Akashi M, Shaw G, Hachiya M, Elstner E, Suzuki G, Koeffler P
Division of Radiation Health, National Institute of Radiological Sciences, Chiba, Japan.
Blood. 1994 Jun 1;83(11):3182-7.
Many RNAs coding for either cytokines or oncogenes are unstable and have a short half-life (t1/2). The AUUUA motif is a highly conserved sequence and is repeated three or more times in the 3' untranslated region (3'UTR) of RNAs encoding many of these short-lived cytokines and oncogenes. These sequences can confer instability. In this study, we investigated the role of number and location of AUUUA motifs in stabilization of RNA. We introduced 1xATTTA, 2xATTTA, ATTTTTTTA (second adenosine of 2xATTTA was substituted with a thymidine), 3xATTTA, 5xATTTA, 7xATTTA [AT-rich sequence from granulocyte-macrophage colony-stimulating factor [GM-CSF] gene (AT-62)], and GC-62 (GC sequences were substituted for ATTTA sequences in the 7xATTTA) into the 3'UTR of rabbit beta-globin (R beta G) gene. This construct also contained the neomycin-resistance gene. These expression vectors were transfected into human lung fibroblasts (W138), which constitutively expressed low levels of GM-CSF mRNA. Stable transfectants were selected by growth in G418. Northern blot analysis of actinomycin D-treated, stably transfected cells demonstrated that the number of AUUUA sequences correlated with rapidity of turnover of the chimeric R beta G mRNA. The rank order of stability was GC-62 = 1xATTTA = 2xATTTA (no RNA decay at 4 hours) > 3xATTTA = 5xATTTA (t1/2, 4 hours) > 7xATTTA (t1/2, 2 hours). Stability of mRNA of R beta G also was reduced (t1/2, 2 to 4 hours) when AT-62 was introduced into the second exon of R beta G gene. In these same cells, the t1/2 of GM-CSF RNA was approximately 10 to 15 minutes, suggesting that the AUUUA motifs cannot alone account for the rapid degradation of this cytokine mRNA. Phorbol diesters, including 12-0-tetradecanoyl phorbol 13-acetate (TPA), stabilize a variety of transiently expressed RNAs, including GM-CSF RNA. We found that TPA markedly increased (> 30-fold) the accumulation of GM-CSF RNA. In contrast, TPA was unable to stimulate the levels of the chimeric R beta G when either 1x, 2x, 3x, or 5xATTTA motifs were fused to 3'UTR, or when either AT-62 or GC-62 control sequences were fused to the second exon. The chimeric beta-globin construct with either AT-62 or ATTTTTTTA in the 3'UTR had only an approximately twofold to threefold increase in accumulation.(ABSTRACT TRUNCATED AT 400 WORDS)
许多编码细胞因子或癌基因的RNA不稳定，半衰期（t1/2）较短。AUUUA基序是一个高度保守的序列，在编码许多这些短命细胞因子和癌基因的RNA的3'非翻译区（3'UTR）中重复三次或更多次。这些序列可导致不稳定性。在本研究中，我们研究了AUUUA基序的数量和位置在RNA稳定化中的作用。我们将1xATTTA、2xATTTA、ATTTTTTTA（2xATTTA的第二个腺苷被胸腺嘧啶取代）、3xATTTA、5xATTTA、7xATTTA [来自粒细胞-巨噬细胞集落刺激因子（GM-CSF）基因的富含AT序列（AT-62）]和GC-62（在7xATTTA中用GC序列取代ATTTA序列）引入兔β-珠蛋白（RβG）基因的3'UTR。该构建体还包含新霉素抗性基因。将这些表达载体转染到人肺成纤维细胞（W138）中，该细胞组成性表达低水平的GM-CSF mRNA。通过在G418中生长选择稳定转染子。对经放线菌素D处理的稳定转染细胞进行Northern印迹分析表明，AUUUA序列的数量与嵌合RβG mRNA的周转速度相关。稳定性的排序为GC-62 = 1xATTTA = 2xATTTA（4小时时无RNA降解）> 3xATTTA = 5xATTTA（t1/2，4小时）> 7xATTTA（t1/2，2小时）。当将AT-62引入RβG基因的第二个外显子时，RβG mRNA的稳定性也降低（t1/2，2至4小时）。在这些相同的细胞中，GM-CSF RNA的t1/2约为10至15分钟，这表明AUUUA基序不能单独解释这种细胞因子mRNA的快速降解。佛波酯，包括12-0-十四酰佛波醇13-乙酸酯（TPA），可稳定多种瞬时表达的RNA，包括GM-CSF RNA。我们发现TPA显著增加（> 30倍）GM-CSF RNA的积累。相反，当1x、2x、3x或5xATTTA基序与3'UTR融合，或当AT-62或GC-62对照序列与第二个外显子融合时，TPA无法刺激嵌合RβG的水平。在3'UTR中带有AT-62或ATTTTTTTA的嵌合β-珠蛋白构建体的积累仅增加约两倍至三倍。（摘要截短于400字）
