| 摘要: |
| Tristetraprolin (TTP)是广泛存在于真核生物的RNA结合蛋白。TTP通过促进mRNA降解或抑制翻译在转录后水平抑制炎症因子表达, 是炎症性疾病的潜在治疗靶点。采用RACE技术获取了大黄鱼(Larimichthys crocea) TTP (命名为LcTTP) cDNA的全长序列。LcTTP全长cDNA 1 508 bp, 包括77 bp的5′-非编码区(5′-UTR)、183 bp的3′-UTR和1 248 bp的开放阅读框(ORF), 编码418个氨基酸残基。推导的LcTTP理论分子量44 897.78 Da, 预测等电点pI 8.37; 哺乳动物TTP的重要功能结构域: N-端核输出序列(NES)、中央串联锌指结构域(TZF)、C-端NOT1-结合结构域(NOT1-BD)也在LcTTP中保守存在; 系统发育分析显示LcTTP和其他脊椎动物TTP聚为一支, 并与哺乳动物ZFP36家族其他成员ZFP36L1、ZFP36L2和ZFP36L3的进化支分离。组织表达特异性分析显示检测的9个组织均表达LcTTP mRNA, 但不同组织间表达水平差异大, 其中肌肉组织表达水平最高。大黄鱼头肾细胞系LYC-hK细胞中过表达LcTTP上调LPS诱导早期(1 h) TNF-α mRNA表达量, 之后TNF-α mRNA表达量快速下调至接近对照组水平(1.5 h); 放线菌D抑制转录后过表达LcTTP上调TNF-α mRNA降解速率, 表明LcTTP可通过促进TNF-α mRNA降解调节TNF-α表达。以上研究结果提示LcTTP可能是大黄鱼炎症反应平衡关键调控蛋白, 在大黄鱼感染性病害防治策略开发中有潜在应用价值。 |
| 关键词: 大黄鱼 RNA结合蛋白 Tristetraprolin蛋白 炎症反应 转录后调控 |
| DOI：10.11693/hyhz20230500103 |
| 分类号:Q789; S965 |
| 基金项目:浙江海洋大学科研启动经费,2020 |
附件 |
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| MOLECULAR CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF TRISTETRAPROLIN FROM LARGE YELLOW CROAKER LARIMICHTHYS CROCEA |
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WANG Hui-Juan1, HE Zhi-Qiao1, SHI Ge1, ZHOU Su-Ming2, ZHANG Xiao-Lin1, SHEN Wang1
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1.Marine Science and Technical College, Zhejiang Ocean University, Zhoushan 316022, China;2.School of Marine Sciences, Ningbo University, Ningbo 315211, China
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| Abstract: |
| Tristetraprolin (TTP) is an RNA binding protein and widely distributed in eukaryotes. In mammals, TTPs decrease the expression of inflammatory factors by promoting mRNA decay or inhibiting translation to regulate the balance of inflammatory response, and has been considered as a potential therapeutic target for inflammatory diseases. A full-length cDNA sequence of TTP in large yellow croaker (Larimichthys crocea; denoted as LcTTP) was obtained by rapid amplification of cDNA ends (RACE) method. The full-length cDNA of LcTTP was 1 508 bp, including a 77-bp 5'-untranslated region (5'-UTR), a 183-bp 3'-UTR, and a 1 248-bp open reading frame (ORF) encoding 418 amino acid residues. The theoretical molecular weight (MW) of LcTTP is 44 897.78 Da, and the predicted isoelectric point (pI) was 8.37. The important functional domains of mammalian TTP are well conserved in LcTTP, including the N-terminal nuclear export sequence (NES), the central CCCH-type tandem zinc finger domain (TZF), and the C-terminal NOT1-binding domain (NOT1-BD). Phylogenetic analysis showed that LcTTP is clustered into one clade with other vertebrate TTPs and separated from the other mammalian ZFP36 family members, including ZFP36L1, ZFP36L2, and ZFP36L3. Tissue expression pattern analysis indicated that the mRNA transcripts of LcTTP were detected in all the 9 examined tissues with the highest expression level in muscle. In LYC-hK cells (a large yellow croaker kidney cell line), the overexpression of LcTTP significantly increased the expression level of TNF-α in the early phase after LPS challenge and peaked in 1 h, and then rapidly decreased near to the control level in 1.5 h. After inhibition of transcription by actinomycin D, the overexpression of LcTTP significantly increased the degradation rate of TNF-α mRNA, indicating that LcTTP can regulate the expression of TNF-α by promoting mRNA decay. These results imply that LcTTP is the key regulator of inflammatory responses and a potential target for the control of infectious diseases in large yellow croaker. |
| Key words: Larimichthys crocea RNA binding protein Tristetraprolin inflammatory response posttranscriptional regulation |
