A stress-responsive RNA switch regulates VEGFA expression

Abstract

Ligand binding to structural elements in the non-coding regions of messenger RNA modulates gene expression. Ligands such as free metabolites or other small molecules directly bind and induce conformational changes in regulatory RNA elements known as riboswitches. Other types of RNA switches are activated by complexed metabolites-for example, RNA-ligated metabolites such as aminoacyl-charged transfer RNA in the T-box system, or protein-bound metabolites in the glucose- or amino-acid-stimulated terminator-anti-terminator systems. All of these switch types are found in bacteria, fungi and plants. Here we report an RNA switch in human vascular endothelial growth factor-A (VEGFA, also known as VEGF) mRNA 3' untranslated region (UTR) that integrates signals from interferon (IFN)-gamma and hypoxia to regulate VEGFA translation in myeloid cells. Analogous to riboswitches, the VEGFA 3' UTR undergoes a binary conformational change in response to environmental signals. However, the VEGFA 3' UTR switch is metabolite-independent, and the conformational change is dictated by mutually exclusive, stimulus-dependent binding of proteins, namely, the IFN-gamma-activated inhibitor of translation complex and heterogeneous nuclear ribonucleoprotein L (HNRNPL, also known as hnRNP L). We speculate that the VEGFA switch represents the founding member of a family of signal-mediated, protein-dependent RNA switches that evolved to regulate gene expression in multicellular animals in which the precise integration of disparate inputs may be more important than the rapidity of response.

Figure 1. Suppression of GAIT-mediated translation silencing of VEGF by hypoxia

a, VEGF in lysates from U937 cells treated with IFN-γ in normoxia (Nmx.) or hypoxia (Hpx.) was determined by immunoblot (IB) and RT-PCR; GAPDH was probed as control. b, In vitro translation of Fluc reporter RNAs bearing VEGF 3’UTR_11-900 (left) or Cp 3’UTR (right) in presence of cytosolic lysates and control Rluc RNA lacking a 3’UTR. c, Schematic of RNA elements in VEGF 3’UTR and HSR. d, In vitro translation of reporter RNAs bearing VEGF HSR (left) or GAIT element (right) in presence of cytosolic lysates. e, U937 cells were nucleofected with pcDNA3-Fluc reporters bearing VEGF HSR (top) or GAIT element (bottom). Cells were co-transfected with a plasmid expressing Rluc under SV40 promoter. Relative luciferase activity (Fluc/Rluc) was expressed as mean ± s.d. (3 experiments).

Figure 2. hnRNP L binding to HSR restores VEGF translation in hypoxia

a, Lysates from IFN-γ-treated cells were subjected to UV-crosslinking with [³²P]UTP-labeled VEGF HSR RNA before and after immunodepletion with anti-hnRNP L (left) or anti-EPRS (right) antibodies. Effective depletion was shown by IB. b, Excess DNA oligomer antisense to hnRNP L binding site (AS_{3’UTR,332-357}) blocks binding of hnRNP L to HSR RNA. c, Inhibition of hnRNP L binding by AS_{3’UTR,332-357} restores translational silencing of reporter RNA in hypoxia. d, siRNA-mediated knockdown of hnRNP L induces translational repression of VEGF in hypoxic cells. Lysates from cells transfected with hnRNP L (left) and control (right) siRNAs were immunoblotted with anti-VEGF, -hnRNP L and -GAPDH antibodies.

Figure 3. hnRNP L is regulated by stimulus-dependent proteasomal degradation

a, Lysates from U937 cells incubated in the absence or presence of IFN-γ were immunoblotted with anti-hnRNP L and -GAPDH antibodies. b, Immunoblot of lysates from cells treated with MG132 (200 nM). c, In vitro translation of VEGF HSR reporter RNA in presence of cell lysates.

Figure 4. Protein-dependent switching of the VEGF 3’UTR HSR

a, Secondary structure of VEGF HSR predicted by Mfold shows GAIT element (green), hnRNP L binding site (red), and stem stability sequence (blue). TP is lowest free energy conformer predicted by Mfold (left). TS conformer was generated by introducing experimentally-determined base-pairing constraints in GAIT element stem (right). Strong and weak RNase cleavage sites are marked by red and blue circles, respectively. Key signature cleavage sites are indicated (*, **). b, ³²P-endlabeled VEGF HSR RNA was probed with RNase A under non-denaturing (lane 2) and denaturing (lane 3) conditions. Cleavages corresponding to predicted signature sites are indicated (*, **). c, RNase A probing of VEGF HSR RNA under non-denaturing conditions, or after the RNA was denatured and renatured. d, VEGF HSR RNA was incubated with cell lysates treated with IFN-γ for 24 h under normoxia or hypoxia, or with lysates immunodepleted with anti-hnRNP L and anti-EPRS antibodies, and subjected to RNase A-mediated cleavage under non-denaturing conditions after protein removal. e, Proposed pathway that switches the VEGF HSR to the TP conformer in the presence of IFN-γ and hypoxia (left), or to the TS conformer in the presence of IFN-γ and normoxia (right). f, Truth table showing AND NOT Boolean logic function of the VEGF RNA switch integrating signals from IFN-γ and hypoxia.