A protein-directed riboswitch in the VEGF-A 3'UTR that regulates translation

描述(申请人提供)：我们的长期目标是了解脊椎动物mRNAs非编码区的元件与它们的同源结合蛋白之间的相互作用，以及它们如何整合来自不同刺激的信号来控制翻译。转录选择性翻译控制是由RNA结合蛋白与非编码区的序列/结构元件相互作用所介导的，最常见的是靶转录物的5‘或3’非翻译区。除了蛋白质-RNA相互作用外，RNA-RNA相互作用还调节基因的表达，例如细菌mRNAs非编码区中的核糖开关包含近端结构元件，这些元件对特定的代谢物做出反应而发生构象变化，并控制翻译。我们实验室最近的实验表明，人血管内皮细胞生长因子(VEGF)-A mRNA包含相邻元件，作为一种新的刺激依赖、蛋白质导向的核糖开关，以两种亚稳态构象存在：翻译沉默构象和翻译允许构象。二进制开关由两个信号积分控制，干扰素-？以及低氧，它们调节结合因子的数量或活性。在干扰素-β的刺激下，Glu-Pro tRNA合成酶(EPRS)从tRNA多合成酶复合体中释放出来，加入到GATIT(干扰素-伽马激活的翻译抑制物)复合体中。EPRS结合一个确定的，29个核苷酸的步态元件，在血管内皮生长因子-A的3‘非编码区，稳定翻译沉默构象，并抑制翻译。而低氧对干扰素-？刺激增加了异质核核糖核蛋白(HnRNP)的水平，L直接结合步态元素上游的富含CA的元素，稳定了翻译允许的构象，并允许血管内皮生长因子-A的表达。我们提出了以下具体假设：髓系细胞整合了来自干扰素？缺氧通过调节hnRNP、L和步态复合体的相对量，进而决定血管内皮生长因子-A 3‘端非编码区的构象，从而允许或抑制血管内皮生长因子-A的mRNA翻译。我们将通过追求以下特定目标来验证这一假说：目标1：确定二元开关功能所需的VEGF-AMRNA3‘UTR中的序列和二级结构。目的2：探讨血管内皮生长因子-A 3‘端非编码区结合蛋白在开关功能中的作用。目的：研究干扰素对血管内皮生长因子-A 3‘端非编码区二元开关的调节作用。和缺氧。我们假设，这种转换是为了维持缺氧、炎症组织中血管内皮生长因子-A的表达和血管生成。肿瘤也居住在缺氧、炎症部位，可能利用血管内皮生长因子-A的开关来刺激向内的血管生长，以提供营养并允许肿瘤生长。因此，血管内皮生长因子-A开关代表了一种新的治疗靶点，可以特异性抑制肿瘤巨噬细胞表达血管内皮生长因子-A。我们还推测，在脊椎动物中，血管内皮生长因子-A开关可能代表了蛋白质导向的核糖开关家族的创始成员，该家族整合了其他生理或病理刺激来控制基因的表达。与公共健康相关某些信使RNA通过改变它们的折叠结构和蛋白质产物的表达速率来响应环境的变化。虽然这些“核糖开关”主要是在细菌中发现的，但我们已经在编码人类血管内皮生长因子(VEGF)的mRNA中发现了类似的开关，这是一种对血管形成至关重要的蛋白质。血管内皮生长因子核糖开关对炎症和缺氧很敏感，这是肿瘤环境中发现的两种情况，了解其机制可能会揭示对肿瘤生长的洞察和抑制这一过程的潜在治疗方法。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the interactions between elements in noncoding regions of vertebrate mRNAs, and their cognate binding proteins, and how they integrate signals from disparate stimuli to control translation. Transcript-selective translational control is mediated by interactions of RNA-binding proteins to sequence/structural elements in non-coding regions, most often the 5'- or 3'-untranslated region (UTR) of the target transcript. In addition to protein-RNA interactions, RNA-RNA interactions also regulate gene expression, e.g., riboswitches in the UTR of bacterial mRNAs contain proximate structural elements that undergo conformational change in response to specific metabolites, and control translation. Recent experiments in our laboratory suggest that human vascular endothelial growth factor (VEGF)-A mRNA contains adjoining elements that function as a novel stimulus-dependent, protein-directed riboswitch that exists in two metastable conformations: a translation-silencing and a translation-permissive conformer. The binary switch is controlled by integration of two signals, interferon (IFN)-? and hypoxia, that regulate the amount or activity of the binding factors. Upon cell stimulation by IFN-?, Glu-Pro tRNA synthetase (EPRS) is released from its residence from the tRNA multisynthetase complex and joins the GAIT (IFN-Gamma-Activated Inhibitor of Translation) complex. EPRS binds a defined, 29-nt GAIT element in the VEGF-A mRNA 3'UTR, stabilizing the translation-silencing conformer and inhibiting translation. However, superimposition of hypoxia on IFN-? stimulation increases the level of heterogeneous nuclear ribonucleoprotein (hnRNP) L that binds a CA-rich element directly upstream of the GAIT element, stabilizing the translation-permissive conformer and allowing VEGF-A expression. We propose the following specific hypothesis: The myeloid cell integrates signals from IFN-? and hypoxia by regulating the relative amounts of hnRNP L and GAIT complex, which in turn dictate the conformation of the VEGF-A 3'UTR to either permit or suppress VEGF-A mRNA translation. We will test this hypothesis by pursuing the following Specific Aims: Aim 1: Determine sequences and secondary structures in the VEGF-A mRNA 3'UTR required for binary switch function. Aim 2: Determine the role of VEGF-A 3'UTR binding proteins in switch function. Aim 3: Investigate regulation of the VEGF-A 3'UTR binary switch by IFN-? and hypoxia. We hypothesize that the switch evolved to maintain VEGF-A expression and angiogenesis in hypoxic, inflammatory tissues. Tumors, also residing in hypoxic, inflammatory sites, may take advantage of the VEGF-A switch to stimulate inward blood vessel growth to provide nourishment and permit tumor growth. Thus, the VEGF-A switch represents a novel therapeutic target to specifically inhibit tumor macrophage expression of VEGF-A. We also speculate that the VEGF-A switch may represent the founding member of a family of protein-directed riboswitches in vertebrates that integrate other physiological or pathological stimuli to control gene expression. PUBLIC HEALTH RELEVANCE Certain messenger RNAs respond to changes in their environment by altering their folding structure and their rate of expression of protein products. Although these "riboswitches" are found primarily in bacteria, we have found a similar switch in the mRNA encoding human vascular endothelial growth factor (VEGF), a protein critical for blood vessel formation. The VEGF riboswitch is sensitive to inflammation and hypoxia, two conditions found in the tumor environment, and an understanding of its mechanism may reveal insights into tumor growth and potential therapies to inhibit the process.