Molecular cloning of AUF1 gene from large yellow croaker (Larimichthys crocea) and analysis of its TNF-α mRNA decay-promoting activity

Hyperinflammation, induced by the overproduction of inflammatory cytokines, is a key pathogenesis in infectious diseases of cultured fish, such as hemorrhagic septicemia. Therefore, the biosynthesis of inflammatory cytokines is precisely regulated by transcriptional and post-transcriptional mechanisms to ensure tight control of the production. The importance of AU-rich element-binding factor 1 (AUF1), a ubiquitously expressed mRNA binding protein also known as heterogeneous nuclear ribonucleoprotein D0 (hnRNPD0), in maintaining the balance of inflammatory response has been well demonstrated in mammals, which it decreases the production of inflammatory cytokines by promoting mRNA decay. However, the regulatory effect of AUF1 on the expression of inflammatory factor mRNA in teleost fish remains unknown. To investigate the regulation of fish AUF1 on the mRNA expression of inflammatory factors, the full-length cDNA of an AUF1 homologue, LcAUF1, was cloned by rapid amplification of cDNA ends (RACE) from large yellow croaker (Larimichthys crocea), and its tissue-specific expression pattern and temporal expression in the spleen and kidney following Vibrio parahaemolyticus infection were investigated by real-time quantitative polymerase chain reaction (RT-qPCR). Additionally, the regulatory effect of LcAUF1 on TNF-α mRNA expression was detected by overexpression of LcAUF1 in RAW264.7 cells using a tetracycline-regulated expression system. The full-length cDNA of LcAUF1 was 1 413 bp in length, containing a 266 bp 5′ untranslated region (5′UTR), a 294 bp 3′UTR, and a 954 bp open reading frame (ORF) encoding a polypeptide of 317 amino acids. Multiple alignment revealed that the amino acid sequences and typical functional domains of mammalian AUF1s are highly conserved in LcAUF1, and LcAUF1 clusters with its homologues from other teleost fishes in the phylogenetic tree. In healthy large yellow croaker, Lcauf1 transcripts were detected in all 9 tested tissues, with the highest level found in the muscle tissue. Upon V. parahaemolyticus challenge, the expression levels of Lcauf1 were significantly down-regulated at 2 h (0.51-fold, P<0.01) and 12 h (0.58-fold, P<0.001) post-infection in the kidney, and at 24 h (0.39-fold, P<0.001) in the spleen. In RAW264.7 cells, overexpression of LcAUF1 significantly decreased the expression level of TNF-α mRNA at 0.5 (0.84-fold, P<0.01), 3.0 (0.70-fold, P<0.001) and 6.0 h (0.77-fold, P<0.01) post LPS challenge. After inhibition of transcription by actinomycin D, overexpression of LcAUF1 in RAW264.7 cells significantly increased the degradation rate of TNF-α mRNA, but had no significant effect on the degradation rate of β-Actin mRNA. These results collectively indicated the structure and function of AUF1 are highly conserved in vertebrates, and LcAUF1 decreases the mRNA expression levels of inflammatory cytokine by promoting mRNA decay, suggesting that LcAUF1 may be an important regulator of inflammatory responses during infection. This study provides reference data for further exploring the role of LcAUF1 in the regulation of inflammatory cytokine expression and the application of LcAUF1 in the prevention and control of infectious diseases in the large yellow croaker industry.