Hormonal Regulation of Messenger RNA Stability

描述（由申请人提供）：mRNA衰变在基因表达中起核心作用，几乎每种mRNA的半衰期都受mRNA及其同源结合蛋白内的调控序列控制。快速周转是编码生长因子、转录因子、细胞因子和细胞信号传导分子的mRNA的特征性特征，并且该过程的选择性调节是控制这些蛋白质的量的一种方式。大多数mRNA的衰变开始于poly（A）尾的缩短、5'帽的去除以及mRNA体的同时5'-3 '和3'-5 '降解。除了poly（A）缩短外，这些过程作用于非降解的mRNA。然而，转录组的一个子集的周转是由核酸内切酶切割催化的，而mRNA是由翻译核糖体参与的。原型mRNA内切核酸酶是PMR 1，一种最初被鉴定为雌激素诱导的核糖核酸酶活性的酶，其在多核糖体上的出现与血清蛋白mRNA的不稳定性一致。核酸内切酶介导的mRNA降解的标志是其对特定mRNA的选择性。这是由含有PMR 1及其翻译底物mRNA的mRNP复合物（称为复合物I）的形成决定的。为了加入这个复合物，PMR 1必须在蛋白质的多核糖体靶向结构域中的酪氨酸残基上磷酸化，并且过去的资助周期将c-Src确定为负责这一关键激活步骤的激酶。这是致癌酪氨酸激酶直接参与mRNA衰变的第一个例子，它提出了PMR 1介导的mRNA衰变可能是癌症中c-Src靶点的可能性。与此相一致，PMR 1与Ena/VASP蛋白结合，Ena/VASP蛋白是肌动蛋白细胞骨架的调节剂，并且在表达催化活性PMR 1的细胞中细胞运动性增加。目的1将使用串联亲和色谱法回收复合物I mRNP，鉴定其组成蛋白，并确定它们在mRNP组装和mRNA降解中的作用。这是破译PMR 1-mRNA衰变的“RNP代码”的第一步。作为将PMR 1募集至mRNP的“看门人”的含有SH 2结构域的蛋白将是特别感兴趣的，因为这些蛋白中没有一个具有已知的RNA结合活性。目标2继续在上一个循环中开始的工作，使用微阵列通过与复合物I的回收来鉴定PMR 1靶mRNA。这些将与通过增加PMR 1的表达而选择性减少并通过其敲低而选择性增加的mRNA进行比较。这些也将用于鉴定与Aim 1中的蛋白质一起定义底物mRNP的共有序列或结构特征。在表达活性PMR 1的细胞中，细胞运动性增加，目的3中的实验将使用细胞运动的成像、定量PCR和免疫荧光来检查运动性、PMR 1与Ena/VASP蛋白的结合及其向复合物I的募集之间的关系。这项工作的长期目标是了解PMR 1介导的mRNA衰变的分子机制，它是如何调节的，以及它在发育过程中控制基因表达的作用，对激素刺激的反应，以及恶性肿瘤。 公共卫生相关性：mRNA衰变是基因调控的关键步骤。PMR 1是一种mRNA核酸内切酶，由致癌酪氨酸激酶c-Src激活，降解不同的mRNA子集。PMR 1介导的mRNA衰变也与细胞运动性增加有关，这提高了这种形式的mRNA衰变、c-Src原癌基因和癌症之间的联系的可能性。这项研究旨在确定PMR 1衰变复合物的成分，确定PMR 1介导的衰变的范围，并将两者与癌细胞的侵袭性生长联系起来。

DESCRIPTION (provided by applicant): mRNA decay plays a central role in gene expression, with the half-life of virtually every mRNA controlled by regulatory sequences within the mRNA and their cognate binding proteins. Rapid turnover is a characteristic feature of mRNAs encoding growth factors, transcription factors, cytokines and cell signaling molecules, and the selective modulation of this process is one way of controlling the amount of these proteins. The decay of most mRNAs begins with shortening of the poly(A) tail, removal of the 5' cap and simultaneous 5'-3' and 3'-5' degradation of the mRNA body. With the exception of poly(A) shortening these processes act on nontranslat- ing mRNAs. However, the turnover of a subset of the transcriptome is catalyzed by endonuclease cleavage while mRNAs are engaged by translating ribosomes. The prototypical mRNA endonuclease is PMR1, an en- zyme that was originally identified as an estrogen-induced ribonuclease activity whose appearance on polysomes coincides with the destabilization of serum protein mRNAs. The hallmark of endonuclease- mediated mRNA decay is its selectivity for specific mRNAs. This is determined by the formation of an mRNP complex (termed Complex I) containing PMR1 and its translating substrate mRNA. To join this complex PMR1 must be phosphorylated on a tyrosine residue in the polysome-targeting domain of the protein, and the past funding cycle identified c-Src as the kinase that is responsible for this key activation step. This is the first example of direct involvement of an oncogenic tyrosine kinase in mRNA decay, and it raises the possibility that PMR1-mediated mRNA decay may be a target of c-Src in cancer. Consistent with this, PMR1 binds to the Ena/VASP proteins, which are regulators of the actin cytoskeleton, and cell motility is increased in cells ex- pressing catalytically-active PMR1. Aim 1 will use tandem affinity chromatography to recover the Complex I mRNP, identify its constituent proteins, and determine their role in mRNP assembly and mRNA decay. This is the first step toward deciphering the 'RNP code' for PMR1-mRNA decay. The SH2 domain containing protein that is the 'gatekeeper' for recruiting PMR1 to the mRNP will be of particular interest, since none of these has known RNA-binding activity. Aim 2 continues work begun in the last cycle using microarrays to identify PMR1 target mRNAs by their recovery with Complex I. These will be compared to mRNAs that are selectively reduced by increasing expression of PMR1 and selectively increased by its knockdown. These will also be used to identify shared sequence or structural features that together with proteins in Aim 1 define the substrate mRNP. Cell motility is increased in cells expressing active PMR1, and the experiments in Aim 3 will use imag- ing of cell movement, quantitative PCR and immunofluorescence to examine the relationship between motility, PMR1 binding to the Ena/VASP proteins, and its recruitment to Complex I. The long-term goal of this work is to understand the molecular mechanisms of PMR1-mediated mRNA decay, how it is regulated and its role in con- trolling gene expression during development, in response to hormonal stimuli, and in malignancy. PUBLIC HEALTH RELEVANCE: mRNA decay is a key step in gene regulation. PMR1 is an mRNA endonuclease that is activated by the oncogenic tyrosine kinase c-Src to degrade a distinct subset of mRNAs. PMR1-mediated mRNA decay is also associated with increased cell motility, raising the possibility of a link between this form of mRNA decay, the c- Src protooncogene and cancer. This research seeks to identify the components of the PMR1 decay complex, identify the scope of PMR1-mediated decay and relate both of these to the invasive growth of cancer cells.