Polyadenylation, a critical step in the production of mature mRNA for translation in most eukaryotes, involves cleavage and poly(A) tail addition at the 3′ end of mRNAs at the polyadenylation site (PAS). Sometimes, one gene can have more than one PAS, which can produce the alternative polyadenylation (APA) phenomenon and affect the stability, localization and translation of the mRNA. In this study, we discovered 28,363 PASs using pig RNAseq data, with 13,033 located in 7,403 genes. Among the genes, 41% were identified to have more than one PAS. PAS distribution analysis indicated that the PAS position was highly variable in genes. Additionally, the analysis of RNAseq data from the liver and testis showed a difference in their PAS number and usage. RT-PCR and qRT-PCR were performed to confirm our findings by detecting the expression of 3′UTR isoforms for five candidate genes. The analysis of RNAseq data under a different androstenone level and salmonella inoculation indicated that the functional usage of PAS might participate in the immune response and may be related to the androstenone level in pigs. This study provides new insights into pig PAS and facilitates further functional research of PAS.
Alternative splicing (AS) plays a significant role in regulating gene expression at the transcriptional level in eukaryotes. Flexibility and diversity of transcriptome and proteome can be significantly increased through alternative splicing of genes. In the present study, transcriptome data of peripheral immune organs including spleen and inguinal lymph nodes (ILN) were used to identify AS difference between PRRSV-resistant Tongcheng (TC) pigs and PRRSV-susceptible Large White (LW) pigs artificially infected with porcine reproductive and respiratory syndrome virus (PRRSV) in vivo. The results showed that PRRSV infection induced global alternative splicing events (ASEs) with different modes. Among them, 373 genes and 595 genes in the spleen and ILN of TC pigs, while 458 genes and 560 genes in the spleen and ILN of LW pigs had significantly differential ASEs. Alternative splicing was subject to tissue-specific and lineage-specific regulation in response to PRRSV infection. Enriched GO terms and pathways showed that genes with differential ASEs played important roles in transcriptional regulation, immune response, metabolism, and apoptosis. Furthermore, a splicing factor associated with apoptosis, SRSF4, was significantly upregulated in LW pigs. Functional analysis on apoptosis associated genes was validated by RT-PCR and DNA sequencing. These findings revealed different response to PRRSV between PRRSV-resistant TC pigs and PRRSV-susceptible LW pigs at the level of alternative splicing, suggesting the potential relationship between AS and disease resistance to PRRSV.
Sex determination and differentiation in ectotherms are very complicated affairs and usually affected by both genetic and environmental factors. Because of their temperature-sensitive expression, heat shock proteins (HSPs) are good candidates for temperature-dependent sex determination (TSD). Similar to most thermosensitive fish species, the male to female ratio increases with temperature in yellow catfish (Pelteobagrus fulvidraco). Yellow catfish is also a type of sexual size dimorphic fish, and the male individuals grow much faster than females of the same age. Therefore, research of sex differentiation in yellow catfish is important in aquiculture. In this attempt, a total of seven HSPs and related genes were identified from transcriptomes of yellow catfish by 454 pyrosequencing and Solexa sequencing that we did previously, including five genes with complete open reading frame (ORF). Phylogenetically, all these genes were compared with their counterparts from other vertebrates. All these genes were sex-biased expressed in gonads. Hspa5, Hip, and Cdc37 were expressed more highly in ovary than in testis, whereas Hsp90α, Hspb2, Hspb8, and Hspbp1 were expressed more highly in testis than in ovary. Additionally, the expression of these genes was assessed after 17α-methyltestosterone (MT) and 17α-ethinylestradiol (EE2) treatment, respectively. Our result showed that working as co-chaperones, these HSPs and related genes may regulate sex steroid receptor activities to influence gonad development in yellow catfish. Our work would help in the understanding of the mechanism of sexual differentiation in teleosts.
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