Characterization of the genomic and transcriptional structure of chicken NRG4 gene.

Neuregulin 4 (NRG4) is an important adipocytokine, which plays crucial roles in maintaining energy balance, regulating glucose and lipid metabolism, and preventing non-alcoholic fatty liver disease in mammals. At present, the genomic organization, transcript and protein isoforms of human NRG4 gene have been fully explored. Previous studies in our laboratory have shown that the NRG4 gene is expressed in chicken adipose tissue, but the chicken NRG4 (cNRG4) genomic structure, transcript and protein isoforms are still unknown. To this end, in this study, the genomic and transcriptional structure of the cNRG4 gene were systematically investigated using rapid amplification of cDNA ends (RACE) and reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the coding region (CDS) of the cNRG4 gene was small, but it had a very complex transcriptional structure characterized by multiple transcription start sites, alternative splicing, intron retention, cryptic exons, and alternative polyadenylation, thus leading to production of four 5?UTR isoforms (cNRG4 A, cNRG4 B, cNRG4 C, and cNRG4 D) and six 3?UTR isoforms (cNRG4 a, cNRG4 b, cNRG4 c, cNRG4 d, cNRG4 e, and cNRG4 f) of the cNRG4 gene. The cNRG4 gene spanned 21,969 bp of genomic DNA (Chr.10:3,490,314~3,512,282) and consisted of 11 exons and 10 introns. Compared with the cNRG4 gene mRNA sequence (NM_001030544.4), two novel exons and one cryptic exon of the cNRG4 gene were identified in this study. Bioinformatics analysis, RT-PCR, cloning and sequencing analysis showed that the cNRG4 gene could encode three protein isoforms (cNRG4-1, cNRG4-2 and cNRG4-3). This study lays a foundation for further research on the function and regulation of the cNRG4 gene.神经调节蛋白4(neuregulin 4，NRG4)是一个重要的脂肪细胞因子，在维持哺乳动物能量平衡、调节糖脂代谢和预防非酒精性脂肪性肝病中起着非常重要的作用。目前，人NRG4基因的基因组结构、转录异构体和蛋白异构体等已有深入的研究。本实验室前期研究显示，鸡脂肪组织也表达NRG4，但是目前有关鸡NRG4(chicken NRG4，cNRG4)基因的基因组结构、转录异构体和蛋白异构体尚不清楚。为此，本研究采用RACE(rapid-amplification of cDNA ends)和RT-PCR(reverse transcription-PCR)等技术，系统开展了cNRG4基因的基因组和转录本的结构分析。结果发现，cNRG4基因编码区很小，但该基因却非常复杂，存在选择性转录起始位点、选择性拼接、内含子滞留、隐匿外显子和选择性多聚腺苷酸化，这导致cNRG4基因产生4种不同的5?UTR异构体(cNRG4 A、cNRG4 B、cNRG4 C和cNRG4 D)和6种不同的3?UTR异构体(cNRG4 a、cNRG4 b、cNRG4 c、cNRG4 d、cNRG4 e和cNRG4 f)。基因组结构分析发现，cNRG4基因跨越基因组21,969 bp (Chr.10: 3,490,314~3,512,282)，由11个外显子和10个内含子构成。与NCBI数据库中的cNRG4基因mRNA序列(NM_001030544.4)相比，本研究发现了cNRG4基因的2个新外显子和1个隐匿外显子。生物信息学分析、RT-PCR、克隆和测序分析发现，cNRG4基因能编码3种蛋白异构体(cNRG4-1、cNRG4-2和 cNRG4-3)。本研究为进一步开展cNRG4基因的功能和调控研究奠定了基础。.