Sheflin L G, Brooks E M, Spaulding S W
The Research Service at the VA Western New York Healthcare System, Buffalo, New York 14215, USA.
Endocrinology. 1996 Jul;137(7):2910-7. doi: 10.1210/endo.137.7.8770913.
Epidermal growth factor (EGF) transcripts that use the terminal polyadenylation signal display a dramatic sex difference in the pattern of polyadenylation in the murine submaxillary gland (SMG), whereas those in the kidney do not. It takes 3 days before testosterone treatment begins to change the polyadenylation pattern in female SMG to resemble the male pattern, a finding that supports previous suggestions that posttranscriptional mechanisms are involved in regulating EGF expression. The conservation of a unique 23-b sequence centered on the terminal polyadenylation signal in all published mammalian EGF sequences suggested that trans-acting factors involved in EGF messenger RNA (mRNA) metabolism might bind to this sequence. To investigate this, we prepared 32P-RNA containing the 3' terminal EGF 23-b sequence plus a short poly-A tail, and incubated it with SMG cytosol. Cytosol retarded the electrophoretic mobility of this RNA as a single prominent band on 8% PAGE, and by UV-cross-linking, a single prominent 47-kDa protein was detected on 10% SDS-PAGE. Trypsin abolished both the gel-retarding and cross-linking activities. Cytosol from female SMGs contained approximately 8 times more of both the RNA binding activities than male cytosol. Injecting testosterone (200 microg QOD) into female mice altered both the RNA binding activities in a biphasic fashion, initially increasing them by about 40% at 2 days, then decreasing them by about 65% > or = 5 days, reaching male levels. Kidney cytosol contained both RNA binding activities but displayed neither sexual dimorphism nor testosterone-responsiveness. The tissue-specific testosterone-dependent changes observed in the 47-kDa protein occur before the increase in EGF mRNA levels and before the change in EGF mRNA polyad-enylation, so this cytosolic protein could be a trans-acting factor involved in EGF polyadenylation.
使用末端多聚腺苷酸化信号的表皮生长因子（EGF）转录本在小鼠颌下腺（SMG）中呈现出显著的多聚腺苷酸化模式性别差异，而在肾脏中则没有。睾酮处理3天后才开始改变雌性SMG中的多聚腺苷酸化模式，使其类似于雄性模式，这一发现支持了先前关于转录后机制参与调节EGF表达的观点。所有已发表的哺乳动物EGF序列中，以末端多聚腺苷酸化信号为中心的独特23碱基序列的保守性表明，参与EGF信使核糖核酸（mRNA）代谢的反式作用因子可能与该序列结合。为了对此进行研究，我们制备了含有3'末端EGF 23碱基序列加短多聚腺苷酸尾的32P-RNA，并将其与SMG胞质溶胶一起孵育。胞质溶胶使该RNA在8%聚丙烯酰胺凝胶电泳（PAGE）上作为一条单一的显著条带出现电泳迁移率阻滞，通过紫外线交联，在10%十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）上检测到一条单一的显著47 kDa蛋白。胰蛋白酶消除了凝胶阻滞和交联活性。来自雌性SMG的胞质溶胶中这两种RNA结合活性大约是雄性胞质溶胶的8倍。给雌性小鼠注射睾酮（200μg隔日一次）以双相方式改变了这两种RNA结合活性，最初在2天时使其增加约40%，然后在≥5天时使其降低约65%，达到雄性水平。肾脏胞质溶胶含有这两种RNA结合活性，但既没有性别二态性也没有睾酮反应性。在47 kDa蛋白中观察到的组织特异性睾酮依赖性变化发生在EGF mRNA水平升高之前以及EGF mRNA多聚腺苷酸化变化之前，因此这种胞质蛋白可能是参与EGF多聚腺苷酸化的反式作用因子。
