A mutation in the Arabidopsis HYL1 gene encoding a dsRNA binding protein affects responses to abscisic acid, auxin, and cytokinin

NASA Technical Reports Server (NTRS)

Lu, C.; Fedoroff, N.

2000-01-01

Both physiological and genetic evidence indicate interconnections among plant responses to different hormones. We describe a pleiotropic recessive Arabidopsis transposon insertion mutation, designated hyponastic leaves (hyl1), that alters the plant's responses to several hormones. The mutant is characterized by shorter stature, delayed flowering, leaf hyponasty, reduced fertility, decreased rate of root growth, and an altered root gravitropic response. It also exhibits less sensitivity to auxin and cytokinin and hypersensitivity to abscisic acid (ABA). The auxin transport inhibitor 2,3,5-triiodobenzoic acid normalizes the mutant phenotype somewhat, whereas another auxin transport inhibitor, N-(1-naph-thyl)phthalamic acid, exacerbates the phenotype. The gene, designated HYL1, encodes a 419-amino acid protein that contains two double-stranded RNA (dsRNA) binding motifs, a nuclear localization motif, and a C-terminal repeat structure suggestive of a protein-protein interaction domain. We present evidence that the HYL1 gene is ABA-regulated and encodes a nuclear dsRNA binding protein. We hypothesize that the HYL1 protein is a regulatory protein functioning at the transcriptional or post-transcriptional level.

Adaptations Required for Mitochondrial Import following Mitochondrial to Nucleus Gene Transfer of Ribosomal Protein S101[w

PubMed Central

Murcha, Monika W.; Rudhe, Charlotta; Elhafez, Dina; Adams, Keith L.; Daley, Daniel O.; Whelan, James

2005-01-01

The minimal requirements to support protein import into mitochondria were investigated in the context of the phenomenon of ongoing gene transfer from the mitochondrion to the nucleus in plants. Ribosomal protein 10 of the small subunit is encoded in the mitochondrion in soybean and many other angiosperms, whereas in several other species it is nuclear encoded and thus must be imported into the mitochondrial matrix to function. When encoded by the nuclear genome, it has adopted different strategies for mitochondrial targeting and import. In lettuce (Lactuca sativa) and carrot (Daucus carota), Rps10 independently gained different N-terminal extensions from other genes, following transfer to the nucleus. (The designation of Rps10 follows the following convention. The gene is indicated in italics. If encoded in the mitochondrion, it is rps10; if encoded in the nucleus, it is Rps10.) Here, we show that the N-terminal extensions of Rps10 in lettuce and carrot are both essential for mitochondrial import. In maize (Zea mays), Rps10 has not acquired an extension upon transfer but can be readily imported into mitochondria. Deletion analysis located the mitochondrial targeting region to the first 20 amino acids. Using site directed mutagenesis, we changed residues in the first 20 amino acids of the mitochondrial encoded soybean (Glycine max) rps10 to the corresponding amino acids in the nuclear encoded maize Rps10 until import was achieved. Changes were required that altered charge, hydrophobicity, predicted ability to form an amphiphatic α-helix, and generation of a binding motif for the outer mitochondrial membrane receptor, translocase of the outer membrane 20. In addition to defining the changes required to achieve mitochondrial localization, the results demonstrate that even proteins that do not present barriers to import can require substantial changes to acquire a mitochondrial targeting signal. PMID:16040655

Identification of fertiity restores for S male-sterile maize: beyond PPRs

USDA-ARS?s Scientific Manuscript database

Nuclear genes are essential for expression of the mitochondrial genome and for the function of mitochondrial protein complexes. Interaction of the plant mitochondrial and nuclear genetic systems is exemplified by mitochondrial-encoded cytoplasmic male sterility (CMS) under the control of nuclear fe...

Mutation of a Conserved Nuclear Export Sequence in Chikungunya Virus Capsid Protein Disrupts Host Cell Nuclear Import.

PubMed

Jacobs, Susan C; Taylor, Adam; Herrero, Lara J; Mahalingam, Suresh; Fazakerley, John K

2017-10-20

Transmitted by mosquitoes; chikungunya virus (CHIKV) is responsible for frequent outbreaks of arthritic disease in humans. CHIKV is an arthritogenic alphavirus of the Togaviridae family. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleus. In encephalitic alphaviruses nuclear translocation induces host cell shut off; however, the role of capsid protein nuclear localisation in arthritogenic alphaviruses remains unclear. Using replicon systems, we investigated a nuclear export sequence (NES) in the N-terminal region of capsid protein; analogous to that found in encephalitic alphavirus capsid but uncharacterised in CHIKV. The chromosomal maintenance 1 (CRM1) export adaptor protein mediated CHIKV capsid protein export from the nucleus and a region within the N-terminal part of CHIKV capsid protein was required for active nuclear targeting. In contrast to encephalitic alphaviruses, CHIKV capsid protein did not inhibit host nuclear import; however, mutating the NES of capsid protein (∆NES) blocked host protein access to the nucleus. Interactions between capsid protein and the nucleus warrant further investigation.

rigor mortis encodes a novel nuclear receptor interacting protein required for ecdysone signaling during Drosophila larval development.

PubMed

Gates, Julie; Lam, Geanette; Ortiz, José A; Losson, Régine; Thummel, Carl S

2004-01-01

Pulses of the steroid hormone ecdysone trigger the major developmental transitions in Drosophila, including molting and puparium formation. The ecdysone signal is transduced by the EcR/USP nuclear receptor heterodimer that binds to specific response elements in the genome and directly regulates target gene transcription. We describe a novel nuclear receptor interacting protein encoded by rigor mortis (rig) that is required for ecdysone responses during larval development. rig mutants display defects in molting, delayed larval development, larval lethality, duplicated mouth parts, and defects in puparium formation--phenotypes that resemble those seen in EcR, usp, E75A and betaFTZ-F1 mutants. Although the expression of these nuclear receptor genes is essentially normal in rig mutant larvae, the ecdysone-triggered switch in E74 isoform expression is defective. rig encodes a protein with multiple WD-40 repeats and an LXXLL motif, sequences that act as specific protein-protein interaction domains. Consistent with the presence of these elements and the lethal phenotypes of rig mutants, Rig protein interacts with several Drosophila nuclear receptors in GST pull-down experiments, including EcR, USP, DHR3, SVP and betaFTZ-F1. The ligand binding domain of betaFTZ-F1 is sufficient for this interaction, which can occur in an AF-2-independent manner. Antibody stains reveal that Rig protein is present in the brain and imaginal discs of second and third instar larvae, where it is restricted to the cytoplasm. In larval salivary gland and midgut cells, however, Rig shuttles between the cytoplasm and nucleus in a spatially and temporally regulated manner, at times that correlate with the major lethal phase of rig mutants and major switches in ecdysone-regulated gene expression. Taken together, these data indicate that rig exerts essential functions during larval development through gene-specific effects on ecdysone-regulated transcription, most likely as a cofactor for one or more nuclear receptors. Furthermore, the dynamic intracellular redistribution of Rig protein suggests that it may act to refine spatial and temporal responses to ecdysone during development.

The phosphoenolpyruvate/phosphate translocator is required for phenolic metabolism, palisade cell development, and plastid-dependent nuclear gene expression.

PubMed

Streatfield, S J; Weber, A; Kinsman, E A; Häusler, R E; Li, J; Post-Beittenmiller, D; Kaiser, W M; Pyke, K A; Flügge, U I; Chory, J

1999-09-01

The Arabidopsis chlorophyll a/b binding protein (CAB) gene underexpressed 1 (cue1) mutant underexpresses light-regulated nuclear genes encoding chloroplast-localized proteins. cue1 also exhibits mesophyll-specific chloroplast and cellular defects, resulting in reticulate leaves. Both the gene underexpression and the leaf cell morphology phenotypes are dependent on light intensity. In this study, we determine that CUE1 encodes the plastid inner envelope phosphoenolpyruvate/phosphate translocator (PPT) and define amino acid residues that are critical for translocator function. The biosynthesis of aromatics is compromised in cue1, and the reticulate phenotype can be rescued by feeding aromatic amino acids. Determining that CUE1 encodes PPT indicates the in vivo role of the translocator in metabolic partitioning and reveals a mesophyll cell-specific requirement for the translocator in Arabidopsis leaves. The nuclear gene expression defects in cue1 suggest that a light intensity-dependent interorganellar signal is modulated through metabolites dependent on a plastid supply of phosphoenolpyruvate.

Identification and characterization of an early gene in the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus

Treesearch

David S. Bischoff; James M. Slavicek

1995-01-01

The Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) gene encoding G22 was cloned and sequenced. The G22 gene codes for a 191 amino acid protein with a predicted Mr of 22000. Expression of G22 in a rabbit reticulocyte system generated a protein with an M...

Mutations in nuclear genes alter post-transcriptional regulation of mitochondrial genes.

USDA-ARS?s Scientific Manuscript database

Nuclear gene products are required for the expression of mitochondrial genes and elaboration of functional mitochondrial protein complexes. To better understand the roles of these nuclear genes, we exploited the mitochondrial encoded S-type of cytoplasmic male sterility (CMS-S) and developed a nove...

Mito-Nuclear Interactions Affecting Lifespan and Neurodegeneration in a Drosophila Model of Leigh Syndrome.

PubMed

Loewen, Carin A; Ganetzky, Barry

2018-04-01

Proper mitochondrial activity depends upon proteins encoded by genes in the nuclear and mitochondrial genomes that must interact functionally and physically in a precisely coordinated manner. Consequently, mito-nuclear allelic interactions are thought to be of crucial importance on an evolutionary scale, as well as for manifestation of essential biological phenotypes, including those directly relevant to human disease. Nonetheless, detailed molecular understanding of mito-nuclear interactions is still lacking, and definitive examples of such interactions in vivo are sparse. Here we describe the characterization of a mutation in Drosophila ND23 , a nuclear gene encoding a highly conserved subunit of mitochondrial complex 1. This characterization led to the discovery of a mito-nuclear interaction that affects the ND23 mutant phenotype. ND23 mutants exhibit reduced lifespan, neurodegeneration, abnormal mitochondrial morphology, and decreased ATP levels. These phenotypes are similar to those observed in patients with Leigh syndrome, which is caused by mutations in a number of nuclear genes that encode mitochondrial proteins, including the human ortholog of ND23 A key feature of Leigh syndrome, and other mitochondrial disorders, is unexpected and unexplained phenotypic variability. We discovered that the phenotypic severity of ND23 mutations varies depending on the maternally inherited mitochondrial background. Sequence analysis of the relevant mitochondrial genomes identified several variants that are likely candidates for the phenotypic interaction with mutant ND23 , including a variant affecting a mitochondrially encoded component of complex I. Thus, our work provides an in vivo demonstration of the phenotypic importance of mito-nuclear interactions in the context of mitochondrial disease. Copyright © 2018 by the Genetics Society of America.

The R35 residue of the influenza A virus NS1 protein has minimal effects on nuclear localization but alters virus replication through disrupting protein dimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lalime, Erin N.; Pekosz, Andrew, E-mail: apekosz@jhsph.edu

The influenza A virus NS1 protein has a nuclear localization sequence (NLS) in the amino terminal region. This NLS overlaps sequences that are important for RNA binding as well as protein dimerization. To assess the significance of the NS1 NLS on influenza virus replication, the NLS amino acids were individually mutated to alanines and recombinant viruses encoding these mutations were rescued. Viruses containing NS1 proteins with mutations at R37, R38 and K41 displayed minimal changes in replication or NS1 protein nuclear localization. Recombinant viruses encoding NS1 R35A were not recovered but viruses containing second site mutations at position D39 inmore » addition to the R35A mutation were isolated. The mutations at position 39 were shown to partially restore NS1 protein dimerization but had minimal effects on nuclear localization. These data indicate that the amino acids in the NS1 NLS region play a more important role in protein dimerization compared to nuclear localization. - Highlights: • Mutations were introduced into influenza NS1 NLS1. • NS1 R37A, R38A, K41A viruses had minimal changes in replication and NS1 localization. • Viruses from NS1 R35A rescue all contained additional mutations at D39. • NS1 R35A D39X mutations recover dimerization lost in NS1 R35A mutations. • These results reaffirm the importance of dimerization for NS1 protein function.« less

C. elegans Nuclear Envelope Proteins Emerin, MAN1, Lamin, and Nucleoporins Reveal Unique Timing of Nuclear Envelope Breakdown during Mitosis

PubMed Central

Lee, Kenneth K.; Gruenbaum, Yosef; Spann, Perah; Liu, Jun; Wilson, Katherine L.

2000-01-01

Emerin, MAN1, and LAP2 are integral membrane proteins of the vertebrate nuclear envelope. They share a 43-residue N-terminal motif termed the LEM domain. We found three putative LEM domain genes in Caenorhabditis elegans, designated emr-1, lem-2, and lem-3. We analyzed emr-l, which encodes Ce-emerin, and lem-2, which encodes Ce-MAN1. Ce-emerin and Ce-MAN1 migrate on SDS-PAGE as 17- and 52-kDa proteins, respectively. Based on their biochemical extraction properties and immunolocalization, both Ce-emerin and Ce-MAN1 are integral membrane proteins localized at the nuclear envelope. We used antibodies against Ce-MAN1, Ce-emerin, nucleoporins, and Ce-lamin to determine the timing of nuclear envelope breakdown during mitosis in C. elegans. The C. elegans nuclear envelope disassembles very late compared with vertebrates and Drosophila. The nuclear membranes remained intact everywhere except near spindle poles during metaphase and early anaphase, fully disassembling only during mid-late anaphase. Disassembly of pore complexes, and to a lesser extent the lamina, depended on embryo age: pore complexes were absent during metaphase in >30-cell embryos but existed until anaphase in 2- to 24-cell embryos. Intranuclear mRNA splicing factors disassembled after prophase. The timing of nuclear disassembly in C. elegans is novel and may reflect its evolutionary position between unicellular and more complex eukaryotes. PMID:10982402

Protein targeting and integration signal for the chloroplastic outer envelope membrane.

PubMed Central

Li, H M; Chen, L J

1996-01-01

Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol. With the exception of most quter envelope membrane proteins, nuclear-encoded chloroplastic proteins are synthesized with N-terminal extensions that contain the chloroplast targeting information of these proteins. Most outer membrane proteins, however, are synthesized without extensions in the cytosol. Therefore, it is not clear where the chloroplastic outer membrane targeting information resides within these polypeptides. We have analyzed a chloroplastic outer membrane protein, OEP14 (outer envelope membrane protein of 14 kD, previously named OM14), and localized its outer membrane targeting and integration signal to the first 30 amino acids of the protein. This signal consists of a positively charged N-terminal portion followed by a hydrophobic core, bearing resemblance to the signal peptides of proteins targeted to the endoplasmic reticulum. However, a chimeric protein containing this signal fused to a passenger protein did not integrate into the endoplasmic reticulum membrane. Furthermore, membrane topology analysis indicated that the signal inserts into the chloroplastic outer membrane in an orientation opposite to that predicted by the "positive inside" rule. PMID:8953775

Nuclear Receptors, RXR, and the Big Bang.

PubMed

Evans, Ronald M; Mangelsdorf, David J

2014-03-27

Isolation of genes encoding the receptors for steroids, retinoids, vitamin D, and thyroid hormone and their structural and functional analysis revealed an evolutionarily conserved template for nuclear hormone receptors. This discovery sparked identification of numerous genes encoding related proteins, termed orphan receptors. Characterization of these orphan receptors and, in particular, of the retinoid X receptor (RXR) positioned nuclear receptors at the epicenter of the "Big Bang" of molecular endocrinology. This Review provides a personal perspective on nuclear receptors and explores their integrated and coordinated signaling networks that are essential for multicellular life, highlighting the RXR heterodimer and its associated ligands and transcriptional mechanism. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification and characterization of a protein kinase gene in the Lymantria dispar nultinucleocapsid nuclear polyhedrosis virus

Treesearch

David S. Bischoff; James M. Slavicek

1994-01-01

The Lymantria dispar multinucleocapsid nuclear polyhedrosis virus (LdMNPV) gene encoding vPK has been cloned and sequenced. LdMNPV vPK shows a 24% amino acid identity to the catalytic domains of the eucaryotic protein kinases nPKC from rabbits, HSPKCE from humans, APLPKCB from Aplysia californica, and dPKC98F from ...

Ectromelia virus encodes a family of Ankyrin/F-box proteins that regulate NFκB

DOE Office of Scientific and Technical Information (OSTI.GOV)

Burles, Kristin, E-mail: burles@ualberta.ca; Buuren, Nicholas van; Barry, Michele

2014-11-15

A notable feature of poxviruses is their ability to inhibit the antiviral response, including the nuclear factor kappa B (NFκB) pathway. NFκB is a transcription factor that is sequestered in the cytoplasm until cell stimulation, and relies on the SCF (Skp1, culllin-1, F-box) ubiquitin ligase to target its inhibitor, IκBα, for degradation. IκBα is recruited to the SCF by the F-box domain-containing protein βTrCP. Here, we show that ectromelia virus, the causative agent of mousepox, encodes four F-box-containing proteins, EVM002, EVM005, EVM154, and EVM165, all of which contain Ankyrin (Ank) domains. The Ank/F-box proteins inhibit NFκB nuclear translocation, and thismore » inhibition is dependent on the F-box domain. We also demonstrate that EVM002, EVM005, EVM154, and EVM165 prevent IκBα degradation, suggesting that they target the SCF. This study identifies a new mechanism by which ectromelia virus inhibits NFκB. - Highlights: • Ectromelia virus encodes four Ank/F-box proteins, EVM002, EVM005, EVM154 and EVM165. • The Ank/F-box proteins inhibit NFκB nuclear translocation, dependent on the F-box. • The Ank/F-box proteins prevent IκBα degradation, suggesting they target the SCF. • Deletion of a single Ank/F-box gene from ECTV does not prevent viral NFκB inhibition. • This study identifies a new mechanism by which ectromelia virus inhibits NFκB.« less

Conservative and compensatory evolution in oxidative phosphorylation complexes of angiosperms with highly divergent rates of mitochondrial genome evolution.

PubMed

Havird, Justin C; Whitehill, Nicholas S; Snow, Christopher D; Sloan, Daniel B

2015-12-01

Interactions between nuclear and mitochondrial gene products are critical for eukaryotic cell function. Nuclear genes encoding mitochondrial-targeted proteins (N-mt genes) experience elevated rates of evolution, which has often been interpreted as evidence of nuclear compensation in response to elevated mitochondrial mutation rates. However, N-mt genes may be under relaxed functional constraints, which could also explain observed increases in their evolutionary rate. To disentangle these hypotheses, we examined patterns of sequence and structural evolution in nuclear- and mitochondrial-encoded oxidative phosphorylation proteins from species in the angiosperm genus Silene with vastly different mitochondrial mutation rates. We found correlated increases in N-mt gene evolution in species with fast-evolving mitochondrial DNA. Structural modeling revealed an overrepresentation of N-mt substitutions at positions that directly contact mutated residues in mitochondrial-encoded proteins, despite overall patterns of conservative structural evolution. These findings support the hypothesis that selection for compensatory changes in response to mitochondrial mutations contributes to the elevated rate of evolution in N-mt genes. We discuss these results in light of theories implicating mitochondrial mutation rates and mitonuclear coevolution as drivers of speciation and suggest comparative and experimental approaches that could take advantage of heterogeneity in rates of mtDNA evolution across eukaryotes to evaluate such theories. © 2015 The Author(s). Evolution © 2015 The Society for the Study of Evolution.

A Comprehensive Analysis of Nuclear-Encoded Mitochondrial Genes in Schizophrenia.

PubMed

Gonçalves, Vanessa F; Cappi, Carolina; Hagen, Christian M; Sequeira, Adolfo; Vawter, Marquis P; Derkach, Andriy; Zai, Clement C; Hedley, Paula L; Bybjerg-Grauholm, Jonas; Pouget, Jennie G; Cuperfain, Ari B; Sullivan, Patrick F; Christiansen, Michael; Kennedy, James L; Sun, Lei

2018-05-01

The genetic risk factors of schizophrenia (SCZ), a severe psychiatric disorder, are not yet fully understood. Multiple lines of evidence suggest that mitochondrial dysfunction may play a role in SCZ, but comprehensive association studies are lacking. We hypothesized that variants in nuclear-encoded mitochondrial genes influence susceptibility to SCZ. We conducted gene-based and gene-set analyses using summary association results from the Psychiatric Genomics Consortium Schizophrenia Phase 2 (PGC-SCZ2) genome-wide association study comprising 35,476 cases and 46,839 control subjects. We applied the MAGMA method to three sets of nuclear-encoded mitochondrial genes: oxidative phosphorylation genes, other nuclear-encoded mitochondrial genes, and genes involved in nucleus-mitochondria crosstalk. Furthermore, we conducted a replication study using the iPSYCH SCZ sample of 2290 cases and 21,621 control subjects. In the PGC-SCZ2 sample, 1186 mitochondrial genes were analyzed, among which 159 had p values < .05 and 19 remained significant after multiple testing correction. A meta-analysis of 818 genes combining the PGC-SCZ2 and iPSYCH samples resulted in 104 nominally significant and nine significant genes, suggesting a polygenic model for the nuclear-encoded mitochondrial genes. Gene-set analysis, however, did not show significant results. In an in silico protein-protein interaction network analysis, 14 mitochondrial genes interacted directly with 158 SCZ risk genes identified in PGC-SCZ2 (permutation p = .02), and aldosterone signaling in epithelial cells and mitochondrial dysfunction pathways appeared to be overrepresented in this network of mitochondrial and SCZ risk genes. This study provides evidence that specific aspects of mitochondrial function may play a role in SCZ, but we did not observe its broad involvement even using a large sample. Copyright © 2018 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Genomic organization of the human mi-er1 gene and characterization of alternatively spliced isoforms: regulated use of a facultative intron determines subcellular localization.

PubMed

Paterno, Gary D; Ding, Zhihu; Lew, Yuan-Y; Nash, Gord W; Mercer, F Corinne; Gillespie, Laura L

2002-07-24

mi-er1 (previously called er1) is a fibroblast growth factor-inducible early response gene activated during mesoderm induction in Xenopus embryos and encoding a nuclear protein that functions as a transcriptional activator. The human orthologue of mi-er1 was shown to be upregulated in breast carcinoma cell lines and breast tumours when compared to normal breast cells. In this report, we investigate the structure of the human mi-er1 (hmi-er1) gene and characterize the alternatively spliced transcripts and protein isoforms. hmi-er1 is a single copy gene located at 1p31.2 and spanning 63 kb. It contains 17 exons and includes one skipped exon, a facultative intron and three polyadenylation signals to produce 12 transcripts encoding six distinct proteins. hmi-er1 transcripts were expressed at very low levels in most human adult tissues and the mRNA isoform pattern varied with the tissue. The 12 transcripts encode proteins containing a common internal sequence with variable N- and C-termini. Three distinct N- and two distinct C-termini were identified, giving rise to six protein isoforms. The two C-termini differ significantly in size and sequence and arise from alternate use of a facultative intron to produce hMI-ER1alpha and hMI-ER1beta. In all tissues except testis, transcripts encoding the beta isoform were predominant. hMI-ER1alpha lacks the predicted nuclear localization signal and transfection assays revealed that, unlike hMI-ER1beta, it is not a nuclear protein, but remains in the cytoplasm. Our results demonstrate that alternate use of a facultative intron regulates the subcellular localization of hMI-ER1 proteins and this may have important implications for hMI-ER1 function.

Global analysis of host-pathogen interactions that regulate early stage HIV-1 replication

PubMed Central

König, Renate; Zhou, Yingyao; Elleder, Daniel; Diamond, Tracy L.; Bonamy, Ghislain M.C.; Irelan, Jeffrey T.; Chiang, Chih-yuan; Tu, Buu P.; De Jesus, Paul D.; Lilley, Caroline E.; Seidel, Shannon; Opaluch, Amanda M.; Caldwell, Jeremy S.; Weitzman, Matthew D.; Kuhen, Kelli L.; Bandyopadhyay, Sourav; Ideker, Trey; Orth, Anthony P.; Miraglia, Loren J.; Bushman, Frederic D.; Young, John A.; Chanda, Sumit K.

2008-01-01

Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA damage response and RNA splicing were identified as important modulators of early stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of post-translational modification, and nucleic acid binding proteins. Finally, fifteen proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multi-scale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate early steps of HIV-1 infection. PMID:18854154

Isolation, sequencing and expression of RED, a novel human gene encoding an acidic-basic dipeptide repeat.

PubMed

Assier, E; Bouzinba-Segard, H; Stolzenberg, M C; Stephens, R; Bardos, J; Freemont, P; Charron, D; Trowsdale, J; Rich, T

1999-04-16

A novel human gene RED, and the murine homologue, MuRED, were cloned. These genes were named after the extensive stretch of alternating arginine (R) and glutamic acid (E) or aspartic acid (D) residues that they contain. We term this the 'RED' repeat. The genes of both species were expressed in a wide range of tissues and we have mapped the human gene to chromosome 5q22-24. MuRED and RED shared 98% sequence identity at the amino acid level. The open reading frame of both genes encodes a 557 amino acid protein. RED fused to a fluorescent tag was expressed in nuclei of transfected cells and localised to nuclear dots. Co-localisation studies showed that these nuclear dots did not contain either PML or Coilin, which are commonly found in the POD or coiled body nuclear compartments. Deletion of the amino terminal 265 amino acids resulted in a failure to sort efficiently to the nucleus, though nuclear dots were formed. Deletion of a further 50 amino acids from the amino terminus generates a protein that can sort to the nucleus but is unable to generate nuclear dots. Neither construct localised to the nucleolus. The characteristics of RED and its nuclear localisation implicate it as a regulatory protein, possibly involved in transcription.

Global Genetic Determinants of Mitochondrial DNA Copy Number

PubMed Central

Zhang, Hengshan; Singh, Keshav K.

2014-01-01

Many human diseases including development of cancer is associated with depletion of mitochondrial DNA (mtDNA) content. These diseases are collectively described as mitochondrial DNA depletion syndrome (MDS). High similarity between yeast and human mitochondria allows genomic study of the budding yeast to be used to identify human disease genes. In this study, we systematically screened the pre-existing respiratory-deficient Saccharomyces cerevisiae yeast strains using fluorescent microscopy and identified 102 nuclear genes whose deletions result in a complete mtDNA loss, of which 52 are not reported previously. Strikingly, these genes mainly encode protein products involved in mitochondrial protein biosynthesis process (54.9%). The rest of these genes either encode protein products associated with nucleic acid metabolism (14.7%), oxidative phosphorylation (3.9%), or other protein products (13.7%) responsible for bud-site selection, mitochondrial intermembrane space protein import, assembly of cytochrome-c oxidase, vacuolar protein sorting, protein-nucleus import, calcium-mediated signaling, heme biosynthesis and iron homeostasis. Thirteen (12.7%) of the genes encode proteins of unknown function. We identified human orthologs of these genes, conducted the interaction between the gene products and linked them to human mitochondrial disorders and other pathologies. In addition, we screened for genes whose defects affect the nuclear genome integrity. Our data provide a systematic view of the nuclear genes involved in maintenance of mitochondrial DNA. Together, our studies i) provide a global view of the genes regulating mtDNA content; ii) provide compelling new evidence toward understanding novel mechanism involved in mitochondrial genome maintenance and iii) provide useful clues in understanding human diseases in which mitochondrial defect and in particular depletion of mitochondrial genome plays a critical role. PMID:25170845

Nuclear-Cytoplasmic Conflict in Pea (Pisum sativum L.) Is Associated with Nuclear and Plastidic Candidate Genes Encoding Acetyl-CoA Carboxylase Subunits

PubMed Central

Bogdanova, Vera S.; Zaytseva, Olga O.; Mglinets, Anatoliy V.; Shatskaya, Natalia V.; Kosterin, Oleg E.; Vasiliev, Gennadiy V.

2015-01-01

In crosses of wild and cultivated peas (Pisum sativum L.), nuclear-cytoplasmic incompatibility frequently occurs manifested as decreased pollen fertility, male gametophyte lethality, sporophyte lethality. High-throughput sequencing of plastid genomes of one cultivated and four wild pea accessions differing in cross-compatibility was performed. Candidate genes for involvement in the nuclear-plastid conflict were searched in the reconstructed plastid genomes. In the annotated Medicago truncatula genome, nuclear candidate genes were searched in the portion syntenic to the pea chromosome region known to harbor a locus involved in the conflict. In the plastid genomes, a substantial variability of the accD locus represented by nucleotide substitutions and indels was found to correspond to the pattern of cross-compatibility among the accessions analyzed. Amino acid substitutions in the polypeptides encoded by the alleles of a nuclear locus, designated as Bccp3, with a complementary function to accD, fitted the compatibility pattern. The accD locus in the plastid genome encoding beta subunit of the carboxyltransferase of acetyl-coA carboxylase and the nuclear locus Bccp3 encoding biotin carboxyl carrier protein of the same multi-subunit enzyme were nominated as candidate genes for main contribution to nuclear-cytoplasmic incompatibility in peas. Existence of another nuclear locus involved in the accD-mediated conflict is hypothesized. PMID:25789472

Complete nucleotide and derived amino acid sequence of cDNA encoding the mitochondrial uncoupling protein of rat brown adipose tissue: lack of a mitochondrial targeting presequence.

PubMed Central

Ridley, R G; Patel, H V; Gerber, G E; Morton, R C; Freeman, K B

1986-01-01

A cDNA clone spanning the entire amino acid sequence of the nuclear-encoded uncoupling protein of rat brown adipose tissue mitochondria has been isolated and sequenced. With the exception of the N-terminal methionine the deduced N-terminus of the newly synthesized uncoupling protein is identical to the N-terminal 30 amino acids of the native uncoupling protein as determined by protein sequencing. This proves that the protein contains no N-terminal mitochondrial targeting prepiece and that a targeting region must reside within the amino acid sequence of the mature protein. Images PMID:3012461

Analyses of pea necrotic yellow dwarf virus-encoded proteins.

PubMed

Krenz, Björn; Schießl, Ingrid; Greiner, Eva; Krapp, Susanna

2017-06-01

Pea necrotic yellow dwarf virus (PNYDV) is a multipartite, circular, single-stranded DNA plant virus. PNYDV encodes eight proteins and the function of three of which remains unknown-U1, U2, and U4. PNYDV proteins cellular localization was analyzed by GFP tagging and bimolecular fluorescence complementation (BiFC) studies. The interactions of all eight PNYDV proteins were tested pairwise in planta (36 combinations in total). Seven interactions were identified and two (M-Rep with CP and MP with U4) were characterized further. MP and U4 complexes appeared as vesicle-like spots and were localized at the nuclear envelope and cell periphery. These vesicle-like spots were associated with the endoplasmatic reticulum. In addition, a nuclear localization signal (NLS) was mapped for U1, and a mutated U1 with NLS disrupted localized at plasmodesmata and therefore might also have a role in movement. Taken together, this study provides evidence for previously undescribed nanovirus protein-protein interactions and their cellular localization with novel findings not only for those proteins with unknown function, but also for characterized proteins such as the CP.

Nuclear localization signal regulates porcine circovirus type 2 capsid protein nuclear export through phosphorylation.

PubMed

Hou, Qiang; Hou, Shaohua; Chen, Qing; Jia, Hong; Xin, Ting; Jiang, Yitong; Guo, Xiaoyu; Zhu, Hongfei

2018-02-15

The open reading frame 2 (ORF2) of Porcine circovirus type 2 (PCV2) encodes the major Capsid (Cap) protein, which self-assembles into virus-like particle (VLP) of similar morphology to the PCV2 virion and accumulates in the nucleus through the N-terminal arginine-rich nuclear localization signal (NLS). In this study, PCV2 Cap protein and its derivates were expressed via the baculovirus expression system, and the cellular localization of the recombinant proteins were investigated using anti-Cap mAb by imaging flow cytometry. Analysis of subcellular localization of Cap protein and its variants demonstrated that NLS mediated Cap protein nuclear export as well as nuclear import, and a phosphorylation site (S17) was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the NLS domain to regulate Cap protein nuclear export. Phosphorylation of NLS regulating the PCV2 Cap protein nuclear export was also demonstrated in PK15 cells by fluorescence microscopy. Moreover, the influence of Rep and Rep' protein on Cap protein subcellular localization was investigated in PK15 cells. Phosphorylation of NLS regulating Cap protein nuclear export provides more detailed knowledge of the PCV2 viral life cycle. Copyright © 2018 Elsevier B.V. All rights reserved.

Positive Selection in Rapidly Evolving Plastid–Nuclear Enzyme Complexes

PubMed Central

Rockenbach, Kate; Havird, Justin C.; Monroe, J. Grey; Triant, Deborah A.; Taylor, Douglas R.; Sloan, Daniel B.

2016-01-01

Rates of sequence evolution in plastid genomes are generally low, but numerous angiosperm lineages exhibit accelerated evolutionary rates in similar subsets of plastid genes. These genes include clpP1 and accD, which encode components of the caseinolytic protease (CLP) and acetyl-coA carboxylase (ACCase) complexes, respectively. Whether these extreme and repeated accelerations in rates of plastid genome evolution result from adaptive change in proteins (i.e., positive selection) or simply a loss of functional constraint (i.e., relaxed purifying selection) is a source of ongoing controversy. To address this, we have taken advantage of the multiple independent accelerations that have occurred within the genus Silene (Caryophyllaceae) by examining phylogenetic and population genetic variation in the nuclear genes that encode subunits of the CLP and ACCase complexes. We found that, in species with accelerated plastid genome evolution, the nuclear-encoded subunits in the CLP and ACCase complexes are also evolving rapidly, especially those involved in direct physical interactions with plastid-encoded proteins. A massive excess of nonsynonymous substitutions between species relative to levels of intraspecific polymorphism indicated a history of strong positive selection (particularly in CLP genes). Interestingly, however, some species are likely undergoing loss of the native (heteromeric) plastid ACCase and putative functional replacement by a duplicated cytosolic (homomeric) ACCase. Overall, the patterns of molecular evolution in these plastid–nuclear complexes are unusual for anciently conserved enzymes. They instead resemble cases of antagonistic coevolution between pathogens and host immune genes. We discuss a possible role of plastid–nuclear conflict as a novel cause of accelerated evolution. PMID:27707788

Molecular analysis of an enhancin gene in the Lymantria dispar nuclear polyhedrosis virus

Treesearch

David S. Bischoff; James M. Slavicek

1997-01-01

A Lymantria dispar nuclear polyhedrosis virus (LdMNPV) gene has been identified that encodes a homolog to the granulovirus (GV) enhancin proteins that are capable of enhancing the infection of other baculoviruses. Enhancin genes have been identified and sequenced for three species of GVs but have not been found in any other nuclear...

The product of the Saccharomyces cerevisiae cell cycle gene DBF2 has homology with protein kinases and is periodically expressed in the cell cycle.

PubMed Central

Johnston, L H; Eberly, S L; Chapman, J W; Araki, H; Sugino, A

1990-01-01

Several Saccharomyces cerevisiae dbf mutants defective in DNA synthesis have been described previously. In this paper, one of them, dbf2, is characterized in detail. The DBF2 gene has been cloned and mapped, and its nucleotide sequence has been determined. This process has identified an open reading frame capable of encoding a protein of molecular weight 64,883 (561 amino acids). The deduced amino acid sequence contains all 11 conserved domains found in various protein kinases. DBF2 was periodically expressed in the cell cycle at a time that clearly differed from the time of expression of either the histone H2A or DNA polymerase I gene. Its first function was completed very near to initiation of DNA synthesis. However, DNA synthesis in the mutant was only delayed at 37 degrees C, and the cells blocked in nuclear division. Consistent with this finding, the execution point occurred about 1 h after DNA synthesis, and the nuclear morphology of the mutant at the restrictive temperature was that of cells blocked in late nuclear division. DBF2 is therefore likely to encode a protein kinase that may function in initiation of DNA synthesis and also in late nuclear division. Images PMID:2181271

Comparative analyses of two Geraniaceae transcriptomes using next-generation sequencing.

PubMed

Zhang, Jin; Ruhlman, Tracey A; Mower, Jeffrey P; Jansen, Robert K

2013-12-29

Organelle genomes of Geraniaceae exhibit several unusual evolutionary phenomena compared to other angiosperm families including accelerated nucleotide substitution rates, widespread gene loss, reduced RNA editing, and extensive genomic rearrangements. Since most organelle-encoded proteins function in multi-subunit complexes that also contain nuclear-encoded proteins, it is likely that the atypical organellar phenomena affect the evolution of nuclear genes encoding organellar proteins. To begin to unravel the complex co-evolutionary interplay between organellar and nuclear genomes in this family, we sequenced nuclear transcriptomes of two species, Geranium maderense and Pelargonium x hortorum. Normalized cDNA libraries of G. maderense and P. x hortorum were used for transcriptome sequencing. Five assemblers (MIRA, Newbler, SOAPdenovo, SOAPdenovo-trans [SOAPtrans], Trinity) and two next-generation technologies (454 and Illumina) were compared to determine the optimal transcriptome sequencing approach. Trinity provided the highest quality assembly of Illumina data with the deepest transcriptome coverage. An analysis to determine the amount of sequencing needed for de novo assembly revealed diminishing returns of coverage and quality with data sets larger than sixty million Illumina paired end reads for both species. The G. maderense and P. x hortorum transcriptomes contained fewer transcripts encoding the PLS subclass of PPR proteins relative to other angiosperms, consistent with reduced mitochondrial RNA editing activity in Geraniaceae. In addition, transcripts for all six plastid targeted sigma factors were identified in both transcriptomes, suggesting that one of the highly divergent rpoA-like ORFs in the P. x hortorum plastid genome is functional. The findings support the use of the Illumina platform and assemblers optimized for transcriptome assembly, such as Trinity or SOAPtrans, to generate high-quality de novo transcriptomes with broad coverage. In addition, results indicated no major improvements in breadth of coverage with data sets larger than six billion nucleotides or when sampling RNA from four tissue types rather than from a single tissue. Finally, this work demonstrates the power of cross-compartmental genomic analyses to deepen our understanding of the correlated evolution of the nuclear, plastid, and mitochondrial genomes in plants.

Comparative analyses of two Geraniaceae transcriptomes using next-generation sequencing

PubMed Central

2013-01-01

Background Organelle genomes of Geraniaceae exhibit several unusual evolutionary phenomena compared to other angiosperm families including accelerated nucleotide substitution rates, widespread gene loss, reduced RNA editing, and extensive genomic rearrangements. Since most organelle-encoded proteins function in multi-subunit complexes that also contain nuclear-encoded proteins, it is likely that the atypical organellar phenomena affect the evolution of nuclear genes encoding organellar proteins. To begin to unravel the complex co-evolutionary interplay between organellar and nuclear genomes in this family, we sequenced nuclear transcriptomes of two species, Geranium maderense and Pelargonium x hortorum. Results Normalized cDNA libraries of G. maderense and P. x hortorum were used for transcriptome sequencing. Five assemblers (MIRA, Newbler, SOAPdenovo, SOAPdenovo-trans [SOAPtrans], Trinity) and two next-generation technologies (454 and Illumina) were compared to determine the optimal transcriptome sequencing approach. Trinity provided the highest quality assembly of Illumina data with the deepest transcriptome coverage. An analysis to determine the amount of sequencing needed for de novo assembly revealed diminishing returns of coverage and quality with data sets larger than sixty million Illumina paired end reads for both species. The G. maderense and P. x hortorum transcriptomes contained fewer transcripts encoding the PLS subclass of PPR proteins relative to other angiosperms, consistent with reduced mitochondrial RNA editing activity in Geraniaceae. In addition, transcripts for all six plastid targeted sigma factors were identified in both transcriptomes, suggesting that one of the highly divergent rpoA-like ORFs in the P. x hortorum plastid genome is functional. Conclusions The findings support the use of the Illumina platform and assemblers optimized for transcriptome assembly, such as Trinity or SOAPtrans, to generate high-quality de novo transcriptomes with broad coverage. In addition, results indicated no major improvements in breadth of coverage with data sets larger than six billion nucleotides or when sampling RNA from four tissue types rather than from a single tissue. Finally, this work demonstrates the power of cross-compartmental genomic analyses to deepen our understanding of the correlated evolution of the nuclear, plastid, and mitochondrial genomes in plants. PMID:24373163

The DNA Helicase Activity of BLM Is Necessary for the Correction of the Genomic Instability of Bloom Syndrome Cells

PubMed Central

Neff, Norma F.; Ellis, Nathan A.; Ye, Tian Zhang; Noonan, James; Huang, Kelly; Sanz, Maureen; Proytcheva, Maria

1999-01-01

Bloom syndrome (BS) is a rare autosomal recessive disorder characterized by growth deficiency, immunodeficiency, genomic instability, and the early development of cancers of many types. BLM, the protein encoded by BLM, the gene mutated in BS, is localized in nuclear foci and absent from BS cells. BLM encodes a DNA helicase, and proteins from three missense alleles lack displacement activity. BLM transfected into BS cells reduces the frequency of sister chromatid exchanges and restores BLM in the nucleus. Missense alleles fail to reduce the sister chromatid exchanges in transfected BS cells or restore the normal nuclear pattern. BLM complements a phenotype of a Saccharomyces cerevisiae sgs1 top3 strain, and the missense alleles do not. This work demonstrates the importance of the enzymatic activity of BLM for its function and nuclear localization pattern. PMID:10069810

Localization of a bacterial group II intron-encoded protein in eukaryotic nuclear splicing-related cell compartments.

PubMed

Nisa-Martínez, Rafael; Laporte, Philippe; Jiménez-Zurdo, José Ignacio; Frugier, Florian; Crespi, Martin; Toro, Nicolás

2013-01-01

Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

Localization of a Bacterial Group II Intron-Encoded Protein in Eukaryotic Nuclear Splicing-Related Cell Compartments

PubMed Central

Nisa-Martínez, Rafael; Laporte, Philippe; Jiménez-Zurdo, José Ignacio; Frugier, Florian; Crespi, Martin; Toro, Nicolás

2013-01-01

Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns. PMID:24391881

Toxic PRn poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export.

PubMed

Shi, Kevin Y; Mori, Eiichiro; Nizami, Zehra F; Lin, Yi; Kato, Masato; Xiang, Siheng; Wu, Leeju C; Ding, Ming; Yu, Yonghao; Gall, Joseph G; McKnight, Steven L

2017-02-14

The toxic proline:arginine (PR n ) poly-dipeptide encoded by the (GGGGCC) n repeat expansion in the C9orf72 form of heritable amyotrophic lateral sclerosis (ALS) binds to the central channel of the nuclear pore and inhibits the movement of macromolecules into and out of the nucleus. The PR n poly-dipeptide binds to polymeric forms of the phenylalanine:glycine (FG) repeat domain, which is shared by several proteins of the nuclear pore complex, including those in the central channel. A method of chemical footprinting was used to characterize labile, cross-β polymers formed from the FG domain of the Nup54 protein. Mutations within the footprinted region of Nup54 polymers blocked both polymerization and binding by the PR n poly-dipeptide. The aliphatic alcohol 1,6-hexanediol melted FG domain polymers in vitro and reversed PR n -mediated enhancement of the nuclear pore permeability barrier. These data suggest that toxicity of the PR n poly-dipeptide results in part from its ability to lock the FG repeats of nuclear pore proteins in the polymerized state. Our study offers a mechanistic interpretation of PR n poly-dipeptide toxicity in the context of a prominent form of ALS.

Subcellular distribution of human RDM1 protein isoforms and their nucleolar accumulation in response to heat shock and proteotoxic stress.

PubMed

Messaoudi, Lydia; Yang, Yun-Gui; Kinomura, Aiko; Stavreva, Diana A; Yan, Gonghong; Bortolin-Cavaillé, Marie-Line; Arakawa, Hiroshi; Buerstedde, Jean-Marie; Hainaut, Pierre; Cavaillé, Jérome; Takata, Minoru; Van Dyck, Eric

2007-01-01

The RDM1 gene encodes a RNA recognition motif (RRM)-containing protein involved in the cellular response to the anti-cancer drug cisplatin in vertebrates. We previously reported a cDNA encoding the full-length human RDM1 protein. Here, we describe the identification of 11 human cDNAs encoding RDM1 protein isoforms. This repertoire is generated by alternative pre-mRNA splicing and differential usage of two translational start sites, resulting in proteins with long or short N-terminus and a great diversity in the exonic composition of their C-terminus. By using tagged proteins and fluorescent microscopy, we examined the subcellular distribution of full-length RDM1 (renamed RDM1alpha), and other RDM1 isoforms. We show that RDM1alpha undergoes subcellular redistribution and nucleolar accumulation in response to proteotoxic stress and mild heat shock. In unstressed cells, the long N-terminal isoforms displayed distinct subcellular distribution patterns, ranging from a predominantly cytoplasmic to almost exclusive nuclear localization, suggesting functional differences among the RDM1 proteins. However, all isoforms underwent stress-induced nucleolar accumulation. We identified nuclear and nucleolar localization determinants as well as domains conferring cytoplasmic retention to the RDM1 proteins. Finally, RDM1 null chicken DT40 cells displayed an increased sensitivity to heat shock, compared to wild-type (wt) cells, suggesting a function for RDM1 in the heat-shock response.

Biogenesis of light harvesting proteins.

PubMed

Dall'Osto, Luca; Bressan, Mauro; Bassi, Roberto

2015-09-01

The LHC family includes nuclear-encoded, integral thylakoid membrane proteins, most of which coordinate chlorophyll and xanthophyll chromophores. By assembling with the core complexes of both photosystems, LHCs form a flexible peripheral moiety for enhancing light-harvesting cross-section, regulating its efficiency and providing protection against photo-oxidative stress. Upon its first appearance, LHC proteins underwent evolutionary diversification into a large protein family with a complex genetic redundancy. Such differentiation appears as a crucial event in the adaptation of photosynthetic organisms to changing environmental conditions and land colonization. The structure of photosystems, including nuclear- and chloroplast-encoded subunits, presented the cell with a number of challenges for the control of the light harvesting function. Indeed, LHC-encoding messages are translated in the cytosol, and pre-proteins imported into the chloroplast, processed to their mature size and targeted to the thylakoids where are assembled with chromophores. Thus, a tight coordination between nuclear and plastid gene expression, in response to environmental stimuli, is required to adjust LHC composition during photoacclimation. In recent years, remarkable progress has been achieved in elucidating structure, function and regulatory pathways involving LHCs; however, a number of molecular details still await elucidation. In this review, we will provide an overview on the current knowledge on LHC biogenesis, ranging from organization of pigment-protein complexes to the modulation of gene expression, import and targeting to the photosynthetic membranes, and regulation of LHC assembly and turnover. Genes controlling these events are potential candidate for biotechnological applications aimed at optimizing light use efficiency of photosynthetic organisms. This article is part of a Special Issue entitled: Chloroplast biogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

Features of cues and processes during chloroplast-mediated retrograde signaling in the alga Chlamydomonas

USDA-ARS?s Scientific Manuscript database

Retrograde signalling is a selective process defined by cues generated in chloroplast/mitochondria which traverse membranes and end up regulating nuclear gene expression and protein synthesis. The coding and encoding of organellar message(s) that alter nuclear gene expression and/or cellular metabo...

Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention

PubMed Central

Yap, Karen; Lim, Zhao Qin; Khandelia, Piyush; Friedman, Brad; Makeyev, Eugene V.

2012-01-01

Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage-specific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and nonneuronal cells. However, in nonneuronal cells, the splicing of 3′-terminal introns within these genes is repressed by the polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these intron-containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery, including the nuclear pore-associated protein Tpr and the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out, thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context. PMID:22661231

Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention.

PubMed

Yap, Karen; Lim, Zhao Qin; Khandelia, Piyush; Friedman, Brad; Makeyev, Eugene V

2012-06-01

Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage-specific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and nonneuronal cells. However, in nonneuronal cells, the splicing of 3'-terminal introns within these genes is repressed by the polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these intron-containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery, including the nuclear pore-associated protein Tpr and the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out, thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context.

Nuclear Proteomics Reveals the Role of Protein Synthesis and Chromatin Structure in Root Tip of Soybean during the Initial Stage of Flooding Stress.

PubMed

Yin, Xiaojian; Komatsu, Setsuko

2016-07-01

To identify the upstream events controlling the regulation of flooding-responsive proteins in soybean, proteomic analysis of nuclear proteins in root tip was performed. By using nuclear fractions, which were highly enriched, a total of 365 nuclear proteins were changed in soybean root tip at initial stage of flooding stress. Four exon-junction complex-related proteins and NOP1/NOP56, which function in upstream of 60S preribosome biogenesis, were decreased in flooded soybean. Furthermore, proteomic analysis of crude protein extract revealed that the protein translation was suppressed by continuous flooding stress. Seventeen chromatin structure-related nuclear proteins were decreased in response to flooding stress. Out of them, histone H3 was clearly decreased with protein abundance and mRNA expression levels at the initial flooding stress. Additionally, a number of protein synthesis-, RNA-, and DNA-related nuclear proteins were decreased in a time-dependent manner. mRNA expressions of genes encoding the significantly changed flooding-responsive nuclear proteins were inhibited by the transcriptional inhibitor, actinomycin D. These results suggest that protein translation is suppressed through inhibition of preribosome biogenesis- and mRNA processing-related proteins in nuclei of soybean root tip at initial flooding stress. In addition, flooding stress may regulate histone variants with gene expression in root tip.

Molecular Characterization of the Calvin Cycle Enzyme Phosphoribulokinase in the Stramenopile Alga Vaucheria litorea and the Plastid Hosting Mollusc Elysia chlorotica

PubMed Central

Rumpho, Mary E.; Pochareddy, Sirisha; Worful, Jared M.; Summer, Elizabeth J.; Bhattacharya, Debashish; Pelletreau, Karen N.; Tyler, Mary S.; Lee, Jungho; Manhart, James R.; Soule, Kara M.

2009-01-01

Phosphoribulokinase (PRK), a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction (Calvin) cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc (sea slug) Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclear-encoded proteins that are directed to complex (i.e. four membrane-bound) algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V. litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V. litorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months. PMID:19995736

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paterson, Carolyn P.; Ayalew, Lisanework E.; Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK S7N 5E3 S7N 5B4 Canada

The L1 region of bovine adenovirus (BAdV)-3 encodes a non-structural protein designated 52K. Anti-52K serum detected a protein of 40 kDa, which localized to the nucleus but not to the nucleolus in BAdV-3-infected or transfected cells. Analysis of mutant 52K proteins suggested that three basic residues ({sup 105}RKR{sup 107}) of the identified domain (amino acids {sup 102}GMPRKRVLT{sup 110}) are essential for nuclear localization of 52K. The nuclear import of a GST-52K fusion protein utilizes the classical importin {alpha}/{beta}-dependent nuclear transport pathway. The 52K protein is preferentially bound to the cellular nuclear import receptor importin {alpha}3. Although deletion of amino acidmore » 102-110 is sufficient to abrogate the nuclear localization of 52K, amino acid 90-133 are required for interaction with importin-{alpha}3 and localizing a cytoplasmic protein to the nucleus. These results suggest that 52K contains a bipartite NLS, which preferentially utilize an importin {alpha}3 nuclear import receptor-mediated pathway to transport 52K to the nucleus.« less

PML nuclear bodies in the pathogenesis of acute promyelocytic leukemia: active players or innocent bystanders?

PubMed

Brown, Nicola J M; Ramalho, Michal; Pedersen, Eva W; Moravcsik, Eva; Solomon, Ellen; Grimwade, David

2009-01-01

The promyelocytic leukemia gene (PML) encodes a protein which localizes to PML-nuclear bodies (NBs), sub-nuclear multi-protein structures, which have been implicated in diverse biological functions such as apoptosis, cell proliferation and senescence. However, the exact biochemical and molecular basis of PML function up until now has not been defined. Strikingly, over a decade ago, PML-NBs were found to be disrupted in acute promyelocytic leukemia (APL) in which PML is fused to the gene encoding retinoic acid receptor alpha (RARA) due to the t(15;17) chromosomal translocation, generating the PML-RARA chimeric protein. The treatment of APL patients with all-transretinoic acid (ATRA) and arsenic trioxide which target the PML-RARA oncoprotein results in clinical remission, associated with blast cell differentiation and reformation of the PML NBs, thus linking NB integrity with disease status. This review focuses on the current theories for molecular and biochemical functions of the PML-NBs, which would imply a role in the pathogenesis of APL, whilst also discussing the intriguing possibility that their disruption may not be in itself a significant oncogenic event.

Insight into the functional organization of nuclear lamins in health and disease.

PubMed

Tatli, Meltem; Medalia, Ohad

2018-05-22

Lamins are the main component of the nuclear lamina, a protein meshwork at the inner nuclear membrane which primarily provide mechanical stability to the nucleus. Lamins, type V intermediate filament proteins, are also involved in many nuclear activities. Structural analysis of nuclei revealed that lamins form 3.5nm thick filaments often interact with nuclear pore complexes. Mutations in the LMNA gene, encoding A-type lamins, have been associated with at least 15 distinct diseases collectively termed laminopathies, including muscle, metabolic and neurological disorders, and premature aging syndrome. It is unclear how laminopathic mutations lead to such a wide array of diseases, essentially affecting almost all tissues. Copyright © 2018 Elsevier Ltd. All rights reserved.

Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of Mg-chelatase H subunit in plastid-to-nucleus signal transduction

PubMed Central

Mochizuki, Nobuyoshi; Brusslan, Judy A.; Larkin, Robert; Nagatani, Akira; Chory, Joanne

2001-01-01

A plastid-derived signal plays an important role in the coordinated expression of both nuclear- and chloroplast-localized genes that encode photosynthesis-related proteins. Arabidopsis GUN (genomes uncoupled) loci have been identified as components of plastid-to-nucleus signal transduction. Unlike wild-type plants, gun mutants have nuclear Lhcb1 expression in the absence of chloroplast development. We observed a synergistic phenotype in some gun double-mutant combinations, suggesting there are at least two independent pathways in plastid-to-nucleus signal transduction. There is a reduction of chlorophyll accumulation in gun4 and gun5 mutant plants, and a gun4gun5 double mutant shows an albino phenotype. We cloned the GUN5 gene, which encodes the ChlH subunit of Mg-chelatase. We also show that gun2 and gun3 are alleles of the known photomorphogenic mutants, hy1 and hy2, which are required for phytochromobilin synthesis from heme. These findings suggest that certain perturbations of the tetrapyrrole biosynthetic pathway generate a signal from chloroplasts that causes transcriptional repression of nuclear genes encoding plastid-localized proteins. The comparison of mutant phenotypes of gun5 and another Mg-chelatase subunit (ChlI) mutant suggests a specific function for ChlH protein in the plastid-signaling pathway. PMID:11172074

Mechanism of protein import across the chloroplast envelope.

PubMed

Chen, K; Chen, X; Schnell, D J

2000-01-01

The development and maintenance of chloroplasts relies on the contribution of protein subunits from both plastid and nuclear genomes. Most chloroplast proteins are encoded by nuclear genes and are post-translationally imported into the organelle across the double membrane of the chloroplast envelope. Protein import into the chloroplast consists of two essential elements: the specific recognition of the targeting signals (transit sequences) of cytoplasmic preproteins by receptors at the outer envelope membrane and the subsequent translocation of preproteins simultaneously across the double membrane of the envelope. These processes are mediated via the co-ordinate action of protein translocon complexes in the outer (Toc apparatus) and inner (Tic apparatus) envelope membranes.

Nuclear targeting of the maize R protein requires two nuclear localization sequences

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shieh, M.W.; Raikhel, N.V.; Wessler, S.R.

1993-02-01

Previous genetic and structural evidence indicates that the maize R gene encodes a nuclear transcriptional activating factor. In-frame carboxyl- and amino-terminal fusions of the R gene to the reporter gene encoding [beta]-glucuronidase (GUS) were sufficient to direct GUS to the nucleus of the transiently transformed onion (Allium cepa) epidermal cells. Further analysis of chimeric constructs containing regions of the R gene fused to the GUS cDNA revealed three specific nuclear localization sequences (NLSs) that were capable of redirecting the GUS protein to the nucleus. Amino-terminal NLS-A (amino acids 100-109, GDRRAAPARP) contained several arginine residues; a similar localization signal is foundmore » in only a few viral proteins. The medial NLS-M (amino acids 419-428, MSERKRREKL) is a simian virus 40 large T antigen-type NLS, and the carboxyl-terminal NLS-C (amino acids 598-610, MISESLRKAIGKR) is a mating type [alpha]2 type. NLSs M and C are independently sufficient to direct the GUS protein to the nucleus when it is fused at the amino terminus of GUS, whereas NLS-A fused to GUS partitioned between the nucleus and cytoplasm. Similar partitioning was observed when localization signals NLS-A and NLS-C were independently fused to the carboxy-terminal portion of GUS. A sequential deletion of the localization signals indicated that the amino-terminal and carboxyl-terminal fusions of R and GUS were redirected to the nucleus only when both NLS-A and -M, or NLS-C and -M, were present. These results indicate that multiple localization signals are necessary for nuclear targeting of this protein. The conservation of the localization signals within the alleles of R and similar proteins from other organisms is also discussed. 45 refs., 6 figs.« less

Mosaic genome of endobacteria in arbuscular mycorrhizal fungi: Transkingdom gene transfer in an ancient mycoplasma-fungus association.

PubMed

Torres-Cortés, Gloria; Ghignone, Stefano; Bonfante, Paola; Schüßler, Arthur

2015-06-23

For more than 450 million years, arbuscular mycorrhizal fungi (AMF) have formed intimate, mutualistic symbioses with the vast majority of land plants and are major drivers in almost all terrestrial ecosystems. The obligate plant-symbiotic AMF host additional symbionts, so-called Mollicutes-related endobacteria (MRE). To uncover putative functional roles of these widespread but yet enigmatic MRE, we sequenced the genome of DhMRE living in the AMF Dentiscutata heterogama. Multilocus phylogenetic analyses showed that MRE form a previously unidentified lineage sister to the hominis group of Mycoplasma species. DhMRE possesses a strongly reduced metabolic capacity with 55% of the proteins having unknown function, which reflects unique adaptations to an intracellular lifestyle. We found evidence for transkingdom gene transfer between MRE and their AMF host. At least 27 annotated DhMRE proteins show similarities to nuclear-encoded proteins of the AMF Rhizophagus irregularis, which itself lacks MRE. Nuclear-encoded homologs could moreover be identified for another AMF, Gigaspora margarita, and surprisingly, also the non-AMF Mortierella verticillata. Our data indicate a possible origin of the MRE-fungus association in ancestors of the Glomeromycota and Mucoromycotina. The DhMRE genome encodes an arsenal of putative regulatory proteins with eukaryotic-like domains, some of them encoded in putative genomic islands. MRE are highly interesting candidates to study the evolution and interactions between an ancient, obligate endosymbiotic prokaryote with its obligate plant-symbiotic fungal host. Our data moreover may be used for further targeted searches for ancient effector-like proteins that may be key components in the regulation of the arbuscular mycorrhiza symbiosis.

The Saccharomyces cerevisiae LOS1 gene involved in pre-tRNA splicing encodes a nuclear protein that behaves as a component of the nuclear matrix.

PubMed

Shen, W C; Selvakumar, D; Stanford, D R; Hopper, A K

1993-09-15

Mutations of the Saccharomyces cerevisiae LOS1 gene cause the accumulation of end matured intron-containing pre-tRNAs at elevated temperatures. In an effort to decipher the role of the LOS1 protein in pre-tRNA splicing, we have analyzed the LOS1 gene and its protein product. The LOS1 gene is located on the left arm of chromosome XI and the order of genes in this area of the chromosome is .... URA1 ... SAC1 TRP3 UBA1 STE6 LOS1 .... FAS1..... The LOS1 open reading frame encodes a putative protein of 1100 amino acids that shows no significant homology to other genes. The LOS1 open reading frame was tagged with the influenza virus hemagglutinin epitope recognized by the 12CA5 antibody. The 12CA5 antibody recognizes an epitope-tagged protein of the size predicted by the LOS1 open reading frame. Using this antibody for indirect immunofluorescence and cell fractionation studies we show that the LOS1 protein is located in nuclei. Los1p cannot be extracted from nuclei by treatment with nucleases, salts, or Triton X-100. This insolubility suggests that Los1p is a component of the nucleoskeleton. We propose that LOS1 mutations may affect pre-tRNA processing via alteration of the nuclear matrix.

Mollusc-Algal Chloroplast Endosymbiosis. Photosynthesis, Thylakoid Protein Maintenance, and Chloroplast Gene Expression Continue for Many Months in the Absence of the Algal Nucleus1

PubMed Central

Green, Brian J.; Li, Wei-Ye; Manhart, James R.; Fox, Theodore C.; Summer, Elizabeth J.; Kennedy, Robert A.; Pierce, Sidney K.; Rumpho, Mary E.

2000-01-01

Early in its life cycle, the marine mollusc Elysia chlorotica Gould forms an intracellular endosymbiotic association with chloroplasts of the chromophytic alga Vaucheria litorea C. Agardh. As a result, the dark green sea slug can be sustained in culture solely by photoautotrophic CO2 fixation for at least 9 months if provided with only light and a source of CO2. Here we demonstrate that the sea slug symbiont chloroplasts maintain photosynthetic oxygen evolution and electron transport activity through photosystems I and II for several months in the absence of any external algal food supply. This activity is correlated to the maintenance of functional levels of chloroplast-encoded photosystem proteins, due in part at least to de novo protein synthesis of chloroplast proteins in the sea slug. Levels of at least one putative algal nuclear encoded protein, a light-harvesting complex protein homolog, were also maintained throughout the 9-month culture period. The chloroplast genome of V. litorea was found to be 119.1 kb, similar to that of other chromophytic algae. Southern analysis and polymerase chain reaction did not detect an algal nuclear genome in the slug, in agreement with earlier microscopic observations. Therefore, the maintenance of photosynthetic activity in the captured chloroplasts is regulated solely by the algal chloroplast and animal nuclear genomes. PMID:10982447

Mollusc-algal chloroplast endosymbiosis. Photosynthesis, thylakoid protein maintenance, and chloroplast gene expression continue for many months in the absence of the algal nucleus.

PubMed

Green, B J; Li, W Y; Manhart, J R; Fox, T C; Summer, E J; Kennedy, R A; Pierce, S K; Rumpho, M E

2000-09-01

Early in its life cycle, the marine mollusc Elysia chlorotica Gould forms an intracellular endosymbiotic association with chloroplasts of the chromophytic alga Vaucheria litorea C. Agardh. As a result, the dark green sea slug can be sustained in culture solely by photoautotrophic CO(2) fixation for at least 9 months if provided with only light and a source of CO(2). Here we demonstrate that the sea slug symbiont chloroplasts maintain photosynthetic oxygen evolution and electron transport activity through photosystems I and II for several months in the absence of any external algal food supply. This activity is correlated to the maintenance of functional levels of chloroplast-encoded photosystem proteins, due in part at least to de novo protein synthesis of chloroplast proteins in the sea slug. Levels of at least one putative algal nuclear encoded protein, a light-harvesting complex protein homolog, were also maintained throughout the 9-month culture period. The chloroplast genome of V. litorea was found to be 119.1 kb, similar to that of other chromophytic algae. Southern analysis and polymerase chain reaction did not detect an algal nuclear genome in the slug, in agreement with earlier microscopic observations. Therefore, the maintenance of photosynthetic activity in the captured chloroplasts is regulated solely by the algal chloroplast and animal nuclear genomes.

Nuclear envelopathies: a complex LINC between nuclear envelope and pathology.

PubMed

Janin, Alexandre; Bauer, Delphine; Ratti, Francesca; Millat, Gilles; Méjat, Alexandre

2017-08-30

Since the identification of the first disease causing mutation in the gene coding for emerin, a transmembrane protein of the inner nuclear membrane, hundreds of mutations and variants have been found in genes encoding for nuclear envelope components. These proteins can be part of the inner nuclear membrane (INM), such as emerin or SUN proteins, outer nuclear membrane (ONM), such as Nesprins, or the nuclear lamina, such as lamins A and C. However, they physically interact with each other to insure the nuclear envelope integrity and mediate the interactions of the nuclear envelope with both the genome, on the inner side, and the cytoskeleton, on the outer side. The core of this complex, called LINC (LInker of Nucleoskeleton to Cytoskeleton) is composed of KASH and SUN homology domain proteins. SUN proteins are INM proteins which interact with lamins by their N-terminal domain and with the KASH domain of nesprins located in the ONM by their C-terminal domain.Although most of these proteins are ubiquitously expressed, their mutations have been associated with a large number of clinically unrelated pathologies affecting specific tissues. Moreover, variants in SUN proteins have been found to modulate the severity of diseases induced by mutations in other LINC components or interactors. For these reasons, the diagnosis and the identification of the molecular explanation of "nuclear envelopathies" is currently challenging.The aim of this review is to summarize the human diseases caused by mutations in genes coding for INM proteins, nuclear lamina, and ONM proteins, and to discuss their potential physiopathological mechanisms that could explain the large spectrum of observed symptoms.

Expression of the ribulose-1,5-bisphosphate carboxylase large subunit gene and three small subunit genes in two cell types of maize leaves

PubMed Central

Sheen, Jenq-Yunn; Bogorad, Lawrence

1986-01-01

Transcripts of three distinct ribulose-1,5-bisphosphate carboxylase (RuBPC) small subunit (SS) genes account for ∼90% of the mRNA for this protein in maize leaves. Transcripts of two of them constitute >80% of the SS mRNA in 24-h greening maize leaves. The third gene contribute ∼10%. Transcripts of all three nuclear-encoded SS genes are detectable in bundle sheath (BSC) and mesophyll cells (MC) of etiolated maize leaves. The level of mRNA for each gene is different in etioplasts of MC but all drop during photoregulated development of chloroplasts in MC and follow a pattern of transitory rise and fall in BSC. The amounts of LS and SS proteins continue to increase steadily well after the mRNA levels reach their peaks in BSC. The molar ratio of mRNA for chloroplast-encoded RuBPC large subunit (LS) to the nuclear genome encoded SS is about 10:1 although LS and SS proteins are present in about equimolar amounts. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6. PMID:16453739

Autoselection of cytoplasmic yeast virus like elements encoding toxin/antitoxin systems involves a nuclear barrier for immunity gene expression.

PubMed

Kast, Alene; Voges, Raphael; Schroth, Michael; Schaffrath, Raffael; Klassen, Roland; Meinhardt, Friedhelm

2015-05-01

Cytoplasmic virus like elements (VLEs) from Kluyveromyces lactis (Kl), Pichia acaciae (Pa) and Debaryomyces robertsiae (Dr) are extremely A/T-rich (>75%) and encode toxic anticodon nucleases (ACNases) along with specific immunity proteins. Here we show that nuclear, not cytoplasmic expression of either immunity gene (PaORF4, KlORF3 or DrORF5) results in transcript fragmentation and is insufficient to establish immunity to the cognate ACNase. Since rapid amplification of 3' ends (RACE) as well as linker ligation of immunity transcripts expressed in the nucleus revealed polyadenylation to occur along with fragmentation, ORF-internal poly(A) site cleavage due to the high A/T content is likely to prevent functional expression of the immunity genes. Consistently, lowering the A/T content of PaORF4 to 55% and KlORF3 to 46% by gene synthesis entirely prevented transcript cleavage and permitted functional nuclear expression leading to full immunity against the respective ACNase toxin. Consistent with a specific adaptation of the immunity proteins to the cognate ACNases, cross-immunity to non-cognate ACNases is neither conferred by PaOrf4 nor KlOrf3. Thus, the high A/T content of cytoplasmic VLEs minimizes the potential of functional nuclear recruitment of VLE encoded genes, in particular those involved in autoselection of the VLEs via a toxin/antitoxin principle.

Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence

USDA-ARS?s Scientific Manuscript database

The human mitochondrial glutamate dehydrogenase isozymes (hGDH1 and 2) are abundant matrix-localized proteins encoded by nuclear genes. The proteins are synthesized in the cytoplasm, with an atypically long N-terminal mitochondrial targeting sequence (MTS). The results of secondary structure predi...

Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants. A case study of endosymbiotic gene transfer.

PubMed

Schnarrenberger, Claus; Martin, William

2002-02-01

The citric acid or tricarboxylic acid cycle is a central element of higher-plant carbon metabolism which provides, among other things, electrons for oxidative phosphorylation in the inner mitochondrial membrane, intermediates for amino-acid biosynthesis, and oxaloacetate for gluconeogenesis from succinate derived from fatty acids via the glyoxylate cycle in glyoxysomes. The tricarboxylic acid cycle is a typical mitochondrial pathway and is widespread among alpha-proteobacteria, the group of eubacteria as defined under rRNA systematics from which mitochondria arose. Most of the enzymes of the tricarboxylic acid cycle are encoded in the nucleus in higher eukaryotes, and several have been previously shown to branch with their homologues from alpha-proteobacteria, indicating that the eukaryotic nuclear genes were acquired from the mitochondrial genome during the course of evolution. Here, we investigate the individual evolutionary histories of all of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle using protein maximum likelihood phylogenies, focusing on the evolutionary origin of the nuclear-encoded proteins in higher plants. The results indicate that about half of the proteins involved in this eukaryotic pathway are most similar to their alpha-proteobacterial homologues, whereas the remainder are most similar to eubacterial, but not specifically alpha-proteobacterial, homologues. A consideration of (a) the process of lateral gene transfer among free-living prokaryotes and (b) the mechanistics of endosymbiotic (symbiont-to-host) gene transfer reveals that it is unrealistic to expect all nuclear genes that were acquired from the alpha-proteobacterial ancestor of mitochondria to branch specifically with their homologues encoded in the genomes of contemporary alpha-proteobacteria. Rather, even if molecular phylogenetics were to work perfectly (which it does not), then some nuclear-encoded proteins that were acquired from the alpha-proteobacterial ancestor of mitochondria should, in phylogenetic trees, branch with homologues that are no longer found in most alpha-proteobacterial genomes, and some should reside on long branches that reveal affinity to eubacterial rather than archaebacterial homologues, but no particular affinity for any specific eubacterial donor.

A nuclear-encoded chloroplast protein harboring a single CRM domain plays an important role in the Arabidopsis growth and stress response.

PubMed

Lee, Kwanuk; Lee, Hwa Jung; Kim, Dong Hyun; Jeon, Young; Pai, Hyun-Sook; Kang, Hunseung

2014-04-16

Although several chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins have been characterized for intron splicing and rRNA processing during chloroplast gene expression, the functional role of a majority of CRM domain proteins in plant growth and development as well as chloroplast RNA metabolism remains largely unknown. Here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single CRM domain (At4g39040), designated CFM4, in Arabidopsis thaliana. Analysis of CFM4-GFP fusion proteins revealed that CFM4 is localized to chloroplasts. The loss-of-function T-DNA insertion mutants for CFM4 (cfm4) displayed retarded growth and delayed senescence, suggesting that CFM4 plays a role in growth and development of plants under normal growth conditions. In addition, cfm4 mutants showed retarded seed germination and seedling growth under stress conditions. No alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16S and 4.5S rRNAs was abnormal in the mutant plants. Importantly, CFM4 was determined to possess RNA chaperone activity. These results suggest that the chloroplast-targeted CFM4, one of two Arabidopsis genes encoding a single CRM domain-containing protein, harbors RNA chaperone activity and plays a role in the Arabidopsis growth and stress response by affecting rRNA processing in chloroplasts.

A nuclear-encoded chloroplast protein harboring a single CRM domain plays an important role in the Arabidopsis growth and stress response

PubMed Central

2014-01-01

Background Although several chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins have been characterized for intron splicing and rRNA processing during chloroplast gene expression, the functional role of a majority of CRM domain proteins in plant growth and development as well as chloroplast RNA metabolism remains largely unknown. Here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single CRM domain (At4g39040), designated CFM4, in Arabidopsis thaliana. Results Analysis of CFM4-GFP fusion proteins revealed that CFM4 is localized to chloroplasts. The loss-of-function T-DNA insertion mutants for CFM4 (cfm4) displayed retarded growth and delayed senescence, suggesting that CFM4 plays a role in growth and development of plants under normal growth conditions. In addition, cfm4 mutants showed retarded seed germination and seedling growth under stress conditions. No alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16S and 4.5S rRNAs was abnormal in the mutant plants. Importantly, CFM4 was determined to possess RNA chaperone activity. Conclusions These results suggest that the chloroplast-targeted CFM4, one of two Arabidopsis genes encoding a single CRM domain-containing protein, harbors RNA chaperone activity and plays a role in the Arabidopsis growth and stress response by affecting rRNA processing in chloroplasts. PMID:24739417

Identification of the WBSCR9 gene, encoding a novel transcriptional regulator, in the Williams-Beuren syndrome deletion at 7q11.23.

PubMed

Peoples, R J; Cisco, M J; Kaplan, P; Francke, U

1998-01-01

We have identified a novel gene (WBSCR9) within the common Williams-Beuren syndrome (WBS) deletion by interspecies sequence conservation. The WBSCR9 gene encodes a roughly 7-kb transcript with an open reading frame of 1483 amino acids and a predicted protein product size of 170.8 kDa. WBSCR9 is comprised of at least 20 exons extending over 60 kb. The transcript is expressed ubiquitously throughout development and is subject to alternative splicing. Functional motifs identified by sequence homology searches include a bromodomain; a PHD, or C4HC3, finger; several putative nuclear localization signals; four nuclear receptor binding motifs; a polyglutamate stretch and two PEST sequences. Bromodomains, PHD motifs and nuclear receptor binding motifs are cardinal features of proteins that are involved in chromatin remodeling and modulation of transcription. Haploinsufficiency for WBSCR9 gene products may contribute to the complex phenotype of WBS by interacting with tissue-specific regulatory factors during development.

Polynucleotides encoding TRF1 binding proteins

DOEpatents

Campisi, Judith; Kim, Sahn-Ho

2002-01-01

The present invention provides a novel telomere associated protein (Trf1-interacting nuclear protein 2 "Tin2") that hinders the binding of Trf1 to its specific telomere repeat sequence and mediates the formation of a Tin2-Trf1-telomeric DNA complex that limits telomerase access to the telomere. Also included are the corresponding nucleic acids that encode the Tin2 of the present invention, as well as mutants of Tin2. Methods of making, purifying and using Tin2 of the present invention are described. In addition, drug screening assays to identify drugs that mimic and/or complement the effect of Tin2 are presented.

Mutational definition of functional domains within the Rev homolog encoded by human endogenous retrovirus K.

PubMed

Bogerd, H P; Wiegand, H L; Yang, J; Cullen, B R

2000-10-01

Nuclear export of the incompletely spliced mRNAs encoded by several complex retroviruses, including human immunodeficiency virus type 1 (HIV-1), is dependent on a virally encoded adapter protein, termed Rev in HIV-1, that directly binds both to a cis-acting viral RNA target site and to the cellular Crm1 export factor. Human endogenous retrovirus K, a family of ancient endogenous retroviruses that is not related to the exogenous retrovirus HIV-1, was recently shown to also encode a Crm1-dependent nuclear RNA export factor, termed K-Rev. Although HIV-1 Rev and K-Rev display little sequence identity, they share the ability not only to bind to Crm1 and to RNA but also to form homomultimers and shuttle between nucleus and cytoplasm. We have used mutational analysis to identify sequences in the 105-amino-acid K-Rev protein required for each of these distinct biological activities. While mutations in K-Rev that inactivate any one of these properties also blocked K-Rev-dependent nuclear RNA export, several K-Rev mutants were comparable to wild type when assayed for any of these individual activities yet nevertheless defective for RNA export. Although several nonfunctional K-Rev mutants acted as dominant negative inhibitors of K-Rev-, but not HIV-1 Rev-, dependent RNA export, these were not defined by their inability to bind to Crm1, as is seen with HIV-1 Rev. In total, this analysis suggests a functional architecture for K-Rev that is similar to, but distinct from, that described for HIV-1 Rev and raises the possibility that viral RNA export mediated by the approximately 25 million-year-old K-Rev protein may require an additional cellular cofactor that is not required for HIV-1 Rev function.

Contrasting patterns of nonneutral evolution in proteins encoded in nuclear and mitochondrial genomes.

PubMed Central

Weinreich, D M; Rand, D M

2000-01-01

We report that patterns of nonneutral DNA sequence evolution among published nuclear and mitochondrially encoded protein-coding loci differ significantly in animals. Whereas an apparent excess of amino acid polymorphism is seen in most (25/31) mitochondrial genes, this pattern is seen in fewer than half (15/36) of the nuclear data sets. This differentiation is even greater among data sets with significant departures from neutrality (14/15 vs. 1/6). Using forward simulations, we examined patterns of nonneutral evolution using parameters chosen to mimic the differences between mitochondrial and nuclear genetics (we varied recombination rate, population size, mutation rate, selective dominance, and intensity of germ line bottleneck). Patterns of evolution were correlated only with effective population size and strength of selection, and no single genetic factor explains the empirical contrast in patterns. We further report that in Arabidopsis thaliana, a highly self-fertilizing plant with effectively low recombination, five of six published nuclear data sets also exhibit an excess of amino acid polymorphism. We suggest that the contrast between nuclear and mitochondrial nonneutrality in animals stems from differences in rates of recombination in conjunction with a distribution of selective effects. If the majority of mutations segregating in populations are deleterious, high linkage may hinder the spread of the occasional beneficial mutation. PMID:10978302

The intriguing plant nuclear lamina.

PubMed

Ciska, Malgorzata; Moreno Díaz de la Espina, Susana

2014-01-01

The nuclear lamina is a complex protein mesh attached to the inner nuclear membrane (INM), which is also associated with nuclear pore complexes. It provides mechanical support to the nucleus and nuclear envelope, and as well as facilitating the connection of the nucleoskeleton to the cytoskeleton, it is also involved in chromatin organization, gene regulation, and signaling. In metazoans, the nuclear lamina consists of a polymeric layer of lamins and other interacting proteins responsible for its association with the INM and chromatin. In plants, field emission scanning electron microscopy of nuclei, and thin section transmission electron microscopy of isolated nucleoskeletons, reveals the lamina to have a similar structure to that of metazoans. Moreover, although plants lack lamin genes and the genes encoding most lamin-binding proteins, the main functions of the lamina are fulfilled in plants. Hence, it would appear that the plant lamina is not based on lamins and that other proteins substitute for lamins in plant cells. The nuclear matrix constituent proteins are the best characterized structural proteins in the plant lamina. Although these proteins do not display strong sequence similarity to lamins, their predicted secondary structure and sub-nuclear distribution, as well as their influence on nuclear size and shape, and on heterochromatin organization, suggest they could be functional lamin analogs. In this review we shall summarize what is currently known about the organization and composition of the plant nuclear lamina and its interacting complexes, and we will discuss the activity of this structure in the plant cell and its nucleus.

Epstein-Barr virus latent membrane protein expression in Hodgkin and Reed-Sternberg cells.

PubMed Central

Herbst, H; Dallenbach, F; Hummel, M; Niedobitek, G; Pileri, S; Müller-Lantzsch, N; Stein, H

1991-01-01

Cryostat sections from lymph nodes of 47 Hodgkin disease patients were examined by immunohistology for the Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP), nuclear antigen 2, and late viral glycoprotein gp350/250. A distinct LMP-specific membrane and cytoplasmic staining was detected exclusively in Hodgkin and Reed-Sternberg cells in 18 patients (38%); EBV nuclear antigen 2 and gp350/250 immunoreactivity was absent in all instances. Thirty-two of 47 (68%) cases contained EBV-specific DNA sequences as detected by PCR, all LMP-positive cases being in this category. Our results confirm previous studies establishing the presence of EBV genomes in Hodgkin and Reed-Sternberg cells by demonstrating expression of an EBV-encoded protein in the tumor-cell population. The finding of LMP expression in the absence of EBV nuclear antigen 2 suggests a pattern of EBV gene expression different from that of B-lymphoblastoid cell lines and Burkitt lymphoma, whereas this finding shows similarities with that seen in undifferentiated nasopharyngeal carcinoma. Because the LMP gene has transforming potential, our findings support the concept of a pathoetiological role of EBV in many cases of Hodgkin disease. Images PMID:1647016

WHITE STRIPE LEAF4 Encodes a Novel P-Type PPR Protein Required for Chloroplast Biogenesis during Early Leaf Development

PubMed Central

Wang, Ying; Ren, Yulong; Zhou, Kunneng; Liu, Linglong; Wang, Jiulin; Xu, Yang; Zhang, Huan; Zhang, Long; Feng, Zhiming; Wang, Liwei; Ma, Weiwei; Wang, Yunlong; Guo, Xiuping; Zhang, Xin; Lei, Cailin; Cheng, Zhijun; Wan, Jianmin

2017-01-01

Pentatricopeptide repeat (PPR) proteins comprise a large family in higher plants and perform diverse functions in organellar RNA metabolism. Despite the rice genome encodes 477 PRR proteins, the regulatory effects of PRR proteins on chloroplast development remains unknown. In this study, we report the functional characterization of the rice white stripe leaf4 (wsl4) mutant. The wsl4 mutant develops white-striped leaves during early leaf development, characterized by decreased chlorophyll content and malformed chloroplasts. Positional cloning of the WSL4 gene, together with complementation and RNA-interference tests, reveal that it encodes a novel P-family PPR protein with 12 PPR motifs, and is localized to chloroplast nucleoids. Quantitative RT-PCR analyses demonstrate that WSL4 is a low temperature response gene abundantly expressed in young leaves. Further expression analyses show that many nuclear- and plastid-encoded genes in the wsl4 mutant are significantly affected at the RNA and protein levels. Notably, the wsl4 mutant causes defects in the splicing of atpF, ndhA, rpl2, and rps12. Our findings identify WSL4 as a novel P-family PPR protein essential for chloroplast RNA group II intron splicing during early leaf development in rice. PMID:28694820

Cytoplasmic and nuclear localizations are important for the hypersensitive response conferred by maize autoactive Rp1-D21 protein

USDA-ARS?s Scientific Manuscript database

Disease resistance (R-) genes have been isolated from many plant species. Most encode nucleotide binding leucine-rich-repeat (NLR) proteins that trigger a rapid localized programmed cell death termed the hypersensitive response (HR) upon pathogen recognition. Despite their structural similarities, d...

Ectromelia virus encodes a family of Ankyrin/F-box proteins that regulate NFκB.

PubMed

Burles, Kristin; van Buuren, Nicholas; Barry, Michele

2014-11-01

A notable feature of poxviruses is their ability to inhibit the antiviral response, including the nuclear factor kappa B (NFκB) pathway. NFκB is a transcription factor that is sequestered in the cytoplasm until cell stimulation, and relies on the SCF (Skp1, culllin-1, F-box) ubiquitin ligase to target its inhibitor, IκBα, for degradation. IκBα is recruited to the SCF by the F-box domain-containing protein βTrCP. Here, we show that ectromelia virus, the causative agent of mousepox, encodes four F-box-containing proteins, EVM002, EVM005, EVM154, and EVM165, all of which contain Ankyrin (Ank) domains. The Ank/F-box proteins inhibit NFκB nuclear translocation, and this inhibition is dependent on the F-box domain. We also demonstrate that EVM002, EVM005, EVM154, and EVM165 prevent IκBα degradation, suggesting that they target the SCF. This study identifies a new mechanism by which ectromelia virus inhibits NFκB. Copyright © 2014 Elsevier Inc. All rights reserved.

The DUF59 Family Gene AE7 Acts in the Cytosolic Iron-Sulfur Cluster Assembly Pathway to Maintain Nuclear Genome Integrity in Arabidopsis[C][W][OA

PubMed Central

Luo, Dexian; Bernard, Delphine G.; Balk, Janneke; Hai, Huang; Cui, Xiaofeng

2012-01-01

Eukaryotic organisms have evolved a set of strategies to safeguard genome integrity, but the underlying mechanisms remain poorly understood. Here, we report that ASYMMETRIC LEAVES1/2 ENHANCER7 (AE7), an Arabidopsis thaliana gene encoding a protein in the evolutionarily conserved Domain of Unknown Function 59 family, participates in the cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway to maintain genome integrity. The severe ae7-2 allele is embryo lethal, whereas plants with the weak ae7 (ae7-1) allele are viable but exhibit highly accumulated DNA damage that activates the DNA damage response to arrest the cell cycle. AE7 is part of a protein complex with CIA1, NAR1, and MET18, which are highly conserved in eukaryotes and are involved in the biogenesis of cytosolic and nuclear Fe-S proteins. ae7-1 plants have lower activities of the cytosolic [4Fe-4S] enzyme aconitase and the nuclear [4Fe-4S] enzyme DNA glycosylase ROS1. Additionally, mutations in the gene encoding the mitochondrial ATP binding cassette transporter ATM3/ABCB25, which is required for the activity of cytosolic Fe-S enzymes in Arabidopsis, also result in defective genome integrity similar to that of ae7-1. These results indicate that AE7 is a central member of the CIA pathway, linking plant mitochondria to nuclear genome integrity through assembly of Fe-S proteins. PMID:23104832

Nuclear Involvement in the Appearance of a Chloroplast-Encoded 32,000 Dalton Thylakoid Membrane Polypeptide Integral to the Photosystem II Complex 1

PubMed Central

Leto, Kenneth J.; Keresztes, Aron; Arntzen, Charles J.

1982-01-01

The genetic locus for the high chlorophyll fluorescent photosystem II-deficient maize mutant hcf*-3 has been definitively located to the nuclear genome. Fluorography of lamellar polypeptides labeled with [35S]methionine in vivo revealed the specific loss of a heavily labeled 32,000 dalton thylakoid membrane polypeptide as well as its chloroplast encoded precursor species at 34,000 daltons. Examination of freeze-fractured mesophyll and bundle sheath thylakoids from hcf*-3 revealed that both plastid types lacked the large EFs particles believed to consist of the photosystem II reaction center-core complex and associated light harvesting chlorophyll-proteins. The present evidence suggests that the synthesis or turnover/integration of the chloroplast-encoded 34,000 to 32,000 dalton polypeptide is under nuclear control, and that these polyipeptides are integral components of photosystem II which may be required for the assembly or structural stabilization of newly formed photosystem II reaction centers in both mesophyll and bundle sheath chloroplasts. Images PMID:16662421

Xenopus LAP2β protein knockdown affects location of lamin B and nucleoporins and has effect on assembly of cell nucleus and cell viability.

PubMed

Dubińska-Magiera, Magda; Chmielewska, Magdalena; Kozioł, Katarzyna; Machowska, Magdalena; Hutchison, Christopher J; Goldberg, Martin W; Rzepecki, Ryszard

2016-05-01

Xenopus LAP2β protein is the single isoform expressed in XTC cells. The protein localizes on heterochromatin clusters both at the nuclear envelope and inside a cell nucleus. The majority of XLAP2β fraction neither colocalizes with TPX2 protein during interphase nor can be immunoprecipitated with XLAP2β antibody. Knockdown of the XLAP2β protein expression in XTC cells by synthetic siRNA and plasmid encoded siRNA resulted in nuclear abnormalities including changes in shape of nuclei, abnormal chromatin structure, loss of nuclear envelope, mislocalization of integral membrane proteins of INM such as lamin B2, mislocalization of nucleoporins, and cell death. Based on timing of cell death, we suggest mechanism associated with nucleus reassembly or with entry into mitosis. This confirms that Xenopus LAP2 protein is essential for the maintenance of cell nucleus integrity and the process of its reassembly after mitosis.

Correlation of mitochondrial protein expression in complexes I to V with natural and induced forms of canine idiopathic dilated cardiomyopathy.

PubMed

Lopes, Rosana; Solter, Philip F; Sisson, D David; Oyama, Mark A; Prosek, Robert

2006-06-01

To identify qualitative and quantitative differences in cardiac mitochondrial protein expression in complexes I to V between healthy dogs and dogs with natural or induced dilated cardiomyopathy (DCM). Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with DCM induced by rapid right ventricular pacing. Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by 2-fold or greater between groups were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry. A total of 22 altered mitochondrial proteins were identified in complexes I to V. Ten and 12 were found in complex I and complexes II to V, respectively. Five were mitochondrial encoded, and 17 were nuclear encoded. Most altered mitochondrial proteins in tissue specimens from dogs with naturally occurring DCM were associated with complexes I and V, whereas in tissue specimens from dogs subjected to rapid ventricular pacing, complexes I and IV were more affected. In the experimentally induced form of DCM, only nuclear-encoded subunits were changed in complex I. In both disease groups, the 22-kd subunit was downregulated. Natural and induced forms of DCM resulted in altered mitochondrial protein expression in complexes I to V. However, subcellular differences between the experimental and naturally occurring forms of DCM may exist.

RNA Nuclear Export: From Neurological Disorders to Cancer.

PubMed

Hautbergue, Guillaume M

2017-01-01

The presence of a nuclear envelope, also known as nuclear membrane, defines the structural framework of all eukaryotic cells by separating the nucleus, which contains the genetic material, from the cytoplasm where the synthesis of proteins takes place. Translation of proteins in Eukaryotes is thus dependent on the active transport of DNA-encoded RNA molecules through pores embedded within the nuclear membrane. Several mechanisms are involved in this process generally referred to as RNA nuclear export or nucleocytoplasmic transport of RNA. The regulated expression of genes requires the nuclear export of protein-coding messenger RNA molecules (mRNAs) as well as non-coding RNAs (ncRNAs) together with proteins and pre-assembled ribosomal subunits. The nuclear export of mRNAs is intrinsically linked to the co-transcriptional processing of nascent transcripts synthesized by the RNA polymerase II. This functional coupling is essential for the survival of cells allowing for timely nuclear export of fully processed transcripts, which could otherwise cause the translation of abnormal proteins such as the polymeric repeat proteins produced in some neurodegenerative diseases. Alterations of the mRNA nuclear export pathways can also lead to genome instability and to various forms of cancer. This chapter will describe the molecular mechanisms driving the nuclear export of RNAs with a particular emphasis on mRNAs. It will also review their known alterations in neurological disorders and cancer, and the recent opportunities they offer for the potential development of novel therapeutic strategies.

Intracellular Distribution of Capsid-Associated pUL77 of Human Cytomegalovirus and Interactions with Packaging Proteins and pUL93.

PubMed

Köppen-Rung, Pánja; Dittmer, Alexandra; Bogner, Elke

2016-07-01

DNA packaging into procapsids is a common multistep process during viral maturation in herpesviruses. In human cytomegalovirus (HCMV), the proteins involved in this process are terminase subunits pUL56 and pUL89, which are responsible for site-specific cleavage and insertion of the DNA into the procapsid via portal protein pUL104. However, additional viral proteins are required for the DNA packaging process. We have shown previously that the plasmid that encodes capsid-associated pUL77 encodes another potential player during capsid maturation. Pulse-chase experiments revealed that pUL77 is stably expressed during HCMV infection. Time course analysis demonstrated that pUL77 is expressed in the early late part of the infectious cycle. The sequence of pUL77 was analyzed to find nuclear localization sequences (NLSs), revealing monopartite NLSm at the N terminus and bipartite NLSb in the middle of pUL77. The potential NLSs were inserted into plasmid pHM829, which encodes a chimeric protein with β-galactosidase and green fluorescent protein. In contrast to pUL56, neither NLSm nor NLSb was sufficient for nuclear import. Furthermore, we investigated by coimmunoprecipitation whether packaging proteins, as well as pUL93, the homologue protein of herpes simplex virus 1 pUL17, are interaction partners of pUL77. The interactions between pUL77 and packaging proteins, as well as pUL93, were verified. We showed that the capsid-associated pUL77 is another potential player during capsid maturation of HCMV. Protein UL77 (pUL77) is a conserved core protein of HCMV. This study demonstrates for the first time that pUL77 has early-late expression kinetics during the infectious cycle and an intrinsic potential for nuclear translocation. According to its proposed functions in stabilization of the capsid and anchoring of the encapsidated DNA during packaging, interaction with further DNA packaging proteins is required. We identified physical interactions with terminase subunits pUL56 and pUL89 and another postulated packaging protein, pUL93, in infected, as well as transfected, cells. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Deficiency of Ferritin Heavy-Chain Nuclear Import in Triple A Syndrome Implies Nuclear Oxidative Damage as the Primary Disease Mechanism

PubMed Central

Storr, Helen L.; Kind, Barbara; Parfitt, David A.; Chapple, J. Paul; Lorenz, M.; Koehler, Katrin; Huebner, Angela; Clark, Adrian J. L.

2009-01-01

Triple A syndrome is a rare autosomal recessive disorder characterized by ACTH-resistant adrenal failure, alacrima, achalasia, and progressive neurological manifestations. The majority of cases are associated with mutations in the AAAS gene, which encodes a novel, 60-kDa WD-repeat nuclear pore protein, alacrima-achalasia-adrenal insufficiency neurological disorder (ALADIN) of unknown function. Our aim was to elucidate the functional role of ALADIN by determining its interacting protein partners using the bacterial two-hybrid (B2-H) technique. Nonidentical cDNA fragments were identified from both a HeLa S-3 cell and human cerebellar cDNA library that encoded the full-length ferritin heavy chain protein (FTH1). This interaction was confirmed by both co-immunoprecipitation and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer studies. Immunoblotting showed that fibroblasts from triple A patients (with known AAAS mutations) lack nuclear FTH1, suggesting that the nuclear translocation of FTH1 is defective. Cells transfected with FTH1 and visualized by confocal microscopy had very little nuclear FTH1, but when cotransfected with AAAS, FTH1 is readily visible in the nuclei. Therefore, FTH1 nuclear translocation is enhanced when ALADIN is coexpressed in these cells. In addition to its well known iron storage role, FTH1 has been shown to protect the nucleus from oxidative damage. Apoptosis of neuronal cells induced by hydrogen peroxide was significantly reduced by transfection of AAAS or by FTH1 or maximally by both genes together. Taken together, this work offers a plausible mechanism for the progressive clinical features of triple A syndrome. PMID:19855093

A Long Terminal Repeat-Containing Retrotransposon of Schizosaccharomyces pombe Expresses a Gag-Like Protein That Assembles into Virus-Like Particles Which Mediate Reverse Transcription

PubMed Central

Teysset, Laure; Dang, Van-Dinh; Kim, Min Kyung; Levin, Henry L.

2003-01-01

The Tf1 element of Schizosaccharomyces pombe is a long terminal repeat-containing retrotransposon that encodes functional protease, reverse transcriptase, and integrase proteins. Although these proteins are known to be necessary for protein processing, reverse transcription, and integration, respectively, the function of the protein thought to be Gag has not been determined. We present here the first electron microscopy of Tf1 particles. We tested whether the putative Gag of Tf1 was required for particle formation, packaging of RNA, and reverse transcription. We generated deletions of 10 amino acids in each of the four hydrophilic domains of the protein and found that all four mutations reduced transposition activity. The N-terminal deletion removed a nuclear localization signal and inhibited nuclear import of the transposon. The two mutations in the center of Gag destabilized the protein and resulted in no virus-like particles. The C-terminal deletion caused a defect in RNA packaging and, as a result, low levels of cDNA. The electron microscopy of cells expressing a truncated Tf1 showed that Gag alone was sufficient for the formation of virus-like particles. Taken together, these results indicate that Tf1 encodes a Gag protein that is a functional equivalent of the Gag proteins of retroviruses. PMID:12692246

A model for the dynamic nuclear/nucleolar/cytoplasmic trafficking of the porcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid protein based on live cell imaging

DOE Office of Scientific and Technical Information (OSTI.GOV)

You, Jae-Hwan; Howell, Gareth; Pattnaik, Asit K.

2008-08-15

Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, in common with many other positive strand RNA viruses, encodes a nucleocapsid (N) protein which can localise not only to the cytoplasm but also to the nucleolus in virus-infected cells and cells over-expressing N protein. The dynamic trafficking of positive strand RNA virus nucleocapsid proteins and PRRSV N protein in particular between the cytoplasm and nucleolus is unknown. In this study live imaging of permissive and non-permissive cell lines, in conjunction with photo-bleaching (FRAP and FLIP), was used to investigate the trafficking of fluorescent labeled (EGFP) PRRSV-N protein. The data indicatedmore » that EGFP-PRRSV-N protein was not permanently sequestered to the nucleolus and had equivalent mobility to cellular nucleolar proteins. Further the nuclear import of N protein appeared to occur faster than nuclear export, which may account for the observed relative distribution of N protein between the cytoplasm and the nucleolus.« less

Microsporidia, amitochondrial protists, possess a 70-kDa heat shock protein gene of mitochondrial evolutionary origin.

PubMed

Peyretaillade, E; Broussolle, V; Peyret, P; Méténier, G; Gouy, M; Vivarès, C P

1998-06-01

An intronless gene encoding a protein of 592 amino acid residues with similarity to 70-kDa heat shock proteins (HSP70s) has been cloned and sequenced from the amitochondrial protist Encephalitozoon cuniculi (phylum Microsporidia). Southern blot analyses show the presence of a single gene copy located on chromosome XI. The encoded protein exhibits an N-terminal hydrophobic leader sequence and two motifs shared by proteobacterial and mitochondrially expressed HSP70 homologs. Phylogenetic analysis using maximum likelihood and evolutionary distances place the E. cuniculi sequence in the cluster of mitochondrially expressed HSP70s, with a higher evolutionary rate than those of homologous sequences. Similar results were obtained after cloning a fragment of the homologous gene in the closely related species E. hellem. The presence of a nuclear targeting signal-like sequence supports a role of the Encephalitozoon HSP70 as a molecular chaperone of nuclear proteins. No evidence for cytosolic or endoplasmic reticulum forms of HSP70 was obtained through PCR amplification. These data suggest that Encephalitozoon species have evolved from an ancestor bearing mitochondria, which is in disagreement with the postulated presymbiotic origin of Microsporidia. The specific role and intracellular localization of the mitochondrial HSP70-like protein remain to be elucidated.

Analysis of Sir2E in the cellular slime mold Dictyostelium discoideum: cellular localization, spatial expression and overexpression.

PubMed

Katayama, Takahiro; Yasukawa, Hiro

2008-10-01

It has been reported that Dictyostelium discoideum encodes four silent information regulator 2 (Sir2) proteins (Sir2A-D) showing sequence similarity to human homologues of Sir2 (SIRT1-3). Further screening in a database revealed that D. discoideum encodes an additional Sir2 homologue (Sir2E). The amino acid sequence of Sir2E is not similar to those of SIRTs but is similar to those of proteins encoded by Giardia lamblia, Cryptosporidium hominis and Cryptosporidium parvum. Fluorescence of Sir2E-green fluorescent protein fusion protein was detected in the D. discoideum nucleus, indicating that Sir2E is a nuclear localizing protein. Reverse transcription-polymerase chain reaction and whole-mount in situ hybridization analyses showed that D. discoideum expressed sir2E in amoebae in the growth phase and in prestalk cells in the developmental phase. D. discoideum overexpressing sir2E grew faster than the wild type. These results indicate that Sir2E plays important roles both in the growth phase and developmental phase of D. discoideum.

Translation and Assembly of Radiolabeled Mitochondrial DNA-Encoded Protein Subunits from Cultured Cells and Isolated Mitochondria.

PubMed

Formosa, Luke E; Hofer, Annette; Tischner, Christin; Wenz, Tina; Ryan, Michael T

2016-01-01

In higher eukaryotes, the mitochondrial electron transport chain consists of five multi-subunit membrane complexes responsible for the generation of cellular ATP. Of these, four complexes are under dual genetic control as they contain subunits encoded by both the mitochondrial and nuclear genomes, thereby adding another layer of complexity to the puzzle of respiratory complex biogenesis. These subunits must be synthesized and assembled in a coordinated manner in order to ensure correct biogenesis of different respiratory complexes. Here, we describe techniques to (1) specifically radiolabel proteins encoded by mtDNA to monitor the rate of synthesis using pulse labeling methods, and (2) analyze the stability, assembly, and turnover of subunits using pulse-chase methods in cultured cells and isolated mitochondria.

Molecular cloning and expression of the gene encoding the kinetoplast-associated type II DNA topoisomerase of Crithidia fasciculata.

PubMed

Pasion, S G; Hines, J C; Aebersold, R; Ray, D S

1992-01-01

A type II DNA topoisomerase, topoIImt, was shown previously to be associated with the kinetoplast DNA of the trypanosomatid Crithidia fasciculata. The gene encoding this kinetoplast-associated topoisomerase has been cloned by immunological screening of a Crithidia genomic expression library with monoclonal antibodies raised against the purified enzyme. The gene CfaTOP2 is a single copy gene and is expressed as a 4.8-kb polyadenylated transcript. The nucleotide sequence of CfaTOP2 has been determined and encodes a predicted polypeptide of 1239 amino acids with a molecular mass of 138,445. The identification of the cloned gene is supported by immunoblot analysis of the beta-galactosidase-CfaTOP2 fusion protein expressed in Escherichia coli and by analysis of tryptic peptide sequences derived from purified topoIImt. CfaTOP2 shares significant homology with nuclear type II DNA topoisomerases of other eukaryotes suggesting that in Crithidia both nuclear and mitochondrial forms of topoisomerase II are encoded by the same gene.

The 21.5-kDa isoform of myelin basic protein has a non-traditional PY-nuclear-localization signal

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Graham S.T.; Seymour, Lauren V.; Boggs, Joan M.

2012-06-15

Highlights: Black-Right-Pointing-Pointer Full-length 21.5-kDa MBP isoform is translocated to the nucleus. Black-Right-Pointing-Pointer We hypothesized that the exon-II-encoded sequence contained the NLS. Black-Right-Pointing-Pointer We mutated this sequence in RFP-tagged constructs and transfected N19-cells. Black-Right-Pointing-Pointer Abolition of two key positively-charged residues resulted in loss of nuclear-trafficking. Black-Right-Pointing-Pointer The 21.5-kDa isoform of classic MBP contains a non-traditional PY-NLS. -- Abstract: The predominant 18.5-kDa classic myelin basic protein (MBP) is mainly responsible for compaction of the myelin sheath in the central nervous system, but is multifunctional, having numerous interactions with Ca{sup 2+}-calmodulin, actin, tubulin, and SH3-domains, and can tether these proteins to a lipidmore » membrane in vitro. The full-length 21.5-kDa MBP isoform has an additional 26 residues encoded by exon-II of the classic gene, which causes it to be trafficked to the nucleus of oligodendrocytes (OLGs). We have performed site-directed mutagenesis of selected residues within this segment in red fluorescent protein (RFP)-tagged constructs, which were then transfected into the immortalized N19-OLG cell line to view protein localization using epifluorescence microscopy. We found that 21.5-kDa MBP contains two non-traditional PY-nuclear-localization signals, and that arginine and lysine residues within these motifs were involved in subcellular trafficking of this protein to the nucleus, where it may have functional roles during myelinogenesis.« less

mRNA localization to the mitochondrial surface allows the efficient translocation inside the organelle of a nuclear recoded ATP6 protein

PubMed Central

Kaltimbacher, Valérie; Bonnet, Crystel; Lecoeuvre, Gaëlle; Forster, Valérie; Sahel, José-Alain; Corral-Debrinski, Marisol

2006-01-01

As previously established in yeast, two sequences within mRNAs are responsible for their specific localization to the mitochondrial surface—the region coding for the mitochondrial targeting sequence and the 3′UTR. This phenomenon is conserved in human cells. Therefore, we decided to use mRNA localization as a tool to address to mitochondria, a protein that is not normally imported. For this purpose, we associated a nuclear recoded ATP6 gene with the mitochondrial targeting sequence and the 3′UTR of the nuclear SOD2 gene, which mRNA exclusively localizes to the mitochondrial surface in HeLa cells. The ATP6 gene is naturally located into the organelle and encodes a highly hydrophobic protein of the respiratory chain complex V. In this study, we demonstrated that hybrid ATP6 mRNAs, as the endogenous SOD2 mRNA, localize to the mitochondrial surface in human cells. Remarkably, fusion proteins localize to mitochondria in vivo. Indeed, ATP6 precursors synthesized in the cytoplasm were imported into mitochondria in a highly efficient way, especially when both the MTS and the 3′UTR of the SOD2 gene were associated with the re-engineered ATP6 gene. Hence, these data indicate that mRNA targeting to the mitochondrial surface represents an attractive strategy for allowing the mitochondrial import of proteins originally encoded by the mitochondrial genome without any amino acid change in the protein that could interfere with its biologic activity. PMID:16751614

DOE Office of Scientific and Technical Information (OSTI.GOV)

Antonacci, R.; Colombo, I.; Volta, M.

The electron-transfer flavoprotein (ETF), located in the mitochondrial matrix, is a nuclear-encoded enzyme delivering to the respiratory chain electrons by straight-chain acyl-CoA dehydrogenases and other dehydrogenases. ETF is composed of a 35-kDa [alpha]-subunit that is cleaved to a 32-kDa protein during mitochondrial import (ETFA) and a [beta]-subunit that reaches the mitochondrion unmodified (ETFB). The cDNA encoding both these subunits has been cloned and sequenced. 14 refs., 1 fig.

Inventory of high-abundance mRNAs in skeletal muscle of normal men.

PubMed

Welle, S; Bhatt, K; Thornton, C A

1999-05-01

G42875rial analysis of gene expression (SAGE) method was used to generate a catalog of 53,875 short (14 base) expressed sequence tags from polyadenylated RNA obtained from vastus lateralis muscle of healthy young men. Over 12,000 unique tags were detected. The frequency of occurrence of each tag reflects the relative abundance of the corresponding mRNA. The mRNA species that were detected 10 or more times, each comprising >/=0.02% of the mRNA population, accounted for 64% of the mRNA mass but <10% of the total number of mRNA species detected. Almost all of the abundant tags matched mRNA or EST sequences cataloged in GenBank. Mitochondrial transcripts accounted for approximately 20% of the polyadenylated RNA. Transcripts encoding proteins of the myofibrils were the most abundant nuclear-encoded mRNAs. Transcripts encoding ribosomal proteins, and those encoding proteins involved in energy metabolism, also were very abundant. The database can be used as a reference for investigations of alterations in gene expression associated with conditions that influence muscle function, such as muscular dystrophies, aging, and exercise.

[Molecular cloning and characterization of cDNA of the rpc10+ gene encoding the smallest subunit of nuclear RNA polymerases of Schizosaccharomyces pombe].

PubMed

Shpakovskiĭ, G V; Lebedenko, E N

1997-05-01

The full-length cDNA of the rpc10+ gene encoding mini-subunit Rpc10, which is common for all three nuclear RNA polymerases of the fission yeast Schizosaccharomyces pombe, was cloned and sequenced. The Rpc10 subunit of Sz. pombe and its homologs from S. cerevisiae and H. sapiens are positively charged proteins with a highly conserved C-terminal region and an invariant zinc-binding domain (Zn-finger) of a typical amino acid composition: YxCx2Cx12RCx2CGxR. Functional tests of heterospecific complementation, using tetrad analysis or plasmid shuffling, showed that the Rpc10 subunit of Sz. pombe can successfully replace the homologous ABC10 alpha subunit in nuclear RNA polymerases I-III of S. cerevisiae.

Drosophila histone locus bodies form by hierarchical recruitment of components

PubMed Central

White, Anne E.; Burch, Brandon D.; Yang, Xiao-cui; Gasdaska, Pamela Y.; Dominski, Zbigniew; Marzluff, William F.

2011-01-01

Nuclear bodies are protein- and RNA-containing structures that participate in a wide range of processes critical to genome function. Molecular self-organization is thought to drive nuclear body formation, but whether this occurs stochastically or via an ordered, hierarchical process is not fully understood. We addressed this question using RNAi and proteomic approaches in Drosophila melanogaster to identify and characterize novel components of the histone locus body (HLB), a nuclear body involved in the expression of replication-dependent histone genes. We identified the transcription elongation factor suppressor of Ty 6 (Spt6) and a homologue of mammalian nuclear protein of the ataxia telangiectasia–mutated locus that is encoded by the homeotic gene multisex combs (mxc) as novel HLB components. By combining genetic manipulation in both cell culture and embryos with cytological observations of Mxc, Spt6, and the known HLB components, FLICE-associated huge protein, Mute, U7 small nuclear ribonucleoprotein, and MPM-2 phosphoepitope, we demonstrated sequential recruitment and hierarchical dependency for localization of factors to HLBs during development, suggesting that ordered assembly can play a role in nuclear body formation. PMID:21576393

A functional TOC complex contributes to gravity signal transduction in Arabidopsis

PubMed Central

Strohm, Allison K.; Barrett-Wilt, Greg A.; Masson, Patrick H.

2014-01-01

Although plastid sedimentation has long been recognized as important for a plant's perception of gravity, it was recently shown that plastids play an additional function in gravitropism. The Translocon at the Outer envelope membrane of Chloroplasts (TOC) complex transports nuclear-encoded proteins into plastids, and a receptor of this complex, Toc132, was previously hypothesized to contribute to gravitropism either by directly functioning as a gravity signal transducer or by indirectly mediating the plastid localization of a gravity signal transducer. Here we show that mutations in multiple genes encoding TOC complex components affect gravitropism in a genetically sensitized background and that the cytoplasmic acidic domain of Toc132 is not required for its involvement in this process. Furthermore, mutations in TOC132 enhance the gravitropic defect of a mutant whose amyloplasts lack starch. Finally, we show that the levels of several nuclear-encoded root proteins are altered in toc132 mutants. These data suggest that the TOC complex indirectly mediates gravity signal transduction in Arabidopsis and support the idea that plastids are involved in gravitropism not only through their ability to sediment but also as part of the signal transduction mechanism. PMID:24795735

A functional TOC complex contributes to gravity signal transduction in Arabidopsis.

PubMed

Strohm, Allison K; Barrett-Wilt, Greg A; Masson, Patrick H

2014-01-01

Although plastid sedimentation has long been recognized as important for a plant's perception of gravity, it was recently shown that plastids play an additional function in gravitropism. The Translocon at the Outer envelope membrane of Chloroplasts (TOC) complex transports nuclear-encoded proteins into plastids, and a receptor of this complex, Toc132, was previously hypothesized to contribute to gravitropism either by directly functioning as a gravity signal transducer or by indirectly mediating the plastid localization of a gravity signal transducer. Here we show that mutations in multiple genes encoding TOC complex components affect gravitropism in a genetically sensitized background and that the cytoplasmic acidic domain of Toc132 is not required for its involvement in this process. Furthermore, mutations in TOC132 enhance the gravitropic defect of a mutant whose amyloplasts lack starch. Finally, we show that the levels of several nuclear-encoded root proteins are altered in toc132 mutants. These data suggest that the TOC complex indirectly mediates gravity signal transduction in Arabidopsis and support the idea that plastids are involved in gravitropism not only through their ability to sediment but also as part of the signal transduction mechanism.

Phylogenetic Analysis of Nuclear-Encoded RNA Maturases

PubMed Central

Malik, Sunita; Upadhyaya, KC; Khurana, SM Paul

2017-01-01

Posttranscriptional processes, such as splicing, play a crucial role in gene expression and are prevalent not only in nuclear genes but also in plant mitochondria where splicing of group II introns is catalyzed by a class of proteins termed maturases. In plant mitochondria, there are 22 mitochondrial group II introns. matR, nMAT1, nMAT2, nMAT3, and nMAT4 proteins have been shown to be required for efficient splicing of several group II introns in Arabidopsis thaliana. Nuclear maturases (nMATs) are necessary for splicing of mitochondrial genes, leading to normal oxidative phosphorylation. Sequence analysis through phylogenetic tree (including bootstrapping) revealed high homology with maturase sequences of A thaliana and other plants. This study shows the phylogenetic relationship of nMAT proteins between A thaliana and other nonredundant plant species taken from BLASTP analysis. PMID:28607538

Distinct Calcium Signaling Pathways Regulate Calmodulin Gene Expression in Tobacco1

PubMed Central

van der Luit, Arnold H.; Olivari, Claudio; Haley, Ann; Knight, Marc R.; Trewavas, Anthony J.

1999-01-01

Cold shock and wind stimuli initiate Ca2+ transients in transgenic tobacco (Nicotiana plumbaginifolia) seedlings (named MAQ 2.4) containing cytoplasmic aequorin. To investigate whether these stimuli initiate Ca2+ pathways that are spatially distinct, stress-induced nuclear and cytoplasmic Ca2+ transients and the expression of a stress-induced calmodulin gene were compared. Tobacco seedlings were transformed with a construct that encodes a fusion protein between nucleoplasmin (a major oocyte nuclear protein) and aequorin. Immunocytochemical evidence indicated targeting of the fusion protein to the nucleus in these plants, which were named MAQ 7.11. Comparison between MAQ 7.11 and MAQ 2.4 seedlings confirmed that wind stimuli and cold shock invoke separate Ca2+ signaling pathways. Partial cDNAs encoding two tobacco calmodulin genes, NpCaM-1 and NpCaM-2, were identified and shown to have distinct nucleotide sequences that encode identical polypeptides. Expression of NpCaM-1, but not NpCaM-2, responded to wind and cold shock stimulation. Comparison of the Ca2+ dynamics with NpCaM-1 expression after stimulation suggested that wind-induced NpCaM-1 expression is regulated by a Ca2+ signaling pathway operational predominantly in the nucleus. In contrast, expression of NpCaM-1 in response to cold shock is regulated by a pathway operational predominantly in the cytoplasm. PMID:10557218

The fanconi anemia proteins FANCA and FANCG stabilize each other and promote the nuclear accumulation of the Fanconi anemia complex.

PubMed

Garcia-Higuera, I; Kuang, Y; Denham, J; D'Andrea, A D

2000-11-01

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with 8 complementation groups. Four of the FA genes have been cloned, and at least 3 of the encoded proteins, FANCA, FANCC, and FANCG/XRCC9, interact in a multisubunit protein complex. The FANCG protein binds directly to the amino terminal nuclear localization sequence (NLS) of FANCA, suggesting that FANCG plays a role in regulating FANCA nuclear accumulation. In the current study the functional consequences of FANCG/FANCA binding were examined. Correction of an FA-G cell line with the FANCG complementary DNA (cDNA) resulted in FANCA/FANCG binding, prolongation of the cellular half-life of FANCA, and an increase in the nuclear accumulation of the FA protein complex. Similar results were obtained upon correction of an FA-A cell line, with a reciprocal increase in the half-life of FANCG. Patient-derived mutant forms of FANCA, containing an intact NLS sequence but point mutations in the carboxy-terminal leucine zipper region, bound FANCG in the cytoplasm. The mutant forms failed to translocate to the nucleus of transduced cells, thereby suggesting a model of coordinated binding and nuclear translocation. These results demonstrate that the FANCA/FANCG interaction is required to maintain the cellular levels of both proteins. Moreover, at least one function of FANCG and FANCA is to regulate the nuclear accumulation of the FA protein complex. Failure to accumulate the nuclear FA protein complex results in the characteristic spectrum of clinical and cellular abnormalities observed in FA.

PRIC320, a transcription coactivator, isolated from peroxisome proliferator-binding protein complex.

PubMed

Surapureddi, Sailesh; Viswakarma, Navin; Yu, Songtao; Guo, Dongsheng; Rao, M Sambasiva; Reddy, Janardan K

2006-05-05

Ciprofibrate, a potent peroxisome proliferator, induces pleiotropic responses in liver by activating peroxisome proliferator-activated receptor alpha (PPARalpha), a nuclear receptor. Transcriptional regulation by liganded nuclear receptors involves the participation of coregulators that form multiprotein complexes possibly to achieve cell and gene specific transcription. SDS-PAGE and matrix-assisted laser desorption/ionization reflection time-of-flight mass spectrometric analyses of ciprofibrate-binding proteins from liver nuclear extracts obtained using ciprofibrate-Sepharose affinity matrix resulted in the identification of a new high molecular weight nuclear receptor coactivator, which we designated PRIC320. The full-length human cDNA encoding this protein has an open-reading frame that codes for a 320kDa protein containing 2882 amino acids. PRIC320 contains five LXXLL signature motifs that mediate interaction with nuclear receptors. PRIC320 binds avidly to nuclear receptors PPARalpha, CAR, ERalpha, and RXR, but only minimally with PPARgamma. PRIC320 also interacts with transcription cofactors CBP, PRIP, and PBP. Immunoprecipitation-immunoblotting as well as cellular localization studies confirmed the interaction between PPARalpha and PRIC320. PRIC320 acts as a transcription coactivator by stimulating PPARalpha-mediated transcription. We conclude that ciprofibrate, a PPARalpha ligand, binds a multiprotein complex and PRIC320 cloned from this complex functions as a nuclear receptor coactivator.

Coordinated Rates of Evolution between Interacting Plastid and Nuclear Genes in Geraniaceae

PubMed Central

Zhang, Jin; Ruhlman, Tracey A.; Sabir, Jamal; Blazier, J. Chris; Jansen, Robert K.

2015-01-01

Although gene coevolution has been widely observed within individuals and between different organisms, rarely has this phenomenon been investigated within a phylogenetic framework. The Geraniaceae is an attractive system in which to study plastid-nuclear genome coevolution due to the highly elevated evolutionary rates in plastid genomes. In plants, the plastid-encoded RNA polymerase (PEP) is a protein complex composed of subunits encoded by both plastid (rpoA, rpoB, rpoC1, and rpoC2) and nuclear genes (sig1-6). We used transcriptome and genomic data for 27 species of Geraniales in a systematic evaluation of coevolution between genes encoding subunits of the PEP holoenzyme. We detected strong correlations of dN (nonsynonymous substitutions) but not dS (synonymous substitutions) within rpoB/sig1 and rpoC2/sig2, but not for other plastid/nuclear gene pairs, and identified the correlation of dN/dS ratio between rpoB/C1/C2 and sig1/5/6, rpoC1/C2 and sig2, and rpoB/C2 and sig3 genes. Correlated rates between interacting plastid and nuclear sequences across the Geraniales could result from plastid-nuclear genome coevolution. Analyses of coevolved amino acid positions suggest that structurally mediated coevolution is not the major driver of plastid-nuclear coevolution. The detection of strong correlation of evolutionary rates between SIG and RNAP genes suggests a plausible explanation for plastome-genome incompatibility in Geraniaceae. PMID:25724640

FHL1B Interacts with Lamin A/C and Emerin at the Nuclear Lamina and is Misregulated in Emery-Dreifuss Muscular Dystrophy.

PubMed

Ziat, Esma; Mamchaoui, Kamel; Beuvin, Maud; Nelson, Isabelle; Azibani, Feriel; Spuler, Simone; Bonne, Gisèle; Bertrand, Anne T

2016-11-29

Emery-Dreifuss muscular dystrophy (EDMD) is associated with mutations in EMD and LMNA genes, encoding for the nuclear envelope proteins emerin and lamin A/C, indicating that EDMD is a nuclear envelope disease. We recently reported mutations in FHL1 gene in X-linked EDMD. FHL1 encodes FHL1A, and the two minor isoforms FHL1B and FHL1C. So far, none have been described at the nuclear envelope. To gain insight into the pathophysiology of EDMD, we focused our attention on the poorly characterized FHL1B isoform. The amount and the localisation of FHL1B were evaluated in control and diseased human primary myoblasts using immunofluorescence and western blotting. We found that in addition to a cytoplasmic localization, this isoform strongly accumulated at the nuclear envelope of primary human myoblasts, like but independently of lamin A/C and emerin. During myoblast differentiation, we observed a major reduction of FHL1B protein expression, especially in the nucleus. Interestingly, we found elevated FHL1B expression level in myoblasts from an FHL1-related EDMD patient where the FHL1 mutation only affects FHL1A, as well as in myoblasts from an LMNA-related EDMD patient. Altogether, the specific localization of FHL1B and its modulation in disease-patient's myoblasts confirmed FHL1-related EDMD as a nuclear envelope disease.

Mutation in the nuclear-encoded mitochondrial isoleucyl-tRNA synthetase IARS2 in patients with cataracts, growth hormone deficiency with short stature, partial sensorineural deafness, and peripheral neuropathy or with Leigh syndrome.

PubMed

Schwartzentruber, Jeremy; Buhas, Daniela; Majewski, Jacek; Sasarman, Florin; Papillon-Cavanagh, Simon; Thiffault, Isabelle; Thiffaut, Isabelle; Sheldon, Katherine M; Massicotte, Christine; Patry, Lysanne; Simon, Mariella; Zare, Amir S; McKernan, Kevin J; Michaud, Jacques; Boles, Richard G; Deal, Cheri L; Desilets, Valerie; Shoubridge, Eric A; Samuels, Mark E

2014-11-01

Mutations in the nuclear-encoded mitochondrial aminoacyl-tRNA synthetases are associated with a range of clinical phenotypes. Here, we report a novel disorder in three adult patients with a phenotype including cataracts, short-stature secondary to growth hormone deficiency, sensorineural hearing deficit, peripheral sensory neuropathy, and skeletal dysplasia. Using SNP genotyping and whole-exome sequencing, we identified a single likely causal variant, a missense mutation in a conserved residue of the nuclear gene IARS2, encoding mitochondrial isoleucyl-tRNA synthetase. The mutation is homozygous in the affected patients, heterozygous in carriers, and absent in control chromosomes. IARS2 protein level was reduced in skin cells cultured from one of the patients, consistent with a pathogenic effect of the mutation. Compound heterozygous mutations in IARS2 were independently identified in a previously unreported patient with a more severe mitochondrial phenotype diagnosed as Leigh syndrome. This is the first report of clinical findings associated with IARS2 mutations. © 2014 WILEY PERIODICALS, INC.

A protein that interacts with members of the nuclear hormone receptor family: identification and cDNA cloning.

PubMed Central

Zeiner, M; Gehring, U

1995-01-01

In search of proteins which interact with activated steroid hormone receptors, we screened a human liver lambda gt11 expression library with the glucocorticoid receptor. We identified and cloned a cDNA sequence of 1322 bp that encodes a protein of 274 aa. This protein consists predominantly of hydrophilic amino acids and contains a putative bipartite nuclear localization signal. The in vitro translated receptor-associating protein runs in SDS/polyacrylamide gels with an apparent molecular mass of 46 kDa. By use of the bacterially expressed fusion protein with glutathione S-transferase we have found that interaction is not limited to the glucocorticoid receptor but included other nuclear receptors--most notably, the estrogen and thyroid receptors. Binding also occurs with the glucocorticoid receptor complexed with the antiglucocorticoid RU 38486, with the estrogen receptor complexed with the antiestrogen 4-hydroxytamoxifen or ICI 164,384, and even with receptors not complexed with ligand. Association with steroid hormone receptors depends on prior receptor activation--i.e., release from heat shock proteins. The sequence identified here appears to be a general partner protein for nuclear hormone receptors, with the gene being expressed in a variety of mammalian tissues. Images Fig. 2 Fig. 3 Fig. 4 PMID:8524784

Human Mitochondrial Protein Database

National Institute of Standards and Technology Data Gateway

SRD 131 Human Mitochondrial Protein Database (Web, free access)   The Human Mitochondrial Protein Database (HMPDb) provides comprehensive data on mitochondrial and human nuclear encoded proteins involved in mitochondrial biogenesis and function. This database consolidates information from SwissProt, LocusLink, Protein Data Bank (PDB), GenBank, Genome Database (GDB), Online Mendelian Inheritance in Man (OMIM), Human Mitochondrial Genome Database (mtDB), MITOMAP, Neuromuscular Disease Center and Human 2-D PAGE Databases. This database is intended as a tool not only to aid in studying the mitochondrion but in studying the associated diseases.

Effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells.

PubMed

Minchenko, O H; Riabovol, O O; Tsymbal, D O; Minchenko, D O; Ratushna, O O

2016-01-01

We have studied the effect of hypoxia on the expression of nuclear genes encoding mitochondrial proteins in U87 glioma cells under the inhibition of IRE1 (inositol requiring enzyme-1), which controls cell proliferation and tumor growth as a central mediator of endoplasmic reticulum stress. It was shown that hypoxia down-regulated gene expression of malate dehydrogenase 2 (MDH2), malic enzyme 2 (ME2), mitochondrial aspartate aminotransferase (GOT2), and subunit B of succinate dehydrogenase (SDHB) in control (transfected by empty vector) glioma cells in a gene specific manner. At the same time, the expression level of mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) and subunit D of succinate dehydrogenase (SDHD) genes in these cells does not significantly change in hypoxic conditions. It was also shown that the inhibition of ІRE1 signaling enzyme function in U87 glioma cells decreases the effect of hypoxia on the expression of ME2, GOT2, and SDHB genes and introduces the sensitivity of IDH2 gene to hypoxia. Furthermore, the expression of all studied genes depends on IRE1-mediated endoplasmic reticulum stress signaling in gene specific manner, because ІRE1 knockdown significantly decreases their expression in normoxic conditions, except for IDH2 gene, which expression level is strongly up-regulated. Therefore, changes in the expression level of nuclear genes encoding ME2, MDH2, IDH2, SDHB, SDHD, and GOT2 proteins possibly reflect metabolic reprogramming of mitochondria by hypoxia and IRE1-mediated endoplasmic reticulum stress signaling and correlate with suppression of glioma cell proliferation under inhibition of the IRE1 enzyme function.

Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

PubMed Central

Kuss, Sharon K.; Mata, Miguel A.; Zhang, Liang; Fontoura, Beatriz M. A.

2013-01-01

Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. PMID:23872491

Glucose-Induced Trophic Shift in an Endosymbiont Dinoflagellate with Physiological and Molecular Consequences1[OPEN

PubMed Central

Jinkerson, Robert E.; Clowez, Sophie; Onishi, Masayuki; Cleves, Phillip A.; Pringle, John R.

2018-01-01

Interactions between the dinoflagellate endosymbiont Symbiodinium and its cnidarian hosts (e.g. corals, sea anemones) are the foundation of coral-reef ecosystems. Carbon flow between the partners is a hallmark of this mutualism, but the mechanisms governing this flow and its impact on symbiosis remain poorly understood. We showed previously that although Symbiodinium strain SSB01 can grow photoautotrophically, it can grow mixotrophically or heterotrophically when supplied with Glc, a metabolite normally transferred from the alga to its host. Here we show that Glc supplementation of SSB01 cultures causes a loss of pigmentation and photosynthetic activity, disorganization of thylakoid membranes, accumulation of lipid bodies, and alterations of cell-surface morphology. We used global transcriptome analyses to determine if these physiological changes were correlated with changes in gene expression. Glc-supplemented cells exhibited a marked reduction in levels of plastid transcripts encoding photosynthetic proteins, although most nuclear-encoded transcripts (including those for proteins involved in lipid synthesis and formation of the extracellular matrix) exhibited little change in their abundances. However, the altered carbon metabolism in Glc-supplemented cells was correlated with modest alterations (approximately 2x) in the levels of some nuclear-encoded transcripts for sugar transporters. Finally, Glc-bleached SSB01 cells appeared unable to efficiently populate anemone larvae. Together, these results suggest links between energy metabolism and cellular physiology, morphology, and symbiotic interactions. However, the results also show that in contrast to many other organisms, Symbiodinium can undergo dramatic physiological changes that are not reflected by major changes in the abundances of nuclear-encoded transcripts and thus presumably reflect posttranscriptional regulatory processes. PMID:29217594

UL31 and UL34 Proteins of Herpes Simplex Virus Type 1 Form a Complex That Accumulates at the Nuclear Rim and Is Required for Envelopment of Nucleocapsids

PubMed Central

Reynolds, Ashley E.; Ryckman, Brent J.; Baines, Joel D.; Zhou, Yuping; Liang, Li; Roller, Richard J.

2001-01-01

The herpes simplex virus type 1 (HSV-1) UL34 protein is likely a type II membrane protein that localizes within the nuclear membrane and is required for efficient envelopment of progeny virions at the nuclear envelope, whereas the UL31 gene product of HSV-1 is a nuclear matrix-associated phosphoprotein previously shown to interact with UL34 protein in HSV-1-infected cell lysates. For these studies, polyclonal antisera directed against purified fusion proteins containing UL31 protein fused to glutathione-S-transferase (UL31-GST) and UL34 protein fused to GST (UL34-GST) were demonstrated to specifically recognize the UL31 and UL34 proteins of approximately 34,000 and 30,000 Da, respectively. The UL31 and UL34 gene products colocalized in a smooth pattern throughout the nuclear rim of infected cells by 10 h postinfection. UL34 protein also accumulated in pleiomorphic cytoplasmic structures at early times and associated with an altered nuclear envelope late in infection. Localization of UL31 protein at the nuclear rim required the presence of UL34 protein, inasmuch as cells infected with a UL34 null mutant virus contained UL31 protein primarily in central intranuclear domains separate from the nuclear rim, and to a lesser extent in the cytoplasm. Conversely, localization of UL34 protein exclusively at the nuclear rim required the presence of the UL31 gene product, inasmuch as UL34 protein was detectable at the nuclear rim, in replication compartments, and in the cytoplasm of cells infected with a UL31 null virus. When transiently expressed in the absence of other viral factors, UL31 protein localized diffusely in the nucleoplasm, whereas UL34 protein localized primarily in the cytoplasm and at the nuclear rim. In contrast, coexpression of the UL31 and UL34 proteins was sufficient to target both proteins exclusively to the nuclear rim. The proteins were also shown to directly interact in vitro in the absence of other viral proteins. In cells infected with a virus lacking the US3-encoded protein kinase, previously shown to phosphorylate the UL34 gene product, UL31 and UL34 proteins colocalized in small punctate areas that accumulated on the nuclear rim. Thus, US3 kinase is required for even distribution of UL31 and UL34 proteins throughout the nuclear rim. Taken together with the similar phenotypes of the UL31 and UL34 deletion mutants, these data strongly suggest that the UL31 and UL34 proteins form a complex that accumulates at the nuclear membrane and plays an important role in nucleocapsid envelopment at the inner nuclear membrane. PMID:11507225

Parallel loss of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases and mtDNA-encoded tRNAs in Cnidaria.

PubMed

Haen, Karri M; Pett, Walker; Lavrov, Dennis V

2010-10-01

Unlike most animal mitochondrial (mt) genomes, which encode a set of 22 transfer RNAs (tRNAs) sufficient for mt protein synthesis, those of cnidarians have only retained one or two tRNA genes. Whether the missing cnidarian mt-tRNA genes relocated outside the main mt chromosome or were lost remains unclear. It is also unknown what impact the loss of tRNA genes had on other components of the mt translational machinery. Here, we explored the nuclear genome of the cnidarian Nematostella vectensis for the presence of mt-tRNA genes and their corresponding mt aminoacyl-tRNA synthetases (mt-aaRS). We detected no candidates for mt-tRNA genes and only two mt-aaRS orthologs. At the same time, we found that all but one cytosolic aaRS appear to be targeted to mitochondria. These results indicate that the loss of mt-tRNAs in Cnidaria is genuine and occurred in parallel with the loss of nuclear-encoded mt-aaRS. Our phylogenetic analyses of individual aaRS revealed that although the nearly total loss of mt-aaRS is rare, aaRS gene deletion and replacement have occurred throughout the evolution of Metazoa.

The arginine methyltransferase Rmt2 is enriched in the nucleus and co-purifies with the nuclear porins Nup49, Nup57 and Nup100

DOE Office of Scientific and Technical Information (OSTI.GOV)

Olsson, Ida; Berrez, Jean-Marc; Leipus, Arunas

2007-05-15

Arginine methylation is a post-translational modification of proteins implicated in RNA processing, protein compartmentalization, signal transduction, transcriptional regulation and DNA repair. In a screen for proteins associated with the nuclear envelope in the yeast Saccharomyces cerevisiae, we have identified the arginine methyltransferase Rmt2, previously shown to methylate the ribosomal protein L12. By indirect immunofluorescence and subcellular fractionations we demonstrate here that Rmt2 has nuclear and cytoplasmic localizations. Biochemical analysis of a fraction enriched in nuclei reveals that nuclear Rmt2 is resistant to extractions with salt and detergent, indicating an association with structural components. This was supported by affinity purification experimentsmore » with TAP-tagged Rmt2. Rmt2 was found to co-purify with the nucleoporins Nup49, Nup57 and Nup100, revealing a novel link between arginine methyltransferases and the nuclear pore complex. In addition, a genome-wide transcription study of the rmt2{delta} mutant shows significant downregulation of the transcription of MYO1, encoding the Type II myosin heavy chain required for cytokinesis and cell separation.« less

Rift Valley fever phlebovirus NSs protein core domain structure suggests molecular basis for nuclear filaments

PubMed Central

Miller, Ona K; Potter, Jane A; Vijayakrishnan, Swetha; Bhella, David; Naismith, James H; Elliott, Richard M

2017-01-01

Rift Valley fever phlebovirus (RVFV) is a clinically and economically important pathogen increasingly likely to cause widespread epidemics. RVFV virulence depends on the interferon antagonist non-structural protein (NSs), which remains poorly characterized. We identified a stable core domain of RVFV NSs (residues 83–248), and solved its crystal structure, a novel all-helical fold organized into highly ordered fibrils. A hallmark of RVFV pathology is NSs filament formation in infected cell nuclei. Recombinant virus encoding the NSs core domain induced intranuclear filaments, suggesting it contains all essential determinants for nuclear translocation and filament formation. Mutations of key crystal fibril interface residues in viruses encoding full-length NSs completely abrogated intranuclear filament formation in infected cells. We propose the fibrillar arrangement of the NSs core domain in crystals reveals the molecular basis of assembly of this key virulence factor in cell nuclei. Our findings have important implications for fundamental understanding of RVFV virulence. PMID:28915104

Rift Valley fever phlebovirus NSs protein core domain structure suggests molecular basis for nuclear filaments.

PubMed

Barski, Michal; Brennan, Benjamin; Miller, Ona K; Potter, Jane A; Vijayakrishnan, Swetha; Bhella, David; Naismith, James H; Elliott, Richard M; Schwarz-Linek, Ulrich

2017-09-15

Rift Valley fever phlebovirus (RVFV) is a clinically and economically important pathogen increasingly likely to cause widespread epidemics. RVFV virulence depends on the interferon antagonist non-structural protein (NSs), which remains poorly characterized. We identified a stable core domain of RVFV NSs (residues 83-248), and solved its crystal structure, a novel all-helical fold organized into highly ordered fibrils. A hallmark of RVFV pathology is NSs filament formation in infected cell nuclei. Recombinant virus encoding the NSs core domain induced intranuclear filaments, suggesting it contains all essential determinants for nuclear translocation and filament formation. Mutations of key crystal fibril interface residues in viruses encoding full-length NSs completely abrogated intranuclear filament formation in infected cells. We propose the fibrillar arrangement of the NSs core domain in crystals reveals the molecular basis of assembly of this key virulence factor in cell nuclei. Our findings have important implications for fundamental understanding of RVFV virulence.

Selective pressure against horizontally acquired prokaryotic genes as a driving force of plastid evolution.

PubMed

Llorente, Briardo; de Souza, Flavio S J; Soto, Gabriela; Meyer, Cristian; Alonso, Guillermo D; Flawiá, Mirtha M; Bravo-Almonacid, Fernando; Ayub, Nicolás D; Rodríguez-Concepción, Manuel

2016-01-11

The plastid organelle comprises a high proportion of nucleus-encoded proteins that were acquired from different prokaryotic donors via independent horizontal gene transfers following its primary endosymbiotic origin. What forces drove the targeting of these alien proteins to the plastid remains an unresolved evolutionary question. To better understand this process we screened for suitable candidate proteins to recapitulate their prokaryote-to-eukaryote transition. Here we identify the ancient horizontal transfer of a bacterial polyphenol oxidase (PPO) gene to the nuclear genome of an early land plant ancestor and infer the possible mechanism behind the plastidial localization of the encoded enzyme. Arabidopsis plants expressing PPO versions either lacking or harbouring a plastid-targeting signal allowed examining fitness consequences associated with its subcellular localization. Markedly, a deleterious effect on plant growth was highly correlated with PPO activity only when producing the non-targeted enzyme, suggesting that selection favoured the fixation of plastid-targeted protein versions. Our results reveal a possible evolutionary mechanism of how selection against heterologous genes encoding cytosolic proteins contributed in incrementing plastid proteome complexity from non-endosymbiotic gene sources, a process that may also impact mitochondrial evolution.

Targeting of cytosolic mRNA to mitochondria: naked RNA can bind to the mitochondrial surface.

PubMed

Michaud, Morgane; Maréchal-Drouard, Laurence; Duchêne, Anne-Marie

2014-05-01

Mitochondria contain hundreds of proteins but only a few are encoded by the mitochondrial genome. The other proteins are nuclear-encoded and imported into mitochondria. These proteins can be translated on free cytosolic polysomes, then targeted and imported into mitochondria. Nonetheless, numerous cytosolic mRNAs encoding mitochondrial proteins are detected at the surface of mitochondria in yeast, plants and animals. The localization of mRNAs to the vicinity of mitochondria would be a way for mitochondrial protein sorting. The mechanisms responsible for mRNA targeting to mitochondria are not clearly identified. Sequences within the mRNA molecules (cis-elements), as well as a few trans-acting factors, have been shown to be essential for targeting of some mRNAs. In order to identify receptors involved in mRNA docking to the mitochondrial surface, we have developed an in vitro mRNA binding assay with isolated plant mitochondria. We show that naked mRNAs are able to bind to isolated mitochondria, and our results strongly suggest that mRNA docking to the plant mitochondrial outer membrane requires at least one component of TOM complex. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Four signature motifs define the first class of structurally related large coiled-coil proteins in plants.

PubMed Central

Gindullis, Frank; Rose, Annkatrin; Patel, Shalaka; Meier, Iris

2002-01-01

Background Animal and yeast proteins containing long coiled-coil domains are involved in attaching other proteins to the large, solid-state components of the cell. One subgroup of long coiled-coil proteins are the nuclear lamins, which are involved in attaching chromatin to the nuclear envelope and have recently been implicated in inherited human diseases. In contrast to other eukaryotes, long coiled-coil proteins have been barely investigated in plants. Results We have searched the completed Arabidopsis genome and have identified a family of structurally related long coiled-coil proteins. Filament-like plant proteins (FPP) were identified by sequence similarity to a tomato cDNA that encodes a coiled-coil protein which interacts with the nuclear envelope-associated protein, MAF1. The FPP family is defined by four novel unique sequence motifs and by two clusters of long coiled-coil domains separated by a non-coiled-coil linker. All family members are expressed in a variety of Arabidopsis tissues. A homolog sharing the structural features was identified in the monocot rice, indicating conservation among angiosperms. Conclusion Except for myosins, this is the first characterization of a family of long coiled-coil proteins in plants. The tomato homolog of the FPP family binds in a yeast two-hybrid assay to a nuclear envelope-associated protein. This might suggest that FPP family members function in nuclear envelope biology. Because the full Arabidopsis genome does not appear to contain genes for lamins, it is of interest to investigate other long coiled-coil proteins, which might functionally replace lamins in the plant kingdom. PMID:11972898

The VBP and a1/EBP leucine zipper factors bind overlapping subsets of avian retroviral long terminal repeat CCAAT/enhancer elements.

PubMed

Smith, C D; Baglia, L A; Curristin, S M; Ruddell, A

1994-10-01

Two long terminal repeat (LTR) enhancer-binding proteins which may regulate high rates of avian leukosis virus (ALV) LTR-enhanced c-myc transcription during bursal lymphomagenesis have been identified (A. Ruddell, M. Linial, and M. Groudine, Mol. Cell. Biol. 9:5660-5668, 1989). The genes encoding the a1/EBP and a3/EBP binding factors were cloned by expression screening of a lambda gt11 cDNA library from chicken bursal lymphoma cells. The a1/EBP cDNA encodes a novel leucine zipper transcription factor (W. Bowers and A. Ruddell, J. Virol. 66:6578-6586, 1992). The partial a3/EBP cDNA clone encodes amino acids 84 to 313 of vitellogenin gene-binding protein (VBP), a leucine zipper factor that binds the avian vitellogenin II gene promoter (S. Iyer, D. Davis, and J. Burch, Mol. Cell. Biol. 11:4863-4875, 1991). Multiple VBP mRNAs are expressed in B cells in a pattern identical to that previously observed for VBP in other cell types. The LTR-binding activities of VBP, a1/EBP, and B-cell nuclear extract protein were compared and mapped by gel shift, DNase I footprinting, and methylation interference assays. The purified VBP and a1/EBP bacterial fusion proteins bind overlapping but distinct subsets of CCAAT/enhancer elements in the closely related ALV and Rous sarcoma virus (RSV) LTR enhancers. Protein binding to these CCAAT/enhancer elements accounts for most of the labile LTR enhancer-binding activity observed in B-cell nuclear extracts. VBP and a1/EBP could mediate the high rates of ALV and RSV LTR-enhanced transcription in bursal lymphoma cells and many other cell types.

Transposon-disruption of a maize nuclear gene, tha1, encoding a chloroplast SecA homologue: in vivo role of cp-SecA in thylakoid protein targeting.

PubMed

Voelker, R; Mendel-Hartvig, J; Barkan, A

1997-02-01

A nuclear mutant of maize, tha1, which exhibited defects in the translocation of proteins across the thylakoid membrane, was described previously. A transposon insertion at the tha1 locus facilitated the cloning of portions of the tha1 gene. Strong sequence similarity with secA genes from bacteria, pea and spinach indicates that tha1 encodes a SecA homologue (cp-SecA). The tha1-ref allele is either null or nearly so, in that tha1 mRNA is undetectable in mutant leaves and cp-SecA accumulation is reduced > or = 40-fold. These results, in conjunction with the mutant phenotype described previously, demonstrate that cp-SecA functions in vivo to facilitate the translocation of OEC33, PSI-F and plastocyanin but does not function in the translocation of OEC23 and OEC16. Our results confirm predictions for cp-SecA function made from the results of in vitro experiments and establish several new functions for cp-SecA, including roles in the targeting of a chloroplast-encoded protein, cytochrome f, and in protein targeting in the etioplast, a nonphotosynthetic plastid type. Our finding that the accumulation of properly targeted plastocyanin and cytochrome f in tha1-ref thylakoid membranes is reduced only a few-fold despite the near or complete absence of cp-SecA suggests that cp-SecA facilitates but is not essential in vivo for their translocation across the membrane.

Epstein–Barr virus latent genes

PubMed Central

Kang, Myung-Soo; Kieff, Elliott

2015-01-01

Latent Epstein–Barr virus (EBV) infection has a substantial role in causing many human disorders. The persistence of these viral genomes in all malignant cells, yet with the expression of limited latent genes, is consistent with the notion that EBV latent genes are important for malignant cell growth. While the EBV-encoded nuclear antigen-1 (EBNA-1) and latent membrane protein-2A (LMP-2A) are critical, the EBNA-leader proteins, EBNA-2, EBNA-3A, EBNA-3C and LMP-1, are individually essential for in vitro transformation of primary B cells to lymphoblastoid cell lines. EBV-encoded RNAs and EBNA-3Bs are dispensable. In this review, the roles of EBV latent genes are summarized. PMID:25613728

Heterologous mitochondrial targeting sequences can deliver functional proteins into mitochondria.

PubMed

Marcus, Dana; Lichtenstein, Michal; Cohen, Natali; Hadad, Rita; Erlich-Hadad, Tal; Greif, Hagar; Lorberboum-Galski, Haya

2016-12-01

Mitochondrial Targeting Sequences (MTSs) are responsible for trafficking nuclear-encoded proteins into mitochondria. Once entering the mitochondria, the MTS is recognized and cleaved off. Some MTSs are long and undergo two-step processing, as in the case of the human frataxin (FXN) protein (80aa), implicated in Friedreich's ataxia (FA). Therefore, we chose the FXN protein to examine whether nuclear-encoded mitochondrial proteins can efficiently be targeted via a heterologous MTS (hMTS) and deliver a functional protein into mitochondria. We examined three hMTSs; that of citrate synthase (cs), lipoamide deydrogenase (LAD) and C6ORF66 (ORF), as classically MTS sequences, known to be removed by one-step processing, to deliver FXN into mitochondria, in the form of fusion proteins. We demonstrate that using hMTSs for delivering FXN results in the production of 4-5-fold larger amounts of the fusion proteins, and at 4-5-fold higher concentrations. Moreover, hMTSs delivered a functional FXN protein into the mitochondria even more efficiently than the native MTSfxn, as evidenced by the rescue of FA patients' cells from oxidative stress; demonstrating a 18%-54% increase in cell survival; and a 13%-33% increase in ATP levels, as compared to the fusion protein carrying the native MTS. One fusion protein with MTScs increased aconitase activity within patients' cells, by 400-fold. The implications form our studies are of vast importance for both basic and translational research of mitochondrial proteins as any mitochondrial protein can be delivered efficiently by an hMTS. Moreover, effective targeting of functional proteins is important for restoration of mitochondrial function and treatment of related disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA.

PubMed

Pereira, Carina F; Read, Eliot K C; Wise, Helen M; Amorim, Maria J; Digard, Paul

2017-08-01

Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export. IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as previously identified roles in antagonizing the innate immune defenses of the cell and directly upregulating translation of viral mRNAs, it also promotes the nuclear export of the viral late gene mRNAs by acting as an adaptor between the viral mRNAs and the cellular mRNA nuclear export machinery. Copyright © 2017 Pereira et al.

Opposite nuclear level and binding activity of STAT5B and STAT3 proteins with rat haptoglobin gene under normal and turpentine induced acute phase conditions.

PubMed

Grigorov, I; Lazić, T; Cvetković, I; Milosavljević, T; Petrović, M

2001-01-01

Transcription of the rat gene encoding haptoglobin (Hp) is highly induced during acute phase (AP) response which has been previously shown to be mediated by inducible STAT3 member of the Signal Transducer and Activators of Transcription (STATs) family proteins. In this study, we observed that under normal but not in the turpentine induced AP conditions, another member of the STAT family proteins, STAT5b is expressed and binds to the hormone regulatory element (HRE) of the rat Hp gene. We found that the nuclear amounts of constitutively active STAT5b in rat liver decreased significantly with time of turpentine treatment as opposed to that of cytosol STAT5b, suggesting possible export of constitutive STAT5b from the nucleus. Nuclear accumulation and binding of inducible STAT3 proteins to the rat Hp gene HRE following turpentine treatment implicated that STAT5b negatively regulates Hp gene expression during normal conditions.

Turnip vein clearing virus movement protein nuclear activity: Do Tobamovirus movement proteins play a role in immune response suppression?

PubMed

Levy, Amit

2015-01-01

Plant viruses' cell-to-cell movement requires the function of virally encoded movement proteins (MPs). The Tobamovirus, Tobacco mosaic virus (TMV) has served as the model virus to study the activities of single MPs. However, since TMV does not infect the model plant Arabidopsis thaliana I have used a related Tobamovirus, Turnip vein-clearing virus (TVCV). I recently showed that, despite belonging to the same genus, the behavior of the 2 viruses MPs differ significantly during infection. Most notably, MP(TVCV), but not MP(TMV), targets the nucleus and induces the formation of F actin-containing filaments that associate with chromatin. Mutational analyses showed that nuclear localization of MP(TVCV) was necessary for TVCV local and systemic infection in both Nicotiana benthamiana and Arabidopsis. In this addendum, I propose possible targets for the MP(TVCV) nuclear activity, and suggest viewing MPs as viral effector-like proteins, playing a role in the inhibition of plant defense.

Mitochondrial and nuclear localization of a novel pea thioredoxin: identification of its mitochondrial target proteins.

PubMed

Martí, María C; Olmos, Enrique; Calvete, Juan J; Díaz, Isabel; Barranco-Medina, Sergio; Whelan, James; Lázaro, Juan J; Sevilla, Francisca; Jiménez, Ana

2009-06-01

Plants contain several genes encoding thioredoxins (Trxs), small proteins involved in the regulation of the activity of many enzymes through dithiol-disulfide exchange. In addition to chloroplastic and cytoplasmic Trx systems, plant mitochondria contain a reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase and a specific Trx o, and to date, there have been no reports of a gene encoding a plant nuclear Trx. We report here the presence in pea (Pisum sativum) mitochondria and nuclei of a Trx isoform (PsTrxo1) that seems to belong to the Trx o group, although it differs from this Trx type by its absence of introns in the genomic sequence. Western-blot analysis with isolated mitochondria and nuclei, immunogold labeling, and green fluorescent protein fusion constructs all indicated that PsTrxo1 is present in both cell compartments. Moreover, the identification by tandem mass spectrometry of the native mitochondrial Trx after gel filtration using the fast-protein liquid chromatography system of highly purified mitochondria and the in vitro uptake assay into isolated mitochondria also corroborated a mitochondrial location for this protein. The recombinant PsTrxo1 protein has been shown to be reduced more effectively by the Saccharomyces cerevisiae mitochondrial Trx reductase Trr2 than by the wheat (Triticum aestivum) cytoplasmic reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase. PsTrxo1 was able to activate alternative oxidase, and it was shown to interact with a number of mitochondrial proteins, including peroxiredoxin and enzymes mainly involved in the photorespiratory process.

Molecular Genetics of Mitochondrial Disorders

ERIC Educational Resources Information Center

Wong, Lee-Jun C.

2010-01-01

Mitochondrial respiratory chain (RC) disorders (RCDs) are a group of genetically and clinically heterogeneous diseases because of the fact that protein components of the RC are encoded by both mitochondrial and nuclear genomes and are essential in all cells. In addition, the biogenesis, structure, and function of mitochondria, including DNA…

Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals.

PubMed

Zlopasa, Livija; Brachner, Andreas; Foisner, Roland

2016-06-01

Ankyrin repeats and LEM domain containing protein 1 (Ankle1) belongs to the LEM protein family, whose members share a chromatin-interacting LEM motif. Unlike most other LEM proteins, Ankle1 is not an integral protein of the inner nuclear membrane but shuttles between the nucleus and the cytoplasm. It contains a GIY-YIG-type nuclease domain, but its function is unknown. The mammalian genome encodes only one other GIY-YIG domain protein, termed Slx1. Slx1 has been described as a resolvase that processes Holliday junctions during homologous recombination-mediated DNA double strand break repair. Resolvase activity is regulated in a spatial and temporal manner during the cell cycle. We hypothesized that Ankle1 may have a similar function and its nucleo-cytoplasmic shuttling may contribute to the regulation of Ankle1 activity. Hence, we aimed at identifying the domains mediating Ankle1 shuttling and investigating whether cellular localization is affected during DNA damage response. Sequence analysis predicts the presence of two canonical nuclear import and export signals in Ankle1. Immunofluorescence microscopy of cells expressing wild-type and various mutated Ankle1-fusion proteins revealed a C-terminally located classical monopartite nuclear localization signal and a centrally located CRM1-dependent nuclear export signal that mediate nucleo-cytoplasmic shuttling of Ankle1. These sequences are also functional in heterologous proteins. The predominant localization of Ankle1 in the cytoplasm, however, does not change upon induction of several DNA damage response pathways throughout the cell cycle. We identified the domains mediating nuclear import and export of Ankle1. Ankle1's cellular localization was not affected following DNA damage.

Thermodynamic properties distinguish human mitochondrial aspartyl-tRNA synthetase from bacterial homolog with same 3D architecture.

PubMed

Neuenfeldt, Anne; Lorber, Bernard; Ennifar, Eric; Gaudry, Agnès; Sauter, Claude; Sissler, Marie; Florentz, Catherine

2013-02-01

In the mammalian mitochondrial translation apparatus, the proteins and their partner RNAs are coded by two genomes. The proteins are nuclear-encoded and resemble their homologs, whereas the RNAs coming from the rapidly evolving mitochondrial genome have lost critical structural information. This raises the question of molecular adaptation of these proteins to their peculiar partner RNAs. The crystal structure of the homodimeric bacterial-type human mitochondrial aspartyl-tRNA synthetase (DRS) confirmed a 3D architecture close to that of Escherichia coli DRS. However, the mitochondrial enzyme distinguishes by an enlarged catalytic groove, a more electropositive surface potential and an alternate interaction network at the subunits interface. It also presented a thermal stability reduced by as much as 12°C. Isothermal titration calorimetry analyses revealed that the affinity of the mitochondrial enzyme for cognate and non-cognate tRNAs is one order of magnitude higher, but with different enthalpy and entropy contributions. They further indicated that both enzymes bind an adenylate analog by a cooperative allosteric mechanism with different thermodynamic contributions. The larger flexibility of the mitochondrial synthetase with respect to the bacterial enzyme, in combination with a preserved architecture, may represent an evolutionary process, allowing nuclear-encoded proteins to cooperate with degenerated organelle RNAs.

Nuclear lamina genetic variants, including a truncated LAP2, in twins and siblings with nonalcoholic fatty liver disease.

PubMed

Brady, Graham F; Kwan, Raymond; Ulintz, Peter J; Nguyen, Phirum; Bassirian, Shirin; Basrur, Venkatesha; Nesvizhskii, Alexey I; Loomba, Rohit; Omary, M Bishr

2018-05-01

Nonalcoholic fatty liver disease (NAFLD) is becoming the major chronic liver disease in many countries. Its pathogenesis is multifactorial, but twin and familial studies indicate significant heritability, which is not fully explained by currently known genetic susceptibility loci. Notably, mutations in genes encoding nuclear lamina proteins, including lamins, cause lipodystrophy syndromes that include NAFLD. We hypothesized that variants in lamina-associated proteins predispose to NAFLD and used a candidate gene-sequencing approach to test for variants in 10 nuclear lamina-related genes in a cohort of 37 twin and sibling pairs: 21 individuals with and 53 without NAFLD. Twelve heterozygous sequence variants were identified in four lamina-related genes (ZMPSTE24, TMPO, SREBF1, SREBF2). The majority of NAFLD patients (>90%) had at least one variant compared to <40% of controls (P < 0.0001). When only insertions/deletions and changes in conserved residues were considered, the difference between the groups was similarly striking (>80% versus <25%; P < 0.0001). Presence of a lamina variant segregated with NAFLD independently of the PNPLA3 I148M polymorphism. Several variants were found in TMPO, which encodes the lamina-associated polypeptide-2 (LAP2) that has not been associated with liver disease. One of these, a frameshift insertion that generates truncated LAP2, abrogated lamin-LAP2 binding, caused LAP2 mislocalization, altered endogenous lamin distribution, increased lipid droplet accumulation after oleic acid treatment in transfected cells, and led to cytoplasmic association with the ubiquitin-binding protein p62/SQSTM1. Several variants in nuclear lamina-related genes were identified in a cohort of twins and siblings with NAFLD; one such variant, which results in a truncated LAP2 protein and a dramatic phenotype in cell culture, represents an association of TMPO/LAP2 variants with NAFLD and underscores the potential importance of the nuclear lamina in NAFLD. (Hepatology 2018;67:1710-1725). © 2017 by the American Association for the Study of Liver Diseases.

Coordinated rates of evolution between interacting plastid and nuclear genes in Geraniaceae.

PubMed

Zhang, Jin; Ruhlman, Tracey A; Sabir, Jamal; Blazier, J Chris; Jansen, Robert K

2015-03-01

Although gene coevolution has been widely observed within individuals and between different organisms, rarely has this phenomenon been investigated within a phylogenetic framework. The Geraniaceae is an attractive system in which to study plastid-nuclear genome coevolution due to the highly elevated evolutionary rates in plastid genomes. In plants, the plastid-encoded RNA polymerase (PEP) is a protein complex composed of subunits encoded by both plastid (rpoA, rpoB, rpoC1, and rpoC2) and nuclear genes (sig1-6). We used transcriptome and genomic data for 27 species of Geraniales in a systematic evaluation of coevolution between genes encoding subunits of the PEP holoenzyme. We detected strong correlations of dN (nonsynonymous substitutions) but not dS (synonymous substitutions) within rpoB/sig1 and rpoC2/sig2, but not for other plastid/nuclear gene pairs, and identified the correlation of dN/dS ratio between rpoB/C1/C2 and sig1/5/6, rpoC1/C2 and sig2, and rpoB/C2 and sig3 genes. Correlated rates between interacting plastid and nuclear sequences across the Geraniales could result from plastid-nuclear genome coevolution. Analyses of coevolved amino acid positions suggest that structurally mediated coevolution is not the major driver of plastid-nuclear coevolution. The detection of strong correlation of evolutionary rates between SIG and RNAP genes suggests a plausible explanation for plastome-genome incompatibility in Geraniaceae. © 2015 American Society of Plant Biologists. All rights reserved.

Overexpression of Nrp/b (nuclear restrict protein in brain) suppresses the malignant phenotype in the C6/ST1 glioma cell line.

PubMed

Degaki, Theri Leica; Demasi, Marcos Angelo Almeida; Sogayar, Mari Cleide

2009-11-01

Upon searching for glucocorticoid-regulated cDNA sequences associated with the transformed to normal phenotypic reversion of C6/ST1 rat glioma cells, we identified Nrp/b (nuclear restrict protein in brain) as a novel rat gene. Here we report on the identification and functional characterization of the complete sequence encoding the rat NRP/B protein. The cloned cDNA presented a 1767 nucleotides open-reading frame encoding a 589 amino acids residues sequence containing a BTB/POZ (broad complex Tramtrack bric-a-brac/Pox virus and zinc finger) domain in its N-terminal region and kelch motifs in its C-terminal region. Sequence analysis indicates that the rat Nrp/b displays a high level of identity with the equivalent gene orthologs from other organisms. Among rat tissues, Nrp/b expression is more pronounced in brain tissue. We show that overexpression of the Nrp/b cDNA in C6/ST1 cells suppresses anchorage independence in vitro and tumorigenicity in vivo, altering their malignant nature towards a more benign phenotype. Therefore, Nrp/b may be postulated as a novel tumor suppressor gene, with possible relevance for glioblastoma therapy.

Functional Activity of the Fanconi Anemia Protein FAA Requires FAC Binding and Nuclear Localization

PubMed Central

Näf, Dieter; Kupfer, Gary M.; Suliman, Ahmed; Lambert, Kathleen; D’Andrea, Alan D.

1998-01-01

Fanconi anemia (FA) is an autosomal recessive disease characterized by genomic instability, cancer susceptibility, and cellular hypersensitivity to DNA-cross-linking agents. Eight complementation groups of FA (FA-A through FA-H) have been identified. Two FA genes, corresponding to complementation groups FA-A and FA-C, have been cloned, but the functions of the encoded FAA and FAC proteins remain unknown. We have recently demonstrated that FAA and FAC interact to form a nuclear complex. In this study, we have analyzed a series of mutant forms of the FAA protein with respect to functional activity, FAC binding, and nuclear localization. Mutation or deletion of the amino-terminal nuclear localization signal (NLS) of FAA results in loss of functional activity, loss of FAC binding, and cytoplasmic retention of FAA. Replacement of the NLS sequence with a heterologous NLS sequence, derived from the simian virus 40 T antigen, results in nuclear localization but does not rescue functional activity or FAC binding. Nuclear localization of the FAA protein is therefore necessary but not sufficient for FAA function. Mutant forms of FAA which fail to bind to FAC also fail to promote the nuclear accumulation of FAC. In addition, wild-type FAC promotes the accumulation of wild-type FAA in the nucleus. Our results suggest that FAA and FAC perform a concerted function in the cell nucleus, required for the maintenance of chromosomal stability. PMID:9742112

Therapeutic implications of Epstein–Barr virus infection for the treatment of nasopharyngeal carcinoma

PubMed Central

Hutajulu, Susanna Hilda; Kurnianda, Johan; Tan, I Bing; Middeldorp, Jaap M

2014-01-01

Nasopharyngeal carcinoma (NPC) is highly endemic in certain regions including the People’s Republic of China and Southeast Asia. Its etiology is unique and multifactorial, involving genetic background, epigenetic, and environment factors, including Epstein–Barr virus (EBV) infection. The presence of EBV in all tumor cells, aberrant pattern of antibodies against EBV antigens in patient sera, and elevated viral DNA in patient circulation as well as nasopharyngeal site underline the role of EBV during NPC development. In NPC tumors, EBV expresses latency type II, where three EBV-encoded proteins, Epstein–Barr nuclear antigen 1, latent membrane protein 1 and 2 (LMP1, 2), are expressed along with BamH1-A rightward reading frame 1, Epstein–Barr virus-encoded small nuclear RNAs, and BamH1-A rightward transcripts. Among all encoded proteins, LMP1 plays a central role in the propagation of NPC. Standard treatment of NPC consists of radiotherapy with or without chemotherapy for early stage, concurrent chemoradiotherapy in locally advanced tumors, and palliative systemic chemotherapy in metastatic disease. However, this standard care has limitations, allowing recurrences and disease progression in a certain proportion of cases. Although the pathophysiological link and molecular process of EBV-induced oncogenesis are not fully understood, therapeutic approaches targeting the virus may increase the cure rate and add clinical benefit. The promising results of early phase clinical trials on EBV-specific immunotherapy, epigenetic therapy, and treatment with viral lytic induction offer new options for treating NPC. PMID:25228810

MitoRes: a resource of nuclear-encoded mitochondrial genes and their products in Metazoa.

PubMed

Catalano, Domenico; Licciulli, Flavio; Turi, Antonio; Grillo, Giorgio; Saccone, Cecilia; D'Elia, Domenica

2006-01-24

Mitochondria are sub-cellular organelles that have a central role in energy production and in other metabolic pathways of all eukaryotic respiring cells. In the last few years, with more and more genomes being sequenced, a huge amount of data has been generated providing an unprecedented opportunity to use the comparative analysis approach in studies of evolution and functional genomics with the aim of shedding light on molecular mechanisms regulating mitochondrial biogenesis and metabolism. In this context, the problem of the optimal extraction of representative datasets of genomic and proteomic data assumes a crucial importance. Specialised resources for nuclear-encoded mitochondria-related proteins already exist; however, no mitochondrial database is currently available with the same features of MitoRes, which is an update of the MitoNuc database extensively modified in its structure, data sources and graphical interface. It contains data on nuclear-encoded mitochondria-related products for any metazoan species for which this type of data is available and also provides comprehensive sequence datasets (gene, transcript and protein) as well as useful tools for their extraction and export. MitoRes http://www2.ba.itb.cnr.it/MitoRes/ consolidates information from publicly external sources and automatically annotates them into a relational database. Additionally, it also clusters proteins on the basis of their sequence similarity and interconnects them with genomic data. The search engine and sequence management tools allow the query/retrieval of the database content and the extraction and export of sequences (gene, transcript, protein) and related sub-sequences (intron, exon, UTR, CDS, signal peptide and gene flanking regions) ready to be used for in silico analysis. The tool we describe here has been developed to support lab scientists and bioinformaticians alike in the characterization of molecular features and evolution of mitochondrial targeting sequences. The way it provides for the retrieval and extraction of sequences allows the user to overcome the obstacles encountered in the integrative use of different bioinformatic resources and the completeness of the sequence collection allows intra- and interspecies comparison at different biological levels (gene, transcript and protein).

Cell lines that support replication of a novel herpes simplex virus 1 U{sub L}31 deletion mutant can properly target U{sub L}34 protein to the nuclear rim in the absence of U{sub L}31

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liang Li; Tanaka, Michiko; Kawaguchi, Yasushi

2004-11-10

Previous results indicated that the herpes simplex virus 1 (HSV-1) U{sub L}31 gene is necessary and sufficient for localization of the U{sub L}34 protein exclusively to the nuclear membrane of infected Hep2 cells. In the current studies, a bacterial artificial chromosome containing the entire HSV-1 strain F genome was used to construct a recombinant viral genome in which a gene encoding kanamycin resistance was inserted in place of 262 codons of the 306 codon U{sub L}31 open reading frame. The deletion virus produced virus titers approximately 10- to 50-fold lower in rabbit skin cells, more than 2000-fold lower in Veromore » cells, and more than 1500-fold lower in CV1 cells, compared to a virus bearing a restored U{sub L}31 gene. The replication of the U{sub L}31 deletion virus was restored on U{sub L}31-complementing cell lines derived either from rabbit skin cells or CV1 cells. Confocal microscopy indicated that the majority of U{sub L}34 protein localized aberrantly in the cytoplasm and nucleoplasm of Vero cells and CV1 cells, whereas U{sub L}34 protein localized at the nuclear membrane in rabbit skin cells, and U{sub L}31 complementing CV1 cells infected with the U{sub L}31 deletion virus. We conclude that rabbit skin cells encode a function that allows proper localization of U{sub L}34 protein to the nuclear membrane. We speculate that this function partially complements that of U{sub L}31 and may explain why U{sub L}31 is less critical for replication in rabbit skin cells as opposed to Vero and CV1 cells.« less

Active Nuclear Import of Membrane Proteins Revisited

PubMed Central

Laba, Justyna K.; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M.

2015-01-01

It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker’s yeast. PMID:26473931

TCOF1 gene encodes a putative nucleolar phosphoprotein that exhibits mutations in Treacher Collins Syndrome throughout its coding region.

PubMed

Wise, C A; Chiang, L C; Paznekas, W A; Sharma, M; Musy, M M; Ashley, J A; Lovett, M; Jabs, E W

1997-04-01

Treacher Collins Syndrome (TCS) is the most common of the human mandibulofacial dysostosis disorders. Recently, a partial TCOF1 cDNA was identified and shown to contain mutations in TCS families. Here we present the entire exon/intron genomic structure and the complete coding sequence of TCOF1. TCOF1 encodes a low complexity protein of 1,411 amino acids, whose predicted protein structure reveals repeated motifs that mirror the organization of its exons. These motifs are shared with nucleolar trafficking proteins in other species and are predicted to be highly phosphorylated by casein kinase. Consistent with this, the full-length TCOF1 protein sequence also contains putative nuclear and nucleolar localization signals. Throughout the open reading frame, we detected an additional eight mutations in TCS families and several polymorphisms. We postulate that TCS results from defects in a nucleolar trafficking protein that is critically required during human craniofacial development.

TCOF1 gene encodes a putative nucleolar phosphoprotein that exhibits mutations in Treacher Collins Syndrome throughout its coding region

PubMed Central

Wise, Carol A.; Chiang, Lydia C.; Paznekas, William A.; Sharma, Mridula; Musy, Maurice M.; Ashley, Jennifer A.; Lovett, Michael; Jabs, Ethylin W.

1997-01-01

Treacher Collins Syndrome (TCS) is the most common of the human mandibulofacial dysostosis disorders. Recently, a partial TCOF1 cDNA was identified and shown to contain mutations in TCS families. Here we present the entire exon/intron genomic structure and the complete coding sequence of TCOF1. TCOF1 encodes a low complexity protein of 1,411 amino acids, whose predicted protein structure reveals repeated motifs that mirror the organization of its exons. These motifs are shared with nucleolar trafficking proteins in other species and are predicted to be highly phosphorylated by casein kinase. Consistent with this, the full-length TCOF1 protein sequence also contains putative nuclear and nucleolar localization signals. Throughout the open reading frame, we detected an additional eight mutations in TCS families and several polymorphisms. We postulate that TCS results from defects in a nucleolar trafficking protein that is critically required during human craniofacial development. PMID:9096354

hnRNP U protein is required for normal pre-mRNA splicing and postnatal heart development and function.

PubMed

Ye, Junqiang; Beetz, Nadine; O'Keeffe, Sean; Tapia, Juan Carlos; Macpherson, Lindsey; Chen, Weisheng V; Bassel-Duby, Rhonda; Olson, Eric N; Maniatis, Tom

2015-06-09

We report that mice lacking the heterogeneous nuclear ribonucleoprotein U (hnRNP U) in the heart develop lethal dilated cardiomyopathy and display numerous defects in cardiac pre-mRNA splicing. Mutant hearts have disorganized cardiomyocytes, impaired contractility, and abnormal excitation-contraction coupling activities. RNA-seq analyses of Hnrnpu mutant hearts revealed extensive defects in alternative splicing of pre-mRNAs encoding proteins known to be critical for normal heart development and function, including Titin and calcium/calmodulin-dependent protein kinase II delta (Camk2d). Loss of hnRNP U expression in cardiomyocytes also leads to aberrant splicing of the pre-mRNA encoding the excitation-contraction coupling component Junctin. We found that the protein product of an alternatively spliced Junctin isoform is N-glycosylated at a specific asparagine site that is required for interactions with specific protein partners. Our findings provide conclusive evidence for the essential role of hnRNP U in heart development and function and in the regulation of alternative splicing.

Cloning and Characterization of the Gene Encoding Alpha-Pinene Oxide Lyase Enzyme (Prα-POL) from Pseudomonas rhodesiae CIP 107491 and Production of the Recombinant Protein in Escherichia coli.

PubMed

Dubessay, Pascal; Larroche, Christian; Fontanille, Pierre

2017-12-28

The alpha-pinene oxide lyase (Prα-POL) from Pseudomonas rhodesiae CIP107491 belongs to catabolic alpha-pinene degradation pathway. In this study, the gene encoding Prα-POL has been identified using mapping approach combined to inverse PCR (iPCR) strategy. The Prα-POL gene included a 609-bp open reading frame encoding 202 amino acids and giving rise to a 23.7 kDa protein, with a theoretical isoelectric point (pI) of 5.23. The amino acids sequence analysis showed homologies with those of proteins with unknown function from GammaProteobacteria group. Identification of a conserved domain in amino acid in positions 18 to 190 permitted to classify Prα-POL among the nuclear transport factor 2 (NTF2) protein superfamily. Heterologous expression of Prα-POL, both under its native form and with a histidin tag, was successfully performed in Escherichia coli, and enzymatic kinetics were analyzed. Bioconversion assay using recombinant E. coli strain allowed to reach a rate of isonovalal production per gramme of biomass about 40-fold higher than the rate obtained with P. rhodesiae.

The matrix peptide exporter HAF-1 signals a mitochondrial unfolded protein response by activating the transcription factor ZC376.7 in C. elegans

PubMed Central

Haynes, Cole M.; Yang, Yun; Blais, Steven P.; Neubert, Thomas A.; Ron, David

2010-01-01

Summary Genetic analyses previously implicated the matrix-localized protease ClpP in signaling the stress of protein misfolding in the mitochondrial matrix to activate nuclear encoded mitochondrial chaperone genes in C. elegans (UPRmt). Here we report that haf-1, a gene encoding a mitochondria-localized ATP-binding cassette protein, is required for signaling within the UPRmt and for coping with misfolded protein stress. Peptide efflux from isolated mitochondria was ATP-dependent and required HAF-1 and the protease ClpP. Defective UPRmt signaling in the haf-1 deleted worms was associated with failure of the bZIP protein, ZC376.7, to localize to nuclei in worms with perturbed mitochondrial protein folding, whereas zc376.7(RNAi) strongly inhibited the UPRmt. These observations suggest a simple model whereby perturbation of the protein-folding environment in the mitochondrial matrix promotes ClpP-mediated generation of peptides whose haf-1-dependent export from the matrix contributes to UPRmt signaling across the mitochondrial inner membrane. PMID:20188671

Mass spectrometric characterization of membrane integral low molecular weight proteins from photosystem II in barley etioplasts.

PubMed

Plöscher, Matthias; Granvogl, Bernhard; Zoryan, Mikael; Reisinger, Veronika; Eichacker, Lutz Andreas

2009-02-01

In Photosystem II (PSII), a high number of plastid encoded and membrane integral low molecular weight proteins smaller than 10 kDa, the proteins PsbE, F, H, I, J, K, L, M, N, Tc, Z and the nuclear encoded PsbW, X, Y1, Y2 proteins have been described. Here we show that all low molecular weight proteins of PSII already accumulate in the etioplast membrane fraction in darkness, whereas PsaI and PsaJ of photosystem I (PSI) represent the only low molecular weight proteins that do not accumulate in darkness. We found by BN-PAGE separation of membrane protein complexes and selective MS that the accumulation of one-helix proteins from PSII is light independent and occurs in etioplasts. In contrast, in chloroplasts isolated from light-grown plants, low molecular weight proteins were found to specifically accumulate in PSI and II complexes. Our results demonstrate how plants grown in darkness prepare for the induction of chlorophyll dependent photosystem assembly upon light perception. We anticipate that our investigation will provide the essential means for the analysis of protein assembly in any membrane utilizing low molecular weight protein subunits.

Dissection of affinity captured LINE-1 macromolecular complexes

PubMed Central

Mita, Paolo; Jiang, Hua; Adney, Emily M; Wudzinska, Aleksandra; Badri, Sana; Ischenko, Dmitry; Eng, George; Burns, Kathleen H; Fenyö, David; Chait, Brian T; Alexeev, Dmitry; Rout, Michael P; Boeke, Jef D

2018-01-01

Long Interspersed Nuclear Element-1 (LINE-1, L1) is a mobile genetic element active in human genomes. L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications, quantitative mass spectrometry, and next generation RNA sequencing, we have characterized the proteins and nucleic acids associated with distinctive, enzymatically active L1 macromolecular complexes. Among them, we describe a cytoplasmic intermediate that we hypothesize to be the canonical ORF1p/ORF2p/L1-RNA-containing RNP, and we describe a nuclear population containing ORF2p, but lacking ORF1p, which likely contains host factors participating in target-primed reverse transcription. PMID:29309035

Expression Analysis of the Theileria parva Subtelomere-Encoded Variable Secreted Protein Gene Family

PubMed Central

Schmied, Stéfanie; Affentranger, Sarah; Parvanova, Iana; Kang'a, Simon; Nene, Vishvanath; Katzer, Frank; McKeever, Declan; Müller, Joachim; Bishop, Richard; Pain, Arnab; Dobbelaere, Dirk A. E.

2009-01-01

Background The intracellular protozoan parasite Theileria parva transforms bovine lymphocytes inducing uncontrolled proliferation. Proteins released from the parasite are assumed to contribute to phenotypic changes of the host cell and parasite persistence. With 85 members, genes encoding subtelomeric variable secreted proteins (SVSPs) form the largest gene family in T. parva. The majority of SVSPs contain predicted signal peptides, suggesting secretion into the host cell cytoplasm. Methodology/Principal Findings We analysed SVSP expression in T. parva-transformed cell lines established in vitro by infection of T or B lymphocytes with cloned T. parva parasites. Microarray and quantitative real-time PCR analysis revealed mRNA expression for a wide range of SVSP genes. The pattern of mRNA expression was largely defined by the parasite genotype and not by host background or cell type, and found to be relatively stable in vitro over a period of two months. Interestingly, immunofluorescence analysis carried out on cell lines established from a cloned parasite showed that expression of a single SVSP encoded by TP03_0882 is limited to only a small percentage of parasites. Epitope-tagged TP03_0882 expressed in mammalian cells was found to translocate into the nucleus, a process that could be attributed to two different nuclear localisation signals. Conclusions Our analysis reveals a complex pattern of Theileria SVSP mRNA expression, which depends on the parasite genotype. Whereas in cell lines established from a cloned parasite transcripts can be found corresponding to a wide range of SVSP genes, only a minority of parasites appear to express a particular SVSP protein. The fact that a number of SVSPs contain functional nuclear localisation signals suggests that proteins released from the parasite could contribute to phenotypic changes of the host cell. This initial characterisation will facilitate future studies on the regulation of SVSP gene expression and the potential biological role of these enigmatic proteins. PMID:19325907

Taperin (c9orf75), a mutated gene in nonsyndromic deafness, encodes a vertebrate specific, nuclear localized protein phosphatase one alpha (PP1α) docking protein

PubMed Central

Ferrar, Tony; Chamousset, Delphine; De Wever, Veerle; Nimick, Mhairi; Andersen, Jens; Trinkle-Mulcahy, Laura; Moorhead, Greg B. G.

2012-01-01

Summary The promiscuous activity of protein phosphatase one (PP1) is controlled in the cell by associated proteins termed regulatory or targeting subunits. Using biochemical and proteomic approaches we demonstrate that the autosomal recessive nonsyndromic hearing loss gene, taperin (C9orf75), encodes a protein that preferentially docks the alpha isoform of PP1. Taperin associates with PP1 through a classic ‘RVxF’ motif and suppresses the general phosphatase activity of the enzyme. The steady-state localization of taperin is predominantly nuclear, however we demonstrate here that the protein can shuttle between the nucleus and cytoplasm and that it is found complexed to PP1 in both of these cellular compartments. Although originally identified as a hearing loss gene, Western blot analyses with taperin-specific antibodies revealed that the protein is widely expressed across mammalian tissues as multiple splice variants. Taperin is a recent proteome addition appearing during the vertebrate lineage with the PP1 binding site embedded within the most conserved region of the protein. Taperin also shares an ancestral relationship with the cytosolic actin binding protein phostensin, another PP1 interacting partner. Quantitative Stable Isotope Labeling by Amino acids in Culture (SILAC)-based mass spectrometry was employed to uncover additional taperin binding partners, and revealed an interaction with the DNA damage response proteins Ku70, Ku80, PARP and topoisomerases I and IIα. Consistent with this, we demonstrate the active recruitment of taperin to sites of DNA damage. This makes taperin a new addition to the family of PP1 targeting subunits involved in the DNA damage repair pathway. PMID:23213405

Neuron-specific specificity protein 4 bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons.

PubMed

Johar, Kaid; Priya, Anusha; Dhar, Shilpa; Liu, Qiuli; Wong-Riley, Margaret T T

2013-11-01

Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1 that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. The present study discovered that among the specificity family of transcription factors, it is the less known neuron-specific Sp4 that regulates the expression of all 13 subunits of mitochondrial cytochrome c oxidase (COX) enzyme in primary neurons. Sp4 also regulates the three mitochondrial transcription factors (TFAM, TFB1M, and TFB2M) and a COX assembly protein SURF-1 in primary neurons. © 2013 International Society for Neurochemistry.

The 87-kD A gamma-globin enhancer-binding protein is a product of the HOXB2(HOX2H) locus.

PubMed

Sengupta, P K; Lavelle, D E; DeSimone, J

1994-03-01

Developmental regulation of globin gene expression may be controlled by developmental stage-specific nuclear proteins that influence interactions between the locus control region and local regulatory sequences near individual globin genes. We previously isolated an 87-kD nuclear protein from K562 cells that bound to DNA sequences in the beta-globin locus control region, gamma-globin promoter, and A gamma-globin enhancer. The presence of this protein in fetal globin-expressing cells and its absence in adult globin-expressing cells suggested that it may be a developmental stage-specific factor. A lambda gt11 K562 cDNA clone encoding a portion of the HOXB2 (formerly HOX2H) homeobox gene was isolated on the basis of the ability of its beta-galactosidase fusion protein to bind to the same DNA sequences as the 87-kD K562 protein. Because no other relationship had been established between the 87-kD K562 protein and the HOXB2 protein other than their ability to bind ot the same DNA sequences, we have investigated whether the two proteins are related antigenically. Our data show that antisera produced against the HOXB2-beta-gal fusion protein and a synthetic HOXB2 decapeptide react specifically with an 87-kD protein from K562 nuclear extract, showing that the 87-kD K562 nuclear protein is a product of the HOXB2 locus, and is the first demonstration of cellular HOXB2 protein.

Interaction of apicoplast-encoded elongation factor (EF) EF-Tu with nuclear-encoded EF-Ts mediates translation in the Plasmodiumfalciparum plastid.

PubMed

Biswas, Subir; Lim, Erin E; Gupta, Ankit; Saqib, Uzma; Mir, Snober S; Siddiqi, Mohammad Imran; Ralph, Stuart A; Habib, Saman

2011-03-01

Protein translation in the plastid (apicoplast) of Plasmodium spp. is of immense interest as a target for potential anti-malarial drugs. However, the molecular data on apicoplast translation needed for optimisation and development of novel inhibitors is lacking. We report characterisation of two key translation elongation factors in Plasmodium falciparum, apicoplast-encoded elongation factor PfEF-Tu and nuclear-encoded PfEF-Ts. Recombinant PfEF-Tu hydrolysed GTP and interacted with its presumed nuclear-encoded partner PfEF-Ts. The EF-Tu inhibitor kirromycin affected PfEF-Tu activity in vitro, indicating that apicoplast EF-Tu is indeed the target of this drug. The predicted PfEF-Ts leader sequence targeted GFP to the apicoplast, confirming that PfEF-Ts functions in this organelle. Recombinant PfEF-Ts mediated nucleotide exchange on PfEF-Tu and homology modeling of the PfEF-Tu:PfEF-Ts complex revealed PfEF-Ts-induced structural alterations that would expedite GDP release from PfEF-Tu. Our results establish functional interaction between two apicoplast translation factors encoded by genes residing in different cellular compartments and highlight the significance of their sequence/structural differences from bacterial elongation factors in relation to inhibitor activity. These data provide an experimental system to study the effects of novel inhibitors targeting PfEF-Tu and PfEF-Tu.PfEF-Ts interaction. Our finding that apicoplast EF-Tu possesses chaperone-related disulphide reductase activity also provides a rationale for retention of the tufA gene on the plastid genome. Copyright © 2010 Australian Society for Parasitology Inc. All rights reserved.

Identification of a Functional, CRM-1-Dependent Nuclear Export Signal in Hepatitis C Virus Core Protein

PubMed Central

Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

2011-01-01

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified. We show here that the aa(109–133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1–173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication. Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection. PMID:22039426

The roles of bile acids and sphingosine-1-phosphate signaling in the hepatobiliary diseases

PubMed Central

Nagahashi, Masayuki; Yuza, Kizuki; Hirose, Yuki; Nakajima, Masato; Ramanathan, Rajesh; Hait, Nitai C.; Hylemon, Phillip B.; Zhou, Huiping; Takabe, Kazuaki; Wakai, Toshifumi

2016-01-01

Based on research carried out over the last decade, it has become increasingly evident that bile acids act not only as detergents, but also as important signaling molecules that exert various biological effects via activation of specific nuclear receptors and cell signaling pathways. Bile acids also regulate the expression of numerous genes encoding enzymes and proteins involved in the synthesis and metabolism of bile acids, glucose, fatty acids, and lipoproteins, as well as energy metabolism. Receptors activated by bile acids include, farnesoid X receptor α, pregnane X receptor, vitamin D receptor, and G protein-coupled receptors, TGR5, muscarinic receptor 2, and sphingosine-1-phosphate receptor (S1PR)2. The ligand of S1PR2, sphingosine-1-phosphate (S1P), is a bioactive lipid mediator that regulates various physiological and pathophysiological cellular processes. We have recently reported that conjugated bile acids, via S1PR2, activate and upregulate nuclear sphingosine kinase 2, increase nuclear S1P, and induce genes encoding enzymes and transporters involved in lipid and sterol metabolism in the liver. Here, we discuss the role of bile acids and S1P signaling in the regulation of hepatic lipid metabolism and in hepatobiliary diseases. PMID:27459945

Identification of Novel Saccharomyces cerevisiae Proteins with Nuclear Export Activity: Cell Cycle-Regulated Transcription Factor Ace2p Shows Cell Cycle-Independent Nucleocytoplasmic Shuttling

PubMed Central

Jensen, Torben Heick; Neville, Megan; Rain, Jean Christophe; McCarthy, Terri; Legrain, Pierre; Rosbash, Michael

2000-01-01

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the NES receptor CRM1/Crm1p. We have carried out a yeast two-hybrid screen with Crm1p as a bait. The Crm1p-interacting clones were subscreened for nuclear export activity in a visual assay utilizing the Crm1p-inhibitor leptomycin B (LMB). This approach identified three Saccharomyces cerevisiae proteins not previously known to have nuclear export activity. These proteins are the 5′ RNA triphosphatase Ctl1p, the cell cycle-regulated transcription factor Ace2p, and a protein encoded by the previously uncharacterized open reading frame YDR499W. Mutagenesis analysis show that YDR499Wp contains an NES that conforms to the consensus sequence for leucine-rich NESs. Mutagenesis of Ctl1p and Ace2p were unable to identify specific NES residues. However, a 29-amino-acid region of Ace2p, rich in hydrophobic residues, contains nuclear export activity. Ace2p accumulates in the nucleus at the end of mitosis and activates early-G1-specific genes. We now provide evidence that Ace2p is nuclear not only in late M-early G1 but also during other stages of the cell cycle. This feature of Ace2p localization explains its ability to activate genes such as CUP1, which are not expressed in a cell cycle-dependent manner. PMID:11027275

Organellar maturases: A window into the evolution of the spliceosome.

PubMed

Schmitz-Linneweber, Christian; Lampe, Marie-Kristin; Sultan, Laure D; Ostersetzer-Biran, Oren

2015-09-01

During the evolution of eukaryotic genomes, many genes have been interrupted by intervening sequences (introns) that must be removed post-transcriptionally from RNA precursors to form mRNAs ready for translation. The origin of nuclear introns is still under debate, but one hypothesis is that the spliceosome and the intron-exon structure of genes have evolved from bacterial-type group II introns that invaded the eukaryotic genomes. The group II introns were most likely introduced into the eukaryotic genome from an α-proteobacterial predecessor of mitochondria early during the endosymbiosis event. These self-splicing and mobile introns spread through the eukaryotic genome and later degenerated. Pieces of introns became part of the general splicing machinery we know today as the spliceosome. In addition, group II introns likely brought intron maturases with them to the nucleus. Maturases are found in most bacterial introns, where they act as highly specific splicing factors for group II introns. In the spliceosome, the core protein Prp8 shows homology to group II intron-encoded maturases. While maturases are entirely intron specific, their descendant of the spliceosomal machinery, the Prp8 protein, is an extremely versatile splicing factor with multiple interacting proteins and RNAs. How could such a general player in spliceosomal splicing evolve from the monospecific bacterial maturases? Analysis of the organellar splicing machinery in plants may give clues on the evolution of nuclear splicing. Plants encode various proteins which are closely related to bacterial maturases. The organellar genomes contain one maturase each, named MatK in chloroplasts and MatR in mitochondria. In addition, several maturase genes have been found in the nucleus as well, which are acting on mitochondrial pre-RNAs. All plant maturases show sequence deviation from their progenitor bacterial maturases, and interestingly are all acting on multiple organellar group II intron targets. Moreover, they seem to function in the splicing of group II introns together with a number of additional nuclear-encoded splicing factors, possibly acting as an organellar proto-spliceosome. Together, this makes them interesting models for the early evolution of nuclear spliceosomal splicing. In this review, we summarize recent advances in our understanding of the role of plant maturases and their accessory factors in plants. This article is part of a Special Issue entitled: Chloroplast Biogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

Phosphorylation and nuclear localization of the varicella-zoster virus gene 63 protein.

PubMed Central

Stevenson, D; Xue, M; Hay, J; Ruyechan, W T

1996-01-01

The protein encoded by varicella-zoster virus open reading frame 63 and carboxy-terminal deletions of the same were expressed either as fusion proteins at the carboxy terminus of the maltose-binding protein in Escherichia coli or independently in transfected mammalian cells. The truncations contained amino acids 1 to 142 (63 delta N) or 1 to 210 (63 delta K) of the complete 278-amino-acid primary sequence. Recombinant casein kinase II phosphorylated the 63F and 63 delta KF fusion proteins in vitro but did not phosphorylate the 63 delta NF fusion protein, implying that phosphorylation occurred between amino acids 142 and 210. Immunoprecipitation of 35S- or 32P-labelled extracts of cells transfected with plasmids expressing 63, 63 delta N, or 63 delta K also indicated that in situ phosphorylation most likely occurred between amino acids 142 and 210. These combined results suggest that casein kinase II plays a significant role in the phosphorylation of the varicella-zoster virus 63 protein. Indirect immunofluorescence of transfected cells indicated nuclear localization of the 63 protein and cytoplasmic localization of 63 delta K and 63 delta N, implying a requirement for sequences between amino acids 210 and 278 for efficient nuclear localization. PMID:8523589

Mitochondrial Genes of Dinoflagellates Are Transcribed by a Nuclear-Encoded Single-Subunit RNA Polymerase.

PubMed

Teng, Chang Ying; Dang, Yunkun; Danne, Jillian C; Waller, Ross F; Green, Beverley R

2013-01-01

Dinoflagellates are a large group of algae that contribute significantly to marine productivity and are essential photosynthetic symbionts of corals. Although these algae have fully-functioning mitochondria and chloroplasts, both their organelle genomes have been highly reduced and the genes fragmented and rearranged, with many aberrant transcripts. However, nothing is known about their RNA polymerases. We cloned and sequenced the gene for the nuclear-encoded mitochondrial polymerase (RpoTm) of the dinoflagellate Heterocapsa triquetra and showed that the protein presequence targeted a GFP construct into yeast mitochondria. The gene belongs to a small gene family, which includes a variety of 3'-truncated copies that may have originated by retroposition. The catalytic C-terminal domain of the protein shares nine conserved sequence blocks with other single-subunit polymerases and is predicted to have the same fold as the human enzyme. However, the N-terminal (promoter binding/transcription initiation) domain is not well-conserved. In conjunction with the degenerate nature of the mitochondrial genome, this suggests a requirement for novel accessory factors to ensure the accurate production of functional mRNAs.

BPF-1, a pathogen-induced DNA-binding protein involved in the plant defense response.

PubMed

da Costa e Silva, O; Klein, L; Schmelzer, E; Trezzini, G F; Hahlbrock, K

1993-07-01

The mechanisms by which plants restrict the growth of pathogens include transient activation of numerous defense-related genes. Box P is a putative cis-acting element of a distinct group of such genes, including those encoding the enzyme phenylalanine ammonialyase (PAL). A DNA-binding activity to Box P was identified in nuclear extracts from cultured parsley cells and a cDNA encoding the protein BPF-1 (Box P-binding Factor) partially characterized. BPF-1 binds to this element with specificity similar to that of the binding activity in nuclear extracts. BPF-1 mRNA accumulates rapidly in elicitor-treated parsley cells and around fungal infection sites on parsley leaves. This accumulation is, at least partly, due to a rapid and transient increase in the transcription rate of BPF-1. Moreover, tight correlation between the relative amounts of BPF-1 and PAL mRNAs was observed in different organs of a parsley plant. These results are consistent with the hypothesis that BPF-1 is involved in disease resistance by modulating plant defense gene expression.

Localization of a bacterial group II intron-encoded protein in human cells.

PubMed

Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; García Pérez, José Luis; Toro, Nicolás

2015-08-05

Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells.

Localization of a bacterial group II intron-encoded protein in human cells

PubMed Central

Reinoso-Colacio, Mercedes; García-Rodríguez, Fernando Manuel; García-Cañadas, Marta; Amador-Cubero, Suyapa; Pérez, José Luis García; Toro, Nicolás

2015-01-01

Group II introns are mobile retroelements that self-splice from precursor RNAs to form ribonucleoparticles (RNP), which can invade new specific genomic DNA sites. This specificity can be reprogrammed, for insertion into any desired DNA site, making these introns useful tools for bacterial genetic engineering. However, previous studies have suggested that these elements may function inefficiently in eukaryotes. We investigated the subcellular distribution, in cultured human cells, of the protein encoded by the group II intron RmInt1 (IEP) and several mutants. We created fusions with yellow fluorescent protein (YFP) and with a FLAG epitope. We found that the IEP was localized in the nucleus and nucleolus of the cells. Remarkably, it also accumulated at the periphery of the nuclear matrix. We were also able to identify spliced lariat intron RNA, which co-immunoprecipitated with the IEP, suggesting that functional RmInt1 RNPs can be assembled in cultured human cells. PMID:26244523

Sense-encoded poly-GR dipeptide repeat proteins correlate to neurodegeneration and uniquely co-localize with TDP-43 in dendrites of repeat-expanded C9orf72 amyotrophic lateral sclerosis.

PubMed

Saberi, Shahram; Stauffer, Jennifer E; Jiang, Jie; Garcia, Sandra Diaz; Taylor, Amy E; Schulte, Derek; Ohkubo, Takuya; Schloffman, Cheyenne L; Maldonado, Marcus; Baughn, Michael; Rodriguez, Maria J; Pizzo, Don; Cleveland, Don; Ravits, John

2018-03-01

Hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (C9 ALS). The main hypothesized pathogenic mechanisms are C9orf72 haploinsufficiency and/or toxicity from one or more of bi-directionally transcribed repeat RNAs and their dipeptide repeat proteins (DPRs) poly-GP, poly-GA, poly-GR, poly-PR and poly-PA. Recently, nuclear import and/or export defects especially caused by arginine-containing poly-GR or poly-PR have been proposed as significant contributors to pathogenesis based on disease models. We quantitatively studied and compared DPRs, nuclear pore proteins and C9orf72 protein in clinically related and clinically unrelated regions of the central nervous system, and compared them to phosphorylated TDP-43 (pTDP-43), the hallmark protein of ALS. Of the five DPRs, only poly-GR was significantly abundant in clinically related areas compared to unrelated areas (p < 0.001), and formed dendritic-like aggregates in the motor cortex that co-localized with pTDP-43 (p < 0.0001). While most poly-GR dendritic inclusions were pTDP-43 positive, only 4% of pTDP-43 dendritic inclusions were poly-GR positive. Staining for arginine-containing poly-GR and poly-PR in nuclei of neurons produced signals that were not specific to C9 ALS. We could not detect significant differences of nuclear markers RanGap, Lamin B1, and Importin β1 in C9 ALS, although we observed subtle nuclear changes in ALS, both C9 and non-C9, compared to control. The C9orf72 protein itself was diffusely expressed in cytoplasm of large neurons and glia, and nearly 50% reduced, in both clinically related frontal cortex and unrelated occipital cortex, but not in cerebellum. In summary, sense-encoded poly-GR DPR was unique, and localized to dendrites and pTDP43 in motor regions of C9 ALS CNS. This is consistent with new emerging ideas about TDP-43 functions in dendrites.

Sense-encoded poly-GR dipeptide repeat proteins correlate to neurodegeneration and uniquely co-localize with TDP-43 in dendrites of repeat expanded C9orf72 amyotrophic lateral sclerosis

PubMed Central

Saberi, Shahram; Stauffer, Jennifer E.; Jiang, Jie; Garcia, Sandra Diaz; Taylor, Amy E; Schulte, Derek; Ohkubo, Takuya; Schloffman, Cheyenne L.; Maldonado, Marcus; Baughn, Michael; Rodriguez, Maria J; Pizzo, Don; Cleveland, Don; Ravits, John

2018-01-01

Hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (C9 ALS). The main hypothesized pathogenic mechanisms are C9orf72 haploinsufficiency and/or toxicity from one or more of bi-directionally transcribed repeat RNAs and their dipeptide repeat proteins (DPRs) poly-GP, poly-GA, poly-GR, poly-PR and poly-PA. Recently, nuclear import and/or export defects especially caused by arginine-containing poly-GR or poly-PR have been proposed as significant contributors to pathogenesis based on disease models. We quantitatively studied and compared DPRs, nuclear pore proteins and C9orf72 protein in clinically-related and clinically-unrelated regions of the central nervous system, and compared them to phosphorylated TDP-43 (pTDP-43), the hallmark protein of ALS. Of the five DPRs, only poly-GR was significantly abundant in clinically-related areas compared to unrelated areas (p<0.001), and formed dendritic-like aggregates in the motor cortex that co-localized with pTDP-43 (p<0.0001). While most poly-GR dendritic inclusions were pTDP-43-positive, only 4% of pTDP-43 dendritic inclusions were poly-GR-positive. Staining for arginine-containing poly-GR and poly-PR in nuclei of neurons produced signals that were not specific to C9 ALS. We could not detect significant differences of nuclear markers RanGap, Lamin B1, and Importin β1 in C9 ALS, although we observed subtle nuclear changes in ALS, both C9 and non-C9, compared to control. The C9orf72 protein itself was diffusely expressed in cytoplasm of large neurons and glia, and nearly 50% reduced, in both clinically-related frontal cortex and unrelated occipital cortex, but not in cerebellum. In summary, sense-encoded poly-GR DPR was unique, and localized to neurites and pTDP43 in motor regions of C9 ALS CNS. This is consistent with new emerging ideas about TDP-43 functions in dendrites. PMID:29196813

Molecular cloning, structural analysis, and expression of a human IRLB, MYC promoter-binding protein: new DENN domain-containing protein family emerges.

PubMed

Semova, Natalia; Kapanadze, Bagrat; Corcoran, Martin; Kutsenko, Alexei; Baranova, Ancha; Semov, Alexandre

2003-09-01

IRLB was originally identified as a partial cDNA clone, encoding a 191-aa protein binding the interferon-stimulated response element (ISRE) in the P2 promoter of human MYC. Here, we cloned the full-size IRLB using different bioinformatics tools and an RT-PCR approach. The full-size gene encompasses 131 kb within chromosome 15q22 and consists of 32 exons. IRLB is transcribed as a 6.6-kb mRNA encoding a protein of 1865 aa. IRLB is ubiquitously expressed and its expression is regulated in a growth- and cell cycle-dependent manner. In addition to the ISRE-binding domain IRLB contains a tripartite DENN domain, a nuclear localization signal, two PPRs, and a calmodulin-binding domain. The presence of DENN domains predicts possible interactions of IRLB with GTPases from the Rab family or regulation of growth-induced MAPKs. Strongly homologous proteins were identified in all available vertebrate genomes as well as in Caenorhabditis elegans and Drosophila melanogaster. In human and mouse a family of IRLB proteins exists, consisting of at least three members.

Identification of Strawberry vein banding virus encoded P6 as an RNA silencing suppressor.

PubMed

Feng, Mingfeng; Zuo, Dengpan; Jiang, Xizi; Li, Shuai; Chen, Jing; Jiang, Lei; Zhou, Xueping; Jiang, Tong

2018-07-01

RNA silencing is a common mechanism that plays a key role in antiviral defense. To overcome host defense responses, plant viruses encode silencing-suppressor proteins to target one or several key steps in the silencing machinery. Here, we report that the P6 protein encoded by Strawberry vein banding virus (SVBV) is an RNA silencing suppressor through Agrobacterium-mediated co-infiltration assays. SVBV P6 protein can suppress green fluorescent protein (GFP) gene silencing induced by single-stranded RNA but not by double-stranded RNA. The P6 protein can also inhibit systemic silencing of GFP through interfering the systemic spread of GFP silencing signal. Subcellular localization study indicated that P6 protein formed irregular bodies and distributed in both cytoplasm and nucleus of Nicotiana benthamiana cells. Furthermore, deletion analysis indicated that a nuclear localization signal (NLS, aa 402-426) in the P6 protein is responsible for the silencing suppression efficiency. In addition, expression of the P6 protein via a Potato virus X (PVX)-based vectors induced more severe mosaic symptoms in N. benthamiana leaves, and transgenic N. benthamiana plants expressing P6 showed obvious vein yellowing as well as severe mosaic symptoms in leaves. Taken together, our results demonstrates that SVBV P6 is a suppressor of RNA silencing, possibly acting at a upstream step for dsRNA generation. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Mutation in TOR1AIP1 encoding LAP1B in a form of muscular dystrophy: a novel gene related to nuclear envelopathies.

PubMed

Kayman-Kurekci, Gulsum; Talim, Beril; Korkusuz, Petek; Sayar, Nilufer; Sarioglu, Turkan; Oncel, Ibrahim; Sharafi, Parisa; Gundesli, Hulya; Balci-Hayta, Burcu; Purali, Nuhan; Serdaroglu-Oflazer, Piraye; Topaloglu, Haluk; Dincer, Pervin

2014-07-01

We performed genome-wide homozygosity mapping and mapped a novel myopathic phenotype to chromosomal region 1q25 in a consanguineous family with three affected individuals manifesting proximal and distal weakness and atrophy, rigid spine and contractures of the proximal and distal interphalangeal hand joints. Additionally, cardiomyopathy and respiratory involvement were noted. DNA sequencing of torsinA-interacting protein 1 (TOR1AIP1) gene encoding lamina-associated polypeptide 1B (LAP1B), showed a homozygous c.186delG mutation that causes a frameshift resulting in a premature stop codon (p.E62fsTer25). We observed that expression of LAP1B was absent in the patient skeletal muscle fibres. Ultrastructural examination showed intact sarcomeric organization but alterations of the nuclear envelope including nuclear fragmentation, chromatin bleb formation and naked chromatin. LAP1B is a type-2 integral membrane protein localized in the inner nuclear membrane that binds to both A- and B-type lamins, and is involved in the regulation of torsinA ATPase. Interestingly, luminal domain-like LAP1 (LULL1)-an endoplasmic reticulum-localized partner of torsinA-was overexpressed in the patient's muscle in the absence of LAP1B. Therefore, the findings suggest that LAP1 and LULL1 might have a compensatory effect on each other. This study expands the spectrum of genes associated with nuclear envelopathies and highlights the critical function for LAP1B in striated muscle. Copyright © 2014 Elsevier B.V. All rights reserved.

cDNA cloning and characterization of a novel gene encoding the MLF1-interacting protein MLF1IP.

PubMed

Hanissian, Silva H; Akbar, Umar; Teng, Bin; Janjetovic, Zorica; Hoffmann, Anne; Hitzler, Johann K; Iscove, Norman; Hamre, Kristin; Du, Xiaoping; Tong, Yiai; Mukatira, Suraj; Robertson, Jon H; Morris, Stephan W

2004-04-29

Myelodysplasia/acute myeloid leukemia (MDS/AML) is characterized by a t(3;5)(q25.1;q34) chromosomal translocation that forms a fusion gene between nucleophosmin (NPM) and MDS/myeloid leukemia factor 1 (MLF1). We identified a novel protein, MLF1-interacting protein (MLF1IP), that specifically associates with MLF1 by yeast two-hybrid analysis and in pulldown assays, and colocalizes with it in both the nuclei and cytoplasm of cells. The MLF1IP gene locus is at chromosome 4q35.1 and is composed of 14 exons spanning 75.8 kb of genomic DNA. The MLF1IP cDNA encodes a 46-kDa protein that contains two bipartite and two classical nuclear localization signals, two nuclear receptor-binding motifs (LXXLL), two leucine zippers, two PEST residues and several potential phosphorylation sites. MLF1IP transcripts are expressed in a variety of tissues (e.g. fetal liver, bone marrow, thymus and testis). MLF1IP appears to be a lineage-specific gene whose expression is confined exclusively to the CFU-E erythroid precursor cells, but not in mature erythrocytes. These observations, together with previous data demonstrating a role for MLF1 in suppressing red cell maturation, suggest a possible role for MLF1IP and MLF1 deregulation in the genesis of erythroleukemias.

Plant parasitic nematode effectors target host defense and nuclear functions to establish feeding cells.

PubMed

Quentin, Michaëel; Abad, Pierre; Favery, Bruno

2013-01-01

Plant parasitic nematodes are microscopic worms, the most damaging species of which have adopted a sedentary lifestyle within their hosts. These obligate endoparasites have a biotrophic relationship with plants, in which they induce the differentiation of root cells into hypertrophied, multinucleate feeding cells (FCs). Effectors synthesized in the esophageal glands of the nematode are injected into the plant cells via the syringe-like stylet and play a key role in manipulating the host machinery. The establishment of specialized FCs requires these effectors to modulate many aspects of plant cell morphogenesis and physiology, including defense responses. This cell reprogramming requires changes to host nuclear processes. Some proteins encoded by parasitism genes target host nuclei. Several of these proteins were immunolocalized within FC nuclei or shown to interact with host nuclear proteins. Comparative genomics and functional analyses are gradually revealing the roles of nematode effectors. We describe here these effectors and their hypothesized roles in the unique feeding behavior of these pests.

Nesprin-1α-Dependent Microtubule Nucleation from the Nuclear Envelope via Akap450 Is Necessary for Nuclear Positioning in Muscle Cells.

PubMed

Gimpel, Petra; Lee, Yin Loon; Sobota, Radoslaw M; Calvi, Alessandra; Koullourou, Victoria; Patel, Rutti; Mamchaoui, Kamel; Nédélec, François; Shackleton, Sue; Schmoranzer, Jan; Burke, Brian; Cadot, Bruno; Gomes, Edgar R

2017-10-09

The nucleus is the main microtubule-organizing center (MTOC) in muscle cells due to the accumulation of centrosomal proteins and microtubule (MT) nucleation activity at the nuclear envelope (NE) [1-4]. The relocalization of centrosomal proteins, including Pericentrin, Pcm1, and γ-tubulin, depends on Nesprin-1, an outer nuclear membrane (ONM) protein that connects the nucleus to the cytoskeleton via its N-terminal region [5-7]. Nesprins are also involved in the recruitment of kinesin to the NE and play a role in nuclear positioning in skeletal muscle cells [8-12]. However, a function for MT nucleation from the NE in nuclear positioning has not been established. Using the proximity-dependent biotin identification (BioID) method [13, 14], we found several centrosomal proteins, including Akap450, Pcm1, and Pericentrin, whose association with Nesprin-1α is increased in differentiated myotubes. We show that Nesprin-1α recruits Akap450 to the NE independently of kinesin and that Akap450, but not other centrosomal proteins, is required for MT nucleation from the NE. Furthermore, we demonstrate that this mechanism is disrupted in congenital muscular dystrophy patient myotubes carrying a nonsense mutation within the SYNE1 gene (23560 G>T) encoding Nesprin-1 [15, 16]. Finally, using computer simulation and cell culture systems, we provide evidence for a role of MT nucleation from the NE on nuclear spreading in myotubes. Our data thus reveal a novel function for Nesprin-1α/Nesprin-1 in nuclear positioning through recruitment of Akap450-mediated MT nucleation activity to the NE. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Autoantibodies in infectious mononucleosis have specificity for the glycine-alanine repeating region of the Epstein-Barr virus nuclear antigen

PubMed Central

1987-01-01

Viruses have been postulated to be involved in the induction of autoantibodies by: autoimmunization with tissue proteins released by virally induced tissue damage; immunization with virally encoded antigens bearing molecular similarities to normal tissue proteins; or nonspecific (polyclonal) B cell stimulation by the infection. Infectious mononucleosis (IM) is an experiment of nature that provides the opportunity for examining these possibilities. We show here that IgM antibodies produced in this disease react with at least nine normal tissue proteins, in addition to the virally encoded Epstein-Barr nuclear antigen (EBNA-1). The antibodies are generated to configurations in the glycine-alanine repeat region of EBNA-1 and are crossreactive with the normal tissue proteins through similar configurations, as demonstrated by the effectiveness of a synthetic glycine-alanine peptide in inhibiting the reactions. The antibodies are absent in preillness sera and gradually disappear over a period of months after illness, being replaced by IgG anti-EBNA-1 antibodies that do not crossreact with the normal tissue proteins but that are still inhibited by the glycine-alanine peptide. These findings are most easily explained by either a molecular mimicry model of IgM autoantibody production or by the polyclonal activation of a germline gene for a crossreactive antibody. It also indicates a selection of highly specific, non-crossreactive anti-EBNA-1 antibodies during IgM to IgG isotype switching. PMID:2435830

Differential mitochondrial DNA and gene expression in inherited retinal dysplasia in miniature Schnauzer dogs.

PubMed

Appleyard, Greg D; Forsyth, George W; Kiehlbauch, Laura M; Sigfrid, Kristen N; Hanik, Heather L J; Quon, Anita; Loewen, Matthew E; Grahn, Bruce H

2006-05-01

To investigate the molecular basis of inherited retinal dysplasia in miniature Schnauzers. Retina and retinal pigment epithelial tissues were collected from canine subjects at the age of 3 weeks. Total RNA isolated from these tissues was reverse transcribed to make representative cDNA pools that were compared for differences in gene expression by using a subtractive hybridization technique referred to as representational difference analysis (RDA). Expression differences identified by RDA were confirmed and quantified by real-time reverse-transcription PCR. Mitochondrial morphology from leukocytes and skeletal muscle of normal and affected miniature Schnauzers was examined by transmission electron microscopy. RDA screening of retinal pigment epithelial cDNA identified differences in mRNA transcript coding for two mitochondrial (mt) proteins--cytochrome oxidase subunit 1 and NADH dehydrogenase subunit 6--in affected dogs. Contrary to expectations, these identified sequences did not contain mutations. Based on the implication of mt-DNA-encoded proteins by the RDA experiments we used real-time PCR to compare the relative amounts of mt-DNA template in white blood cells from normal and affected dogs. White blood cells of affected dogs contained less than 30% of the normal amount of two specific mtDNA sequences, compared with the content of the nuclear-encoded glyceraldehyde-3-phosphate dehydrogenase (GA-3-PDH) reference gene. Retina and RPE tissue from affected dogs had reduced mRNA transcript levels for the two mitochondrial genes detected in the RDA experiment. Transcript levels for another mtDNA-encoded gene as well as the nuclear-encoded mitochondrial Tfam transcription factor were reduced in these tissues in affected dogs. Mitochondria from affected dogs were reduced in number and size and were unusually electron dense. Reduced levels of nuclear and mitochondrial transcripts in the retina and RPE of miniature Schnauzers affected with retinal dysplasia suggest that the pathogenesis of the disorder may arise from a lowered energy supply to the retina and RPE.

Identification of cis-elements for ethylene and circadian regulation of the Solanum melongena gene encoding cysteine proteinase.

PubMed

Rawat, Reetika; Xu, Zeng-Fu; Yao, Kwok-Ming; Chye, Mee-Len

2005-03-01

We have previously shown that the expression of SmCP which encodes Solanum melongena cysteine proteinase is ethylene-inducible and is under circadian control. To understand the regulation of SmCP, a 1.34-kb SmCP 5'-flanking region and its deletion derivatives were analyzed for cis-elements using GUS and luc fusions and by in vitro binding assays. Analysis of transgenic tobacco transformed with SmCP promoter-GUS constructs confirmed that the promoter region -415/+54 containing Ethylene Responsive Element ERE(-355/-348) conferred threefold ethylene-induction of GUS expression, while -827/+54 which also contains ERE(-683/-676), produced fivefold induction. Using gel mobility shift assays, we demonstrated that each ERE binds nuclear proteins from both ethephon-treated and untreated 5-week-old seedlings, suggesting that different transcriptions factors bind each ERE under varying physiological conditions. Binding was also observed in extracts from senescent, but not young, fruits. The variation in binding at the EREs in fruits and seedlings imply that organ-specific factors may participate in binding. Analysis of transgenic tobacco expressing various SmCP promoter-luc constructs containing wild-type or mutant Evening Elements (EEs) confirmed that both conserved EEs at -795/-787 and -785/-777 are important in circadian control. We confirmed the binding of total nuclear proteins to EEs in gel mobility shift assays and in DNase I footprinting. Our results suggest that multiple proteins bind the EEs which are conserved in plants other than Arabidopsis and that functional EEs and EREs are present in the 5'-flanking region of a gene encoding cysteine proteinase.

Light-modulated abundance of an mRNA encoding a calmodulin-regulated, chromatin-associated NTPase in pea

NASA Technical Reports Server (NTRS)

Hsieh, H. L.; Tong, C. G.; Thomas, C.; Roux, S. J.

1996-01-01

A CDNA encoding a 47 kDa nucleoside triphosphatase (NTPase) that is associated with the chromatin of pea nuclei has been cloned and sequenced. The translated sequence of the cDNA includes several domains predicted by known biochemical properties of the enzyme, including five motifs characteristic of the ATP-binding domain of many proteins, several potential casein kinase II phosphorylation sites, a helix-turn-helix region characteristic of DNA-binding proteins, and a potential calmodulin-binding domain. The deduced primary structure also includes an N-terminal sequence that is a predicted signal peptide and an internal sequence that could serve as a bipartite-type nuclear localization signal. Both in situ immunocytochemistry of pea plumules and immunoblots of purified cell fractions indicate that most of the immunodetectable NTPase is within the nucleus, a compartment proteins typically reach through nuclear pores rather than through the endoplasmic reticulum pathway. The translated sequence has some similarity to that of human lamin C, but not high enough to account for the earlier observation that IgG against human lamin C binds to the NTPase in immunoblots. Northern blot analysis shows that the NTPase MRNA is strongly expressed in etiolated plumules, but only poorly or not at all in the leaf and stem tissues of light-grown plants. Accumulation of NTPase mRNA in etiolated seedlings is stimulated by brief treatments with both red and far-red light, as is characteristic of very low-fluence phytochrome responses. Southern blotting with pea genomic DNA indicates the NTPase is likely to be encoded by a single gene.

Alterations in the mitochondrial regulatory pathways constituted by the nuclear co-factors PGC-1alpha or PGC-1beta and mitofusin 2 in skeletal muscle in type 2 diabetes.

PubMed

Zorzano, Antonio; Hernández-Alvarez, María Isabel; Palacín, Manuel; Mingrone, Geltrude

2010-01-01

Muscle mitochondrial metabolism is regulated by a number of factors, many of which are responsible for the transcription of nuclear genes encoding mitochondrial proteins such as PPARdelta, PGC-1alpha or PGC-1beta. Recent evidence indicates that proteins participating in mitochondrial dynamics also regulate mitochondrial metabolism. Thus, in cultured cells the mitochondrial fusion protein mitofusin 2 (Mfn2) stimulates respiration, substrate oxidation and the expression of subunits involved in respiratory complexes. Mitochondrial dysfunction has been reported in skeletal muscle of type 2 diabetic patients. Reduced mitochondrial mass and defective activity has been proposed to explain this dysfunction. Alterations in mitochondrial metabolism may be crucial to account for some of the pathophysiological traits that characterize type 2 diabetes. Skeletal muscle of type 2 diabetic patients shows reduced expression of PGC-1alpha, PGC-1beta, and Mfn2. In addition, a differential response to bilio-pancreatic diversion-induced weight loss in non-diabetic and type 2 diabetic patients has been reported. While non-diabetic morbidly obese subjects showed an increased expression of genes encoding Mfn2, PGC-1alpha, PGC-1beta, PPARdelta or SIRT1 in response to bariatric surgery-induced weight loss, no effect was detected in type 2 diabetic patients. These observations suggest the existence of a heritable component responsible for the abnormal control of the expression of genes encoding for modulators of mitochondrial biogenesis/metabolism, and which may participate in the development of the disease. Copyright © 2010 Elsevier B.V. All rights reserved.

Medicago truncatula contains a second gene encoding a plastid located glutamine synthetase exclusively expressed in developing seeds.

PubMed

Seabra, Ana R; Vieira, Cristina P; Cullimore, Julie V; Carvalho, Helena G

2010-08-19

Nitrogen is a crucial nutrient that is both essential and rate limiting for plant growth and seed production. Glutamine synthetase (GS), occupies a central position in nitrogen assimilation and recycling, justifying the extensive number of studies that have been dedicated to this enzyme from several plant sources. All plants species studied to date have been reported as containing a single, nuclear gene encoding a plastid located GS isoenzyme per haploid genome. This study reports the existence of a second nuclear gene encoding a plastid located GS in Medicago truncatula. This study characterizes a new, second gene encoding a plastid located glutamine synthetase (GS2) in M. truncatula. The gene encodes a functional GS isoenzyme with unique kinetic properties, which is exclusively expressed in developing seeds. Based on molecular data and the assumption of a molecular clock, it is estimated that the gene arose from a duplication event that occurred about 10 My ago, after legume speciation and that duplicated sequences are also present in closely related species of the Vicioide subclade. Expression analysis by RT-PCR and western blot indicate that the gene is exclusively expressed in developing seeds and its expression is related to seed filling, suggesting a specific function of the enzyme associated to legume seed metabolism. Interestingly, the gene was found to be subjected to alternative splicing over the first intron, leading to the formation of two transcripts with similar open reading frames but varying 5' UTR lengths, due to retention of the first intron. To our knowledge, this is the first report of alternative splicing on a plant GS gene. This study shows that Medicago truncatula contains an additional GS gene encoding a plastid located isoenzyme, which is functional and exclusively expressed during seed development. Legumes produce protein-rich seeds requiring high amounts of nitrogen, we postulate that this gene duplication represents a functional innovation of plastid located GS related to storage protein accumulation exclusive to legume seed metabolism.

Protein and DNA Modifications: Evolutionary Imprints of Bacterial Biochemical Diversification and Geochemistry on the Provenance of Eukaryotic Epigenetics

PubMed Central

Aravind, L.; Burroughs, A. Maxwell; Zhang, Dapeng; Iyer, Lakshminarayan M.

2014-01-01

Epigenetic information, which plays a major role in eukaryotic biology, is transmitted by covalent modifications of nuclear proteins (e.g., histones) and DNA, along with poorly understood processes involving cytoplasmic/secreted proteins and RNAs. The origin of eukaryotes was accompanied by emergence of a highly developed biochemical apparatus for encoding, resetting, and reading covalent epigenetic marks in proteins such as histones and tubulins. The provenance of this apparatus remained unclear until recently. Developments in comparative genomics show that key components of eukaryotic epigenetics emerged as part of the extensive biochemical innovation of secondary metabolism and intergenomic/interorganismal conflict systems in prokaryotes, particularly bacteria. These supplied not only enzymatic components for encoding and removing epigenetic modifications, but also readers of some of these marks. Diversification of these prokaryotic systems and subsequently eukaryotic epigenetics appear to have been considerably influenced by the great oxygenation event in the Earth’s history. PMID:24984775

Protein and DNA modifications: evolutionary imprints of bacterial biochemical diversification and geochemistry on the provenance of eukaryotic epigenetics.

PubMed

Aravind, L; Burroughs, A Maxwell; Zhang, Dapeng; Iyer, Lakshminarayan M

2014-07-01

Epigenetic information, which plays a major role in eukaryotic biology, is transmitted by covalent modifications of nuclear proteins (e.g., histones) and DNA, along with poorly understood processes involving cytoplasmic/secreted proteins and RNAs. The origin of eukaryotes was accompanied by emergence of a highly developed biochemical apparatus for encoding, resetting, and reading covalent epigenetic marks in proteins such as histones and tubulins. The provenance of this apparatus remained unclear until recently. Developments in comparative genomics show that key components of eukaryotic epigenetics emerged as part of the extensive biochemical innovation of secondary metabolism and intergenomic/interorganismal conflict systems in prokaryotes, particularly bacteria. These supplied not only enzymatic components for encoding and removing epigenetic modifications, but also readers of some of these marks. Diversification of these prokaryotic systems and subsequently eukaryotic epigenetics appear to have been considerably influenced by the great oxygenation event in the Earth's history. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Epstein–Barr virus nuclear antigen 3C interact with p73: Interplay between a viral oncoprotein and cellular tumor suppressor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sahu, Sushil Kumar; Mohanty, Suchitra; Kumar, Amit

The p73 protein has structural and functional homology with the tumor suppressor p53, which plays an important role in cell cycle regulation, apoptosis, and DNA repair. The p73 locus encodes both a tumor suppressor (TAp73) and a putative oncogene (ΔNp73). p73 May play a significant role in p53-deficient lymphomas infected with Epstein–Barr virus (EBV). EBV produces an asymptomatic infection in the majority of the global population, but it is associated with several human B-cell malignancies. The EBV-encoded Epstein–Barr virus nuclear antigen 3C (EBNA3C) is thought to disrupt the cell cycle checkpoint by interacting directly with p53 family proteins. Doxorubicin, amore » commonly used chemotherapeutic agent, induces apoptosis through p53 and p73 signaling such that the lowΔNp73 level promotes the p73-mediated intrinsic pathway of apoptosis. In this report, we investigated the mechanism by which EBV infection counters p73α-induced apoptosis through EBNA3C. - Highlights: • EBV-encoded EBNA3C suppresses doxorubicin-induced apoptosis in B-cell lymphomas. • EBNA3C binds to p73 to suppress its apoptotic effect. • EBNA3C maintains latency by regulating downstream mitochondrial pathways.« less

Three new members of the RNP protein family in Xenopus.

PubMed Central

Good, P J; Rebbert, M L; Dawid, I B

1993-01-01

Many RNP proteins contain one or more copies of the RNA recognition motif (RRM) and are thought to be involved in cellular RNA metabolism. We have previously characterized in Xenopus a nervous system specific gene, nrp1, that is more similar to the hnRNP A/B proteins than to other known proteins (K. Richter, P. J. Good, and I. B. Dawid (1990), New Biol. 2, 556-565). PCR amplification with degenerate primers was used to identify additional cDNAs encoding two RRMs in Xenopus. Three previously uncharacterized genes were identified. Two genes encode hnRNP A/B proteins with two RRMs and a glycine-rich domain. One of these is the Xenopus homolog of the human A2/B1 gene; the other, named hnRNP A3, is similar to both the A1 and A2 hnRNP genes. The Xenopus hnRNP A1, A2 and A3 genes are expressed throughout development and in all adult tissues. Multiple protein isoforms for the hnRNP A2 gene are predicted that differ by the insertion of short peptide sequences in the glycine-rich domain. The third newly isolated gene, named xrp1, encodes a protein that is related by sequence to the nrp1 protein but is expressed ubiquitously. Despite the similarity to nuclear RNP proteins, both the nrp1 and xrp1 proteins are localized to the cytoplasm in the Xenopus oocyte. The xrp1 gene may have a function in all cells that is similar to that executed by nrp1 specifically within the nervous system. Images PMID:8451200

Mechanism of Protein Biosynthesis in Mammalian Mitochondria

PubMed Central

Christian, Brooke E.; Spremulli, Linda L.

2011-01-01

Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided. PMID:22172991

The PB2 Subunit of the Influenza Virus RNA Polymerase Affects Virulence by Interacting with the Mitochondrial Antiviral Signaling Protein and Inhibiting Expression of Beta Interferon▿

PubMed Central

Graef, Katy M.; Vreede, Frank T.; Lau, Yuk-Fai; McCall, Amber W.; Carr, Simon M.; Subbarao, Kanta; Fodor, Ervin

2010-01-01

The PB2 subunit of the influenza virus RNA polymerase is a major virulence determinant of influenza viruses. However, the molecular mechanisms involved remain unknown. It was previously shown that the PB2 protein, in addition to its nuclear localization, also accumulates in the mitochondria. Here, we demonstrate that the PB2 protein interacts with the mitochondrial antiviral signaling protein, MAVS (also known as IPS-1, VISA, or Cardif), and inhibits MAVS-mediated beta interferon (IFN-β) expression. In addition, we show that PB2 proteins of influenza viruses differ in their abilities to associate with the mitochondria. In particular, the PB2 proteins of seasonal human influenza viruses localize to the mitochondria while PB2 proteins of avian influenza viruses are nonmitochondrial. This difference in localization is caused by a single amino acid polymorphism in the PB2 mitochondrial targeting signal. In order to address the functional significance of the mitochondrial localization of the PB2 protein in vivo, we have generated two recombinant human influenza viruses encoding either mitochondrial or nonmitochondrial PB2 proteins. We found that the difference in the mitochondrial localization of the PB2 proteins does not affect the growth of these viruses in cell culture. However, the virus encoding the nonmitochondrial PB2 protein induces higher levels of IFN-β and, in an animal model, is attenuated compared to the isogenic virus encoding a mitochondrial PB2. Overall this study implicates the PB2 protein in the regulation of host antiviral innate immune pathways and suggests an important role for the mitochondrial association of the PB2 protein in determining virulence. PMID:20538852

NUCLEAR PORE ANCHOR, the Arabidopsis Homolog of Tpr/Mlp1/Mlp2/Megator, Is Involved in mRNA Export and SUMO Homeostasis and Affects Diverse Aspects of Plant Development[W

PubMed Central

Xu, Xianfeng Morgan; Rose, Annkatrin; Muthuswamy, Sivaramakrishnan; Jeong, Sun Yong; Venkatakrishnan, Sowmya; Zhao, Qiao; Meier, Iris

2007-01-01

Vertebrate Tpr and its yeast homologs Mlp1/Mlp2, long coiled-coil proteins of nuclear pore inner basket filaments, are involved in mRNA export, telomere organization, spindle pole assembly, and unspliced RNA retention. We identified Arabidopsis thaliana NUCLEAR PORE ANCHOR (NUA) encoding a 237-kD protein with similarity to Tpr. NUA is located at the inner surface of the nuclear envelope in interphase and in the vicinity of the spindle in prometaphase. Four T-DNA insertion lines were characterized, which comprise an allelic series of increasing severity for several correlating phenotypes, such as early flowering under short days and long days, increased abundance of SUMO conjugates, altered expression of several flowering regulators, and nuclear accumulation of poly(A)+ RNA. nua mutants phenocopy mutants of EARLY IN SHORT DAYS4 (ESD4), an Arabidopsis SUMO protease concentrated at the nuclear periphery. nua esd4 double mutants resemble nua and esd4 single mutants, suggesting that the two proteins act in the same pathway or complex, supported by yeast two-hybrid interaction. Our data indicate that NUA is a component of nuclear pore-associated steps of sumoylation and mRNA export in plants and that defects in these processes affect the signaling events of flowering time regulation and additional developmental processes. PMID:17513499

Localization of human T-cell lymphotropic virus type II Tax protein is dependent upon a nuclear localization determinant in the N-terminal region.

PubMed

Turci, Marco; Romanelli, Maria Grazia; Lorenzi, Pamela; Righi, Paola; Bertazzoni, Umberto

2006-01-03

Human T-cell lymphotropic viruses (HTLV) types I and II are closely related oncogenic retroviruses that have been associated with lymphoproliferative and neurological disorders. The proviral genome encodes a trans-regulatory Tax protein that activates viral genes and upregulates various cellular genes involved in both cell growth and transformation. Tax proteins of HTLV-I (Tax-I) and HTLV-II (Tax-II) exhibit more than 77% aa homology and expression of either Tax-I or Tax-II is sufficient for immortalization of cultured T lymphocytes. Tax-I shuttles from the nucleus to the cytoplasm and accumulates within the nucleus, whereas Tax-II is found mainly in the cytoplasm. In the present study we have used recombinant vectors to analyze the size and structure of the nuclear localization domain within the Tax-II protein sequence. The Tax-II protein was expressed in HeLa cells either as the complete protein, or regions thereof, that were individually fused to the green fluorescent protein (GFP). Immunoblot analysis of the fused Tax-II products confirmed their expression and size. Fluorescence microscopy studies indicated that the complete Tax-II as well as N-truncated forms presented a punctuate cytoplasmic distribution and that a nuclear localization determinant is confined to within the first 60 aa of Tax-II. Accordingly, site directed mutagenesis and deletion of specific sequences within the first 60 aa showed that the nuclear determinant lies within the first 41 residues of Tax-II. These results point to a direct involvement of the amino-terminal residues of Tax-II protein in determining its nuclear functionality.

Expression of almond KNOTTED1 homologue (PdKn1) anticipates adventitious shoot initiation

USDA-ARS?s Scientific Manuscript database

Background and Aims: The transcription factor encoded by the gene Knotted1 is a nuclear homeodomain protein, regulating meristematic cells at the shoot apical meristem. It has been proven that Knotted1 (KN1) has a role in the switch from an indeterminate to determinate cell fate and as such this gen...

Arabidopsis TOBAMOVIRUS MULTIPLICATION (TOM) 2 locus encodes a transmembrane protein that interacts with TOM1

PubMed Central

Tsujimoto, Yayoi; Numaga, Takuro; Ohshima, Kiyoshi; Yano, Masa-aki; Ohsawa, Ryuji; Goto, Derek B.; Naito, Satoshi; Ishikawa, Masayuki

2003-01-01

The tom2-1 mutation of Arabidopsis thaliana reduces the efficiency of intracellular multiplication of tobamoviruses. The tom2-1 mutant was derived from fast-neutron-irradiated seeds, and the original mutant line also carries ttm1, a dominant modifier that increases tobamovirus multiplication efficiency in a tobamovirus-strain-specific manner in the tom2-1 genetic background. Here, we show that the tom2-1 mutation involved a deletion of ∼20 kb in the nuclear genome. The deleted region included two genes named TOM2A and TOM2B that were both associated with the tom2-1 phenotype, whereas ttm1 corresponded to the translocation of part of the deleted region that included intact TOM2B but not TOM2A. TOM2A encodes a 280 amino acid putative four-pass transmembrane protein with a C-terminal farnesylation signal, while TOM2B encodes a 122 amino acid basic protein. The split-ubiquitin assay demonstrated an interaction of TOM2A both with itself and with TOM1, an integral membrane protein of A.thaliana presumed to be an essential constituent of tobamovirus replication complex. The data presented here suggest that TOM2A is also an integral part of the tobamovirus replication complex. PMID:12514139

Generation of a variety of stable Influenza A reporter viruses by genetic engineering of the NS gene segment

PubMed Central

Reuther, Peter; Göpfert, Kristina; Dudek, Alexandra H.; Heiner, Monika; Herold, Susanne; Schwemmle, Martin

2015-01-01

Influenza A viruses (IAV) pose a constant threat to the human population and therefore a better understanding of their fundamental biology and identification of novel therapeutics is of upmost importance. Various reporter-encoding IAV were generated to achieve these goals, however, one recurring difficulty was the genetic instability especially of larger reporter genes. We employed the viral NS segment coding for the non-structural protein 1 (NS1) and nuclear export protein (NEP) for stable expression of diverse reporter proteins. This was achieved by converting the NS segment into a single open reading frame (ORF) coding for NS1, the respective reporter and NEP. To allow expression of individual proteins, the reporter genes were flanked by two porcine Teschovirus-1 2A peptide (PTV-1 2A)-coding sequences. The resulting viruses encoding luciferases, fluorescent proteins or a Cre recombinase are characterized by a high genetic stability in vitro and in mice and can be readily employed for antiviral compound screenings, visualization of infected cells or cells that survived acute infection. PMID:26068081

An Essential Role for COPI in mRNA Localization to Mitochondria and Mitochondrial Function.

PubMed

Zabezhinsky, Dmitry; Slobodin, Boris; Rapaport, Doron; Gerst, Jeffrey E

2016-04-19

Nuclear-encoded mRNAs encoding mitochondrial proteins (mMPs) can localize directly to the mitochondrial surface, yet how mMPs target mitochondria and whether RNA targeting contributes to protein import into mitochondria and cellular metabolism are unknown. Here, we show that the COPI vesicle coat complex is necessary for mMP localization to mitochondria and mitochondrial function. COPI inactivation leads to reduced mMP binding to COPI itself, resulting in the dissociation of mMPs from mitochondria, a reduction in mitochondrial membrane potential, a decrease in protein import in vivo and in vitro, and severe deficiencies in mitochondrial respiration. Using a model mMP (OXA1), we observed that COPI inactivation (or mutation of the potential COPI-interaction site) led to altered mRNA localization and impaired cellular respiration. Overall, COPI-mediated mMP targeting is critical for mitochondrial protein import and function, and transcript delivery to the mitochondria or endoplasmic reticulum is regulated by cis-acting RNA sequences and trans-acting proteins. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

The NS2 proteins of parvovirus minute virus of mice are required for efficient nuclear egress of progeny virions in mouse cells.

PubMed

Eichwald, Virginie; Daeffler, Laurent; Klein, Michèle; Rommelaere, Jean; Salomé, Nathalie

2002-10-01

The small nonstructural NS2 proteins of parvovirus minute virus of mice (MVMp) were previously shown to interact with the nuclear export receptor Crm1. We report here the analysis of two MVM mutant genomic clones generating NS2 proteins that are unable to interact with Crm1 as a result of amino acid substitutions within their nuclear export signal (NES) sequences. Upon transfection of human and mouse cells, the MVM-NES21 and MVM-NES22 mutant genomic clones were proficient in synthesis of the four virus-encoded proteins. While the MVM-NES22 clone was further able to produce infectious mutant virions, no virus could be recovered from cells transfected with the MVM-NES21 clone. Whereas the defect of MVM-NES21 appeared to be complex, the phenotype of MVM-NES22 could be traced back to a novel distinct NS2 function. Infection of mouse cells with the MVM-NES22 mutant led to stronger nuclear retention not only of the NS2 proteins but also of infectious progeny MVM particles. This nuclear sequestration correlated with a severe delay in the release of mutant virions in the medium and with prolonged survival of the infected cell populations compared with wild-type virus-treated cultures. This defect could explain, at least in part, the small size of the plaques generated by the MVM-NES22 mutant when assayed on mouse indicator cells. Altogether, our data indicate that the interaction of MVMp NS2 proteins with the nuclear export receptor Crm1 plays a critical role at a late stage of the parvovirus life cycle involved in release of progeny viruses.

The NS2 Proteins of Parvovirus Minute Virus of Mice Are Required for Efficient Nuclear Egress of Progeny Virions in Mouse Cells

PubMed Central

Eichwald, Virginie; Daeffler, Laurent; Klein, Michèle; Rommelaere, Jean; Salomé, Nathalie

2002-01-01

The small nonstructural NS2 proteins of parvovirus minute virus of mice (MVMp) were previously shown to interact with the nuclear export receptor Crm1. We report here the analysis of two MVM mutant genomic clones generating NS2 proteins that are unable to interact with Crm1 as a result of amino acid substitutions within their nuclear export signal (NES) sequences. Upon transfection of human and mouse cells, the MVM-NES21 and MVM-NES22 mutant genomic clones were proficient in synthesis of the four virus-encoded proteins. While the MVM-NES22 clone was further able to produce infectious mutant virions, no virus could be recovered from cells transfected with the MVM-NES21 clone. Whereas the defect of MVM-NES21 appeared to be complex, the phenotype of MVM-NES22 could be traced back to a novel distinct NS2 function. Infection of mouse cells with the MVM-NES22 mutant led to stronger nuclear retention not only of the NS2 proteins but also of infectious progeny MVM particles. This nuclear sequestration correlated with a severe delay in the release of mutant virions in the medium and with prolonged survival of the infected cell populations compared with wild-type virus-treated cultures. This defect could explain, at least in part, the small size of the plaques generated by the MVM-NES22 mutant when assayed on mouse indicator cells. Altogether, our data indicate that the interaction of MVMp NS2 proteins with the nuclear export receptor Crm1 plays a critical role at a late stage of the parvovirus life cycle involved in release of progeny viruses. PMID:12239307

Selective Inhibitor of Nuclear Export (SINE) Compounds Alter New World Alphavirus Capsid Localization and Reduce Viral Replication in Mammalian Cells.

PubMed

Lundberg, Lindsay; Pinkham, Chelsea; de la Fuente, Cynthia; Brahms, Ashwini; Shafagati, Nazly; Wagstaff, Kylie M; Jans, David A; Tamir, Sharon; Kehn-Hall, Kylene

2016-11-01

The capsid structural protein of the New World alphavirus, Venezuelan equine encephalitis virus (VEEV), interacts with the host nuclear transport proteins importin α/β1 and CRM1. Novel selective inhibitor of nuclear export (SINE) compounds, KPT-185, KPT-335 (verdinexor), and KPT-350, target the host's primary nuclear export protein, CRM1, in a manner similar to the archetypical inhibitor Leptomycin B. One major limitation of Leptomycin B is its irreversible binding to CRM1; which SINE compounds alleviate because they are slowly reversible. Chemically inhibiting CRM1 with these compounds enhanced capsid localization to the nucleus compared to the inactive compound KPT-301, as indicated by immunofluorescent confocal microscopy. Differences in extracellular versus intracellular viral RNA, as well as decreased capsid in cell free supernatants, indicated the inhibitors affected viral assembly, which led to a decrease in viral titers. The decrease in viral replication was confirmed using a luciferase-tagged virus and through plaque assays. SINE compounds had no effect on VEEV TC83_Cm, which encodes a mutated form of capsid that is unable to enter the nucleus. Serially passaging VEEV in the presence of KPT-185 resulted in mutations within the nuclear localization and nuclear export signals of capsid. Finally, SINE compound treatment also reduced the viral titers of the related eastern and western equine encephalitis viruses, suggesting that CRM1 maintains a common interaction with capsid proteins across the New World alphavirus genus.

Nipah virus sequesters inactive STAT1 in the nucleus via a P gene-encoded mechanism.

PubMed

Ciancanelli, Michael J; Volchkova, Valentina A; Shaw, Megan L; Volchkov, Viktor E; Basler, Christopher F

2009-08-01

The Nipah virus (NiV) phosphoprotein (P) gene encodes the C, P, V, and W proteins. P, V, and W, have in common an amino-terminal domain sufficient to bind STAT1, inhibiting its interferon (IFN)-induced tyrosine phosphorylation. P is also essential for RNA-dependent RNA polymerase function. C is encoded by an alternate open reading frame (ORF) within the common amino-terminal domain. Mutations within residues 81 to 113 of P impaired its polymerase cofactor function, as assessed by a minireplicon assay, but these mutants retained STAT1 inhibitory function. Mutations within the residue 114 to 140 region were identified that abrogated interaction with and inhibition of STAT1 by P, V, and W without disrupting P polymerase cofactor function. Recombinant NiVs were then generated. A G121E mutation, which abrogated inhibition of STAT1, was introduced into a C protein knockout background (C(ko)) because the mutation would otherwise also alter the overlapping C ORF. In cell culture, relative to the wild-type virus, the C(ko) mutation proved attenuating but the G121E mutant virus replicated identically to the C(ko) virus. In cells infected with the wild-type and C(ko) viruses, STAT1 was nuclear despite the absence of tyrosine phosphorylation. This latter observation mirrors what has been seen in cells expressing NiV W. In the G121E mutant virus-infected cells, STAT1 was not phosphorylated and was cytoplasmic in the absence of IFN stimulation but became tyrosine phosphorylated and nuclear following IFN addition. These data demonstrate that the gene for NiV P encodes functions that sequester inactive STAT1 in the nucleus, preventing its activation and suggest that the W protein is the dominant inhibitor of STAT1 in NiV-infected cells.

Plastid-targeting peptides from the chlorarachniophyte Bigelowiella natans.

PubMed

Rogers, Matthew B; Archibald, John M; Field, Matthew A; Li, Catherine; Striepen, Boris; Keeling, Patrick J

2004-01-01

Chlorarachniophytes are marine amoeboflagellate protists that have acquired their plastid (chloroplast) through secondary endosymbiosis with a green alga. Like other algae, most of the proteins necessary for plastid function are encoded in the nuclear genome of the secondary host. These proteins are targeted to the organelle using a bipartite leader sequence consisting of a signal peptide (allowing entry in to the endomembrane system) and a chloroplast transit peptide (for transport across the chloroplast envelope membranes). We have examined the leader sequences from 45 full-length predicted plastid-targeted proteins from the chlorarachniophyte Bigelowiella natans with the goal of understanding important features of these sequences and possible conserved motifs. The chemical characteristics of these sequences were compared with a set of 10 B. natans endomembrane-targeted proteins and 38 cytosolic or nuclear proteins, which show that the signal peptides are similar to those of most other eukaryotes, while the transit peptides differ from those of other algae in some characteristics. Consistent with this, the leader sequence from one B. natans protein was tested for function in the apicomplexan parasite, Toxoplasma gondii, and shown to direct the secretion of the protein.

GTF2IRD2 from the Williams–Beuren critical region encodes a mobile-element-derived fusion protein that antagonizes the action of its related family members

PubMed Central

Palmer, Stephen J.; Taylor, Kylie M.; Santucci, Nicole; Widagdo, Jocelyn; Chan, Yee-Ka Agnes; Yeo, Jen-Li; Adams, Merritt; Gunning, Peter W.; Hardeman, Edna C.

2012-01-01

Summary GTF2IRD2 belongs to a family of transcriptional regulators (including TFII-I and GTF2IRD1) that are responsible for many of the key features of Williams–Beuren syndrome (WBS). Sequence evidence suggests that GTF2IRD2 arose in eutherian mammals by duplication and divergence from the gene encoding TFII-I. However, in GTF2IRD2, most of the C-terminal domain has been lost and replaced by the domesticated remnant of an in-frame hAT-transposon mobile element. In this first experimental analysis of function, we show that transgenic expression of each of the three family members in skeletal muscle causes significant fiber type shifts, but the GTF2IRD2 protein causes an extreme shift in the opposite direction to the two other family members. Mating of GTF2IRD1 and GTF2IRD2 mice restores the fiber type balance, indicating an antagonistic relationship between these two paralogs. In cells, GTF2IRD2 localizes to cytoplasmic microtubules and discrete speckles in the nuclear periphery. We show that it can interact directly with TFII-Iβ and GTF2IRD1, and upon co-transfection changes the normal distribution of these two proteins into a punctate nuclear pattern typical of GTF2IRD2. These data suggest that GTF2IRD2 has evolved as a regulator of GTF2IRD1 and TFII-I; inhibiting their function by direct interaction and sequestration into inactive nuclear zones. PMID:22899722

The effect of ethidium bromide and chloramphenicol on mitochondrial biogenesis in primary human fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kao, Li-Pin; Ovchinnikov, Dmitry; Wolvetang, Ernst, E-mail: e.wolvetang@uq.edu.au

2012-05-15

The expression of mitochondrial components is controlled by an intricate interplay between nuclear transcription factors and retrograde signaling from mitochondria. The role of mitochondrial DNA (mtDNA) and mtDNA-encoded proteins in mitochondrial biogenesis is, however, poorly understood and thus far has mainly been studied in transformed cell lines. We treated primary human fibroblasts with ethidium bromide (EtBr) or chloramphenicol for six weeks to inhibit mtDNA replication or mitochondrial protein synthesis, respectively, and investigated how the cells recovered from these insults two weeks after removal of the drugs. Although cellular growth and mitochondrial gene expression were severely impaired after both inhibitor treatmentsmore » we observed marked differences in mitochondrial structure, membrane potential, glycolysis, gene expression, and redox status between fibroblasts treated with EtBr and chloramphenicol. Following removal of the drugs we further detected clear differences in expression of both mtDNA-encoded genes and nuclear transcription factors that control mitochondrial biogenesis, suggesting that the cells possess different compensatory mechanisms to recover from drug-induced mitochondrial dysfunction. Our data reveal new aspects of the interplay between mitochondrial retrograde signaling and the expression of nuclear regulators of mitochondrial biogenesis, a process with direct relevance to mitochondrial diseases and chloramphenicol toxicity in humans. -- Highlights: ► Cells respond to certain environmental toxins by increasing mitochondrial biogenesis. ► We investigated the effect of Chloramphenicol and EtBr in primary human fibroblasts. ► Inhibiting mitochondrial protein synthesis or DNA replication elicit different effects. ► We provide novel insights into the cellular responses toxins and antibiotics.« less

Extreme heterogeneity of polyadenylation sites in mRNAs encoding chloroplast RNA-binding proteins in Nicotiana plumbaginifolia.

PubMed

Klahre, U; Hemmings-Mieszczak, M; Filipowicz, W

1995-06-01

We have previously characterized nuclear cDNA clones encoding two RNA binding proteins, CP-RBP30 and CP-RBP-31, which are targeted to chloroplasts in Nicotiana plumbaginifolia. In this report we describe the analysis of the 3'-untranslated regions (3'-UTRs) in 22 CP-RBP30 and 8 CP-RBP31 clones which reveals that mRNAs encoding both proteins have a very complex polyadenylation pattern. Fourteen distinct poly(A) sites were identified among CP-RBP30 clones and four sites among the CP-RBP31 clones. The authenticity of the sites was confirmed by RNase A/T1 mapping of N. plumbaginifolia RNA. CP-RBP30 provides an extreme example of the heterogeneity known to be a feature of mRNA polyadenylation in higher plants. Using PCR we have demonstrated that CP-RBP genes in N. plumbaginifolia and N. sylvestris, in addition to the previously described introns interrupting the coding region, contain an intron located in the 3' non-coding part of the gene. In the case of the CP-RBP31, we have identified one polyadenylation event occurring in this intron.

[Expression and significance of respiratory chain enzyme of cells in urine sediment in MELAS syndrome].

PubMed

Wu, Hai-rong; Ma, Yi-nan; Qi, Yu; Liu, Hong-gang

2013-04-23

To explore the expression and significance of respiratory chain enzyme of cells in urine sediment in mitochondrial encephalopathy myopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome. Through enzyme histochemistry, the authors analyzed the changes of respiratory chain enzyme in urine sediment in 20 MELAS patients due to mitochondrial A3243G mutation (MELAS group) and 20 health peoples (control group). And the impact on the expression of protein encoded by nuclear DNA (A21347) and mitochondrial DNA (A6404) was detected by immunochemistry. Image pro Plus 6.0 software was used for analysis of absorbance (A) of staining images as staining intensity. The data were expressed as M (Q1, Q3) and analyzed through statistical software. The staining intensity of complexes Iin the MELAS group was lower than that in the control group (0.06(0.01, 0.12) vs 0.12(0.01, 0.62), P = 0.010). The intergroup staining intensity of complex II showed no marked difference. Increased density of blue particle and cytoplasmic gathering was found in 13 cased (65%) of the MELAS group under light microscope. The staining intensity of complexes IV was expressed at a low level in the MELAS group (0.14(0.03, 0.32) vs 0.23(0.06, 0.43), P = 0.038). The expression of protein encoded by nuclear DNA (A21347) was lower than that in the control group (0.05(0.02, 0.45) vs 0.17(0.03, 0.70), P = 0.000). The expression of protein encoded by mitochondrial DNA (A6404) was also lower than that in the control group (0.03(0.01, 0.07) vs 0.15 (0.09, 0.23), P = 0.000). Abnormal change of respiratory chain enzyme in urine sediment in MELAS due to mitochondrial A3243G mutation and a low expression of proteins encoded by two kinds of DNA in complexes IV can help to confirm the genetic diagnosis of mitochondrial encephalomyopathies so that different subtypes may be classified and its pathogenesis elucidated.

Structural Protein 4.1 in the Nucleus of Human Cells: Dynamic Rearrangements during Cell Division

PubMed Central

Krauss, Sharon Wald; Larabell, Carolyn A.; Lockett, Stephen; Gascard, Philippe; Penman, Sheldon; Mohandas, Narla; Chasis, Joel Anne

1997-01-01

Structural protein 4.1, first identified as a crucial 80-kD protein in the mature red cell membrane skeleton, is now known to be a diverse family of protein isoforms generated by complex alternative mRNA splicing, variable usage of translation initiation sites, and posttranslational modification. Protein 4.1 epitopes are detected at multiple intracellular sites in nucleated mammalian cells. We report here investigations of protein 4.1 in the nucleus. Reconstructions of optical sections of human diploid fibroblast nuclei using antibodies specific for 80-kD red cell 4.1 and for 4.1 peptides showed 4.1 immunofluorescent signals were intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes were detected in nuclear matrix both by immunofluorescence light microscopy and resinless section immunoelectron microscopy. Western blot analysis of fibroblast nuclear matrix protein fractions, isolated under identical extraction conditions as those for microscopy, revealed several polypeptide bands reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 was detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes were detected throughout the cell cycle but underwent dynamic spatial rearrangements during cell division. Protein 4.1 was observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function. PMID:9128242

Loss of the mitochondrial protein-only ribonuclease P complex causes aberrant tRNA processing and lethality in Drosophila.

PubMed

Sen, Aditya; Karasik, Agnes; Shanmuganathan, Aranganathan; Mirkovic, Elena; Koutmos, Markos; Cox, Rachel T

2016-07-27

Proteins encoded by mitochondrial DNA are translated using mitochondrially encoded tRNAs and rRNAs. As with nuclear encoded tRNAs, mitochondrial tRNAs must be processed to become fully functional. The mitochondrial form of ribonuclease P (mt:RNase P) is responsible for 5'-end maturation and is comprised of three proteins; mitochondrial RNase P protein (MRPP) 1 and 2 together with proteinaceous RNase P (PRORP). However, its mechanism and impact on development is not yet known. Using homology searches, we have identified the three proteins composing Drosophila mt:RNase P: Mulder (PRORP), Scully (MRPP2) and Roswell (MRPP1). Here, we show that each protein is essential and localizes with mitochondria. Furthermore, reducing levels of each causes mitochondrial deficits, which appear to be due at least in part to defective mitochondrial tRNA processing. Overexpressing two members of the complex, Mulder and Roswell, is also lethal, and in the case of Mulder, causes abnormal mitochondrial morphology. These data are the first evidence that defective mt:RNase P causes mitochondrial dysfunction, lethality and aberrant mitochondrial tRNA processing in vivo, underscoring its physiological importance. This in vivo mt:RNase P model will advance our understanding of how loss of mitochondrial tRNA processing causes tissue failure, an important aspect of human mitochondrial disease. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Nucleoporins as components of the nuclear pore complex core structure and Tpr as the architectural element of the nuclear basket.

PubMed

Krull, Sandra; Thyberg, Johan; Björkroth, Birgitta; Rackwitz, Hans-Richard; Cordes, Volker C

2004-09-01

The vertebrate nuclear pore complex (NPC) is a macromolecular assembly of protein subcomplexes forming a structure of eightfold radial symmetry. The NPC core consists of globular subunits sandwiched between two coaxial ring-like structures of which the ring facing the nuclear interior is capped by a fibrous structure called the nuclear basket. By postembedding immunoelectron microscopy, we have mapped the positions of several human NPC proteins relative to the NPC core and its associated basket, including Nup93, Nup96, Nup98, Nup107, Nup153, Nup205, and the coiled coil-dominated 267-kDa protein Tpr. To further assess their contributions to NPC and basket architecture, the genes encoding Nup93, Nup96, Nup107, and Nup205 were posttranscriptionally silenced by RNA interference (RNAi) in HeLa cells, complementing recent RNAi experiments on Nup153 and Tpr. We show that Nup96 and Nup107 are core elements of the NPC proper that are essential for NPC assembly and docking of Nup153 and Tpr to the NPC. Nup93 and Nup205 are other NPC core elements that are important for long-term maintenance of NPCs but initially dispensable for the anchoring of Nup153 and Tpr. Immunogold-labeling for Nup98 also results in preferential labeling of NPC core regions, whereas Nup153 is shown to bind via its amino-terminal domain to the nuclear coaxial ring linking the NPC core structures and Tpr. The position of Tpr in turn is shown to coincide with that of the nuclear basket, with different Tpr protein domains corresponding to distinct basket segments. We propose a model in which Tpr constitutes the central architectural element that forms the scaffold of the nuclear basket.

Venture from the Interior-Herpesvirus pUL31 Escorts Capsids from Nucleoplasmic Replication Compartments to Sites of Primary Envelopment at the Inner Nuclear Membrane.

PubMed

Bailer, Susanne M.

2017-11-25

Herpesviral capsid assembly is initiated in the nucleoplasm of the infected cell. Size constraints require that newly formed viral nucleocapsids leave the nucleus by an evolutionarily conserved vescular transport mechanism called nuclear egress. Mature capsids released from the nucleoplasm are engaged in a membrane-mediated budding process, composed of primary envelopment at the inner nuclear membrane and de-envelopment at the outer nuclear membrane. Once in the cytoplasm, the capsids receive their secondary envelope for maturation into infectious virions. Two viral proteins conserved throughout the herpesvirus family, the integral membrane protein pUL34 and the phosphoprotein pUL31, form the nuclear egress complex required for capsid transport from the infected nucleus to the cytoplasm. Formation of the nuclear egress complex results in budding of membrane vesicles revealing its function as minimal virus-encoded membrane budding and scission machinery. The recent structural analysis unraveled details of the heterodimeric nuclear egress complex and the hexagonal coat it forms at the inside of budding vesicles to drive primary envelopment. With this review, I would like to present the capsid-escort-model where pUL31 associates with capsids in nucleoplasmic replication compartments for escort to sites of primary envelopment thereby coupling capsid maturation and nuclear egress.

Predicting Drug-Target Interaction Networks Based on Functional Groups and Biological Features

PubMed Central

Shi, Xiao-He; Hu, Le-Le; Kong, Xiangyin; Cai, Yu-Dong; Chou, Kuo-Chen

2010-01-01

Background Study of drug-target interaction networks is an important topic for drug development. It is both time-consuming and costly to determine compound-protein interactions or potential drug-target interactions by experiments alone. As a complement, the in silico prediction methods can provide us with very useful information in a timely manner. Methods/Principal Findings To realize this, drug compounds are encoded with functional groups and proteins encoded by biological features including biochemical and physicochemical properties. The optimal feature selection procedures are adopted by means of the mRMR (Maximum Relevance Minimum Redundancy) method. Instead of classifying the proteins as a whole family, target proteins are divided into four groups: enzymes, ion channels, G-protein- coupled receptors and nuclear receptors. Thus, four independent predictors are established using the Nearest Neighbor algorithm as their operation engine, with each to predict the interactions between drugs and one of the four protein groups. As a result, the overall success rates by the jackknife cross-validation tests achieved with the four predictors are 85.48%, 80.78%, 78.49%, and 85.66%, respectively. Conclusion/Significance Our results indicate that the network prediction system thus established is quite promising and encouraging. PMID:20300175

CsSPL functions as an adaptor between HD-ZIP III and CsWUS transcription factors regulating anther and ovule development in cucumber

USDA-ARS?s Scientific Manuscript database

Anthers and ovules are the sex organs that produce male and female spores, respectively. The SPOROCYTELESS (SPL)/NOZZLE (NZZ) encodes a nuclear protein controlling the sporogenesis in sex organs. However, the function of SPL in unisexual plants has never been explored, and the upstream regulators of...

Reconstructing the phylogeny of Scolytinae and close allies: Major obstacles and prospects for a solution

Treesearch

Bjarte H. Jordal

2007-01-01

To enable the resolution of deep phylogenetic divergence in Scolytinae and closely related weevils, several new molecular markers were screened for their phylogenetic potential. The nuclear protein encoding genes, CAD and Arginine Kinase, were particularly promising and will be added to future phylogenetic studies in combination with 28S, COI, and Elongation Factor 1...

Transcriptomic evidence that longevity of acquired plastids in the photosynthetic slugs Elysia timida and Plakobranchus ocellatus does not entail lateral transfer of algal nuclear genes.

PubMed

Wägele, Heike; Deusch, Oliver; Händeler, Katharina; Martin, Rainer; Schmitt, Valerie; Christa, Gregor; Pinzger, Britta; Gould, Sven B; Dagan, Tal; Klussmann-Kolb, Annette; Martin, William

2011-01-01

Sacoglossan sea slugs are unique in the animal kingdom in that they sequester and maintain active plastids that they acquire from the siphonaceous algae upon which they feed, making the animals photosynthetic. Although most sacoglossan species digest their freshly ingested plastids within hours, four species from the family Plakobranchidae retain their stolen plastids (kleptoplasts) in a photosynthetically active state on timescales of weeks to months. The molecular basis of plastid maintenance within the cytosol of digestive gland cells in these photosynthetic metazoans is yet unknown but is widely thought to involve gene transfer from the algal food source to the slugs based upon previous investigations of single genes. Indeed, normal plastid development requires hundreds of nuclear-encoded proteins, with protein turnover in photosystem II in particular known to be rapid under various conditions. Moreover, only algal plastids, not the algal nuclei, are sequestered by the animals during feeding. If algal nuclear genes are transferred to the animal either during feeding or in the germ line, and if they are expressed, then they should be readily detectable with deep-sequencing methods. We have sequenced expressed mRNAs from actively photosynthesizing, starved individuals of two photosynthetic sea slug species, Plakobranchus ocellatus Van Hasselt, 1824 and Elysia timida Risso, 1818. We find that nuclear-encoded, algal-derived genes specific to photosynthetic function are expressed neither in P. ocellatus nor in E. timida. Despite their dramatic plastid longevity, these photosynthetic sacoglossan slugs do not express genes acquired from algal nuclei in order to maintain plastid function.

Specific binding of a HeLa cell nuclear protein to RNA sequences in the human immunodeficiency virus transactivating region.

PubMed Central

Gaynor, R; Soultanakis, E; Kuwabara, M; Garcia, J; Sigman, D S

1989-01-01

The transactivator protein, tat, encoded by the human immunodeficiency virus is a key regulator of viral transcription. Activation by the tat protein requires sequences downstream of the transcription initiation site called the transactivating region (TAR). RNA derived from the TAR is capable of forming a stable stem-loop structure and the maintenance of both the stem structure and the loop sequences located between +19 and +44 is required for complete in vivo activation by tat. Gel retardation assays with RNA from both wild-type and mutant TAR constructs generated in vitro with SP6 polymerase indicated specific binding of HeLa nuclear proteins to the TAR. To characterize this RNA-protein interaction, a method of chemical "imprinting" has been developed using photoactivated uranyl acetate as the nucleolytic agent. This reagent nicks RNA under physiological conditions at all four nucleotides in a reaction that is independent of sequence and secondary structure. Specific interaction of cellular proteins with TAR RNA could be detected by enhanced cleavages or imprints surrounding the loop region. Mutations that either disrupted stem base-pairing or extensively changed the primary sequence resulted in alterations in the cleavage pattern of the TAR RNA. Structural features of the TAR RNA stem-loop essential for tat activation are also required for specific binding of the HeLa cell nuclear protein. Images PMID:2544877

Identification of a subunit of NADH-dehydrogenase as a p49/STRAP-binding protein.

PubMed

Zhang, Xiaomin; Azhar, Gohar; Helms, Scott; Zhong, Ying; Wei, Jeanne Y

2008-01-29

The p49/STRAP (or SRFBP1) protein was recently identified in our laboratory as a cofactor of serum response factor that contributes to the regulation of SRF target genes in the heart. In the present study, we report that NDUFAB1, a nuclear encoded subunit of NADH dehydrogenase, represented the majority of the cDNA clones that interacted with p49/STRAP in multiple screenings using the yeast two-hybrid system. The p49/STRAP and NDUFAB1 proteins interacted and co-localized with each other in the cell. The p49/STRAP protein contains four classic nuclear localization sequence motifs, and it was observed to be present predominantly in the nucleus. Overexpression of p49/STRAP altered the intracellular level of NAD, and reduced the NAD/NADH ratio. Overexpression of p49/STRAP also induced the deacetylation of serum response factor. These data suggest that p49/STRAP plays a role in the regulation of intracellular processes such as cardiac cellular metabolism, gene expression, and possibly aging.

Orphan spin operators enable the acquisition of multiple 2D and 3D magic angle spinning solid-state NMR spectra

NASA Astrophysics Data System (ADS)

Gopinath, T.; Veglia, Gianluigi

2013-05-01

We propose a general method that enables the acquisition of multiple 2D and 3D solid-state NMR spectra for U-13C, 15N-labeled proteins. This method, called MEIOSIS (Multiple ExperIments via Orphan SpIn operatorS), makes it possible to detect four coherence transfer pathways simultaneously, utilizing orphan (i.e., neglected) spin operators of nuclear spin polarization generated during 15N-13C cross polarization (CP). In the MEIOSIS experiments, two phase-encoded free-induction decays are decoded into independent nuclear polarization pathways using Hadamard transformations. As a proof of principle, we show the acquisition of multiple 2D and 3D spectra of U-13C, 15N-labeled microcrystalline ubiquitin. Hadamard decoding of CP coherences into multiple independent spin operators is a new concept in solid-state NMR and is extendable to many other multidimensional experiments. The MEIOSIS method will increase the throughput of solid-state NMR techniques for microcrystalline proteins, membrane proteins, and protein fibrils.

Mutations in a signal sequence for the thylakoid membrane identify multiple protein transport pathways and nuclear suppressors

PubMed Central

1994-01-01

The apparatus that permits protein translocation across the internal thylakoid membranes of chloroplasts is completely unknown, even though these membranes have been the subject of extensive biochemical analysis. We have used a genetic approach to characterize the translocation of Chlamydomonas cytochrome f, a chloroplast-encoded protein that spans the thylakoid once. Mutations in the hydrophobic core of the cytochrome f signal sequence inhibit the accumulation of cytochrome f, lead to an accumulation of precursor, and impair the ability of Chlamydomonas cells to grow photosynthetically. One hydrophobic core mutant also reduces the accumulation of other thylakoid membrane proteins, but not those that translocate completely across the membrane. These results suggest that the signal sequence of cytochrome f is required and is involved in one of multiple insertion pathways. Suppressors of two signal peptide mutations describe at least two nuclear genes whose products likely describe the translocation apparatus, and selected second-site chloroplast suppressors further define regions of the cytochrome f signal peptide. PMID:8034740

Genome-wide screen uncovers novel pathways for tRNA processing and nuclear-cytoplasmic dynamics.

PubMed

Wu, Jingyan; Bao, Alicia; Chatterjee, Kunal; Wan, Yao; Hopper, Anita K

2015-12-15

Transfer ribonucleic acids (tRNAs) are essential for protein synthesis. However, key gene products involved in tRNA biogenesis and subcellular movement remain to be discovered. We conducted the first comprehensive unbiased analysis of the role of nearly an entire proteome in tRNA biology and describe 162 novel and 12 previously known Saccharomyces cerevisiae gene products that function in tRNA processing, turnover, and subcellular movement. tRNA nuclear export is of particular interest because it is essential, but the known tRNA exporters (Los1 [exportin-t] and Msn5 [exportin-5]) are unessential. We report that mutations of CRM1 (Exportin-1), MEX67/MTR2 (TAP/p15), and five nucleoporins cause accumulation of unspliced tRNA, a hallmark of defective tRNA nuclear export. CRM1 mutation genetically interacts with los1Δ and causes altered tRNA nuclear-cytoplasmic distribution. The data implicate roles for the protein and mRNA nuclear export machineries in tRNA nuclear export. Mutations of genes encoding actin cytoskeleton components and mitochondrial outer membrane proteins also cause accumulation of unspliced tRNA, likely due to defective splicing on mitochondria. Additional gene products, such as chromatin modification enzymes, have unanticipated effects on pre-tRNA end processing. Thus, this genome-wide screen uncovered putative novel pathways for tRNA nuclear export and extensive links between tRNA biology and other aspects of cell physiology. © 2015 Wu et al.; Published by Cold Spring Harbor Laboratory Press.

LL5beta: a regulator of postsynaptic differentiation identified in a screen for synaptically enriched transcripts at the neuromuscular junction.

PubMed

Kishi, Masashi; Kummer, Terrance T; Eglen, Stephen J; Sanes, Joshua R

2005-04-25

In both neurons and muscle fibers, specific mRNAs are concentrated beneath and locally translated at synaptic sites. At the skeletal neuromuscular junction, all synaptic RNAs identified to date encode synaptic components. Using microarrays, we compared RNAs in synapse-rich and -free regions of muscles, thereby identifying transcripts that are enriched near synapses and that encode soluble membrane and nuclear proteins. One gene product, LL5beta, binds to both phosphoinositides and a cytoskeletal protein, filamin, one form of which is concentrated at synaptic sites. LL5beta is itself associated with the cytoplasmic face of the postsynaptic membrane; its highest levels border regions of highest acetylcholine receptor (AChR) density, which suggests a role in "corraling" AChRs. Consistent with this idea, perturbing LL5beta expression in myotubes inhibits AChR aggregation. Thus, a strategy designed to identify novel synaptic components led to identification of a protein required for assembly of the postsynaptic apparatus.

Butyrate modulating effects on pro-inflammatory pathways in human intestinal epithelial cells.

PubMed

Elce, A; Amato, F; Zarrilli, F; Calignano, A; Troncone, R; Castaldo, G; Canani, R B

2017-10-13

Butyrate acts as energy source for intestinal epithelial cells and as key mediator of several immune processes, modulating gene expression mainly through histone deacetylation inhibition. Thanks to these effects, butyrate has been proposed for the treatment of many intestinal diseases. Aim of this study was to investigate the effect of butyrate on the expression of a large series of target genes encoding proteins involved in pro-inflammatory pathways. We performed quantitative real-time-PCR analysis of the expression of 86 genes encoding proteins bearing to pro-inflammatory pathways, before and after butyrate exposure, in primary epithelial cells derived from human small intestine and colon. Butyrate significantly down-regulated the expression of genes involved in inflammatory response, among which nuclear factor kappa beta, interferon-gamma, Toll like 2 receptor and tumour necrosis factor-alpha. Further confirmations of these data, including studies at protein level, would support the use of butyrate as effective therapeutic strategy in intestinal inflammatory disorders.

Peroxisome Proliferator-Activated Receptor γ Target Gene Encoding a Novel Angiopoietin-Related Protein Associated with Adipose Differentiation

PubMed Central

Yoon, J. Cliff; Chickering, Troy W.; Rosen, Evan D.; Dussault, Barry; Qin, Yubin; Soukas, Alexander; Friedman, Jeffrey M.; Holmes, William E.; Spiegelman, Bruce M.

2000-01-01

The nuclear receptor peroxisome proliferator-activated receptor γ regulates adipose differentiation and systemic insulin signaling via ligand-dependent transcriptional activation of target genes. However, the identities of the biologically relevant target genes are largely unknown. Here we describe the isolation and characterization of a novel target gene induced by PPARγ ligands, termed PGAR (for PPARγ angiopoietin related), which encodes a novel member of the angiopoietin family of secreted proteins. The transcriptional induction of PGAR follows a rapid time course typical of immediate-early genes and occurs in the absence of protein synthesis. The expression of PGAR is predominantly localized to adipose tissues and placenta and is consistently elevated in genetic models of obesity. Hormone-dependent adipocyte differentiation coincides with a dramatic early induction of the PGAR transcript. Alterations in nutrition and leptin administration are found to modulate the PGAR expression in vivo. Taken together, these data suggest a possible role for PGAR in the regulation of systemic lipid metabolism or glucose homeostasis. PMID:10866690

Two host cytoplasmic effectors are required for pathogenesis of Phytophthora sojae by suppression of host defenses.

PubMed

Liu, Tingli; Ye, Wenwu; Ru, Yanyan; Yang, Xinyu; Gu, Biao; Tao, Kai; Lu, Shan; Dong, Suomeng; Zheng, Xiaobo; Shan, Weixing; Wang, Yuanchao; Dou, Daolong

2011-01-01

Phytophthora sojae encodes hundreds of putative host cytoplasmic effectors with conserved FLAK motifs following signal peptides, termed crinkling- and necrosis-inducing proteins (CRN) or Crinkler. Their functions and mechanisms in pathogenesis are mostly unknown. Here, we identify a group of five P. sojae-specific CRN-like genes with high levels of sequence similarity, of which three are putative pseudogenes. Functional analysis shows that the two functional genes encode proteins with predicted nuclear localization signals that induce contrasting responses when expressed in Nicotiana benthamiana and soybean (Glycine max). PsCRN63 induces cell death, while PsCRN115 suppresses cell death elicited by the P. sojae necrosis-inducing protein (PsojNIP) or PsCRN63. Expression of CRN fragments with deleted signal peptides and FLAK motifs demonstrates that the carboxyl-terminal portions of PsCRN63 or PsCRN115 are sufficient for their activities. However, the predicted nuclear localization signal is required for PsCRN63 to induce cell death but not for PsCRN115 to suppress cell death. Furthermore, silencing of the PsCRN63 and PsCRN115 genes in P. sojae stable transformants leads to a reduction of virulence on soybean. Intriguingly, the silenced transformants lose the ability to suppress host cell death and callose deposition on inoculated plants. These results suggest a role for CRN effectors in the suppression of host defense responses.

The relationships between the isoelectric point and: length of proteins, taxonomy and ecology of organisms

PubMed Central

Kiraga, Joanna; Mackiewicz, Pawel; Mackiewicz, Dorota; Kowalczuk, Maria; Biecek, Przemysław; Polak, Natalia; Smolarczyk, Kamila; Dudek, Miroslaw R; Cebrat, Stanislaw

2007-01-01

Background The distribution of isoelectric point (pI) of proteins in a proteome is universal for all organisms. It is bimodal dividing the proteome into two sets of acidic and basic proteins. Different species however have different abundance of acidic and basic proteins that may be correlated with taxonomy, subcellular localization, ecological niche of organisms and proteome size. Results We have analysed 1784 proteomes encoded by chromosomes of Archaea, Bacteria, Eukaryota, and also mitochondria, plastids, prokaryotic plasmids, phages and viruses. We have found significant correlation in more than 95% of proteomes between the protein length and pI in proteomes – positive for acidic proteins and negative for the basic ones. Plastids, viruses and plasmids encode more basic proteomes while chromosomes of Archaea, Bacteria, Eukaryota, mitochondria and phages more acidic ones. Mitochondrial proteomes of Viridiplantae, Protista and Fungi are more basic than Metazoa. It results from the presence of basic proteins in the former proteomes and their absence from the latter ones and is related with reduction of metazoan genomes. Significant correlation was found between the pI bias of proteomes encoded by prokaryotic chromosomes and proteomes encoded by plasmids but there is no correlation between eukaryotic nuclear-coded proteomes and proteomes encoded by organelles. Detailed analyses of prokaryotic proteomes showed significant relationships between pI distribution and habitat, relation to the host cell and salinity of the environment, but no significant correlation with oxygen and temperature requirements. The salinity is positively correlated with acidicity of proteomes. Host-associated organisms and especially intracellular species have more basic proteomes than free-living ones. The higher rate of mutations accumulation in the intracellular parasites and endosymbionts is responsible for the basicity of their tiny proteomes that explains the observed positive correlation between the decrease of genome size and the increase of basicity of proteomes. The results indicate that even conserved proteins subjected to strong selectional constraints follow the global trend in the pI distribution. Conclusion The distribution of pI of proteins in proteomes shows clear relationships with length of proteins, subcellular localization, taxonomy and ecology of organisms. The distribution is also strongly affected by mutational pressure especially in intracellular organisms. PMID:17565672

Immunogenicity of nuclear-encoded LTB:ST fusion protein from Escherichia coli expressed in tobacco plants.

PubMed

Rosales-Mendoza, Sergio; Soria-Guerra, Ruth E; Moreno-Fierros, Leticia; Govea-Alonso, Dania O; Herrera-Díaz, Areli; Korban, Schuyler S; Alpuche-Solís, Ángel G

2011-06-01

Enterotoxigenic Escherichia coli (ETEC) is one of the main causative agents of diarrhea in infants and for travelers. Inclusion of a heat-stable (ST) toxin into vaccine formulations is mandatory as most ETEC strains can produce both heat-labile (LT) and ST enterotoxins. In this study, a genetic fusion gene encoding for an LTB:ST protein has been constructed and transferred into tobacco via Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco plants carrying the LTB:ST gene are then subjected to GM1-ELISA revealing that the LTB:ST has assembled into pentamers and displays antigenic determinants from both LTB and ST. Protein accumulation of up to 0.05% total soluble protein is detected. Subsequently, mucosal and systemic humoral responses are elicited in mice orally dosed with transgenic tobacco leaves. This has suggested that the plant-derived LTB:ST is immunogenic via the oral route. These findings are critical for the development of a plant-based vaccine capable of eliciting broader protection against ETEC and targeting both LTB and ST. Features of this platform in comparison to transplastomic approaches are discussed.

Nup93, a Vertebrate Homologue of Yeast Nic96p, Forms a Complex with a Novel 205-kDa Protein and Is Required for Correct Nuclear Pore Assembly

PubMed Central

Grandi, Paola; Dang, Tam; Pané, Nelly; Shevchenko, Andrej; Mann, Matthias; Forbes, Douglass; Hurt, Ed

1997-01-01

Yeast and vertebrate nuclear pores display significant morphological similarity by electron microscopy, but sequence similarity between the respective proteins has been more difficult to observe. Herein we have identified a vertebrate nucleoporin, Nup93, in both human and Xenopus that has proved to be an evolutionarily related homologue of the yeast nucleoporin Nic96p. Polyclonal antiserum to human Nup93 detects corresponding proteins in human, rat, and Xenopus cells. Immunofluorescence and immunoelectron microscopy localize vertebrate Nup93 at the nuclear basket and at or near the nuclear entry to the gated channel of the pore. Immunoprecipitation from both mammalian and Xenopus cell extracts indicates that a small fraction of Nup93 physically interacts with the nucleoporin p62, just as yeast Nic96p interacts with the yeast p62 homologue. However, a large fraction of vertebrate Nup93 is extracted from pores and is also present in Xenopus egg extracts in complex with a newly discovered 205-kDa protein. Mass spectrometric sequencing of the human 205-kDa protein reveals that this protein is encoded by an open reading frame, KIAAO225, present in the human database. The putative human nucleoporin of 205 kDa has related sequence homologues in Caenorhabditis elegans and Saccharomyces cerevisiae. To analyze the role of the Nup93 complex in the pore, nuclei were assembled that lack the Nup93 complex after immunodepletion of a Xenopus nuclear reconstitution extract. The Nup93-complex–depleted nuclei are clearly defective for correct nuclear pore assembly. From these experiments, we conclude that the vertebrate and yeast pore have significant homology in their functionally important cores and that, with the identification of Nup93 and the 205-kDa protein, we have extended the knowledge of the nearest-neighbor interactions of this core in both yeast and vertebrates. PMID:9348540

Rye B chromosomes encode a functional Argonaute-like protein with in vitro slicer activities similar to its A chromosome paralog.

PubMed

Ma, Wei; Gabriel, Tobias Sebastian; Martis, Mihaela Maria; Gursinsky, Torsten; Schubert, Veit; Vrána, Jan; Doležel, Jaroslav; Grundlach, Heidrun; Altschmied, Lothar; Scholz, Uwe; Himmelbach, Axel; Behrens, Sven-Erik; Banaei-Moghaddam, Ali Mohammad; Houben, Andreas

2017-01-01

B chromosomes (Bs) are supernumerary, dispensable parts of the nuclear genome, which appear in many different species of eukaryote. So far, Bs have been considered to be genetically inert elements without any functional genes. Our comparative transcriptome analysis and the detection of active RNA polymerase II (RNAPII) in the proximity of B chromatin demonstrate that the Bs of rye (Secale cereale) contribute to the transcriptome. In total, 1954 and 1218 B-derived transcripts with an open reading frame were expressed in generative and vegetative tissues, respectively. In addition to B-derived transposable element transcripts, a high percentage of short transcripts without detectable similarity to known proteins and gene fragments from A chromosomes (As) were found, suggesting an ongoing gene erosion process. In vitro analysis of the A- and B-encoded AGO4B protein variants demonstrated that both possess RNA slicer activity. These data demonstrate unambiguously the presence of a functional AGO4B gene on Bs and that these Bs carry both functional protein coding genes and pseudogene copies. Thus, B-encoded genes may provide an additional level of gene control and complexity in combination with their related A-located genes. Hence, physiological effects, associated with the presence of Bs, may partly be explained by the activity of B-located (pseudo)genes. © 2016 IPK Gatersleben. New Phytologist © 2016 New Phytologist Trust.

Correlation of rare coding variants in the gene encoding human glucokinase regulatory protein with phenotypic, cellular, and kinetic outcomes.

PubMed

Rees, Matthew G; Ng, David; Ruppert, Sarah; Turner, Clesson; Beer, Nicola L; Swift, Amy J; Morken, Mario A; Below, Jennifer E; Blech, Ilana; Mullikin, James C; McCarthy, Mark I; Biesecker, Leslie G; Gloyn, Anna L; Collins, Francis S

2012-01-01

Defining the genetic contribution of rare variants to common diseases is a major basic and clinical science challenge that could offer new insights into disease etiology and provide potential for directed gene- and pathway-based prevention and treatment. Common and rare nonsynonymous variants in the GCKR gene are associated with alterations in metabolic traits, most notably serum triglyceride levels. GCKR encodes glucokinase regulatory protein (GKRP), a predominantly nuclear protein that inhibits hepatic glucokinase (GCK) and plays a critical role in glucose homeostasis. The mode of action of rare GCKR variants remains unexplored. We identified 19 nonsynonymous GCKR variants among 800 individuals from the ClinSeq medical sequencing project. Excluding the previously described common missense variant p.Pro446Leu, all variants were rare in the cohort. Accordingly, we functionally characterized all variants to evaluate their potential phenotypic effects. Defects were observed for the majority of the rare variants after assessment of cellular localization, ability to interact with GCK, and kinetic activity of the encoded proteins. Comparing the individuals with functional rare variants to those without such variants showed associations with lipid phenotypes. Our findings suggest that, while nonsynonymous GCKR variants, excluding p.Pro446Leu, are rare in individuals of mixed European descent, the majority do affect protein function. In sum, this study utilizes computational, cell biological, and biochemical methods to present a model for interpreting the clinical significance of rare genetic variants in common disease.

Small-molecule control of protein function through Staudinger reduction

NASA Astrophysics Data System (ADS)

Luo, Ji; Liu, Qingyang; Morihiro, Kunihiko; Deiters, Alexander

2016-11-01

Using small molecules to control the function of proteins in live cells with complete specificity is highly desirable, but challenging. Here we report a small-molecule switch that can be used to control protein activity. The approach uses a phosphine-mediated Staudinger reduction to activate protein function. Genetic encoding of an ortho-azidobenzyloxycarbonyl amino acid using a pyrrolysyl transfer RNA synthetase/tRNACUA pair in mammalian cells enables the site-specific introduction of a small-molecule-removable protecting group into the protein of interest. Strategic placement of this group renders the protein inactive until deprotection through a bioorthogonal Staudinger reduction delivers the active wild-type protein. This developed methodology was applied to the conditional control of several cellular processes, including bioluminescence (luciferase), fluorescence (enhanced green fluorescent protein), protein translocation (nuclear localization sequence), DNA recombination (Cre) and gene editing (Cas9).

The early UL31 gene of equine herpesvirus 1 encodes a single-stranded DNA-binding protein that has a nuclear localization signal sequence at the C-terminus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Seongman; Chul Ahn, Byung; O'Callaghan, Dennis J.

2012-10-25

The amino acid sequence of the UL31 protein (UL31P) of equine herpesvirus 1 (EHV-1) has homology to that of the ICP8 of herpes simplex virus type 1 (HSV-1). Here we show that the UL31 gene is synergistically trans-activated by the IEP and the UL5P (EICP27). Detection of the UL31 RNA transcript and the UL31P in EHV-1-infected cells at 6 h post-infection (hpi) as well as metabolic inhibition assays indicated that UL31 is an early gene. The UL31P preferentially bound to single-stranded DNA over double-stranded DNA in gel shift assays. Subcellular localization of the green fluorescent protein (GFP)-UL31 fusion proteins revealedmore » that the C-terminal 32 amino acid residues of the UL31P are responsible for the nuclear localization. These findings may contribute to defining the role of the UL31P single-stranded DNA-binding protein in EHV-1 DNA replication.« less

Suppression of HTLV-1 replication by Tax-mediated rerouting of the p13 viral protein to nuclear speckles

PubMed Central

Andresen, Vibeke; Pise-Masison, Cynthia A.; Sinha-Datta, Uma; Bellon, Marcia; Valeri, Valerio; Washington Parks, Robyn; Cecchinato, Valentina; Fukumoto, Risaku; Nicot, Christophe

2011-01-01

Disease development in human T-cell leukemia virus type 1 (HTLV-1)–infected individuals is positively correlated with the level of integrated viral DNA in T cells. HTLV-1 replication is positively regulated by Tax and Rex and negatively regulated by the p30 and HBZ proteins. In the present study, we demonstrate that HTLV-1 encodes another negative regulator of virus expression, the p13 protein. Expressed separately, p13 localizes to the mitochondria, whereas in the presence of Tax, part of it is ubiquitinated, stabilized, and rerouted to the nuclear speckles. The p13 protein directly binds Tax, decreases Tax binding to the CBP/p300 transcriptional coactivator, and, by reducing Tax transcriptional activity, suppresses viral expression. Because Tax stabilizes its own repressor, these findings suggest that HTLV-1 has evolved a complex mechanism to control its own replication. Further, these results highlight the importance of studying the function of the HTLV-1 viral proteins, not only in isolation, but also in the context of full viral replication. PMID:21677314

How protein targeting to primary plastids via the endomembrane system could have evolved? A new hypothesis based on phylogenetic studies.

PubMed

Gagat, Przemysław; Bodył, Andrzej; Mackiewicz, Paweł

2013-07-11

It is commonly assumed that a heterotrophic ancestor of the supergroup Archaeplastida/Plantae engulfed a cyanobacterium that was transformed into a primary plastid; however, it is still unclear how nuclear-encoded proteins initially were imported into the new organelle. Most proteins targeted to primary plastids carry a transit peptide and are transported post-translationally using Toc and Tic translocons. There are, however, several proteins with N-terminal signal peptides that are directed to higher plant plastids in vesicles derived from the endomembrane system (ES). The existence of these proteins inspired a hypothesis that all nuclear-encoded, plastid-targeted proteins initially carried signal peptides and were targeted to the ancestral primary plastid via the host ES. We present the first phylogenetic analyses of Arabidopsis thaliana α-carbonic anhydrase (CAH1), Oryza sativa nucleotide pyrophosphatase/phosphodiesterase (NPP1), and two O. sativa α-amylases (αAmy3, αAmy7), proteins that are directed to higher plant primary plastids via the ES. We also investigated protein disulfide isomerase (RB60) from the green alga Chlamydomonas reinhardtii because of its peculiar dual post- and co-translational targeting to both the plastid and ES. Our analyses show that these proteins all are of eukaryotic rather than cyanobacterial origin, and that their non-plastid homologs are equipped with signal peptides responsible for co-translational import into the host ES. Our results indicate that vesicular trafficking of proteins to primary plastids evolved long after the cyanobacterial endosymbiosis (possibly only in higher plants) to permit their glycosylation and/or transport to more than one cellular compartment. The proteins we analyzed are not relics of ES-mediated protein targeting to the ancestral primary plastid. Available data indicate that Toc- and Tic-based translocation dominated protein import into primary plastids from the beginning. Only a handful of host proteins, which already were targeted through the ES, later were adapted to reach the plastid via the vesicular trafficking. They represent a derived class of higher plant plastid-targeted proteins with an unusual evolutionary history.

Impact of light intensity and quality on chromatophore and nuclear gene expression in Paulinella chromatophora, an amoeba with nascent photosynthetic organelles.

PubMed

Zhang, Ru; Nowack, Eva C M; Price, Dana C; Bhattacharya, Debashish; Grossman, Arthur R

2017-04-01

Plastid evolution has been attributed to a single primary endosymbiotic event that occurred about 1.6 billion years ago (BYA) in which a cyanobacterium was engulfed and retained by a eukaryotic cell, although early steps in plastid integration are poorly understood. The photosynthetic amoeba Paulinella chromatophora represents a unique model for the study of plastid evolution because it contains cyanobacterium-derived photosynthetic organelles termed 'chromatophores' that originated relatively recently (0.09-0.14 BYA). The chromatophore genome is about a third the size of the genome of closely related cyanobacteria, but 10-fold larger than most plastid genomes. Several genes have been transferred from the chromatophore genome to the host nuclear genome through endosymbiotic gene transfer (EGT). Some EGT-derived proteins could be imported into chromatophores for function. Two photosynthesis-related genes (psaI and csos4A) are encoded by both the nuclear and chromatophore genomes, suggesting that EGT in Paulinella chromatophora is ongoing. Many EGT-derived genes encode proteins that function in photosynthesis and photoprotection, including an expanded family of high-light-inducible (ncHLI) proteins. Cyanobacterial hli genes are high-light induced and required for cell viability under excess light. We examined the impact of light on Paulinella chromatophora and found that this organism is light sensitive and lacks light-induced transcriptional regulation of chromatophore genes and most EGT-derived nuclear genes. However, several ncHLI genes have reestablished light-dependent regulation, which appears analogous to what is observed in cyanobacteria. We postulate that expansion of the ncHLI gene family and its regulation may reflect the light/oxidative stress experienced by Paulinella chromatophora as a consequence of the as yet incomplete integration of host and chromatophore metabolisms. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Plant rhabdoviruses.

PubMed

Redinbaugh, M G; Hogenhout, S A

2005-01-01

This chapter provides an overview of plant rhabdovirus structure and taxonomy, genome structure, protein function, and insect and plant infection. It is focused on recent research and unique aspects of rhabdovirus biology. Plant rhabdoviruses are transmitted by aphid, leafhopper or planthopper vectors, and the viruses replicate in both their insect and plant hosts. The two plant rhabdovirus genera, Nucleorhabdovirus and Cytorhabdovirus, can be distinguished on the basis of their intracellular site of morphogenesis in plant cells. All plant rhabdoviruses carry analogs of the five core genes: the nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G) and large or polymerase (L). However, compared to vesiculoviruses that are composed of the five core genes, all plant rhabdoviruses encode more than these five genes, at least one of which is inserted between the P and M genes in the rhabdoviral genome. Interestingly, while these extra genes are not similar among plant rhabdoviruses, two encode proteins with similarity to the 30K superfamily of plant virus movement proteins. Analysis of nucleorhabdoviral protein sequences revealed nuclear localization signals for the N, P, M and L proteins, consistent with virus replication and morphogenesis of these viruses in the nucleus. Plant and insect factors that limit virus infection and transmission are discussed.

Exaptation of Bornavirus-Like Nucleoprotein Elements in Afrotherians

PubMed Central

Kobayashi, Yuki; Horie, Masayuki; Nakano, Ayumi; Murata, Koichi; Itou, Takuya; Suzuki, Yoshiyuki

2016-01-01

Endogenous bornavirus-like nucleoprotein elements (EBLNs), the nucleotide sequence elements derived from the nucleoprotein gene of ancient bornavirus-like viruses, have been identified in many animal genomes. Here we show evidence that EBLNs encode functional proteins in their host. Some afrotherian EBLNs were observed to have been maintained for more than 83.3 million years under negative selection. Splice variants were expressed from the genomic loci of EBLNs in elephant, and some were translated into proteins. The EBLN proteins appeared to be localized to the rough endoplasmic reticulum in African elephant cells, in contrast to the nuclear localization of bornavirus N. These observations suggest that afrotherian EBLNs have acquired a novel function in their host. Interestingly, genomic sequences of the first exon and its flanking regions in these EBLN loci were homologous to those of transmembrane protein 106B (TMEM106B). The upstream region of the first exon in the EBLN loci exhibited a promoter activity, suggesting that the ability of these EBLNs to be transcribed in the host cell was gained through capturing a partial duplicate of TMEM106B. In conclusion, our results strongly support for exaptation of EBLNs to encode host proteins in afrotherians. PMID:27518265

High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase.

PubMed

Kim, Sung Bae; Ozawa, Takeaki; Watanabe, Shigeaki; Umezawa, Yoshio

2004-08-10

Nucleocytoplasmic trafficking of functional proteins plays a key role in regulating gene expressions in response to extracellular signals. We developed a genetically encoded bioluminescent indicator for monitoring the nuclear trafficking of target proteins in vitro and in vivo. The principle is based on reconstitution of split fragments of Renilla reniformis (Rluc) by protein splicing with a DnaE intein (a catalytic subunit of DNA polymerase III). A target cytosolic protein fused to the N-terminal half of Rluc is expressed in mammalian cells. If the protein translocates into the nucleus, the Rluc moiety meets the C-terminal half of Rluc, and full-length Rluc is reconstituted by protein splicing. We demonstrated quantitative cell-based in vitro sensing of ligand-induced translocation of androgen receptor, which allowed high-throughput screening of exo- and endogenous agonists and antagonists. Furthermore, the indicator enabled noninvasive in vivo imaging of the androgen receptor translocation in the brains of living mice with a charge-coupled device imaging system. These rapid and quantitative analyses in vitro and in vivo provide a wide variety of applications for screening pharmacological or toxicological compounds and testing them in living animals.

The amino-terminal region of the retinoblastoma gene product binds a novel nuclear matrix protein that co-localizes to centers for RNA processing

PubMed Central

1994-01-01

The tumor suppressing capacity of the retinoblastoma protein (p110RB) is dependent on interactions made with cellular proteins through its carboxy-terminal domains. How the p110RB amino-terminal region contributes to this activity is unclear, though evidence now indicates it is important for both growth suppression and regulation of the full- length protein. We have used the yeast two-hybrid system to screen for cellular proteins which bind to the first 300 amino acids of p110RB. The only gene isolated from this screen encodes a novel 84-kD nuclear matrix protein that localizes to subnuclear regions associated with RNA processing. This protein, p84, requires a structurally defined domain in the amino terminus of p110RB for binding. Furthermore, both in vivo and in vitro experiments demonstrate that p84 binds preferentially to the functionally active, hypophosphorylated form of p110RB. Thus, the amino terminus of p110RB may function in part to facilitate the binding of growth promoting factors at subnuclear regions actively involved in RNA metabolism. PMID:7525595

The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

PubMed

Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

2016-05-17

Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Mechanism of protein biosynthesis in mammalian mitochondria.

PubMed

Christian, Brooke E; Spremulli, Linda L

2012-01-01

Protein synthesis in mammalian mitochondria produces 13 proteins that are essential subunits of the oxidative phosphorylation complexes. This review provides a detailed outline of each phase of mitochondrial translation including initiation, elongation, termination, and ribosome recycling. The roles of essential proteins involved in each phase are described. All of the products of mitochondrial protein synthesis in mammals are inserted into the inner membrane. Several proteins that may help bind ribosomes to the membrane during translation are described, although much remains to be learned about this process. Mutations in mitochondrial or nuclear genes encoding components of the translation system often lead to severe deficiencies in oxidative phosphorylation, and a summary of these mutations is provided. This article is part of a Special Issue entitled: Mitochondrial Gene Expression. Copyright © 2011 Elsevier B.V. All rights reserved.

Early stages in the biogenesis of eukaryotic β-barrel proteins.

PubMed

Jores, Tobias; Rapaport, Doron

2017-09-01

The endosymbiotic organelles mitochondria and chloroplasts harbour, similarly to their prokaryotic progenitors, β-barrel proteins in their outer membrane. These proteins are encoded on nuclear DNA, translated on cytosolic ribosomes and imported into their target organelles by a dedicated machinery. Recent studies have provided insights into the import into the organelles and the membrane insertion of these proteins. Although the cytosolic stages of their biogenesis are less well defined, it is speculated that upon their synthesis, chaperones prevent β-barrel proteins from aggregation and keep them in an import-competent conformation. In this Review, we summarize the current knowledge about the biogenesis of β-barrel proteins, focusing on the early stages from the translation on cytosolic ribosomes to the recognition on the surface of the organelle. © 2017 Federation of European Biochemical Societies.

The C3H-type zinc finger protein GDS1/C3H42 is a nuclear-speckle-localized protein that is essential for normal growth and development in Arabidopsis.

PubMed

Kim, Dae Won; Jeon, Su Jeong; Hwang, Sung Min; Hong, Jong Chan; Bahk, Jeong Dong

2016-09-01

Eukaryotic C3H-type zinc finger proteins (Znfs) comprise a large family of regulatory proteins involved in many aspects of plant stress response, growth and development. However, compared to mammalian, only a few plant Znfs have been functionally characterized. Here, T-DNA inserted gds1 (growth, development and splicing 1) mutant, displayed abnormal growth throughout the lifecycle owing to the reduction of cell size and number. Inverse PCR analysis revealed that the abnormal growth was caused by the disruption of At3g47120, which encodes a C3H42 protein belonging to the C-X7-C-X5-C-X3-H class of the Znf family. GDS1 was ubiquitously transcribed, but shows high levels of expression in young seedling and unexpanded new leaves. In gds1, the transcripts of many growth- and development-related genes were down-regulated, and the auxin response was dramatically reduced. A fluorescence-based assay revealed that the GDS1 protein was localized to the nucleus, prominently in the speckle compartments. Its arginine/serine dipeptide-rich-like (RS-like) domain was essential for nuclear localization. In addition, the SR1, SRm102 and U1-70K components of the U1 spliceosome interacted with GDS1 in the nuclear speckle compartments. Taken together, these suggest that GDS1, a nuclear-speckle-associated Znf, might play a significant role in splicing during plant growth and development. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Cloning of the cDNA for U1 small nuclear ribonucleoprotein particle 70K protein from Arabidopsis thaliana

NASA Technical Reports Server (NTRS)

Reddy, A. S.; Czernik, A. J.; An, G.; Poovaiah, B. W.

1992-01-01

We cloned and sequenced a plant cDNA that encodes U1 small nuclear ribonucleoprotein (snRNP) 70K protein. The plant U1 snRNP 70K protein cDNA is not full length and lacks the coding region for 68 amino acids in the amino-terminal region as compared to human U1 snRNP 70K protein. Comparison of the deduced amino acid sequence of the plant U1 snRNP 70K protein with the amino acid sequence of animal and yeast U1 snRNP 70K protein showed a high degree of homology. The plant U1 snRNP 70K protein is more closely related to the human counter part than to the yeast 70K protein. The carboxy-terminal half is less well conserved but, like the vertebrate 70K proteins, is rich in charged amino acids. Northern analysis with the RNA isolated from different parts of the plant indicates that the snRNP 70K gene is expressed in all of the parts tested. Southern blotting of genomic DNA using the cDNA indicates that the U1 snRNP 70K protein is coded by a single gene.

Abnormal retention of nuclear lamina and disorganization of chromatin-related proteins in spermatozoa from DPY19L2-deleted globozoospermic patients.

PubMed

Paci, Marine; Elkhatib, Razan; Longepied, Guy; Hennebicq, Sylviane; Bessonat, Julien; Courbière, Blandine; Bourgeois, Patrice; Levy, Nicolas; Mitchell, Michael J; Metzler-Guillemain, Catherine

2017-11-01

The aim of this study was to characterize the nuclear lamina (NL) and lamin chromatin-partners in spermatozoa from four DPY19L2-deleted globozoospermic patients. We tested for spermatid transcripts encoding lamins and their chromatin-partners emerin, LAP2α, BAF and BAF-L, by reverse transcriptase-PCR using spermatozoa RNA. We also determined the localization of lamin B1, BAF and BAF-L by immunofluorescent analysis of spermatozoa from all patients. In RNA from globozoospermic and control spermatozoa we detected transcripts encoding lamin B1, lamin B3, emerin, LAP2α and BAF-L, but not A-type lamins. In contrast, BAF transcripts were detected in globozoospermic but not control spermatozoa. The NL was immature in human globozoospermic spermatozoa: lamin B1 signal was detected in the nuclei of globozoospermic spermatozoa in significantly higher proportions than the control (P < 0.05; 56-91% versus 40%) and was predominantly observed at the whole nuclear periphery, not polarized as in control spermatozoa. Conversely, BAF and BAF-L were detected in control, but not globozoospermic spermatozoa. Our results strongly emphasize the importance of the NL and associated proteins during human spermiogenesis. In globozoospermia, the lack of maturation of the NL, and the modifications in expression and location of chromatin-partners, could explain the chromatin defects observed in this rare phenotype. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Division of Labor Among the Yeast Sol Proteins Implicated in tRNA Nuclear Export and Carbohydrate Metabolism

PubMed Central

Stanford, D. R.; Whitney, M. L.; Hurto, R. L.; Eisaman, D. M.; Shen, W.-C.; Hopper, A. K.

2004-01-01

SOL1, the founding member of the S. cerevisiae SOL family, was previously identified as a multi-copy suppressor of the los1 defect in tRNA-mediated nonsense suppression. Here we report that the four-member SOL family is not essential and that individual family members appear to have distinct functions. SOL1–SOL4 are homologous to genes encoding 6-phosphogluconolactonase (6Pgl) involved in the pentose phosphate pathway. Both Sol3p and Sol4p affect this activity. However, Sol4p does not act as a los1 multi-copy suppressor. In contrast, neither Sol1p nor Sol2p, both of which correct the los1 defect in nonsense suppression, possess detectable 6Pgl activity. Rather, Sol1p and Sol2p appear to function in tRNA nuclear export as sol1 and sol2 mutants possess elevated levels of nuclear tRNA. Members of the Sol protein family appear to have different subcellular distributions. Thus, Sol3p and Sol4p likely function in carbohydrate metabolism, while Sol1p and Sol2p appear to have roles in tRNA function and nuclear export, thereby defining an unusual protein family whose individual members are biochemically distinct and spatially dispersed. PMID:15454531

Division of labor among the yeast Sol proteins implicated in tRNA nuclear export and carbohydrate metabolism.

PubMed

Stanford, D R; Whitney, M L; Hurto, R L; Eisaman, D M; Shen, W-C; Hopper, A K

2004-09-01

SOL1, the founding member of the S. cerevisiae SOL family, was previously identified as a multi-copy suppressor of the los1 defect in tRNA-mediated nonsense suppression. Here we report that the four-member SOL family is not essential and that individual family members appear to have distinct functions. SOL1-SOL4 are homologous to genes encoding 6-phosphogluconolactonase (6Pgl) involved in the pentose phosphate pathway. Both Sol3p and Sol4p affect this activity. However, Sol4p does not act as a los1 multi-copy suppressor. In contrast, neither Sol1p nor Sol2p, both of which correct the los1 defect in nonsense suppression, possess detectable 6Pgl activity. Rather, Sol1p and Sol2p appear to function in tRNA nuclear export as sol1 and sol2 mutants possess elevated levels of nuclear tRNA. Members of the Sol protein family appear to have different subcellular distributions. Thus, Sol3p and Sol4p likely function in carbohydrate metabolism, while Sol1p and Sol2p appear to have roles in tRNA function and nuclear export, thereby defining an unusual protein family whose individual members are biochemically distinct and spatially dispersed.

Abscisic acid affects transcription of chloroplast genes via protein phosphatase 2C-dependent activation of nuclear genes: repression by guanosine-3'-5'-bisdiphosphate and activation by sigma factor 5.

PubMed

Yamburenko, Maria V; Zubo, Yan O; Börner, Thomas

2015-06-01

Abscisic acid (ABA) represses the transcriptional activity of chloroplast genes (determined by run-on assays), with the exception of psbD and a few other genes in wild-type Arabidopsis seedlings and mature rosette leaves. Abscisic acid does not influence chloroplast transcription in the mutant lines abi1-1 and abi2-1 with constitutive protein phosphatase 2C (PP2C) activity, suggesting that ABA affects chloroplast gene activity by binding to the pyrabactin resistance (PYR)/PYR1-like or regulatory component of ABA receptor protein family (PYR/PYL/RCAR) and signaling via PP2Cs and sucrose non-fermenting protein-related kinases 2 (SnRK2s). Further we show by quantitative PCR that ABA enhances the transcript levels of RSH2, RSH3, PTF1 and SIG5. RelA/SpoT homolog 2 (RSH2) and RSH3 are known to synthesize guanosine-3'-5'-bisdiphosphate (ppGpp), an inhibitor of the plastid-gene-encoded chloroplast RNA polymerase. We propose, therefore, that ABA leads to an inhibition of chloroplast gene expression via stimulation of ppGpp synthesis. On the other hand, sigma factor 5 (SIG5) and plastid transcription factor 1 (PTF1) are known to be necessary for the transcription of psbD from a specific light- and stress-induced promoter (the blue light responsive promoter, BLRP). We demonstrate that ABA activates the psbD gene by stimulation of transcription initiation at BLRP. Taken together, our data suggest that ABA affects the transcription of chloroplast genes by a PP2C-dependent activation of nuclear genes encoding proteins involved in chloroplast transcription. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Biogenesis of the mitochondrial TOM complex: Mim1 promotes insertion and assembly of signal-anchored receptors.

PubMed

Becker, Thomas; Pfannschmidt, Sylvia; Guiard, Bernard; Stojanovski, Diana; Milenkovic, Dusanka; Kutik, Stephan; Pfanner, Nikolaus; Meisinger, Chris; Wiedemann, Nils

2008-01-04

The translocase of the outer membrane (TOM complex) is the central entry gate for nuclear-encoded mitochondrial precursor proteins. All Tom proteins are also encoded by nuclear genes and synthesized as precursors in the cytosol. The channel-forming beta-barrel protein Tom40 is targeted to mitochondria via Tom receptors and inserted into the outer membrane by the sorting and assembly machinery (SAM complex). A further outer membrane protein, Mim1, plays a less defined role in assembly of Tom40 into the TOM complex. The three receptors Tom20, Tom22, and Tom70 are anchored in the outer membrane by a single transmembrane alpha-helix, located at the N terminus in the case of Tom20 and Tom70 (signal-anchored) or in the C-terminal portion in the case of Tom22 (tail-anchored). Insertion of the precursor of Tom22 into the outer membrane requires pre-existing Tom receptors while the import pathway of the precursors of Tom20 and Tom70 is only poorly understood. We report that Mim1 is required for efficient membrane insertion and assembly of Tom20 and Tom70, but not Tom22. We show that Mim1 associates with SAM(core) components to a large SAM complex, explaining its role in late steps of the assembly pathway of Tom40. We conclude that Mim1 is not only required for biogenesis of the beta-barrel protein Tom40 but also for membrane insertion and assembly of signal-anchored Tom receptors. Thus, Mim1 plays an important role in the efficient assembly of the mitochondrial TOM complex.

Characterization and subcellular localization of an RNA silencing suppressor encoded by Rice stripe tenuivirus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiong Ruyi; Wu Jianxiang; Zhou Yijun

2009-04-25

Rice stripe virus (RSV) is a single-stranded (ss) RNA virus belonging to the genus Tenuivirus. RSV is present in many East Asian countries and causes severe diseases in rice fields, especially in China. In this study, we analyzed six proteins encoded by the virus for their abilities to suppress RNA silencing in plant using a green fluorescent protein (GFP)-based transient expression assay. Our results indicate that NS3 encoded by RSV RNA3, but not other five RSV encoded proteins, can strongly suppress local GFP silencing in agroinfiltrated Nicotiana benthamiana leaves. NS3 can reverse the GFP silencing, it can also prevent longmore » distance spread of silencing signals which have been reported to be necessary for inducing systemic silencing in host plants. The NS3 protein can significantly reduce the levels of small interfering RNAs (siRNAs) in silencing cells, and was found to bind 21-nucleotide ss-siRNA, siRNA duplex and long ssRNA but not long double-stranded (ds)-RNA. Both N and C terminal of the NS3 protein are critical for silencing suppression, and mutation of the putative nuclear localization signal decreases its local silencing suppression efficiency and blocks its systemic silencing suppression. The NS3-GFP fusion protein and NS3 were shown to accumulate predominantly in nuclei of onion, tobacco and rice cells through transient expression assay or immunocytochemistry and electron microscopy. In addition, transgenic rice and tobacco plants expressing the NS3 did not show any apparent alteration in plant growth and morphology, although NS3 was proven to be a pathogenicity determinant in the PVX heterogenous system. Taken together, our results demonstrate that RSV NS3 is a suppressor of RNA silencing in planta, possibly through sequestering siRNA molecules generated in cells that are undergoing gene silencing.« less

Differential expression of the nuclear-encoded mitochondrial transcriptome in pediatric septic shock.

PubMed

Weiss, Scott L; Cvijanovich, Natalie Z; Allen, Geoffrey L; Thomas, Neal J; Freishtat, Robert J; Anas, Nick; Meyer, Keith; Checchia, Paul A; Shanley, Thomas P; Bigham, Michael T; Fitzgerald, Julie; Banschbach, Sharon; Beckman, Eileen; Howard, Kelli; Frank, Erin; Harmon, Kelli; Wong, Hector R

2014-11-19

Increasing evidence supports a role for mitochondrial dysfunction in organ injury and immune dysregulation in sepsis. Although differential expression of mitochondrial genes in blood cells has been reported for several diseases in which bioenergetic failure is a postulated mechanism, there are no data about the blood cell mitochondrial transcriptome in pediatric sepsis. We conducted a focused analysis using a multicenter genome-wide expression database of 180 children ≤ 10 years of age with septic shock and 53 healthy controls. Using total RNA isolated from whole blood within 24 hours of PICU admission for septic shock, we evaluated 296 nuclear-encoded mitochondrial genes using a false discovery rate of 1%. A series of bioinformatic approaches were applied to compare differentially expressed genes across previously validated gene expression-based subclasses (groups A, B, and C) of pediatric septic shock. In total, 118 genes were differentially regulated in subjects with septic shock compared to healthy controls, including 48 genes that were upregulated and 70 that were downregulated. The top scoring canonical pathway was oxidative phosphorylation, with general downregulation of the 51 genes corresponding to the electron transport system (ETS). The top two gene networks were composed primarily of mitochondrial ribosomal proteins highly connected to ETS complex I, and genes encoding for ETS complexes I, II, and IV that were highly connected to the peroxisome proliferator activated receptor (PPAR) family. There were 162 mitochondrial genes differentially regulated between groups A, B, and C. Group A, which had the highest maximum number of organ failures and mortality, exhibited a greater downregulation of mitochondrial genes compared to groups B and C. Based on a focused analysis of a pediatric septic shock transcriptomic database, nuclear-encoded mitochondrial genes were differentially regulated early in pediatric septic shock compared to healthy controls, as well as across genotypic and phenotypic distinct pediatric septic shock subclasses. The nuclear genome may be an important mechanism contributing to alterations in mitochondrial bioenergetic function and outcomes in pediatric sepsis.

Transcriptome analysis of the epidermis of the purple quail-like (q-lp) mutant of silkworm, Bombyx mori.

PubMed

Wang, Pingyang; Qiu, Zhiyong; Xia, Dingguo; Tang, Shunming; Shen, Xingjia; Zhao, Qiaoling

2017-01-01

A new purple quail-like (q-lp) mutant found from the plain silkworm strain 932VR has pigment dots on the epidermis similar to the pigment mutant quail (q). In addition, q-lp mutant larvae are inactive, consume little and grow slowly, with a high death rate and other developmental abnormalities. Pigmentation of the silkworm epidermis consists of melanin, ommochrome and pteridine. Silkworm development is regulated by ecdysone and juvenile hormone. In this study, we performed RNA-Seq on the epidermis of the q-lp mutant in the 4th instar during molting, with 932VR serving as the control. The results showed 515 differentially expressed genes, of which 234 were upregulated and 281 downregulated in q-lp. BLASTGO analysis indicated that the downregulated genes mainly encode protein-binding proteins, membrane components, oxidation/reduction enzymes, and proteolytic enzymes, whereas the upregulated genes largely encode cuticle structural constituents, membrane components, transport related proteins, and protein-binding proteins. Quantitative reverse transcription PCR was used to verify the accuracy of the RNA-Seq data, focusing on key genes for biosynthesis of the three pigments and chitin as well as genes encoding cuticular proteins and several related nuclear receptors, which are thought to play key roles in the q-lp mutant. We drew three conclusions based on the results: 1) melanin, ommochrome and pteridine pigments are all increased in the q-lp mutant; 2) more cuticle proteins are expressed in q-lp than in 932VR, and the number of upregulated cuticular genes is significantly greater than downregulated genes; 3) the downstream pathway regulated by ecdysone is blocked in the q-lp mutant. Our research findings lay the foundation for further research on the developmental changes responsible for the q-lp mutant.

Nuclear relocalization of polyadenylate binding protein during rift valley fever virus infection involves expression of the NSs gene.

PubMed

Copeland, Anna Maria; Altamura, Louis A; Van Deusen, Nicole M; Schmaljohn, Connie S

2013-11-01

Rift Valley fever virus (RVFV), an ambisense member of the family Bunyaviridae, genus Phlebovirus, is the causative agent of Rift Valley fever, an important zoonotic infection in Africa and the Middle East. Phlebovirus proteins are translated from virally transcribed mRNAs that, like host mRNA, are capped but, unlike host mRNAs, are not polyadenylated. Here, we investigated the role of PABP1 during RVFV infection of HeLa cells. Immunofluorescence studies of infected cells demonstrated a gross relocalization of PABP1 to the nucleus late in infection. Immunofluorescence microscopy studies of nuclear proteins revealed costaining between PABP1 and markers of nuclear speckles. PABP1 relocalization was sharply decreased in cells infected with a strain of RVFV lacking the gene encoding the RVFV nonstructural protein S (NSs). To determine whether PABP1 was required for RVFV infection, we measured the production of nucleocapsid protein (N) in cells transfected with small interfering RNAs (siRNAs) targeting PABP1. We found that the overall percentage of RVFV N-positive cells was not changed by siRNA treatment, indicating that PABP1 was not required for RVFV infection. However, when we analyzed populations of cells producing high versus low levels of PABP1, we found that the percentage of RVFV N-positive cells was decreased in cell populations producing physiologic levels of PABP1 and increased in cells with reduced levels of PABP1. Together, these results suggest that production of the NSs protein during RVFV infection leads to sequestration of PABP1 in the nuclear speckles, creating a state within the cell that favors viral protein production.

A Novel Lamin A Mutant Responsible for Congenital Muscular Dystrophy Causes Distinct Abnormalities of the Cell Nucleus.

PubMed

Barateau, Alice; Vadrot, Nathalie; Vicart, Patrick; Ferreiro, Ana; Mayer, Michèle; Héron, Delphine; Vigouroux, Corinne; Buendia, Brigitte

2017-01-01

A-type lamins, the intermediate filament proteins participating in nuclear structure and function, are encoded by LMNA. LMNA mutations can lead to laminopathies such as lipodystrophies, premature aging syndromes (progeria) and muscular dystrophies. Here, we identified a novel heterozygous LMNA p.R388P de novo mutation in a patient with a non-previously described severe phenotype comprising congenital muscular dystrophy (L-CMD) and lipodystrophy. In culture, the patient's skin fibroblasts entered prematurely into senescence, and some nuclei showed a lamina honeycomb pattern. C2C12 myoblasts were transfected with a construct carrying the patient's mutation; R388P-lamin A (LA) predominantly accumulated within the nucleoplasm and was depleted at the nuclear periphery, altering the anchorage of the inner nuclear membrane protein emerin and the nucleoplasmic protein LAP2-alpha. The mutant LA triggered a frequent and severe nuclear dysmorphy that occurred independently of prelamin A processing, as well as increased histone H3K9 acetylation. Nuclear dysmorphy was not significantly improved when transfected cells were treated with drugs disrupting microtubules or actin filaments or modifying the global histone acetylation pattern. Therefore, releasing any force exerted at the nuclear envelope by the cytoskeleton or chromatin did not rescue nuclear shape, in contrast to what was previously shown in Hutchinson-Gilford progeria due to other LMNA mutations. Our results point to the specific cytotoxic effect of the R388P-lamin A mutant, which is clinically related to a rare and severe multisystemic laminopathy phenotype.

The ANGULATA7 gene encodes a DnaJ-like zinc finger-domain protein involved in chloroplast function and leaf development in Arabidopsis.

PubMed

Muñoz-Nortes, Tamara; Pérez-Pérez, José Manuel; Ponce, María Rosa; Candela, Héctor; Micol, José Luis

2017-03-01

The characterization of mutants with altered leaf shape and pigmentation has previously allowed the identification of nuclear genes that encode plastid-localized proteins that perform essential functions in leaf growth and development. A large-scale screen previously allowed us to isolate ethyl methanesulfonate-induced mutants with small rosettes and pale green leaves with prominent marginal teeth, which were assigned to a phenotypic class that we dubbed Angulata. The molecular characterization of the 12 genes assigned to this phenotypic class should help us to advance our understanding of the still poorly understood relationship between chloroplast biogenesis and leaf morphogenesis. In this article, we report the phenotypic and molecular characterization of the angulata7-1 (anu7-1) mutant of Arabidopsis thaliana, which we found to be a hypomorphic allele of the EMB2737 gene, which was previously known only for its embryonic-lethal mutations. ANU7 encodes a plant-specific protein that contains a domain similar to the central cysteine-rich domain of DnaJ proteins. The observed genetic interaction of anu7-1 with a loss-of-function allele of GENOMES UNCOUPLED1 suggests that the anu7-1 mutation triggers a retrograde signal that leads to changes in the expression of many genes that normally function in the chloroplasts. Many such genes are expressed at higher levels in anu7-1 rosettes, with a significant overrepresentation of those required for the expression of plastid genome genes. Like in other mutants with altered expression of plastid-encoded genes, we found that anu7-1 exhibits defects in the arrangement of thylakoidal membranes, which appear locally unappressed. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Nesprin-2 epsilon: A novel nesprin isoform expressed in human ovary and Ntera-2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lam, Le Thanh; Boehm, Sabrina V.; Roberts, Roland G.

2011-08-26

Highlights: {yields} A novel epsilon isoform of nesprin-2 has been discovered. {yields} This 120 kDa protein was predicted by bioinformatic analysis, but has not previously been observed. {yields} It is the main isoform expressed in a teratocarcinoma cell line and is also found in ovary. {yields} Like other nesprins, it is located at the nuclear envelope. {yields} We suggest it may have a role in very early development or in some ovary-specific function. -- Abstract: The nuclear envelope-associated cytoskeletal protein, nesprin-2, is encoded by a large gene containing several internal promoters that produce shorter isoforms. In a study of Ntera-2more » teratocarcinoma cells, a novel isoform, nesprin-2-epsilon, was found to be the major mRNA and protein product of the nesprin-2 gene. Its existence was predicted by bioinformatic analysis, but this is the first direct demonstration of both the mRNA and the 120 kDa protein which is located at the nuclear envelope. In a panel of 21 adult and foetal human tissues, the nesprin-2-epsilon mRNA was strongly expressed in ovary but was a minor isoform elsewhere. The expression pattern suggests a possible link with very early development and a likely physiological role in ovary.« less

Laminopathies and the long strange trip from basic cell biology to therapy

PubMed Central

Worman, Howard J.; Fong, Loren G.; Muchir, Antoine; Young, Stephen G.

2009-01-01

The main function of the nuclear lamina, an intermediate filament meshwork lying primarily beneath the inner nuclear membrane, is to provide structural scaffolding for the cell nucleus. However, the lamina also serves other functions, such as having a role in chromatin organization, connecting the nucleus to the cytoplasm, gene transcription, and mitosis. In somatic cells, the main protein constituents of the nuclear lamina are lamins A, C, B1, and B2. Interest in the nuclear lamins increased dramatically in recent years with the realization that mutations in LMNA, the gene encoding lamins A and C, cause a panoply of human diseases (“laminopathies”), including muscular dystrophy, cardiomyopathy, partial lipodystrophy, and progeroid syndromes. Here, we review the laminopathies and the long strange trip from basic cell biology to therapeutic approaches for these diseases. PMID:19587457

Topological and organizational properties of the products of house-keeping and tissue-specific genes in protein-protein interaction networks.

PubMed

Lin, Wen-Hsien; Liu, Wei-Chung; Hwang, Ming-Jing

2009-03-11

Human cells of various tissue types differ greatly in morphology despite having the same set of genetic information. Some genes are expressed in all cell types to perform house-keeping functions, while some are selectively expressed to perform tissue-specific functions. In this study, we wished to elucidate how proteins encoded by human house-keeping genes and tissue-specific genes are organized in human protein-protein interaction networks. We constructed protein-protein interaction networks for different tissue types using two gene expression datasets and one protein-protein interaction database. We then calculated three network indices of topological importance, the degree, closeness, and betweenness centralities, to measure the network position of proteins encoded by house-keeping and tissue-specific genes, and quantified their local connectivity structure. Compared to a random selection of proteins, house-keeping gene-encoded proteins tended to have a greater number of directly interacting neighbors and occupy network positions in several shortest paths of interaction between protein pairs, whereas tissue-specific gene-encoded proteins did not. In addition, house-keeping gene-encoded proteins tended to connect with other house-keeping gene-encoded proteins in all tissue types, whereas tissue-specific gene-encoded proteins also tended to connect with other tissue-specific gene-encoded proteins, but only in approximately half of the tissue types examined. Our analysis showed that house-keeping gene-encoded proteins tend to occupy important network positions, while those encoded by tissue-specific genes do not. The biological implications of our findings were discussed and we proposed a hypothesis regarding how cells organize their protein tools in protein-protein interaction networks. Our results led us to speculate that house-keeping gene-encoded proteins might form a core in human protein-protein interaction networks, while clusters of tissue-specific gene-encoded proteins are attached to the core at more peripheral positions of the networks.

Cloning of cDNA encoding the nuclear form of chicken sterol response element binding protein-2 (SREBP-2), chromosomal localization, and tissue expression of chicken SREBP-1 and -2 genes.

PubMed

Assaf, S; Hazard, D; Pitel, F; Morisson, M; Alizadeh, M; Gondret, F; Diot, C; Vignal, A; Douaire, M; Lagarrigue, S

2003-01-01

Sterol regulatory element binding protein-1 and -2 (SREBP-1 and -2) are key transcription factors involved in the biosynthesis of cholesterol and fatty adds. The SREBP have mainly been studied in rodents in which lipogenesis is regulated in both liver and adipose tissue. There is, however, a paucity of information on birds, in which lipogenesis occurs essentially in the liver as in humans. As a prelude to the investigation of the role of SREBP in lipid metabolism regulation in chicken, we sequenced the cDNA, encoding the mature nuclear form of chicken SREBP-2 protein, mapped SREBP-1 and -2 genes and studied their tissue expressions. The predicted chicken SREBP-2 amino acid sequence shows a 77 to 79% identity with human, mouse, and hamster homologues, with a nearly perfect conservation in all the important functional motifs, basic, helix-loop-helix, and leucine zipper (bHLH-Zip) region as well as cleavage sites. As in the human genome, SREBP-1 and SREBP-2 chicken genes are located on two separate chromosomes, respectively microchromosome 14 and macrochromosome 1. Tissue expression data show that SREBP-1 and SREBP-2 are expressed in a wide variety of tissues in chicken. However, unlike SREBP-2, SREBP-1 is expressed preferentially in the liver and uropygial gland, suggesting an important role of SREBP-1 in the regulation of lipogenesis in avian species.

Phylogenetic Analysis of Mitochondrial Outer Membrane β-Barrel Channels

PubMed Central

Wojtkowska, Małgorzata; Jąkalski, Marcin; Pieńkowska, Joanna R.; Stobienia, Olgierd; Karachitos, Andonis; Przytycka, Teresa M.; Weiner, January; Kmita, Hanna; Makałowski, Wojciech

2012-01-01

Transport of molecules across mitochondrial outer membrane is pivotal for a proper function of mitochondria. The transport pathways across the membrane are formed by ion channels that participate in metabolite exchange between mitochondria and cytoplasm (voltage-dependent anion-selective channel, VDAC) as well as in import of proteins encoded by nuclear genes (Tom40 and Sam50/Tob55). VDAC, Tom40, and Sam50/Tob55 are present in all eukaryotic organisms, encoded in the nuclear genome, and have β-barrel topology. We have compiled data sets of these protein sequences and studied their phylogenetic relationships with a special focus on the position of Amoebozoa. Additionally, we identified these protein-coding genes in Acanthamoeba castellanii and Dictyostelium discoideum to complement our data set and verify the phylogenetic position of these model organisms. Our analysis show that mitochondrial β-barrel channels from Archaeplastida (plants) and Opisthokonta (animals and fungi) experienced many duplication events that resulted in multiple paralogous isoforms and form well-defined monophyletic clades that match the current model of eukaryotic evolution. However, in representatives of Amoebozoa, Chromalveolata, and Excavata (former Protista), they do not form clearly distinguishable clades, although they locate basally to the plant and algae branches. In most cases, they do not posses paralogs and their sequences appear to have evolved quickly or degenerated. Consequently, the obtained phylogenies of mitochondrial outer membrane β-channels do not entirely reflect the recent eukaryotic classification system involving the six supergroups: Chromalveolata, Excavata, Archaeplastida, Rhizaria, Amoebozoa, and Opisthokonta. PMID:22155732

Olfactory Proteins Mediating Chemical Communication in the Navel Orangeworm Moth, Amyelois transitella

PubMed Central

Leal, Walter S.; Ishida, Yuko; Pelletier, Julien; Xu, Wei; Rayo, Josep; Xu, Xianzhong; Ames, James B.

2009-01-01

Background The navel orangeworm, Amyelois transitella Walker (Lepidoptera: Pyralidae), is the most serious insect pest of almonds and pistachios in California for which environmentally friendly alternative methods of control — like pheromone-based approaches — are highly desirable. Some constituents of the sex pheromone are unstable and could be replaced with parapheromones, which may be designed on the basis of molecular interaction of pheromones and pheromone-detecting olfactory proteins. Methodology By analyzing extracts from olfactory and non-olfactory tissues, we identified putative olfactory proteins, obtained their N-terminal amino acid sequences by Edman degradation, and used degenerate primers to clone the corresponding cDNAs by SMART RACE. Additionally, we used degenerate primers based on conserved sequences of known proteins to fish out other candidate olfactory genes. We expressed the gene encoding a newly identified pheromone-binding protein, which was analyzed by circular dichroism, fluorescence, and nuclear magnetic resonance, and used in a binding assay to assess affinity to pheromone components. Conclusion We have cloned nine cDNAs encoding olfactory proteins from the navel orangeworm, including two pheromone-binding proteins, two general odorant-binding proteins, one chemosensory protein, one glutathione S-transferase, one antennal binding protein X, one sensory neuron membrane protein, and one odorant receptor. Of these, AtraPBP1 is highly enriched in male antennae. Fluorescence, CD and NMR studies suggest a dramatic pH-dependent conformational change, with high affinity to pheromone constituents at neutral pH and no binding at low pH. PMID:19789654

Retrograde movement of tRNAs from the cytoplasm to the nucleus in Saccharomyces cerevisiae

PubMed Central

Shaheen, Hussam H.; Hopper, Anita K.

2005-01-01

In eukaryotes, tRNAs transcribed in the nucleus function in cytoplasmic protein synthesis. The Ran-GTP-binding exportin, Los1p/Xpo-t, and additional pathway(s) mediate tRNA transport to the cytoplasm. Although tRNA movement was thought to be unidirectional, recent reports that yeast precursor tRNA splicing occurs in the cytoplasm, whereas fully spliced tRNAs can reside in the nucleus, require that either the precursor tRNA splicing machinery or mature tRNAs move from the cytoplasm to the nucleus. Our data argue against the first possibility and strongly support the second. Combining heterokaryon analysis with fluorescence in situ hybridization, we show that a foreign tRNA encoded by one nucleus can move from the cytoplasm to a second nucleus that does not encode the tRNA. We also discovered nuclear accumulation of endogenous cytoplasmic tRNAs in haploid yeast cells in response to nutritional deprivation. Nuclear accumulation of cytoplasmic tRNA requires Ran and the Mtr10/Kap111 member of the importin-β family. Retrograde tRNA nuclear import may provide a novel mechanism to regulate gene expression in eukaryotes. PMID:16040803

Retrograde movement of tRNAs from the cytoplasm to the nucleus in Saccharomyces cerevisiae.

PubMed

Shaheen, Hussam H; Hopper, Anita K

2005-08-09

In eukaryotes, tRNAs transcribed in the nucleus function in cytoplasmic protein synthesis. The Ran-GTP-binding exportin, Los1p/Xpo-t, and additional pathway(s) mediate tRNA transport to the cytoplasm. Although tRNA movement was thought to be unidirectional, recent reports that yeast precursor tRNA splicing occurs in the cytoplasm, whereas fully spliced tRNAs can reside in the nucleus, require that either the precursor tRNA splicing machinery or mature tRNAs move from the cytoplasm to the nucleus. Our data argue against the first possibility and strongly support the second. Combining heterokaryon analysis with fluorescence in situ hybridization, we show that a foreign tRNA encoded by one nucleus can move from the cytoplasm to a second nucleus that does not encode the tRNA. We also discovered nuclear accumulation of endogenous cytoplasmic tRNAs in haploid yeast cells in response to nutritional deprivation. Nuclear accumulation of cytoplasmic tRNA requires Ran and the Mtr10/Kap111 member of the importin-beta family. Retrograde tRNA nuclear import may provide a novel mechanism to regulate gene expression in eukaryotes.

Nutrient-sensing nuclear receptors PPARα and FXR control liver energy balance.

PubMed

Preidis, Geoffrey A; Kim, Kang Ho; Moore, David D

2017-04-03

The nuclear receptors PPARα (encoded by NR1C1) and farnesoid X receptor (FXR, encoded by NR1H4) are activated in the liver in the fasted and fed state, respectively. PPARα activation induces fatty acid oxidation, while FXR controls bile acid homeostasis, but both nuclear receptors also regulate numerous other metabolic pathways relevant to liver energy balance. Here we review evidence that they function coordinately to control key nutrient pathways, including fatty acid oxidation and gluconeogenesis in the fasted state and lipogenesis and glycolysis in the fed state. We have also recently reported that these receptors have mutually antagonistic impacts on autophagy, which is induced by PPARα but suppressed by FXR. Secretion of multiple blood proteins is a major drain on liver energy and nutrient resources, and we present preliminary evidence that the liver secretome may be directly suppressed by PPARα, but induced by FXR. Finally, previous studies demonstrated a striking deficiency in bile acid levels in malnourished mice that is consistent with results in malnourished children. We present evidence that hepatic targets of PPARα and FXR are dysregulated in chronic undernutrition. We conclude that PPARα and FXR function coordinately to integrate liver energy balance.

Identification of a subunit of NADH-dehydrogenase as a p49/STRAP-binding protein

PubMed Central

Zhang, Xiaomin; Azhar, Gohar; Helms, Scott; Zhong, Ying; Wei, Jeanne Y

2008-01-01

Background The p49/STRAP (or SRFBP1) protein was recently identified in our laboratory as a cofactor of serum response factor that contributes to the regulation of SRF target genes in the heart. Results In the present study, we report that NDUFAB1, a nuclear encoded subunit of NADH dehydrogenase, represented the majority of the cDNA clones that interacted with p49/STRAP in multiple screenings using the yeast two-hybrid system. The p49/STRAP and NDUFAB1 proteins interacted and co-localized with each other in the cell. The p49/STRAP protein contains four classic nuclear localization sequence motifs, and it was observed to be present predominantly in the nucleus. Overexpression of p49/STRAP altered the intracellular level of NAD, and reduced the NAD/NADH ratio. Overexpression of p49/STRAP also induced the deacetylation of serum response factor. Conclusion These data suggest that p49/STRAP plays a role in the regulation of intracellular processes such as cardiac cellular metabolism, gene expression, and possibly aging. PMID:18230186

C. elegans EAK-3 inhibits dauer arrest via nonautonomous regulation of nuclear DAF-16/FoxO activity

PubMed Central

Zhang, Yanmei; Xu, Jinling; Puscau, Cristina; Kim, Yongsoon; Wang, Xi; Alam, Hena; Hu, Patrick J.

2008-01-01

SUMMARY Insulin regulates development, metabolism, and lifespan via a conserved PI3K/Akt pathway that promotes cytoplasmic sequestration of FoxO transcription factors. The regulation of nuclear FoxO is poorly understood. In the nematode Caenorhabditis elegans, insulin-like signaling functions in larvae to inhibit dauer arrest and acts during adulthood to regulate lifespan. In a screen for genes that modulate C. elegans insulin-like signaling, we identified eak-3, which encodes a novel protein that is specifically expressed in the two endocrine XXX cells. The dauer arrest phenotype of eak-3 mutants is fully suppressed by mutations in daf-16/FoxO, which encodes the major target of C. elegans insulin-like signaling, and daf-12, which encodes a nuclear receptor regulated by steroid hormones known as dafachronic acids. eak-3 mutation does not affect DAF-16/FoxO subcellular localization but enhances expression of the direct DAF-16/FoxO target sod-3 in a daf-16/FoxO- and daf-12-dependent manner. eak-3 mutants have normal lifespans, suggesting that EAK-3 decouples insulin-like regulation of development and longevity. We propose that EAK-3 activity in the XXX cells promotes the synthesis and/or secretion of a hormone that acts in parallel to AKT-1 to inhibit the expression of DAF-16/FoxO target genes. Similar hormonal pathways may regulate FoxO target gene expression in mammals. PMID:18241854

Quantitative microscopy uncovers ploidy changes during mitosis in live Drosophila embryos and their effect on nuclear size.

PubMed

Puah, Wee Choo; Chinta, Rambabu; Wasser, Martin

2017-03-15

Time-lapse microscopy is a powerful tool to investigate cellular and developmental dynamics. In Drosophila melanogaster , it can be used to study division cycles in embryogenesis. To obtain quantitative information from 3D time-lapse data and track proliferating nuclei from the syncytial stage until gastrulation, we developed an image analysis pipeline consisting of nuclear segmentation, tracking, annotation and quantification. Image analysis of maternal-haploid ( mh ) embryos revealed that a fraction of haploid syncytial nuclei fused to give rise to nuclei of higher ploidy (2n, 3n, 4n). Moreover, nuclear densities in mh embryos at the mid-blastula transition varied over threefold. By tracking synchronized nuclei of different karyotypes side-by-side, we show that DNA content determines nuclear growth rate and size in early interphase, while the nuclear to cytoplasmic ratio constrains nuclear growth during late interphase. mh encodes the Drosophila ortholog of human Spartan, a protein involved in DNA damage tolerance. To explore the link between mh and chromosome instability, we fluorescently tagged Mh protein to study its subcellular localization. We show Mh-mKO2 localizes to nuclear speckles that increase in numbers as nuclei expand in interphase. In summary, quantitative microscopy can provide new insights into well-studied genes and biological processes. © 2017. Published by The Company of Biologists Ltd.

The baculovirus-integrated retrotransposon TED encodes gag and pol proteins that assemble into viruslike particles with reverse transcriptase.

PubMed Central

Lerch, R A; Friesen, P D

1992-01-01

TED is a lepidopteran retrotransposon found inserted within the DNA genome of the Autographa californica nuclear polyhedrosis virus mutant, FP-D. To examine the proteins and functions encoded by this representative of the gypsy family of retrotransposons, the gag- and pol-like open reading frames (ORFs 1 and 2) were expressed in homologous lepidopteran cells by using recombinant baculovirus vectors. Expression of ORF 1 resulted in synthesis of an abundant TED-specific protein (Pr55gag) that assembled into viruslike particles with a diameter of 55 to 60 nm. Expression of ORF 2, requiring a -1 translational frameshift, resulted in synthesis of a protease that mediated cleavage of Pr55gag to generate p37, the major protein component of the resulting particles. Expression of ORF 2 also produced reverse transcriptase that associated with these particles. Both protease and reverse transcriptase activities mapped to domains within ORF 2 that contain sequence similarities with the corresponding functional domains of the pol gene of the vertebrate retroviruses. These results indicated that TED ORFs 1 and 2 functionally resemble the retrovirus gag and pol genes and demonstrated for the first time that an invertebrate member of the gypsy family of elements encodes active forms of the structural and enzymatic functions necessary for transposition via an RNA intermediate. TED integration within the baculovirus genome thus represents one of the first examples of transposon-mediated transfer of host-derived genes to an eukaryotic virus. Images PMID:1371168

Properties of genes essential for mouse development

PubMed Central

Kabir, Mitra; Barradas, Ana; Tzotzos, George T.; Hentges, Kathryn E.

2017-01-01

Essential genes are those that are critical for life. In the specific case of the mouse, they are the set of genes whose deletion means that a mouse is unable to survive after birth. As such, they are the key minimal set of genes needed for all the steps of development to produce an organism capable of life ex utero. We explored a wide range of sequence and functional features to characterise essential (lethal) and non-essential (viable) genes in mice. Experimental data curated manually identified 1301 essential genes and 3451 viable genes. Very many sequence features show highly significant differences between essential and viable mouse genes. Essential genes generally encode complex proteins, with multiple domains and many introns. These genes tend to be: long, highly expressed, old and evolutionarily conserved. These genes tend to encode ligases, transferases, phosphorylated proteins, intracellular proteins, nuclear proteins, and hubs in protein-protein interaction networks. They are involved with regulating protein-protein interactions, gene expression and metabolic processes, cell morphogenesis, cell division, cell proliferation, DNA replication, cell differentiation, DNA repair and transcription, cell differentiation and embryonic development. Viable genes tend to encode: membrane proteins or secreted proteins, and are associated with functions such as cellular communication, apoptosis, behaviour and immune response, as well as housekeeping and tissue specific functions. Viable genes are linked to transport, ion channels, signal transduction, calcium binding and lipid binding, consistent with their location in membranes and involvement with cell-cell communication. From the analysis of the composite features of essential and viable genes, we conclude that essential genes tend to be required for intracellular functions, and viable genes tend to be involved with extracellular functions and cell-cell communication. Knowledge of the features that are over-represented in essential genes allows for a deeper understanding of the functions and processes implemented during mammalian development. PMID:28562614

Characterization of rtSH3p13 gene encoding a development protein involved in vesicular traffic in spermiogenesis.

PubMed

Qiao, Yuan; Yang, Ju Xiang; Zhang, Xiao Dong L; Liu, Yu; Zhang, Jian Chao; Zong, Shu Dong; Miao, Shi Ying; Wang, Lin Fang; Koide, Samuel S

2004-06-01

A cDNA, designated as rtSH3p13, was isolated from a rat testis cDNA library. It consists of 1463 bp nuclear acids, which encodes a protein of 312 amino acids and was assigned the GenBank accession number AF227439. The deduced rtSH3p13 protein is a truncated isoform of SH3p13 as a result of mRNA alternative splicing. It is mainly expressed in the rat testis, detected in spermatids at the steps 8-19 of spermiogenesis, and found around the acrosome. During postnatal development, rtSH3p13 appears on day 18 and reaches maximum on day 60. Further experimental results suggested that rtSH3p13 forms a complex with activated epidermal growth factor receptor (EGFR) and interacts with synaptojanin I. Surprisingly, similar to SH3 domain, the V region of rtSH3p13 also inhibits endocytosis in CHO cells. Our results reveal a link between an rtSH3p13-synaptojanin-clathrin complex-mediated formation of pits and the process of spermiogenesis.

Mitochondrial disease associated with complex I (NADH-CoQ oxidoreductase) deficiency.

PubMed

Scheffler, Immo E

2015-05-01

Mitochondrial diseases due to a reduced capacity for oxidative phosphorylation were first identified more than 20 years ago, and their incidence is now recognized to be quite significant. In a large proportion of cases the problem can be traced to a complex I (NADH-CoQ oxidoreductase) deficiency (Phenotype MIM #252010). Because the complex consists of 44 subunits, there are many potential targets for pathogenic mutations, both on the nuclear and mitochondrial genomes. Surprisingly, however, almost half of the complex I deficiencies are due to defects in as yet unidentified genes that encode proteins other than the structural proteins of the complex. This review attempts to summarize what we know about the molecular basis of complex I deficiencies: mutations in the known structural genes, and mutations in an increasing number of genes encoding "assembly factors", that is, proteins required for the biogenesis of a functional complex I that are not found in the final complex I. More such genes must be identified before definitive genetic counselling can be applied in all cases of affected families.

Mam33 promotes cytochrome c oxidase subunit I translation in Saccharomyces cerevisiae mitochondria.

PubMed

Roloff, Gabrielle A; Henry, Michael F

2015-08-15

Three mitochondrial DNA-encoded proteins, Cox1, Cox2, and Cox3, comprise the core of the cytochrome c oxidase complex. Gene-specific translational activators ensure that these respiratory chain subunits are synthesized at the correct location and in stoichiometric ratios to prevent unassembled protein products from generating free oxygen radicals. In the yeast Saccharomyces cerevisiae, the nuclear-encoded proteins Mss51 and Pet309 specifically activate mitochondrial translation of the largest subunit, Cox1. Here we report that Mam33 is a third COX1 translational activator in yeast mitochondria. Mam33 is required for cells to adapt efficiently from fermentation to respiration. In the absence of Mam33, Cox1 translation is impaired, and cells poorly adapt to respiratory conditions because they lack basal fermentative levels of Cox1. © 2015 Roloff and Henry. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Solanum tuberosum ZPR1 encodes a light-regulated nuclear DNA-binding protein adjusting the circadian expression of StBBX24 to light cycle.

PubMed

Kiełbowicz-Matuk, Agnieszka; Czarnecka, Jagoda; Banachowicz, Ewa; Rey, Pascal; Rorat, Tadeusz

2017-03-01

ZPR1 proteins belong to the C4-type of zinc finger coordinators known in animal cells to interact with other proteins and participate in cell growth and proliferation. In contrast, the current knowledge regarding plant ZPR1 proteins is very scarce. Here, we identify a novel potato nuclear factor belonging to this family and named StZPR1. StZPR1 is specifically expressed in photosynthetic organs during the light period, and the ZPR1 protein is located in the nuclear chromatin fraction. From modelling and experimental analyses, we reveal the StZPR1 ability to bind the circadian DNA cis motif 'CAACAGCATC', named CIRC and present in the promoter of the clock-controlled double B-box StBBX24 gene, the expression of which peaks in the middle of the day. We found that transgenic lines silenced for StZPR1 expression still display a 24 h period for the oscillation of StBBX24 expression but delayed by 4 h towards the night. Importantly, other BBX genes exhibit altered circadian regulation in these lines. Our data demonstrate that StZPR1 allows fitting of the StBBX24 circadian rhythm to the light period and provide evidence that ZPR1 is a novel clock-associated protein in plants necessary for the accurate rhythmic expression of specific circadian-regulated genes. © 2016 John Wiley & Sons Ltd.

Brassica rapa Has Three Genes That Encode Proteins Associated with Different Neutral Lipids in Plastids of Specific Tissues1

PubMed Central

Kim, Hyun Uk; Wu, Sherry S.H.; Ratnayake, Chandra; Huang, Anthony H.C.

2001-01-01

Plastid lipid-associated protein (PAP), a predominant structural protein associated with carotenoids and other non-green neutral lipids in plastids, was shown to be encoded by a single nuclear gene in several species. Here we report three PAP genes in the diploid Brassica rapa; the three PAPs are associated with different lipids in specific tissues. Pap1 and Pap2 are more similar to each other (84% amino acid sequence identity) than to Pap3 (46% and 44%, respectively) in the encoded mature proteins. Pap1 transcript was most abundant in the maturing anthers (tapetum) and in lesser amounts in leaves, fruit coats, seeds, and sepals; Pap2 transcript was abundant only in the petals; and Pap3 transcript had a wide distribution, but at minimal levels in numerous organs. Immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that most organs had several nanograms of PAP1 or PAP2 per milligram of total protein, the highest amounts being in the anthers (10.9 μg mg−1 PAP1) and petals (6.6 μg mg−1 PAP2), and that they had much less PAP3 (<0.02 μg mg−1). In these organs PAP was localized in isolated plastid fractions. Plants were subjected to abiotic stresses; drought and ozone reduced the levels of the three Pap transcripts, whereas mechanical wounding and altering the light intensity enhanced their levels. We conclude that the PAP gene family consists of several members whose proteins are associated with different lipids and whose expressions are controlled by distinct mechanisms. Earlier reports of the expression of one Pap gene in various organs in a species need to be re-examined. PMID:11351096

The plastid ribosomal proteins. Identification of all the proteins in the 30 S subunit of an organelle ribosome (chloroplast).

PubMed

Yamaguchi, K; von Knoblauch, K; Subramanian, A R

2000-09-15

Identification of all the protein components of a plastid (chloroplast) ribosomal 30 S subunit has been achieved, using two-dimensional gel electropholesis, high performance liquid chromatography purification, N-terminal sequencing, polymerase chain reaction-based screening of cDNA library, nucleotide sequencing, and mass spectrometry (electrospray ionization, matrix-assisted laser desorption/ionization time-of-flight, and reversed-phase HPLC coupled with electrospray ionization mass spectrometry). 25 proteins were identified, of which 21 are orthologues of all Escherichia coli 30 S ribosomal proteins (S1-S21), and 4 are plastid-specific ribosomal proteins (PSRPs) that have no homologues in the mitochondrial, archaebacterial, or cytosolic ribosomal protein sequences in data bases. 12 of the 25 plastid 30 S ribosomal proteins (PRPs) are encoded in the plastid genome, whereas the remaining 13 are encoded by the nuclear genome. Post-translational transit peptide cleavage sites for the maturation of the 13 cytosolically synthesized PRPs, and post-translational N-terminal processing in the maturation of the 12 plastid synthesized PRPs are described. Post-translational modifications in several PRPs were observed: alpha-N-acetylation of S9, N-terminal processings leading to five mature forms of S6 and two mature forms of S10, C-terminal and/or internal modifications in S1, S14, S18, and S19, leading to two distinct forms differing in mass and/or charge (the corresponding modifications are not observed in E. coli). The four PSRPs in spinach plastid 30 S ribosomal subunit (PSRP-1, 26.8 kDa, pI 6.2; PSRP-2, 21.7 kDa, pI 5.0; PSRP-3, 13.8 kDa, pI 4.9; PSRP-4, 5.2 kDa, pI 11.8) comprise 16% (67.6 kDa) of the total protein mass of the 30 S subunit (429.3 kDa). PSRP-1 and PSRP-3 show sequence similarities with hypothetical photosynthetic bacterial proteins, indicating their possible origins in photosynthetic bacteria. We propose the hypothesis that PSRPs form a "plastid translational regulatory module" on the 30 S ribosomal subunit structure for the possible mediation of nuclear factors on plastid translation.

Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development.

PubMed

Liebers, Monique; Grübler, Björn; Chevalier, Fabien; Lerbs-Mache, Silva; Merendino, Livia; Blanvillain, Robert; Pfannschmidt, Thomas

2017-01-01

Plastids display a high morphological and functional diversity. Starting from an undifferentiated small proplastid, these plant cell organelles can develop into four major forms: etioplasts in the dark, chloroplasts in green tissues, chromoplasts in colored flowers and fruits and amyloplasts in roots. The various forms are interconvertible into each other depending on tissue context and respective environmental condition. Research of the last two decades uncovered that each plastid type contains its own specific proteome that can be highly different from that of the other types. Composition of these proteomes largely defines the enzymatic functionality of the respective plastid. The vast majority of plastid proteins is encoded in the nucleus and must be imported from the cytosol. However, a subset of proteins of the photosynthetic and gene expression machineries are encoded on the plastid genome and are transcribed by a complex transcriptional apparatus consisting of phage-type nuclear-encoded RNA polymerases and a bacterial-type plastid-encoded RNA polymerase. Both types recognize specific sets of promoters and transcribe partly over-lapping as well as specific sets of genes. Here we summarize the current knowledge about the sequential activity of these plastid RNA polymerases and their relative activities in different types of plastids. Based on published plastid gene expression profiles we hypothesize that each conversion from one plastid type into another is either accompanied or even preceded by significant changes in plastid transcription suggesting that these changes represent important determinants of plastid morphology and protein composition and, hence, the plastid type.

Regulatory Shifts in Plastid Transcription Play a Key Role in Morphological Conversions of Plastids during Plant Development

PubMed Central

Liebers, Monique; Grübler, Björn; Chevalier, Fabien; Lerbs-Mache, Silva; Merendino, Livia; Blanvillain, Robert; Pfannschmidt, Thomas

2017-01-01

Plastids display a high morphological and functional diversity. Starting from an undifferentiated small proplastid, these plant cell organelles can develop into four major forms: etioplasts in the dark, chloroplasts in green tissues, chromoplasts in colored flowers and fruits and amyloplasts in roots. The various forms are interconvertible into each other depending on tissue context and respective environmental condition. Research of the last two decades uncovered that each plastid type contains its own specific proteome that can be highly different from that of the other types. Composition of these proteomes largely defines the enzymatic functionality of the respective plastid. The vast majority of plastid proteins is encoded in the nucleus and must be imported from the cytosol. However, a subset of proteins of the photosynthetic and gene expression machineries are encoded on the plastid genome and are transcribed by a complex transcriptional apparatus consisting of phage-type nuclear-encoded RNA polymerases and a bacterial-type plastid-encoded RNA polymerase. Both types recognize specific sets of promoters and transcribe partly over-lapping as well as specific sets of genes. Here we summarize the current knowledge about the sequential activity of these plastid RNA polymerases and their relative activities in different types of plastids. Based on published plastid gene expression profiles we hypothesize that each conversion from one plastid type into another is either accompanied or even preceded by significant changes in plastid transcription suggesting that these changes represent important determinants of plastid morphology and protein composition and, hence, the plastid type. PMID:28154576

Discerning regulation of cis- and trans-presentation of CD8+ T-cell epitopes by EBV-encoded oncogene LMP-1 through self-aggregation.

PubMed

Smith, Corey; Wakisaka, Naohiro; Crough, Tania; Peet, Jesse; Yoshizaki, Tomokazu; Beagley, Leone; Khanna, Rajiv

2009-06-11

Activation of the nuclear factor-kappaB pathway by Epstein-Barr virus-encoded latent membrane protein-1 (LMP-1) leads to an up-regulation of the major histocompatibility complex class I antigen-processing pathway. Paradoxically, LMP-1 itself induces a subdominant CD8+ T-cell response and appears to have evolved to avoid immune recognition. Here we show that, although expression of LMP-1 in human cells dramatically enhanced the trans-presentation of CD8+ T-cell epitopes, cis-presentation of LMP-1-derived epitopes was severely impaired. Testing of a series of LMP-1 mutants revealed that deletion of the first transmembrane domain of LMP-1, which prevented self-aggregation, significantly enhanced cis-presentation of T-cell epitopes from this protein, whereas it lost its ability to up-regulate trans-presentation. Interestingly, we also found that cis-presentation of LMP-1 epitopes was rescued by blocking the proteasome function. Taken together, these results delineate a novel mechanism of immune evasion, which renders a virally encoded oncogene inaccessible to the conventional major histocompatibility complex class I pathway limiting its cis-presentation to effector cells.

Genome-wide screen uncovers novel pathways for tRNA processing and nuclear–cytoplasmic dynamics

PubMed Central

Wu, Jingyan; Bao, Alicia; Chatterjee, Kunal; Wan, Yao; Hopper, Anita K.

2015-01-01

Transfer ribonucleic acids (tRNAs) are essential for protein synthesis. However, key gene products involved in tRNA biogenesis and subcellular movement remain to be discovered. We conducted the first comprehensive unbiased analysis of the role of nearly an entire proteome in tRNA biology and describe 162 novel and 12 previously known Saccharomyces cerevisiae gene products that function in tRNA processing, turnover, and subcellular movement. tRNA nuclear export is of particular interest because it is essential, but the known tRNA exporters (Los1 [exportin-t] and Msn5 [exportin-5]) are unessential. We report that mutations of CRM1 (Exportin-1), MEX67/MTR2 (TAP/p15), and five nucleoporins cause accumulation of unspliced tRNA, a hallmark of defective tRNA nuclear export. CRM1 mutation genetically interacts with los1Δ and causes altered tRNA nuclear–cytoplasmic distribution. The data implicate roles for the protein and mRNA nuclear export machineries in tRNA nuclear export. Mutations of genes encoding actin cytoskeleton components and mitochondrial outer membrane proteins also cause accumulation of unspliced tRNA, likely due to defective splicing on mitochondria. Additional gene products, such as chromatin modification enzymes, have unanticipated effects on pre-tRNA end processing. Thus, this genome-wide screen uncovered putative novel pathways for tRNA nuclear export and extensive links between tRNA biology and other aspects of cell physiology. PMID:26680305

batman Interacts with polycomb and trithorax group genes and encodes a BTB/POZ protein that is included in a complex containing GAGA factor.

PubMed

Faucheux, M; Roignant, J-Y; Netter, S; Charollais, J; Antoniewski, C; Théodore, L

2003-02-01

Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila melanogaster development. We have previously identified the batman gene as a Polycomb group candidate since its function is necessary for the repression of Sex combs reduced. However, our present genetic analysis indicates functions of batman in both activation and repression of homeotic genes. The 127-amino-acid Batman protein is almost reduced to a BTB/POZ domain, an evolutionary conserved protein-protein interaction domain found in a large protein family. We show that this domain is involved in the interaction between Batman and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Batman codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Batman, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. Together, our results suggest that batman belongs to a subset of the Polycomb/trithorax group of genes that includes Trithorax-like, whose products are involved in both activation and repression of homeotic genes.

Identification of NAB1, a repressor of NGFI-A- and Krox20-mediated transcription.

PubMed Central

Russo, M W; Sevetson, B R; Milbrandt, J

1995-01-01

NGFI-A (also called Egr1, Zif268, or Krox24) and the closely related proteins Krox20, NGFI-C, and Egr3 are zinc-finger transcription factors encoded by immediate-early genes which are induced by a wide variety of extracellular stimuli. NGFI-A has been implicated in cell proliferation, macrophage differentiation, synaptic activation, and long-term potentiation, whereas Krox20 is critical for proper hindbrain segmentation and peripheral nerve myelination. In previous work, a structure/function analysis of NGFI-A revealed a 34-aa inhibitory domain that was hypothesized to be the target of a cellular factor that represses NGFI-A transcriptional activity. Using the yeast two-hybrid system, we have isolated a cDNA clone which encodes a protein that interacts with this inhibitory domain and inhibits the ability of NGFI-A to activate transcription. This NGFI-A-binding protein, NAB1, is a 570-aa nuclear protein that bears no obvious sequence homology to known proteins. NAB1 also represses Krox20 activity, but it does not influence Egr3 or NGFI-G, thus providing a mechanism for the differential regulation of this family of immediate-early transcription factors. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7624335

Conserved herpesvirus protein kinases

PubMed Central

Gershburg, Edward; Pagano, Joseph S.

2008-01-01

Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however along with a conserved role, individual kinases may have unique functions in the context of viral infection in such a way that they are only partially replaceable even by close homologues. Recent studies demonstrated that CHPKs are crucial for viral infection and suggested their involvement in regulation of numerous processes at various infection steps (primary infection, nuclear egress, tegumentation), although the mechanisms of this regulation remain unknown. Notwithstanding, recent advances in discovery of new CHPK targets, and studies of CHPK knockout phenotypes have raised their attractiveness as targets for antiviral therapy. A number of compounds have been shown to inhibit the activity of human cytomegalovirus (HCMV)-encoded UL97 protein kinase and exhibit a pronounced antiviral effect, although the same compounds are inactive against Epstein-Barr Virus (EBV)-encoded protein kinase BGLF4, illustrating the fact that low homology between the members of this group complicates development of compounds targeting the whole group, and suggesting that individualized, structure-based inhibitor design will be more effective. Determination of CHPK structures will greatly facilitate this task. PMID:17881303

batman Interacts with Polycomb and trithorax Group Genes and Encodes a BTB/POZ Protein That Is Included in a Complex Containing GAGA Factor

PubMed Central

Faucheux, M.; Roignant, J.-Y.; Netter, S.; Charollais, J.; Antoniewski, C.; Théodore, L.

2003-01-01

Polycomb and trithorax group genes maintain the appropriate repressed or activated state of homeotic gene expression throughout Drosophila melanogaster development. We have previously identified the batman gene as a Polycomb group candidate since its function is necessary for the repression of Sex combs reduced. However, our present genetic analysis indicates functions of batman in both activation and repression of homeotic genes. The 127-amino-acid Batman protein is almost reduced to a BTB/POZ domain, an evolutionary conserved protein-protein interaction domain found in a large protein family. We show that this domain is involved in the interaction between Batman and the DNA binding GAGA factor encoded by the Trithorax-like gene. The GAGA factor and Batman codistribute on polytene chromosomes, coimmunoprecipitate from nuclear embryonic and larval extracts, and interact in the yeast two-hybrid assay. Batman, together with the GAGA factor, binds to MHS-70, a 70-bp fragment of the bithoraxoid Polycomb response element. This binding, like that of the GAGA factor, requires the presence of d(GA)n sequences. Together, our results suggest that batman belongs to a subset of the Polycomb/trithorax group of genes that includes Trithorax-like, whose products are involved in both activation and repression of homeotic genes. PMID:12556479

The clp1 gene of the mushroom Coprinus cinereus is essential for A-regulated sexual development.

PubMed Central

Inada, K; Morimoto, Y; Arima, T; Murata, Y; Kamada, T

2001-01-01

Sexual development in the mushroom Coprinus cinereus is under the control of the A and B mating-type loci, both of which must be different for a compatible, dikaryotic mycelium to form between two parents. The A genes, encoding proteins with homeodomain motifs, regulate conjugate division of the two nuclei from each mating partner and promote the formation of clamp connections. The latter are hyphal configurations required for the maintenance of the nuclear status in the dikaryotic phase of basidiomycetes. The B genes encode pheromones and pheromone receptors. They regulate the cellular fusions that complete clamp connections during growth, as well as the nuclear migration required for dikaryosis. The AmutBmut strain (326) of C. cinereus, in which both A- and B-regulated pathways are constitutively activated by mutations, produces, without mating, dikaryon-like, fertile hyphae with clamp connections. In this study we isolated and characterized clampless1-1 (clp1-1), a mutation that blocks clamp formation, an essential step in A-regulated sexual development, in the AmutBmut background. A genomic DNA fragment that rescues the clp1-1 mutation was identified by transformations. Sequencing of the genomic DNA, together with RACE experiments, identified an ORF interrupted by one intron, encoding a novel protein of 365 amino acids. The clp1-1 mutant allele carries a deletion of four nucleotides, which is predicted to cause elimination of codon 128 and frameshifts thereafter. The clp1 transcript was normally detected only in the presence of the A protein heterodimer formed when homokaryons with compatible A genes were mated. Forced expression of clp1 by promoter replacements induced clamp development without the need for a compatible A gene combination. These results indicate that expression of clp1 is necessary and sufficient for induction of the A-regulated pathway that leads to clamp development. PMID:11139497

An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fowlkes, Jason Davidson; Owens, Elizabeth T; Standaert, Robert F

2009-01-01

Identifying and characterizing protein interactions are fundamental steps towards understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the co-localization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecularmore » tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin (Imp ) and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified co-localization assay is sensitive enough to detect protein interactions with Kd values that span over four orders of magnitude (1nM to 15 M). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris« less

Mitochondrial Molecular Abnormalities Revealed by Proteomic Analysis of Hippocampal Organelles of Mice Triple Transgenic for Alzheimer Disease

PubMed Central

Yu, Haitao; Lin, Xuemei; Wang, Dian; Zhang, Zaijun; Guo, Yi; Ren, Xiaohu; Xu, Benhong; Yuan, Jianhui; Liu, Jianjun; Spencer, Peter S.; Wang, Jian-Zhi; Yang, Xifei

2018-01-01

Mitochondrial dysfunction is implicated in the pathogenesis of Alzheimer’s disease (AD). However, the precise mitochondrial molecular deficits in AD remain poorly understood. Mitochondrial and nuclear proteomic analysis in mature male triple transgenic AD mice (PS1M146V/APPSwe/TauP301L) by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with MALDI-TOF-MS/MS, bio-informatics analysis and immunofluorescent staining were performed in this study. In addition to impaired spatial memory impairment and intracellular accumulation of amyloid 1–42 (Aβ1–42) in the 3xTg-AD mice, a well-accepted mouse model of the human disease, we also found significantly increased DNA oxidative damage in entorhinal cortex, hippocampal CA1, CA3 and dental gyrus (DG), as evidenced by the positive staining of 8-hydroxyguanosine, a biomarker of mild cognitive impairment early in AD. We identified significant differences in 27 hippocampal mitochondrial proteins (11 increased and 16 decreased), and 37 hippocampal nuclear proteins (12 increased and 25 decreased) in 3xTg-AD mice compared with the wild-type (WT) mice. Differentially expressed mitochondrial and nuclear proteins were mainly involved in energy metabolism (>55%), synapses, DNA damage, apoptosis and oxidative stress. Two proteins were differentially expressed in both hippocampal mitochondria and nuclei, namely electron transport chain (ETC)-related protein ATP synthase subunit d (ATP5H) was significantly decreased, and apoptosis-related dynamin-1 (DYN1), a pre-synaptic and mitochondrial division-regulated protein that was significantly increased. In sum, perturbations of hippocampus mitochondrial energy metabolism-related proteins responsible for ATP generation via oxidation phosphorylation (OXPHOS), especially nuclear-encoded OXPHOS proteins, correlated with the amyloid-associated cognitive deficits of this murine AD model. The molecular changes in respiratory chain-related proteins and DYN1 may represent novel biomarkers of AD. PMID:29593495

Assignment of electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) to human chromosome 4q33 by fluorescence in situ hybridization and somatic cell hybridization.

PubMed

Spector, E B; Seltzer, W K; Goodman, S I

1999-08-01

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a nuclear-encoded protein located in the inner mitochondrial membrane. Inherited defects of ETF-QO cause glutaric acidemia type II. We here describe the localization of the ETF-QO gene to human chromosome 4q33 by somatic cell hybridization and fluorescence in situ hybridization. Copyright 1999 Academic Press.

Salvaging hope: Is increasing NAD(+) a key to treating mitochondrial myopathy?

PubMed

Lightowlers, Robert N; Chrzanowska-Lightowlers, Zofia M A

2014-06-01

Mitochondrial diseases can arise from mutations either in mitochondrial DNA or in nuclear DNA encoding mitochondrially destined proteins. Currently, there is no cure for these diseases although treatments to ameliorate a subset of the symptoms are being developed. In this issue of EMBO Molecular Medicine, Khan et al (2014) use a mouse model to test the efficacy of a simple dietary supplement of nicotinamide riboside to treat and prevent mitochondrial myopathies.

The C2 protein of tomato leaf curl Taiwan virus is a pathogenicity determinant that interferes with expression of host genes encoding chromomethylases.

PubMed

Tu, Yu-Ching; Tsai, Wen-Shi; Wei, Jyuan-Yu; Chang, Kai-Ya; Tien, Chang-Ching; Hsiao, Hui-Yu; Fu, Shih-Feng

2017-12-01

Tomato (Solanum lycopersicum) is one of the most important crops worldwide and is severely affected by geminiviruses. Tomato leaf curl Taiwan virus (ToLCTWV), belonging to the geminiviruses, was isolated in Taiwan and causes tremendous crop loss. The geminivirus-encoded C2 proteins are crucial for a successful interaction between the virus and host plants. However, the exact functions of the viral C2 protein of ToLCTWV have not been investigated. We analyzed the molecular function(s) of the C2 protein by transient or stable expression in tomato cv. Micro-Tom and Nicotiana benthamiana. Severe stunting of tomato and N. benthamiana plants infected with ToLCTWV was observed. Expression of ToLCTWV C2-green fluorescent protein (GFP) fusion protein was predominately located in the nucleus and contributed to activation of a coat protein promoter. Notably, the C2-GFP fluorescence was distributed in nuclear aggregates. Tomato and N. benthamiana plants inoculated with potato virus X (PVX)-C2 displayed chlorotic lesions and stunted growth. PVX-C2 elicited hypersensitive responses accompanied by production of reactive oxygen species in N. benthamiana plants, which suggests that the viral C2 was a potential recognition target to induce host-defense responses. In tomato and N. benthamiana, ToLCTWV C2 was found to interfere with expression of genes encoding chromomethylases. N. benthamiana plants with suppressed NbCMT3-2 expression were more susceptible to ToLCTWV infection. Transgenic N. benthamiana plants expressing the C2 protein showed decreased expression of the NbCMT3-2 gene and pNbCMT3-2::GUS (β-glucuronidase) promoter activity. C2 protein is an important pathogenicity determinant of ToLCTWV and interferes with host components involved in DNA methylation. © 2017 Scandinavian Plant Physiology Society.

Characterization of Coffee ringspot virus-Lavras: A model for an emerging threat to coffee production and quality

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramalho, T.O.; Figueira, A.R.; Sotero, A.J.

2014-09-15

The emergence of viruses in Coffee (Coffea arabica and Coffea canephora), the most widely traded agricultural commodity in the world, is of critical concern. The RNA1 (6552 nt) of Coffee ringspot virus is organized into five open reading frames (ORFs) capable of encoding the viral nucleocapsid (ORF1p), phosphoprotein (ORF2p), putative cell-to-cell movement protein (ORF3p), matrix protein (ORF4p) and glycoprotein (ORF5p). Each ORF is separated by a conserved intergenic junction. RNA2 (5945 nt), which completes the bipartite genome, encodes a single protein (ORF6p) with homology to RNA-dependent RNA polymerases. Phylogenetic analysis of L protein sequences firmly establishes CoRSV as a membermore » of the recently proposed Dichorhavirus genus. Predictive algorithms, in planta protein expression, and a yeast-based nuclear import assay were used to determine the nucleophillic character of five CoRSV proteins. Finally, the temperature-dependent ability of CoRSV to establish systemic infections in an initially local lesion host was quantified. - Highlights: • We report genome sequence determination for Coffee ringspot virus (CoRSV). • CoRSV should be considered a member of the proposed Dichorhavirus genus. • We report temperature-dependent systemic infection of an initially local lesion host. • We report in planta protein and localization data for five CoRSV proteins. • In silico predictions of the CoRSV proteins were validated using in vivo assays.« less

Negative transcriptional regulation of mitochondrial transcription factor A (TFAM) by nuclear TFAM

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Eun Jin; Kang, Young Cheol; Park, Wook-Ha

2014-07-18

Highlights: • TFAM localizes in nuclei and mitochondria of neuronal cells. • Nuclear TFAM does not bind the Tfam promoter. • Nuclear TFAM reduced the Tfam promoter activity via suppressing NRF-1 activity. • A novel self-negative feedback regulation of Tfam gene expression is explored. • FAM may play different roles depending on its subcellular localizations. - Abstract: The nuclear DNA-encoded mitochondrial transcription factor A (TFAM) is synthesized in cytoplasm and transported into mitochondria. TFAM enhances both transcription and replication of mitochondrial DNA. It is unclear, however, whether TFAM plays a role in regulating nuclear gene expression. Here, we demonstrated thatmore » TFAM was localized to the nucleus and mitochondria by immunostaining, subcellular fractionation, and TFAM-green fluorescent protein hybrid protein studies. In HT22 hippocampal neuronal cells, human TFAM (hTFAM) overexpression suppressed human Tfam promoter-mediated luciferase activity in a dose-dependent manner. The mitochondria targeting sequence-deficient hTFAM also repressed Tfam promoter activity to the same degree as hTFAM. It indicated that nuclear hTFAM suppressed Tfam expression without modulating mitochondrial activity. The repression required for nuclear respiratory factor-1 (NRF-1), but hTFAM did not bind to the NRF-1 binding site of its promoter. TFAM was co-immunoprecipitated with NRF-1. Taken together, we suggest that nuclear TFAM down-regulate its own gene expression as a NRF-1 repressor, showing that TFAM may play different roles depending on its subcellular localizations.« less

Uncoupling of the hnRNP Npl3p from mRNAs during the stress-induced block in mRNA export.

PubMed

Krebber, H; Taura, T; Lee, M S; Silver, P A

1999-08-01

Npl3p, the major mRNA-binding protein of the yeast Saccharomyces cerevisiae shuttles between the nucleus and the cytoplasm. A single amino acid change in the carboxyl terminus of Npl3p (E409 --> K) renders the mutant protein largely cytoplasmic because of a delay in its import into the nucleus. This import defect can be reversed by increasing the intracellular concentration of Mtr10p, the nuclear import receptor for Npl3p. Conversely, using this mutant, we show that Npl3p and mRNA export out of the nucleus is significantly slowed in cells bearing mutations in XPO1/CRM1, which encodes the export receptor for NES-containing proteins and in RAT7, which encodes an essential nucleoporin. Interestingly, following induction of stress by heat shock, high salt, or ethanol, conditions under which most mRNA export is blocked, Npl3p is still exported from the nucleus. The stress-induced export of Npl3p is independent of both the activity of Xpo1p and the continued selective export of heat-shock mRNAs that occurs following stress. UV-cross-linking experiments show that Npl3p is bound to mRNA under normal conditions, but is no longer RNA associated in stressed cells. Taken together, we suggest that the uncoupling of Npl3p and possibly other mRNA-binding proteins from mRNAs in the nucleus provides a general switch that regulates mRNA export. By this model, under normal conditions Npl3p is a major component of an export-competent RNP complex. However, under conditions of stress, Npl3p no longer associates with the export complex, rendering it export incompetent and thus nuclear.

Physiological and Pathological Aging Affects Chromatin Dynamics, Structure and Function at the Nuclear Edge

PubMed Central

Robin, Jérôme D.; Magdinier, Frédérique

2016-01-01

Lamins are intermediate filaments that form a complex meshwork at the inner nuclear membrane. Mammalian cells express two types of Lamins, Lamins A/C and Lamins B, encoded by three different genes, LMNA, LMNB1, and LMNB2. Mutations in the LMNA gene are associated with a group of phenotypically diverse diseases referred to as laminopathies. Lamins interact with a large number of binding partners including proteins of the nuclear envelope but also chromatin-associated factors. Lamins not only constitute a scaffold for nuclear shape, rigidity and resistance to stress but also contribute to the organization of chromatin and chromosomal domains. We will discuss here the impact of A-type Lamins loss on alterations of chromatin organization and formation of chromatin domains and how disorganization of the lamina contributes to the patho-physiology of premature aging syndromes. PMID:27602048

Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes.

PubMed

Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

2015-01-01

TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene.

How protein targeting to primary plastids via the endomembrane system could have evolved? A new hypothesis based on phylogenetic studies

PubMed Central

2013-01-01

Background It is commonly assumed that a heterotrophic ancestor of the supergroup Archaeplastida/Plantae engulfed a cyanobacterium that was transformed into a primary plastid; however, it is still unclear how nuclear-encoded proteins initially were imported into the new organelle. Most proteins targeted to primary plastids carry a transit peptide and are transported post-translationally using Toc and Tic translocons. There are, however, several proteins with N-terminal signal peptides that are directed to higher plant plastids in vesicles derived from the endomembrane system (ES). The existence of these proteins inspired a hypothesis that all nuclear-encoded, plastid-targeted proteins initially carried signal peptides and were targeted to the ancestral primary plastid via the host ES. Results We present the first phylogenetic analyses of Arabidopsis thaliana α-carbonic anhydrase (CAH1), Oryza sativa nucleotide pyrophosphatase/phosphodiesterase (NPP1), and two O. sativa α-amylases (αAmy3, αAmy7), proteins that are directed to higher plant primary plastids via the ES. We also investigated protein disulfide isomerase (RB60) from the green alga Chlamydomonas reinhardtii because of its peculiar dual post- and co-translational targeting to both the plastid and ES. Our analyses show that these proteins all are of eukaryotic rather than cyanobacterial origin, and that their non-plastid homologs are equipped with signal peptides responsible for co-translational import into the host ES. Our results indicate that vesicular trafficking of proteins to primary plastids evolved long after the cyanobacterial endosymbiosis (possibly only in higher plants) to permit their glycosylation and/or transport to more than one cellular compartment. Conclusions The proteins we analyzed are not relics of ES-mediated protein targeting to the ancestral primary plastid. Available data indicate that Toc- and Tic-based translocation dominated protein import into primary plastids from the beginning. Only a handful of host proteins, which already were targeted through the ES, later were adapted to reach the plastid via the vesicular trafficking. They represent a derived class of higher plant plastid-targeted proteins with an unusual evolutionary history. Reviewers This article was reviewed by Prof. William Martin, Dr. Philippe Deschamps (nominated by Dr. Purificacion Lopez-Garcia) and Dr Simonetta Gribaldo. PMID:23845039

Evolution Analysis of the Aux/IAA Gene Family in Plants Shows Dual Origins and Variable Nuclear Localization Signals.

PubMed

Wu, Wentao; Liu, Yaxue; Wang, Yuqian; Li, Huimin; Liu, Jiaxi; Tan, Jiaxin; He, Jiadai; Bai, Jingwen; Ma, Haoli

2017-10-08

The plant hormone auxin plays pivotal roles in many aspects of plant growth and development. The auxin/indole-3-acetic acid (Aux/IAA) gene family encodes short-lived nuclear proteins acting on auxin perception and signaling, but the evolutionary history of this gene family remains to be elucidated. In this study, the Aux/IAA gene family in 17 plant species covering all major lineages of plants is identified and analyzed by using multiple bioinformatics methods. A total of 434 Aux/IAA genes was found among these plant species, and the gene copy number ranges from three ( Physcomitrella patens ) to 63 ( Glycine max ). The phylogenetic analysis shows that the canonical Aux/IAA proteins can be generally divided into five major clades, and the origin of Aux/IAA proteins could be traced back to the common ancestor of land plants and green algae. Many truncated Aux/IAA proteins were found, and some of these truncated Aux/IAA proteins may be generated from the C-terminal truncation of auxin response factor (ARF) proteins. Our results indicate that tandem and segmental duplications play dominant roles for the expansion of the Aux/IAA gene family mainly under purifying selection. The putative nuclear localization signals (NLSs) in Aux/IAA proteins are conservative, and two kinds of new primordial bipartite NLSs in P. patens and Selaginella moellendorffii were discovered. Our findings not only give insights into the origin and expansion of the Aux/IAA gene family, but also provide a basis for understanding their functions during the course of evolution.

Evolution Analysis of the Aux/IAA Gene Family in Plants Shows Dual Origins and Variable Nuclear Localization Signals

PubMed Central

Wu, Wentao; Liu, Yaxue; Wang, Yuqian; Li, Huimin; Liu, Jiaxi; Tan, Jiaxin; He, Jiadai; Bai, Jingwen

2017-01-01

The plant hormone auxin plays pivotal roles in many aspects of plant growth and development. The auxin/indole-3-acetic acid (Aux/IAA) gene family encodes short-lived nuclear proteins acting on auxin perception and signaling, but the evolutionary history of this gene family remains to be elucidated. In this study, the Aux/IAA gene family in 17 plant species covering all major lineages of plants is identified and analyzed by using multiple bioinformatics methods. A total of 434 Aux/IAA genes was found among these plant species, and the gene copy number ranges from three (Physcomitrella patens) to 63 (Glycine max). The phylogenetic analysis shows that the canonical Aux/IAA proteins can be generally divided into five major clades, and the origin of Aux/IAA proteins could be traced back to the common ancestor of land plants and green algae. Many truncated Aux/IAA proteins were found, and some of these truncated Aux/IAA proteins may be generated from the C-terminal truncation of auxin response factor (ARF) proteins. Our results indicate that tandem and segmental duplications play dominant roles for the expansion of the Aux/IAA gene family mainly under purifying selection. The putative nuclear localization signals (NLSs) in Aux/IAA proteins are conservative, and two kinds of new primordial bipartite NLSs in P. patens and Selaginella moellendorffii were discovered. Our findings not only give insights into the origin and expansion of the Aux/IAA gene family, but also provide a basis for understanding their functions during the course of evolution. PMID:28991190

Periodic expression of nuclear and mitochondrial DNA replication genes during the trypanosomatid cell cycle.

PubMed

Pasion, S G; Brown, G W; Brown, L M; Ray, D S

1994-12-01

In trypanosomatids, DNA replication in the nucleus and in the single mitochondrion (or kinetoplast) initiates nearly simultaneously, suggesting that the DNA synthesis (S) phases of the nucleus and the mitochondrion are coordinately regulated. To investigate the basis for the temporal link between nuclear and mitochondrial DNA synthesis phases the expression of the genes encoding DNA ligase I, the 51 and 28 kDa subunits of replication protein A, dihydrofolate reductase and the mitochondrial type II topoisomerase were analyzed during the cell cycle progression of synchronous cultures of Crithidia fasciculata. These DNA replication genes were all expressed periodically, with peak mRNA levels occurring just prior to or at the peak of DNA synthesis in the synchronized cultures. A plasmid clone (pdN-1) in which TOP2, the gene encoding the mitochondrial topoisomerase, was disrupted by the insertion of a NEO drug-resistance cassette was found to express both a truncated TOP2 mRNA and a truncated topoisomerase polypeptide. The truncated mRNA was also expressed periodically coordinate with the expression of the endogenous TOP2 mRNA indicating that cis elements necessary for periodic expression are contained within cloned sequences. The expression of both TOP2 and nuclear DNA replication genes at the G1/S boundary suggests that regulated expression of these genes may play a role in coordinating nuclear and mitochondrial S phases in trypanosomatids.

Tyrosine 110 in the measles virus phosphoprotein is required to block STAT1 phosphorylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Devaux, Patricia; Messling, Veronika von; Songsungthong, Warangkhana

2007-03-30

The measles virus (MV) P gene encodes three proteins: P, an essential polymerase cofactor, and C and V, which have multiple functions including immune evasion. We show here that the MV P protein also contributes to immune evasion, and that tyrosine 110 is required to block nuclear translocation of the signal transducer and activator of transcription factors (STAT) after interferon type I treatment. In particular, MV P inhibits STAT1 phosphorylation. This is shown not only by transient expression but also by reverse genetic analyses based on a new functional infectious cDNA derived from a MV vaccine vial (Moraten strain). Ourmore » study also identifies a conserved sequence around P protein tyrosine 110 as a candidate interaction site with a cellular protein.« less

Nuclear magnetic resonance-based model of a TF1/HmU-DNA complex.

PubMed

Silva, M V; Pasternack, L B; Kearns, D R

1997-12-15

Transcription factor 1 (TF1), a type II DNA-binding protein encoded by the Bacillus subtilis bacteriophage SPO1, has the capacity for sequence-selective DNA binding and a preference for 5-hydroxymethyl-2'-deoxyuridine (HmU)-containing DNA. In NMR studies of the TF1/HmU-DNA complex, intermolecular NOEs indicate that the flexible beta-ribbon and C-terminal alpha-helix are involved in the DNA-binding site of TF1, placing it in the beta-sheet category of DNA-binding proteins proposed to bind by wrapping two beta-ribbon "arms" around the DNA. Intermolecular and intramolecular NOEs were used to generate an energy-minimized model of the protein-DNA complex in which both DNA bending and protein structure changes are evident.

Methods to study the biogenesis of membrane proteins in yeast mitochondria.

PubMed

Weckbecker, Daniel; Herrmann, Johannes M

2013-01-01

The biogenesis of mitochondrial membrane proteins is an intricate process that relies on the import and submitochondrial sorting of nuclear-encoded preproteins and on the synthesis of mitochondrial translation products in the matrix. Subsequently, these polypeptides need to be inserted into the outer and the inner membranes of the organelle where many of them assemble into multisubunit complexes. In this chapter we provide established protocols to study these different processes experimentally using mitochondria of budding yeast. In particular, methods are described in detail to purify mitochondria, to study mitochondrial protein synthesis, to follow the import of radiolabeled preproteins into isolated mitochondria, and to assess membrane association and the aggregation of mitochondrial proteins by fractionation. These protocols and a list of dos and don'ts shall enable beginners and experienced scientists to address the targeting and assembly of mitochondrial membrane proteins.

Reduction of nuclear encoded enzymes of mitochondrial energy metabolism in cells devoid of mitochondrial DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mueller, Edith E., E-mail: ed.mueller@salk.at; Mayr, Johannes A., E-mail: h.mayr@salk.at; Zimmermann, Franz A., E-mail: f.zimmermann@salk.at

2012-01-20

Highlights: Black-Right-Pointing-Pointer We examined OXPHOS and citrate synthase enzyme activities in HEK293 cells devoid of mtDNA. Black-Right-Pointing-Pointer Enzymes partially encoded by mtDNA show reduced activities. Black-Right-Pointing-Pointer Also the entirely nuclear encoded complex II and citrate synthase exhibit reduced activities. Black-Right-Pointing-Pointer Loss of mtDNA induces a feedback mechanism that downregulates complex II and citrate synthase. -- Abstract: Mitochondrial DNA (mtDNA) depletion syndromes are generally associated with reduced activities of oxidative phosphorylation (OXPHOS) enzymes that contain subunits encoded by mtDNA. Conversely, entirely nuclear encoded mitochondrial enzymes in these syndromes, such as the tricarboxylic acid cycle enzyme citrate synthase (CS) and OXPHOS complexmore » II, usually exhibit normal or compensatory enhanced activities. Here we report that a human cell line devoid of mtDNA (HEK293 {rho}{sup 0} cells) has diminished activities of both complex II and CS. This finding indicates the existence of a feedback mechanism in {rho}{sup 0} cells that downregulates the expression of entirely nuclear encoded components of mitochondrial energy metabolism.« less

tRNA travels from the cytoplasm to organelles

PubMed Central

Rubio, Mary Anne T.; Hopper, Anita K.

2011-01-01

Transfer RNAs (tRNAs) encoded by the nuclear genome are surprisingly dynamic. Although tRNAs function in protein synthesis occurring on cytoplasmic ribosomes, tRNAs can transit from the cytoplasm to the nucleus and then again return to the cytoplasm by a process known as the tRNA retrograde process. Subsets of the cytoplasmic tRNAs are also imported into mitochondria and function in mitochondrial protein synthesis. The numbers of tRNA species that are imported into mitchondria differ among organisms, ranging from just a few to the entire set needed to decode mitochondrially encoded mRNAs. For some tRNAs, import is dependent on the mitochondrial protein import machinery, whereas the majority of tRNA mitochondrial import is independent of this machinery. Although cytoplasmic proteins and proteins located on the mitochondrial surface participating in the tRNA import process have been described for several organisms, the identity of these proteins differ among organisms. Likewise, the tRNA determinants required for mitochondrial import differ among tRNA species and organisms. Here, we present an overview and discuss the current state of knowledge regarding the mechanisms involved in the tRNA retrograde process and continue with an overview of tRNA import into mitochondria. Finally, we highlight areas of future research to understand the function and regulation of movement of tRNAs between the cytoplasm and organelles. PMID:21976284

Mutations in glycyl-tRNA synthetase impair mitochondrial metabolism in neurons.

PubMed

Boczonadi, Veronika; Meyer, Kathrin; Gonczarowska-Jorge, Humberto; Griffin, Helen; Roos, Andreas; Bartsakoulia, Marina; Bansagi, Boglarka; Ricci, Giulia; Palinkas, Fanni; Zahedi, René P; Bruni, Francesco; Kaspar, Brian; Lochmüller, Hanns; Boycott, Kym M; Müller, Juliane S; Horvath, Rita

2018-06-15

The nuclear-encoded glycyl-tRNA synthetase gene (GARS) is essential for protein translation in both cytoplasm and mitochondria. In contrast, different genes encode the mitochondrial and cytosolic forms of most other tRNA synthetases. Dominant GARS mutations were described in inherited neuropathies, while recessive mutations cause severe childhood-onset disorders affecting skeletal muscle and heart. The downstream events explaining tissue-specific phenotype-genotype relations remained unclear. We investigated the mitochondrial function of GARS in human cell lines and in the GarsC210R mouse model. Human-induced neuronal progenitor cells (iNPCs) carrying dominant and recessive GARS mutations showed alterations of mitochondrial proteins, which were more prominent in iNPCs with dominant, neuropathy-causing mutations. Although comparative proteomic analysis of iNPCs showed significant changes in mitochondrial respiratory chain complex subunits, assembly genes, Krebs cycle enzymes and transport proteins in both recessive and dominant mutations, proteins involved in fatty acid oxidation were only altered by recessive mutations causing mitochondrial cardiomyopathy. In contrast, significant alterations of the vesicle-associated membrane protein-associated protein B (VAPB) and its downstream pathways such as mitochondrial calcium uptake and autophagy were detected in dominant GARS mutations. The role of VAPB has been supported by similar results in the GarsC210R mice. Our data suggest that altered mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) may be important disease mechanisms leading to neuropathy in this condition.

Cleavage of bovine adenovirus type 3 non-structural 100K protein by protease is required for nuclear localization in infected cells but is not essential for virus replication.

PubMed

Makadiya, Nirajkumar; Gaba, Amit; Tikoo, Suresh K

2015-09-01

The L6 region of bovine adenovirus type 3 (BAdV-3) encodes a non-structural protein named 100K. Rabbit antiserum raised against BAdV-3 100K recognized a protein of 130 kDa at 12-24 h and proteins of 130, 100, 95 and 15 kDa at 36-48 h after BAdV-3 infection. The 100K species localized to the nucleus and the cytoplasm of BAdV-3-infected cells. In contrast, 100K localized predominantly to the cytoplasm of the transfected cells. However, BAdV-3 infection of cells transfected with 100K-enhanced yellow fluorescent protein-expressing plasmid detected fluorescent protein in the nucleus of the cells, suggesting that other viral proteins may be required for the nuclear localization of 100K. Interaction of BAdV-3 100K with BAdV-3 33K protein did not alter the cytoplasmic localization of 100K. However, co-expression of BAdV-3 100K and BAdV-3 protease localized 100K to the nucleolus of the transfected cells. Subsequent analysis suggested that BAdV-3 protease cleaves 100K at two identified potential protease cleavage sites (aa 740-745 and 781-786) in transfected or BAdV-3-infected cells. The cleaved C terminus (107 aa) was localized to the nucleolus of the transfected cells. Further analysis suggested that the cleaved C terminus contains a bipartite nuclear localization signal and utilizes import receptor importin-α3 of the classical importin-α/β transport pathway for nuclear transport. Successful isolation of recombinant BAdV-3 expressing mutant 100K (substitution of alanine for glycine in the potential protease cleavage site) suggested that cytoplasmic cleavage of BAdV-3 100K by adenoviral protease is not essential for virus replication.

FANCJ/BRIP1 recruitment and regulation of FANCD2 in DNA damage responses

PubMed Central

Zhang, Fan; Fan, Qiang; Ren, Keqin; Auerbach, Arleen D.; Andreassen, Paul R.

2016-01-01

FANCJ/BRIP1 encodes a helicase that has been implicated in the maintenance of genomic stability. Here, to better understand FANCJ function in DNA damage responses, we have examined the regulation of its cellular localization. FANCJ nuclear foci assemble spontaneously during S phase and are induced by various stresses. FANCJ foci colocalize with the replication fork following treatment with hydroxyurea, but not spontaneously. Using FANCJ mutants, we find that FANCJ helicase activity and the capacity to bind BRCA1 are both involved in FANCJ recruitment. Given similarities to the recruitment of another Fanconi anemia protein, FANCD2, we tested for colocalization of FANCJ and FANCD2. Importantly, these proteins show substantial colocalization, and FANCJ promotes the assembly of FANCD2 nuclear foci. This process is linked to the proper localization of FANCJ itself since both FANCJ and FANCD2 nuclear foci are compromised by FANCJ mutants that abrogate its helicase activity or interaction with BRCA1. Our results suggest that FANCJ is recruited in response to replication stress and that FANCJ/BRIP1 may serve to link FANCD2 to BRCA1. PMID:20676667

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bandyopadhyay, Anindya; Kopperud, Kristin; Anderson, Gavin

The genome of Potato yellow dwarf virus (PYDV; Nucleorhabdovirus type species) was determined to be 12,875 nucleotides (nt). The antigenome is organized into seven open reading frames (ORFs) ordered 3'-N-X-P-Y-M-G-L-5', which likely encode the nucleocapsid, phospho, movement, matrix, glyco and RNA-dependent RNA polymerase proteins, respectively, except for X, which is of unknown function. The ORFs are flanked by a 3' leader RNA of 149 nt and a 5' trailer RNA of 97 nt, and are separated by conserved intergenic junctions. Phylogenetic analyses indicated that PYDV is closely related to other leafhopper-transmitted rhabdoviruses. Functional protein assays were used to determine themore » subcellular localization of PYDV proteins. Surprisingly, the M protein was able to induce the intranuclear accumulation of the inner nuclear membrane in the absence of any other viral protein. Finally, bimolecular fluorescence complementation was used to generate the most comprehensive protein interaction map for a plant-adapted rhabdovirus to date.« less

Protein Tertiary Structure Prediction Based on Main Chain Angle Using a Hybrid Bees Colony Optimization Algorithm

NASA Astrophysics Data System (ADS)

Mahmood, Zakaria N.; Mahmuddin, Massudi; Mahmood, Mohammed Nooraldeen

Encoding proteins of amino acid sequence to predict classified into their respective families and subfamilies is important research area. However for a given protein, knowing the exact action whether hormonal, enzymatic, transmembranal or nuclear receptors does not depend solely on amino acid sequence but on the way the amino acid thread folds as well. This study provides a prototype system that able to predict a protein tertiary structure. Several methods are used to develop and evaluate the system to produce better accuracy in protein 3D structure prediction. The Bees Optimization algorithm which inspired from the honey bees food foraging method, is used in the searching phase. In this study, the experiment is conducted on short sequence proteins that have been used by the previous researches using well-known tools. The proposed approach shows a promising result.

The primary structure of L37--a rat ribosomal protein with a zinc finger-like motif.

PubMed

Chan, Y L; Paz, V; Olvera, J; Wool, I G

1993-04-30

The amino acid sequence of the rat 60S ribosomal subunit protein L37 was deduced from the sequence of nucleotides in a recombinant cDNA. Ribosomal protein L37 has 96 amino acids, the NH2-terminal methionine is removed after translation of the mRNA, and has a molecular weight of 10,939. Ribosomal protein L37 has a single zinc finger-like motif of the C2-C2 type. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 13 or 14 copies of the L37 gene. The mRNA for the protein is about 500 nucleotides in length. Rat L37 is related to Saccharomyces cerevisiae ribosomal protein YL35 and to Caenorhabditis elegans L37. We have identified in the data base a DNA sequence that encodes the chicken homolog of rat L37.

The unusually large Plasmodium telomerase reverse-transcriptase localizes in a discrete compartment associated with the nucleolus

PubMed Central

Figueiredo, Luisa M.; Rocha, Eduardo P. C.; Mancio-Silva, Liliana; Prevost, Christine; Hernandez-Verdun, Danièle; Scherf, Artur

2005-01-01

Telomerase replicates chromosome ends, a function necessary for maintaining genome integrity. We have identified the gene that encodes the catalytic reverse transcriptase (RT) component of this enzyme in the malaria parasite Plasmodium falciparum (PfTERT) as well as the orthologous genes from two rodent and one simian malaria species. PfTERT is predicted to encode a basic protein that contains the major sequence motifs previously identified in known telomerase RTs (TERTs). At ∼2500 amino acids, PfTERT is three times larger than other characterized TERTs. We observed remarkable sequence diversity between TERT proteins of different Plasmodial species, with conserved domains alternating with hypervariable regions. Immunofluorescence analysis revealed that PfTERT is expressed in asexual blood stage parasites that have begun DNA synthesis. Surprisingly, rather than at telomere clusters, PfTERT typically localizes into a discrete nuclear compartment. We further demonstrate that this compartment is associated with the nucleolus, hereby defined for the first time in P.falciparum. PMID:15722485

Inhibition of U snRNP assembly by a virus-encoded proteinase.

PubMed

Almstead, Laura L; Sarnow, Peter

2007-05-01

It has been proposed that defects in the assembly of spliceosomal uridine-rich small nuclear ribonucleoprotein (U snRNP) complexes could account for the death of motor neurons in spinal muscular atrophy (SMA). We discovered that infection of cultured cells with poliovirus results in the specific cleavage of the host factor Gemin3 by a virus-encoded proteinase, 2A(pro). Gemin3 is a component of the macromolecular SMN complex that mediates assembly of U snRNP complexes by aiding the heptameric oligomerization of Sm proteins onto U snRNAs. Using in vitro Sm core assembly assays, we found that lowering the intracellular amounts of Gemin3 by either poliovirus infection or small interfering RNA (siRNA)-mediated knockdown of Gemin3 resulted in reduced assembly of U snRNPs. Immunofluorescence analyses revealed a specific redistribution of Sm proteins from the nucleoplasm to the cytoplasmic periphery of the nucleus in poliovirus-infected cells. We propose that defects in U snRNP assembly may be shared features of SMA and poliomyelitis.

Developmental Regulation of Genes Encoding Universal Stress Proteins in Schistosoma mansoni

PubMed Central

Isokpehi, Raphael D.; Mahmud, Ousman; Mbah, Andreas N.; Simmons, Shaneka S.; Avelar, Lívia; Rajnarayanan, Rajendram V.; Udensi, Udensi K.; Ayensu, Wellington K.; Cohly, Hari H.; Brown, Shyretha D.; Dates, Centdrika R.; Hentz, Sonya D.; Hughes, Shawntae J.; Smith-McInnis, Dominique R.; Patterson, Carvey O.; Sims, Jennifer N.; Turner, Kelisha T.; Williams, Baraka S.; Johnson, Matilda O.; Adubi, Taiwo; Mbuh, Judith V.; Anumudu, Chiaka I.; Adeoye, Grace O.; Thomas, Bolaji N.; Nashiru, Oyekanmi; Oliveira, Guilherme

2011-01-01

The draft nuclear genome sequence of the snail-transmitted, dimorphic, parasitic, platyhelminth Schistosoma mansoni revealed eight genes encoding proteins that contain the Universal Stress Protein (USP) domain. Schistosoma mansoni is a causative agent of human schistosomiasis, a severe and debilitating Neglected Tropical Disease (NTD) of poverty, which is endemic in at least 76 countries. The availability of the genome sequences of Schistosoma species presents opportunities for bioinformatics and genomics analyses of associated gene families that could be targets for understanding schistosomiasis ecology, intervention, prevention and control. Proteins with the USP domain are known to provide bacteria, archaea, fungi, protists and plants with the ability to respond to diverse environmental stresses. In this research investigation, the functional annotations of the USP genes and predicted nucleotide and protein sequences were initially verified. Subsequently, sequence clusters and distinctive features of the sequences were determined. A total of twelve ligand binding sites were predicted based on alignment to the ATP-binding universal stress protein from Methanocaldococcus jannaschii. In addition, six USP sequences showed the presence of ATP-binding motif residues indicating that they may be regulated by ATP. Public domain gene expression data and RT-PCR assays confirmed that all the S. mansoni USP genes were transcribed in at least one of the developmental life cycle stages of the helminth. Six of these genes were up-regulated in the miracidium, a free-swimming stage that is critical for transmission to the snail intermediate host. It is possible that during the intra-snail stages, S. mansoni gene transcripts for universal stress proteins are low abundant and are induced to perform specialized functions triggered by environmental stressors such as oxidative stress due to hydrogen peroxide that is present in the snail hemocytes. This report serves to catalyze the formation of a network of researchers to understand the function and regulation of the universal stress proteins encoded in genomes of schistosomes and their snail intermediate hosts. PMID:22084571

Salvaging hope: Is increasing NAD+ a key to treating mitochondrial myopathy?

PubMed Central

Lightowlers, Robert N; Chrzanowska-Lightowlers, Zofia MA

2014-01-01

Mitochondrial diseases can arise from mutations either in mitochondrial DNA or in nuclear DNA encoding mitochondrially destined proteins. Currently, there is no cure for these diseases although treatments to ameliorate a subset of the symptoms are being developed. In this issue of EMBO Molecular Medicine, Khan et al (2014) use a mouse model to test the efficacy of a simple dietary supplement of nicotinamide riboside to treat and prevent mitochondrial myopathies. PMID:24838280

Genome-wide analysis of signal transducers and regulators of mitochondrial dysfunction in Saccharomyces cerevisiae.

PubMed

Singh, Keshav K; Rasmussen, Anne Karin; Rasmussen, Lene Juel

2004-04-01

Mitochondrial dysfunction is a hallmark of cancer cells. However, genetic response to mitochondrial dysfunction during carcinogenesis is unknown. To elucidate genetic response to mitochondrial dysfunction we used Saccharomyces cerevisiae as a model system. We analyzed genome-wide expression of nuclear genes involved in signal transduction and transcriptional regulation in a wild-type yeast and a yeast strain lacking the mitochondrial genome (rho(0)). Our analysis revealed that the gene encoding cAMP-dependent protein kinase subunit 3 (PKA3) was upregulated. However, the gene encoding cAMP-dependent protein kinase subunit 2 (PKA2) and the VTC1, PTK2, TFS1, CMK1, and CMK2 genes, involved in signal transduction, were downregulated. Among the known transcriptional factors, OPI1, MIG2, INO2, and ROX1 belonged to the upregulated genes, whereas MSN4, MBR1, ZMS1, ZAP1, TFC3, GAT1, ADR1, CAT8, and YAP4 including RFA1 were downregulated. RFA1 regulates DNA repair genes at the transcriptional level. RFA is also involved directly in DNA recombination, DNA replication, and DNA base excision repair. Downregulation of RFA1 in rho(0) cells is consistent with our finding that mitochondrial dysfunction leads to instability of the nuclear genome. Together, our data suggest that gene(s) involved in mitochondria-to-nucleus communication play a role in mutagenesis and may be implicated in carcinogenesis.

Genome Sequence and Transcriptome Analyses of Chrysochromulina tobin: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae)

PubMed Central

Hovde, Blake T.; Deodato, Chloe R.; Hunsperger, Heather M.; Ryken, Scott A.; Yost, Will; Jha, Ramesh K.; Patterson, Johnathan; Monnat, Raymond J.; Barlow, Steven B.; Starkenburg, Shawn R.; Cattolico, Rose Ann

2015-01-01

Haptophytes are recognized as seminal players in aquatic ecosystem function. These algae are important in global carbon sequestration, form destructive harmful blooms, and given their rich fatty acid content, serve as a highly nutritive food source to a broad range of eco-cohorts. Haptophyte dominance in both fresh and marine waters is supported by the mixotrophic nature of many taxa. Despite their importance the nuclear genome sequence of only one haptophyte, Emiliania huxleyi (Isochrysidales), is available. Here we report the draft genome sequence of Chrysochromulina tobin (Prymnesiales), and transcriptome data collected at seven time points over a 24-hour light/dark cycle. The nuclear genome of C. tobin is small (59 Mb), compact (∼40% of the genome is protein coding) and encodes approximately 16,777 genes. Genes important to fatty acid synthesis, modification, and catabolism show distinct patterns of expression when monitored over the circadian photoperiod. The C. tobin genome harbors the first hybrid polyketide synthase/non-ribosomal peptide synthase gene complex reported for an algal species, and encodes potential anti-microbial peptides and proteins involved in multidrug and toxic compound extrusion. A new haptophyte xanthorhodopsin was also identified, together with two “red” RuBisCO activases that are shared across many algal lineages. The Chrysochromulina tobin genome sequence provides new information on the evolutionary history, ecology and economic importance of haptophytes. PMID:26397803

Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export.

PubMed

Behrens, Ryan T; Aligeti, Mounavya; Pocock, Ginger M; Higgins, Christina A; Sherer, Nathan M

2017-02-01

HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding. HIV-1 infects more than 34 million people worldwide causing >1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place. Copyright © 2017 American Society for Microbiology.

Nuclear Export Signal Masking Regulates HIV-1 Rev Trafficking and Viral RNA Nuclear Export

PubMed Central

Behrens, Ryan T.; Aligeti, Mounavya; Pocock, Ginger M.; Higgins, Christina A.

2016-01-01

ABSTRACT HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding. IMPORTANCE HIV-1 infects more than 34 million people worldwide causing >1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place. PMID:27852860

A heterogeneous population of nuclear-encoded mitochondrial mRNAs is present in the axons of primary sympathetic neurons.

PubMed

Aschrafi, Armaz; Kar, Amar N; Gale, Jenna R; Elkahloun, Abdel G; Vargas, Jose Noberto S; Sales, Naomi; Wilson, Gabriel; Tompkins, Miranda; Gioio, Anthony E; Kaplan, Barry B

2016-09-01

Mitochondria are enriched in subcellular regions of high energy consumption, such as axons and pre-synaptic nerve endings. Accumulating evidence suggests that mitochondrial maintenance in these distal structural/functional domains of the neuron depends on the "in-situ" translation of nuclear-encoded mitochondrial mRNAs. In support of this notion, we recently provided evidence for the axonal targeting of several nuclear-encoded mRNAs, such as cytochrome c oxidase, subunit 4 (COXIV) and ATP synthase, H+ transporting and mitochondrial Fo complex, subunit C1 (ATP5G1). Furthermore, we showed that axonal trafficking and local translation of these mRNAs plays a critical role in the generation of axonal ATP. Using a global gene expression analysis, this study identified a highly diverse population of nuclear-encoded mRNAs that were enriched in the axon and presynaptic nerve terminals. Among this population of mRNAs, fifty seven were found to be at least two-fold more abundant in distal axons, as compared with the parental cell bodies. Gene ontology analysis of the nuclear-encoded mitochondrial mRNAs suggested functions for these gene products in molecular and biological processes, including but not limited to oxidoreductase and electron carrier activity and proton transport. Based on these results, we postulate that local translation of nuclear-encoded mitochondrial mRNAs present in the axons may play an essential role in local energy production and maintenance of mitochondrial function. Published by Elsevier B.V.

Axonal localization and mitochondrial association of precursor microRNA 338

PubMed Central

Vargas, Jose Norberto S.; Kar, Amar N.; Kowalak, Jeffrey A.; Gale, Jenna R.; Aschrafi, Armaz; Chen, Cai-Yun; Gioio, Anthony E.; Kaplan, Barry B.

2016-01-01

microRNAs (miRNAs) selectively localize to subcompartments of the neuron, such as dendrites, axons and presynaptic terminals, where they regulate the local protein synthesis of their putative target genes. In addition to mature miRNAs, precursor miRNAs (pre-miRNAs) have also been shown to localize to somatodendritic and axonal compartments. miRNA-338 (miR-338) regulates the local expression of several nuclear-encoded mitochondrial mRNAs within axons of sympathetic neurons. Previous work has shown that precursor miR-338 (pre-miR-338) introduced into the axon can be locally processed into mature miR-338, where it can regulate local ATP synthesis. However, the mechanisms underlying the localization of pre-miRNAs to the axonal compartment remain unknown. In this study, we investigated the axonal localization of pre-miR-338. Using proteomic and biochemical approaches, we provide evidence for the localization of pre-miR-338 to distal neuronal compartments and identify several constituents of the pre-miR-338 ribonucleoprotein complex. Furthermore, we found that pre-miR-338 is associated with the mitochondria in axons of superior cervical ganglion (SCG) neurons. The maintenance of mitochondrial function within axons requires the precise spatio-temporal synthesis of nuclear-encoded mRNAs, some of which are regulated by miR-338. Therefore, the association of pre-miR-338 with axonal mitochondria could serve as a reservoir of mature, biologically active miRNAs, which could coordinate the intra-axonal expression of multiple nuclear-encoded mitochondrial mRNAs. PMID:27229124

Inhibition of stress-inducible HSP70 impairs mitochondrial proteostasis and function.

PubMed

Leu, Julia I-Ju; Barnoud, Thibaut; Zhang, Gao; Tian, Tian; Wei, Zhi; Herlyn, Meenhard; Murphy, Maureen E; George, Donna L

2017-07-11

Protein quality control is an important component of survival for all cells. The use of proteasome inhibitors for cancer therapy derives from the fact that tumor cells generally exhibit greater levels of proteotoxic stress than do normal cells, and thus cancer cells tend to be more sensitive to proteasome inhibition. However, this approach has been limited in some cases by toxicity to normal cells. Recently, the concept of inhibiting proteostasis in organelles for cancer therapy has been advanced, in part because it is predicted to have reduced toxicity for normal cells. Here we demonstrate that a fraction of the major stress-induced chaperone HSP70 (also called HSPA1A or HSP72, but hereafter HSP70) is abundantly present in mitochondria of tumor cells, but is expressed at quite low or undetectable levels in mitochondria of most normal tissues and non-tumor cell lines. We show that treatment of tumor cells with HSP70 inhibitors causes a marked change in mitochondrial protein quality control, loss of mitochondrial membrane potential, reduced oxygen consumption rate, and loss of ATP production. We identify several nuclear-encoded mitochondrial proteins, including polyadenylate binding protein-1 (PABPC1), which exhibit decreased abundance in mitochondria following treatment with HSP70 inhibitors. We also show that targeting HSP70 function leads to reduced levels of several mitochondrial-encoded RNA species that encode components of the electron transport chain. Our data indicate that small molecule inhibitors of HSP70 represent a new class of organelle proteostasis inhibitors that impair mitochondrial function in cancer cells, and therefore constitute novel therapeutics.

Virus-Like Particles Derived from HIV-1 for Delivery of Nuclear Proteins: Improvement of Production and Activity by Protein Engineering.

PubMed

Robert, Marc-André; Lytvyn, Viktoria; Deforet, Francis; Gilbert, Rénald; Gaillet, Bruno

2017-01-01

Virus-like particles (VLPs) derived from retroviruses and lentiviruses can be used to deliver recombinant proteins without the fear of causing insertional mutagenesis to the host cell genome. In this study we evaluate the potential of an inducible lentiviral vector packaging cell line for VLP production. The Gag gene from HIV-1 was fused to a gene encoding a selected protein and it was transfected into the packaging cells. Three proteins served as model: the green fluorescent protein and two transcription factors-the cumate transactivator (cTA) of the inducible CR5 promoter and the human Krüppel-like factor 4 (KLF4). The sizes of the VLPs were 120-150 nm in diameter and they were resistant to freeze/thaw cycles. Protein delivery by the VLPs reached up to 100% efficacy in human cells and was well tolerated. Gag-cTA triggered up to 1100-fold gene activation of the reporter gene in comparison to the negative control. Protein engineering was required to detect Gag-KLF4 activity. Thus, insertion of the VP16 transactivation domain increased the activity of the VLPs by eightfold. An additional 2.4-fold enhancement was obtained by inserting nuclear export signal. In conclusion, our platform produced VLPs capable of efficient protein transfer, and it was shown that protein engineering can be used to improve the activity of the delivered proteins as well as VLP production.

A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif

NASA Technical Reports Server (NTRS)

Reddy, A. S.; Reddy, V. S.; Golovkin, M.

2000-01-01

Calmodulin (CaM), a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that interacts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcription factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similarity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high sequence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.

Selective nuclear export of specific classes of mRNA from mammalian nuclei is promoted by GANP

PubMed Central

Wickramasinghe, Vihandha O.; Andrews, Robert; Ellis, Peter; Langford, Cordelia; Gurdon, John B.; Stewart, Murray; Venkitaraman, Ashok R.; Laskey, Ronald A.

2014-01-01

The nuclear phase of the gene expression pathway culminates in the export of mature messenger RNAs (mRNAs) to the cytoplasm through nuclear pore complexes. GANP (germinal- centre associated nuclear protein) promotes the transfer of mRNAs bound to the transport factor NXF1 to nuclear pore complexes. Here, we demonstrate that GANP, subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex, promotes selective nuclear export of a specific subset of mRNAs whose transport depends on NXF1. Genome-wide gene expression profiling showed that half of the transcripts whose nuclear export was impaired following NXF1 depletion also showed reduced export when GANP was depleted. GANP-dependent transcripts were highly expressed, yet short-lived, and were highly enriched in those encoding central components of the gene expression machinery such as RNA synthesis and processing factors. After injection into Xenopus oocyte nuclei, representative GANP-dependent transcripts showed faster nuclear export kinetics than representative transcripts that were not influenced by GANP depletion. We propose that GANP promotes the nuclear export of specific classes of mRNAs that may facilitate rapid changes in gene expression. PMID:24510098

The Thoc1 Encoded Ribonucleoprotein Is Required for Myeloid Progenitor Cell Homeostasis in the Adult Mouse

PubMed Central

Chinnam, Meenalakshmi; Povinelli, Benjamin J.; Fisher, Daniel T.; Golding, Michelle; Appenheimer, Michelle M.; Nemeth, Michael J.; Evans, Sharon; Goodrich, David W.

2014-01-01

Co-transcriptionally assembled ribonucleoprotein (RNP) complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover. PMID:24830368

The Thoc1 encoded ribonucleoprotein is required for myeloid progenitor cell homeostasis in the adult mouse.

PubMed

Pitzonka, Laura; Ullas, Sumana; Chinnam, Meenalakshmi; Povinelli, Benjamin J; Fisher, Daniel T; Golding, Michelle; Appenheimer, Michelle M; Nemeth, Michael J; Evans, Sharon; Goodrich, David W

2014-01-01

Co-transcriptionally assembled ribonucleoprotein (RNP) complexes are critical for RNA processing and nuclear export. RNPs have been hypothesized to contribute to the regulation of coordinated gene expression, and defects in RNP biogenesis contribute to genome instability and disease. Despite the large number of RNPs and the importance of the molecular processes they mediate, the requirements for individual RNP complexes in mammalian development and tissue homeostasis are not well characterized. THO is an evolutionarily conserved, nuclear RNP complex that physically links nascent transcripts with the nuclear export apparatus. THO is essential for early mouse embryonic development, limiting characterization of the requirements for THO in adult tissues. To address this shortcoming, a mouse strain has been generated allowing inducible deletion of the Thoc1 gene which encodes an essential protein subunit of THO. Bone marrow reconstitution was used to generate mice in which Thoc1 deletion could be induced specifically in the hematopoietic system. We find that granulocyte macrophage progenitors have a cell autonomous requirement for Thoc1 to maintain cell growth and viability. Lymphoid lineages are not detectably affected by Thoc1 loss under the homeostatic conditions tested. Myeloid lineages may be more sensitive to Thoc1 loss due to their relatively high rate of proliferation and turnover.

Psr1, a nuclear localized protein that regulates phosphorus metabolism in Chlamydomonas.

PubMed

Wykoff, D D; Grossman, A R; Weeks, D P; Usuda, H; Shimogawara, K

1999-12-21

Understanding the ways in which phosphorus metabolism is regulated in photosynthetic eukaryotes is critical for optimizing crop productivity and managing aquatic ecosystems in which phosphorus can be a major source of pollution. Here we describe a gene encoding a regulator of phosphorus metabolism, designated Psr1 (phosphorus starvation response), from a photosynthetic eukaryote. The Psr1 protein is critical for acclimation of the unicellular green alga Chlamydomonas reinhardtii to phosphorus starvation. The N-terminal half of Psr1 contains a region similar to myb DNA-binding domains and the C-terminal half possesses glutamine-rich sequences characteristic of transcriptional activators. The level of Psr1 increases at least 10-fold upon phosphate starvation, and immunocytochemical studies demonstrate that this protein is nuclear-localized under both nutrient-replete and phosphorus-starvation conditions. Finally, Psr1 and angiosperm proteins have domains that are similar, suggesting a possible role for Psr1 homologs in the control of phosphorus metabolism in vascular plants. With the identification of regulators such as Psr1 it may become possible to engineer photosynthetic organisms for more efficient utilization of phosphorus and to establish better practices for the management of agricultural lands and natural ecosystems.

Identification of a functional element in the promoter of the silkworm (Bombyx mori) fat body-specific gene Bmlp3.

PubMed

Xu, Hanfu; Deng, Dangjun; Yuan, Lin; Wang, Yuancheng; Wang, Feng; Xia, Qingyou

2014-08-01

30K proteins are a group of structurally related proteins that play important roles in the life cycle of the silkworm Bombyx mori and are largely synthesized and regulated in a time-dependent manner in the fat body. Little is known about the upstream regulatory elements associated with the genes encoding these proteins. In the present study, the promoter of Bmlp3, a fat body-specific gene encoding a 30K protein family member, was characterized by joining sequences containing the Bmlp3 promoter with various amounts of 5' upstream sequences to a luciferase reporter gene. The results indicated that the sequences from -150 to -250bp and -597 to -675bp upstream of the Bmlp3 transcription start site were necessary for high levels of luciferase activity. Further analysis showed that a 21-bp sequence located between -230 and -250 was specifically recognized by nuclear factors from silkworm fat bodies and BmE cells, and could enhance luciferase reporter-gene expression 2.8-fold in BmE cells. This study provides new insights into the Bmlp3 promoter and contributes to the further clarification of the function and developmental regulation of Bmlp3. Copyright © 2014. Published by Elsevier B.V.

Recent advances in understanding hematopoiesis in Fanconi Anemia

PubMed Central

Bagby, Grover

2018-01-01

Fanconi anemia is an inherited disease characterized by genomic instability, hypersensitivity to DNA cross-linking agents, bone marrow failure, short stature, skeletal abnormalities, and a high relative risk of myeloid leukemia and epithelial malignancies. The 21 Fanconi anemia genes encode proteins involved in multiple nuclear biochemical pathways that effect DNA interstrand crosslink repair. In the past, bone marrow failure was attributed solely to the failure of stem cells to repair DNA. Recently, non-canonical functions of many of the Fanconi anemia proteins have been described, including modulating responses to oxidative stress, viral infection, and inflammation as well as facilitating mitophagic responses and enhancing signals that promote stem cell function and survival. Some of these functions take place in non-nuclear sites and do not depend on the DNA damage response functions of the proteins. Dysfunctions of the canonical and non-canonical pathways that drive stem cell exhaustion and neoplastic clonal selection are reviewed, and the potential therapeutic importance of fully investigating the scope and interdependences of the canonical and non-canonical pathways is emphasized. PMID:29399332

Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica

PubMed Central

Rumpho, Mary E.; Worful, Jared M.; Lee, Jungho; Kannan, Krishna; Tyler, Mary S.; Bhattacharya, Debashish; Moustafa, Ahmed; Manhart, James R.

2008-01-01

The sea slug Elysia chlorotica acquires plastids by ingestion of its algal food source Vaucheria litorea. Organelles are sequestered in the mollusc's digestive epithelium, where they photosynthesize for months in the absence of algal nucleocytoplasm. This is perplexing because plastid metabolism depends on the nuclear genome for >90% of the needed proteins. Two possible explanations for the persistence of photosynthesis in the sea slug are (i) the ability of V. litorea plastids to retain genetic autonomy and/or (ii) more likely, the mollusc provides the essential plastid proteins. Under the latter scenario, genes supporting photosynthesis have been acquired by the animal via horizontal gene transfer and the encoded proteins are retargeted to the plastid. We sequenced the plastid genome and confirmed that it lacks the full complement of genes required for photosynthesis. In support of the second scenario, we demonstrated that a nuclear gene of oxygenic photosynthesis, psbO, is expressed in the sea slug and has integrated into the germline. The source of psbO in the sea slug is V. litorea because this sequence is identical from the predator and prey genomes. Evidence that the transferred gene has integrated into sea slug nuclear DNA comes from the finding of a highly diverged psbO 3′ flanking sequence in the algal and mollusc nuclear homologues and gene absence from the mitochondrial genome of E. chlorotica. We demonstrate that foreign organelle retention generates metabolic novelty (“green animals”) and is explained by anastomosis of distinct branches of the tree of life driven by predation and horizontal gene transfer. PMID:19004808

SPK1 is an essential S-phase-specific gene of Saccharomyces cerevisiae that encodes a nuclear serine/threonine/tyrosine kinase.

PubMed

Zheng, P; Fay, D S; Burton, J; Xiao, H; Pinkham, J L; Stern, D F

1993-09-01

SPK1 was originally discovered in an immunoscreen for tyrosine-protein kinases in Saccharomyces cerevisiae. We have used biochemical and genetic techniques to investigate the function of this gene and its encoded protein. Hybridization of an SPK1 probe to an ordered genomic library showed that SPK1 is adjacent to PEP4 (chromosome XVI L). Sporulation of spk1/+ heterozygotes gave rise to spk1 spores that grew into microcolonies but could not be further propagated. These colonies were greatly enriched for budded cells, especially those with large buds. Similarly, eviction of CEN plasmids bearing SPK1 from cells with a chromosomal SPK1 disruption yielded viable cells with only low frequency. Spk1 protein was identified by immunoprecipitation and immunoblotting. It was associated with protein-Ser, Thr, and Tyr kinase activity in immune complex kinase assays. Spk1 was localized to the nucleus by immunofluorescence. The nucleotide sequence of the SPK1 5' noncoding region revealed that SPK1 contains two MluI cell cycle box elements. These elements confer S-phase-specific transcription to many genes involved in DNA synthesis. Northern (RNA) blotting of synchronized cells verified that the SPK1 transcript is coregulated with other MluI box-regulated genes. The SPK1 upstream region also includes a domain highly homologous to sequences involved in induction of RAD2 and other excision repair genes by agents that induce DNA damage. spk1 strains were hypersensitive to UV irradiation. Taken together, these findings indicate that SPK1 is a dual-specificity (Ser/Thr and Tyr) protein kinase that is essential for viability. The cell cycle-dependent transcription, presence of DNA damage-related sequences, requirement for UV resistance, and nuclear localization of Spk1 all link this gene to a crucial S-phase-specific role, probably as a positive regulator of DNA synthesis.

Emerin plays a crucial role in nuclear invagination and in the nuclear calcium transient

PubMed Central

Shimojima, Masaya; Yuasa, Shinsuke; Motoda, Chikaaki; Yozu, Gakuto; Nagai, Toshihiro; Ito, Shogo; Lachmann, Mark; Kashimura, Shin; Takei, Makoto; Kusumoto, Dai; Kunitomi, Akira; Hayashiji, Nozomi; Seki, Tomohisa; Tohyama, Shugo; Hashimoto, Hisayuki; Kodaira, Masaki; Egashira, Toru; Hayashi, Kenshi; Nakanishi, Chiaki; Sakata, Kenji; Yamagishi, Masakazu; Fukuda, Keiichi

2017-01-01

Alteration of the nuclear Ca2+ transient is an early event in cardiac remodeling. Regulation of the nuclear Ca2+ transient is partly independent of the cytosolic Ca2+ transient in cardiomyocytes. One nuclear membrane protein, emerin, is encoded by EMD, and an EMD mutation causes Emery-Dreifuss muscular dystrophy (EDMD). It remains unclear whether emerin is involved in nuclear Ca2+ homeostasis. The aim of this study is to elucidate the role of emerin in rat cardiomyocytes by means of hypertrophic stimuli and in EDMD induced pluripotent stem (iPS) cell-derived cardiomyocytes in terms of nuclear structure and the Ca2+ transient. The cardiac hypertrophic stimuli increased the nuclear area, decreased nuclear invagination, and increased the half-decay time of the nuclear Ca2+ transient in cardiomyocytes. Emd knockdown cardiomyocytes showed similar properties after hypertrophic stimuli. The EDMD-iPS cell-derived cardiomyocytes showed increased nuclear area, decreased nuclear invagination, and increased half-decay time of the nuclear Ca2+ transient. An autopsied heart from a patient with EDMD also showed increased nuclear area and decreased nuclear invagination. These data suggest that Emerin plays a crucial role in nuclear structure and in the nuclear Ca2+ transient. Thus, emerin and the nuclear Ca2+ transient are possible therapeutic targets in heart failure and EDMD. PMID:28290476

Structure of a group II intron in complex with its reverse transcriptase.

PubMed

Qu, Guosheng; Kaushal, Prem Singh; Wang, Jia; Shigematsu, Hideki; Piazza, Carol Lyn; Agrawal, Rajendra Kumar; Belfort, Marlene; Wang, Hong-Wei

2016-06-01

Bacterial group II introns are large catalytic RNAs related to nuclear spliceosomal introns and eukaryotic retrotransposons. They self-splice, yielding mature RNA, and integrate into DNA as retroelements. A fully active group II intron forms a ribonucleoprotein complex comprising the intron ribozyme and an intron-encoded protein that performs multiple activities including reverse transcription, in which intron RNA is copied into the DNA target. Here we report cryo-EM structures of an endogenously spliced Lactococcus lactis group IIA intron in its ribonucleoprotein complex form at 3.8-Å resolution and in its protein-depleted form at 4.5-Å resolution, revealing functional coordination of the intron RNA with the protein. Remarkably, the protein structure reveals a close relationship between the reverse transcriptase catalytic domain and telomerase, whereas the active splicing center resembles the spliceosomal Prp8 protein. These extraordinary similarities hint at intricate ancestral relationships and provide new insights into splicing and retromobility.

A Family of Zinc Finger Proteins Is Required forChromosome-specific Pairing and Synapsis during Meiosis in C.elegans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Phillips, Carolyn M.; Dernburg, Abby F.

2006-06-07

Homologous chromosome pairing and synapsis are prerequisitefor accurate chromosome segregation during meiosis. Here, we show that afamily of four related C2H2 zinc-finger proteins plays a central role inthese events in C. elegans. These proteins are encoded within a tandemgene cluster. In addition to the X-specific HIM-8 protein, threeadditional paralogs collectively mediate the behavior of the fiveautosomes. Each chromosome relies on a specific member of the family topair and synapse with its homolog. These "ZIM" proteins concentrate atspecial regions called meiotic pairing centers on the correspondingchromosomes. These sites are dispersed along the nuclear envelope duringearly meiotic prophase, suggesting a role analogousmore » to thetelomere-mediated meiotic bouquet in other organisms. To gain insightinto the evolution of these components, wecharacterized homologs in C.briggsae and C. remanei, which revealed changes in copy number of thisgene family within the nematode lineage.« less

Extreme disorder in an ultrahigh-affinity protein complex

NASA Astrophysics Data System (ADS)

Borgia, Alessandro; Borgia, Madeleine B.; Bugge, Katrine; Kissling, Vera M.; Heidarsson, Pétur O.; Fernandes, Catarina B.; Sottini, Andrea; Soranno, Andrea; Buholzer, Karin J.; Nettels, Daniel; Kragelund, Birthe B.; Best, Robert B.; Schuler, Benjamin

2018-03-01

Molecular communication in biology is mediated by protein interactions. According to the current paradigm, the specificity and affinity required for these interactions are encoded in the precise complementarity of binding interfaces. Even proteins that are disordered under physiological conditions or that contain large unstructured regions commonly interact with well-structured binding sites on other biomolecules. Here we demonstrate the existence of an unexpected interaction mechanism: the two intrinsically disordered human proteins histone H1 and its nuclear chaperone prothymosin-α associate in a complex with picomolar affinity, but fully retain their structural disorder, long-range flexibility and highly dynamic character. On the basis of closely integrated experiments and molecular simulations, we show that the interaction can be explained by the large opposite net charge of the two proteins, without requiring defined binding sites or interactions between specific individual residues. Proteome-wide sequence analysis suggests that this interaction mechanism may be abundant in eukaryotes.

A high-throughput screening system targeting the nuclear export pathway via the third nuclear export signal of influenza A virus nucleoprotein.

PubMed

Kakisaka, Michinori; Mano, Takafumi; Aida, Yoko

2016-06-02

Two classes of antiviral drugs, M2 channel inhibitors and neuraminidase (NA) inhibitors, are currently approved for the treatment of influenza; however, the development of resistance against these agents limits their efficacy. Therefore, the identification of new targets and the development of new antiviral drugs against influenza are urgently needed. The third nuclear export signal (NES3) of nucleoprotein (NP) is the most important for viral replication among seven NESs encoded by four viral proteins, NP, M1, NS1, and NS2. NP-NES3 is critical for the nuclear export of NP, and targeting NP-NES3 is therefore a promising strategy that may lead to the development of antiviral drugs. However, a high-throughput screening (HTS) system to identify inhibitors of NP nuclear export has not been established. Here, we developed a novel HTS system to evaluate the inhibitory effects of compounds on the nuclear export pathway mediated by NP-NES3 using a MDCK cell line stably expressing NP-NES3 fused to a green fluorescent protein from aequorea coerulescens (AcGFP-NP-NES3) and a cell imaging analyzer. This HTS system was used to screen a 9600-compound library, leading to the identification of several hit compounds with inhibitory activity against the nuclear export of AcGFP-NP-NES3. The present HTS system provides a useful strategy for the identification of inhibitors targeting the nuclear export of NP via its NES3 sequence. Copyright © 2016. Published by Elsevier B.V.

Lamina-independent lamins in the nuclear interior serve important functions.

PubMed

Dechat, T; Gesson, K; Foisner, R

2010-01-01

Nuclear lamins were originally described as the main constituents of the nuclear lamina, a filamentous meshwork closely associated with the inner nuclear membrane. However, within recent years, it has become increasingly evident that a fraction of lamins also resides throughout the nuclear interior. As intermediate-filament-type proteins, lamins have been suggested to fulfill mainly structural functions such as providing shape and mechanical stability to the nucleus. But recent findings show that both peripheral and nucleoplasmic lamins also have important roles in essential cellular processes such as transcription, DNA replication, cell cycle progression, and chromatin organization. Furthermore, more than 300 mutations in the gene encoding A-type lamins have been associated with several human diseases now generally termed laminopathies and comprising muscular dystrophies, lipodystrophies, cardiomyopathies, and premature aging diseases. This review focuses on the lamina-independent pool of lamins in the nuclear interior, which surprisingly has not been studied in much detail so far. We discuss the properties and regulation of nucleoplasmic lamins during the cell cycle, their interaction partners, and their potential involvement in cellular processes and the development of laminopathies.

The general mitochondrial processing peptidase from potato is an integral part of cytochrome c reductase of the respiratory chain.

PubMed Central

Braun, H P; Emmermann, M; Kruft, V; Schmitz, U K

1992-01-01

The major mitochondrial processing activity removing presequences from nuclear encoded precursor proteins is present in the soluble fraction of fungal and mammalian mitochondria. We found that in potato, this activity resides in the inner mitochondrial membrane. Surprisingly, the proteolytic activity co-purifies with cytochrome c reductase, a protein complex of the respiratory chain. The purified complex is bifunctional, as it has the ability to transfer electrons from ubiquinol to cytochrome c and to cleave off the presequences of mitochondrial precursor proteins. In contrast to the nine subunit fungal complex, cytochrome c reductase from potato comprises 10 polypeptides. Protein sequencing of peptides from individual subunits and analysis of corresponding cDNA clones reveals that subunit III of cytochrome c reductase (51 kDa) represents the general mitochondrial processing peptidase. Images PMID:1324169

Altered nuclear tRNA metabolism in La-deleted Schizosaccharomyces pombe is accompanied by a nutritional stress response involving Atf1p and Pcr1p that is suppressible by Xpo-t/Los1p.

PubMed

Cherkasova, Vera; Maury, Luis Lopez; Bacikova, Dagmar; Pridham, Kevin; Bähler, Jürg; Maraia, Richard J

2012-02-01

Deletion of the sla1(+) gene, which encodes a homologue of the human RNA-binding protein La in Schizosaccharomyces pombe, causes irregularities in tRNA processing, with altered distribution of pre-tRNA intermediates. We show, using mRNA profiling, that cells lacking sla1(+) have increased mRNAs from amino acid metabolism (AAM) genes and, furthermore, exhibit slow growth in Edinburgh minimal medium. A subset of these AAM genes is under control of the AP-1-like, stress-responsive transcription factors Atf1p and Pcr1p. Although S. pombe growth is resistant to rapamycin, sla1-Δ cells are sensitive, consistent with deficiency of leucine uptake, hypersensitivity to NH4, and genetic links to the target of rapamycin (TOR) pathway. Considering that perturbed intranuclear pre-tRNA metabolism and apparent deficiency in tRNA nuclear export in sla1-Δ cells may trigger the AAM response, we show that modest overexpression of S. pombe los1(+) (also known as Xpo-t), encoding the nuclear exportin for tRNA, suppresses the reduction in pre-tRNA levels, AAM gene up-regulation, and slow growth of sla1-Δ cells. The conclusion that emerges is that sla1(+) regulates AAM mRNA production in S. pombe through its effects on nuclear tRNA processing and probably nuclear export. Finally, the results are discussed in the context of stress response programs in Saccharomyces cerevisiae.

Altered nuclear tRNA metabolism in La-deleted Schizosaccharomyces pombe is accompanied by a nutritional stress response involving Atf1p and Pcr1p that is suppressible by Xpo-t/Los1p

PubMed Central

Cherkasova, Vera; Lopez Maury, Luis; Bacikova, Dagmar; Pridham, Kevin; Bähler, Jürg; Maraia, Richard J.

2012-01-01

Deletion of the sla1+ gene, which encodes a homologue of the human RNA-binding protein La in Schizosaccharomyces pombe, causes irregularities in tRNA processing, with altered distribution of pre-tRNA intermediates. We show, using mRNA profiling, that cells lacking sla1+ have increased mRNAs from amino acid metabolism (AAM) genes and, furthermore, exhibit slow growth in Edinburgh minimal medium. A subset of these AAM genes is under control of the AP-1–like, stress-responsive transcription factors Atf1p and Pcr1p. Although S. pombe growth is resistant to rapamycin, sla1-Δ cells are sensitive, consistent with deficiency of leucine uptake, hypersensitivity to NH4, and genetic links to the target of rapamycin (TOR) pathway. Considering that perturbed intranuclear pre-tRNA metabolism and apparent deficiency in tRNA nuclear export in sla1-Δ cells may trigger the AAM response, we show that modest overexpression of S. pombe los1+ (also known as Xpo-t), encoding the nuclear exportin for tRNA, suppresses the reduction in pre-tRNA levels, AAM gene up-regulation, and slow growth of sla1-Δ cells. The conclusion that emerges is that sla1+ regulates AAM mRNA production in S. pombe through its effects on nuclear tRNA processing and probably nuclear export. Finally, the results are discussed in the context of stress response programs in Saccharomyces cerevisiae. PMID:22160596

Arabidopsis ASYMMETRIC LEAVES2 protein required for leaf morphogenesis consistently forms speckles during mitosis of tobacco BY-2 cells via signals in its specific sequence.

PubMed

Luo, Lilan; Ando, Sayuri; Sasabe, Michiko; Machida, Chiyoko; Kurihara, Daisuke; Higashiyama, Tetsuya; Machida, Yasunori

2012-09-01

Leaf primordia with high division and developmental competencies are generated around the periphery of stem cells at the shoot apex. Arabidopsis ASYMMETRIC-LEAVES2 (AS2) protein plays a key role in the regulation of many genes responsible for flat symmetric leaf formation. The AS2 gene, expressed in leaf primordia, encodes a plant-specific nuclear protein containing an AS2/LOB domain with cysteine repeats (C-motif). AS2 proteins are present in speckles in and around the nucleoli, and in the nucleoplasm of some leaf epidermal cells. We used the tobacco cultured cell line BY-2 expressing the AS2-fused yellow fluorescent protein to examine subnuclear localization of AS2 in dividing cells. AS2 mainly localized to speckles (designated AS2 bodies) in cells undergoing mitosis and distributed in a pairwise manner during the separation of sets of daughter chromosomes. Few interphase cells contained AS2 bodies. Deletion analyses showed that a short stretch of the AS2 amino-terminal sequence and the C-motif play negative and positive roles, respectively, in localizing AS2 to the bodies. These results suggest that AS2 bodies function to properly distribute AS2 to daughter cells during cell division in leaf primordia; and this process is controlled at least partially by signals encoded by the AS2 sequence itself.

Endogenous and ectopic expression of telomere regulating genes in chicken embryonic fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Michailidis, Georgios; Saretzki, Gabriele; Hall, Judith

In this study, we compared the endogenous expression of genes encoding telomere regulating proteins in cultured chicken embryonic fibroblasts (CEFs) and 10-day-old chicken embryos. CEFs maintained in vitro senesced and senescence was accompanied by reduced telomere length, telomerase activity, and expression of the chicken (c) TRF1 gene. There was no change in TRF2 gene expression although the major TRF2 transcript identified in 10-day-old chicken embryos encoded a truncated TRF2 protein (TRF2'), containing an N-terminal dimerisation domain but lacking a myb-related DNA binding domain and nuclear localisation signal. Senescence of the CEFs in vitro was associated with the loss of themore » TRF2' transcript, indicative of a novel function for the encoded protein. Senescence was also coupled with decreased expression of RAD51, but increased RAD52 expression. These data support that RAD51 independent recombination mechanisms do not function in vitro to maintain chicken telomeres. To attempt to rescue the CEFs from replicative senescence, we stably transfected passage 3 CEFs with the human telomerase reverse transcriptase (hTERT) catalytic subunit. While hTERT expression was detected in the stable transfectants neither telomerase activity nor the stabilisation of telomere length was observed, and the transfectant cells senesced at the same passage number as the untransfected cells. These data indicate that the human TERT is incompatible with the avian telomere maintenance apparatus and suggest the functioning of a species specific telomere system in the avian.« less

Rat1p and Xrn1p are functionally interchangeable exoribonucleases that are restricted to and required in the nucleus and cytoplasm, respectively.

PubMed Central

Johnson, A W

1997-01-01

XRN1 encodes an abundant cytoplasmic exoribonuclease, Xrn1p, responsible for mRNA turnover in yeast. A screen for bypass suppressors of the inviability of xrn1 ski2 double mutants identified dominant alleles of RAT1, encoding an exoribonuclease homologous with Xrn1p. These RAT1 alleles restored XRN1-like functions, including cytoplasmic RNA turnover, wild-type sensitivity to the microtubule-destabilizing drug benomyl, and sporulation. The mutations were localized to a region of the RAT1 gene encoding a putative bipartite nuclear localization sequence (NLS). Fusions to green fluorescent protein were used to demonstrate that wild-type Rat1p is localized to the nucleus and that the mutant alleles result in mislocalization of Rat1p to the cytoplasm. Conversely, targeting Xrn1p to the nucleus by the addition of the simian virus 40 large-T-antigen NLS resulted in complementation of the temperature sensitivity of a rat1-1 strain. These results indicate that Xrn1p and Rat1p are functionally interchangeable exoribonucleases that function in and are restricted to the cytoplasm and nucleus, respectively. It is likely that the higher eukaryotic homologs of these proteins will function similarly in the cytoplasm and nucleus. PMID:9315672

Serine 192 in the tiny RS repeat of the adenoviral L4-33K splicing enhancer protein is essential for function and reorganization of the protein to the periphery of viral replication centers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oestberg, Sara, E-mail: sara.ostberg@imbim.uu.se; Toermaenen Persson, Heidi, E-mail: heidi.tormanen.persson@imbim.uu.se; Akusjaervi, Goeran, E-mail: goran.akusjarvi@imbim.uu.se

2012-11-25

The adenovirus L4-33K protein is a key regulator involved in the temporal shift from early to late pattern of mRNA expression from the adenovirus major late transcription unit. L4-33K is a virus-encoded alternative splicing factor, which enhances processing of 3 Prime splice sites with a weak sequence context. Here we show that L4-33K expressed from a plasmid is localized at the nuclear margin of uninfected cells. During an infection L4-33K is relocalized to the periphery of E2A-72K containing viral replication centers. We also show that serine 192 in the tiny RS repeat of the conserved carboxy-terminus of L4-33K, which ismore » critical for the splicing enhancer function of L4-33K, is necessary for the nuclear localization and redistribution of the protein to viral replication sites. Collectively, our results show a good correlation between the activity of L4-33K as a splicing enhancer protein and its localization to the periphery of viral replication centers.« less

LAF1, a MYB transcription activator for phytochrome A signaling

PubMed Central

Ballesteros, María L.; Bolle, Cordelia; Lois, Luisa M.; Moore, James M.; Vielle-Calzada, Jean-Philippe; Grossniklaus, Ueli; Chua, Nam-Hai

2001-01-01

The photoreceptor phytochrome (phy) A has a well-defined role in regulating gene expression in response to specific light signals. Here, we describe a new Arabidopsis mutant, laf1 (long after far-red light 1) that has an elongated hypocotyl specifically under far-red light. Gene expression studies showed that laf1 has reduced responsiveness to continuous far-red light but retains wild-type responses to other light wavelengths. As far-red light is only perceived by phyA, our results suggest that LAF1 is specifically involved in phyA signal transduction. Further analyses revealed that laf1 is affected in a subset of phyA-dependent responses and the phenotype is more severe at low far-red fluence rates. LAF1 encodes a nuclear protein with strong homology with the R2R3–MYB family of DNA-binding proteins. Experiments using yeast cells identified a transactivation domain in the C-terminal portion of the protein. LAF1 is constitutively targeted to the nucleus by signals in its N-terminal portion, and the full-length protein accumulates in distinct nuclear speckles. This accumulation in speckles is abolished by a point mutation in a lysine residue (K258R), which might serve as a modification site by a small ubiquitin-like protein (SUMO). PMID:11581165

Nuclear localization of Klotho in brain: an anti-aging protein

PubMed Central

German, Dwight C.; Khobahy, Ida; Pastor, Johanne; Kuro-o, Makoto; Liu, Xinran

2011-01-01

Klotho is a putative age-suppressing gene whose over-expression in mice results in extension of life span. The klotho gene encodes a single-pass transmembrane protein whose extracellular domain is shed and released into blood, urine, and cerebrospinal fluid, potentially functioning as a humoral factor. The extracellular domain of Klotho has an activity that increases the expression of anti-oxidant enzymes and confers resistance to oxidative stress in cultured cells and in whole animals. The transmembrane form of the Klotho protein directly binds to multiple fibroblast growth factor receptors and modifies their ligand affinity and specificity. The purpose of the present study was to determine the precise cellular localization of Klotho in the mouse brain. Using light microscopic immunohistochemical methods, we found the highest levels of Klotho immunoreactivity in two brain regions: the choroid plexus, and cerebellar Purkinje cells. In the choroid plexus cells, Klotho was found not only on the plasma membrane but also in large amounts near the nuclear membrane. Likewise, in the Purkinje cell Klotho was found throughout the cell including dendrites, axon and soma with large amounts near the nuclear membrane. Using immunoelectron microscopy, we found Klotho in the cell membrane, but the highest concentration was localized in the peripheral portion of the nucleus and the nucleolus in both cell types. This new finding suggests that in addition to Klotho being secreted from cells in brain, it also has a nuclear function. PMID:22245317

Transgenic cells with increased plastoquinone levels and methods of use

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sayre, Richard T.; Subramanian, Sowmya; Cahoon, Edgar

Disclosed herein are transgenic cells expressing a heterologous nucleic acid encoding a prephenate dehydrogenase (PDH) protein, a heterologous nucleic acid encoding a homogentisate solanesyl transferase (HST) protein, a heterologous nucleic acid encoding a deoxyxylulose phosphate synthase (DXS) protein, or a combination of two or more thereof. In particular examples, the disclosed transgenic cells have increased plastoquinone levels. Also disclosed are methods of increasing cell growth rates or production of biomass by cultivating transgenic cells expressing a heterologous nucleic acid encoding a PDH protein, a heterologous nucleic acid encoding an HST protein, a heterologous nucleic acid encoding a DXS protein, ormore » a combination of two or more thereof under conditions sufficient to produce cell growth or biomass.« less

Epstein-Barr virus-derived EBNA2 regulates STAT3 activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muromoto, Ryuta; Ikeda, Osamu; Okabe, Kanako

The Epstein-Barr virus (EBV)-encoded latency protein EBNA2 is a nuclear transcriptional activator that is essential for EBV-induced cellular transformation. Here, we show that EBNA2 interacts with STAT3, a signal transducer for an interleukin-6 family cytokine, and enhances the transcriptional activity of STAT3 by influencing its DNA-binding activity. Furthermore, EBNA2 cooperatively acts on STAT3 activation with LMP1. These data demonstrate that EBNA2 acts as a transcriptional coactivator of STAT3.

Efficient identification of tubby-binding proteins by an improved system of T7 phage display.

PubMed

Caberoy, Nora B; Zhou, Yixiong; Jiang, Xiaoyu; Alvarado, Gabriela; Li, Wei

2010-01-01

Mutation in the tubby gene causes adult-onset obesity, progressive retinal, and cochlear degeneration with unknown mechanism. In contrast, mutations in tubby-like protein 1 (Tulp1), whose C-terminus is highly homologous to tubby, only lead to retinal degeneration. We speculate that their diverse N-terminus may define their distinct disease profile. To elucidate the binding partners of tubby, we used tubby N-terminus (tubby-N) as bait to identify unknown binding proteins with open-reading-frame (ORF) phage display. T7 phage display was engineered with three improvements: high-quality ORF phage display cDNA library, specific phage elution by protease cleavage, and dual phage display for sensitive high throughput screening. The new system is capable of identifying unknown bait-binding proteins in as fast as approximately 4-7 days. While phage display with conventional cDNA libraries identifies high percentage of out-of-frame unnatural short peptides, all 28 tubby-N-binding clones identified by ORF phage display were ORFs. They encode 16 proteins, including 8 nuclear proteins. Fourteen proteins were analyzed by yeast two-hybrid assay and protein pull-down assay with ten of them independently verified. Comparative binding analyses revealed several proteins binding to both tubby and Tulp1 as well as one tubby-specific binding protein. These data suggest that tubby-N is capable of interacting with multiple nuclear and cytoplasmic protein binding partners. These results demonstrated that the newly-engineered ORF phage display is a powerful technology to identify unknown protein-protein interactions. (c) 2009 John Wiley & Sons, Ltd.

Is ftsH the Key to Plastid Longevity in Sacoglossan Slugs?

PubMed Central

de Vries, Jan; Habicht, Jörn; Woehle, Christian; Huang, Changjie; Christa, Gregor; Wägele, Heike; Nickelsen, Jörg; Martin, William F.; Gould, Sven B.

2013-01-01

Plastids sequestered by sacoglossan sea slugs have long been a puzzle. Some sacoglossans feed on siphonaceous algae and can retain the plastids in the cytosol of their digestive gland cells. There, the stolen plastids (kleptoplasts) can remain photosynthetically active in some cases for months. Kleptoplast longevity itself challenges current paradigms concerning photosystem turnover, because kleptoplast photosystems remain active in the absence of nuclear algal genes. In higher plants, nuclear genes are essential for plastid maintenance, in particular, for the constant repair of the D1 protein of photosystem II. Lateral gene transfer was long suspected to underpin slug kleptoplast longevity, but recent transcriptomic and genomic analyses show that no algal nuclear genes are expressed from the slug nucleus. Kleptoplast genomes themselves, however, appear expressed in the sequestered state. Here we present sequence data for the chloroplast genome of Acetabularia acetabulum, the food source of the sacoglossan Elysia timida, which can maintain Acetabularia kleptoplasts in an active state for months. The data reveal what might be the key to sacoglossan kleptoplast longevity: plastids that remain photosynthetically active within slugs for periods of months share the property of encoding ftsH, a D1 quality control protease that is essential for photosystem II repair. In land plants, ftsH is always nuclear encoded, it was transferred to the nucleus from the plastid genome when Charophyta and Embryophyta split. A replenishable supply of ftsH could, in principle, rescue kleptoplasts from D1 photodamage, thereby influencing plastid longevity in sacoglossan slugs. PMID:24336424

Protein Structure Prediction by Protein Threading

NASA Astrophysics Data System (ADS)

Xu, Ying; Liu, Zhijie; Cai, Liming; Xu, Dong

The seminal work of Bowie, Lüthy, and Eisenberg (Bowie et al., 1991) on "the inverse protein folding problem" laid the foundation of protein structure prediction by protein threading. By using simple measures for fitness of different amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. Their follow-up work (Elofsson et al., 1996; Fischer and Eisenberg, 1996; Fischer et al., 1996a,b) and the work by Jones, Taylor, and Thornton (Jones et al., 1992) on protein fold recognition led to the development of a new brand of powerful tools for protein structure prediction, which we now term "protein threading." These computational tools have played a key role in extending the utility of all the experimentally solved structures by X-ray crystallography and nuclear magnetic resonance (NMR), providing structural models and functional predictions for many of the proteins encoded in the hundreds of genomes that have been sequenced up to now.

Proteomic screening of variola virus reveals a unique NF-kappaB inhibitor that is highly conserved among pathogenic orthopoxviruses.

PubMed

Mohamed, Mohamed R; Rahman, Masmudur M; Lanchbury, Jerry S; Shattuck, Donna; Neff, Chris; Dufford, Max; van Buuren, Nick; Fagan, Katharine; Barry, Michele; Smith, Scott; Damon, Inger; McFadden, Grant

2009-06-02

Identification of the binary interactions between viral and host proteins has become a valuable tool for investigating viral tropism and pathogenesis. Here, we present the first systematic protein interaction screening of the unique variola virus proteome by using yeast 2-hybrid screening against a variety of human cDNA libraries. Several protein-protein interactions were identified, including an interaction between variola G1R, an ankryin/F-box containing protein, and human nuclear factor kappa-B1 (NF-kappaB1)/p105. This represents the first direct interaction between a pathogen-encoded protein and NF-kappaB1/p105. Orthologs of G1R are present in a variety of pathogenic orthopoxviruses, but not in vaccinia virus, and expression of any one of these viral proteins blocks NF-kappaB signaling in human cells. Thus, proteomic screening of variola virus has the potential to uncover modulators of the human innate antiviral responses.

Prohibitin 2: At a communications crossroads.

PubMed

Bavelloni, Alberto; Piazzi, Manuela; Raffini, Mirco; Faenza, Irene; Blalock, William L

2015-04-01

Prohibitins (PHBs) are a highly conserved class of proteins first discovered as inhibitors of cellular proliferation. Since then PHBs have been found to have a significant role in transcription, nuclear signaling, mitochondrial structural integrity, cell division, and cellular membrane metabolism, placing these proteins among the key regulators of pathologies such as cancer, neuromuscular degeneration, and other metabolic diseases. The human genome encodes two PHB proteins, prohibitin 1 (PHB1) and prohibitin 2 (PHB2), which function not only as a heterodimeric complex, but also independently. While many previous reviews have focused on the better characterized prohibitin, PHB1, this review focuses on PHB2 and new data concerning its cellular functions both in complex with PHB1 and independent of PHB1. © 2015 International Union of Biochemistry and Molecular Biology.

Valosin-Containing Protein (VCP/p97) Is an Activator of Wild-Type Ataxin-3

PubMed Central

Laço, Mário N.; Cortes, Luisa; Travis, Sue M.; Paulson, Henry L.; Rego, A. Cristina

2012-01-01

Alterations in the ubiquitin-proteasome system (UPS) have been reported in several neurodegenerative disorders characterized by protein misfolding and aggregation, including the polylgutamine diseases. Machado-Joseph disease (MJD) or Spinocerebellar Ataxia type 3 is caused by a polyglutamine-encoding CAG expansion in the ATXN3 gene, which encodes a 42 kDa deubiquitinating enzyme (DUB), ataxin-3. We investigated ataxin-3 deubiquitinating activity and the functional relevance of ataxin-3 interactions with two proteins previously described to interact with ataxin-3, hHR23A and valosin-containing protein (VCP/p97). We confirmed ataxin-3 affinity for both hHR23A and VCP/p97. hHR23A and ataxin-3 were shown to co-localize in discrete nuclear foci, while VCP/p97 was primarily cytoplasmic. hHR23A and VCP/p97 recombinant proteins were added, separately or together, to normal and expanded ataxin-3 in in vitro deubiquitination assays to evaluate their influence on ataxin-3 activity. VCP/p97 was shown to be an activator specifically of wild-type ataxin-3, exhibiting no effect on expanded ataxin-3, In contrast, we observed no significant alterations in ataxin-3 enzyme kinetics or substrate preference in the presence of hHR23A alone or in combination with VCP. Based on our results we propose a model where ataxin-3 normally functions with its interactors to specify the cellular fate of ubiquitinated proteins. PMID:22970133

HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors.

PubMed

Pocock, Ginger M; Becker, Jordan T; Swanson, Chad M; Ahlquist, Paul; Sherer, Nathan M

2016-04-01

Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent "burst-like" transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm.

HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors

PubMed Central

Pocock, Ginger M.; Becker, Jordan T.; Swanson, Chad M.; Ahlquist, Paul; Sherer, Nathan M.

2016-01-01

Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent “burst-like” transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm. PMID:27070420

Transcriptional Activity and Nuclear Localization of Cabut, the Drosophila Ortholog of Vertebrate TGF-β-Inducible Early-Response Gene (TIEG) Proteins

PubMed Central

Belacortu, Yaiza; Weiss, Ron; Kadener, Sebastian; Paricio, Nuria

2012-01-01

Background Cabut (Cbt) is a C2H2-class zinc finger transcription factor involved in embryonic dorsal closure, epithelial regeneration and other developmental processes in Drosophila melanogaster. Cbt orthologs have been identified in other Drosophila species and insects as well as in vertebrates. Indeed, Cbt is the Drosophila ortholog of the group of vertebrate proteins encoded by the TGF-ß-inducible early-response genes (TIEGs), which belong to Sp1-like/Krüppel-like family of transcription factors. Several functional domains involved in transcriptional control and subcellular localization have been identified in the vertebrate TIEGs. However, little is known of whether these domains and functions are also conserved in the Cbt protein. Methodology/Principal Findings To determine the transcriptional regulatory activity of the Drosophila Cbt protein, we performed Gal4-based luciferase assays in S2 cells and showed that Cbt is a transcriptional repressor and able to regulate its own expression. Truncated forms of Cbt were then generated to identify its functional domains. This analysis revealed a sequence similar to the mSin3A-interacting repressor domain found in vertebrate TIEGs, although located in a different part of the Cbt protein. Using β-Galactosidase and eGFP fusion proteins, we also showed that Cbt contains the bipartite nuclear localization signal (NLS) previously identified in TIEG proteins, although it is non-functional in insect cells. Instead, a monopartite NLS, located at the amino terminus of the protein and conserved across insects, is functional in Drosophila S2 and Spodoptera exigua Sec301 cells. Last but not least, genetic interaction and immunohistochemical assays suggested that Cbt nuclear import is mediated by Importin-α2. Conclusions/Significance Our results constitute the first characterization of the molecular mechanisms of Cbt-mediated transcriptional control as well as of Cbt nuclear import, and demonstrate the existence of similarities and differences in both aspects of Cbt function between the insect and the vertebrate TIEG proteins. PMID:22359651

Metabolic Adaptation in Transplastomic Plants Massively Accumulating Recombinant Proteins

PubMed Central

Bally, Julia; Job, Claudette; Belghazi, Maya; Job, Dominique

2011-01-01

Background Recombinant chloroplasts are endowed with an astonishing capacity to accumulate foreign proteins. However, knowledge about the impact on resident proteins of such high levels of recombinant protein accumulation is lacking. Methodology/Principal Findings Here we used proteomics to characterize tobacco (Nicotiana tabacum) plastid transformants massively accumulating a p-hydroxyphenyl pyruvate dioxygenase (HPPD) or a green fluorescent protein (GFP). While under the conditions used no obvious modifications in plant phenotype could be observed, these proteins accumulated to even higher levels than ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the most abundant protein on the planet. This accumulation occurred at the expense of a limited number of leaf proteins including Rubisco. In particular, enzymes involved in CO2 metabolism such as nuclear-encoded plastidial Calvin cycle enzymes and mitochondrial glycine decarboxylase were found to adjust their accumulation level to these novel physiological conditions. Conclusions/Significance The results document how protein synthetic capacity is limited in plant cells. They may provide new avenues to evaluate possible bottlenecks in recombinant protein technology and to maintain plant fitness in future studies aiming at producing recombinant proteins of interest through chloroplast transformation. PMID:21966485

Plastid, nuclear and reverse transcriptase sequences in the mitochondrial genome of Oenothera: is genetic information transferred between organelles via RNA?

PubMed Central

Schuster, W; Brennicke, A

1987-01-01

We describe an open reading frame (ORF) with high homology to reverse transcriptase in the mitochondrial genome of Oenothera. This ORF displays all the characteristics of an active plant mitochondrial gene with a possible ribosome binding site and 39% T in the third codon position. It is located between a sequence fragment from the plastid genome and one of nuclear origin downstream from the gene encoding subunit 5 of the NADH dehydrogenase. The nuclear derived sequence consists of 528 nucleotides from the small ribosomal RNA and contains an expansion segment unique to nuclear rRNAs. The plastid sequence contains part of the ribosomal protein S4 and the complete tRNA(Ser). The observation that only transcribed sequences have been found i more than one subcellular compartment in higher plants suggests that interorganellar transfer of genetic information may occur via RNA and subsequent local reverse transcription and genomic integration. PMID:14650433

MNF, an ankyrin repeat protein of myxoma virus, is part of a native cellular SCF complex during viral infection

PubMed Central

2010-01-01

Myxoma virus (MYXV), a member of the Poxviridae family, is the agent responsible for myxomatosis, a fatal disease in the European rabbit (Oryctolagus cuniculus). Like all poxviruses, MYXV is known for encoding multiple proteins that regulate cellular signaling pathways. Among them, four proteins share the same ANK/PRANC structure: M148R, M149R, MNF (Myxoma Nuclear factor) and M-T5, all of them described as virulence factors. This family of poxvirus proteins, recently identified, has drawn considerable attention for its potential role in modulating the host ubiquitin-proteasome system during viral infection. To date, many members of this novel protein family have been shown to interact with SCF components, in vitro. Here, we focus on MNF gene, which has been shown to express a nuclear protein presenting nine ANK repeats, one of which has been identified as a nuclear localization signal. In transfection, MNF has been shown to colocalise with the transcription factor NF-κB in the nucleus of TNFα-stimulated cells. Functionally, MNF is a critical virulence factor since its deletion generates an almost apathogenic virus. In this study, to pursue the investigation of proteins interacting with MNF and of its mechanism of action, we engineered a recombinant MYXV expressing a GFP-linked MNF under the control of MNF native promoter. Infection of rabbits with MYXV-GFPMNF recombinant virus provided the evidence that the GFP fusion does not disturb the main function of MNF. Hence, cells were infected with MYXV-GFPMNF and immunoprecipitation of the GFPMNF fusion protein was performed to identify MNF's partners. For the first time, endogenous components of SCF (Cullin-1 and Skp1) were co-precipitated with an ANK myxoma virus protein, expressed in an infectious context, and without over-expression of any protein. PMID:20211013

Evidence for a Posttranscriptional Role of a TFIIICα-like Protein in Chironomus tentans

PubMed Central

Sabri, Nafiseh; Farrants, Ann-Kristin Östlund; Hellman, Ulf; Visa, Neus

2002-01-01

We have cloned and sequenced a cDNA that encodes for a nuclear protein of 238 kDa in the dipteran Chironomus tentans. This protein, that we call p2D10, is structurally similar to the α subunit of the general transcription factor TFIIIC. Using immunoelectron microscopy we have shown that a fraction of p2D10 is located at sites of transcription, which is consistent with a possible role of this protein in transcription initiation. We have also found that a large fraction of p2D10 is located in the nucleoplasm and in the nuclear pore complexes. Using gel filtration chromatography and coimmunoprecipitation methods, we have identified and characterized two p2D10-containing complexes that differ in molecular mass and composition. The heavy p2D10-containing complex contains at least one other component of the TFIIIC complex, TFIIIC-ε. Based on its molecular mass and composition, the heavy p2D10-containing complex may be the Pol III holoenzyme. The light p2D10-containing complex contains RNA together with at least two proteins that are thought to be involved in mRNA trafficking, RAE1 and hrp65. The observations reported here suggest that this new TFIIIC-α-like protein is involved in posttranscriptional steps of premRNA metabolism in Chironomus tentans. PMID:12006668

NRIP is newly identified as a Z-disc protein, activating calmodulin signaling for skeletal muscle contraction and regeneration.

PubMed

Chen, Hsin-Hsiung; Chen, Wen-Pin; Yan, Wan-Lun; Huang, Yuan-Chun; Chang, Szu-Wei; Fu, Wen-Mei; Su, Ming-Jai; Yu, I-Shing; Tsai, Tzung-Chieh; Yan, Yu-Ting; Tsao, Yeou-Ping; Chen, Show-Li

2015-11-15

Nuclear receptor interaction protein (NRIP, also known as DCAF6 and IQWD1) is a Ca(2+)-dependent calmodulin-binding protein. In this study, we newly identify NRIP as a Z-disc protein in skeletal muscle. NRIP-knockout mice were generated and found to have reduced muscle strength, susceptibility to fatigue and impaired adaptive exercise performance. The mechanisms of NRIP-regulated muscle contraction depend on NRIP being downstream of Ca(2+) signaling, where it stimulates activation of both 'calcineurin-nuclear factor of activated T-cells, cytoplasmic 1' (CaN-NFATc1; also known as NFATC1) and calmodulin-dependent protein kinase II (CaMKII) through interaction with calmodulin (CaM), resulting in the induction of mitochondrial activity and the expression of genes encoding the slow class of myosin, and in the regulation of Ca(2+) homeostasis through the internal Ca(2+) stores of the sarcoplasmic reticulum. Moreover, NRIP-knockout mice have a delayed regenerative capacity. The amount of NRIP can be enhanced after muscle injury and is responsible for muscle regeneration, which is associated with the increased expression of myogenin, desmin and embryonic myosin heavy chain during myogenesis, as well as for myotube formation. In conclusion, NRIP is a novel Z-disc protein that is important for skeletal muscle strength and regenerative capacity. © 2015. Published by The Company of Biologists Ltd.

RNA-binding protein HuR autoregulates its expression by promoting alternative polyadenylation site usage

PubMed Central

Dai, Weijun; Zhang, Gen; Makeyev, Eugene V.

2012-01-01

RNA-binding protein HuR modulates the stability and translational efficiency of messenger RNAs (mRNAs) encoding essential components of the cellular proliferation, growth and survival pathways. Consistent with these functions, HuR levels are often elevated in cancer cells and reduced in senescent and quiescent cells. However, the molecular mechanisms that control HuR expression are poorly understood. Here we show that HuR protein autoregulates its abundance through a negative feedback loop that involves interaction of the nuclear HuR protein with a GU-rich element (GRE) overlapping with the HuR major polyadenylation signal (PAS2). An increase in the cellular HuR protein levels stimulates the expression of long HuR mRNA species containing an AU-rich element (ARE) that destabilizes the mRNAs and thus reduces the protein production output. The PAS2 read-through occurs due to a reduced recruitment of the CstF-64 subunit of the pre-mRNA cleavage stimulation factor in the presence of the GRE-bound HuR. We propose that this mechanism maintains HuR homeostasis in proliferating cells. Since only the nuclear HuR is expected to contribute to the auto-regulation, our model may explain the longstanding observation that the increase in the total HuR expression in cancer cells often correlates with the accumulation of its substantial fraction in the cytoplasm. PMID:21948791

RNA-binding protein HuR autoregulates its expression by promoting alternative polyadenylation site usage.

PubMed

Dai, Weijun; Zhang, Gen; Makeyev, Eugene V

2012-01-01

RNA-binding protein HuR modulates the stability and translational efficiency of messenger RNAs (mRNAs) encoding essential components of the cellular proliferation, growth and survival pathways. Consistent with these functions, HuR levels are often elevated in cancer cells and reduced in senescent and quiescent cells. However, the molecular mechanisms that control HuR expression are poorly understood. Here we show that HuR protein autoregulates its abundance through a negative feedback loop that involves interaction of the nuclear HuR protein with a GU-rich element (GRE) overlapping with the HuR major polyadenylation signal (PAS2). An increase in the cellular HuR protein levels stimulates the expression of long HuR mRNA species containing an AU-rich element (ARE) that destabilizes the mRNAs and thus reduces the protein production output. The PAS2 read-through occurs due to a reduced recruitment of the CstF-64 subunit of the pre-mRNA cleavage stimulation factor in the presence of the GRE-bound HuR. We propose that this mechanism maintains HuR homeostasis in proliferating cells. Since only the nuclear HuR is expected to contribute to the auto-regulation, our model may explain the longstanding observation that the increase in the total HuR expression in cancer cells often correlates with the accumulation of its substantial fraction in the cytoplasm.

The Yersinia pseudotuberculosis and Yersinia pestis toxin complex is active against cultured mammalian cells.

PubMed

Hares, Michelle C; Hinchliffe, Stewart J; Strong, Philippa C R; Eleftherianos, Ioannis; Dowling, Andrea J; ffrench-Constant, Richard H; Waterfield, Nick

2008-11-01

The toxin complex (Tc) genes were first identified in the insect pathogen Photorhabdus luminescens and encode approximately 1 MDa protein complexes which are toxic to insect pests. Subsequent genome sequencing projects have revealed the presence of tc orthologues in a range of bacterial pathogens known to be associated with insects. Interestingly, members of the mammalian-pathogenic yersiniae have also been shown to encode Tc orthologues. Studies in Yersinia enterocolitica have shown that divergent tc loci either encode insect-active toxins or play a role in colonization of the gut in gastroenteritis models of rats. So far little is known about the activity of the Tc proteins in the other mammalian-pathogenic yersiniae. Here we present work to suggest that Tc proteins in Yersinia pseudotuberculosis and Yersinia pestis are not insecticidal toxins but have evolved for mammalian pathogenicity. We show that Tc is secreted by Y. pseudotuberculosis strain IP32953 during growth in media at 28 degrees C and 37 degrees C. We also demonstrate that oral toxicity of strain IP32953 to Manduca sexta larvae is not due to Tc expression and that lysates of Escherichia coli BL21 expressing the Yersinia Tc proteins are not toxic to Sf9 insect cells but are toxic to cultured mammalian cell lines. Cell lysates of E. coli BL21 expressing the Y. pseudotuberculosis Tc proteins caused actin ruffles, vacuoles and multi-nucleation in cultured human gut cells (Caco-2); similar morphology was observed after application of a lysate of E. coli BL21 expressing the Y. pestis Tc proteins to mouse fibroblast NIH3T3 cells, but not Caco-2 cells. Finally, transient expression of the individual Tc proteins in Caco-2 and NIH3T3 cell lines reproduced the actin and nuclear rearrangement observed with the topical applications. Together these results add weight to the growing hypothesis that the Tc proteins in Y. pseudotuberculosis and Y. pestis have been adapted for mammalian pathogenicity. We further conclude that Tc proteins from Y. pseudotuberculosis and Y. pestis display differential mammalian cell specificity in their toxicity.

Solid lipid nanoparticle-based vectors intended for the treatment of X-linked juvenile retinoschisis by gene therapy: In vivo approaches in Rs1h-deficient mouse model.

PubMed

Apaolaza, P S; Del Pozo-Rodríguez, A; Torrecilla, J; Rodríguez-Gascón, A; Rodríguez, J M; Friedrich, U; Weber, B H F; Solinís, M A

2015-11-10

X-linked juvenile retinoschisis (XLRS), which results from mutations in the gene RS1 that encodes the protein retinoschisin, is a retinal degenerative disease affecting between 1/5000 and 1/25,000 people worldwide. Currently, there is no cure for this disease and the treatment is based on the application of low-vision aids. The aim of the present work was the in vitro and in vivo evaluation of two different non-viral vectors based on solid lipid nanoparticles (SLNs), protamine and two anionic polysaccharides, hyaluronic acid (HA) or dextran (DX), for the treatment of XLRS. First, the vectors containing a plasmid which encodes both the reporter green fluorescent protein (GFP) and the therapeutic protein retinoschisin, under the control of CMV promoters, were characterized in vitro. Then, the vectors were subretinally or intravitreally administrated to C57BL/6 wild type mice. One week later, GFP was detected in all treated mice and in all retinal layers except in the Outer Nuclear Layer (ONL) and the Inner Nuclear Layer (INL), regardless of the administration route and the vector employed. Finally, two weeks after subretinal or intravitreal injection to Rs1h-deficient mice, GFP and retinoschisin expression was detected in all retinal layers, except in the ONL, which was maintained for at least two months after subretinal administration. The structural analysis of the treated Rs1h-deficient eyes showed a partial recovery of the retina related to the production of retinoschisin. This work shows for the first time a successful RS1 gene transfer to Rs1h-deficient animals using non-viral nanocarriers, with promising results that point to non-viral gene therapy as a feasible future therapeutic tool for retinal disorders.

Novel detection schemes of nuclear magnetic resonance and magnetic resonance imaging: applications from analytical chemistry to molecular sensors.

PubMed

Harel, Elad; Schröder, Leif; Xu, Shoujun

2008-01-01

Nuclear magnetic resonance (NMR) is a well-established analytical technique in chemistry. The ability to precisely control the nuclear spin interactions that give rise to the NMR phenomenon has led to revolutionary advances in fields as diverse as protein structure determination and medical diagnosis. Here, we discuss methods for increasing the sensitivity of magnetic resonance experiments, moving away from the paradigm of traditional NMR by separating the encoding and detection steps of the experiment. This added flexibility allows for diverse applications ranging from lab-on-a-chip flow imaging and biological sensors to optical detection of magnetic resonance imaging at low magnetic fields. We aim to compare and discuss various approaches for a host of problems in material science, biology, and physics that differ from the high-field methods routinely used in analytical chemistry and medical imaging.

Subcellular localization of Mitf in monocytic cells.

PubMed

Lu, Ssu-Yi; Wan, Hsiao-Ching; Li, Mengtao; Lin, Yi-Ling

2010-06-01

Microphthalmia-associated transcription factor (Mitf) is a transcription factor that plays an important role in regulating the development of several cell lineages. The subcellular localization of Mitf is dynamic and is associated with its transcription activity. In this study, we examined factors that affect its subcellular localization in cells derived from the monocytic lineage since Mitf is present abundantly in these cells. We identified a domain encoded by Mitf exon 1B1b to be important for Mitf to commute between the cytoplasm and the nucleus. Deletion of this domain disrupts the shuttling of Mitf to the cytoplasm and results in its retention in the nucleus. M-CSF and RANKL both induce nuclear translocation of Mitf. We showed that Mitf nuclear transport is greatly influenced by ratio of M-CSF/Mitf protein expression. In addition, cell attachment to a solid surface also is needed for the nuclear transport of Mitf.

Phosphorylation of the RSRSP stretch is critical for splicing regulation by RNA-Binding Motif Protein 20 (RBM20) through nuclear localization.

PubMed

Murayama, Rie; Kimura-Asami, Mariko; Togo-Ohno, Marina; Yamasaki-Kato, Yumiko; Naruse, Taeko K; Yamamoto, Takeshi; Hayashi, Takeharu; Ai, Tomohiko; Spoonamore, Katherine G; Kovacs, Richard J; Vatta, Matteo; Iizuka, Mai; Saito, Masumi; Wani, Shotaro; Hiraoka, Yuichi; Kimura, Akinori; Kuroyanagi, Hidehito

2018-06-12

RBM20 is a major regulator of heart-specific alternative pre-mRNA splicing of TTN encoding a giant sarcomeric protein titin. Mutation in RBM20 is linked to autosomal-dominant familial dilated cardiomyopathy (DCM), yet most of the RBM20 missense mutations in familial and sporadic cases were mapped to an RSRSP stretch in an arginine/serine-rich region of which function remains unknown. In the present study, we identified an R634W missense mutation within the stretch and a G1031X nonsense mutation in cohorts of DCM patients. We demonstrate that the two serine residues in the RSRSP stretch are constitutively phosphorylated and mutations in the stretch disturb nuclear localization of RBM20. Rbm20 S637A knock-in mouse mimicking an S635A mutation reported in a familial case showed a remarkable effect on titin isoform expression like in a patient carrying the mutation. These results revealed the function of the RSRSP stretch as a critical part of a nuclear localization signal and offer the Rbm20 S637A mouse as a good model for in vivo study.

Genome Sequence and Transcriptome Analyses of Chrysochromulina tobin: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae)

DOE PAGES

Hovde, Blake T.; Deodato, Chloe R.; Hunsperger, Heather M.; ...

2015-09-23

Haptophytes are recognized as seminal players in aquatic ecosystem function. These algae are important in global carbon sequestration, form destructive harmful blooms, and given their rich fatty acid content, serve as a highly nutritive food source to a broad range of eco-cohorts. Haptophyte dominance in both fresh and marine waters is supported by the mixotrophic nature of many taxa. Despite their importance the nuclear genome sequence of only one haptophyte, Emiliania huxleyi (Isochrysidales), is available. Here we report the draft genome sequence of Chrysochromulina tobin (Prymnesiales), and transcriptome data collected at seven time points over a 24-hour light/dark cycle. Themore » nuclear genome of C. tobin is small (59 Mb), compact (~40% of the genome is protein coding) and encodes approximately 16,777 genes. Genes important to fatty acid synthesis, modification, and catabolism show distinct patterns of expression when monitored over the circadian photoperiod. The C. tobin genome harbors the first hybrid polyketide synthase/non-ribosomal peptide synthase gene complex reported for an algal species, and encodes potential anti-microbial peptides and proteins involved in multidrug and toxic compound extrusion. A new haptophyte xanthorhodopsin was also identified, together with two “red” RuBisCO activases that are shared across many algal lineages. In conclusion, the Chrysochromulina tobin genome sequence provides new information on the evolutionary history, ecology and economic importance of haptophytes.« less

An Out-of-frame Overlapping Reading Frame in the Ataxin-1 Coding Sequence Encodes a Novel Ataxin-1 Interacting Protein*

PubMed Central

Bergeron, Danny; Lapointe, Catherine; Bissonnette, Cyntia; Tremblay, Guillaume; Motard, Julie; Roucou, Xavier

2013-01-01

Spinocerebellar ataxia type 1 is an autosomal dominant cerebellar ataxia associated with the expansion of a polyglutamine tract within the ataxin-1 (ATXN1) protein. Recent studies suggest that understanding the normal function of ATXN1 in cellular processes is essential to decipher the pathogenesis mechanisms in spinocerebellar ataxia type 1. We found an alternative translation initiation ATG codon in the +3 reading frame of human ATXN1 starting 30 nucleotides downstream of the initiation codon for ATXN1 and ending at nucleotide 587. This novel overlapping open reading frame (ORF) encodes a 21-kDa polypeptide termed Alt-ATXN1 (Alternative ATXN1) with a completely different amino acid sequence from ATXN1. We introduced a hemagglutinin tag in-frame with Alt-ATXN1 in ATXN1 cDNA and showed in cell culture the co-expression of both ATXN1 and Alt-ATXN1. Remarkably, Alt-ATXN1 colocalized and interacted with ATXN1 in nuclear inclusions. In contrast, in the absence of ATXN1 expression, Alt-ATXN1 displays a homogenous nucleoplasmic distribution. Alt-ATXN1 interacts with poly(A)+ RNA, and its nuclear localization is dependent on RNA transcription. Polyclonal antibodies raised against Alt-ATXN1 confirmed the expression of Alt-ATXN1 in human cerebellum expressing ATXN1. These results demonstrate that human ATXN1 gene is a dual coding sequence and that ATXN1 interacts with and controls the subcellular distribution of Alt-ATXN1. PMID:23760502

Genome Sequence and Transcriptome Analyses of Chrysochromulina tobin: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hovde, Blake T.; Deodato, Chloe R.; Hunsperger, Heather M.

Haptophytes are recognized as seminal players in aquatic ecosystem function. These algae are important in global carbon sequestration, form destructive harmful blooms, and given their rich fatty acid content, serve as a highly nutritive food source to a broad range of eco-cohorts. Haptophyte dominance in both fresh and marine waters is supported by the mixotrophic nature of many taxa. Despite their importance the nuclear genome sequence of only one haptophyte, Emiliania huxleyi (Isochrysidales), is available. Here we report the draft genome sequence of Chrysochromulina tobin (Prymnesiales), and transcriptome data collected at seven time points over a 24-hour light/dark cycle. Themore » nuclear genome of C. tobin is small (59 Mb), compact (~40% of the genome is protein coding) and encodes approximately 16,777 genes. Genes important to fatty acid synthesis, modification, and catabolism show distinct patterns of expression when monitored over the circadian photoperiod. The C. tobin genome harbors the first hybrid polyketide synthase/non-ribosomal peptide synthase gene complex reported for an algal species, and encodes potential anti-microbial peptides and proteins involved in multidrug and toxic compound extrusion. A new haptophyte xanthorhodopsin was also identified, together with two “red” RuBisCO activases that are shared across many algal lineages. In conclusion, the Chrysochromulina tobin genome sequence provides new information on the evolutionary history, ecology and economic importance of haptophytes.« less

Loss of the smallest subunit of cytochrome c oxidase, COX8A, causes Leigh-like syndrome and epilepsy.

PubMed

Hallmann, Kerstin; Kudin, Alexei P; Zsurka, Gábor; Kornblum, Cornelia; Reimann, Jens; Stüve, Burkhard; Waltz, Stephan; Hattingen, Elke; Thiele, Holger; Nürnberg, Peter; Rüb, Cornelia; Voos, Wolfgang; Kopatz, Jens; Neumann, Harald; Kunz, Wolfram S

2016-02-01

Isolated cytochrome c oxidase (complex IV) deficiency is one of the most frequent respiratory chain defects in humans and is usually caused by mutations in proteins required for assembly of the complex. Mutations in nuclear-encoded structural subunits are very rare. In a patient with Leigh-like syndrome presenting with leukodystrophy and severe epilepsy, we identified a homozygous splice site mutation in COX8A, which codes for the ubiquitously expressed isoform of subunit VIII, the smallest nuclear-encoded subunit of complex IV. The mutation, affecting the last nucleotide of intron 1, leads to aberrant splicing, a frame-shift in the highly conserved exon 2, and decreased amount of the COX8A transcript. The loss of the wild-type COX8A protein severely impairs the stability of the entire cytochrome c oxidase enzyme complex and manifests in isolated complex IV deficiency in skeletal muscle and fibroblasts, similar to the frequent c.845_846delCT mutation in the assembly factor SURF1 gene. Stability and activity of complex IV could be rescued in the patient's fibroblasts by lentiviral expression of wild-type COX8A. Our findings demonstrate that COX8A is indispensable for function of human complex IV and its mutation causes human disease. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Molecular Cloning of a cDNA Encoding for Taenia solium TATA-Box Binding Protein 1 (TsTBP1) and Study of Its Interactions with the TATA-Box of Actin 5 and Typical 2-Cys Peroxiredoxin Genes

PubMed Central

Rodríguez-Lima, Oscar; García-Gutierrez, Ponciano; Jiménez, Lucía; Zarain-Herzberg, Ángel; Lazzarini, Roberto; Landa, Abraham

2015-01-01

TATA-box binding protein (TBP) is an essential regulatory transcription factor for the TATA-box and TATA-box-less gene promoters. We report the cloning and characterization of a full-length cDNA that encodes a Taenia solium TATA-box binding protein 1 (TsTBP1). Deduced amino acid composition from its nucleotide sequence revealed that encodes a protein of 238 residues with a predicted molecular weight of 26.7 kDa, and a theoretical pI of 10.6. The NH2-terminal domain shows no conservation when compared with to pig and human TBP1s. However, it shows high conservation in size and amino acid identity with taeniids TBP1s. In contrast, the TsTBP1 COOH-terminal domain is highly conserved among organisms, and contains the amino acids involved in interactions with the TATA-box, as well as with TFIIA and TFIIB. In silico TsTBP1 modeling reveals that the COOH-terminal domain forms the classical saddle structure of the TBP family, with one α-helix at the end, not present in pig and human. Native TsTBP1 was detected in T. solium cysticerci´s nuclear extract by western blot using rabbit antibodies generated against two synthetic peptides located in the NH2 and COOH-terminal domains of TsTBP1. These antibodies, through immunofluorescence technique, identified the TBP1 in the nucleus of cells that form the bladder wall of cysticerci of Taenia crassiceps, an organism close related to T. solium. Electrophoretic mobility shift assays using nuclear extracts from T. solium cysticerci and antibodies against the NH2-terminal domain of TsTBP1 showed the interaction of native TsTBP1 with the TATA-box present in T. solium actin 5 (pAT5) and 2-Cys peroxiredoxin (Ts2-CysPrx) gene promoters; in contrast, when antibodies against the anti-COOH-terminal domain of TsTBP1 were used, they inhibited the binding of TsTBP1 to the TATA-box of the pAT5 promoter gene. PMID:26529408

The translation initiation factor 3 subunit eIF3K interacts with PML and associates with PML nuclear bodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Salsman, Jayme; Pinder, Jordan; Tse, Brenda

2013-10-15

The promyelocytic leukemia protein (PML) is a tumor suppressor protein that regulates a variety of important cellular processes, including gene expression, DNA repair and cell fate decisions. Integral to its function is the ability of PML to form nuclear bodies (NBs) that serve as hubs for the interaction and modification of over 90 cellular proteins. There are seven canonical isoforms of PML, which encode diverse C-termini generated by alternative pre-mRNA splicing. Recruitment of specific cellular proteins to PML NBs is mediated by protein–protein interactions with individual PML isoforms. Using a yeast two-hybrid screen employing peptide sequences unique to PML isoformmore » I (PML-I), we identified an interaction with the eukaryotic initiation factor 3 subunit K (eIF3K), and in the process identified a novel eIF3K isoform, which we term eIF3K-2. We further demonstrate that eIF3K and PML interact both in vitro via pull-down assays, as well as in vivo within human cells by co-immunoprecipitation and co-immunofluorescence. In addition, eIF3K isoform 2 (eIF3K-2) colocalizes to PML bodies, particularly those enriched in PML-I, while eIF3K isoform 1 associates poorly with PML NBs. Thus, we report eIF3K as the first known subunit of the eIF3 translation pre-initiation complex to interact directly with the PML protein, and provide data implicating alternative splicing of both PML and eIF3K as a possible regulatory mechanism for eIF3K localization at PML NBs. - Highlights: • The PML-I C-terminus, encoded by exon 9, interacts with translation factor eIF3K. • We identify a novel eIF3K isoform that excludes exon 2 (eIF3K-2). • eIF3K-2 preferentially associates with PML bodies enriched in PML-I vs. PML-IV. • Alternative splicing of eIF3K regulates association with PML bodies.« less

The Rieske Iron-Sulfur Protein: Import and Assembly into the Cytochrome bc(1) Complex of Yeast Mitochondria.

PubMed

Conte, Laura; Zara, Vincenzo

2011-01-01

The Rieske iron-sulfur protein, one of the catalytic subunits of the cytochrome bc(1) complex, is involved in electron transfer at the level of the inner membrane of yeast mitochondria. The Rieske iron-sulfur protein is encoded by nuclear DNA and, after being synthesized in the cytosol, is imported into mitochondria with the help of a cleavable N-terminal presequence. The imported protein, besides incorporating the 2Fe-2S cluster, also interacts with other catalytic and non-catalytic subunits of the cytochrome bc(1) complex, thereby assembling into the mature and functional respiratory complex. In this paper, we summarize the most recent findings on the import and assembly of the Rieske iron-sulfur protein into Saccharomyces cerevisiae mitochondria, also discussing a possible role of this protein both in the dimerization of the cytochrome bc(1) complex and in the interaction of this homodimer with other complexes of the mitochondrial respiratory chain.

The Rieske Iron-Sulfur Protein: Import and Assembly into the Cytochrome bc 1 Complex of Yeast Mitochondria

PubMed Central

Conte, Laura; Zara, Vincenzo

2011-01-01

The Rieske iron-sulfur protein, one of the catalytic subunits of the cytochrome bc 1 complex, is involved in electron transfer at the level of the inner membrane of yeast mitochondria. The Rieske iron-sulfur protein is encoded by nuclear DNA and, after being synthesized in the cytosol, is imported into mitochondria with the help of a cleavable N-terminal presequence. The imported protein, besides incorporating the 2Fe-2S cluster, also interacts with other catalytic and non-catalytic subunits of the cytochrome bc 1 complex, thereby assembling into the mature and functional respiratory complex. In this paper, we summarize the most recent findings on the import and assembly of the Rieske iron-sulfur protein into Saccharomyces cerevisiae mitochondria, also discussing a possible role of this protein both in the dimerization of the cytochrome bc 1 complex and in the interaction of this homodimer with other complexes of the mitochondrial respiratory chain. PMID:21716720

Intracellular Localization of Arabidopsis Sulfurtransferases1

PubMed Central

Bauer, Michael; Dietrich, Christof; Nowak, Katharina; Sierralta, Walter D.; Papenbrock, Jutta

2004-01-01

Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyze the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. In all organisms analyzed to date, small gene families encoding Str proteins have been identified. The gene products were localized to different compartments of the cells. Our interest concerns the localization of Str proteins encoded in the nuclear genome of Arabidopsis. Computer-based prediction methods revealed localization in different compartments of the cell for six putative AtStrs. Several methods were used to determine the localization of the AtStr proteins experimentally. For AtStr1, a mitochondrial localization was demonstrated by immunodetection in the proteome of isolated mitochondria resolved by one- and two-dimensional gel electrophoresis and subsequent blotting. The respective mature AtStr1 protein was identified by mass spectrometry sequencing. The same result was obtained by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts, whereas AtStr2 was exclusively localized to the cytoplasm by this method. Three members of the single-domain AtStr were localized in the chloroplasts as demonstrated by transient expression of green fluorescent protein fusions in protoplasts and stomata, whereas the single-domain AtStr18 was shown to be cytoplasmic. The remarkable subcellular distribution of AtStr15 was additionally analyzed by transmission electron immunomicroscopy using a monospecific antibody against green fluorescent protein, indicating an attachment to the thylakoid membrane. The knowledge of the intracellular localization of the members of this multiprotein family will help elucidate their specific functions in the organism. PMID:15181206

Intracellular localization of Arabidopsis sulfurtransferases.

PubMed

Bauer, Michael; Dietrich, Christof; Nowak, Katharina; Sierralta, Walter D; Papenbrock, Jutta

2004-06-01

Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyze the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. In all organisms analyzed to date, small gene families encoding Str proteins have been identified. The gene products were localized to different compartments of the cells. Our interest concerns the localization of Str proteins encoded in the nuclear genome of Arabidopsis. Computer-based prediction methods revealed localization in different compartments of the cell for six putative AtStrs. Several methods were used to determine the localization of the AtStr proteins experimentally. For AtStr1, a mitochondrial localization was demonstrated by immunodetection in the proteome of isolated mitochondria resolved by one- and two-dimensional gel electrophoresis and subsequent blotting. The respective mature AtStr1 protein was identified by mass spectrometry sequencing. The same result was obtained by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts, whereas AtStr2 was exclusively localized to the cytoplasm by this method. Three members of the single-domain AtStr were localized in the chloroplasts as demonstrated by transient expression of green fluorescent protein fusions in protoplasts and stomata, whereas the single-domain AtStr18 was shown to be cytoplasmic. The remarkable subcellular distribution of AtStr15 was additionally analyzed by transmission electron immunomicroscopy using a monospecific antibody against green fluorescent protein, indicating an attachment to the thylakoid membrane. The knowledge of the intracellular localization of the members of this multiprotein family will help elucidate their specific functions in the organism.

Multi-Omics Driven Assembly and Annotation of the Sandalwood (Santalum album) Genome.

PubMed

Mahesh, Hirehally Basavarajegowda; Subba, Pratigya; Advani, Jayshree; Shirke, Meghana Deepak; Loganathan, Ramya Malarini; Chandana, Shankara Lingu; Shilpa, Siddappa; Chatterjee, Oishi; Pinto, Sneha Maria; Prasad, Thottethodi Subrahmanya Keshava; Gowda, Malali

2018-04-01

Indian sandalwood ( Santalum album ) is an important tropical evergreen tree known for its fragrant heartwood-derived essential oil and its valuable carving wood. Here, we applied an integrated genomic, transcriptomic, and proteomic approach to assemble and annotate the Indian sandalwood genome. Our genome sequencing resulted in the establishment of a draft map of the smallest genome for any woody tree species to date (221 Mb). The genome annotation predicted 38,119 protein-coding genes and 27.42% repetitive DNA elements. In-depth proteome analysis revealed the identities of 72,325 unique peptides, which confirmed 10,076 of the predicted genes. The addition of transcriptomic and proteogenomic approaches resulted in the identification of 53 novel proteins and 34 gene-correction events that were missed by genomic approaches. Proteogenomic analysis also helped in reassigning 1,348 potential noncoding RNAs as bona fide protein-coding messenger RNAs. Gene expression patterns at the RNA and protein levels indicated that peptide sequencing was useful in capturing proteins encoded by nuclear and organellar genomes alike. Mass spectrometry-based proteomic evidence provided an unbiased approach toward the identification of proteins encoded by organellar genomes. Such proteins are often missed in transcriptome data sets due to the enrichment of only messenger RNAs that contain poly(A) tails. Overall, the use of integrated omic approaches enhanced the quality of the assembly and annotation of this nonmodel plant genome. The availability of genomic, transcriptomic, and proteomic data will enhance genomics-assisted breeding, germplasm characterization, and conservation of sandalwood trees. © 2018 American Society of Plant Biologists. All Rights Reserved.

Inactivation of retinoblastoma protein does not overcome the requirement for human cytomegalovirus UL97 in lamina disruption and nuclear egress.

PubMed

Reim, Natalia I; Kamil, Jeremy P; Wang, Depeng; Lin, Alison; Sharma, Mayuri; Ericsson, Maria; Pesola, Jean M; Golan, David E; Coen, Donald M

2013-05-01

Human cytomegalovirus (HCMV) encodes one conventional protein kinase, UL97. During infection, UL97 phosphorylates the retinoblastoma tumor suppressor protein (pRb) on sites ordinarily phosphorylated by cyclin-dependent kinases (CDK), inactivating the ability of pRb to repress host genes required for cell cycle progression to S phase. UL97 is important for viral DNA synthesis in quiescent cells, but this function can be replaced by human papillomavirus type 16 E7, which targets pRb for degradation. However, viruses in which E7 replaces UL97 are still defective for virus production. UL97 is also required for efficient nuclear egress of viral nucleocapsids, which is associated with disruption of the nuclear lamina during infection, and phosphorylation of lamin A/C on serine 22, which antagonizes lamin polymerization. We investigated whether inactivation of pRb might overcome the requirement of UL97 for these roles, as pRb inactivation induces CDK1, and CDK1 phosphorylates lamin A/C on serine 22. We found that lamin A/C serine 22 phosphorylation during HCMV infection correlated with expression of UL97 and was considerably delayed in UL97-null mutants, even when E7 was expressed. E7 failed to restore gaps in the nuclear lamina seen in wild-type but not UL97-null virus infections. In electron microscopy analyses, a UL97-null virus expressing E7 was as impaired as a UL97-null mutant in cytoplasmic accumulation of viral nucleocapsids. Our results demonstrate that pRb inactivation is insufficient to restore efficient viral nuclear egress of HCMV in the absence of UL97 and instead argue further for a direct role of UL97 in this stage of the infectious cycle.

Pod Corn Is Caused by Rearrangement at the Tunicate1 Locus[W][OA

PubMed Central

Han, Jong-Jin; Jackson, David; Martienssen, Robert

2012-01-01

Pod corn (Zea mays var tunicata) was once regarded as ancestral to cultivated maize, and was prized by pre-Columbian cultures for its magical properties. Tunicate1 (Tu1) is a dominant pod corn mutation in which kernels are completely enclosed in leaflike glumes. Here we show that Tu1 encodes a MADS box transcription factor expressed in leaves whose 5′ regulatory region is fused by a 1.8-Mb chromosomal inversion to the 3′ region of a gene expressed in the inflorescence. Both genes are further duplicated, accounting for classical derivative alleles isolated by recombination, and Tu1 transgenes interact with these derivative alleles in a dose-dependent manner. In young ear primordia, TU1 proteins are nuclearly localized in specific cells at the base of spikelet pair meristems. Tu1 branch determination defects resemble those in ramosa mutants, which encode regulatory proteins expressed in these same cells, accounting for synergism in double mutants discovered almost 100 years ago. The Tu1 rearrangement is not found in ancestral teosinte and arose after domestication of maize. PMID:22829149

Saccharomyces cerevisiae possesses a stress-inducible glycyl-tRNA synthetase gene.

PubMed

Chen, Shun-Jia; Wu, Yi-Hua; Huang, Hsiao-Yun; Wang, Chien-Chia

2012-01-01

Aminoacyl-tRNA synthetases are a large family of housekeeping enzymes that are pivotal in protein translation and other vital cellular processes. Saccharomyces cerevisiae possesses two distinct nuclear glycyl-tRNA synthetase (GlyRS) genes, GRS1 and GRS2. GRS1 encodes both cytoplasmic and mitochondrial activities, while GRS2 is essentially silent and dispensable under normal conditions. We herein present evidence that expression of GRS2 was drastically induced upon heat shock, ethanol or hydrogen peroxide addition, and high pH, while expression of GRS1 was somewhat repressed under those conditions. In addition, GlyRS2 (the enzyme encoded by GRS2) had a higher protein stability and a lower K(M) value for yeast tRNA(Gly) under heat shock conditions than under normal conditions. Moreover, GRS2 rescued the growth defect of a GRS1 knockout strain when highly expressed by a strong promoter at 37 °C, but not at the optimal temperature of 30 °C. These results suggest that GRS2 is actually an inducible gene that may function to rescue the activity of GRS1 under stress conditions.

HTLV-1 Tax upregulates early growth response protein 1 through nuclear factor-κB signaling.

PubMed

Huang, Qingsong; Niu, Zhiguo; Han, Jingxian; Liu, Xihong; Lv, Zhuangwei; Li, Huanhuan; Yuan, Lixiang; Li, Xiangping; Sun, Shuming; Wang, Hui; Huang, Xinxiang

2017-08-01

Human T cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that causes adult T cell leukemia (ATL) in susceptible individuals. The HTLV-1-encoded oncoprotein Tax induces persistent activation of the nuclear factor-κB (NF-κB) pathway. Early growth response protein 1 (EGR1) is overexpressed in HTLV-1-infected T cell lines and ATL cells. Here, we showed that both Tax expression and HTLV-1 infection promoted EGR1 overexpression. Loss of the NF-κB binding site in the EGR1 promotor or inhibition of NF-κB activation reduced Tax-induced EGR1 upregulation. Tax mutants unable to activate NF-κB induced only slight EGR1 upregulation as compared with wild-type Tax, confirming NF-κB pathway involvement in EGR1 regulation. Tax also directly interacted with the EGR1 protein and increased endogenous EGR1 stability. Elevated EGR1 in turn promoted p65 nuclear translocation and increased NF-κB activation. These results demonstrate a positive feedback loop between EGR1 expression and NF-κB activation in HTLV-1-infected and Tax-expressing cells. Both NF-κB activation and Tax-induced EGR1 stability upregulated EGR1, which in turn enhanced constitutive NF-κB activation and facilitated ATL progression in HTLV-1-infected cells. These findings suggest EGR1 may be an effective anti-ATL therapeutic target.

A-type Lamins Form Distinct Filamentous Networks with Differential Nuclear Pore Complex Associations.

PubMed

Xie, Wei; Chojnowski, Alexandre; Boudier, Thomas; Lim, John S Y; Ahmed, Sohail; Ser, Zheng; Stewart, Colin; Burke, Brian

2016-10-10

The nuclear lamina is a universal feature of metazoan nuclear envelopes (NEs) [1]. In mammalian cells, it appears as a 10-30 nm filamentous layer at the nuclear face of the inner nuclear membrane (INM) and is composed primarily of A- and B-type lamins, members of the intermediate filament family [2]. While providing structural integrity to the NE, the lamina also represents an important signaling and regulatory platform [3]. Two A-type lamin isoforms, lamins A and C (LaA and LaC), are expressed in most adult human cells. Encoded by a single gene, these proteins are largely identical, diverging only in their C-terminal tail domains. By contrast with that of LaC, the unique LaA tail undergoes extensive processing, including farnesylation and endo-proteolysis [4, 5]. However, functional differences between LaA and LaC are still unclear. Compounding this uncertainty, the structure of the lamina remains ill defined. In this study, we used BioID, an in vivo proximity-labeling method to identify differential interactors of A-type lamins [6]. One of these, Tpr, a nuclear pore complex (NPC) protein, is highlighted by its selective association with LaC. By employing superresolution microscopy, we demonstrate that this Tpr association is mirrored in enhanced interaction of LaC with NPCs. Further superresolution studies visualizing both endogenous A- and B-type lamins have allowed us to construct a nanometer-scale model of the mammalian nuclear lamina. Our data indicate that different A- and B-type lamin species assemble into separate filament networks that together form an extended composite structure at the nuclear periphery providing attachment sites for NPCs, thereby regulating their distribution. Copyright © 2016 Elsevier Ltd. All rights reserved.

Perturbations of carotenoid and tetrapyrrole biosynthetic pathways result in differential alterations in chloroplast function and plastid signaling.

PubMed

Park, Joon-Heum; Jung, Sunyo

2017-01-22

In this study, we used the biosynthetic inhibitors of carotenoid and tetrapyrrole biosynthetic pathways, norflurazon (NF) and oxyfluorfen (OF), as tools to gain insight into mechanisms of photooxidation in rice plants. NF resulted in bleaching symptom on leaves of the treated plants, whereas OF treatment developed a fast symptom of an apparent necrotic phenotype. Both plants exhibited decreases in photosynthetic efficiency, as indicated by F v /F m . NF caused severe disruption in thylakoid membranes, whereas OF-treated plants exhibited disruption of chloroplast envelope and plasma membrane. Levels of Lhca and Lhcb proteins in photosystem I (PSI) and PSII were reduced by photooxidative stress in NF- and OF-treated plants, with a greater decrease in NF plants. The down-regulation of nuclear-encoded photosynthesis genes Lhcb and rbcS was also found in both NF- and OF-treated plants, whereas plastid-encoded photosynthetic genes including RbcL, PsaC, and PsbD accumulated normally in NF plants but decreased drastically in OF plants. This proposes that the plastids in NF plants retain their potential to develop thylakoid membranes and that photobleaching is mainly controlled by nuclear genes. Distinct photooxidation patterns between NF- and OF-treated plants developed differential signaling, which might enable the plant to coordinate the expression of photosynthetic genes from the nuclear and plastidic genomes. Copyright © 2016 Elsevier Inc. All rights reserved.

Potential Functional Replacement of the Plastidic Acetyl-CoA Carboxylase Subunit (accD) Gene by Recent Transfers to the Nucleus in Some Angiosperm Lineages1[W][OA

PubMed Central

Rousseau-Gueutin, Mathieu; Huang, Xun; Higginson, Emily; Ayliffe, Michael; Day, Anil; Timmis, Jeremy N.

2013-01-01

Eukaryotic cells originated when an ancestor of the nucleated cell engulfed bacterial endosymbionts that gradually evolved into the mitochondrion and the chloroplast. Soon after these endosymbiotic events, thousands of ancestral prokaryotic genes were functionally transferred from the endosymbionts to the nucleus. This process of functional gene relocation, now rare in eukaryotes, continues in angiosperms. In this article, we show that the chloroplastic acetyl-CoA carboxylase subunit (accD) gene that is present in the plastome of most angiosperms has been functionally relocated to the nucleus in the Campanulaceae. Surprisingly, the nucleus-encoded accD transcript is considerably smaller than the plastidic version, consisting of little more than the carboxylase domain of the plastidic accD gene fused to a coding region encoding a plastid targeting peptide. We verified experimentally the presence of a chloroplastic transit peptide by showing that the product of the nuclear accD fused to green fluorescent protein was imported in the chloroplasts. The nuclear gene regulatory elements that enabled the erstwhile plastidic gene to become functional in the nuclear genome were identified, and the evolution of the intronic and exonic sequences in the nucleus is described. Relocation and truncation of the accD gene is a remarkable example of the processes underpinning endosymbiotic evolution. PMID:23435694

The Popeye Domain Containing Genes and Their Function in Striated Muscle

PubMed Central

Schindler, Roland F. R.; Scotton, Chiara; French, Vanessa; Ferlini, Alessandra; Brand, Thomas

2016-01-01

The Popeye domain containing (POPDC) genes encode a novel class of cAMP effector proteins, which are abundantly expressed in heart and skeletal muscle. Here, we will review their role in striated muscle as deduced from work in cell and animal models and the recent analysis of patients carrying a missense mutation in POPDC1. Evidence suggests that POPDC proteins control membrane trafficking of interacting proteins. Furthermore, we will discuss the current catalogue of established protein-protein interactions. In recent years, the number of POPDC-interacting proteins has been rising and currently includes ion channels (TREK-1), sarcolemma-associated proteins serving functions in mechanical stability (dystrophin), compartmentalization (caveolin 3), scaffolding (ZO-1), trafficking (NDRG4, VAMP2/3) and repair (dysferlin) or acting as a guanine nucleotide exchange factor for Rho-family GTPases (GEFT). Recent evidence suggests that POPDC proteins might also control the cellular level of the nuclear proto-oncoprotein c-Myc. These data suggest that this family of cAMP-binding proteins probably serves multiple roles in striated muscle. PMID:27347491

Alternative function for the mitochondrial SAM complex in biogenesis of alpha-helical TOM proteins.

PubMed

Stojanovski, Diana; Guiard, Bernard; Kozjak-Pavlovic, Vera; Pfanner, Nikolaus; Meisinger, Chris

2007-12-03

The mitochondrial outer membrane contains two preprotein translocases: the general translocase of outer membrane (TOM) and the beta-barrel-specific sorting and assembly machinery (SAM). TOM functions as the central entry gate for nuclear-encoded proteins. The channel-forming Tom40 is a beta-barrel protein, whereas all Tom receptors and small Tom proteins are membrane anchored by a transmembrane alpha-helical segment in their N- or C-terminal portion. Synthesis of Tom precursors takes place in the cytosol, and their import occurs via preexisting TOM complexes. The precursor of Tom40 is then transferred to SAM for membrane insertion and assembly. Unexpectedly, we find that the biogenesis of alpha-helical Tom proteins with a membrane anchor in the C-terminal portion is SAM dependent. Each SAM protein is necessary for efficient membrane integration of the receptor Tom22, whereas assembly of the small Tom proteins depends on Sam37. Thus, the substrate specificity of SAM is not restricted to beta-barrel proteins but also includes the majority of alpha-helical Tom proteins.

A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5′ End or within the Genome of Feline Parvovirus and Can Modify Virus Replication

PubMed Central

Wang, Dai; Parrish, Colin R.

1999-01-01

Phage display of cDNA clones prepared from feline cells was used to identify host cell proteins that bound to DNA-containing feline panleukopenia virus (FPV) capsids but not to empty capsids. One gene found in several clones encoded a heterogeneous nuclear ribonucleoprotein (hnRNP)-related protein (DBP40) that was very similar in sequence to the A/B-type hnRNP proteins. DBP40 bound specifically to oligonucleotides representing a sequence near the 5′ end of the genome which is exposed on the outside of the full capsid but did not bind most other terminal sequences. Adding purified DBP40 to an in vitro fill-in reaction using viral DNA as a template inhibited the production of the second strand after nucleotide (nt) 289 but prior to nt 469. DBP40 bound to various regions of the viral genome, including a region between nt 295 and 330 of the viral genome which has been associated with transcriptional attenuation of the parvovirus minute virus of mice, which is mediated by a stem-loop structure of the DNA and cellular proteins. Overexpression of the protein in feline cells from a plasmid vector made them largely resistant to FPV infection. Mutagenesis of the protein binding site within the 5′ end viral genome did not affect replication of the virus. PMID:10438866

Characterization of the extrinsic apoptotic pathway in the basal chordate amphioxus.

PubMed

Yuan, Shaochun; Liu, Huiling; Gu, Ming; Xu, Liqun; Huang, Shengfeng; Ren, Zhenhua; Xu, Anlong

2010-09-14

The death receptor (DR)-mediated apoptosis pathway is thought to be unique to vertebrates. However, the presence of DR-encoding genes in the sea urchin and the basal chordate amphioxus prompted us to reconsider, especially given that amphioxus contains 14 DR proteins and hundreds of death domain (DD)-containing adaptor proteins. To understand how the extrinsic apoptotic pathway was originally established and what the differences in signaling are between invertebrates and vertebrates, we performed functional studies of several genes that encode DDs in the amphioxus Branchiostoma belcheri tsingtauense (Bbt). First, we observed that the increased abundance of Bbt Fas-associated death domain 1 (BbtFADD1) in HeLa cells resulted in the formation of death effector filamentous structures in the cytoplasm and the activation of the nuclear factor κB pathway, whereas BbtFADD2 protein was restricted to the nucleus, although its death effector domain induced apoptosis when in the cytoplasm. We further demonstrated that formation of a FADD-caspase-8 complex recruited amphioxus DR1 (BbtDR1), which bound to the adaptor proteins CRADD or TRAF6 (tumor necrosis factor receptor-associated factor 6) to convey distinct signals, ranging from apoptosis to gene activation. Thus, our study not only reveals the evolutionary origin of the extrinsic apoptotic pathway in a basal chordate but also adds to our understanding of the similarities and differences between invertebrate and vertebrate FADD signaling.

T:G mismatch-specific thymine-DNA glycosylase (TDG) as a coregulator of transcription interacts with SRC1 family members through a novel tyrosine repeat motif

PubMed Central

Lucey, Marie J.; Chen, Dongsheng; Lopez-Garcia, Jorge; Hart, Stephen M.; Phoenix, Fladia; Al-Jehani, Rajai; Alao, John P.; White, Roger; Kindle, Karin B.; Losson, Régine; Chambon, Pierre; Parker, Malcolm G.; Schär, Primo; Heery, David M.; Buluwela, Lakjaya; Ali, Simak

2005-01-01

Gene activation involves protein complexes with diverse enzymatic activities, some of which are involved in chromatin modification. We have shown previously that the base excision repair enzyme thymine DNA glycosylase (TDG) acts as a potent coactivator for estrogen receptor-α. To further understand how TDG acts in this context, we studied its interaction with known coactivators of nuclear receptors. We find that TDG interacts in vitro and in vivo with the p160 coactivator SRC1, with the interaction being mediated by a previously undescribed motif encoding four equally spaced tyrosine residues in TDG, each tyrosine being separated by three amino acids. This is found to interact with two motifs in SRC1 also containing tyrosine residues separated by three amino acids. Site-directed mutagenesis shows that the tyrosines encoded in these motifs are critical for the interaction. The related p160 protein TIF2 does not interact with TDG and has the altered sequence, F-X-X-X-Y, at the equivalent positions relative to SRC1. Substitution of the phenylalanines to tyrosines is sufficient to bring about interaction of TIF2 with TDG. These findings highlight a new protein–protein interaction motif based on Y-X-X-X-Y and provide new insight into the interaction of diverse proteins in coactivator complexes. PMID:16282588

Analysis of Autoregulation at the Level of Pre-mRNA Splicing of the Suppressor-of-White-Apricot Gene in Drosophila

PubMed Central

Zachar, Z.; Chou, T. B.; Kramer, J.; Mims, I. P.; Bingham, P. M.

1994-01-01

The Drosophila suppressor-of-white-apricot [su(w(a))] protein regulates/modulates at least two somatic RNA processing events. It is a potent regulator of its own expression. We report here new studies of this autoregulatory circuit. Among other things, our studies show the following. First, new evidence that su(w(a)) expression is autoregulated at the level of pre-mRNA splicing is reported. su(w(a)) protein represses accumulation of the fully spliced su(w(a)) mRNA encoding it and promotes accumulation of high levels of incompletely spliced su(w(a)) pre-mRNA. Second, the fully spliced su(w(a)) mRNA is sufficient for all known su(w(a)) genetic functions indicating that it encodes the sole su(w(a)) protein. Third, the incompletely spliced su(w(a)) pre-mRNAs resulting from autoregulation are not translated (probably as a result of nuclear retention) and apparently represent nonfunctional by-products. Fourth, the special circumstances of su(w(a)) expression during oogenesis allows maternal deposition exclusively of fully spliced su(w(a)) mRNA. Fifth, su(w(a)) protein immunolocalizes to nuclei consistent with its being a direct regulator of pre-mRNA processing. We discuss the implications of our results for mechanisms of splicing regulation and for developmental control of su(w(a)) expression. PMID:8056305

The Reverse Transcriptase/RNA Maturase Protein MatR Is Required for the Splicing of Various Group II Introns in Brassicaceae Mitochondria

PubMed Central

Sultan, Laure D.; Grewe, Felix; Rolle, Katarzyna; Abudraham, Sivan; Shevtsov, Sofia; Klipcan, Liron; Barciszewski, Jan; Dietrich, André

2016-01-01

Group II introns are large catalytic RNAs that are ancestrally related to nuclear spliceosomal introns. Sequences corresponding to group II RNAs are found in many prokaryotes and are particularly prevalent within plants organellar genomes. Proteins encoded within the introns themselves (maturases) facilitate the splicing of their own host pre-RNAs. Mitochondrial introns in plants have diverged considerably in sequence and have lost their maturases. In angiosperms, only a single maturase has been retained in the mitochondrial DNA: the matR gene found within NADH dehydrogenase 1 (nad1) intron 4. Its conservation across land plants and RNA editing events, which restore conserved amino acids, indicates that matR encodes a functional protein. However, the biological role of MatR remains unclear. Here, we performed an in vivo investigation of the roles of MatR in Brassicaceae. Directed knockdown of matR expression via synthetically designed ribozymes altered the processing of various introns, including nad1 i4. Pull-down experiments further indicated that MatR is associated with nad1 i4 and several other intron-containing pre-mRNAs. MatR may thus represent an intermediate link in the gradual evolutionary transition from the intron-specific maturases in bacteria into their versatile spliceosomal descendants in the nucleus. The similarity between maturases and the core spliceosomal Prp8 protein further supports this intriguing theory. PMID:27760804

A Fungal Effector With Host Nuclear Localization and DNA-Binding Properties Is Required for Maize Anthracnose Development.

PubMed

Vargas, Walter A; Sanz-Martín, José M; Rech, Gabriel E; Armijos-Jaramillo, Vinicio D; Rivera, Lina P; Echeverria, María Mercedes; Díaz-Mínguez, José M; Thon, Michael R; Sukno, Serenella A

2016-02-01

Plant pathogens have the capacity to manipulate the host immune system through the secretion of effectors. We identified 27 putative effector proteins encoded in the genome of the maize anthracnose pathogen Colletotrichum graminicola that are likely to target the host's nucleus, as they simultaneously contain sequence signatures for secretion and nuclear localization. We functionally characterized one protein, identified as CgEP1. This protein is synthesized during the early stages of disease development and is necessary for anthracnose development in maize leaves, stems, and roots. Genetic, molecular, and biochemical studies confirmed that this effector targets the host's nucleus and defines a novel class of double-stranded DNA-binding protein. We show that CgEP1 arose from a gene duplication in an ancestor of a lineage of monocot-infecting Colletotrichum spp. and has undergone an intense evolution process, with evidence for episodes of positive selection. We detected CgEP1 homologs in several species of a grass-infecting lineage of Colletotrichum spp., suggesting that its function may be conserved across a large number of anthracnose pathogens. Our results demonstrate that effectors targeted to the host nucleus may be key elements for disease development and aid in the understanding of the genetic basis of anthracnose development in maize plants.

Epstein-Barr virus-encoded EBNA-5 binds to Epstein-Barr virus-induced Fte1/S3a protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kashuba, Elena; Yurchenko, Mariya; Szirak, Krisztina

Epstein-Barr virus (EBV) transforms resting human B cells into immortalized immunoblasts. EBV-encoded nuclear antigens EBNA-5 (also called EBNA-LP) is one of the earliest viral proteins expressed in freshly infected B cells. We have recently shown that EBNA-5 binds p14ARF, a nucleolar protein that regulates the p53 pathway. Here, we report the identification of another protein with partially nucleolar localization, the v-fos transformation effector Fte-1 (Fte-1/S3a), as an EBNA-5 binding partner. In transfected cells, Fte-1/S3a and EBNA-5 proteins showed high levels of colocalization in extranucleolar inclusions. Fte-1/S3a has multiple biological functions. It enhances v-fos-mediated cellular transformation and is part of themore » small ribosomal subunit. It also interacts with the transcriptional factor CHOP and apoptosis regulator poly(ADP-ribose) polymerase (PARP). Fte-1/S3a is regularly expressed at high levels in both tumors and cancer cell lines. Its high expression favors the maintenance of malignant phenotype and undifferentiated state, whereas its down-regulation is associated with cellular differentiation and growth arrest. Here, we show that EBV-induced B cell transformation leads to the up-regulation of Fte-1/S3a. We suggest that EBNA-5 through binding may influence the growth promoting, differentiation inhibiting, or apoptosis regulating functions of Fte-1/S3a.« less

Group I introns are inherited through common ancestry in the nuclear-encoded rRNA of Zygnematales (Charophyceae).

PubMed Central

Bhattacharya, D; Surek, B; Rüsing, M; Damberger, S; Melkonian, M

1994-01-01

Group I introns are found in organellar genomes, in the genomes of eubacteria and phages, and in nuclear-encoded rRNAs. The origin and distribution of nuclear-encoded rRNA group I introns are not understood. To elucidate their evolutionary relationships, we analyzed diverse nuclear-encoded small-subunit rRNA group I introns including nine sequences from the green-algal order Zygnematales (Charophyceae). Phylogenetic analyses of group I introns and rRNA coding regions suggest that lateral transfers have occurred in the evolutionary history of group I introns and that, after transfer, some of these elements may form stable components of the host-cell nuclear genomes. The Zygnematales introns, which share a common insertion site (position 1506 relative to the Escherichia coli small-subunit rRNA), form one subfamily of group I introns that has, after its origin, been inherited through common ancestry. Since the first Zygnematales appear in the middle Devonian within the fossil record, the "1506" group I intron presumably has been a stable component of the Zygnematales small-subunit rRNA coding region for 350-400 million years. PMID:7937917

Sequential Actions of SIRT1-RELB-SIRT3 Coordinate Nuclear-Mitochondrial Communication during Immunometabolic Adaptation to Acute Inflammation and Sepsis*

PubMed Central

Liu, Tie Fu; Vachharajani, Vidula; Millet, Patrick; Bharadwaj, Manish S.; Molina, Anthony J.; McCall, Charles E.

2015-01-01

We reported that NAD+-dependent SIRT1, RELB, and SIRT6 nuclear proteins in monocytes regulate a switch from the glycolysis-dependent acute inflammatory response to fatty acid oxidation-dependent sepsis adaptation. We also found that disrupting SIRT1 activity during adaptation restores immunometabolic homeostasis and rescues septic mice from death. Here, we show that nuclear SIRT1 guides RELB to differentially induce SIRT3 expression and also increases mitochondrial biogenesis, which alters bioenergetics during sepsis adaptation. We constructed this concept using TLR4-stimulated THP1 human promonocytes, a model that mimics the initiation and adaptation stages of sepsis. Following increased expression, mitochondrial SIRT3 deacetylase activates the rate-limiting tricarboxylic acid cycle (TCA) isocitrate dehydrogenase 2 and superoxide dismutase 2, concomitant with increases in citrate synthase activity. Mitochondrial oxygen consumption rate increases early and decreases during adaptation, parallel with modifications to membrane depolarization, ATP generation, and production of mitochondrial superoxide and whole cell hydrogen peroxide. Evidence of SIRT1-RELB induction of mitochondrial biogenesis included increases in mitochondrial mass, mitochondrial-to-nuclear DNA ratios, and both nuclear and mitochondrial encoded proteins. We confirmed the SIRT-RELB-SIRT3 adaptation link to mitochondrial bioenergetics in both TLR4-stimulated normal and sepsis-adapted human blood monocytes and mouse splenocytes. We also found that SIRT1 inhibition ex vivo reversed the sepsis-induced changes in bioenergetics. PMID:25404738

Kobuvirus VP3 protein restricts the IFN-β-triggered signaling pathway by inhibiting STAT2-IRF9 and STAT2-STAT2 complex formation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peng, Qianqian; Lan, Xi; Wang, Chen

Emerged porcine kobuvirus (PKV) has adversely affected the global swine industry since 2008, but the etiological biology of PKV is unclear. Screening PKV-encoded structural and non-structural proteins with a type I IFN-responsive luciferase reporter showed that PKV VP3 protein inhibited the IFN-β-triggered signaling pathway, resulting in the decrease of VSV-GFP replication. QPCR data showed that IFN-β downstream cytokine genes were suppressed without cell-type specificity as well. The results from biochemical experiments indicated that PKV VP3 associated with STAT2 and IRF9, and interfered with the formation of the STAT2-IRF9 and STAT2-STAT2 complex, impairing nuclear translocation of STAT2 and IRF9. Taken together,more » these data reveal a new mechanism for immune evasion of PKV. - Highlights: •PKV VP3 inhibits the IFN-β-triggered signaling pathway. •VP3 associates with STAT2 and IRF9. •VP3 blocks the STAT2-IRF9 nuclear translocation. •VP3 utilizes a novel strategy for innate immune evasion.« less

Arsenic degrades PML or PML-RARalpha through a SUMO-triggered RNF4/ubiquitin-mediated pathway.

PubMed

Lallemand-Breitenbach, Valérie; Jeanne, Marion; Benhenda, Shirine; Nasr, Rihab; Lei, Ming; Peres, Laurent; Zhou, Jun; Zhu, Jun; Raught, Brian; de Thé, Hugues

2008-05-01

In acute promyelocytic leukaemia (APL), arsenic trioxide induces degradation of the fusion protein encoded by the PML-RARA oncogene, differentiation of leukaemic cells and produces clinical remissions. SUMOylation of its PML moiety was previously implicated, but the nature of the degradation pathway involved and the role of PML-RARalpha catabolism in the response to therapy have both remained elusive. Here, we demonstrate that arsenic-induced PML SUMOylation triggers its Lys 48-linked polyubiquitination and proteasome-dependent degradation. When exposed to arsenic, SUMOylated PML recruits RNF4, the human orthologue of the yeast SUMO-dependent E3 ubiquitin-ligase, as well as ubiquitin and proteasomes onto PML nuclear bodies. Arsenic-induced differentiation is impaired in cells transformed by a non-degradable PML-RARalpha SUMOylation mutant or in APL cells transduced with a dominant-negative RNF4, directly implicating PML-RARalpha catabolism in the therapeutic response. We thus identify PML as the first protein degraded by SUMO-dependent polyubiquitination. As PML SUMOylation recruits not only RNF4, ubiquitin and proteasomes, but also many SUMOylated proteins onto PML nuclear bodies, these domains could physically integrate the SUMOylation, ubiquitination and degradation pathways.

Expression of membrane targeted aequorin in Xenopus laevis oocytes.

PubMed

Daguzan, C; Nicolas, M T; Mazars, C; Leclerc, C; Moreau, M

1995-08-01

We described here a system for high level of expression of the calcium activated photoprotein aequorin. This protein has been targeted to the plasma membrane of Xenopus oocyte by nuclear microinjection of a plasmid containing a construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and the 5-HT1A receptor. The expression of this fusion protein is placed under the control of RSV promoter. Functional photoprotein was reconstituted in the oocyte by incubation with coelenterazine. The amount of photoprotein 24 h after nuclear microinjection of the plasmid was sufficient to trigger a detectable light emission following calcium entry. The efficiency of the expression is correlated with the dose of plasmid injected. Intracytoplasmic injection of the plasmid always failed in photoprotein expression. Targeting of the apoprotein was demonstrated by immunolocalization under confocal microscopy. In our experimental conditions, the apoprotein was always localized at the animal pole above the nucleus. We never observed expression and targeting to the plasma membrane of the vegetal pole. WE suggest that such expression might be of great interest for the study of numerous problems of developmental biology, in which calcium-dependent pathways are involved.

A reference map of the Arabidopsis thaliana mature pollen proteome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Noir, Sandra; Braeutigam, Anne; Colby, Thomas

The male gametophyte (or pollen) plays an obligatory role during sexual reproduction of higher plants. The extremely reduced complexity of this organ renders pollen a valuable experimental system for studying fundamental aspects of plant biology such as cell fate determination, cell-cell interactions, cell polarity, and tip-growth. Here, we present the first reference map of the mature pollen proteome of the dicotyledonous model plant species, Arabidopsis thaliana. Based on two-dimensional gel electrophoresis, matrix-assisted laser desorption/ionization time-of-flight, and electrospray quadrupole time-of-flight mass spectrometry, we reproducibly identified 121 different proteins in 145 individual spots. The presence, subcellular localization, and functional classification of themore » identified proteins are discussed in relation to the pollen transcriptome and the full protein complement encoded by the nuclear Arabidopsis genome.« less

Extending the Applicability of Exact Nuclear Overhauser Enhancements to Large Proteins and RNA.

PubMed

Nichols, Parker; Born, Alexandra; Henen, Morkos; Strotz, Dean; Chi, Celestine N; Güntert, Peter; Vögeli, Beat Rolf

2018-06-08

Distance-dependent NOEs are one of the most popular and important experimental restraints for calculating NMR structures. Despite this, they are mostly employed as semi-quantitative upper distance bounds, which discards a wealth of information that is encoded in the cross-relaxation rate constant. Information that is lost includes exact distances between protons and dynamics that occur on the sub-millisecond time-scale. Our recently introduced exact measurement of the NOE (eNOE) requires little additional experimental effort relative to other NMR observables. So far, we have used eNOEs to calculate multi-state ensembles of proteins up to ~150 residues. Here, we briefly revisit the eNOE methodology and present two new directions for the use of eNOEs: Applications to large proteins and RNA. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Drosophila mitochondrial translation elongation factor G1 contains a nuclear localization signal and inhibits growth and DPP signaling.

PubMed

Trivigno, Catherine; Haerry, Theodor E

2011-02-25

Mutations in the human mitochondrial elongation factor G1 (EF-G1) are recessive lethal and cause death shortly after birth. We have isolated mutations in iconoclast (ico), which encodes the highly conserved Drosophila orthologue of EF-G1. We find that EF-G1 is essential during fly development, but its function is not required in every tissue. In contrast to null mutations, missense mutations exhibit stronger, possibly neomorphic phenotypes that lead to premature death during embryogenesis. Our experiments show that EF-G1 contains a secondary C-terminal nuclear localization signal. Expression of missense mutant forms of EF-G1 can accumulate in the nucleus and cause growth and patterning defects and animal lethality. We find that transgenes that encode mutant human EF-G1 proteins can rescue ico mutants, indicating that the underlying problem of the human disease is not just the loss of enzymatic activity. Our results are consistent with a model where EF-G1 acts as a retrograde signal from mitochondria to the nucleus to slow down cell proliferation if mitochondrial energy output is low.

The Drosophila Mitochondrial Translation Elongation Factor G1 Contains a Nuclear Localization Signal and Inhibits Growth and DPP Signaling

PubMed Central

Trivigno, Catherine; Haerry, Theodor E.

2011-01-01

Mutations in the human mitochondrial elongation factor G1 (EF-G1) are recessive lethal and cause death shortly after birth. We have isolated mutations in iconoclast (ico), which encodes the highly conserved Drosophila orthologue of EF-G1. We find that EF-G1 is essential during fly development, but its function is not required in every tissue. In contrast to null mutations, missense mutations exhibit stronger, possibly neomorphic phenotypes that lead to premature death during embryogenesis. Our experiments show that EF-G1 contains a secondary C-terminal nuclear localization signal. Expression of missense mutant forms of EF-G1 can accumulate in the nucleus and cause growth and patterning defects and animal lethality. We find that transgenes that encode mutant human EF-G1 proteins can rescue ico mutants, indicating that the underlying problem of the human disease is not just the loss of enzymatic activity. Our results are consistent with a model where EF-G1 acts as a retrograde signal from mitochondria to the nucleus to slow down cell proliferation if mitochondrial energy output is low. PMID:21364917

The Complete Plastome Sequence of an Antarctic Bryophyte Sanionia uncinata (Hedw.) Loeske

PubMed Central

Park, Mira; Park, Hyun; Lee, Hyoungseok; Lee, Byeong-ha

2018-01-01

Organellar genomes of bryophytes are poorly represented with chloroplast genomes of only four mosses, four liverworts and two hornworts having been sequenced and annotated. Moreover, while Antarctic vegetation is dominated by the bryophytes, there are few reports on the plastid genomes for the Antarctic bryophytes. Sanionia uncinata (Hedw.) Loeske is one of the most dominant moss species in the maritime Antarctic. It has been researched as an important marker for ecological studies and as an extremophile plant for studies on stress tolerance. Here, we report the complete plastome sequence of S. uncinata, which can be exploited in comparative studies to identify the lineage-specific divergence across different species. The complete plastome of S. uncinata is 124,374 bp in length with a typical quadripartite structure of 114 unique genes including 82 unique protein-coding genes, 37 tRNA genes and four rRNA genes. However, two genes encoding the α subunit of RNA polymerase (rpoA) and encoding the cytochrome b6/f complex subunit VIII (petN) were absent. We could identify nuclear genes homologous to those genes, which suggests that rpoA and petN might have been relocated from the chloroplast genome to the nuclear genome. PMID:29494552

PmVRP15, a novel viral responsive protein from the black tiger shrimp, Penaeus monodon, promoted white spot syndrome virus replication.

PubMed

Vatanavicharn, Tipachai; Prapavorarat, Adisak; Jaree, Phattarunda; Somboonwiwat, Kunlaya; Tassanakajon, Anchalee

2014-01-01

Suppression subtractive hybridization of Penaeus monodon hemocytes challenged with white spot syndrome virus (WSSV) has identified the viral responsive gene, PmVRP15, as the highest up-regulated gene ever reported in shrimps. Expression analysis by quantitative real time RT-PCR revealed 9410-fold up-regulated level at 48 h post WSSV injection. Tissue distribution analysis showed that PmVRP15 transcript was mainly expressed in the hemocytes of shrimp. The full-length cDNA of PmVRP15 transcript was obtained and showed no significant similarity to any known gene in the GenBank database. The predicted open reading frame of PmVRP15 encodes for a deduced 137 amino acid protein containing a putative transmembrane helix. Immunofluorescent localization of the PmVRP15 protein revealed it accumulated around the nuclear membrane in all three types of shrimp hemocytes and that the protein was highly up-regulated in WSSV-infected shrimps. Double-stranded RNA interference-mediated gene silencing of PmVRP15 in P. monodon significantly decreased WSSV propagation compared to the control shrimps (injected with GFP dsRNA). The significant decrease in cumulative mortality rate of WSSV-infected shrimp following PmVRP15 knockdown was observed. These results suggest that PmVRP15 is likely to be a nuclear membrane protein and that it acts as a part of WSSV propagation pathway.

A novel firefly luciferase biosensor enhances the detection of apoptosis induced by ESAT-6 family proteins of Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Junwei; Zhang, Huan; Fang, Liurong

Highlights: • We developed a novel firefly luciferase based biosensor to detect apoptosis. • The novel biosensor 233-DnaE-DEVDG was reliable, sensitive and convenient. • 233-DnaE-DEVDG faithfully indicated ESAT-6 family proteins of Mycobacterium tuberculosis induced apoptosis. • EsxA, esxT and esxL in ESAT-6 family proteins induced apoptosis. • Activation of nuclear factor-κB (NF-κB) participated in esxT-induced apoptosis. - Abstract: The activation of caspase-3 is a key surrogate marker for detecting apoptosis. To quantitate caspase-3 activity, we constructed a biosensor comprising a recombinant firefly luciferase containing a caspase-3 cleavage site. When apoptosis was induced, caspase-3 cleavage of the biosensor activated firefly luciferasemore » by a factor greater than 25. The assay conveniently detected apoptosis in real time, indicating that it will facilitate drug discovery. We screened ESAT-6 family proteins of Mycobacterium tuberculosis and found that esxA, esxT and esxL induced apoptosis. Further, activation of nuclear factor-κB (NF-κB) and the NF-κB-regulated genes encoding tumor necrosis factor-α (TNF-α) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) participated in esxT-induced apoptosis. We conclude that this assay is useful for high-throughput screening to identify and characterize proteins and drugs that regulate apoptosis.« less

The Potyviral P3 Protein Targets Eukaryotic Elongation Factor 1A to Promote the Unfolded Protein Response and Viral Pathogenesis1[OPEN

PubMed Central

Shine, M.B.; Cui, Xiaoyan; Chen, Xin; Ma, Na; Kachroo, Pradeep; Zhi, Haijan; Kachroo, Aardra

2016-01-01

The biochemical function of the potyviral P3 protein is not known, although it is known to regulate virus replication, movement, and pathogenesis. We show that P3, the putative virulence determinant of soybean mosaic virus (SMV), targets a component of the translation elongation complex in soybean. Eukaryotic elongation factor 1A (eEF1A), a well-known host factor in viral pathogenesis, is essential for SMV virulence and the associated unfolded protein response (UPR). Silencing GmEF1A inhibits accumulation of SMV and another ER-associated virus in soybean. Conversely, endoplasmic reticulum (ER) stress-inducing chemicals promote SMV accumulation in wild-type, but not GmEF1A-knockdown, plants. Knockdown of genes encoding the eEF1B isoform, which is important for eEF1A function in translation elongation, has similar effects on UPR and SMV resistance, suggesting a link to translation elongation. P3 and GmEF1A promote each other’s nuclear localization, similar to the nuclear-cytoplasmic transport of eEF1A by the Human immunodeficiency virus 1 Nef protein. Our results suggest that P3 targets host elongation factors resulting in UPR, which in turn facilitates SMV replication and place eEF1A upstream of BiP in the ER stress response during pathogen infection. PMID:27356973

Adaptive antioxidant methionine accumulation in respiratory chain complexes explains the use of a deviant genetic code in mitochondria.

PubMed

Bender, Aline; Hajieva, Parvana; Moosmann, Bernd

2008-10-28

Humans and most other animals use 2 different genetic codes to translate their hereditary information: the standard code for nuclear-encoded proteins and a modern variant of this code in mitochondria. Despite the pivotal role of the genetic code for cell biology, the functional significance of the deviant mitochondrial code has remained enigmatic since its first description in 1979. Here, we show that profound and functionally beneficial alterations on the encoded protein level were causative for the AUA codon reassignment from isoleucine to methionine observed in most mitochondrial lineages. We demonstrate that this codon reassignment leads to a massive accumulation of the easily oxidized amino acid methionine in the highly oxidative inner mitochondrial membrane. This apparently paradoxical outcome can yet be smoothly settled if the antioxidant surface chemistry of methionine is taken into account, and we present direct experimental evidence that intramembrane accumulation of methionine exhibits antioxidant and cytoprotective properties in living cells. Our results unveil that methionine is an evolutionarily selected antioxidant building block of respiratory chain complexes. Collective protein alterations can thus constitute the selective advantage behind codon reassignments, which authenticates the "ambiguous decoding" hypothesis of genetic code evolution. Oxidative stress has shaped the mitochondrial genetic code.

Molecular characterization of the major membrane skeletal protein in the ciliate Tetrahymena pyriformis suggests n-plication of an early evolutionary intermediate filament protein subdomain.

PubMed

Bouchard, P; Chomilier, J; Ravet, V; Mornon, J P; Viguès, B

2001-01-01

Epiplasmin C is the major protein component of the membrane skeleton in the ciliate Tetrahymena pyriformis. Cloning and analysis of the gene encoding epiplasmin C showed this protein to be a previously unrecognized protein. In particular, epiplasmin C was shown to lack the canonical features of already known epiplasmic proteins in ciliates and flagellates. By means of hydrophobic cluster analysis (HCA), it has been shown that epiplasmin C is constituted of a repeat of 25 domains of 40 residues each. These domains are related and can be grouped in two families called types I and types II. Connections between types I and types II present rules that can be evidenced in the sequence itself, thus enforcing the validity of the splitting of the domains. Using these repeated domains as queries, significant structural similarities were demonstrated with an extra six heptads shared by nuclear lamins and invertebrate cytoplasmic intermediate filament proteins and deleted in the cytoplasmic intermediate filament protein lineage at the protostome-deuterostome branching in the eukaryotic phylogenetic tree.

QconCATs: design and expression of concatenated protein standards for multiplexed protein quantification.

PubMed

Simpson, Deborah M; Beynon, Robert J

2012-09-01

Systems biology requires knowledge of the absolute amounts of proteins in order to model biological processes and simulate the effects of changes in specific model parameters. Quantification concatamers (QconCATs) are established as a method to provide multiplexed absolute peptide standards for a set of target proteins in isotope dilution standard experiments. Two or more quantotypic peptides representing each of the target proteins are concatenated into a designer gene that is metabolically labelled with stable isotopes in Escherichia coli or other cellular or cell-free systems. Co-digestion of a known amount of QconCAT with the target proteins generates a set of labelled reference peptide standards for the unlabelled analyte counterparts, and by using an appropriate mass spectrometry platform, comparison of the intensities of the peptide ratios delivers absolute quantification of the encoded peptides and in turn the target proteins for which they are surrogates. In this review, we discuss the criteria and difficulties associated with surrogate peptide selection and provide examples in the design of QconCATs for quantification of the proteins of the nuclear factor κB pathway.

Peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP) but not PPAR-interacting protein (PRIP) is required for nuclear translocation of constitutive androstane receptor in mouse liver.

PubMed

Guo, Dongsheng; Sarkar, Joy; Ahmed, Mohamed R; Viswakarma, Navin; Jia, Yuzhi; Yu, Songtao; Sambasiva Rao, M; Reddy, Janardan K

2006-08-25

The constitutive androstane receptor (CAR) regulates transcription of phenobarbital-inducible genes that encode xenobiotic-metabolizing enzymes in liver. CAR is localized to the hepatocyte cytoplasm but to be functional, it translocates into the nucleus in the presence of phenobarbital-like CAR ligands. We now demonstrate that adenovirally driven EGFP-CAR, as expected, translocates into the nucleus of normal wild-type hepatocytes following phenobarbital treatment under both in vivo and in vitro conditions. Using this approach we investigated the role of transcription coactivators PBP and PRIP in the translocation of EGFP-CAR into the nucleus of PBP and PRIP liver conditional null mouse hepatocytes. We show that coactivator PBP is essential for nuclear translocation of CAR but not PRIP. Adenoviral expression of both PBP and EGFP-CAR restored phenobarbital-mediated nuclear translocation of exogenously expressed CAR in PBP null livers in vivo and in PBP null primary hepatocytes in vitro. CAR translocation into the nucleus of PRIP null livers resulted in the induction of CAR target genes such as CYP2B10, necessary for the conversion of acetaminophen to its hepatotoxic intermediate metabolite, N-acetyl-p-benzoquinone imine. As a consequence, PRIP-deficiency in liver did not protect from acetaminophen-induced hepatic necrosis, unlike that exerted by PBP deficiency. These results establish that transcription coactivator PBP plays a pivotal role in nuclear localization of CAR, that it is likely that PBP either enhances nuclear import or nuclear retention of CAR in hepatocytes, and that PRIP is redundant for CAR function.

Comparative analysis and molecular characterization of a gene BANF1 encoded a DNA-binding protein during mitosis from the Giant Panda and Black Bear.

PubMed

Zeng, Yichun; Hou, Yi-Ling; Ding, Xiang; Hou, Wan-Ru; Li, Jian

2014-01-01

Barrier to autointegration factor 1 (BANF1) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mitosis. The cDNA and the genomic sequence of BANF1 were cloned from the Giant Panda (Ailuropoda melanoleuca) and Black Bear (Ursus thibetanus mupinensis) using RT-PCR technology and Touchdown-PCR, respectively. The cDNA of the BANF1 cloned from Giant Panda and Black Bear is 297 bp in size, containing an open reading frame of 270 bp encoding 89 amino acids. The length of the genomic sequence from Giant Panda is 521 bp, from Black Bear is 536 bp, which were found both to possess 2 exons. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to some mammalian species studied. Topology prediction showed there is one Protein kinase C phosphorylation site, one Casein kinase II phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Giant Panda, and there is one Protein kinase C phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Black Bear. The BANF1 gene can be readily expressed in E. coli. Results showed that the protein BANF1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 14 kD polypeptide that formed inclusion bodies. The expression products obtained could be used to purify the proteins and study their function further.

Autophagy is induced through the ROS-TP53-DRAM1 pathway in response to mitochondrial protein synthesis inhibition.

PubMed

Xie, Xiaolei; Le, Li; Fan, Yanxin; Lv, Lin; Zhang, Junjie

2012-07-01

Mitoribosome in mammalian cells is responsible for synthesis of 13 mtDNA-encoded proteins, which are integral parts of four mitochondrial respiratory chain complexes (I, III, IV and V). ERAL1 is a nuclear-encoded GTPase important for the formation of the 28S small mitoribosomal subunit. Here, we demonstrate that knockdown of ERAL1 by RNA interference inhibits mitochondrial protein synthesis and promotes reactive oxygen species (ROS) generation, leading to autophagic vacuolization in HeLa cells. Cells that lack ERAL1 expression showed a significant conversion of LC3-I to LC3-II and an enhanced accumulation of autophagic vacuoles carrying the LC3 marker, all of which were blocked by the autophagy inhibitor 3-MA as well as by the ROS scavenger NAC. Inhibition of mitochondrial protein synthesis either by ERAL1 siRNA or chloramphenicol (CAP), a specific inhibitor of mitoribosomes, induced autophagy in HTC-116 TP53 (+/+) cells, but not in HTC-116 TP53 (-/-) cells, indicating that tumor protein 53 (TP53) is essential for the autophagy induction. The ROS elevation resulting from mitochondrial protein synthesis inhibition induced TP53 expression at transcriptional levels by enhancing TP53 promoter activity, and increased TP53 protein stability by suppressing TP53 ubiquitination through MAPK14/p38 MAPK-mediated TP53 phosphorylation. Upregulation of TP53 and its downstream target gene DRAM1, but not CDKN1A/p21, was required for the autophagy induction in ERAL1 siRNA or CAP-treated cells. Altogether, these data indicate that autophagy is induced through the ROS-TP53-DRAM1 pathway in response to mitochondrial protein synthesis inhibition.

Distribution and Evolution of Yersinia Leucine-Rich Repeat Proteins

PubMed Central

Hu, Yueming; Huang, He; Hui, Xinjie; Cheng, Xi; White, Aaron P.

2016-01-01

Leucine-rich repeat (LRR) proteins are widely distributed in bacteria, playing important roles in various protein-protein interaction processes. In Yersinia, the well-characterized type III secreted effector YopM also belongs to the LRR protein family and is encoded by virulence plasmids. However, little has been known about other LRR members encoded by Yersinia genomes or their evolution. In this study, the Yersinia LRR proteins were comprehensively screened, categorized, and compared. The LRR proteins encoded by chromosomes (LRR1 proteins) appeared to be more similar to each other and different from those encoded by plasmids (LRR2 proteins) with regard to repeat-unit length, amino acid composition profile, and gene expression regulation circuits. LRR1 proteins were also different from LRR2 proteins in that the LRR1 proteins contained an E3 ligase domain (NEL domain) in the C-terminal region or an NEL domain-encoding nucleotide relic in flanking genomic sequences. The LRR1 protein-encoding genes (LRR1 genes) varied dramatically and were categorized into 4 subgroups (a to d), with the LRR1a to -c genes evolving from the same ancestor and LRR1d genes evolving from another ancestor. The consensus and ancestor repeat-unit sequences were inferred for different LRR1 protein subgroups by use of a maximum parsimony modeling strategy. Structural modeling disclosed very similar repeat-unit structures between LRR1 and LRR2 proteins despite the different unit lengths and amino acid compositions. Structural constraints may serve as the driving force to explain the observed mutations in the LRR regions. This study suggests that there may be functional variation and lays the foundation for future experiments investigating the functions of the chromosomally encoded LRR proteins of Yersinia. PMID:27217422

Complete Mitochondrial Genome of the Medicinal Mushroom Ganoderma lucidum

PubMed Central

Chen, Haimei; Chen, Xiangdong; Lan, Jin; Liu, Chang

2013-01-01

Ganoderma lucidum is one of the well-known medicinal basidiomycetes worldwide. The mitochondrion, referred to as the second genome, is an organelle found in most eukaryotic cells and participates in critical cellular functions. Elucidating the structure and function of this genome is important to understand completely the genetic contents of G. lucidum. In this study, we assembled the mitochondrial genome of G. lucidum and analyzed the differential expressions of its encoded genes across three developmental stages. The mitochondrial genome is a typical circular DNA molecule of 60,630 bp with a GC content of 26.67%. Genome annotation identified genes that encode 15 conserved proteins, 27 tRNAs, small and large rRNAs, four homing endonucleases, and two hypothetical proteins. Except for genes encoding trnW and two hypothetical proteins, all genes were located on the positive strand. For the repeat structure analysis, eight forward, two inverted, and three tandem repeats were detected. A pair of fragments with a total length around 5.5 kb was found in both the nuclear and mitochondrial genomes, which suggests the possible transfer of DNA sequences between two genomes. RNA-Seq data for samples derived from three stages, namely, mycelia, primordia, and fruiting bodies, were mapped to the mitochondrial genome and qualified. The protein-coding genes were expressed higher in mycelia or primordial stages compared with those in the fruiting bodies. The rRNA abundances were significantly higher in all three stages. Two regions were transcribed but did not contain any identified protein or tRNA genes. Furthermore, three RNA-editing sites were detected. Genome synteny analysis showed that significant genome rearrangements occurred in the mitochondrial genomes. This study provides valuable information on the gene contents of the mitochondrial genome and their differential expressions at various developmental stages of G. lucidum. The results contribute to the understanding of the functions and evolution of fungal mitochondrial DNA. PMID:23991034

A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants

NASA Technical Reports Server (NTRS)

Yang, Tianbao; Poovaiah, B. W.

2002-01-01

We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein (Yang, T., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 38467-38473). Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H(2)O(2), and salicylic acid. Hence, they were designated as AtSR1-6 (Arabidopsis thaliana signal-responsive genes). Ca(2+)/calmodulin binds to all AtSRs, and their calmodulin-binding regions are located on a conserved basic amphiphilic alpha-helical motif in the C terminus. AtSR1 targets the nucleus and specifically recognizes a novel 6-bp CGCG box (A/C/G)CGCG(G/T/C). The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. The DNA-binding domain in AtSR1 is located on the N-terminal 146 bp where all AtSR1-related proteins share high similarity but have no similarity to other known DNA-binding proteins. The calmodulin-binding nuclear proteins isolated from wounded leaves exhibit specific CGCG box DNA binding activities. These results suggest that the AtSR gene family encodes a family of calmodulin-binding/DNA-binding proteins involved in multiple signal transduction pathways in plants.

Specific targeting of proteins to outer envelope membranes of endosymbiotic organelles, chloroplasts, and mitochondria

PubMed Central

Lee, Junho; Kim, Dae Heon; Hwang, Inhwan

2014-01-01

Chloroplasts and mitochondria are endosymbiotic organelles thought to be derived from endosymbiotic bacteria. In present-day eukaryotic cells, these two organelles play pivotal roles in photosynthesis and ATP production. In addition to these major activities, numerous reactions, and cellular processes that are crucial for normal cellular functions occur in chloroplasts and mitochondria. To function properly, these organelles constantly communicate with the surrounding cellular compartments. This communication includes the import of proteins, the exchange of metabolites and ions, and interactions with other organelles, all of which heavily depend on membrane proteins localized to the outer envelope membranes. Therefore, correct and efficient targeting of these membrane proteins, which are encoded by the nuclear genome and translated in the cytosol, is critically important for organellar function. In this review, we summarize the current knowledge of the mechanisms of protein targeting to the outer membranes of mitochondria and chloroplasts in two different directions, as well as targeting signals and cytosolic factors. PMID:24808904

The role of porcine reproductive and respiratory syndrome (PRRS) virus structural and non-structural proteins in virus pathogenesis.

PubMed

Music, Nedzad; Gagnon, Carl A

2010-12-01

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating viral disease affecting the swine industry worldwide. The etiological agent, PRRS virus (PRRSV), possesses a RNA viral genome with nine open reading frames (ORFs). The ORF1a and ORF1b replicase-associated genes encode the polyproteins pp1a and pp1ab, respectively. The pp1a is processed in nine non-structural proteins (nsps): nsp1α, nsp1β, and nsp2 to nsp8. Proteolytic cleavage of pp1ab generates products nsp9 to nsp12. The proteolytic pp1a cleavage products process and cleave pp1a and pp1ab into nsp products. The nsp9 to nsp12 are involved in virus genome transcription and replication. The 3' end of the viral genome encodes four minor and three major structural proteins. The GP(2a), GP₃ and GP₄ (encoded by ORF2a, 3 and 4), are glycosylated membrane associated minor structural proteins. The fourth minor structural protein, the E protein (encoded by ORF2b), is an unglycosylated membrane associated protein. The viral envelope contains two major structural proteins: a glycosylated major envelope protein GP₅ (encoded by ORF5) and an unglycosylated membrane M protein (encoded by ORF6). The third major structural protein is the nucleocapsid N protein (encoded by ORF7). All PRRSV non-structural and structural proteins are essential for virus replication, and PRRSV infectivity is relatively intolerant to subtle changes within the structural proteins. PRRSV virulence is multigenic and resides in both the non-structural and structural viral proteins. This review discusses the molecular characteristics, biological and immunological functions of the PRRSV structural and nsps and their involvement in the virus pathogenesis.

Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers

PubMed Central

Quiroz, Felipe García; Chilkoti, Ashutosh

2015-01-01

Proteins and synthetic polymers that undergo aqueous phase transitions mediate self-assembly in nature and in man-made material systems. Yet little is known about how the phase behaviour of a protein is encoded in its amino acid sequence. Here, by synthesizing intrinsically disordered, repeat proteins to test motifs that we hypothesized would encode phase behaviour, we show that the proteins can be designed to exhibit tunable lower or upper critical solution temperature (LCST and UCST, respectively) transitions in physiological solutions. We also show that mutation of key residues at the repeat level abolishes phase behaviour or encodes an orthogonal transition. Furthermore, we provide heuristics to identify, at the proteome level, proteins that might exhibit phase behaviour and to design novel protein polymers consisting of biologically active peptide repeats that exhibit LCST or UCST transitions. These findings set the foundation for the prediction and encoding of phase behaviour at the sequence level. PMID:26390327

The ribosome as a missing link in prebiotic evolution II: Ribosomes encode ribosomal proteins that bind to common regions of their own mRNAs and rRNAs.

PubMed

Root-Bernstein, Robert; Root-Bernstein, Meredith

2016-05-21

We have proposed that the ribosome may represent a missing link between prebiotic chemistries and the first cells. One of the predictions that follows from this hypothesis, which we test here, is that ribosomal RNA (rRNA) must have encoded the proteins necessary for ribosomal function. In other words, the rRNA also functioned pre-biotically as mRNA. Since these ribosome-binding proteins (rb-proteins) must bind to the rRNA, but the rRNA also functioned as mRNA, it follows that rb-proteins should bind to their own mRNA as well. This hypothesis can be contrasted to a "null" hypothesis in which rb-proteins evolved independently of the rRNA sequences and therefore there should be no necessary similarity between the rRNA to which rb-proteins bind and the mRNA that encodes the rb-protein. Five types of evidence reported here support the plausibility of the hypothesis that the mRNA encoding rb-proteins evolved from rRNA: (1) the ubiquity of rb-protein binding to their own mRNAs and autogenous control of their own translation; (2) the higher-than-expected incidence of Arginine-rich modules associated with RNA binding that occurs in rRNA-encoded proteins; (3) the fact that rRNA-binding regions of rb-proteins are homologous to their mRNA binding regions; (4) the higher than expected incidence of rb-protein sequences encoded in rRNA that are of a high degree of homology to their mRNA as compared with a random selection of other proteins; and (5) rRNA in modern prokaryotes and eukaryotes encodes functional proteins. None of these results can be explained by the null hypothesis that assumes independent evolution of rRNA and the mRNAs encoding ribosomal proteins. Also noteworthy is that very few proteins bind their own mRNAs that are not associated with ribosome function. Further tests of the hypothesis are suggested: (1) experimental testing of whether rRNA-encoded proteins bind to rRNA at their coding sites; (2) whether tRNA synthetases, which are also known to bind to their own mRNAs, are encoded by the tRNA sequences themselves; (3) and the prediction that archaeal and prokaryotic (DNA-based) genomes were built around rRNA "genes" so that rRNA-related sequences will be found to make up an unexpectedly high proportion of these genomes. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Evolutionary Characteristics of Missing Proteins: Insights into the Evolution of Human Chromosomes Related to Missing-Protein-Encoding Genes.

PubMed

Xu, Aishi; Li, Guang; Yang, Dong; Wu, Songfeng; Ouyang, Hongsheng; Xu, Ping; He, Fuchu

2015-12-04

Although the "missing protein" is a temporary concept in C-HPP, the biological information for their "missing" could be an important clue in evolutionary studies. Here we classified missing-protein-encoding genes into two groups, the genes encoding PE2 proteins (with transcript evidence) and the genes encoding PE3/4 proteins (with no transcript evidence). These missing-protein-encoding genes distribute unevenly among different chromosomes, chromosomal regions, or gene clusters. In the view of evolutionary features, PE3/4 genes tend to be young, spreading at the nonhomology chromosomal regions and evolving at higher rates. Interestingly, there is a higher proportion of singletons in PE3/4 genes than the proportion of singletons in all genes (background) and OTCSGs (organ, tissue, cell type-specific genes). More importantly, most of the paralogous PE3/4 genes belong to the newly duplicated members of the paralogous gene groups, which mainly contribute to special biological functions, such as "smell perception". These functions are heavily restricted into specific type of cells, tissues, or specific developmental stages, acting as the new functional requirements that facilitated the emergence of the missing-protein-encoding genes during evolution. In addition, the criteria for the extremely special physical-chemical proteins were first set up based on the properties of PE2 proteins, and the evolutionary characteristics of those proteins were explored. Overall, the evolutionary analyses of missing-protein-encoding genes are expected to be highly instructive for proteomics and functional studies in the future.

Transcriptomic analysis of Arabidopsis developing stems: a close-up on cell wall genes

PubMed Central

Minic, Zoran; Jamet, Elisabeth; San-Clemente, Hélène; Pelletier, Sandra; Renou, Jean-Pierre; Rihouey, Christophe; Okinyo, Denis PO; Proux, Caroline; Lerouge, Patrice; Jouanin, Lise

2009-01-01

Background Different strategies (genetics, biochemistry, and proteomics) can be used to study proteins involved in cell biogenesis. The availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. Although the expression patterns of some Arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. Using transcriptomic and bioinformatic tools, we studied the regulation of cell wall genes in Arabidopsis stems, i.e. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins. Results Transcriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. Many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. A total of 345 genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. The encoded proteins were distributed into 8 classes, based on the presence of predicted functional domains. Proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes. Other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. Particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. Finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. Only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. Conversely, only a few proteins encoded by genes having moderate or high level of transcripts were identified by proteomics. Conclusion Analysis of the genes predicted to encode cell wall proteins revealed that about 345 genes had moderate or high levels of transcripts. Among them, we identified many new genes possibly involved in cell wall biogenesis. The discrepancies observed between results of this transcriptomic study and a previous proteomic study on the same material revealed post-transcriptional mechanisms of regulation of expression of genes encoding cell wall proteins. PMID:19149885

Specific nuclear localizing sequence directs two myosin isoforms to the cell nucleus in calmodulin-sensitive manner.

PubMed

Dzijak, Rastislav; Yildirim, Sukriye; Kahle, Michal; Novák, Petr; Hnilicová, Jarmila; Venit, Tomáš; Hozák, Pavel

2012-01-01

Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the "cytoplasmic" myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. We have shown that the novel specific NLS brings to the cell nucleus not only the "nuclear" isoform of myosin I (NM1 protein) but also its "cytoplasmic" isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus.

Targeted Quantitative Screening of Chromosome 18 Encoded Proteome in Plasma Samples of Astronaut Candidates.

PubMed

Kopylov, Artur T; Ilgisonis, Ekaterina V; Moysa, Alexander A; Tikhonova, Olga V; Zavialova, Maria G; Novikova, Svetlana E; Lisitsa, Andrey V; Ponomarenko, Elena A; Moshkovskii, Sergei A; Markin, Andrey A; Grigoriev, Anatoly I; Zgoda, Victor G; Archakov, Alexander I

2016-11-04

This work was aimed at estimating the concentrations of proteins encoded by human chromosome 18 (Chr 18) in plasma samples of 54 healthy male volunteers (aged 20-47). These young persons have been certified by the medical evaluation board as healthy subjects ready for space flight training. Over 260 stable isotope-labeled peptide standards (SIS) were synthesized to perform the measurements of proteins encoded by Chr 18. Selected reaction monitoring (SRM) with SIS allowed an estimate of the levels of 84 of 276 proteins encoded by Chr 18. These proteins were quantified in whole and depleted plasma samples. Concentration of the proteins detected varied from 10 -6 M (transthyretin, P02766) to 10 -11 M (P4-ATPase, O43861). A minor part of the proteins (mostly representing intracellular proteins) was characterized by extremely high inter individual variations. The results provide a background for studies of a potential biomarker in plasma among proteins encoded by Chr 18. The SRM raw data are available in ProteomeXchange repository (PXD004374).

Phaseolin expression in tobacco chloroplast reveals an autoregulatory mechanism in heterologous protein translation.

PubMed

De Marchis, Francesca; Bellucci, Michele; Pompa, Andrea

2016-02-01

Plastid DNA engineering is a well-established research area of plant biotechnology, and plastid transgenes often give high expression levels. However, it is still almost impossible to predict the accumulation rate of heterologous protein in transplastomic plants, and there are many cases of unsuccessful transgene expression. Chloroplasts regulate their proteome at the post-transcriptional level, mainly through translation control. One of the mechanisms to modulate the translation has been described in plant chloroplasts for the chloroplast-encoded subunits of multiprotein complexes, and the autoregulation of the translation initiation of these subunits depends on the availability of their assembly partners [control by epistasy of synthesis (CES)]. In Chlamydomonas reinhardtii, autoregulation of endogenous proteins recruited in the assembly of functional complexes has also been reported. In this study, we revealed a self-regulation mechanism triggered by the accumulation of a soluble recombinant protein, phaseolin, in the stroma of chloroplast-transformed tobacco plants. Immunoblotting experiments showed that phaseolin could avoid this self-regulation mechanism when targeted to the thylakoids in transplastomic plants. To inhibit the thylakoid-targeted phaseolin translation as well, this protein was expressed in the presence of a nuclear version of the phaseolin gene with a transit peptide. Pulse-chase and polysome analysis revealed that phaseolin mRNA translation on plastid ribosomes was repressed due to the accumulation in the stroma of the same soluble polypeptide imported from the cytosol. We suggest that translation autoregulation in chloroplast is not limited to heteromeric protein subunits but also involves at least some of the foreign soluble recombinant proteins, leading to the inhibition of plastome-encoded transgene expression in chloroplast. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Viral mimicry of Cdc2/cyclin-dependent kinase 1 mediates disruption of nuclear lamina during human cytomegalovirus nuclear egress.

PubMed

Hamirally, Sofia; Kamil, Jeremy P; Ndassa-Colday, Yasmine M; Lin, Alison J; Jahng, Wan Jin; Baek, Moon-Chang; Noton, Sarah; Silva, Laurie A; Simpson-Holley, Martha; Knipe, David M; Golan, David E; Marto, Jarrod A; Coen, Donald M

2009-01-01

The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser(22)). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser(22) is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle.

Coevolution study of mitochondria respiratory chain proteins: toward the understanding of protein--protein interaction.

PubMed

Yang, Ming; Ge, Yan; Wu, Jiayan; Xiao, Jingfa; Yu, Jun

2011-05-20

Coevolution can be seen as the interdependency between evolutionary histories. In the context of protein evolution, functional correlation proteins are ever-present coordinated evolutionary characters without disruption of organismal integrity. As to complex system, there are two forms of protein--protein interactions in vivo, which refer to inter-complex interaction and intra-complex interaction. In this paper, we studied the difference of coevolution characters between inter-complex interaction and intra-complex interaction using "Mirror tree" method on the respiratory chain (RC) proteins. We divided the correlation coefficients of every pairwise RC proteins into two groups corresponding to the binary protein--protein interaction in intra-complex and the binary protein--protein interaction in inter-complex, respectively. A dramatical discrepancy is detected between the coevolution characters of the two sets of protein interactions (Wilcoxon test, p-value = 4.4 × 10(-6)). Our finding reveals some critical information on coevolutionary study and assists the mechanical investigation of protein--protein interaction. Furthermore, the results also provide some unique clue for supramolecular organization of protein complexes in the mitochondrial inner membrane. More detailed binding sites map and genome information of nuclear encoded RC proteins will be extraordinary valuable for the further mitochondria dynamics study. Copyright © 2011. Published by Elsevier Ltd.

Molecular mechanisms for protein-encoded inheritance

PubMed Central

Wiltzius, Jed J. W.; Landau, Meytal; Nelson, Rebecca; Sawaya, Michael R.; Apostol, Marcin I.; Goldschmidt, Lukasz; Soriaga, Angela B.; Cascio, Duilio; Rajashankar, Kanagalaghatta; Eisenberg, David

2013-01-01

Strains are phenotypic variants, encoded by nucleic acid sequences in chromosomal inheritance and by protein “conformations” in prion inheritance and transmission. But how is a protein “conformation” stable enough to endure transmission between cells or organisms? Here new polymorphic crystal structures of segments of prion and other amyloid proteins offer structural mechanisms for prion strains. In packing polymorphism, prion strains are encoded by alternative packings (polymorphs) of β-sheets formed by the same segment of a protein; in a second mechanism, segmental polymorphism, prion strains are encoded by distinct β-sheets built from different segments of a protein. Both forms of polymorphism can produce enduring “conformations,” capable of encoding strains. These molecular mechanisms for transfer of information into prion strains share features with the familiar mechanism for transfer of information by nucleic acid inheritance, including sequence specificity and recognition by non-covalent bonds. PMID:19684598

Thionin-D4E1 chimeric protein protects plants against bacterial infections

DOEpatents

Stover, Eddie W; Gupta, Goutam; Hao, Guixia

2017-08-08

The generation of a chimeric protein containing a first domain encoding either a pro-thionon or thionin, a second domain encoding D4E1 or pro-D4E1, and a third domain encoding a peptide linker located between the first domain and second domain is described. Either the first domain or the second domain is located at the amino terminal of the chimeric protein and the other domain (second domain or first domain, respectively) is located at the carboxyl terminal. The chimeric protein has antibacterial activity. Genetically altered plants and their progeny expressing a polynucleotide encoding the chimeric protein resist diseases caused by bacteria.

Nuclear pore complex integrity requires Lnp1, a regulator of cortical endoplasmic reticulum

PubMed Central

Casey, Amanda K.; Chen, Shuliang; Novick, Peter; Ferro-Novick, Susan; Wente, Susan R.

2015-01-01

The nuclear envelope (NE) and endoplasmic reticulum (ER) are components of the same contiguous membrane system and yet have distinct cellular functions. Mounting evidence suggests roles for some ER proteins in the NE for proper nuclear pore complex (NPC) structure and function. In this study, we identify a NE role in Saccharomyces cerevisiae for Lnp1 and Sey1, proteins required for proper cortical ER formation. Both lnp1Δ and sey1Δ mutants exhibit synthetic genetic interactions with mutants in genes encoding key NPC structural components. Both Lnp1 and Sey1 physically associate with other ER components that have established NPC roles, including Rtn1, Yop1, Pom33, and Per33. Of interest, lnp1Δ rtn1Δ mutants but not rtn1Δ sey1Δ mutants exhibit defects in NPC distribution. Furthermore, the essential NPC assembly factor Ndc1 has altered interactions in the absence of Sey1. Lnp1 dimerizes in vitro via its C-terminal zinc finger motif, a property that is required for proper ER structure but not NPC integrity. These findings suggest that Lnp1's role in NPC integrity is separable from functions in the ER and is linked to Ndc1 and Rtn1 interactions. PMID:26041935

The identification of FANCD2 DNA binding domains reveals nuclear localization sequences.

PubMed

Niraj, Joshi; Caron, Marie-Christine; Drapeau, Karine; Bérubé, Stéphanie; Guitton-Sert, Laure; Coulombe, Yan; Couturier, Anthony M; Masson, Jean-Yves

2017-08-21

Fanconi anemia (FA) is a recessive genetic disorder characterized by congenital abnormalities, progressive bone-marrow failure, and cancer susceptibility. The FA pathway consists of at least 21 FANC genes (FANCA-FANCV), and the encoded protein products interact in a common cellular pathway to gain resistance against DNA interstrand crosslinks. After DNA damage, FANCD2 is monoubiquitinated and accumulates on chromatin. FANCD2 plays a central role in the FA pathway, using yet unidentified DNA binding regions. By using synthetic peptide mapping and DNA binding screen by electromobility shift assays, we found that FANCD2 bears two major DNA binding domains predominantly consisting of evolutionary conserved lysine residues. Furthermore, one domain at the N-terminus of FANCD2 bears also nuclear localization sequences for the protein. Mutations in the bifunctional DNA binding/NLS domain lead to a reduction in FANCD2 monoubiquitination and increase in mitomycin C sensitivity. Such phenotypes are not fully rescued by fusion with an heterologous NLS, which enable separation of DNA binding and nuclear import functions within this domain that are necessary for FANCD2 functions. Collectively, our results enlighten the importance of DNA binding and NLS residues in FANCD2 to activate an efficient FA pathway. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

HTLV-1 Tax upregulates early growth response protein 1 through nuclear factor-κB signaling

PubMed Central

Han, Jingxian; Liu, Xihong; Lv, Zhuangwei; Li, Huanhuan; Yuan, Lixiang; Li, Xiangping; Sun, Shuming; Wang, Hui; Huang, Xinxiang

2017-01-01

Human T cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that causes adult T cell leukemia (ATL) in susceptible individuals. The HTLV-1-encoded oncoprotein Tax induces persistent activation of the nuclear factor-κB (NF-κB) pathway. Early growth response protein 1 (EGR1) is overexpressed in HTLV-1-infected T cell lines and ATL cells. Here, we showed that both Tax expression and HTLV-1 infection promoted EGR1 overexpression. Loss of the NF-κB binding site in the EGR1 promotor or inhibition of NF-κB activation reduced Tax-induced EGR1 upregulation. Tax mutants unable to activate NF-κB induced only slight EGR1 upregulation as compared with wild-type Tax, confirming NF-κB pathway involvement in EGR1 regulation. Tax also directly interacted with the EGR1 protein and increased endogenous EGR1 stability. Elevated EGR1 in turn promoted p65 nuclear translocation and increased NF-κB activation. These results demonstrate a positive feedback loop between EGR1 expression and NF-κB activation in HTLV-1-infected and Tax-expressing cells. Both NF-κB activation and Tax-induced EGR1 stability upregulated EGR1, which in turn enhanced constitutive NF-κB activation and facilitated ATL progression in HTLV-1-infected cells. These findings suggest EGR1 may be an effective anti-ATL therapeutic target. PMID:28881635

The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic Ribosomal Proteins and the Phosphorylation of the Cytosolic Ribosomal Protein S6

PubMed Central

Dobrenel, Thomas; Mancera-Martínez, Eder; Forzani, Céline; Azzopardi, Marianne; Davanture, Marlène; Moreau, Manon; Schepetilnikov, Mikhail; Chicher, Johana; Langella, Olivier; Zivy, Michel; Robaglia, Christophe; Ryabova, Lyubov A.; Hanson, Johannes; Meyer, Christian

2016-01-01

Protein translation is an energy consuming process that has to be fine-tuned at both the cell and organism levels to match the availability of resources. The target of rapamycin kinase (TOR) is a key regulator of a large range of biological processes in response to environmental cues. In this study, we have investigated the effects of TOR inactivation on the expression and regulation of Arabidopsis ribosomal proteins at different levels of analysis, namely from transcriptomic to phosphoproteomic. TOR inactivation resulted in a coordinated down-regulation of the transcription and translation of nuclear-encoded mRNAs coding for plastidic ribosomal proteins, which could explain the chlorotic phenotype of the TOR silenced plants. We have identified in the 5′ untranslated regions (UTRs) of this set of genes a conserved sequence related to the 5′ terminal oligopyrimidine motif, which is known to confer translational regulation by the TOR kinase in other eukaryotes. Furthermore, the phosphoproteomic analysis of the ribosomal fraction following TOR inactivation revealed a lower phosphorylation of the conserved Ser240 residue in the C-terminal region of the 40S ribosomal protein S6 (RPS6). These results were confirmed by Western blot analysis using an antibody that specifically recognizes phosphorylated Ser240 in RPS6. Finally, this antibody was used to follow TOR activity in plants. Our results thus uncover a multi-level regulation of plant ribosomal genes and proteins by the TOR kinase. PMID:27877176

Genomics and functional genomics in Chlamydomonas reinhardtii

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blaby, Ian K.; Blaby-Haas, Crysten E.

The availability of the Chlamydomonas reinhardtii nuclear genome sequence continues to enable researchers to address biological questions relevant to algae, land plants and animals in unprecedented ways. As we continue to characterize and understand biological processes in C. reinhardtii and translate that knowledge to other systems, we are faced with the realization that many genes encode proteins without a defined function. The field of functional genomics aims to close this gap between genome sequence and protein function. Transcriptomes, proteomes and phenomes can each provide layers of gene-specific functional data while supplying a global snapshot of cellular behavior under different conditions.more » Herein we present a brief history of functional genomics, the present status of the C. reinhardtii genome, how genome-wide experiments can aid in supplying protein function inferences, and provide an outlook for functional genomics in C. reinhardtii.« less

Genomics and functional genomics in Chlamydomonas reinhardtii

DOE PAGES

Blaby, Ian K.; Blaby-Haas, Crysten E.

2017-03-21

The availability of the Chlamydomonas reinhardtii nuclear genome sequence continues to enable researchers to address biological questions relevant to algae, land plants and animals in unprecedented ways. As we continue to characterize and understand biological processes in C. reinhardtii and translate that knowledge to other systems, we are faced with the realization that many genes encode proteins without a defined function. The field of functional genomics aims to close this gap between genome sequence and protein function. Transcriptomes, proteomes and phenomes can each provide layers of gene-specific functional data while supplying a global snapshot of cellular behavior under different conditions.more » Herein we present a brief history of functional genomics, the present status of the C. reinhardtii genome, how genome-wide experiments can aid in supplying protein function inferences, and provide an outlook for functional genomics in C. reinhardtii.« less

Herpes simplex virus-1 infection causes the secretion of a type I interferon-antagonizing protein and inhibits signaling at or before Jak-1 activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Karen E.; Knipe, David M., E-mail: david_knipe@hms.harvard.ed

2010-01-05

Host cells respond to viral infection by the production of type I interferons (IFNs), which induce the expression of antiviral genes. Herpes simplex virus I (HSV-1) encodes many mechanisms that inhibit the type I IFN response, including the ICP27-dependent inhibition of type I IFN signaling. Here we show inhibition of Stat-1 nuclear accumulation in cells that express ICP27. ICP27 expression also induces the secretion of a small, heat-stable type I IFN antagonizing protein that inhibits Stat-1 nuclear accumulation. We show that the inhibition of IFN-induced Stat-1 phosphorylation occurs at or upstream of Jak-1 phosphorylation. Finally, we show that ISG15 expressionmore » is induced after IFNalpha treatment in mock-infected cells, but not cells infected with WT HSV-1 or ICP27{sup -} HSV-1. These data suggest that HSV-1 has evolved multiple mechanisms to inhibit IFN signaling not only in infected cells, but also in neighboring cells, thereby allowing for increased viral replication and spread.« less

Evolutionary changes in lamin expression in the vertebrate lineage

PubMed Central

Stick, Reimer; Peter, Annette

2017-01-01

ABSTRACT The nuclear lamina is involved in fundamental nuclear functions and provides mechanical stability to the nucleus. Lamin filaments form a meshwork closely apposed to the inner nuclear membrane and a small fraction of lamins exist in the nuclear interior. Mutations in lamin genes cause severe hereditary diseases, the laminopathies. During vertebrate evolution the lamin protein family has expanded. While most vertebrate genomes contain 4 lamin genes, encoding the lamins A, B1, B2, and LIII, the majority of non-vertebrate genomes harbor only a single lamin gene. We have collected lamin gene and cDNA sequence information for representatives of the major vertebrate lineages. With the help of RNA-seq data we have determined relative lamin expression levels for representative tissues for species of 9 different gnathostome lineages. Here we report that the level of lamin A expression is low in cartilaginous fishes and ancient fishes and increases toward the mammals. Lamin B1 expression shows an inverse tendency to that of lamin A. Possible implications for the change in the lamin A to B ratio is discussed in the light of its role in nuclear mechanics. PMID:28430006

Novel RepA-MCM proteins encoded in plasmids pTAU4, pORA1 and pTIK4 from Sulfolobus neozealandicus

PubMed Central

Greve, Bo; Jensen, Susanne; Phan, Hoa; Brügger, Kim; Zillig, Wolfram; She, Qunxin; Garrett, Roger A.

2005-01-01

Three plasmids isolated from the crenarchaeal thermoacidophile Sulfolobus neozealandicus were characterized. Plasmids pTAU4 (7,192 bp), pORA1 (9,689 bp) and pTIK4 (13,638 bp) show unusual properties that distinguish them from previously characterized cryptic plasmids of the genus Sulfolobus. Plasmids pORA1 and pTIK4 encode RepA proteins, only the former of which carries the novel polymerase–primase domain of other known Sulfolobus plasmids. Plasmid pTAU4 encodes a mini-chromosome maintenance protein homolog and no RepA protein; the implications for DNA replication are considered. Plasmid pORA1 is the first Sulfolobus plasmid to be characterized that does not encode the otherwise highly conserved DNA-binding PlrA protein. Another encoded protein appears to be specific for the New Zealand plasmids. The three plasmids should provide useful model systems for functional studies of these important crenarchaeal proteins. PMID:15876565

Rotating-frame gradient fields for magnetic resonance imaging and nuclear magnetic resonance in low fields

DOEpatents

Bouchard, Louis-Serge; Pines, Alexander; Demas, Vasiliki

2014-01-21

A system and method for Fourier encoding a nuclear magnetic resonance (NMR) signal is disclosed. A static magnetic field B.sub.0 is provided along a first direction. An NMR signal from the sample is Fourier encoded by applying a rotating-frame gradient field B.sub.G superimposed on the B.sub.0, where the B.sub.G comprises a vector component rotating in a plane perpendicular to the first direction at an angular frequency .omega.in a laboratory frame. The Fourier-encoded NMR signal is detected.

Nuclear export of human hepatitis B virus core protein and pregenomic RNA depends on the cellular NXF1-p15 machinery.

PubMed

Yang, Ching-Chun; Huang, Er-Yi; Li, Hung-Cheng; Su, Pei-Yi; Shih, Chiaho

2014-01-01

Hepatitis B virus (HBV) core protein (HBc) can shuttle between nucleus and cytoplasm. Cytoplasm-predominant HBc is clinically associated with severe liver inflammation. Previously, we found that HBc arginine-rich domain (ARD) can associate with a host factor NXF1 (TAP) by coimmunoprecipitation. It is well known that NXF1-p15 heterodimer can serve as a major export receptor of nuclear mRNA as a ribonucleoprotein complex (RNP). In the NXF1-p15 pathway, TREX (transcription/export) complex plays an important role in coupling nuclear pre-mRNA processing with mRNA export in mammalian cells. Here, we tested the hypothesis whether HBc and HBV specific RNA can be exported via the TREX and NXF1-p15 mediated pathway. We demonstrated here that HBc can physically and specifically associate with TREX components, and the NXF1-p15 export receptor by coimmunoprecipitation. Accumulation of HBc protein in the nucleus can be induced by the interference with TREX and NXF1-p15 mediated RNA export machinery. HBV transcripts encodes a non-spliced 3.5 kb pregenomic RNA (pgRNA) which can serve as a template for reverse transcription. Cytoplasmic HBV pgRNA appeared to be reduced by siRNA treatment specific for the NXF1-p15 complex by quantitative RT-qPCR and Northern blot analyses. This result suggests that the pgRNA was also exported via the NXF1-p15 machinery. We entertain the hypothesis that HBc protein can be exported as an RNP cargo via the mRNA export pathway by hijacking the TREX and NXF1-p15 complex. In our current and previous studies, HBc is not required for pgRNA accumulation in the cytoplasm. Furthermore, HBc ARD can mediate nuclear export of a chimeric protein containing HBc ARD in a pgRNA-independent manner. Taken together, it suggests that while both pgRNA and HBc protein exports are dependent on NXF1-p15, they are using the same export machinery in a manner independent of each other.

A spontaneous tRNA suppressor of a mutation in the Chlamydomonas reinhardtii nuclear MCD1 gene required for stability of the chloroplast petD mRNA

PubMed Central

Murakami, Shinya; Kuehnle, Katrin; Stern, David B.

2005-01-01

Numerous nuclear gene products are required for the correct expression of organellar genes. One such gene in the green alga Chlamydomonas reinhardtii is MCD1, whose product is required for stability of the chloroplast-encoded petD mRNA. In mcd1 mutants, which are non-photosynthetic, petD mRNA is degraded by a 5′–3′ exonuclease activity, resulting in a failure to synthesize its product, subunit IV of the cytochrome b 6/f complex. Here, we report the sequence of the wild-type MCD1 gene, which encodes a large and novel putative protein. Analysis of three mutant alleles showed that two harbored large deletions, but that one allele, mcd1-2, had a single base change resulting in a nonsense codon near the N-terminus. This same mutant allele can be suppressed by a second-site mutation in the nuclear MCD2 gene, whereas mcd2-1 cannot suppress the deletion in mcd1-1 (Esposito,D. Higgs,D.C. Drager,R.G. Stern, D.B. and Girard-Bascou,J. (2001) Curr. Genet., 39, 40–48). We report the cloning of mcd2-1, and show that the mutation lies in a tRNASer(CGA), which has been modified to translate the nonsense codon in mcd1-2. We discuss how the existence of a large tRNASer gene family may permit this suppression without pleiotropic consequences. PMID:15947135

Retinoic Acid Inducible Gene 1 Protein (RIG1)-like Receptor Pathway is Required for Efficient Nuclear Reprogramming

PubMed Central

Sayed, Nazish; Ospino, Frank; Himmati, Farhan; Lee, Jieun; Chanda, Palas; Mocarski, Edward S.; Cooke, John P.

2017-01-01

We have revealed a critical role for innate immune signaling in nuclear reprogramming to pluripotency, and in the nuclear reprogramming required for somatic cell transdifferentiation. Activation of innate immune signaling causes global changes in the expression and activity of epigenetic modifiers to promote epigenetic plasticity. In our previous papers, we focused on the role of toll-like receptor 3 (TLR3) in this signaling pathway. Here we define the role of another innate immunity pathway known to participate in the response to viral RNA, the retinoic acid-inducible gene 1 receptor (RIG-1)-like receptor (RLR) pathway. This pathway is represented by the sensors of viral RNA, RIG-1, LGP2 and MDA5. We first found that TLR3 deficiency only causes a partial inhibition of nuclear reprogramming to pluripotency in mouse tail-tip fibroblasts, which motivated us to determine the contribution of RLR. We found that knockdown of iPS-1, the common adaptor protein for the RLR family, substantially reduced nuclear reprogramming induced by retroviral or by mmRNA expression of Oct 4, Sox2, KLF4 and cMYC (OSKM). Importantly a double knockdown of both RLR and TLR3 pathway led to a further decrease in iPSC colonies suggesting an additive effect of both these pathways on nuclear reprogramming. Furthermore, in murine embryonic fibroblasts expressing a dox-inducible cassette of the genes encoding OSKM, an RLR agonist increased the yield of iPSCs. Similarly, the RLR agonist enhanced nuclear reprogramming by cell permeant peptides of the Yamanaka factors. Finally, in the dox-inducible system, RLR activation promotes activating histone marks in the promoter region of pluripotency genes. To conclude, innate immune signaling mediated by RLR plays a critical role in nuclear reprogramming. Manipulation of innate immune signaling may facilitate nuclear reprogramming to achieve pluripotency. PMID:28276156

NADH:ubiquinone oxidoreductase from bovine heart mitochondria. cDNA sequences of the import precursors of the nuclear-encoded 39 kDa and 42 kDa subunits.

PubMed Central

Fearnley, I M; Finel, M; Skehel, J M; Walker, J E

1991-01-01

The 39 kDa and 42 kDa subunits of NADH:ubiquinone oxidoreductase from bovine heart mitochondria are nuclear-coded components of the hydrophobic protein fraction of the enzyme. Their amino acid sequences have been deduced from the sequences of overlapping cDNA clones. These clones were amplified from total bovine heart cDNA by means of the polymerase chain reaction, with the use of complex mixtures of oligonucleotide primers based upon fragments of protein sequence determined at the N-terminals of the proteins and at internal sites. The protein sequences of the 39 kDa and 42 kDa subunits are 345 and 320 amino acid residues long respectively, and their calculated molecular masses are 39,115 Da and 36,693 Da. Both proteins are predominantly hydrophilic, but each contains one or two hydrophobic segments that could possibly be folded into transmembrane alpha-helices. The bovine 39 kDa protein sequence is related to that of a 40 kDa subunit from complex I from Neurospora crassa mitochondria; otherwise, it is not related significantly to any known sequence, including redox proteins and two polypeptides involved in import of proteins into mitochondria, known as the mitochondrial processing peptidase and the processing-enhancing protein. Therefore the functions of the 39 kDa and 42 kDa subunits of complex I are unknown. The mitochondrial gene product, ND4, a hydrophobic component of complex I with an apparent molecular mass of about 39 kDa, has been identified in preparations of the enzyme. This subunit stains faintly with Coomassie Blue dye, and in many gel systems it is not resolved from the nuclearcoded 36 kDa subunit. Images Fig. 1. PMID:1832859

The rice blast resistance gene Ptr encodes an atypical protein required for broad spectrum disease resistance

USDA-ARS?s Scientific Manuscript database

Plant resistance (R) genes typically encode proteins with nucleotide binding site-leucine rich repeat (NLR) domains. We identified a novel, broad-spectrum rice blast R gene, Ptr, encoding a non-NLR protein with four Armadillo repeats. Ptr was originally identified by fast neutron mutagenesis as a ...

Alternative intronic promoters in development and disease.

PubMed

Vacik, Tomas; Raska, Ivan

2017-05-01

Approximately 20,000 mammalian genes are estimated to encode between 250 thousand and 1 million different proteins. This enormous diversity of the mammalian proteome is caused by the ability of a single-gene locus to encode multiple protein isoforms. Protein isoforms encoded by one gene locus can be functionally distinct, and they can even have antagonistic functions. One of the mechanisms involved in creating this proteome complexity is alternative promoter usage. Alternative intronic promoters are located downstream from their canonical counterparts and drive the expression of alternative RNA isoforms that lack upstream exons. These upstream exons can encode some important functional domains, and proteins encoded by alternative mRNA isoforms can be thus functionally distinct from the full-length protein encoded by canonical mRNA isoforms. Since any misbalance of functionally distinct protein isoforms is likely to have detrimental consequences for the cell and the whole organism, their expression must be precisely regulated. Misregulation of alternative intronic promoters is frequently associated with various developmental defects and diseases including cancer, and it is becoming increasingly clear that this phenomenon deserves more attention.

Fine mapping of Restorer-of-fertility in pepper (Capsicum annuum L.) identified a candidate gene encoding a pentatricopeptide repeat (PPR)-containing protein.

PubMed

Jo, Yeong Deuk; Ha, Yeaseong; Lee, Joung-Ho; Park, Minkyu; Bergsma, Alex C; Choi, Hong-Il; Goritschnig, Sandra; Kloosterman, Bjorn; van Dijk, Peter J; Choi, Doil; Kang, Byoung-Cheorl

2016-10-01

Using fine mapping techniques, the genomic region co-segregating with Restorer - of - fertility ( Rf ) in pepper was delimited to a region of 821 kb in length. A PPR gene in this region, CaPPR6 , was identified as a strong candidate for Rf based on expression pattern and characteristics of encoding sequence. Cytoplasmic-genic male sterility (CGMS) has been used for the efficient production of hybrid seeds in peppers (Capsicum annuum L.). Although the mitochondrial candidate genes that might be responsible for cytoplasmic male sterility (CMS) have been identified, the nuclear Restorer-of-fertility (Rf) gene has not been isolated. To identify the genomic region co-segregating with Rf in pepper, we performed fine mapping using an Rf-segregating population consisting of 1068 F2 individuals, based on BSA-AFLP and a comparative mapping approach. Through six cycles of chromosome walking, the co-segregating region harboring the Rf locus was delimited to be within 821 kb of sequence. Prediction of expressed genes in this region based on transcription analysis revealed four candidate genes. Among these, CaPPR6 encodes a pentatricopeptide repeat (PPR) protein with PPR motifs that are repeated 14 times. Characterization of the CaPPR6 protein sequence, based on alignment with other homologs, showed that CaPPR6 is a typical Rf-like (RFL) gene reported to have undergone diversifying selection during evolution. A marker developed from a sequence near CaPPR6 showed a higher prediction rate of the Rf phenotype than those of previously developed markers when applied to a panel of breeding lines of diverse origin. These results suggest that CaPPR6 is a strong candidate for the Rf gene in pepper.

Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2-8) in Normal and Cancerous Breast and Prostate Cells.

PubMed

Hu, Dong Gui; McKinnon, Ross A; Hulin, Julie-Ann; Mackenzie, Peter I; Meech, Robyn

2016-12-27

Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2-8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer ( PCGEM1 , PEG3 , EPHA3 , and EFNB2 ) or other types of human cancers ( TOX3 , ST8SIA4 , and SLITRK3 ), and genes that are diagnostic/prognostic biomarkers of prostate cancer ( GRINA3 , and BCHE ).

Molecular cloning and characterization of a membrane associated NAC family gene, SiNAC from foxtail millet [Setaria italica (L.) P. Beauv].

PubMed

Puranik, Swati; Bahadur, Ranjit Prasad; Srivastava, Prem S; Prasad, Manoj

2011-10-01

The plant-specific NAC (NAM, ATAF, and CUC) transcription factors have diverse role in development and stress regulation. A transcript encoding NAC protein, termed SiNAC was identified from a salt stress subtractive cDNA library of S. italica seedling (Puranik et al., J Plant Physiol 168:280-287, 2011). This single/low copy gene containing four exons and four introns within the genomic-sequence encoded a protein of 462 amino acids. Structural analysis revealed that highly divergent C terminus contains a transmembrane domain. The NAC domain consisted of a twisted antiparallel beta-sheet packing against N terminal alpha helix on one side and a shorter helix on the other side. The domain was predicted to homodimerize and control DNA-binding specificity. The physicochemical features of the SiNAC homodimer interface justified the dimeric form of the predicted model. A 1539 bp fragment upstream to the start codon of SiNAC gene was cloned and in silico analysis revealed several putative cis-acting regulatory elements within the promoter sequence. Transactivation analysis indicated that SiNAC activated expression of reporter gene and the activation domain lied at the C terminal. The SiNAC:GFP was detected in the nucleus and cytoplasm while SiNAC ΔC(1-158):GFP was nuclear localized in onion epidermal cells. SiNAC transcripts mostly accumulated in young spikes and were strongly induced by dehydration, salinity, ethephon, and methyl jasmonate. These results suggest that SiNAC encodes a membrane associated NAC-domain protein that may function as a transcriptional activator in response to stress and developmental regulation in plants.

Overexpression of OsRAN2 in rice and Arabidopsis renders transgenic plants hypersensitive to salinity and osmotic stress

PubMed Central

Zang, Aiping; Xu, Xiaojie; Neill, Steven; Cai, Weiming

2010-01-01

Nucleo-cytoplasmic partitioning of regulatory proteins is increasingly being recognized as a major control mechanism for the regulation of signalling in plants. Ras-related nuclear protein (Ran) GTPase is required for regulating transport of proteins and RNA across the nuclear envelope and also has roles in mitotic spindle assembly and nuclear envelope (NE) assembly. However, thus far little is known of any Ran functions in the signalling pathways in plants in response to changing environmental stimuli. The OsRAN2 gene, which has high homology (77% at the amino acid level) with its human counterpart, was isolated here. Subcellular localization results showed that OsRan2 is mainly localized in the nucleus, with some in the cytoplasm. Transcription of OsRAN2 was reduced by salt, osmotic, and exogenous abscisic acid (ABA) treatments, as determined by real-time PCR. Overexpression of OsRAN2 in rice resulted in enhanced sensitivity to salinity, osmotic stress, and ABA. Seedlings of transgenic Arabidopsis thaliana plants overexpressing OsRAN2 were overly sensitive to salinity stress and exogenous ABA treatment. Furthermore, three ABA- or stress-responsive genes, AtNCED3, AtPLC1, and AtMYB2, encoding a key enzyme in ABA synthesis, a phospholipase C homologue, and a putative transcriptional factor, respectively, were shown to have differentially induced expression under salinity and ABA treatments in transgenic and wild-type Arabidopsis plants. OsRAN2 overexpression in tobacco epidermal leaf cells disturbed the nuclear import of a maize (Zea mays L.) leaf colour transcription factor (Lc). In addition, gene-silenced rice plants generated via RNA interference (RNAi) displayed pleiotropic developmental abnormalities and were male sterile. PMID:20018899

The Reverse Transcriptase/RNA Maturase Protein MatR Is Required for the Splicing of Various Group II Introns in Brassicaceae Mitochondria.

PubMed

Sultan, Laure D; Mileshina, Daria; Grewe, Felix; Rolle, Katarzyna; Abudraham, Sivan; Głodowicz, Paweł; Niazi, Adnan Khan; Keren, Ido; Shevtsov, Sofia; Klipcan, Liron; Barciszewski, Jan; Mower, Jeffrey P; Dietrich, André; Ostersetzer-Biran, Oren

2016-11-01

Group II introns are large catalytic RNAs that are ancestrally related to nuclear spliceosomal introns. Sequences corresponding to group II RNAs are found in many prokaryotes and are particularly prevalent within plants organellar genomes. Proteins encoded within the introns themselves (maturases) facilitate the splicing of their own host pre-RNAs. Mitochondrial introns in plants have diverged considerably in sequence and have lost their maturases. In angiosperms, only a single maturase has been retained in the mitochondrial DNA: the matR gene found within NADH dehydrogenase 1 (nad1) intron 4. Its conservation across land plants and RNA editing events, which restore conserved amino acids, indicates that matR encodes a functional protein. However, the biological role of MatR remains unclear. Here, we performed an in vivo investigation of the roles of MatR in Brassicaceae. Directed knockdown of matR expression via synthetically designed ribozymes altered the processing of various introns, including nad1 i4. Pull-down experiments further indicated that MatR is associated with nad1 i4 and several other intron-containing pre-mRNAs. MatR may thus represent an intermediate link in the gradual evolutionary transition from the intron-specific maturases in bacteria into their versatile spliceosomal descendants in the nucleus. The similarity between maturases and the core spliceosomal Prp8 protein further supports this intriguing theory. © 2016 American Society of Plant Biologists. All rights reserved.

[Transcription factors NF-kB, HIF-1, HIF-2, growth factor VEGF, VEGFR2 and carboanhydrase IX mRNA and protein level in the development of kidney cancer metastasis].

PubMed

Spirina, L V; Usynin, Y A; Yurmazov, Z A; Slonimskaya, E M; Kolegova, E S; Kondakova, I V

2017-01-01

Here, we have investigated the participation of nuclear factors NF-kB, HIF-1 and HIF-2, VEGF, VEGFR2, and carboanhydrase IX in clear-cell renal cancer. We have determined the expression and protein level of transcription factors, VEGF, VEGFR2, and carboanhydrase IX in tumor and normal tissues of 30 patients with kidney cancer. The Real-Time PCR and ELISA were used in the study. The low levels of HIF-1 mRNA expression associated with high levels of HIF-1 protein were also associated with metastasis. The expression levels of VEGF, VEGFR2, and their protein levels are increased in primary tumors of patients with disseminated kidney cancer compared to nonmetastatic cancer. No correlation was revealed between the content of mRNA and encoded proteins in the kidney cancer tissues. The changes in the ratios of mRNA levels and the respective proteins (HIF-1α, HIF-2, NF-kB, VEGF, VEGFR2, and carboanhydrase IX) may contribute to kidney-cancer metastasis.

ERAD components in organisms with complex red plastids suggest recruitment of a preexisting protein transport pathway for the periplastid membrane.

PubMed

Felsner, Gregor; Sommer, Maik S; Gruenheit, Nicole; Hempel, Franziska; Moog, Daniel; Zauner, Stefan; Martin, William; Maier, Uwe G

2011-01-01

The plastids of cryptophytes, haptophytes, and heterokontophytes (stramenopiles) (together once known as chromists) are surrounded by four membranes, reflecting the origin of these plastids through secondary endosymbiosis. They share this trait with apicomplexans, which are alveolates, the plastids of which have been suggested to stem from the same secondary symbiotic event and therefore form a phylogenetic clade, the chromalveolates. The chromists are quantitatively the most important eukaryotic contributors to primary production in marine ecosystems. The mechanisms of protein import across their four plastid membranes are still poorly understood. Components of an endoplasmic reticulum-associated degradation (ERAD) machinery in cryptophytes, partially encoded by the reduced genome of the secondary symbiont (the nucleomorph), are implicated in protein transport across the second outermost plastid membrane. Here, we show that the haptophyte Emiliania huxleyi, like cryptophytes, stramenopiles, and apicomplexans, possesses a nuclear-encoded symbiont-specific ERAD machinery (SELMA, symbiont-specific ERAD-like machinery) in addition to the host ERAD system, with targeting signals that are able to direct green fluorescent protein or yellow fluorescent protein to the predicted cellular localization in transformed cells of the stramenopile Phaeodactylum tricornutum. Phylogenies of the duplicated ERAD factors reveal that all SELMA components trace back to a red algal origin. In contrast, the host copies of cryptophytes and haptophytes associate with the green lineage to the exclusion of stramenopiles and alveolates. Although all chromalveolates with four membrane-bound plastids possess the SELMA system, this has apparently not arisen in a single endosymbiotic event. Thus, our data do not support the chromalveolate hypothesis.

[Cloning of cDNA for RNA polymerase subunit from the fission yeast Schizosaccharomyces pombe by heterospecific complementation in Saccharomyces cerevisiae].

PubMed

Shpakovskiĭ, G V; Lebedenko, E N; Thuriaux, P

1997-02-01

The rpb10 cDNA of the fission yeast Schizosaccharomyces pombe, encoding one of the five small subunits common to all three nuclear DNA-dependent RNA polymerases, was isolated from an expression cDNA library by two independent approaches: PCR-based screening and direct suppression by means of heterospecific complementation of a temperature-sensitive mutant defective in the corresponding gene of Saccharomyces cerevisiae. The cloned Sz. pombe cDNA encodes a protein Rpb10 of 71 amino acids with an M of 8,275 Da, sharing 51 amino acids (71% identity) with the subunit ABC10 beta of RNA polymerases I-III from S. cerevisiae. All eukaryotic members of this protein family have the same general organization featuring two highly conserved motifs (RCFT/SCGK and RYCCRRM) around an atypical zinc finger and an additional invariant HVDLIEK motif toward the C-terminal end. The last motif is only characteristics for homologs from eukaryotes. In keeping with this remarkable structural conservation, the Sz. pombe cDNA also fully complemented a S. cerevisiae deletion mutant lacking subunit ABC10 beta (null allele rpb10-delta 1::HIS3).

Respiromics - An integrative analysis linking mitochondrial bioenergetics to molecular signatures.

PubMed

Walheim, Ellen; Wiśniewski, Jacek R; Jastroch, Martin

2018-03-01

Energy metabolism is challenged upon nutrient stress, eventually leading to a variety of metabolic diseases that represent a major global health burden. Here, we combine quantitative mitochondrial respirometry (Seahorse technology) and proteomics (LC-MS/MS-based total protein approach) to understand how molecular changes translate to changes in mitochondrial energy transduction during diet-induced obesity (DIO) in the liver. The integrative analysis reveals that significantly increased palmitoyl-carnitine respiration is supported by an array of proteins enriching lipid metabolism pathways. Upstream of the respiratory chain, the increased capacity for ATP synthesis during DIO associates strongest to mitochondrial uptake of pyruvate, which is routed towards carboxylation. At the respiratory chain, robust increases of complex I are uncovered by cumulative analysis of single subunit concentrations. Specifically, nuclear-encoded accessory subunits, but not mitochondrial-encoded or core units, appear to be permissive for enhanced lipid oxidation. Our integrative analysis, that we dubbed "respiromics", represents an effective tool to link molecular changes to functional mechanisms in liver energy metabolism, and, more generally, can be applied for mitochondrial analysis in a variety of metabolic and mitochondrial disease models. Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.

Mechanism of Dual Targeting of the Phytochrome Signaling Component HEMERA/pTAC12 to Plastids and the Nucleus.

PubMed

Nevarez, P Andrew; Qiu, Yongjian; Inoue, Hitoshi; Yoo, Chan Yul; Benfey, Philip N; Schnell, Danny J; Chen, Meng

2017-04-01

HEMERA (HMR) is a nuclear and plastidial dual-targeted protein. While it functions in the nucleus as a transcriptional coactivator in phytochrome signaling to regulate a distinct set of light-responsive, growth-relevant genes, in plastids it is known as pTAC12, which associates with the plastid-encoded RNA polymerase, and is essential for inducing the plastomic photosynthetic genes and initiating chloroplast biogenesis. However, the mechanism of targeting HMR to the nucleus and plastids is still poorly understood. Here, we show that HMR can be directly imported into chloroplasts through a transit peptide residing in the N-terminal 50 amino acids. Upon cleavage of the transit peptide and additional proteolytic processing, mature HMR, which begins from Lys-58, retains its biochemical properties in phytochrome signaling. Unexpectedly, expression of mature HMR failed to rescue not only the plastidial but also the nuclear defects of the hmr mutant. This is because the predicted nuclear localization signals of HMR are nonfunctional, and therefore mature HMR is unable to accumulate in either plastids or the nucleus. Surprisingly, fusing the transit peptide of the small subunit of Rubisco with mature HMR rescues both its plastidial and nuclear localization and functions. These results, combined with the observation that the nuclear form of HMR has the same reduced molecular mass as plastidial HMR, support a retrograde protein translocation mechanism in which HMR is targeted first to plastids, processed to the mature form, and then relocated to the nucleus. © 2017 American Society of Plant Biologists. All Rights Reserved.

Analysis of a developmentally regulated nuclear localization signal in Xenopus

PubMed Central

1992-01-01

The 289 residue nuclear oncoprotein encoded by the adenovirus 5 Ela gene contains two peptide sequences that behave as nuclear localization signals (NLS). One signal, located at the carboxy terminus, is like many other known NLSs in that it consists of a short stretch of basic residues (KRPRP) and is constitutively active in cells. The second signal resides within an internal 45 residue region of E1a that contains few basic residues or sequences that resemble other known NLSs. Moreover, this internal signal functions in injected Xenopus oocytes, but not in transfected Xenopus A6 cells, suggesting that it could be regulated developmentally (Slavicek et al. 1989. J. Virol. 63:4047). In this study, we show that the activity of this signal is sensitive to ATP depletion in vivo, efficiently directs the import of a 50 kD fusion protein and can compete with the E1a carboxy-terminal NLS for nuclear import. In addition, we have delineated the precise amino acid residues that comprise the second E1a NLS, and have assessed its utilization during Xenopus embryogenesis. Using amino acid deletion and substitution analyses, we show that the signal consists of the sequence FV(X)7-20MXSLXYM(X)4MF. By expressing in Xenopus embryos a truncated E1a protein that contains only the second NLS and by monitoring its cytoplasmic/nuclear distribution during development with indirect immunofluorescence, we find that the second NLS is utilized up to the early neurula stage. In addition, there appears to be a hierarchy among the embryonic germ layers as to when the second NLS becomes nonfunctional. For this reason, we refer to this NLS as the developmentally regulated nuclear localization signal (drNLS). The implications of these findings for early development are discussed. PMID:1387407

Expression and one-step purification of recombinant Arabidopsis thaliana frataxin homolog (AtFH).

PubMed

Maliandi, Maria V; Busi, Maria V; Clemente, Marina; Zabaleta, Eduardo J; Araya, Alejandro; Gomez-Casati, Diego F

2007-02-01

Frataxin, a nuclear-encoded mitochondrial protein, has been proposed to participate in Fe-S cluster assembly, mitochondrial energy metabolism, respiration, and iron homeostasis. However, its precise function remains elusive. Frataxin is highly conserved in living organisms with no major structural changes, in particular at the C-terminal protein domain, suggesting that it plays a key function in all organisms. Recently, a plant gene, AtFH, with significant homology to other members of the frataxin family has been described. To gain insight on the frataxin role in plants, the frataxin domain was expressed in Escherichia coli BL21-codonPlus (DE3)-RIL cells and purified using a Ni-chelating column. The purified protein, added to a mixture containing Fe(II) and H2O2, attenuates the Fenton reaction indicating that the recombinant plant frataxin is functional. The procedure described here produced high yield of 99% pure protein through only one chromatographic step, suitable for further structure-function studies.

Human I-mfa domain proteins specifically interact with KSHV LANA and affect its regulation of Wnt signaling-dependent transcription

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kusano, Shuichi, E-mail: skusano@m2.kufm.kagoshima-u.ac.jp; Eizuru, Yoshito

2010-06-04

Kaposi's sarcoma-associated herpes virus (KSHV)-encoded latency-associated nuclear antigen (LANA) protein has been reported to interact with glycogen synthase kinase 3{beta} (GSK-3{beta}) and to negatively regulate its activity, leading to stimulation of GSK-3{beta}-dependent {beta}-catenin degradation. We show here that the I-mfa domain proteins, HIC (human I-mfa domain-containing protein) and I-mfa (inhibitor of MyoD family a), interacted in vivo with LANA through their C-terminal I-mfa domains. This interaction affected the intracellular localization of HIC, inhibited the LANA-dependent transactivation of a {beta}-catenin-regulated reporter construct, and decreased the level of the LANA.GSK-3{beta} complex. These data reveal for the first time that I-mfa domain proteinsmore » interact with LANA and negatively regulate LANA-mediated activation of Wnt signaling-dependent transcription by inhibiting the formation of the LANA.GSK-3{beta} complex.« less

Small Molecule Disrupts Abnormal Gene Fusion Associated with Leukemia | Center for Cancer Research

Cancer.gov

Rare chromosomal abnormalities, called chromosomal translocations, in which part of a chromosome breaks off and becomes attached to another chromosome, can result in the generation of chimeric proteins. These aberrant proteins have unpredictable, and sometimes harmful, functions, including uncontrolled cell growth that can lead to cancer. One type of translocation, in which a portion of the gene encoding nucleoporin 98 (NUP98)—one of about 50 proteins comprising the nuclear pore complex through which proteins are shuttled into and out of the nucleus—fuses with another gene, has been shown to result in improper histone modifications. These abnormalities alter the gene expression patterns of certain types of hematopoietic, or blood-forming, stem cells, resulting primarily in overexpression of the Hoxa7, Hoxa9,and Hoxa10 genes. NUP98 chromosomal translocations have been associated with many types of leukemia, including acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia in blast crisis (CML-bc), and myelodysplastic syndrome (MDS).

Multifunctional Mitochondrial AAA Proteases

PubMed Central

Glynn, Steven E.

2017-01-01

Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle. PMID:28589125

Multifunctional Mitochondrial AAA Proteases.

PubMed

Glynn, Steven E

2017-01-01

Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle.

Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly

PubMed Central

Cook, Jeremy D.; Kondapalli, Kalyan C.; Rawat, Swati; Childs, William C.; Murugesan, Yogapriya; Dancis, Andrew; Stemmler, Timothy L.

2010-01-01

Frataxin, a conserved nuclear encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich’s ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two: Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone n the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural module to better understand the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry (ITC). Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into a Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly. PMID:20815377

PmVRP15, a Novel Viral Responsive Protein from the Black Tiger Shrimp, Penaeus monodon, Promoted White Spot Syndrome Virus Replication

PubMed Central

Vatanavicharn, Tipachai; Prapavorarat, Adisak; Jaree, Phattarunda; Somboonwiwat, Kunlaya; Tassanakajon, Anchalee

2014-01-01

Suppression subtractive hybridization of Penaeus monodon hemocytes challenged with white spot syndrome virus (WSSV) has identified the viral responsive gene, PmVRP15, as the highest up-regulated gene ever reported in shrimps. Expression analysis by quantitative real time RT-PCR revealed 9410–fold up-regulated level at 48 h post WSSV injection. Tissue distribution analysis showed that PmVRP15 transcript was mainly expressed in the hemocytes of shrimp. The full-length cDNA of PmVRP15 transcript was obtained and showed no significant similarity to any known gene in the GenBank database. The predicted open reading frame of PmVRP15 encodes for a deduced 137 amino acid protein containing a putative transmembrane helix. Immunofluorescent localization of the PmVRP15 protein revealed it accumulated around the nuclear membrane in all three types of shrimp hemocytes and that the protein was highly up-regulated in WSSV-infected shrimps. Double-stranded RNA interference-mediated gene silencing of PmVRP15 in P. monodon significantly decreased WSSV propagation compared to the control shrimps (injected with GFP dsRNA). The significant decrease in cumulative mortality rate of WSSV-infected shrimp following PmVRP15 knockdown was observed. These results suggest that PmVRP15 is likely to be a nuclear membrane protein and that it acts as a part of WSSV propagation pathway. PMID:24637711

Suppression of cell division by pKi-67 antisense-RNA and recombinant protein.

PubMed

Duchrow, M; Schmidt, M H; Zingler, M; Anemüller, S; Bruch, H P; Broll, R

2001-01-01

The human antigen defined by the monoclonal antibody Ki-67 (pKi-67) is a human nuclear protein strongly associated with cell proliferation and found in all tissues studied. It is widely used as a marker of proliferating cells, yet its function is unknown. To investigate its function we suppressed pKi-67 expression by antisense RNA and overexpressed a partial structure of pKi-67 in HeLa cells. A BrdU-incorporation assay showed a significant decrease in DNA synthesis after antisense inhibition. Cell cycle analysis indicated a higher proportion of cells in G1 phase and a lower proportion of cells in S phase while the number of G(2)/M phase cells remained constant. Overexpression of a recombinant protein encoding three of the repetitive elements from exon 13 of pKi-67 had a similar effect to that obtained by antisense inhibition. The similarity of the effect of expressing 'Ki-67 repeats' and pKi-67 antisense RNA could be explained by a negative effect on the folding of the endogenous protein in the endoplasmatic reticulum. Furthermore excessive self-association of pKi-67 via the repeat structure could inhibit its nuclear transport, preventing it from getting to its presumptive site of action. We conclude that the Ki-67 protein has an important role in the regulation of the cell cycle, which is mediated in part by its repetitive elements. Copyright 2001 S. Karger AG, Basel

Molecular Details of the Yeast Frataxin-Isu1 Interaction during Mitochondrial Fe-S Cluster Assembly

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, J.; Kondapalli, K; Rawat, S

2010-01-01

Frataxin, a conserved nuclear-encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two, Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone in the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural modulemore » to improve our understanding of the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry. Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into an Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly.« less

Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly.

PubMed

Cook, Jeremy D; Kondapalli, Kalyan C; Rawat, Swati; Childs, William C; Murugesan, Yogapriya; Dancis, Andrew; Stemmler, Timothy L

2010-10-12

Frataxin, a conserved nuclear-encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two, Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone in the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural module to improve our understanding of the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry. Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into an Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly.

[Primary investigation of the relationship between glucocorticoid induced leucine zipper and inflammatory reaction].

PubMed

Bai, Xiang-jun; Li, Bo; Wang, Hai-ping; Yang, Zhao-hui; Li, Si-qi

2007-01-01

To investigate the mechanism of the action of glucocorticoid induced leucine zipper (GILZ) in inflammatory reaction. Human monocyte cell line THP-1 cells were divided into two groups and cultured in non-serum RPMI1640 medium.In one group the cells were treated with dexamethasone (DEX). Twelve hours later total RNA and total protein were abstracted in both two groups. The mRNA encoding for expression of GILZ was semiquantitatively detected by reserve transcriptase-polymerase chain reaction (RT-PCR). Protein expression of nuclear factor-KappaB (NF-KappaB) p65 and activator protein-1 (AP-1) were assessed by Western blotting. Peripheral blood of 10 trauma patients [injury severity score (ISS) >or=16 scores] were collected and the leukocytes were isolated within 24 hours after trauma. The leukocytes were divided into two groups and cultured in non-serum medium. In one group the cells were treated with DEX. Twelve hours later total RNA and total protein were abstracted in both two groups. The mRNA encoding for expression of GILZ was semiquantitatively detected by RT-PCR. Protein expression of NF-KappaB p65 and AP-1 were assessed by Western blotting. Stimulated by DEX, the expression of GILZ mRNA was increased both in THP-1 cells and the leukocytes of trauma patients compared with those of control groups (both P<0.01). Whereas, protein expressions of NF-KappaB p65 and AP-1 of THP-1 cells and leukocytes in peripheral blood of trauma patients were decreased in the stimulation groups compared with those of control groups (all P<0.01). The expression of GILZ gene is up-regulated by glucocorticoid. Overexpression of GILZ inhibits NF-KappaB and AP-1 activities, suggesting that GILZ possesses anti-inflammatory function.

Herpes Simplex Virus Is Equipped with RNA- and Protein-Based Mechanisms To Repress Expression of ATRX, an Effector of Intrinsic Immunity

PubMed Central

Jurak, Igor; Silverstein, Leah B.; Sharma, Mayuri

2012-01-01

Intrinsic immunity is a first-line intracellular defense against virus infection, and viruses have evolved mechanisms to counteract it. During herpes simplex virus (HSV) infection, nuclear domain 10 (ND10) components localize adjacent to incoming viral genomes and generate a repressive environment for viral gene expression. Here, we found that the ND10 component, alpha-thalassemia/mental retardation syndrome X-linked (ATRX) protein, is predicted to be a target of HSV-1 miR-H1 and HSV-2 miR-H6. These microRNAs (miRNAs) share a seed sequence and are abundant during lytic infection. Mimics of both miRNAs could deplete endogenous ATRX, and an miR-H1 mimic could repress the expression of a reporter linked to the 3′ untranslated region of ATRX mRNA, identifying a cellular mRNA targeted by an HSV miRNA. Interestingly, ATRX protein and its mRNA were depleted in cells lytically infected with HSV, and ATRX protein was also depleted in cells infected with human cytomegalovirus. However, infection with an HSV-1 mutant lacking miR-H1 still resulted in ATRX depletion. This depletion was sensitive to a proteasome inhibitor and was largely ablated by a deletion of the gene encoding the immediate-early ICP0 protein. Additionally, a deletion of the gene encoding the tegument protein Vhs ablated most of the depletion of ATRX mRNA. Thus, HSV is equipped with multiple mechanisms to limit the expression of ATRX. As ATRX is implicated in repression of lytic viral gene expression, our results suggest roles for these different mechanisms during various phases of HSV infection. PMID:22787211

Herpes simplex virus is equipped with RNA- and protein-based mechanisms to repress expression of ATRX, an effector of intrinsic immunity.

PubMed

Jurak, Igor; Silverstein, Leah B; Sharma, Mayuri; Coen, Donald M

2012-09-01

Intrinsic immunity is a first-line intracellular defense against virus infection, and viruses have evolved mechanisms to counteract it. During herpes simplex virus (HSV) infection, nuclear domain 10 (ND10) components localize adjacent to incoming viral genomes and generate a repressive environment for viral gene expression. Here, we found that the ND10 component, alpha-thalassemia/mental retardation syndrome X-linked (ATRX) protein, is predicted to be a target of HSV-1 miR-H1 and HSV-2 miR-H6. These microRNAs (miRNAs) share a seed sequence and are abundant during lytic infection. Mimics of both miRNAs could deplete endogenous ATRX, and an miR-H1 mimic could repress the expression of a reporter linked to the 3' untranslated region of ATRX mRNA, identifying a cellular mRNA targeted by an HSV miRNA. Interestingly, ATRX protein and its mRNA were depleted in cells lytically infected with HSV, and ATRX protein was also depleted in cells infected with human cytomegalovirus. However, infection with an HSV-1 mutant lacking miR-H1 still resulted in ATRX depletion. This depletion was sensitive to a proteasome inhibitor and was largely ablated by a deletion of the gene encoding the immediate-early ICP0 protein. Additionally, a deletion of the gene encoding the tegument protein Vhs ablated most of the depletion of ATRX mRNA. Thus, HSV is equipped with multiple mechanisms to limit the expression of ATRX. As ATRX is implicated in repression of lytic viral gene expression, our results suggest roles for these different mechanisms during various phases of HSV infection.

Reverse Genetic Analysis of Ourmiaviruses Reveals the Nucleolar Localization of the Coat Protein in Nicotiana benthamiana and Unusual Requirements for Virion Formation ▿ † ‡

PubMed Central

Crivelli, Giulia; Ciuffo, Marina; Genre, Andrea; Masenga, Vera; Turina, Massimo

2011-01-01

Ourmia melon virus (OuMV) is the type member of the genus Ourmiavirus. These viruses have a trisegmented genome, each part of which encodes a single protein. Ourmiaviruses share a distant similarity with other plant viruses only in their movement proteins (MP), whereas their RNA-dependent RNA polymerase (RdRP) shares features only with fungal viruses of the family Narnaviridae. Thus, ourmiaviruses are in a unique phylogenetic position among existing plant viruses. Here, we developed an agroinoculation system to launch infection in Nicotiana benthamiana plants. Using different combinations of the three segments, we demonstrated that RNA1 is necessary and sufficient for cis-acting replication in the agroinfiltrated area. RNA2 and RNA3, encoding the putative movement protein and the coat protein (CP), respectively, are both necessary for successful systemic infection of N. benthamiana. The CP is dispensable for long-distance transport of the virus through vascular tissues, but its absence prevents efficient systemic infection at the exit sites. Virion formation occurred only when the CP was translated from replication-derived RNA3. Transient expression of a green fluorescent protein-MP (GFP-MP) fusion via agroinfiltration showed that the MP is present in cytoplasmic connections across plant cell walls; in protoplasts the GFP-MP fusion stimulates the formation of tubular protrusions. Expression through agroinfiltration of a GFP-CP fusion displays most of the fluorescence inside the nucleus and within the nucleolus in particular. Nuclear localization of the CP was also confirmed through Western blot analysis of purified nuclei. The significance of several unusual properties of OuMV for replication, virion assembly, and movement is discussed in relation to other positive-strand RNA viruses. PMID:21411534

Encoding of contextual fear memory requires de novo proteins in the prelimbic cortex

PubMed Central

Rizzo, Valerio; Touzani, Khalid; Raveendra, Bindu L.; Swarnkar, Supriya; Lora, Joan; Kadakkuzha, Beena M.; Liu, Xin-An; Zhang, Chao; Betel, Doron; Stackman, Robert W.; Puthanveettil, Sathyanarayanan V.

2016-01-01

Background Despite our understanding of the significance of the prefrontal cortex in the consolidation of long-term memories (LTM), its role in the encoding of LTM remains elusive. Here we investigated the role of new protein synthesis in the mouse medial prefrontal cortex (mPFC) in encoding contextual fear memory. Methods Because a change in the association of mRNAs to polyribosomes is an indicator of new protein synthesis, we assessed the changes in polyribosome-associated mRNAs in the mPFC following contextual fear conditioning (CFC) in the mouse. Differential gene expression in mPFC was identified by polyribosome profiling (n = 18). The role of new protein synthesis in mPFC was determined by focal inhibition of protein synthesis (n = 131) and by intra-prelimbic cortex manipulation (n = 56) of Homer 3, a candidate identified from polyribosome profiling. Results We identified several mRNAs that are differentially and temporally recruited to polyribosomes in the mPFC following CFC. Inhibition of protein synthesis in the prelimbic (PL), but not in the anterior cingulate cortex (ACC) region of the mPFC immediately after CFC disrupted encoding of contextual fear memory. Intriguingly, inhibition of new protein synthesis in the PL 6 hours after CFC did not impair encoding. Furthermore, expression of Homer 3, an mRNA enriched in polyribosomes following CFC, in the PL constrained encoding of contextual fear memory. Conclusions Our studies identify several molecular substrates of new protein synthesis in the mPFC and establish that encoding of contextual fear memories require new protein synthesis in PL subregion of mPFC. PMID:28503670

Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Deep Raj; Sunkaria, Aditya; Wani, Willayat Yousuf

The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10 mg/kg b.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) andmore » Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits–NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases. - Highlights: • Aluminium decreases the mRNA levels of mitochondrial and nuclear encoded subunits. • It decreases the mtDNA copy number and mitochondrial content in rat brain. • It down-regulates the mRNA and protein levels of PGC-1α, NRF-1, NRF-2 and Tfam. • It also disturbs the mitochondrial or nuclear architecture of neurons. • Finally it also decreases mitochondrial number in HC and CS regions of rat brain.« less

Pnrc2 regulates 3'UTR-mediated decay of segmentation clock-associated transcripts during zebrafish segmentation.

PubMed

Gallagher, Thomas L; Tietz, Kiel T; Morrow, Zachary T; McCammon, Jasmine M; Goldrich, Michael L; Derr, Nicolas L; Amacher, Sharon L

2017-09-01

Vertebrate segmentation is controlled by the segmentation clock, a molecular oscillator that regulates gene expression and cycles rapidly. The expression of many genes oscillates during segmentation, including hairy/Enhancer of split-related (her or Hes) genes, which encode transcriptional repressors that auto-inhibit their own expression, and deltaC (dlc), which encodes a Notch ligand. We previously identified the tortuga (tor) locus in a zebrafish forward genetic screen for genes involved in cyclic transcript regulation and showed that cyclic transcripts accumulate post-splicing in tor mutants. Here we show that cyclic mRNA accumulation in tor mutants is due to loss of pnrc2, which encodes a proline-rich nuclear receptor co-activator implicated in mRNA decay. Using an inducible in vivo reporter system to analyze transcript stability, we find that the her1 3'UTR confers Pnrc2-dependent instability to a heterologous transcript. her1 mRNA decay is Dicer-independent and likely employs a Pnrc2-Upf1-containing mRNA decay complex. Surprisingly, despite accumulation of cyclic transcripts in pnrc2-deficient embryos, we find that cyclic protein is expressed normally. Overall, we show that Pnrc2 promotes 3'UTR-mediated decay of developmentally-regulated segmentation clock transcripts and we uncover an additional post-transcriptional regulatory layer that ensures oscillatory protein expression in the absence of cyclic mRNA decay. Copyright © 2017 Elsevier Inc. All rights reserved.

Burkholderia Mallei tssM Encodes a Secreted Deubiquitinase that is Expressed Inside Infected RAW 264.7 Murine Macrophages

DTIC Science & Technology

2008-10-13

Furthermore, the encoded protein of this gene is only 30 kDa. A potential GTG start codon at position 625 also encodes a protein that is too small...horizontal bar and putative alternate translation initiation sites (ATG, GTG , and TTG) are indicated. The sizes and locations of the proteins encoded... gray line with rounded rectangles showing sequence features and motifs, including the Ala- and Pro-rich N-terminal region and the C-terminal Cys and

The majority of total nuclear-encoded non-ribosomal RNA in a human cell is 'dark matter' un-annotated RNA.

PubMed

Kapranov, Philipp; St Laurent, Georges; Raz, Tal; Ozsolak, Fatih; Reynolds, C Patrick; Sorensen, Poul H B; Reaman, Gregory; Milos, Patrice; Arceci, Robert J; Thompson, John F; Triche, Timothy J

2010-12-21

Discovery that the transcriptional output of the human genome is far more complex than predicted by the current set of protein-coding annotations and that most RNAs produced do not appear to encode proteins has transformed our understanding of genome complexity and suggests new paradigms of genome regulation. However, the fraction of all cellular RNA whose function we do not understand and the fraction of the genome that is utilized to produce that RNA remain controversial. This is not simply a bookkeeping issue because the degree to which this un-annotated transcription is present has important implications with respect to its biologic function and to the general architecture of genome regulation. For example, efforts to elucidate how non-coding RNAs (ncRNAs) regulate genome function will be compromised if that class of RNAs is dismissed as simply 'transcriptional noise'. We show that the relative mass of RNA whose function and/or structure we do not understand (the so called 'dark matter' RNAs), as a proportion of all non-ribosomal, non-mitochondrial human RNA (mt-RNA), can be greater than that of protein-encoding transcripts. This observation is obscured in studies that focus only on polyA-selected RNA, a method that enriches for protein coding RNAs and at the same time discards the vast majority of RNA prior to analysis. We further show the presence of a large number of very long, abundantly-transcribed regions (100's of kb) in intergenic space and further show that expression of these regions is associated with neoplastic transformation. These overlap some regions found previously in normal human embryonic tissues and raises an interesting hypothesis as to the function of these ncRNAs in both early development and neoplastic transformation. We conclude that 'dark matter' RNA can constitute the majority of non-ribosomal, non-mitochondrial-RNA and a significant fraction arises from numerous very long, intergenic transcribed regions that could be involved in neoplastic transformation.

Identification of Circular RNAs from the Parental Genes Involved in Multiple Aspects of Cellular Metabolism in Barley

PubMed Central

Darbani, Behrooz; Noeparvar, Shahin; Borg, Søren

2016-01-01

RNA circularization made by head-to-tail back-splicing events is involved in the regulation of gene expression from transcriptional to post-translational levels. By exploiting RNA-Seq data and down-stream analysis, we shed light on the importance of circular RNAs in plants. The results introduce circular RNAs as novel interactors in the regulation of gene expression in plants and imply the comprehensiveness of this regulatory pathway by identifying circular RNAs for a diverse set of genes. These genes are involved in several aspects of cellular metabolism as hormonal signaling, intracellular protein sorting, carbohydrate metabolism and cell-wall biogenesis, respiration, amino acid biosynthesis, transcription and translation, and protein ubiquitination. Additionally, these parental loci of circular RNAs, from both nuclear and mitochondrial genomes, encode for different transcript classes including protein coding transcripts, microRNA, rRNA, and long non-coding/microprotein coding RNAs. The results shed light on the mitochondrial exonic circular RNAs and imply the importance of circular RNAs for regulation of mitochondrial genes. Importantly, we introduce circular RNAs in barley and elucidate their cellular-level alterations across tissues and in response to micronutrients iron and zinc. In further support of circular RNAs' functional roles in plants, we report several cases where fluctuations of circRNAs do not correlate with the levels of their parental-loci encoded linear transcripts. PMID:27375638

The RING finger/B-box factor TAM-1 and a retinoblastoma-like protein LIN-35 modulate context-dependent gene silencing in Caenorhabditis elegans.

PubMed

Hsieh, J; Liu, J; Kostas, S A; Chang, C; Sternberg, P W; Fire, A

1999-11-15

Context-dependent gene silencing is used by many organisms to stably modulate gene activity for large chromosomal regions. We have used tandem array transgenes as a model substrate in a screen for Caenorhabditis elegans mutants that affect context-dependent gene silencing in somatic tissues. This screen yielded multiple alleles of a previously uncharacterized gene, designated tam-1 (for tandem-array-modifier). Loss-of-function mutations in tam-1 led to a dramatic reduction in the activity of numerous highly repeated transgenes. These effects were apparently context dependent, as nonrepetitive transgenes retained activity in a tam-1 mutant background. In addition to the dramatic alterations in transgene activity, tam-1 mutants showed modest alterations in expression of a subset of endogenous cellular genes. These effects include genetic interactions that place tam-1 into a group called the class B synMuv genes (for a Synthetic Multivulva phenotype); this family plays a negative role in the regulation of RAS pathway activity in C. elegans. Loss-of-function mutants in other members of the class-B synMuv family, including lin-35, which encodes a protein similar to the tumor suppressor Rb, exhibit a hypersilencing in somatic transgenes similar to that of tam-1 mutants. Molecular analysis reveals that tam-1 encodes a broadly expressed nuclear protein with RING finger and B-box motifs.

Prevalence of mutations in hepatitis C virus core protein associated with alteration of NF-kappaB activation.

PubMed

Mann, Elizabeth A; Stanford, Sandra; Sherman, Kenneth E

2006-10-01

The hepatitis C virus (HCV) core protein is a key structural element of the virion but also affects a number of cellular pathways, including nuclear factor kappaB (NF-kappaB) signaling. NF-kappaB is a transcription factor that regulates both anti-apoptotic and pro-inflammatory genes and its activation may contribute to HCV-mediated pathogenesis. Amino acid sequence divergence in core is seen at the genotype level as well as within patient isolates. Recent work has implicated amino acids 9-11 of core in the modulation of NF-kappaB activation. We report that the sequence RKT is highly conserved (93%) at this position across all HCV genotypes, based on sequences collected in the Los Alamos HCV database. Of the 13 types of variants present in the database, the two most prevalent substitutions are RQT and RKP. We further show that core encoding RKP fails to activate NF-kappaB signaling in vitro while NF-kappaB activation by core encoding RQT does not differ from control RKT core. The effect of RKP core is specific to NF-kappaB signaling as activator protein 1 (AP-1) activity is not altered. Further studies are needed to assess potential associations between specific amino acid substitutions at positions 9-11 and liver disease progression and/or response to treatment in individual patients.

Conserved Locus-Specific Silencing Functions of Schizosaccharomyces pombe sir2+

PubMed Central

Freeman-Cook, Lisa L.; Gómez, Eliana B.; Spedale, Erik J.; Marlett, John; Forsburg, Susan L.; Pillus, Lorraine; Laurenson, Patricia

2005-01-01

In Schizosaccharomyces pombe, three genes, sir2+, hst2+, and hst4+, encode members of the Sir2 family of conserved NAD+-dependent protein deacetylases. The S. pombe sir2+ gene encodes a nuclear protein that is not essential for viability or for resistance to treatment with UV or a microtubule-destabilizing agent. However, sir2+ is essential for full transcriptional silencing of centromeres, telomeres, and the cryptic mating-type loci. Chromatin immunoprecipitation results suggest that the Sir2 protein acts directly at these chromosomal regions. Enrichment of Sir2p at silenced regions does not require the HP1 homolog Swi6p; instead, Swi6-GFP localization to telomeres depends in part on Sir2p. The phenotype of sir2 swi6 double mutants supports a model whereby Sir2p functions prior to Swi6p at telomeres and the silent mating-type loci. However, Sir2p does not appear to be essential for the localization of Swi6p to centromeric foci. Cross-complementation experiments showed that the Saccharomyces cerevisiae SIR2 gene can function in place of S. pombe sir2+, suggesting overlapping deacetylation substrates in both species. These results also suggest that, despite differences in most of the other molecules required, the two distantly related yeast species share a mechanism for targeting Sir2p homologs to silent chromatin. PMID:15545655

Antiviral Defense Involves AGO4 in an Arabidopsis-Potexvirus Interaction.

PubMed

Brosseau, Chantal; El Oirdi, Mohamed; Adurogbangba, Ayooluwa; Ma, Xiaofang; Moffett, Peter

2016-11-01

In plants, RNA silencing regulates gene expression through the action of Dicer-like (DCL) and Argonaute (AGO) proteins via micro RNAs and RNA-dependent DNA methylation (RdDM). In addition, RNA silencing functions as an antiviral defense mechanism by targeting virus-derived double-stranded RNA. Plants encode multiple AGO proteins with specialized functions, including AGO4-like proteins that affect RdDM and AGO2, AGO5, and AGO1, which have antiviral activities. Here, we show that AGO4 is also required for defense against the potexvirus Plantago asiatica mosaic virus (PlAMV), most likely independent of RdDM components such as DCL3, Pol IV, and Pol V. Transient assays showed that AGO4 has direct antiviral activity on PlAMV and, unlike RdDM, this activity does not require nuclear localization of AGO4. Furthermore, although PlAMV infection causes a decrease in AGO4 expression, PlAMV causes a change in AGO4 localization from a largely nuclear to a largely cytoplasmic distribution. These results indicate an important role for AGO4 in targeting plant RNA viruses as well as demonstrating novel mechanisms of regulation of and by AGO4, independent of its canonical role in regulating gene expression by RdDM.

An in silico argument for mitochondrial microRNA as a determinant of primary non function in liver transplantation.

PubMed

Khorsandi, Shirin Elizabeth; Salehi, Siamak; Cortes, Miriam; Vilca-Melendez, Hector; Menon, Krishna; Srinivasan, Parthi; Prachalias, Andreas; Jassem, Wayel; Heaton, Nigel

2018-02-15

Mitochondria have their own genomic, transcriptomic and proteomic machinery but are unable to be autonomous, needing both nuclear and mitochondrial genomes. The aim of this work was to use computational biology to explore the involvement of Mitochondrial microRNAs (MitomiRs) and their interactions with the mitochondrial proteome in a clinical model of primary non function (PNF) of the donor after cardiac death (DCD) liver. Archival array data on the differential expression of miRNA in DCD PNF was re-analyzed using a number of publically available computational algorithms. 10 MitomiRs were identified of importance in DCD PNF, 7 with predicted interaction of their seed sequence with the mitochondrial transcriptome that included both coding, and non coding areas of the hypervariability region 1 (HVR1) and control region. Considering miRNA regulation of the nuclear encoded mitochondrial proteome, 7 hypothetical small proteins were identified with homolog function that ranged from co-factor for formation of ATP Synthase, REDOX balance and an importin/exportin protein. In silico, unconventional seed interactions, both non canonical and alternative seed sites, appear to be of greater importance in MitomiR regulation of the mitochondrial genome. Additionally, a number of novel small proteins of relevance in transplantation have been identified which need further characterization.

The Fn14 cytoplasmic tail binds tumour-necrosis-factor-receptor-associated factors 1, 2, 3 and 5 and mediates nuclear factor-kappaB activation.

PubMed Central

Brown, Sharron A N; Richards, Christine M; Hanscom, Heather N; Feng, Sheau-Line Y; Winkles, Jeffrey A

2003-01-01

Fn14 is a growth-factor-inducible immediate-early-response gene encoding a 102-amino-acid type I transmembrane protein. The human Fn14 protein was recently identified as a cell-surface receptor for the tumour necrosis factor (TNF) superfamily member named TWEAK (TNF-like weak inducer of apoptosis). In the present paper, we report that the human TWEAK extracellular domain can also bind the murine Fn14 protein. Furthermore, site-specific mutagenesis and directed yeast two-hybrid interaction assays revealed that the TNFR-associated factor (TRAF) 1, 2, 3 and 5 adaptor molecules bind the murine Fn14 cytoplasmic tail at an overlapping, but non-identical, amino acid sequence motif. We also found that TWEAK treatment of quiescent NIH 3T3 cells stimulates inhibitory kappaBalpha phosphorylation and transcriptional activation of a nuclear factor-kappaB (NF-kappaB) enhancer/luciferase reporter construct. Fn14 overexpression in transiently transfected NIH 3T3 cells also promotes NF-kappaB activation, and this cellular response requires an intact TRAF binding site. These results indicate that Fn14 is a functional TWEAK receptor that can associate with four distinct TRAF family members and stimulate the NF-kappaB transcription factor signalling pathway. PMID:12529173

Oncoprotein AEG-1 is an endoplasmic reticulum RNA-binding protein whose interactome is enriched in organelle resident protein-encoding mRNAs.

PubMed

Hsu, Jack C-C; Reid, David W; Hoffman, Alyson M; Sarkar, Devanand; Nicchitta, Christopher V

2018-05-01

Astrocyte elevated gene-1 (AEG-1), an oncogene whose overexpression promotes tumor cell proliferation, angiogenesis, invasion, and enhanced chemoresistance, is thought to function primarily as a scaffolding protein, regulating PI3K/Akt and Wnt/β-catenin signaling pathways. Here we report that AEG-1 is an endoplasmic reticulum (ER) resident integral membrane RNA-binding protein (RBP). Examination of the AEG-1 RNA interactome by HITS-CLIP and PAR-CLIP methodologies revealed a high enrichment for endomembrane organelle-encoding transcripts, most prominently those encoding ER resident proteins, and within this cohort, for integral membrane protein-encoding RNAs. Cluster mapping of the AEG-1/RNA interaction sites demonstrated a normalized rank order interaction of coding sequence >5' untranslated region, with 3' untranslated region interactions only weakly represented. Intriguingly, AEG-1/membrane protein mRNA interaction sites clustered downstream from encoded transmembrane domains, suggestive of a role in membrane protein biogenesis. Secretory and cytosolic protein-encoding mRNAs were also represented in the AEG-1 RNA interactome, with the latter category notably enriched in genes functioning in mRNA localization, translational regulation, and RNA quality control. Bioinformatic analyses of RNA-binding motifs and predicted secondary structure characteristics indicate that AEG-1 lacks established RNA-binding sites though shares the property of high intrinsic disorder commonly seen in RBPs. These data implicate AEG-1 in the localization and regulation of secretory and membrane protein-encoding mRNAs and provide a framework for understanding AEG-1 function in health and disease. © 2018 Hsu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Solenopsis invicta virus 3: mapping of structural proteins, ribosomal frameshifting, and similarities to Acyrthosiphon pisum virus and Kelp fly virus.

PubMed

Valles, Steven M; Bell, Susanne; Firth, Andrew E

2014-01-01

Solenopsis invicta virus 3 (SINV-3) is a positive-sense single-stranded RNA virus that infects the red imported fire ant, Solenopsis invicta. We show that the second open reading frame (ORF) of the dicistronic genome is expressed via a frameshifting mechanism and that the sequences encoding the structural proteins map to both ORF2 and the 3' end of ORF1, downstream of the sequence that encodes the RNA-dependent RNA polymerase. The genome organization and structural protein expression strategy resemble those of Acyrthosiphon pisum virus (APV), an aphid virus. The capsid protein that is encoded by the 3' end of ORF1 in SINV-3 and APV is predicted to have a jelly-roll fold similar to the capsid proteins of picornaviruses and caliciviruses. The capsid-extension protein that is produced by frameshifting, includes the jelly-roll fold domain encoded by ORF1 as its N-terminus, while the C-terminus encoded by the 5' half of ORF2 has no clear homology with other viral structural proteins. A third protein, encoded by the 3' half of ORF2, is associated with purified virions at sub-stoichiometric ratios. Although the structural proteins can be translated from the genomic RNA, we show that SINV-3 also produces a subgenomic RNA encoding the structural proteins. Circumstantial evidence suggests that APV may also produce such a subgenomic RNA. Both SINV-3 and APV are unclassified picorna-like viruses distantly related to members of the order Picornavirales and the family Caliciviridae. Within this grouping, features of the genome organization and capsid domain structure of SINV-3 and APV appear more similar to caliciviruses, perhaps suggesting the basis for a "Calicivirales" order.

Missing genes, multiple ORFs, and C-to-U type RNA editing in Acrasis kona (Heterolobosea, Excavata) mitochondrial DNA.

PubMed

Fu, Cheng-Jie; Sheikh, Sanea; Miao, Wei; Andersson, Siv G E; Baldauf, Sandra L

2014-08-21

Discoba (Excavata) is an ancient group of eukaryotes with great morphological and ecological diversity. Unlike the other major divisions of Discoba (Jakobida and Euglenozoa), little is known about the mitochondrial DNAs (mtDNAs) of Heterolobosea. We have assembled a complete mtDNA genome from the aggregating heterolobosean amoeba, Acrasis kona, which consists of a single circular highly AT-rich (83.3%) molecule of 51.5 kb. Unexpectedly, A. kona mtDNA is missing roughly 40% of the protein-coding genes and nearly half of the transfer RNAs found in the only other sequenced heterolobosean mtDNAs, those of Naegleria spp. Instead, over a quarter of A. kona mtDNA consists of novel open reading frames. Eleven of the 16 protein-coding genes missing from A. kona mtDNA were identified in its nuclear DNA and polyA RNA, and phylogenetic analyses indicate that at least 10 of these 11 putative nuclear-encoded mitochondrial (NcMt) proteins arose by direct transfer from the mitochondrion. Acrasis kona mtDNA also employs C-to-U type RNA editing, and 12 homologs of DYW-type pentatricopeptide repeat (PPR) proteins implicated in plant organellar RNA editing are found in A. kona nuclear DNA. A mapping of mitochondrial gene content onto a consensus phylogeny reveals a sporadic pattern of relative stasis and rampant gene loss in Discoba. Rampant loss occurred independently in the unique common lineage leading to Heterolobosea + Tsukubamonadida and later in the unique lineage leading to Acrasis. Meanwhile, mtDNA gene content appears to be remarkably stable in the Acrasis sister lineage leading to Naegleria and in their distant relatives Jakobida. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Achieving a golden mean: mechanisms by which coronaviruses ensure synthesis of the correct stoichiometric ratios of viral proteins.

PubMed

Plant, Ewan P; Rakauskaite, Rasa; Taylor, Deborah R; Dinman, Jonathan D

2010-05-01

In retroviruses and the double-stranded RNA totiviruses, the efficiency of programmed -1 ribosomal frameshifting is critical for ensuring the proper ratios of upstream-encoded capsid proteins to downstream-encoded replicase enzymes. The genomic organizations of many other frameshifting viruses, including the coronaviruses, are very different, in that their upstream open reading frames encode nonstructural proteins, the frameshift-dependent downstream open reading frames encode enzymes involved in transcription and replication, and their structural proteins are encoded by subgenomic mRNAs. The biological significance of frameshifting efficiency and how the relative ratios of proteins encoded by the upstream and downstream open reading frames affect virus propagation has not been explored before. Here, three different strategies were employed to test the hypothesis that the -1 PRF signals of coronaviruses have evolved to produce the correct ratios of upstream- to downstream-encoded proteins. Specifically, infectious clones of the severe acute respiratory syndrome (SARS)-associated coronavirus harboring mutations that lower frameshift efficiency decreased infectivity by >4 orders of magnitude. Second, a series of frameshift-promoting mRNA pseudoknot mutants was employed to demonstrate that the frameshift signals of the SARS-associated coronavirus and mouse hepatitis virus have evolved to promote optimal frameshift efficiencies. Finally, we show that a previously described frameshift attenuator element does not actually affect frameshifting per se but rather serves to limit the fraction of ribosomes available for frameshifting. The findings of these analyses all support a "golden mean" model in which viruses use both programmed ribosomal frameshifting and translational attenuation to control the relative ratios of their encoded proteins.

Mitochondrial-nuclear crosstalk, haplotype and copy number variation distinct in muscle fiber type, mitochondrial respiratory and metabolic enzyme activities.

PubMed

Liu, Xuan; Trakooljul, Nares; Hadlich, Frieder; Murani, Eduard; Wimmers, Klaus; Ponsuksili, Siriluck

2017-10-25

Genes expressed in mitochondria work in concert with those expressed in the nucleus to mediate oxidative phosphorylation (OXPHOS), a process that is relevant for muscle metabolism and meat quality. Mitochondrial genome activity can be efficiently studied and compared in Duroc and Pietrain pigs, which harbor different mitochondrial haplotypes and distinct muscle fiber types, mitochondrial respiratory activities, and fat content. Pietrain pigs homozygous-positive for malignant hyperthermia susceptibility (PiPP) carried only haplotype 8 and showed the lowest absolute mtDNA copy number accompanied by a decrease transcript abundance of mitochondrial-encoded subunits ND1, ND6, and ATP6 and nuclear-encoded subunits NDUFA11 and NDUFB8. In contrast, we found that haplotype 4 of Duroc pigs had significantly higher mitochondrial DNA (mtDNA) copy numbers and an increase transcript abundance of mitochondrial-encoded subunits ND1, ND6, and ATP6. These results suggest that the variation in mitochondrial and nuclear genetic background among these animals has an effect on mitochondrial content and OXPHOS system subunit expression. We observed the co-expression pattern of mitochondrial and nuclear encoded OXPHOS subunits suggesting that the mitochondrial-nuclear crosstalk functionally involves in muscle metabolism. The findings provide valuable information for understanding muscle biology processes and energy metabolism, and may direct use for breeding strategies to improve meat quality and animal health.

Mutation in the factor VII hepatocyte nuclear factor 4α-binding site contributes to factor VII deficiency.

PubMed

Zheng, Xing-Wu; Kudaravalli, Rama; Russell, Theresa T; DiMichele, Donna M; Gibb, Constance; Russell, J Eric; Margaritis, Paris; Pollak, Eleanor S

2011-10-01

Severe coagulant factor VII (FVII) deficiency in postpubertal dizygotic twin males results from two point mutations in the FVII gene, a promoter region T→C transition at -60 and a His-to-Arg substitution at amino acid 348; both mutations prevent persistence of plasma functional FVII. This report documents longitudinal laboratory measurements from infancy to adulthood of FVII coagulant activity (FVII:C) in the twin FVII-deficient patients; it also details specific biochemical analyses of the -60 T→C mutation. The results revealed FVII:C levels of less than 1% in infancy that remain severely decreased through puberty and into adulthood. In-vitro analyses utilizing hepatocyte nuclear factor 4α (HNF4α) co-transfection and a chromatin immunoprecipitation assay indicate that the -60 T→C mutation severely diminishes functional interaction between the FVII promoter and transcription factor HNF4α. The importance of interaction between the FVII gene and HNF4α in normal FVII expression provides an in-vivo illustration of the regulated expression of an autosomal gene encoding a coagulation protein. The constancy of FVII:C and peripubertal patient symptomatology reported here illustrates androgen-independent expression in contrast to expression with an analogous mutation in the promoter region of the gene encoding coagulation FIX.

Genome Analysis Reveals Interplay between 5′UTR Introns and Nuclear mRNA Export for Secretory and Mitochondrial Genes

PubMed Central

Cenik, Can; Chua, Hon Nian; Zhang, Hui; Tarnawsky, Stefan P.; Akef, Abdalla; Derti, Adnan; Tasan, Murat; Moore, Melissa J.; Palazzo, Alexander F.; Roth, Frederick P.

2011-01-01

In higher eukaryotes, messenger RNAs (mRNAs) are exported from the nucleus to the cytoplasm via factors deposited near the 5′ end of the transcript during splicing. The signal sequence coding region (SSCR) can support an alternative mRNA export (ALREX) pathway that does not require splicing. However, most SSCR–containing genes also have introns, so the interplay between these export mechanisms remains unclear. Here we support a model in which the furthest upstream element in a given transcript, be it an intron or an ALREX–promoting SSCR, dictates the mRNA export pathway used. We also experimentally demonstrate that nuclear-encoded mitochondrial genes can use the ALREX pathway. Thus, ALREX can also be supported by nucleotide signals within mitochondrial-targeting sequence coding regions (MSCRs). Finally, we identified and experimentally verified novel motifs associated with the ALREX pathway that are shared by both SSCRs and MSCRs. Our results show strong correlation between 5′ untranslated region (5′UTR) intron presence/absence and sequence features at the beginning of the coding region. They also suggest that genes encoding secretory and mitochondrial proteins share a common regulatory mechanism at the level of mRNA export. PMID:21533221

The Varicella-zoster virus DNA encapsidation genes: Identification and characterization of the putative terminase subunits

PubMed Central

Visalli, Robert J.; Nicolosi, Denise M.; Irven, Karen L.; Goshorn, Bradley; Khan, Tamseel; Visalli, Melissa A.

2007-01-01

The putative DNA encapsidation genes encoded by open reading frames (ORFs) 25, 26, 30, 34, 43, 45/42 and 54 were cloned from Varicella-zoster virus (VZV) strain Ellen. Sequencing revealed that the Ellen ORFs were highly conserved at the amino acid level when compared to those of nineteen previously published VZV isolates. Additionally, RT-PCR provided the first evidence that ORF45/42 was expressed as a spliced transcript in VZV-infected cells. All seven ORFs were expressed in vitro and full length products were identified using a C-terminal V5 epitope tag. The in vitro products of the putative VZV terminase subunits encoded by ORFs 30 and 45/42 proved useful in protein-protein interaction assays. Previous studies have reported the formation of a heterodimeric terminase complex involved in DNA encapsidation for both herpes simplex virus-type 1 (HSV-1) and human cytomegalovirus (HCMV). Here we report that the C-terminal portion of exon II of ORF45/42 (ORF42-C269) interacted in GST-pull down experiments with in vitro synthesized ORF30 and ORF45/42. The interactions were maintained in the presence of anionic detergents and in buffers of increasing ionic strength. Cells transiently transfected with epitope tagged ORF45/42 or ORF30 showed primarily cytoplasmic staining. In contrast, an antiserum directed to the N-terminal portion of ORF45 showed nearly exclusive nuclear localization of the ORF45/42 gene product in infected cells. An ORF30 specific antiserum detected an 87 kDa protein in both the cytoplasmic and nuclear fractions of VZV infected cells. The results were consistent with the localization and function of herpesviral terminase subunits. This is the first study aimed at the identification and characterization of the VZV DNA encapsidation gene products. PMID:17868947

Elevated progranulin contributes to synaptic and learning deficit due to loss of fragile X mental retardation protein.

PubMed

Zhang, Kun; Li, Yu-Jiao; Guo, Yanyan; Zheng, Kai-Yin; Yang, Qi; Yang, Le; Wang, Xin-Shang; Song, Qian; Chen, Tao; Zhuo, Min; Zhao, Ming-Gao

2017-12-01

Fragile X syndrome is an inheritable form of intellectual disability caused by loss of fragile X mental retardation protein (FMRP, encoded by the FMR1 gene). Absence of FMRP caused overexpression of progranulin (PGRN, encoded by GRN), a putative tumour necrosis factor receptor ligand. In the present study, we found that progranulin mRNA and protein were upregulated in the medial prefrontal cortex of Fmr1 knock-out mice. In Fmr1 knock-out mice, elevated progranulin caused insufficient dendritic spine pruning and late-phase long-term potentiation in the medial prefrontal cortex of Fmr1 knock-out mice. Partial progranulin knock-down restored spine morphology and reversed behavioural deficits, including impaired fear memory, hyperactivity, and motor inflexibility in Fmr1 knock-out mice. Progranulin increased levels of phosphorylated glutamate ionotropic receptor GluA1 and nuclear factor kappa B in cultured wild-type neurons. Tumour necrosis factor receptor 2 antibody perfusion blocked the effects of progranulin on GluA1 phosphorylation; this result indicates that tumour necrosis factor receptor 2 is required for progranulin-mediated GluA1 phosphorylation and late-phase long-term potentiation expression. However, high basal level of progranulin in Fmr1 knock-out mice prevented further facilitation of synaptic plasticity by exogenous progranulin. Partial downregulation of progranulin or tumour necrosis factor receptor 2/nuclear factor kappa B signalling restored synaptic plasticity and memory deficits in Fmr1 knock-out mice. These findings suggest that elevated PGRN is linked to cognitive deficits of fragile X syndrome, and the progranulin/tumour necrosis factor receptor 2 signalling pathway may be a putative therapeutic target for improving cognitive deficits in fragile X syndrome. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Characterization of SIS1, a Saccharomyces cerevisiae homologue of bacterial dnaJ proteins

PubMed Central

1991-01-01

The Saccharomyces cerevisiae SIS1 gene was identified as a high copy number suppressor of the slow growth phenotype of strains containing mutations in the SIT4 gene, which encodes a predicted serine/threonine protein phosphatase. The SIS1 protein is similar to bacterial dnaJ proteins in the amino-terminal third and carboxyl-terminal third of the proteins. In contrast, the middle third of SIS1 is not similar to dnaJ proteins. This region of SIS1 contains a glycine/methionine-rich region which, along with more amino-terminal sequences, is required for SIS1 to associate with a protein of apparent molecular mass of 40 kD. The SIS1 gene is essential. Strains limited for the SIS1 protein accumulate cells that appear blocked for migration of the nucleus from the mother cell into the daughter cell. In addition, many of the cells become very large and contain a large vacuole. The SIS1 protein is localized throughout the cell but is more concentrated at the nucleus. About one- fourth of the SIS1 protein is released from a nuclear fraction upon treatment with RNase. We also show that overexpression of YDJ1, another yeast protein with similarity to bacterial dnaJ proteins, can not substitute for SIS1. PMID:1714460

Expression and testing in plants of ArcLight, a genetically-encoded voltage indicator used in neuroscience research.

PubMed

Matzke, Antonius J M; Matzke, Marjori

2015-10-12

It is increasingly appreciated that electrical controls acting at the cellular and supra-cellular levels influence development and initiate rapid responses to environmental cues. An emerging method for non-invasive optical imaging of electrical activity at cell membranes uses genetically-encoded voltage indicators (GEVIs). Developed by neuroscientists to chart neuronal circuits in animals, GEVIs comprise a fluorescent protein that is fused to a voltage-sensing domain. One well-known GEVI, ArcLight, undergoes strong shifts in fluorescence intensity in response to voltage changes in mammalian cells. ArcLight consists of super-ecliptic (SE) pHluorin (pH-sensitive fluorescent protein) with an A227D substitution, which confers voltage sensitivity in neurons, fused to the voltage-sensing domain of the voltage-sensing phosphatase of C iona i ntestinalis (Ci-VSD). In an ongoing effort to adapt tools of optical electrophysiology for plants, we describe here the expression and testing of ArcLight and various derivatives in different membranes of root cells in Arabidopsis thaliana. Transgenic constructs were designed to express ArcLight and various derivatives targeted to the plasma membrane and nuclear membranes of Arabidopsis root cells. In transgenic seedlings, changes in fluorescence intensity of these reporter proteins following extracellular ATP (eATP) application were monitored using a fluorescence microscope equipped with a high speed camera. Coordinate reductions in fluorescence intensity of ArcLight and Ci-VSD-containing derivatives were observed at both the plasma membrane and nuclear membranes following eATP treatments. However, similar responses were observed for derivatives lacking the Ci-VSD. The dispensability of the Ci-VSD suggests that in plants, where H(+) ions contribute substantially to electrical activities, the voltage-sensing ability of ArcLight is subordinate to the pH sensitivity of its SEpHluorin base. The transient reduction of ArcLight fluorescence triggered by eATP most likely reflects changes in pH and not membrane voltage. The pH sensitivity of ArcLight precludes its use as a direct sensor of membrane voltage in plants. Nevertheless, ArcLight and derivatives situated in the plasma membrane and nuclear membranes may offer robust, fluorescence intensity-based pH indicators for monitoring concurrent changes in pH at these discrete membrane systems. Such tools will assist analyses of pH as a signal and/or messenger at the cell surface and the nuclear periphery in living plants.

Human AZU-1 gene, variants thereof and expressed gene products

DOEpatents

Chen, Huei-Mei; Bissell, Mina

2004-06-22

A human AZU-1 gene, mutants, variants and fragments thereof. Protein products encoded by the AZU-1 gene and homologs encoded by the variants of AZU-1 gene acting as tumor suppressors or markers of malignancy progression and tumorigenicity reversion. Identification, isolation and characterization of AZU-1 and AZU-2 genes localized to a tumor suppressive locus at chromosome 10q26, highly expressed in nonmalignant and premalignant cells derived from a human breast tumor progression model. A recombinant full length protein sequences encoded by the AZU-1 gene and nucleotide sequences of AZU-1 and AZU-2 genes and variant and fragments thereof. Monoclonal or polyclonal antibodies specific to AZU-1, AZU-2 encoded protein and to AZU-1, or AZU-2 encoded protein homologs.

Interactive Effects of Dietary Lipid and Phenotypic Feed Efficiency on the Expression of Nuclear and Mitochondrial Genes Involved in the Mitochondrial Electron Transport Chain in Rainbow Trout

PubMed Central

Eya, Jonathan C.; Ukwuaba, Vitalis O.; Yossa, Rodrigue; Gannam, Ann L.

2015-01-01

A 2 × 3 factorial study was conducted to evaluate the effects of dietary lipid level on the expression of mitochondrial and nuclear genes involved in electron transport chain in all-female rainbow trout Oncorhynchus mykiss. Three practical diets with a fixed crude protein content of 40%, formulated to contain 10% (40/10), 20% (40/20) and 30% (40/30) dietary lipid, were fed to apparent satiety to triplicate groups of either low-feed efficient (F120; 217.66 ± 2.24 g initial average mass) or high-feed efficient (F136; 205.47 ± 1.27 g) full-sib families of fish, twice per day, for 90 days. At the end of the experiment, the results showed that there is an interactive effect of the dietary lipid levels and the phenotypic feed efficiency (growth rate and feed efficiency) on the expression of the mitochondrial genes nd1 (NADH dehydrogenase subunit 1), cytb (Cytochrome b), cox1 (Cytochrome c oxidase subunits 1), cox2 (Cytochrome c oxidase subunits 2) and atp6 (ATP synthase subunit 6) and nuclear genes ucp2α (uncoupling proteins 2 alpha), ucp2β (uncoupling proteins 2 beta), pparα (peroxisome proliferator-activated receptor alpha), pparβ (peroxisome proliferatoractivated receptor beta) and ppargc1α (proliferator-activated receptor gamma coactivator 1 alpha) in fish liver, intestine and muscle, except on ppargc1α in the muscle which was affected by the diet and the family separately. Also, the results revealed that the expression of mitochondrial genes is associated with that of nuclear genes involved in electron transport chain in fish liver, intestine and muscle. Furthermore, this work showed that the expression of mitochondrial genes parallels with the expression of genes encoding uncoupling proteins (UCP) in the liver and the intestine of rainbow trout. This study for the first time presents the molecular basis of the effects of dietary lipid level on mitochondrial and nuclear genes involved in mitochondrial electron transport chain in fish. PMID:25853266

The Bean pod mottle virus RNA2-encoded 58-kilodalton protein P58 is required in cis for RNA2 accumulation

USDA-ARS?s Scientific Manuscript database

Bean pod mottle virus (BPMV) is a bipartite, positive sense (+) RNA plant virus in the Secoviridae family. Its RNA1 encodes proteins required for genome replication, whereas RNA2 primarily encodes proteins needed for virion assembly and cell-to-cell movement. However, the function of a 58 kilo-dalto...

Characterization of the 5’- and 3’-terminal subgenomic RNAs produced by a capillovirus: evidence for a CP subgenomic RNA

USDA-ARS?s Scientific Manuscript database

The members of Capillovirus genus encode two overlapping open reading frames (ORFs): ORF1 encodes a large polyprotein containing the domains of replication-associated proteins plus a coat protein (CP), and ORF2 encodes a movement protein, located within ORF1 in a different reading frame. Organizatio...

Protection of Mice from Fatal Measles Encephalitis by Vaccination with Vaccinia Virus Recombinants Encoding Either the Hemagglutinin or the Fusion Protein

NASA Astrophysics Data System (ADS)

Drillien, Robert; Spehner, Daniele; Kirn, Andre; Giraudon, Pascale; Buckland, Robin; Wild, Fabian; Lecocq, Jean-Pierre

1988-02-01

Vaccinia virus recombinants encoding the hemagglutinin or fusion protein of measles virus have been constructed. Infection of cell cultures with the recombinants led to the synthesis of authentic measles proteins as judged by their electrophoretic mobility, recognition by antibodies, glycosylation, proteolytic cleavage, and presentation on the cell surface. Mice vaccinated with a single dose of the recombinant encoding the hemagglutinin protein developed antibodies capable of both inhibiting hemagglutination activity and neutralizing measles virus, whereas animals vaccinated with the recombinant encoding the fusion protein developed measles neutralizing antibodies. Mice vaccinated with either of the recombinants resisted a normally lethal intracerebral inoculation of a cell-associated measles virus subacute sclerosing panencephalitis strain.

Cytochrome b5 gene and protein of Candida tropicalis and methods relating thereto

DOEpatents

Craft, David L.; Madduri, Krishna M.; Loper, John C.

2003-01-01

A novel gene has been isolated which encodes cytochrome b5 (CYTb5) protein of the .omega.-hydroxylase complex of C. tropicalis 20336. Vectors including this gene, and transformed host cells are provided. Methods of increasing the production of a CYTb5 protein are also provided which involve transforming a host cell with a gene encoding this protein and culturing the cells. Methods of increasing the production of a dicarboxylic acid are also provided which involve increasing in the host cell the number of genes encoding this protein.

Investigating the Production of Foreign Membrane Proteins in Tobacco Chloroplasts: Expression of an Algal Plastid Terminal Oxidase

PubMed Central

Ahmad, Niaz; Michoux, Franck; Nixon, Peter J.

2012-01-01

Chloroplast transformation provides an inexpensive, easily scalable production platform for expression of recombinant proteins in plants. However, this technology has been largely limited to the production of soluble proteins. Here we have tested the ability of tobacco chloroplasts to express a membrane protein, namely plastid terminal oxidase 1 from the green alga Chlamydomonas reinhardtii (Cr-PTOX1), which is predicted to function as a plastoquinol oxidase. A homoplastomic plant containing a codon-optimised version of the nuclear gene encoding PTOX1, driven by the 16S rRNA promoter and 5′UTR of gene 10 from phage T7, was generated using a particle delivery system. Accumulation of Cr-PTOX1 was shown by immunoblotting and expression in an enzymatically active form was confirmed by using chlorophyll fluorescence to measure changes in the redox state of the plastoquinone pool in leaves. Growth of Cr-PTOX1 expressing plants was, however, more sensitive to high light than WT. Overall our results confirm the feasibility of using plastid transformation as a means of expressing foreign membrane proteins in the chloroplast. PMID:22848578

Characterization of L1 ORF1p self-interaction and cellular localization using a mammalian two-hybrid system.

PubMed

Sokolowski, Mark; deHaro, Dawn; Christian, Claiborne M; Kines, Kristine J; Belancio, Victoria P

2013-01-01

Long INterspersed Element-1 (LINE-1, L1) is an active retrotransposon that mobilizes using a ribonucleoprotein particle (RNP) intermediate composed of the full-length bicistronic L1 mRNA and the two proteins (ORF1p and ORF2p) encoded by that mRNA. ORF1p and ORF2p demonstrate cis-preference for their encoding mRNA. Previous studies of ORF1p, purified from bacterial and insect cells demonstrated that this protein forms trimers in vitro. While valuable for understanding ORF1p function, these in vitro approaches do not provide any information on ORF1p self-interaction in the context of mammalian cells. We used a mammalian two-hybrid (M2H) system in order to study L1 ORF1p self-interaction in human and mouse cells. We demonstrate that the M2H system successfully detects human and mouse ORF1p self-interactions in transiently transfected mammalian cells. We also generated mouse and human ORF1p-specific antibodies to characterize the expression of ORF1p fusion proteins used in the M2H system. Using these antibodies, we demonstrate that ORF1p interaction in trans leads to the formation of heterodimers that are expected to produce a positive signal in the M2H system. Although the role for L1 ORF1p cis-preference in L1 mobilization is established, the impact of ability of ORF1pto interact in trans on the L1 replication cycle is not known. Furthermore, western blot analysis of ORF1p generated by a full-length L1, wild type ORF1, or a codon-optimized ORF1 expression vector is detected in the nucleus. In contrast, the addition of a tag to the N-terminus of the mouse and human ORF1 proteins can significantly alter the subcellular localization in a tag-specific manner. These data support that nuclear localization of ORF1p may contribute to L1 (and potentially the SINE Alu) RNP nuclear access in the host cell.

Molecular characterization and expression of the M6 gene of grass carp hemorrhage virus (GCHV), an aquareovirus.

PubMed

Qiu, T; Lu, R H; Zhang, J; Zhu, Z Y

2001-07-01

The complete nucleotide sequence of M6 gene of grass carp hemorrhage virus (GCHV) was determined. It is 2039 nucleotides in length and contains a single large open reading frame that could encode a protein of 648 amino acids with predicted molecular mass of 68.7 kDa. Amino acid sequence comparison revealed that the protein encoded by GCHV M6 is closely related to the protein mu1 of mammalian reovirus. The M6 gene, encoding the major outer-capsid protein, was expressed using the pET fusion protein vector in Escherichia coli and detected by Western blotting using chicken anti-GCHV immunoglobulin (IgY). The result indicates that the protein encoded by M6 may share a putative Asn-42-Pro-43 proteolytic cleavage site with mu1.

DNA encoding a DNA repair protein

DOEpatents

Petrini, John H.; Morgan, William Francis; Maser, Richard Scott; Carney, James Patrick

2006-08-15

An isolated and purified DNA molecule encoding a DNA repair protein, p95, is provided, as is isolated and purified p95. Also provided are methods of detecting p95 and DNA encoding p95. The invention further provides p95 knock-out mice.

Challenges in NMR-based structural genomics

NASA Astrophysics Data System (ADS)

Sue, Shih-Che; Chang, Chi-Fon; Huang, Yao-Te; Chou, Ching-Yu; Huang, Tai-huang

2005-05-01

Understanding the functions of the vast number of proteins encoded in many genomes that have been completely sequenced recently is the main challenge for biologists in the post-genomics era. Since the function of a protein is determined by its exact three-dimensional structure it is paramount to determine the 3D structures of all proteins. This need has driven structural biologists to undertake the structural genomics project aimed at determining the structures of all known proteins. Several centers for structural genomics studies have been established throughout the world. Nuclear magnetic resonance (NMR) spectroscopy has played a major role in determining protein structures in atomic details and in a physiologically relevant solution state. Since the number of new genes being discovered daily far exceeds the number of structures determined by both NMR and X-ray crystallography, a high-throughput method for speeding up the process of protein structure determination is essential for the success of the structural genomics effort. In this article we will describe NMR methods currently being employed for protein structure determination. We will also describe methods under development which may drastically increase the throughput, as well as point out areas where opportunities exist for biophysicists to make significant contribution in this important field.