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

描述（由申请人提供）：我们的长期目标是了解脊椎动物mRNA非编码区元件与其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以控制翻译。转录选择性翻译控制由RNA结合蛋白与非编码区中的序列/结构元件的相互作用介导，最常见的是靶转录物的5 '-或3'-非翻译区（UTR）。除了蛋白质-RNA相互作用之外，RNA-RNA相互作用还调节基因表达，例如，在细菌mRNA的UTR中的核糖开关包含响应于特定代谢物而经历构象变化并控制翻译的邻近结构元件。我们实验室最近的实验表明，人血管内皮生长因子（VEGF）-A mRNA包含相邻的元素，作为一种新的刺激依赖性，蛋白质导向的核糖开关，存在于两个亚稳态构象：抑制沉默和抑制允许构象。二进制开关控制的两个信号，干扰素（IFN）-？和缺氧，其调节结合因子的量或活性。在IFN-？刺激细胞后，Glu-Pro tRNA合成酶（EPRS）从tRNA多合成酶复合物中释放出来，并加入GAIT（IFN-γ激活的翻译抑制剂）复合物。EPRS结合VEGF-A mRNA 3 'UTR中的一个确定的29-nt GAIT元件，稳定抑制-沉默构象异构体并抑制翻译。然而，叠加缺氧IFN-？刺激增加了异质核核糖核蛋白（hnRNP）L的水平，其结合GAIT元件直接上游的富含CA的元件，稳定了抑制允许构象异构体并允许VEGF-A表达。我们提出以下具体假设：骨髓细胞整合信号从IFN-？通过调节hnRNP L和GAIT复合物的相对量来调节VEGF-A 3 'UTR的构象，从而允许或抑制VEGF-A mRNA翻译。我们将通过以下具体目标来验证这一假设：目标1：确定二元开关功能所需的VEGF-A mRNA 3 'UTR中的序列和二级结构。目的2：确定VEGF-A 3 'UTR结合蛋白在开关功能中的作用。目的3：研究IFN-？和缺氧。我们推测，开关进化到维持VEGF-A的表达和缺氧，炎症组织中的血管生成。肿瘤也位于缺氧、炎症部位，可以利用VEGF-A开关刺激向内的血管生长，以提供营养并允许肿瘤生长。因此，VEGF-A开关代表了特异性抑制肿瘤巨噬细胞表达VEGF-A的新的治疗靶点。我们还推测，VEGF-A开关可能代表了脊椎动物中整合其他生理或病理刺激以控制基因表达的蛋白质导向核糖开关家族的创始成员。某些信使RNA通过改变其折叠结构和蛋白质产物的表达速率来响应其环境的变化。虽然这些“核糖开关”主要存在于细菌中，但我们在编码人血管内皮生长因子（VEGF）的mRNA中发现了类似的开关，VEGF是一种对血管形成至关重要的蛋白质。VEGF核糖开关对炎症和缺氧敏感，这是肿瘤环境中发现的两种情况，对其机制的理解可能揭示肿瘤生长和抑制该过程的潜在疗法的见解。

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.