Our long-term goal is to understand how interactions between elements in noncoding regions of vertebrate mRNAs and their cognate binding proteins integrate signals from disparate stimuli to control translation. Tran- script-selective translationl control is mediated by interactions of RNA-binding proteins to sequence/structural elements in the 5'- or 3'-untranslated region (UTR) of target transcripts. Recently, an additional layer of com plexity has been recognized in which element pairs act as condition-dependent RNA switches. For example, riboswitches are proximate structural elements in the UTR of multiple bacterial mRNAs that undergo conforma- tional changes in response to specific metabolites. We have reported an analogous, stimulus-dependent switch in the 3'UTR of human vascular endothelial growth factor (VEGF)-A mRNA. VEGF-A mRNA contains adjoining elements that function as a novel stimulus-dependent, protein-directed RNA switch 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-?, phosphorylation of Glu-Pro tRNA synthetase (EPRS) initiates formation of the GAIT (IFN-Gamma-Activated Inhibitor of Translation) complex. EPRS binds a defined, GAIT element in the VEGF-A mRNA 3'UTR, stabilizing the translation-silencing conformer and inhibiting translation. However, superimposition of hypoxia on IFN-? stimulation induces phosphorylation of hnRNP L at Tyr359 that initiates assembly of a newly discovered 3-component HILDA complex 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: Myeloid cells integrate signals from IFN-? and hypoxia by inducing Tyr359 phosphorylation of hnRNP L and assembly of the HILDA complex, which in turn directs an RNA switch in the 3'-UTR of VEGF-A and other inflammation-related mRNAs to regulate translation. We will test this hypothesis by pursuing the following Specific Aims:
Aim 1 : Investigate molecular mechanisms regulating hnRNP L expression and localization;
Aim 2 : Determine the functions of HILDA components in regulating the RNA switch;
Aim 3 : Identify novel transcripts controlled by protein-directed RNA switches. We suggest 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 RNA switches in vertebrates that integrate physiological or pathological stimuli to control gene expression.
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 molecular mechanism may reveal insights into tumor growth and potential therapies to inhibit the process.
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