Butcher Neville J, Broadhurst Gysell M, Minchin Rodney F
Department of Physiology and Pharmacology, School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland 4072, Australia.
Department of Physiology and Pharmacology, School of Biomedical Sciences, University of Queensland, St. Lucia, Queensland 4072, Australia.
J Biol Chem. 2007 Sep 28;282(39):28530-28539. doi: 10.1074/jbc.M701265200. Epub 2007 Aug 9.
Spermidine-spermine N(1)-acetyltransferase (SSAT) is induced in response to an elevation in intracellular polyamine pools. The increased enzyme activity is the result of an increase in gene transcription, mRNA translation, and protein stability. Induction of SSAT by polyamine analogues can lead to intracellular polyamine depletion and apoptosis. The mechanism by which polyamines alter the translational efficiency of SSAT mRNA is not well understood. In this study, we investigated the regulation of SSAT translation by the polyamine analogue N(1),N(11)-diethylnorspermine (DENSPM). DENSPM induced expression of both FLAG-tagged SSAT and SSAT fused to Renilla luciferase in a time- and concentration-dependent manner. This effect was not inhibited by actinomycin D indicating that changes in gene transcription did not explain the enhanced expression in the presence of DENSPM. Furthermore, because FLAG-SSAT did not contain the 5'- or 3'-untranslated regions of SSAT, translational regulation involved the coding sequence only. By contrast, cycloheximide completely inhibited induction by DENSPM, indicating a requirement for new protein synthesis. Deletion constructs identified two regions of the SSAT protein-coding RNA sequence that conferred polyamine responsiveness. Using these regions as probes in RNA electrophoretic mobility shift assays, we observed specific binding of a cytoplasmic protein. In addition, we found that the interaction between the RNA probes and the binding protein could be inhibited by DENSPM in a concentration-dependent manner. These results suggest that polyamines regulate SSAT mRNA translational efficiency by inhibiting a repressor protein from binding to regions of the coding sequence of the SSAT transcript.
亚精胺 - 精胺N(1)-乙酰基转移酶（SSAT）会因细胞内多胺池水平升高而被诱导。酶活性增加是基因转录、mRNA翻译和蛋白质稳定性增加的结果。多胺类似物诱导SSAT可导致细胞内多胺耗竭和细胞凋亡。多胺改变SSAT mRNA翻译效率的机制尚不清楚。在本研究中，我们研究了多胺类似物N(1),N(11)-二乙基亚精胺（DENSPM）对SSAT翻译的调控。DENSPM以时间和浓度依赖性方式诱导FLAG标记的SSAT和与海肾荧光素酶融合的SSAT的表达。放线菌素D未抑制这种效应，表明基因转录的变化并不能解释在DENSPM存在下表达增强的原因。此外，由于FLAG - SSAT不包含SSAT的5'或3'非翻译区，翻译调控仅涉及编码序列。相比之下，环己酰亚胺完全抑制了DENSPM的诱导作用，表明需要新的蛋白质合成。缺失构建体鉴定出SSAT蛋白质编码RNA序列中赋予多胺反应性的两个区域。在RNA电泳迁移率变动分析中使用这些区域作为探针，我们观察到一种细胞质蛋白的特异性结合。此外，我们发现RNA探针与结合蛋白之间的相互作用可被DENSPM以浓度依赖性方式抑制。这些结果表明，多胺通过抑制阻遏蛋白与SSAT转录本编码序列区域的结合来调节SSAT mRNA的翻译效率。
