Identification of secondary structure in the 5′-untranslated region of the human adrenomedullin mRNA with implications for the regulation of mRNA translation
Identification of secondary structure in the 5′-untranslated region of the human adrenomedullin mRNA with implications for the regulation of mRNA translation
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DOI：
10.1038/sj.onc.1209672
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发表时间：
2006-10-19
期刊：
影响因子：
8
通讯作者：
Ouafik, L. H.
中科院分区：
文献类型：
--
作者：
Brenet, F.;Dussault, N.;Ouafik, L. H.
肾上腺髓质素(adrenomedullin，AM)是一种多功能调节肽，具有重要的血管生成和有丝分裂特性。在此，我们在人AM mRNA的5‘-非翻译区(50非编码区)中发现了一个稳定的二级结构区域。50个非编码区的逆转录酶-聚合酶链式反应一致地导致第二个产物，除了预期大小为155个碱基对(BP)的产物外，在50个编码区的中心区有类似65个碱基对的缺失，这表明存在二级结构。用对单链或双链RNA具有选择性的RNase探测50个非编码区，证实了茎环结构的存在。我们研究了这种茎环结构在荧光素酶报告基因在培养细胞系中表达的作用。使用结合荧光素酶和AM mRNA的50个非编码区的嵌合mRNA进行的报告分析表明，由于翻译减少，报告活性急剧下降，而位于CAPITE+31和+95之间的茎环结构的缺失导致活性恢复。使用哺乳动物细胞系胞浆提取物的凝胶迁移位移分析表明，胞浆蛋白与含有AM的50UTR区的核探针特异结合，但不与对应于消息的其他区域或含有50UTR区但缺乏二级结构区域的核探针特异性结合。尽管我们得出结论，人AM mRNA的50UTR可以调节AM mRNA在体内的翻译，并且预测的茎环结构是这种抑制所必需的，但顺式元件结合活性的功能后果仍有待确定。
Adrenomedullin (AM) is a multifunctional regulatory peptide with important angiogenic and mitogenic properties. Here we identify a region of stable secondary structure in the 5'-untranslated region ( 50 UTR) of human AM mRNA. Reverse transcriptase-polymerase chain reaction of the 50 UTR consistently resulted, in addition to the product with the expected size of 155 base pair ( bp), in a second product with an similar to 65-bp deletion from the central region of the 50 UTR, suggesting the presence of a secondary structure. The presence of a stem-loop structure was confirmed by probing the 50 UTR with RNases with selectivity for single- or double-stranded RNA. We investigated the role of this stem-loop structure in expression of luciferase reporter gene in cultured cell lines. Reporter assays using a chimeric mRNA that combined luciferase and the 50 UTR of AM mRNA demonstrated a dramatic decrease of the reporter activity owing to a decreased translation, whereas the deletion of the stem-loop structure localized between nt + 31 and + 95 from the capsite led to the recovery of activity. Gel migration shift assays using cytosolic extracts from mammalian cell lines demonstrate a specific binding of a cytosolic protein to riboprobes containing the 50 UTR of AM but not to riboprobes either corresponding to other areas of the message or containing the 50 UTR but lacking the region of secondary structure. Although we conclude that the 50 UTR of the human AM mRNA can modulate the translation of AM mRNA in vivo, and that the predicted stem-loop structure is necessary for this inhibition, the functional consequences of the cis element-binding activity remain to be determined.