Dearth Lawrence R, DeWille James
Molecular, Cellular and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH 43210-1093, USA.
Biochem Biophys Res Commun. 2003 May 2;304(2):344-50. doi: 10.1016/s0006-291x(03)00597-7.
Posttranscriptional regulation at the level of mRNA stability is becoming increasingly recognized as an important mechanism to control the levels of mRNAs that encode key cell fate determining proteins. Previous work from our laboratory demonstrated that C/EBPdelta is a highly unstable mRNA in G(0) growth arrested mammary epithelial cells. In this report we investigated trans-acting factor binding to the C/EBPdelta 3'-UTR and identified a cis-acting element important for this interaction. RNA electromobility shift assays (REMSAs) demonstrate that the C/EBPdelta mRNA 3'-UTR binds trans-acting factor(s) present in G(0) growth arrested mammary epithelial cell lysates. This binding was not detected in the presence of lysates from growing cells. UV-binding analysis detected a RNA/protein complex of approximately 35kDa following incubation of the full-length C/EBPdelta 3'UTR with lysates from G(0) growth arrested mammary epithelial cells. Competition assays indicate that a specific AU-rich region (U1) is necessary for trans-acting factor binding to the C/EBPdelta 3'-UTR. These studies have identified an AU-rich element located within the C/EBPdelta 3'-UTR that interacts with a putative G(0) growth arrest-specific trans-acting factor(s), which may regulate C/EBPdelta mRNA decay.
在mRNA稳定性水平上的转录后调控日益被认为是控制编码关键细胞命运决定蛋白的mRNA水平的重要机制。我们实验室之前的工作表明,C/EBPδ在G(0)期生长停滞的乳腺上皮细胞中是一种高度不稳定的mRNA。在本报告中,我们研究了与C/EBPδ 3'-UTR结合的反式作用因子,并鉴定了对这种相互作用很重要的顺式作用元件。RNA电泳迁移率变动分析(REMSAs)表明,C/EBPδ mRNA 3'-UTR与G(0)期生长停滞的乳腺上皮细胞裂解物中存在的反式作用因子结合。在生长细胞的裂解物存在下未检测到这种结合。紫外线结合分析在将全长C/EBPδ 3'UTR与G(0)期生长停滞的乳腺上皮细胞裂解物孵育后,检测到一个约35kDa的RNA/蛋白质复合物。竞争分析表明,一个特定的富含AU的区域(U1)对于反式作用因子与C/EBPδ 3'-UTR的结合是必需的。这些研究鉴定了位于C/EBPδ 3'-UTR内的一个富含AU的元件,它与一种假定的G(0)期生长停滞特异性反式作用因子相互作用,这可能调节C/EBPδ mRNA的降解。