Hormonal Regulation of Messenger RNA Stability

描述(申请人提供)：信使核糖核酸衰变在基因表达中起核心作用，几乎每个信使核糖核酸的半衰期由信使核糖核酸及其同源结合蛋白中的调控序列控制。快速周转是编码生长因子、转录因子、细胞因子和细胞信号分子的mRNAs的一个特征，对这一过程的选择性调节是控制这些蛋白质数量的一种方式。大多数mRNAs的衰变始于Poly(A)尾巴的缩短，5‘端帽子的去掉，以及mRNA体的5’-3‘和3’-5‘同时降解。除了Poly(A)缩短外，这些过程作用于非翻译的mRNA。然而，转录组的一部分的周转是由核酸内切酶催化的，而mRNAs是通过翻译核糖体进行的。其原型是PMR1，一种最初被鉴定为雌激素诱导的核糖核酸酶活性的酶，其在多聚体上的出现与血清蛋白mRNAs的不稳定相一致。核酸内切酶介导的信使核糖核酸衰变的特点是对特定的信使核糖核酸具有选择性。这是由含有PMR1及其翻译底物mRNA的mRNP复合体(称为复合体I)的形成所决定的。为了加入这个复合体，PMR1必须在蛋白质的多聚体靶向结构域的酪氨酸残基上被磷酸化，而过去的资金周期确定c-Src是负责这一关键激活步骤的激酶。这是第一个直接涉及致癌酪氨酸激酶参与mRNA衰变的例子，它增加了PMR1介导的mRNA衰变可能是c-Src在癌症中的靶点的可能性。与此一致的是，PMR1与作为肌动蛋白细胞骨架调节因子的Ena/Vasp蛋白结合，在表达催化活性PMR1的细胞中细胞运动性增加。目的1将利用串联亲和层析回收复合体I mRNP，鉴定其组成蛋白，并确定它们在mRNP组装和mRNA衰退中的作用。这是破译PMR1-信使核糖核酸衰变的“RNP密码”的第一步。含有SH2结构域的蛋白质是将PMR1招募到mRNP的“守门人”，这将是人们特别感兴趣的，因为这些蛋白质中没有一个具有已知的RNA结合活性。AIM 2继续上一个周期开始的工作，使用微阵列通过与复合体I的恢复来识别PMR1靶mRNAs。这些mRNAs将与通过增加PMR1的表达而选择性地减少和通过其敲除而选择性地增加的mRNAs进行比较。这些也将被用来识别共同的序列或结构特征，这些特征与目标1中的蛋白质一起定义底物mRNP。在表达活性PMR1的细胞中，细胞的运动性增加，目标3中的实验将使用细胞运动成像、定量PCR和免疫荧光来检测运动性、PMR1与Ena/Vasp蛋白的结合以及它与复合体I的募集之间的关系。这项工作的长期目标是了解PMR1介导的mRNA衰退的分子机制，它是如何调节的，以及它在发育过程中、对激素刺激的反应以及在恶性肿瘤中控制基因表达的作用。 与公共健康相关：信使核糖核酸的衰退是基因调控的关键一步。PMR1是一种mRNA内切酶，被致癌的酪氨酸激酶c-Src激活，以降解不同的mRNAs亚群。PMR1介导的信使核糖核酸衰变也与细胞活力增加有关，增加了这种形式的信使核糖核酸衰变、c-Src原癌基因与癌症之间联系的可能性。这项研究试图确定PMR1衰变复合体的成分，确定PMR1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.