V1, V2, V3, V4 and V5 are displayed. The red parts are signal peptides, orderly followed by three Ig domains, TM domains and TIR domains. Dotted boxes are the unconcerned portion of corresponding proteins; (c) Fold changes of the splice variants in KR (comparing to KS) and SR (comparing to SS), tested by RT-qPCR. (d) Relative expression of V1, V2 and V3 in different samples. *t-test P value < 0.05; **t-test P value < 0.01; ***t-test P < 0.001; ‘NS’, not significant, t-test P > 0.05.
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f6: Splicing transcripts of IL-1R1 gene and their expressions in the head-kidney and spleen of C. idella.(a) Illustration of gene structures of the splice variants. Primers for RT-qPCR validation of the gene expression are showed on the corresponding structures. Primer ILF1666, ILF1703 and ILR1707 were designed at the corresponding junctions; (b) The FPKM values of the 12 splice transcripts in RNA-seq dataset, and the schematic diagram of suppositional proteins ‘encoded’ by V1, V2, V3, V4 and V5 are displayed. The red parts are signal peptides, orderly followed by three Ig domains, TM domains and TIR domains. Dotted boxes are the unconcerned portion of corresponding proteins; (c) Fold changes of the splice variants in KR (comparing to KS) and SR (comparing to SS), tested by RT-qPCR. (d) Relative expression of V1, V2 and V3 in different samples. *t-test P value < 0.05; **t-test P value < 0.01; ***t-test P < 0.001; ‘NS’, not significant, t-test P > 0.05.

Mentions: In order to validate the AS events, certain representative genes were selected from 322 DETs mentioned above. IL-12p40 gene and IL-1R1 gene were picked out due to their coordinating function in innate and adaptive immunity2829 and also, ILLRs were selected for their differential expression in the myeloid and lymphoid lineages of D. rerio30. In case of the sequencing and assembling errors, those assembly sequences should be validated by Sanger sequencing. Hence, 3′-, 5′-terminal and genomic DNA regions of these representative genes were cloned and sequenced. The comparison between the genomic DNA regions and the unisequences of these genes revealed that the IL-12p40 gene possessed 3 splicing transcripts (Fig. 5a), that the IL-1R1 gene contained 12 splicing transcripts (Fig. 6a), and that the ILLR1, ILLR2, ILLR3, ILLR4 respectively possessed 1, 8, 6, and 2 splicing transcripts (Fig. 7a). IL-12p40a encodes one kind of novel p40 (375 aa, IL-12p40α), while IL-12p40b and IL-12p40c encode another one (330 aa, IL-12p40β). Furthermore, the protein structures of IL-12p40α and IL-12p40β were predicted by using SCRATCH protein predictor (free trial version, http://scratch.proteomics.ics.uci.edu/index.html) (Fig. 8). As for these representative genes, approximately 30.8% of AS events were alternative donor, 7.7% were alternative acceptor; and 12.8% were exon skipping, while 48.7% were intron retention. According to the AS validation results, it can be deduced that over 20% of C. idella genes are alternatively spliced during GCRV infection, which is approximate to the AS event ratio in zebrafish (D. rerio) (17.0%), but lower than the AS event ratios in medaka (Oryzias latipes) (31.2%), stickleback fish (Gasterosteus aculeatus) (32.4%), Japanese puffer fish (Takifugu rubripes) (43.2%), and human (Homo sapiens) (94%)731.


Transcriptome analysis provides insights into the regulatory function of alternative splicing in antiviral immunity in grass carp (Ctenopharyngodon idella).

Wan Q, Su J - Sci Rep (2015)

Splicing transcripts of IL-1R1 gene and their expressions in the head-kidney and spleen of C. idella.(a) Illustration of gene structures of the splice variants. Primers for RT-qPCR validation of the gene expression are showed on the corresponding structures. Primer ILF1666, ILF1703 and ILR1707 were designed at the corresponding junctions; (b) The FPKM values of the 12 splice transcripts in RNA-seq dataset, and the schematic diagram of suppositional proteins ‘encoded’ by V1, V2, V3, V4 and V5 are displayed. The red parts are signal peptides, orderly followed by three Ig domains, TM domains and TIR domains. Dotted boxes are the unconcerned portion of corresponding proteins; (c) Fold changes of the splice variants in KR (comparing to KS) and SR (comparing to SS), tested by RT-qPCR. (d) Relative expression of V1, V2 and V3 in different samples. *t-test P value < 0.05; **t-test P value < 0.01; ***t-test P < 0.001; ‘NS’, not significant, t-test P > 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4528194&req=5

f6: Splicing transcripts of IL-1R1 gene and their expressions in the head-kidney and spleen of C. idella.(a) Illustration of gene structures of the splice variants. Primers for RT-qPCR validation of the gene expression are showed on the corresponding structures. Primer ILF1666, ILF1703 and ILR1707 were designed at the corresponding junctions; (b) The FPKM values of the 12 splice transcripts in RNA-seq dataset, and the schematic diagram of suppositional proteins ‘encoded’ by V1, V2, V3, V4 and V5 are displayed. The red parts are signal peptides, orderly followed by three Ig domains, TM domains and TIR domains. Dotted boxes are the unconcerned portion of corresponding proteins; (c) Fold changes of the splice variants in KR (comparing to KS) and SR (comparing to SS), tested by RT-qPCR. (d) Relative expression of V1, V2 and V3 in different samples. *t-test P value < 0.05; **t-test P value < 0.01; ***t-test P < 0.001; ‘NS’, not significant, t-test P > 0.05.
Mentions: In order to validate the AS events, certain representative genes were selected from 322 DETs mentioned above. IL-12p40 gene and IL-1R1 gene were picked out due to their coordinating function in innate and adaptive immunity2829 and also, ILLRs were selected for their differential expression in the myeloid and lymphoid lineages of D. rerio30. In case of the sequencing and assembling errors, those assembly sequences should be validated by Sanger sequencing. Hence, 3′-, 5′-terminal and genomic DNA regions of these representative genes were cloned and sequenced. The comparison between the genomic DNA regions and the unisequences of these genes revealed that the IL-12p40 gene possessed 3 splicing transcripts (Fig. 5a), that the IL-1R1 gene contained 12 splicing transcripts (Fig. 6a), and that the ILLR1, ILLR2, ILLR3, ILLR4 respectively possessed 1, 8, 6, and 2 splicing transcripts (Fig. 7a). IL-12p40a encodes one kind of novel p40 (375 aa, IL-12p40α), while IL-12p40b and IL-12p40c encode another one (330 aa, IL-12p40β). Furthermore, the protein structures of IL-12p40α and IL-12p40β were predicted by using SCRATCH protein predictor (free trial version, http://scratch.proteomics.ics.uci.edu/index.html) (Fig. 8). As for these representative genes, approximately 30.8% of AS events were alternative donor, 7.7% were alternative acceptor; and 12.8% were exon skipping, while 48.7% were intron retention. According to the AS validation results, it can be deduced that over 20% of C. idella genes are alternatively spliced during GCRV infection, which is approximate to the AS event ratio in zebrafish (D. rerio) (17.0%), but lower than the AS event ratios in medaka (Oryzias latipes) (31.2%), stickleback fish (Gasterosteus aculeatus) (32.4%), Japanese puffer fish (Takifugu rubripes) (43.2%), and human (Homo sapiens) (94%)731.

Bottom Line: Characterization of the transcriptomic response to infection is an effective approach to understanding the immune mechanisms.Furthermore, the splicing transcripts of IL-12p40 and IL-1R1 are firstly found to play diverse roles in the antiviral response of fishes.This study provides a complete transcriptome dataset of C. idella, which is valuable for the studies of immune complexity and, moreover, throws light on the regulatory role of AS in antiviral immunity.

View Article: PubMed Central - PubMed

Affiliation: 1] College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China [2] Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070, China.

ABSTRACT
Characterization of the transcriptomic response to infection is an effective approach to understanding the immune mechanisms. Herein we challenged grass carp (Ctenopharyngodon idella) with grass carp reovirus (GCRV) and sequenced four cDNA libraries obtained from head-kidney and spleen by using Illumina Miseq. As a result, we gained a total of 21.52 Gb clean data with 107.96 million reads, and de novo assembled 55,199 unigenes with an average length of 1,470 bp. Comparative transcriptome analysis reveals that 217 unigenes are differentially expressed (fold-change of at least 4) between resistant and susceptible fish in both head-kidney and spleen, and of which 36 unigenes were validated by RT-qPCR experiment. The expression profile of immune-related genes demonstrates that the immune response of spleen is more intense than that of head-kidney. Remarkably, 11,811 unigenes contain multiple transcripts, of which 322 unigenes possess notably differentially expressed transcripts between the four transcriptomic datasets. Furthermore, the splicing transcripts of IL-12p40 and IL-1R1 are firstly found to play diverse roles in the antiviral response of fishes. This study provides a complete transcriptome dataset of C. idella, which is valuable for the studies of immune complexity and, moreover, throws light on the regulatory role of AS in antiviral immunity.

No MeSH data available.


Related in: MedlinePlus