Molecular characterization of the RNA-binding protein, TRN-1 in the lepidopteran insects

TRN-1是一种与哺乳动物T细胞胞内抗原-1同源的RNA结合蛋白，在鳞翅目昆虫桑尺蠖和草地贪夜蛾的各个组织中都有表达。TRN-1分子已显示具有由N-末端的三个重复潜在RNA识别基序（RRM）和C-末端的辅助结构域组成的结构域结构，与TIA-1家族相似，并且通过研究在BmN 4细胞中外源重组蛋白和转录物的产生，TRN-1分子作为转录物的调节蛋白发挥作用（Kotani，et.例如，Gene 320，67-79，2003）。为了更深入地了解TRN-1的细胞功能，通过对过表达GFP-TRN-1融合蛋白和缺乏C-末端辅助结构域和RRM结构域的GFP-TRN-1的细胞进行共聚焦荧光显微镜分析，研究了TRN-1的每个结构域在培养细胞中的亚细胞定位和作用。结果表明，重组GFP融合蛋白具有与poly（U）RNA结合的活性。缺失C-末端辅助结构域和GFP-C-末端结构域的GFP-TRN-1可能定位于细胞核，而全长GFP-TRN-1既定位于细胞核又定位于细胞质。然而，GFP-TRN-1在AcNPV感染细胞后，观察到明确的细胞核定位。此外，GFP-TRN-1的过表达可导致重组杆状病毒在感染过程中产生的荧光素酶活性发生变化，从而表明鳞翅目昆虫TRN-1可能控制杆状病毒和细胞转录物在病毒感染引起的应激反应中的功能。

TRN-1, an RNA-binding protein homologous with mammalian T-cell intracellular antigen-1 has been found to express in every tissue of the Iepidopteran, Bombyx mori and Spodoptera frugiperda. TRN-1 molecule has been shown to possess the domain structure consisting of three-repetitive potential RNA-recognition motifs (RRMs) in the N-terminal followed by auxiliary domain in the C-terminal as similar with TIA-1 family, and to function as a modulator protein for transcripts by investigating the production of foreign recombinant proteins and transcripts in the Bombyx TRN-1 knock-down BmN4 cells (Kotani, et. al., Gene 320, 67-79, 2003). Subcellular localization and role of each domain of the Bombyx TRN-1 in the cultured cells has been investigated by the confocal fluorescence microscopy analyses on the cells over-expressing GFP-TRN-1 fusion protein and GFP-TRN-1 lacking C-terminal auxiliary domain and RRM domain in order to gain more insight into cellular function of the TRN-1. The recombinant GFP-fusion proteins with RRMs were indicated to assuredly have the poly (U) RNA binding activity. It was also shown that GFP-TRN-1 lacking C-terminal auxiliary domain and GFP-C-terminal domain were likely to be localized in the nucleus, while full-length GFP-TRN-1 was localized both in the nucleus and cytoplasm. However, clear localization of GFP-TRN-1 in the nucleus was observed after AcNPV was infected into the cells. In addition, it was indicated that GFP-TRN-1 over-expression could cause change of the recombinant baculovirus-produced luciferase activity during its infection, thus demonstrating that lepidopteran TRN-1 may control the function of both baculovirus and cellular transcripts in the response to stress caused by the viral infection.