Chen C Y, Chen T M, Shyu A B
Department of Biochemistry and Molecular Biology, University of Texas Houston Health Science Center Medical School 77030.
Mol Cell Biol. 1994 Jan;14(1):416-26. doi: 10.1128/mcb.14.1.416-426.1994.
AU-rich elements (ARE) in the 3' untranslated region of many highly labile mRNAs for proto-oncogenes, lymphokines, and cytokines can act as an RNA-destabilizing element. The absence of a clear understanding of the key sequence and structural features of the ARE that are required for its destabilizing function has precluded the further elucidation of its mode of action and the basis of its specificity. Combining extensive mutagenesis of the c-fos ARE with in vivo analysis of mRNA stability, we were able to identify mutations that exhibited kinetic phenotypes consistent with the biphasic decay characteristic of a two-step mechanism: accelerated poly(A) shortening and subsequent decay of the transcribed portion of the mRNA. These mutations, which affected either an individual step or both steps, all changed the mRNA stability. Our experiments further revealed the existence of two structurally distinct and functionally interdependent domains that constitute the c-fos ARE. Domain I, which is located within the 5' 49-nucleotide segment of the ARE and contains the three AUUUA motifs, can function as an RNA destabilizer by itself. It forms the essential core unit necessary for the ARE-destabilizing function. Domain II is a 20-nucleotide U-rich sequence which is located within the 3' part of the c-fos ARE. Although it alone can not act as an RNA destabilizer, this domain serves two critical roles: (i) its presence enhances the destabilizing ability of domain I by accelerating the deadenylation step, and (ii) it has a novel capacity of buffering decay-impeding effects exerted by mutations introduced within domain I. A model is proposed to explain how these critical structural features may be involved in the c-fos ARE-directed mRNA decay pathway. These findings have important implications for furthering our understanding of the molecular basis of differential mRNA decay mediated by different AREs.
许多原癌基因、淋巴因子和细胞因子的高不稳定mRNA的3'非翻译区中的富含AU元件（ARE）可作为一种RNA不稳定元件。由于对ARE发挥其不稳定功能所需的关键序列和结构特征缺乏清晰的认识，阻碍了对其作用方式及其特异性基础的进一步阐明。通过将c-fos ARE的广泛诱变与mRNA稳定性的体内分析相结合，我们能够鉴定出表现出与两步机制的双相衰变特征一致的动力学表型的突变：加速多聚腺苷酸化缩短以及随后mRNA转录部分的衰变。这些影响单个步骤或两个步骤的突变均改变了mRNA的稳定性。我们的实验进一步揭示了构成c-fos ARE的两个结构上不同且功能上相互依赖的结构域的存在。结构域I位于ARE的5'端49个核苷酸片段内，包含三个AUUUA基序，其自身可作为RNA去稳定剂发挥作用。它形成了ARE去稳定功能所必需的核心单元。结构域II是一个富含20个核苷酸的U序列，位于c-fos ARE的3'部分。尽管它本身不能作为RNA去稳定剂发挥作用，但该结构域发挥两个关键作用：（i）它的存在通过加速去腺苷酸化步骤增强了结构域I的去稳定能力，（ii）它具有一种新的能力，可缓冲结构域I内引入的突变所产生的衰变阻碍作用。本文提出了一个模型来解释这些关键结构特征可能如何参与c-fos ARE指导的mRNA衰变途径。这些发现对于进一步理解不同ARE介导的差异性mRNA衰变的分子基础具有重要意义。
