TDP-43 stabilises the processing intermediates of mitochondrial transcripts

The 43-kDa trans-activating response region DNA-binding protein 43 (TDP-43) is a product of a causative gene for amyotrophic lateral sclerosis (ALS). Despite of accumulating evidence that mitochondrial dysfunction underlies the pathogenesis of TDP-43–related ALS, the roles of wild-type TDP-43 in mitochondria are unknown. Here, we show that the small TDP-43 population present in mitochondria binds directly to a subset of mitochondrial tRNAs and precursor RNA encoded in L-strand mtDNA. Upregulated expression of TDP-43 stabilised the processing intermediates of mitochondrial polycistronic transcripts and their products including the components of electron transport and 16S mt-rRNA, similar to the phenotype observed in cells deficient for mitochondrial RNase P. Conversely, TDP-43 deficiency reduced the population of processing intermediates and impaired mitochondrial function. We propose that TDP-43 has a novel role in maintaining mitochondrial homeostasis by regulating the processing of mitochondrial transcripts

Jagged1 DNA Copy Number Variation Is Associated with Poor Outcome in Liver Cancer

Notch signaling abnormalities are reported to be involved in the acceleration of malignancy in solid tumors and stem cell formation or regeneration in various organs. We analyzed specific genes for DNA copy number variations in liver cancer cells and investigated whether these factors relate to clinical outcome. Chromosome 20p, which includes the ligand for Notch pathways, Jagged1, was found to be amplified in several types of hepatoma cells, and its mRNA was up-regulated according to α-fetoprotein gene expression levels. Notch inhibition using Jagged1 shRNA and γ-secretase inhibitors produced significant suppression of cell growth in α-fetoprotein–producing cells with suppression of downstream genes. Using in vivo hepatoma models, the administration of γ-secretase inhibitors resulted in reduced tumor sizes and effective Notch inhibition with widespread apoptosis and necrosis of viable tumor cells. The γ-secretase inhibitors suppressed cell growth of the epithelial cell adhesion molecule–positive fraction in hepatoma cells, indicating that Notch inhibitors could suppress the stem cell features of liver cancer cells. Even in clinical liver cancer samples, the expression of α-fetoprotein and Jagged1 showed significant correlation, and amplification of the copy number of Jagged1 was associated with Jagged1 mRNA expression and poor survival after liver cancer surgical resection. In conclusion, amplification of Jagged1 contributed to mRNA expression that activates the Jagged1-Notch signaling pathway in liver cancer and led to poor outcome. © 2016 American Society for Investigative PathologyEmbargo Period 12 month

Structural basis of hydroxycarboxylic acid receptor signaling mechanisms through ligand binding

Abstract Hydroxycarboxylic acid receptors (HCA) are expressed in various tissues and immune cells. HCA2 and its agonist are thus important targets for treating inflammatory and metabolic disorders. Only limited information is available, however, on the active-state binding of HCAs with agonists. Here, we present cryo-EM structures of human HCA2-Gi and HCA3-Gi signaling complexes binding with multiple compounds bound. Agonists were revealed to form a salt bridge with arginine, which is conserved in the HCA family, to activate these receptors. Extracellular regions of the receptors form a lid-like structure that covers the ligand-binding pocket. Although transmembrane (TM) 6 in HCAs undergoes dynamic conformational changes, ligands do not directly interact with amino acids in TM6, suggesting that indirect signaling induces a slight shift in TM6 to activate Gi proteins. Structural analyses of agonist-bound HCA2 and HCA3 together with mutagenesis and molecular dynamics simulation provide molecular insights into HCA ligand recognition and activation mechanisms

Comparison of gene expression data obtained by RNAseq and by RT-qPCR.

<p>Expression level of selected genes in whole roots of ice plant subjected to 0 mM, 140 mM, 250 mM, or 500 mM NaCl treatment for 24 h as measured by RT-qPCR (white boxes). Gene expression was normalized against the housekeeping gene, <i>PolyUBQ10</i>. (n = 3, ±S.D.; **and * indicate <i>p</i><0.001 and <i>p</i><0.05 by Student’s <i>t</i>-test, respectively, the comparison is between 0 mM NaCl and the remaining NaCl concentrations). Gray boxes (NGS) indicate the level of gene expression obtained from RNAseq data. The RNAseq data are shown as the level of expression relative to 0 mM NaCl treatment which was set at a value of 1.</p

Flowchart of the experimental design used to obtain transcriptome data of the response of ice plant to salt stress.

<p>Trinity software was used for the assembly and blastx against the <i>Arabidopsis</i> protein database was used for annotation of the assembled ice plant genes.</p

Effect of NaCl on root growth in ice plants.

<p>(A-D) Five-day-old ice plant seedlings grown on the MS medium prior to NaCl treatment. (E-H) five-day-old ice plant seedlings after 24 h on MS containing 0 mM NaCl (E), 140 mM NaCl (F), 250 mM NaCl (G), or 500 mM NaCl (H). Bar = 1 cm. (I) Root length in five-day-old ice plant and <i>Arabidopsis thaliana</i> Col-0 ecotype seedlings (white boxes and white boxes with diagonal lines) and that of the same roots after 24 h on plates containing 140 mM NaCl (gray boxes and gray boxes with diagonal lines). Mean ± standard deviation (SD) (n = 15): *<i>p</i><0.001, as determined by a Student’s <i>t</i>-test. (J) Root length in five-day-old ice plants (white boxes) and that of the same roots after 24 h on the indicated concentration of NaCl (gray boxes). Mean ± standard deviation (SD) (n = 15): *<i>p</i><0.001, as determined by a Student’s <i>t</i>-test. (K-N) Morphological changes in root hairs on roots of ice plant seedlings treated with 0 mM (K), 140 mM (L), 250 mM (M) or 500 mM NaCl (N) for 24 h. Bar = 1 mm.</p

Comparison of salt-responsive genes in <i>Arabidopsis</i> and ice plant.

<p>Venn diagram of the genes that were up-regulated in <i>Arabidopsis</i> (obtained from microarray data) and ice plant (obtained from RNAseq data) (A). Enriched Gene Ontology (GO) categories of biological functions within the list of genes found to be significantly up-regulated by NaCl treatment (B). Venn diagram of the genes that were down-regulated in <i>Arabidopsis</i> and ice plant (C). GO categories of biological functions within the list of genes found to be significantly down-regulated by NaCl treatment (D).</p

RNA-Seq Analysis of the Response of the Halophyte, <i>Mesembryanthemum crystallinum</i> (Ice Plant) to High Salinity

<div><p>Understanding the molecular mechanisms that convey salt tolerance in plants is a crucial issue for increasing crop yield. The ice plant (<i>Mesembryanthemum crystallinum</i>) is a halophyte that is capable of growing under high salt conditions. For example, the roots of ice plant seedlings continue to grow in 140 mM NaCl, a salt concentration that completely inhibits <i>Arabidopsis thaliana</i> root growth. Identifying the molecular mechanisms responsible for this high level of salt tolerance in a halophyte has the potential of revealing tolerance mechanisms that have been evolutionarily successful. In the present study, deep sequencing (RNAseq) was used to examine gene expression in ice plant roots treated with various concentrations of NaCl. Sequencing resulted in the identification of 53,516 contigs, 10,818 of which were orthologs of <i>Arabidopsis</i> genes. In addition to the expression analysis, a web-based ice plant database was constructed that allows broad public access to the data. The results obtained from an analysis of the RNAseq data were confirmed by RT-qPCR. Novel patterns of gene expression in response to high salinity within 24 hours were identified in the ice plant when the RNAseq data from the ice plant was compared to gene expression data obtained from <i>Arabidopsis</i> plants exposed to high salt. Although ABA responsive genes and a sodium transporter protein (HKT1), are up-regulated and down-regulated respectively in both <i>Arabidopsis</i> and the ice plant; peroxidase genes exhibit opposite responses. The results of this study provide an important first step towards analyzing environmental tolerance mechanisms in a non-model organism and provide a useful dataset for predicting novel gene functions.</p></div