Post-transcriptional regulation in cranial neural crest cells expands developmental potential.

Developmental potential is progressively restricted after germ layer specification during gastrulation. However, cranial neural crest cells challenge this paradigm, as they develop from anterior ectoderm, yet give rise to both ectodermal derivatives of the peripheral nervous system and ectomesenchymal bone and cartilage. How cranial neural crest cells differentiate into multiple lineages is poorly understood. Here, we demonstrate that cranial neural crest cells possess a transient state of increased chromatin accessibility. We profile the spatiotemporal emergence of premigratory neural crest and find evidence of lineage bias toward either a neuronal or ectomesenchymal fate, with each expressing distinct factors from earlier stages of development. We identify the miR-302 miRNA family to be highly expressed in cranial neural crest cells and genetic deletion leads to precocious specification of the ectomesenchymal lineage. Loss of mir-302 results in reduced chromatin accessibility in the neuronal progenitor lineage of neural crest and a reduction in peripheral neuron differentiation. Mechanistically, we find that mir-302 directly targets Sox9 to slow the timing of ectomesenchymal neural crest specification and represses multiple genes involved in chromatin condensation to promote accessibility required for neuronal differentiation. Our findings reveal a posttranscriptional mechanism governed by miRNAs to expand developmental potential of cranial neural crest.

LIG4 mediates Wnt signalling-induced radioresistance.

Despite the implication of Wnt signalling in radioresistance, the underlying mechanisms are unknown. Here we find that high Wnt signalling is associated with radioresistance in colorectal cancer (CRC) cells and intestinal stem cells (ISCs). We find that LIG4, a DNA ligase in DNA double-strand break repair, is a direct target of ß-catenin. Wnt signalling enhances non-homologous end-joining repair in CRC, which is mediated by LIG4 transactivated by ß-catenin. During radiation-induced intestinal regeneration, LIG4 mainly expressed in the crypts is conditionally upregulated in ISCs, accompanied by Wnt/ß-catenin signalling activation. Importantly, among the DNA repair genes, LIG4 is highly upregulated in human CRC cells, in correlation with ß-catenin hyperactivation. Furthermore, blocking LIG4 sensitizes CRC cells to radiation. Our results reveal the molecular mechanism of Wnt signalling-induced radioresistance in CRC and ISCs, and further unveils the unexpected convergence between Wnt signalling and DNA repair pathways in tumorigenesis and tissue regeneration.

miR-543 and miR-590-3p regulate human mesenchymal stem cell aging via direct targeting of AIMP3/p18.

Previously, AIMP3 (aminoacyl-tRNAsynthetase-interacting multifunctional protein-3) was shown to be involved in the macromolecular tRNA synthetase complex or to act as a tumor suppressor. In this study, we report a novel role of AIMP3/p18 in the cellular aging of human mesenchymal stem cells (hMSCs). We found that AIMP3/p18 expression significantly increased in senescent hMSCs and in aged mouse bone marrow-derived MSCs (mBM-MSCs). AIMP3/p18 overexpression is sufficient to induce the cellular senescence phenotypes with compromised clonogenicity and adipogenic differentiation potential. To identify the upstream regulators of AIMP3/p18 during senescence, we screened for potential epigenetic regulators and for miRNAs. We found that the levels of miR-543 and miR-590-3p significantly decreased under senescence-inducing conditions, whereas the AIMP3/p18 protein levels increased. We demonstrate for the first time that miR-543 and miR-590-3p are able to decrease AIMP3/p18 expression levels through direct binding to the AIMP/p18 transcripts, which further compromised the induction of the senescence phenotype. Taken together, our data demonstrate that AIMP3/p18 regulates cellular aging in hMSCs possibly through miR-543 and miR-590-3p.

Promiscuous methionyl-tRNA synthetase mediates adaptive mistranslation to protect cells against oxidative stress.

Aminoacyl-tRNA synthetases (ARSs) acylate transfer (t)RNAs with amino acids. Charging tRNAs with the right amino acids is the first step in translation; therefore, the accurate and error-free functioning of ARSs is an essential prerequisite for translational fidelity. A recent study found that methionine (Met) can be incorporated into non-Met residues of proteins through methionylation of non-cognate tRNAs under conditions of oxidative stress. However, it was not understood how this mis-methionylation is achieved. Here, we report that methionyl-tRNA synthetase (MRS) is phosphorylated at Ser209 and Ser825 by extracellular signal-related kinase (ERK1/2) under conditions of stress caused by reactive oxygen species (ROS), and that this phosphorylated MRS shows increased affinity for non-cognate tRNAs with lower affinity for tRNA(Met), leading to an increase in Met residues in cellular proteins. The expression of a mutant MRS containing the substitutions S209D and S825D, mimicking dual phosphorylation, reduced ROS levels and cell death. This controlled inaccuracy of MRS seems to serve as a defense mechanism against ROS-mediated damage at the cost of translational fidelity.

Smad6 inhibits non-canonical TGF-ß1 signalling by recruiting the deubiquitinase A20 to TRAF6.

Transforming growth factor (TGF)-ß, a pivotal cytokine involved in a variety of cellular functions, transmits signals through Smad-dependent canonical and Smad-independent noncanonical pathways. In contrast to the canonical TGF-ß pathway, it is unknown how noncanonical TGF-ß pathways are negatively regulated. Here we demonstrate that the inhibitory Smad Smad6, but not Smad7, negatively regulates TGF-ß1-induced activation of the TRAF6-TAK1-p38 MAPK/JNK pathway, a noncanonical TGF-ß pathway. TGF-ß1-induced Smad6 abolishes K63-linked polyubiquitination of TRAF6 by recruiting the A20 deubiquitinating enzyme in AML-12 mouse liver cells and primary hepatocytes. In addition, the knockdown of Smad6 or A20 in an animal model or cell culture system maintains TAK1 and p38 MAPK/JNK phosphorylation and increases apoptosis, emphasizing the crucial role of the Smad6-A20 axis in negative regulation of the TGF-ß1-TRAF6-TAK1-p38 MAPK/JNK pathway. Therefore, our findings provide insight into the molecular mechanisms underlying negative regulation of noncanonical TGF-ß pathways.

Chemical suppression of an oncogenic splicing variant of AIMP2 induces tumour regression.

AIMP2 (aminoacyl-tRNA synthetase-interacting multifunctional protein 2) is a potent tumour suppressor that induces apoptosis in response to various oncogenic signals. AIMP2-DX2, an exon2-deleted splicing variant of AIMP2, is up-regulated in lung cancer and competitively suppresses the pro-apoptotic activity of AIMP2, resulting in tumorigenesis. In the present study we report that BC-DXI01, a synthetic compound, specifically reduces the cellular levels of AIMP2-DX2 through selective degradation of the AIMP2-DX2 mRNA transcript. We found that BC-DXI01-mediated cell death positively correlates with AIMP2-DX2 expression in the lung cancer cell lines tested. Administration of BC-DXI01 in a AIMP2-DX2-driven tumour xenograft mice model led to reduced tumour sizes and volumes of up to 60% in comparison with vehicle-treated mice group, consistent with decreases in AIMP2-DX2 transcript and protein levels. Taken together, our findings suggest that tumorigenic activity of AIMP2-DX2 can be controlled by the small chemical BC-DXI01, which can selectively suppress the AIMP2-DX2 mRNA transcript.

Rational drug repositioning guided by an integrated pharmacological network of protein, disease and drug.

BACKGROUND: The process of drug discovery and development is time-consuming and costly, and the probability of success is low. Therefore, there is rising interest in repositioning existing drugs for new medical indications. When successful, this process reduces the risk of failure and costs associated with de novo drug development. However, in many cases, new indications of existing drugs have been found serendipitously. Thus there is a clear need for establishment of rational methods for drug repositioning. RESULTS: In this study, we have established a database we call "PharmDB" which integrates data associated with disease indications, drug development, and associated proteins, and known interactions extracted from various established databases. To explore linkages of known drugs to diseases of interest from within PharmDB, we designed the Shared Neighborhood Scoring (SNS) algorithm. And to facilitate exploration of tripartite (Drug-Protein-Disease) network, we developed a graphical data visualization software program called phExplorer, which allows us to browse PharmDB data in an interactive and dynamic manner. We validated this knowledge-based tool kit, by identifying a potential application of a hypertension drug, benzthiazide (TBZT), to induce lung cancer cell death. CONCLUSIONS: By combining PharmDB, an integrated tripartite database, with Shared Neighborhood Scoring (SNS) algorithm, we developed a knowledge platform to rationally identify new indications for known FDA approved drugs, which can be customized to specific projects using manual curation. The data in PharmDB is open access and can be easily explored with phExplorer and accessed via BioMart web service (http://www.i-pharm.org/, http://biomart.i-pharm.org/).

Interaction of two translational components, lysyl-tRNA synthetase and p40/37LRP, in plasma membrane promotes laminin-dependent cell migration.

Although human lysyl-tRNA synthetase (KRS), an enzyme for protein synthesis, is often highly expressed in various cancer cells, its pathophysiological implications have not been understood. Here we found that KRS induces cancer cell migration through interaction with the 67-kDa laminin receptor (67LR) that is converted from ribosomal subunit p40. On laminin signal, KRS was phosphorylated at the T52 residue by p38MAPK and dissociated from the cytosolic multi-tRNA synthetase complex for membrane translocation. The importance of T52 phosphorylation for membrane translocation of KRS was confirmed by site-directed mutagenesis. In the membrane, turnover of 67LR was controlled by Nedd4-mediated ubiquitination, and KRS inhibited ubiquitin-dependent degradation of 67LR, thereby enhancing laminin-induced cell migration. This work thus unveiled a unique function of KRS in the control of cell migration and its pathological implication in metastasis.

Dual role of methionyl-tRNA synthetase in the regulation of translation and tumor suppressor activity of aminoacyl-tRNA synthetase-interacting multifunctional protein-3.

Mammalian methionyl-tRNA synthetase (MRS) plays an essential role in initiating translation by transferring Met to initiator tRNA (tRNA(i)(Met)). MRS also provides a cytosolic anchoring site for aminoacyl-tRNA synthetase-interacting multifunctional protein-3 (AIMP3)/p18, a potent tumor suppressor that is translocated to the nucleus for DNA repair upon DNA damage. However, the mechanism by which this enzyme mediates these two seemingly unrelated functions is unknown. Here we demonstrate that AIMP3 is released from MRS by UV irradiation-induced stress. Dissociation was induced by phosphorylation of MRS at Ser662 by general control nonrepressed-2 (GCN2) following UV irradiation. Substitution of Ser662 to Asp (S662D) induced a conformational change in MRS and significantly reduced its interaction with AIMP3. This mutant possessed significantly reduced MRS catalytic activity because of loss of tRNA(Met) binding, resulting in down-regulation of global translation. According to the Met incorporation assay using stable HeLa cells expressing MRS S662A or eukaryotic initiation factor-2 subunit-α (eIF2α) S51A, inactivation of GCN2-induced phosphorylation at eIF2α or MRS augmented the role of the other, suggesting a cross-talk between MRS and eIF2α for efficient translational inhibition. This work reveals a unique mode of regulation of global translation as mediated by aminoacyl-tRNA synthetase, specifically MRS, which we herein identified as a previously unidentified GCN2 substrate. In addition, our research suggests a dual role for MRS: (i) as a coregulator with eIF2α for GCN2-mediated translational inhibition; and (ii) as a coupler of translational inhibition and DNA repair following DNA damage by releasing bound tumor suppressor AIMP3 for its nuclear translocation.

Downregulation of lamin A by tumor suppressor AIMP3/p18 leads to a progeroid phenotype in mice.

Although AIMP3/p18 is normally associated with the macromolecular tRNA synthetase complex, recent reports have revealed a new role of AIMP3 in tumor suppression. In this study, we generated a transgenic mouse that overexpresses AIMP3 and characterized the associated phenotype in vivo and in vitro. Surprisingly, the AIMP3 transgenic mouse exhibited a progeroid phenotype, and the cells that overexpressed AIMP3 showed accelerated senescence and defects in nuclear morphology. We found that overexpression of AIMP3 resulted in proteasome-dependent degradation of mature lamin A, but not of lamin C, prelamin A, or progerin. The resulting imbalance in the protein levels of lamin A isoforms, namely altered stoichiometry of prelamin A and progerin to lamin A, appeared to be responsible for a phenotype that resembled progeria. An increase in the level of endogenous AIMP3 has been observed in aged human tissues and cells. The findings in this report suggest that AIMP3 is a specific regulator of mature lamin A and imply that enhanced expression of AIMP3 might be a factor driving cellular and/or organismal aging.

AIMP2/p38, the scaffold for the multi-tRNA synthetase complex, responds to genotoxic stresses via p53.

AIMP2/p38 is a scaffolding protein required for the assembly of the macromolecular tRNA synthetase complex. Here, we describe a previously unknown function for AIMP2 as a positive regulator of p53 in response to genotoxic stresses. Depletion of AIMP2 increased resistance to DNA damage-induced apoptosis, and introduction of AIMP2 into AIMP2-deficient cells restored the susceptibility to apoptosis. Upon DNA damage, AIMP2 was phosphorylated, dissociated from the multi-tRNA synthetase complex, and translocated into the nuclei of cells. AIMP2 directly interacts with p53, thereby preventing MDM2-mediated ubiquitination and degradation of p53. Mutations in AIMP2, affecting its interaction with p53, hampered its ability to activate p53. Nutlin-3 recovered the level of p53 and the susceptibility to UV-induced cell death in AIMP2-deficient cells. This work demonstrates that AIMP2, a component of the translational machinery, functions as proapoptotic factor via p53 in response to DNA damage.

Determination of three-dimensional structure and residues of the novel tumor suppressor AIMP3/p18 required for the interaction with ATM.

Although AIMP3/p18 is normally associated with the multi-tRNA synthetase complex via its specific interaction with methionyl-tRNA synthetase, it also works as a tumor suppressor by interacting with ATM, the upstream kinase of p53. To understand the molecular interactions of AIMP3 and the mechanisms involved, we determined the crystal structure of AIMP3 at 2.0-angstroms resolution and identified its potential sites of interaction with ATM. AIMP3 contains two distinct domains linked by a 7-amino acid (Lys57-Ser63) peptide, which contains a 3(10) helix. The 56-amino acid N-terminal domain consists of two helices into which three antiparallel beta strands are inserted, and the 111-amino acid C-terminal domain contains a bundle of five helices (Thr64-Tyr152) followed by a coiled region (Pro153-Leu169). Structural analyses revealed homologous proteins such as yeast glutamyl-tRNA synthetase, Arc1p, EF1Bgamma, and glutathione S-transferase and suggested two potential molecular binding sites. Moreover, mutations at the C-terminal putative binding site abolished the interaction between AIMP3 and ATM and the ability of AIMP3 to activate p53. Thus, this work identified the two potential molecular interaction sites of AIMP3 and determined the residues critical for its tumor-suppressive activity through the interaction with ATM.

AIMP3 haploinsufficiency disrupts oncogene-induced p53 activation and genomic stability.

AIMP3 (previously known as p18) was shown to up-regulate p53 in response to DNA damage. Here, we show that AIMP3 couples oncogenic stresses to p53 activation to prevent cell transformation. Growth factor- or Ras-dependent induction of p53 was blocked by single allelic loss of AIMP3 as well as by suppression of AIMP3. AIMP3 heterozygous cells became susceptible to cell transformation induced by oncogenes such as Ras or Myc alone. The transformed AIMP3+/- cells showed severe abnormality in cell division and chromosomal structure. Thus, AIMP3 plays crucial roles in p53-mediated tumor-suppressive response against oncogenic stresses via differential activation of ATM and ATR, and in the maintenance of genomic stability.