SUMMARY Translational readthrough, observed primarily in less complex organisms from viruses to Drosophila, expands the proteome by translating select transcripts beyond the canonical stop codon. Here we show that vascular endothelial growth factor-A (VEGFA) mRNA in mammalian endothelial cells undergoes programmed translational readthrough (PTR) generating VEGF-Ax, an isoform containing a unique 22-amino acid C-terminus extension. A cis-acting element in the VEGFA 3′UTR serves a dual function, not only encoding the appended peptide, but also directing the PTR by decoding the UGA stop codon as serine. Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 binds this element and promotes readthrough. Remarkably, VEGF-Ax exhibits anti-angiogenic activity in contrast to the pro-angiogenic activity of VEGF-A. Pathophysiological significance of VEGF-Ax is indicated by robust expression in multiple human tissues, but depletion in colon adenocarcinoma. Furthermore, genome-wide analysis revealed AGO1 and MTCH2 as authentic readthrough targets. Overall, our studies reveal a novel protein-regulated PTR event in a vertebrate system.

Metabolic pathways contributing to adiposity and aging are activated by the mammalian target of rapamycin complex 1 (mTORC1) and p70 ribosomal protein S6 kinase 1 (S6K1) axis1–3. However, known mTORC1-S6K1 targets do not account for observed loss-of-function phenotypes, suggesting additional downstream effectors4–6. Here we identify glutamyl-prolyl tRNA synthetase (EPRS) as an mTORC1-S6K1 target that contributes importantly to adiposity and aging. EPRS phosphorylation at Ser999 by mTORC1-S6K1 induces its release from the aminoacyl tRNA multisynthetase complex (MSC), required for execution of noncanonical functions beyond protein synthesis7,8. To investigate physiological function of EPRS phosphorylation, we generated EPRS knock-in mice bearing phospho-deficient Ser999-to-Ala (S999A) and phospho-mimetic (S999D) mutations. Homozygous S999A mice exhibited low body weight, reduced adipose tissue mass, and increased lifespan, thereby displaying notable similarities with S6K1-deficient mice9–11 and mice with adipocyte-specific deficiency of raptor, an mTORC1 constituent12. Substitution of the EPRS S999D allele in S6K1-deficient mice normalized body mass and adiposity, indicating EPRS phosphorylation mediates S6K1-dependent metabolic responses. In adipocytes, insulin stimulated S6K1-dependent EPRS phosphorylation and release from the MSC. Interaction screening revealed phospho-EPRS binds Slc27a1 (i.e., fatty acid transport protein 1, FATP1)13–15, inducing its translocation to the plasma membrane and long-chain fatty acid uptake. Thus, EPRS and FATP1 are terminal mTORC1-S6K1 axis effectors critical for metabolic phenotypes.

No abstract

SUMMARY Post-transcriptional regulatory mechanisms superimpose “fine-tuning” control upon “on-off” switches characteristic of gene transcription. We have exploited computational modeling with experimental validation to resolve an anomalous relationship between mRNA expression and protein synthesis. Differential GAIT (Gamma-interferon Activated Inhibitor of Translation) complex activation repressed VEGF-A synthesis to a low, constant rate despite high, variable VEGFA mRNA expression. Dynamic model simulations indicated the presence of an unidentified, inhibitory GAIT element-interacting factor. We discovered a truncated form of glutamyl-prolyl tRNA synthetase (EPRS), the GAIT constituent that binds the 3’-UTR GAIT element in target transcripts. The truncated protein, EPRSN1, prevents binding of functional GAIT complex. EPRSN1 mRNA is generated by a remarkable polyadenylation-directed conversion of a Tyr codon in the EPRS coding sequence to a stop codon (PAY*). By low-level protection of GAIT element-bearing transcripts, EPRSN1 imposes a robust “translational trickle” of target protein expression. Genome-wide analysis shows PAY* generates multiple truncated transcripts thereby contributing to transcriptome expansion.

No abstract

SUMMARY The tumor vascular microenvironment supports tumorigenesis by supplying not only oxygen and diffusible nutrients but also by secreting soluble factors that promote tumorigenesis. Here we identify a feed-forward mechanism in which endothelial cells (EC), in response to tumor-derived mediators, release angiocrines driving aberrant vascularization and glioblastoma multiforme (GBM) progression through a hypoxia-independent induction of hypoxia-inducible factor (HIF)-1α. Phosphorylation of profilin-1 (Pfn-1) at Tyr129 in EC induces binding to tumor suppressor protein von Hippel-Lindau (VHL), prevents VHL-mediated degradation of prolyl-hydroxylated HIF-1α, culminating in HIF-1α accumulation even in normoxia. Elevated HIF-1α induces expression of multiple angiogenic factors, leading to vascular abnormality and tumor progression. In a genetic model of GBM, mice with an EC-specific defect in Pfn-1 phosphorylation exhibit reduced tumor angiogenesis, normalized vasculature, and improved survival. Moreover, EC-specific Pfn-1 phosphorylation is associated with tumor aggressiveness in human glioma. These findings suggest that targeting Pfn-1 phosphorylation may offer a selective strategy for therapeutic intervention of malignant solid tumors.

The HILDA complex coordinates three regulatory elements located in the 3′ UTR of the VEGFA mRNA in a RNA switch that controls translation in response to inflammation and hypoxia.

Cell migration paths of mammary epithelial cells (expressing different versions of the promigratory tyrosine kinase receptor Her2/Neu) were analyzed within a bimodal framework that is a generalization of the run-and-tumble description applicable to bacterial migration. The mammalian cell trajectories were segregated into two types of alternating modes, namely, the “directional-mode” (mode I, the more persistent mode, analogous to the bacterial run phase) and the “re-orientation-mode” (mode II, the less persistent mode, analogous to the bacterial tumble phase). Higher resolution (more pixel information, relative to cell size) and smaller sampling intervals (time between images) were found to give a better estimate of the deduced single cell dynamics (such as directional-mode time and turn angle distribution) of the various cell types from the bimodal analysis. The bimodal analysis tool permits the deduction of short-time dynamics of cell motion such as the turn angle distributions and turn frequencies during the course of cell migration compared to standard methods of cell migration analysis. We find that the two-hour mammalian cell tracking data do not fall into the diffusive regime implying that the often-used random motility expressions for mammalian cell motion (based on assuming diffusive motion) are invalid over the time steps (fraction of minute) typically used in modeling mammalian cell migration.