De Moor C H, Jansen M, Bonte E J, Thomas A A, Sussenbach J S, Van den Brande J L
Department of Pediatrics, Utrecht University, The Netherlands.
Eur J Biochem. 1994 Dec 15;226(3):1039-47. doi: 10.1111/j.1432-1033.1994.t01-1-01039.x.
The human insulin-like-growth-factor-2 (IGF-2) gene generates mRNAs with four different leader sequences, but with identical coding and trailing regions. Previous research has revealed that the leader-2-containing and leader-4-containing mRNAs are completely polysomal, whereas mRNAs possessing leader-3 are predominantly present in the untranslated free messenger ribonucleoprotein particle (mRNP), both in cell lines and in foetal liver tissue. To investigate the influence of the IGF-2 leader sequences on expression of the gene, IGF-2 leader-luciferase and leader-chloramphenicol acetyltransferase fusion constructs were transfected transiently into different cell lines. In these experiments, the levels of expression obtained by constructs with leader-1, leader-2 and leader-4 were very similar, both at the level of mRNA and protein. Leader-3, however, strongly repressed the expression of the fusion mRNA via an unknown mechanism. This repression appeared to be confined to nucleotides at positions 328-906 of the leader sequence. The remaining 5' part of the leader sequence was efficient both in RNA expression and in translation, but the 3' part of the leader (nucleotides 906-1180) again moderately repressed luciferase expression, possibly due to endonucleolytic cleavage in this region of the RNA. To evaluate the effect of the IGF-2 leaders on in vitro translation, leader-chloramphenicol acetyltransferase fusion mRNAs were synthesized and translated in reticulocyte lysates. Compared to a chloramphenicol acetyltransferase control RNA, leader-1-chloramphenicol acetyltransferase mRNA translated over 20-fold less efficiently, whereas leader-2 repressed translation of its chloramphenicol acetyltransferase mRNA moderately (3-5 fold). Despite a general improvement of the translation efficiency upon translation in HeLa lysate, these discrepancies with the transfection data persisted. Translation of leader-3-containing mRNAs in reticulocyte lysates was barely detectable. The whole 5' region of leader-3, up to nucleotide 614, could be shown to be repressive. Only leader-4 directed translation of the chloramphenicol acetyltransferase open reading frame efficiently. As with leader-1 and leader-2, this L4-chloramphenicol acetyltransferase mRNA translated in a cap-dependent manner under the conditions of our experiments; translation of this mRNA was relatively resistant to addition of cap analogue. We conclude that all four IGF-2 leader sequences differ in their translational properties. This makes it likely that changes in the translational machinery will affect the expression of the various IGF-2 mRNAs differentially.
人类胰岛素样生长因子2（IGF-2）基因可产生具有四种不同前导序列但编码区和尾随区相同的mRNA。先前的研究表明，含前导序列2和前导序列4的mRNA完全存在于多核糖体中，而具有前导序列3的mRNA在细胞系和胎儿肝脏组织中主要存在于未翻译的游离信使核糖核蛋白颗粒（mRNP）中。为了研究IGF-2前导序列对该基因表达的影响，将IGF-2前导序列-荧光素酶和前导序列-氯霉素乙酰转移酶融合构建体瞬时转染到不同的细胞系中。在这些实验中，含前导序列1、前导序列2和前导序列4的构建体在mRNA和蛋白质水平上获得的表达水平非常相似。然而，前导序列3通过未知机制强烈抑制融合mRNA的表达。这种抑制作用似乎局限于前导序列328 - 906位的核苷酸。前导序列剩余的5'部分在RNA表达和翻译方面均有效，但前导序列的3'部分（核苷酸906 - 1180）再次适度抑制荧光素酶表达，这可能是由于该区域RNA发生了核酸内切酶切割。为了评估IGF-2前导序列对体外翻译的影响，合成了前导序列-氯霉素乙酰转移酶融合mRNA并在网织红细胞裂解物中进行翻译。与氯霉素乙酰转移酶对照RNA相比，前导序列1-氯霉素乙酰转移酶mRNA的翻译效率低20多倍，而前导序列2适度抑制其氯霉素乙酰转移酶mRNA的翻译（3 - 5倍）。尽管在HeLa裂解物中翻译时总体翻译效率有所提高，但这些与转染数据的差异仍然存在。在网织红细胞裂解物中几乎检测不到含前导序列3的mRNA的翻译。前导序列3的整个5'区域（直至核苷酸614）都具有抑制作用。只有前导序列4能有效指导氯霉素乙酰转移酶开放阅读框的翻译。与前导序列1和前导序列2一样，在我们的实验条件下，这种L4-氯霉素乙酰转移酶mRNA以帽依赖性方式进行翻译；该mRNA的翻译对添加帽类似物相对不敏感。我们得出结论，所有四种IGF-2前导序列在翻译特性上存在差异。这使得翻译机制的变化可能会对各种IGF-2 mRNA的表达产生不同的影响。
