产生应激颗粒是生物体遭受不利环境时细胞产生的一种自我保护机制,基于此,本研究开展了高温胁迫下刺参(Apostichopus japonicus)肠道细胞中应激颗粒(Stress granule)标记蛋白基因TIA-1表达特征的研究.采用RACE技术克隆了刺参T细胞胞内抗原-1基因(TIA-1)全长cDNA序列.该基因cDNA全长为3108 bp,包括16 bp的5'UTR,1284 bp的开放阅读框(ORF),1808 bp的3'UTR,编码427个氨基酸.结构分析表明,刺参TIA-1基因编码3个N末端RNA识别基序(RRM)和2个类似蛋白聚集区的C末端区域(low complexity).系统进化树分析表明,刺参TIA-1与软体动物门鸭嘴海豆芽(Lingula anatina)TIAR聚为一支,具有最近的亲缘关系.使用Western blotting技术检测目的蛋白TIA-1在全细胞和亚细胞成分中的表达差异,并且利用细胞免疫荧光技术检测细胞在高温胁迫下TIA-1的定位情况.研究结果表明,刺参肠道细胞TIA-1在高温胁迫下在细胞质中表达量显著上升(P<0.05),且高温胁迫下TIA-1定位表达发生变化,多分布在细胞核周围,由此推断TIA-1在刺参高温应激时可能参与了应激颗粒的生成,从而对刺参肠道细胞起到了保护作用.%In response to environmental stresses, such as heat shock, oxidative stress, and viral infection, arrest of translation initiation reduces energy-expensive cellular processes and conserves the energy required for the repair of cellular damage. Untranslated mRNAs that accumulate in these cells move to discrete cytoplasmic foci known as stress granules (SGs). SGs are characterized by the presence of translational initiation factors such as eIF4E, eIF4G, eIF4A, eIF4B, eIF3, eIF2, poly(A)-binding protein, and stalled 40S ribosomal units. The best-known pro-teins integral to SG formation are T-cell intracellular antigen (TIA) proteins, which are considered robust markers of SGs. The assembly of SGs, one of the most important post-transcriptional mechanisms, helps cells to survive under adverse environmental conditions. Currently, there is no information on the cell-protective roles of SGs in marine invertebrates under environmental stress. In the present study, we hypothesized that TIA-1, an mRNA-binding protein that can aggregate within granules, may play a significant role in the sea cucumber Apos-tichopus japonicus in response to heat stress. We characterized the amino sequence of A. japonicus TIA-1. The full-length cDNA of TIA-1 is 3108 bp, comprising a 5′-untranslated region (UTR) of 16 bp, 3′-UTR of 1808 bp, and open reading frame (ORF) of 1284 bp. The ORF encodes 427 amino acids, with a calculated molecular mass of 48.07 kD and 6.19 theoretical isoelectric point. Structural analysis showed that A. japonicus TIA-1 has three N-terminal RNA-recognition motifs and two C-terminal low-complexity regions, which are related to prion pro-teins and have the capacity to form reversible aggregates. Phylogenetic analyses revealed that A. japonicus TIA-1 is closely related to Lingula anatina TIAR. Moreover, we detected whole-cell and subcellular protein expression levels of TIA-1 by using western blotting. The localization of TIA-1 to subnuclear structures was assessed using immunofluorescence analysis. The results showed that the expression level of TIA-1 in cytoplasmic fractions un-der heat stress (25℃) was significantly increased when compared with the control group (15℃). The fluorescence signals of TIA-1 were observed to be translocated from the nucleus to the cytoplasm in response to heat stress, which suggests that TIA-1 may be involved in the formation of SGs to protect the stressed intestinal cells of A. japonicus under high temperature conditions. We identified a potential novel cytoprotective mechanism in re-sponse to heat stress in A. japonicus from the viewpoint of post-transcriptional regulation, which may play an im-portant role in reducing the translational rates and/or mRNA processing under unfavorable environmental condi-tions in marine invertebrates.
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