TGFβ induces epithelial-mesenchymal transdifferentiation (EMT) accompanied by cellular differentiation and migration. Despite extensive transcriptomic profiling, identification of TGFβ-inducible, EMT-specific genes has met with limited success. Here, we identify a post-transcriptional pathway by which TGFβ modulates expression of EMT-specific proteins, and EMT itself. We show that heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) binds a structural, 33 nucleotides (nt) TGF beta-activated translation (BAT) element in the 3’-UTR of disabled-2 (Dab2) and interleukin-like EMT inducer (ILEI) transcripts, and repress their translation. TGFβ activation leads to phosphorylation at Ser43 of hnRNP E1 by protein kinase Bβ/Akt2, inducing its release from the BAT element and translational activation of Dab2 and ILEI mRNAs. Modulation of hnRNP E1 expression or its post-translational modification alters TGFβ-mediated reversal of translational silencing of the target transcripts and EMT. These results suggest the existence of a TGFβ-inducible post-transcriptional regulon that controls EMT during development and metastatic progression of tumors.

The p53 tumour suppressor protein has a crucial role in cell-cycle arrest and apoptosis. Previous reports show that the p53 messenger RNA is translated to produce an amino-terminaldeleted isoform (DN-p53) from an internal initiation codon, which acts as a dominant-negative inhibitor of full-length p53. Here, we show that two internal ribosome entry sites (IRESs) mediate the translation of both full-length and DN-p53 isoforms. The IRES directing the translation of full-length p53 is in the 5 0 -untranslated region of the mRNA, whereas the IRES mediating the translation of DN-p53 extends into the protein-coding region. The two IRESs show distinct cell-cycle phase-dependent activity, with the IRES for full-length p53 being active at the G2-M transition and the IRES for DN-p53 showing highest activity at the G1-S transition. These results indicate a novel translational control of p53 gene expression and activity.

Ligand binding to structural elements in noncoding regions of mRNA modulates gene expression1,2. Ligands such as free metabolites or other small molecules directly bind and induce conformational changes in regulatory RNA elements known as riboswitches1-4. Other types of RNA switches are activated by complexed metabolites, e.g., RNA-ligated metabolites such as aminoacyl-charged tRNA in the T-box system5, or protein-bound metabolites in the glucose- or amino acid-stimulated terminator-antiterminator systems6,7. All of these switch types are found in bacteria, fungi, and plants8-10. Here, we report an RNA switch in human vascular endothelial growth factor-A (VEGF) mRNA 3’UTR that integrates signals from interferon (IFN)-γ and hypoxia to regulate VEGF translation in myeloid cells. Analogous to riboswitches, the VEGF 3’UTR undergoes a binary conformational change in response to environmental signals. However, the VEGF 3’UTR switch is metabolite-independent, and the conformational change is dictated by mutually exclusive, stimulus-dependent binding of proteins, namely, the IFN-γ-activated inhibitor of translation (GAIT) complex11,12 and heterogenous nuclear ribonucleoprotein (hnRNP) L. We speculate the VEGF switch represents the founding member of a family of signal-mediated, protein-dependent RNA switches that evolved to regulate gene expression in multicellular animals where precise integration of disparate inputs may be more important than rapidity of response.

Functionally related genes are coregulated by specific RNA–protein interactions that direct transcript-selective translational control. In myeloid cells, interferon (IFN)-γ induces formation of the heterotetrameric, IFN-γ-activated inhibitor of translation (GAIT) complex comprising glutamyl-prolyl tRNA synthetase (EPRS), NS1-associated protein 1 (NSAP1), ribosomal protein L13a and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This complex binds defined 3′ untranslated region elements within a family of inflammatory mRNAs and suppresses their translation. IFN-γ-dependent phosphorylation, and consequent release of EPRS and L13a from the tRNA multisynthetase complex and 60S ribosomal subunit, respectively, regulates GAIT complex assembly. EPRS recognizes and binds target mRNAs, NSAP1 negatively regulates RNA binding, and L13a inhibits translation initiation by binding eukaryotic initiation factor 4G. Repression of a post-transcriptional regulon by the GAIT system might contribute to the resolution of chronic inflammation.

Phosphorylation of ribosomal protein L13a is essential for translational repression of inflammatory genes by the interferon (IFN)-gamma-activated inhibitor of translation (GAIT) complex. Here we show IFN-γ activates a kinase cascade in which death-associated protein kinase-1 (DAPK) activates zipper-interacting protein kinase (ZIPK), culminating in L13a phosphorylation on Ser77, L13a release from the ribosome, and translational silencing of GAIT element-bearing target mRNAs. Remarkably, both kinase mRNAs contain functional 3’UTR GAIT elements and thus the same inhibitory pathway activated by the kinases is co-opted to suppress their expression. Inhibition of DAPK and ZIPK facilitates cell restoration to the basal state and allows renewed induction of GAIT target transcripts by repeated stimulation. Thus, the DAPK-ZIPK-L13a axis forms a unique regulatory module that first represses, then re-permits inflammatory gene expression. We propose the module presents an important checkpoint in the macrophage “resolution of inflammation” program, and that pathway defects may contribute to chronic inflammatory disorders.

The heterotetrameric GAIT complex suppresses translation of selected mRNAs in interferon-gamma-activated monocytic cells. Specificity is dictated by glutamyl-prolyl tRNA synthetase (EPRS) binding to a 3'UTR element in target mRNAs. EPRS consists of two synthetase cores joined by a linker containing three WHEP domains of unknown function. Here we show the critical role of EPRS WHEP domains in targeting and regulating GAIT complex binding to RNA. The upstream WHEP pair directs high-affinity binding to GAIT element-bearing mRNAs, while the overlapping, downstream pair binds NSAP1, which inhibits mRNA binding. Interaction of EPRS with ribosomal protein L13a and GAPDH induces a conformational switch that rescues mRNA binding and restores translational control. Total reconstitution from purified components indicates that the four GAIT proteins are necessary and sufficient for self-assembly of a functional complex. Our results establish the essentiality of WHEP domains in the noncanonical function of EPRS in regulating inflammatory gene expression.

The p53 tumor suppressor protein plays a key role in maintaining genomic integrity. Enhanced expression of p53 during genotoxic stress is due to both increased protein stability and translational upregulation. Previous reports have shown that p53 mRNA is translated from an alternative initiation codon to produce N-terminal truncated isoform (ΔN-p53) besides fulllength p53. We have demonstrated that two internal ribosome entry sites (IRESs) regulate the translation of p53 and ΔN-p53 in a distinct cell cycle phase-dependent manner. Here, we report that polypyrimidine tract-binding protein (PTB) is a p53 IRES interacting trans-acting factor. PTB protein binds specifically to both the p53 IRESs but with differential affinity. siRNA-mediated knockdown of PTB protein results in reduction of activity of both IRESs and also the levels of both the isoforms. It is well known that DNA-damaging agents such as doxorubicin enhance the expression of p53. Our results indicate that during doxorubicin treatment, PTB protein translocates from nucleus to the cytoplasm, probably to facilitate IRES mediated p53 translation. These observations suggest that the relative cytoplasmic abundance of PTB protein, under DNA-damaging conditions, might contribute to regulating the coordinated expression of the p53 isoforms, owing to the differential affinity of PTB binding to the two p53 IRESs.

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