Role of the 5'-untranslated region of mRNA in the synthesis of S-adenosylmethionine decarboxylase and its regulation by spermine.
Role of the 5'-untranslated region of mRNA in the synthesis of S-adenosylmethionine decarboxylase and its regulation by spermine.
复制标题
mRNA 5-非翻译区在 S-腺苷甲硫氨酸脱羧酶合成中的作用及其受精胺的调节。
作者：
L. Shantz;R. Viswanath;A. Pegg
S-腺苷甲硫氨酸脱羧酶（S-Adenosylmethionine decarboxylase，S-MetDC）是多胺生物合成的限速酶，受多胺在转录和翻译水平的调控。MetDC mRNA的两个不寻常的特征是长（320 nt）的5 '非翻译区（5' UTR），其被认为包含广泛的二级结构，以及在5 'UTR内的短（15 nt）开放阅读框（ORF）。我们已经研究了改变这些元素的影响，表达的n-butyl-1，3-diaminopropane（BDAP），精胺合成酶抑制剂，和它的调节。将在5 'UTR中含有改变的人CD 3 MetDC cDNA，以及其中CD 3 MetDC 5' UTR插入在辅酶A酶编码区之前的嵌合构建体转染到COS-7细胞中。含有所有5 'UTR、CD 3 MetDC蛋白编码区和3' UTR的构建体pSAM 320表达较差（超过内源活性2倍）。几乎整个5 'UTR的缺失，留下nt -12至-1，增加表达59倍，表明5' UTR作为负调节剂。当去除最末端5'端的27个nt（pSAM 293，增加47倍）时，或当通过将ATG改变为CGA而破坏存在于该区域的内部ORF（pSAM 320-ATG，增加38倍）时，观察到相同的效果。具有非常少的预测二级结构的pSAM 44（通过缺失nt -288至-12制备）的表达和调节与pSAM 320的表达和调节非常相似，表明包括内部ORF的末端27 nt而不是广泛的二级结构可能是导致低基础水平的pSAM MetDC表达的原因。使用荧光素酶构建体证实的这些结果表明，对表达的负面影响主要是由于内部ORF。BDAP消耗精胺使pSAM 320的表达增加5倍以上，而不影响pSAM MetDC mRNA水平。来自pSAM 293的表达不受精胺消耗的影响，而来自pSAM 320-ATG的表达增加了2.5倍。这些结果表明在5 'UTR的前27 nt中存在精胺应答元件，其可能包括但不完全是由于内部ORF。
S-Adenosylmethionine decarboxylase (AdoMetDC), a rate-limiting enzyme in polyamine biosynthesis, is regulated by polyamines at the levels of both transcription and translation. Two unusual features of AdoMetDC mRNA are a long (320 nt) 5'-untranslated region (5'UTR), which is thought to contain extensive secondary structure, and a short (15 nt) open reading frame (ORF) within the 5'UTR. We have studied the effects of altering these elements on both the expression of AdoMetDC and its regulation by n-butyl-1,3-diaminopropane (BDAP), a spermine synthase inhibitor. Human AdoMetDC cDNAs containing alterations in the 5'UTR, as well as chimaeric constructs in which the AdoMetDC 5'UTR was inserted ahead of the luciferase-coding region, were transfected into COS-7 cells. Construct pSAM320, which contains all of the 5'UTR, the AdoMetDC protein-coding region and the 3'UTR, was expressed poorly (2-fold over the endogenous activity). Deletion of virtually the entire 5'UTR, leaving nt -12 to -1, increased expression 59-fold, suggesting that 5'UTR acts as a negative regulator. The same effect was seen when the 27 nt at the extreme 5' end were removed (pSAM293, 47-fold increase), or when the internal ORF which is present in this region was destroyed by changing the ATG to CGA (pSAM320-ATG, 38-fold increase). The expression and regulation of pSAM44 (made by deleting nt -288 to -12), which has very little predicted secondary strucutre, was very similar to that of pSAM320 indicating that the terminal 27 nt including the internal ORF rather than extensive secondary structure may be responsible for the low basal levels of AdoMetDC expression. These results, confirmed using luciferase constructs, suggest that the negative effect on expression is predominantly due to the internal ORF. Depletion of spermine by BDAP increased the expression from pSAM320 more than 5-fold without affecting AdoMetDC mRNA levels. Expression from pSAM293 was unchanged by spermine depletion, whereas that from pSAM320-ATG was increased 2.5-fold. These results indicate the presence of a spermine response element in the first 27 nt of the 5'UTR that may include but is not entirely due to the internal ORF.