Kabnick K S, Housman D E
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139.
Mol Cell Biol. 1988 Aug;8(8):3244-50. doi: 10.1128/mcb.8.8.3244-3250.1988.
We have analyzed the contributions to cytoplasmic stability in an mRNA species with a very short half-life (human c-fos) and an mRNA species with a very long half-life (human beta-globin). When the human c-fos promoter was used to drive the expression of human c-fos, beta-globin, and chimeric DNAs between c-fos and beta-globin in transfected cells, a pulse of mRNA synthesis was obtained following induction of transcription by refeeding quiescent cells with medium containing 15% calf serum. The mRNA half-life was determined by using Northern (RNA) blot analysis of mRNAs prepared at various times following the pulse of transcription. Under these conditions human c-fos mRNA exhibited a half-life of 6.6 min and human beta-globin mRNA exhibited a half-life of 17.5 h. Replacement of the 3' end of the c-fos mRNA with the 3' end of the beta-globin mRNA increased the half-life of the resultant RNA from 6.6 to 34 min. The reciprocal chimera had a half-life of 34.6 min compared with the 17.5-h half-life of beta-globin mRNA. These results suggest that sequences which make a major contribution to mRNA stability reside in the 3' end of either or both molecules. A chimera in which the 5' untranslated region of globin was replaced by part of the 5' untranslated region of fos led to destabilization of the encoded mRNA. This construct produced an mRNA with a half-life of 6.8 h instead of the 17.5-h half-life of globin. This result suggests that additional determinants of stability reside in the 5' end of these mRNA molecules. Substitution of part of the 5' untranslated region of fos by the 5' untranslated region of beta-globin yielded an mRNA with stability similar to fos mRNA. These results suggest that interactions among sequences within each mRNA contribute to the stability of the respective molecules.
我们分析了对半衰期非常短的mRNA种类（人c-fos）和半衰期非常长的mRNA种类（人β-珠蛋白）细胞质稳定性的贡献。当使用人c-fos启动子驱动人c-fos、β-珠蛋白以及c-fos与β-珠蛋白之间的嵌合DNA在转染细胞中表达时，通过用含15%小牛血清的培养基重新喂养静止细胞诱导转录后，获得了一个mRNA合成脉冲。通过对转录脉冲后不同时间制备的mRNA进行Northern（RNA）印迹分析来确定mRNA半衰期。在这些条件下，人c-fos mRNA的半衰期为6.6分钟，人β-珠蛋白mRNA的半衰期为17.5小时。用β-珠蛋白mRNA的3'末端替换c-fos mRNA的3'末端，使所得RNA的半衰期从6.6分钟增加到34分钟。与之相反的嵌合体半衰期为34.6分钟，而β-珠蛋白mRNA的半衰期为17.5小时。这些结果表明，对mRNA稳定性起主要作用的序列存在于这两种分子中任一种或两种分子的3'末端。一种嵌合体，其中珠蛋白的5'非翻译区被fos的部分5'非翻译区取代，导致编码的mRNA不稳定。该构建体产生的mRNA半衰期为6.8小时，而不是珠蛋白的17.5小时半衰期。这一结果表明，这些mRNA分子的5'末端存在其他稳定性决定因素。用β-珠蛋白的5'非翻译区替换fos的部分5'非翻译区，产生了一种稳定性与人c-fos mRNA相似的mRNA。这些结果表明，每个mRNA内序列之间的相互作用有助于各自分子的稳定性。
