Motohashi Hiromi, Mukudai Yoshiki, Ito Chihiro, Kato Kosuke, Shimane Toshikazu, Kondo Seiji, Shirota Tatsuo
Department of Oral and Maxillofacial Surgery, School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Ota-ku, Tokyo 145-8515, Japan.
Department of Oral and Maxillofacial Surgery, School of Dentistry, Showa University, 2-1-1 Kitasenzoku, Ota-ku, Tokyo 145-8515, Japan
Biochem J. 2017 May 4;474(10):1669-1687. doi: 10.1042/BCJ20160942.
Although tumor protein D52 (TPD52) family proteins were first identified nearly 20 years ago, their molecular regulatory mechanisms remain unclear. Therefore, we investigated the post-transcriptional regulation of TPD52 family genes. An RNA immunoprecipitation (RIP) assay showed the potential binding ability of TPD52 family mRNAs to several RNA-binding proteins, and an RNA degradation assay revealed that TPD52 is subject to more prominent post-transcriptional regulation than are TPD53 and TPD54. We subsequently focused on the 3'-untranslated region (3'-UTR) of TPD52 as a -acting element in post-transcriptional gene regulation. Several deletion mutants of the 3'-UTR of TPD52 mRNA were constructed and ligated to the 3'-end of a reporter green fluorescence protein gene. An RNA degradation assay revealed that a minimal -acting region, located in the 78-280 region of the 5'-proximal region of the 3'-UTR, stabilized the reporter mRNA. Biotin pull-down and RIP assays revealed specific binding of the region to T-cell intracellular antigen 1 (TIA-1) and TIA-1-related protein (TIAR). Knockdown of TIA-1/TIAR decreased not only the expression, but also the stability of TPD52 mRNA; it also decreased the expression and stability of the reporter gene ligated to the 3'-end of the 78-280 fragment. Stimulation of transforming growth factor-β and epidermal growth factor decreased the binding ability of these factors, resulting in decreased mRNA stability. These results indicate that the 78-280 fragment and TIA-1/TIAR concordantly contribute to mRNA stability as a -acting element and -acting factor(s), respectively. Thus, we here report the specific interactions between these elements in the post-transcriptional regulation of the TPD52 gene.
尽管肿瘤蛋白D52（TPD52）家族蛋白在近20年前就首次被发现，但其分子调控机制仍不清楚。因此，我们研究了TPD52家族基因的转录后调控。RNA免疫沉淀（RIP）分析显示TPD52家族mRNA与几种RNA结合蛋白具有潜在的结合能力，RNA降解分析表明TPD52比TPD53和TPD54受到更显著的转录后调控。随后，我们将TPD52的3'-非翻译区（3'-UTR）作为转录后基因调控中的一个顺式作用元件进行研究。构建了TPD52 mRNA 3'-UTR的几个缺失突变体，并将其连接到报告绿色荧光蛋白基因的3'-末端。RNA降解分析表明，位于3'-UTR 5'-近端区域78-280区域的一个最小顺式作用区域可稳定报告mRNA。生物素下拉和RIP分析显示该区域与T细胞胞内抗原1（TIA-1）和TIA-1相关蛋白（TIAR）特异性结合。敲低TIA-1/TIAR不仅降低了TPD52 mRNA的表达，还降低了其稳定性；它还降低了连接到78-280片段3'-末端的报告基因的表达和稳定性。转化生长因子-β和表皮生长因子的刺激降低了这些因子的结合能力，导致mRNA稳定性下降。这些结果表明，78-280片段和TIA-1/TIAR分别作为顺式作用元件和顺式作用因子共同促进mRNA稳定性。因此，我们在此报告了这些元件在TPD52基因转录后调控中的特异性相互作用。
