Partho Sarothi Ray

A stress-responsive RNA switch regulates VEGFA expression.

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)-γ 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-γ-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.

TGF-beta-mediated phosphorylation of hnRNP E1 induces EMT via transcript-selective translational induction of Dab2 and ILEI.

Transforming growth factor-β (TGF-β) induces epithelial–mesenchymal transdifferentiation (EMT) accompanied by cellular differentiation and migration. Despite extensive transcriptomic profiling, the identification of TGF-β-inducible, EMT-specific genes has met with limited success. Here we identify a post-transcriptional pathway by which TGF-β modulates the expression of EMT-specific proteins and of EMT itself. We show that heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) binds a structural, 33-nucleotide TGF-β-activated translation (BAT) element in the 3′ untranslated region of disabled-2 (Dab2) and interleukin-like EMT inducer (ILEI) transcripts, and represses their translation. TGF-β activation leads to phosphorylation at Ser 43 of hnRNP E1 by protein kinase Bβ/Akt2, inducing its release from the BAT element and translational activation of Dab2 and ILEI messenger RNAs. Modulation of hnRNP E1 expression or its post-translational modification alters the TGF-β-mediated reversal of translational silencing of the target transcripts and EMT. These results suggest the existence of a TGF-β-inducible post-transcriptional regulon that controls EMT during the development and metastatic progression of tumours.

A post-transcriptional pathway represses monocyte VEGF-A expression and angiogenic activity

Monocyte‐macrophage activation by interferon (IFN)‐γ is a key initiating event in inflammation. Usually, the macrophage response is self‐limiting and inflammation resolves. Here, we describe a mechanism by which IFN‐γ contributes to inflammation resolution by suppressing expression of vascular endothelial growth factor‐A (VEGF‐A), a macrophage product that stimulates angiogenesis during chronic inflammation and tumorigenesis. VEGF‐A was identified as a candidate target of the IFN‐γ‐activated inhibitor of translation (GAIT) complex by bioinformatic analysis, and experimentally validated by messenger RNA–protein interaction studies. Although IFN‐γ induced persistent VEGF‐A mRNA expression, translation was suppressed by delayed binding of the GAIT complex to a specific element delineated in the 3′UTR. Translational silencing resulted in decreased VEGF‐A synthesis and angiogenic activity. Our results describe a unique anti‐inflammatory pathway in which IFN‐γ‐dependent induction of VEGF‐A mRNA is translationally silenced by the same stimulus, and they suggest the GAIT system directs a post‐transcriptional operon that contributes to inflammation resolution.

The HILDA Complex Coordinates a Conditional Switch in the 3′-Untranslated Region of the VEGFA mRNA

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.

Evolution of function of a fused metazoan tRNA synthetase

The origin and evolution of multidomain proteins are driven by diverse processes including fusion/fission, domain shuffling, and alternative splicing. The 20 aminoacyl-tRNA synthetases (AARS) constitute an ancient conserved family of multidomain proteins. The glutamyl-prolyl tRNA synthetase (EPRS) of bilaterian animals is unique among AARSs, containing two functional enzymes catalyzing ligation of glutamate and proline to their cognate transfer RNAs (tRNAs). The ERS and PRS catalytic domains in multiple bilaterian taxa are linked by variable number of helix-turn-helix domains referred to as WHEP-TRS domains. In addition to its canonical aminoacylation activities, human EPRS exhibits a noncanonical function as an inflammation-responsive regulator of translation. Recently, we have shown that the WHEP domains direct this auxiliary function of human EPRS by interacting with an mRNA stem-loop element (interferon-gamma-activated inhibitor of translation [GAIT] element). Here, we show that EPRS is present in the cnidarian Nematostella vectensis, which pushes the origin of the fused protein back to the cnidarian-bilaterian ancestor, 50-75 My before the origin of the Bilateria. Remarkably, the Nematostella EPRS mRNA is alternatively spliced to yield three isoforms with variable number and sequence of WHEP domains and with distinct RNA-binding activities. Whereas one isoform containing a single WHEP domain binds tRNA, a second binds both tRNA and GAIT element RNA. However, the third isoform contains two WHEP domains and like the human ortholog binds specifically to GAIT element RNA. These results suggest that alternative splicing of WHEP domains in the EPRS gene of the cnidarian-bilaterian ancestor gave rise to a novel molecular function of EPRS conserved during metazoan evolution.

Interplay between RNA-binding Protein HuR and microRNA-125b Regulates p53 mRNA Translation in Response to Genotoxic Stress

ABSTRACT Tumor suppressor protein p53 plays a crucial role in maintaining genomic integrity in response to DNA damage. Regulation of translation of p53 mRNA is a major mode of regulation of p53 expression under genotoxic stress. The AU/U-rich element-binding protein HuR has been shown to bind to p53 mRNA 3′UTR and enhance translation in response to DNA-damaging UVC radiation. On the other hand, the microRNA miR-125b is reported to repress p53 expression and stress-induced apoptosis. Here, we show that UVC radiation causes an increase in miR-125b level in a biphasic manner, as well as nuclear cytoplasmic translocation of HuR. Binding of HuR to the p53 mRNA 3′UTR, especially at a site adjacent to the miR-125b target site, causes dissociation of the p53 mRNA from the RNA-induced silencing complex (RISC) and inhibits the miR-125b-mediated translation repression of p53. HuR prevents the oncogenic effect of miR-125b by reversing the decrease in apoptosis and increase in cell proliferation caused by the overexpression of miR-125b. The antagonistic interplay between miR-125b and HuR might play an important role in fine-tuning p53 gene expression at the post-transcriptional level, and thereby regulate the cellular response to genotoxic stress.

Origin and evolution of glutamyl-prolyl tRNA synthetase WHEP domains reveal evolutionary relationships within Holozoa.

Repeated domains in proteins that have undergone duplication or loss, and sequence divergence, are especially informative about phylogenetic relationships. We have exploited divergent repeats of the highly structured, 50-amino acid WHEP domains that join the catalytic subunits of bifunctional glutamyl-prolyl tRNA synthetase (EPRS) as a sequence-informed repeat (SIR) to trace the origin and evolution of EPRS in holozoa. EPRS is the only fused tRNA synthetase, with two distinct aminoacylation activities, and a non-canonical translation regulatory function mediated by the WHEP domains in the linker. Investigating the duplications, deletions and divergence of WHEP domains, we traced the bifunctional EPRS to choanozoans and identified the fusion event leading to its origin at the divergence of ichthyosporea and emergence of filozoa nearly a billion years ago. Distribution of WHEP domains from a single species in two or more distinct clades suggested common descent, allowing the identification of linking organisms. The discrete assortment of choanoflagellate WHEP domains with choanozoan domains as well as with those in metazoans supported the phylogenetic position of choanoflagellates as the closest sister group to metazoans. Analysis of clustering and assortment of WHEP domains provided unexpected insights into phylogenetic relationships amongst holozoan taxa. Furthermore, observed gaps in the transition between WHEP domain groupings in distant taxa allowed the prediction of undiscovered or extinct evolutionary intermediates. Analysis based on SIR domains can provide a phylogenetic counterpart to palaentological approaches of discovering “missing links” in the tree of life.

The GAIT translational control system

The interferon (IFN)‐γ‐activated inhibitor of translation (GAIT) system directs transcript‐selective translational control of functionally related genes. In myeloid cells, IFN‐γ induces formation of a multiprotein GAIT complex that binds structural GAIT elements in the 3′‐untranslated regions (UTRs) of multiple inflammation‐related mRNAs, including ceruloplasmin and VEGF‐A, and represses their translation. The human GAIT complex is a heterotetramer containing glutamyl‐prolyl tRNA synthetase (EPRS), NS1‐associated protein 1 (NSAP1), ribosomal protein L13a (L13a), and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). A network of IFN‐γ‐stimulated kinases regulates recruitment and assembly of GAIT complex constituents. Activation of cyclin‐dependent kinase 5 (Cdk5), mammalian target of rapamycin complex 1 (mTORC1), and S6K1 kinases induces EPRS release from its parental multiaminoacyl tRNA synthetase complex to join NSAP1 in a ‘pre‐GAIT’ complex. Subsequently, the DAPK‐ZIPK kinase axis phosphorylates L13a, inducing release from the 60S ribosomal subunit and binding to GAPDH. The subcomplexes join to form the functional GAIT complex. Each constituent has a distinct role in the GAIT system. EPRS binds the GAIT element in target mRNAs, NSAP1 negatively regulates mRNA binding, L13a binds eIF4G to block ribosome recruitment, and GAPDH shields L13a from proteasomal degradation. The GAIT system is susceptible to genetic and condition‐specific regulation. An N‐terminus EPRS truncate is a dominant‐negative inhibitor ensuring a ‘translational trickle’ of target transcripts. Also, hypoxia and oxidatively modified lipoproteins regulate GAIT activity. Mouse models exhibiting absent or genetically modified GAIT complex constituents are beginning to elucidate the physiological role of the GAIT system, particularly in the resolution of chronic inflammation. Finally, GAIT‐like systems in proto‐chordates suggests an evolutionarily conserved role of the pathway in innate immunity. WIREs RNA 2018, 9:e1441. doi: 10.1002/wrna.1441

29 Noncanonical Functions of Aminoacyl-tRNA Synthetases in Translational Control

Aminoacyl-tRNA synthetases (AARSs) are ancient enzymes, ubiquitous in the three domains of life, that catalyze the ligation of amino acids to cognate tRNAs (Ibba and Soll 2000; Ribas de Pouplana and Schimmel 2001). They are uniquely responsible for deciphering the genetic code, reading the genetic information in the tRNA anticodon, and ligating the appropriate amino acid to the terminal ribose of the conserved CCA sequence at the 3′ end of the tRNA. In most prokaryotes, there are 20 AARSs, one for each major amino acid. Lower eukaryotes have separate cytoplasmic and nuclear-encoded mitochondrial (as well as chloroplastic) AARSs (Sissler et al. 2005). In all vertebrates, and in some invertebrates, the 20 cytoplasmic AARS activities are contained in 19 proteins; the bifunctional GluProRS expresses two enzyme activities in a single polypeptide chain. All synthetases contain catalytic and tRNA anticodon recognition sites in separate domains, and belong to one of two structurally distinct classes (Ibba and Soll 2000). The 10 Class I enzymes have a Rossman fold in the active site, bind the minor groove of the tRNA acceptor stem, and aminoacylate ribose at the 2′-OH position. In contrast, the 10 Class II enzymes have an antiparallel β-sheet in the active site, bind the major groove of the acceptor stem, and aminoacylate ribose at 3′-OH. Class I and II enzymes can be further grouped into subclasses that exhibit additional structural similarities and that recognize related amino acid substrates. In vertebrate cells, 9 AARS activities in 8 enzymes (including the bifunctional GluProRS, also...

Equality of the sexes: found in translation.

Male fruit flies (and humans) have half the number of X chromosomes that females have, yet the expression of X-linked proteins in males is the same because of a sex-specific compensatory mechanism. In this issue of Cell, report that a unique two-stage translation silencing mechanism thwarts synthesis of a male-specific transcription activator in female flies, ensuring that gene dosage compensation occurs only in males.