Yang L, Steussy C N, Fuhrer D K, Hamilton J, Yang Y C
Department of Biochemistry and Molecular Biology, Walter Oncology Center, Indiana University School of Medicine, Indianapolis, 46202, USA.
Mol Cell Biol. 1996 Jul;16(7):3300-7. doi: 10.1128/MCB.16.7.3300.
12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulation of PU-34 cells, a primate bone marrow stromal cell line, resulted in a prolonged elevation of interleukin-11 (IL-11) mRNA, which can be inhibited by protein synthesis inhibitors. Nuclear run-on assays and actinomycin D experiments demonstrated that the up-regulation of IL-11 gene expression is mainly controlled at the posttranscriptional level through the protein kinase C (PKC) pathway. Inhibition of PKC activity by calphostin C generated an IL-11 mRNA degradation intermediate in TPA-stimulated PU-34 cells. This intermediate retains the 5' untranslated region (5'UTR) and coding region of the IL-11 mRNA but has lost the poly(A) tail and the 3'UTR. The mechanisms underlying IL-11 mRNA stabilization were further investigated by transfections with a variety of chimeric IL-11 constructs and deletion mutants. Two important observations were made from these transient expression experiments: (i) the same 3'UTR of IL-11 mRNA shown to confer instability in one chimeric transcript may not function as a destabilizer in another chimeric RNA, and (ii) the 5'UTR, coding region, and 3'UTR all contribute to IL-11 mRNA decay, and labile IL-11 deletion transcripts are not necessarily stabilized by TPA stimulation. Our study suggests that multiple regions within the IL-11 mRNA are involved in TPA-stimulated IL-11 mRNA stabilization, possibly through a unique RNA folding conformation involving interactions of various RNA sequences within the IL-11 mRNA molecule.
用12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）刺激灵长类骨髓基质细胞系PU - 34细胞，导致白细胞介素 - 11（IL - 11）mRNA长期升高，而这种升高可被蛋白质合成抑制剂抑制。核转录分析和放线菌素D实验表明，IL - 11基因表达的上调主要在转录后水平通过蛋白激酶C（PKC）途径受到调控。钙磷蛋白C抑制PKC活性在TPA刺激的PU - 34细胞中产生了IL - 11 mRNA降解中间体。该中间体保留了IL - 11 mRNA的5'非翻译区（5'UTR）和编码区，但失去了多聚腺苷酸尾和3'UTR。通过用多种嵌合IL - 11构建体和缺失突变体进行转染，进一步研究了IL - 11 mRNA稳定化的机制。从这些瞬时表达实验中得到了两个重要发现：（i）在一个嵌合转录本中显示会导致不稳定的相同IL - 11 mRNA的3'UTR，在另一个嵌合RNA中可能不起去稳定化作用；（ii）5'UTR、编码区和3'UTR都对IL - 11 mRNA的降解有贡献，并且不稳定的IL - 11缺失转录本不一定会被TPA刺激稳定化。我们的研究表明，IL - 11 mRNA中的多个区域参与了TPA刺激的IL - 11 mRNA稳定化过程，可能是通过一种独特的RNA折叠构象，该构象涉及IL - 11 mRNA分子内各种RNA序列的相互作用。
