mRNA–mRNA duplexes that auto-elicit Staufen1-mediated mRNA decay

PubMed Central

Gong, Chenguang; Tang, Yalan; Maquat, Lynne E.

2013-01-01

We report a new mechanism by which human mRNAs crosstalk: an Alu element in the 3'-untranslated region (3' UTR) of one mRNA can base-pair with a partially complementary Alu element in the 3' UTR of a different mRNA thereby creating a Staufen1 (STAU1)-binding site (SBS). STAU1 binding to a 3' UTR SBS was previously shown to trigger STAU1-mediated mRNA decay (SMD) by directly recruiting the ATP-dependent RNA helicase UPF1, which is also a key factor in the mechanistically related nonsense-mediated mRNA decay (NMD) pathway. In the case of a 3' UTR SBS created via mRNA–mRNA base-pairing, we show that SMD targets both mRNAs in the duplex provided that both mRNAs are translated. If only one mRNA is translated, then it alone is targeted for SMD. We demonstrate the importance of mRNA–mRNA-triggered SMD to the processes of cell migration and invasion. PMID:24056942

Glucocorticoid receptor interacts with PNRC2 in a ligand-dependent manner to recruit UPF1 for rapid mRNA degradation.

PubMed

Cho, Hana; Park, Ok Hyun; Park, Joori; Ryu, Incheol; Kim, Jeonghan; Ko, Jesang; Kim, Yoon Ki

2015-03-31

Glucocorticoid receptor (GR), which was originally known to function as a nuclear receptor, plays a role in rapid mRNA degradation by acting as an RNA-binding protein. The mechanism by which this process occurs remains unknown. Here, we demonstrate that GR, preloaded onto the 5'UTR of a target mRNA, recruits UPF1 through proline-rich nuclear receptor coregulatory protein 2 (PNRC2) in a ligand-dependent manner, so as to elicit rapid mRNA degradation. We call this process GR-mediated mRNA decay (GMD). Although GMD, nonsense-mediated mRNA decay (NMD), and staufen-mediated mRNA decay (SMD) share upstream frameshift 1 (UPF1) and PNRC2, we find that GMD is mechanistically distinct from NMD and SMD. We also identify de novo cellular GMD substrates using microarray analysis. Intriguingly, GMD functions in the chemotaxis of human monocytes by targeting chemokine (C-C motif) ligand 2 (CCL2) mRNA. Thus, our data provide molecular evidence of a posttranscriptional role of the well-studied nuclear hormone receptor, GR, which is traditionally considered a transcription factor.

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.

Inhibition and Avoidance of mRNA Degradation by RNA Viruses

PubMed Central

Moon, Stephanie L.; Barnhart, Michael D.; Wilusz, Jeffrey

2012-01-01

The cellular mRNA decay machinery plays a major role in regulating the quality and quantity of gene expression in cells. This machinery involves multiple enzymes and pathways that converge to promote the exonucleolytic decay of mRNAs. The transcripts made by RNA viruses are susceptible to degradation by this machinery and, in fact, can be actively targeted. Thus, to maintain gene expression and replication, RNA viruses have evolved a number of strategies to avoid and/or inactivate aspects of the cellular mRNA decay machinery. Recent work uncovering the mechanisms used by RNA viruses to maintain the stability of their transcripts is described below. PMID:22626865

Interrelations between translation and general mRNA degradation in yeast

PubMed Central

Huch, Susanne; Nissan, Tracy

2014-01-01

Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, alternatively, embark on a program of enhanced mRNA decay en masse. Finally, recent results suggest that mRNA decay is not limited to non-translating messages and can occur when ribosomes are not initiating but are still elongating on mRNA. This review will discuss the models for the mechanisms of these processes and recent developments in understanding the relationship between translation and general mRNA degradation, with a focus on yeast as a model system. How to cite this article: WIREs RNA 2014, 5:747–763. doi: 10.1002/wrna.1244 PMID:24944158

Interrelations between translation and general mRNA degradation in yeast.

PubMed

Huch, Susanne; Nissan, Tracy

2014-01-01

Messenger RNA (mRNA) degradation is an important element of gene expression that can be modulated by alterations in translation, such as reductions in initiation or elongation rates. Reducing translation initiation strongly affects mRNA degradation by driving mRNA toward the assembly of a decapping complex, leading to decapping. While mRNA stability decreases as a consequence of translational inhibition, in apparent contradiction several external stresses both inhibit translation initiation and stabilize mRNA. A key difference in these processes is that stresses induce multiple responses, one of which stabilizes mRNAs at the initial and rate-limiting step of general mRNA decay. Because this increase in mRNA stability is directly induced by stress, it is independent of the translational effects of stress, which provide the cell with an opportunity to assess its response to changing environmental conditions. After assessment, the cell can store mRNAs, reinitiate their translation or, alternatively, embark on a program of enhanced mRNA decay en masse. Finally, recent results suggest that mRNA decay is not limited to non-translating messages and can occur when ribosomes are not initiating but are still elongating on mRNA. This review will discuss the models for the mechanisms of these processes and recent developments in understanding the relationship between translation and general mRNA degradation, with a focus on yeast as a model system. © 2014 The Authors. WIREs RNA published by John Wiley & Sons, Ltd.

The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation.

PubMed Central

Zubiaga, A M; Belasco, J G; Greenberg, M E

1995-01-01

Labile mRNAs that encode cytokine and immediate-early gene products often contain AU-rich sequences within their 3' untranslated region (UTR). These AU-rich sequences appear to be key determinants of the short half-lives of these mRNAs, although the sequence features of these elements and the mechanism by which they target mRNAs for rapid decay have not been fully defined. We have examined the features of AU-rich elements (AREs) that are crucial for their function as determinants of mRNA instability in mammalian cells by testing the ability of various mutant c-fos AREs and synthetic AREs to direct rapid mRNA deadenylation and decay when inserted within the 3' UTR of the normally stable beta-globin mRNA. Evidence is presented that the pentamer AUUUA, which previously was suggested to be the minimal determinant of instability present in mammalian AREs, cannot direct rapid mRNA deadenylation and decay. Instead, the nonomer UUAUUUAUU is the elemental AU-rich sequence motif that destabilizes mRNA. Removal of one uridine residue from either end of the nonamer (UUAUUUAU or UAUUUAUU) results in a decrease of potency of the element, while removal of a uridine residue from both ends of the nonamer (UAUUUAU) eliminates detectable destabilizing activity. The inclusion of an additional uridine residue at both ends of the nonamer (UUUAUUUAUUU) does not further increase the efficacy of the element. Taken together, these findings suggest that the nonamer UUAUUUAUU is the minimal AU-rich motif that effectively destabilizes mRNA. Additional ARE potency is achieved by combining multiple copies of this nonamer in a single mRNA 3' UTR. Furthermore, analysis of poly(A) shortening rates for ARE-containing mRNAs reveals that the UUAUUUAUU sequence also accelerates mRNA deadenylation and suggests that the UUAUUUAUU motif targets mRNA for rapid deadenylation as an early step in the mRNA decay process. PMID:7891716

Regulation of p21/CIP1/WAF-1 mediated cell-cycle arrest by RNase L and tristetraprolin, and involvement of AU-rich elements

PubMed Central

Al-Haj, Latifa; Blackshear, Perry J.; Khabar, Khalid S.A.

2012-01-01

The p21Cip1/WAF1 plays an important role in cell-cycle arrest. Here, we find that RNase L regulates p21-mediated G1 growth arrest in AU-rich elements-dependent manner. We found a significant loss of p21 mRNA expression in RNASEL−/− MEFs and that the overexpression of RNase L in HeLa cells induces p21 mRNA expression. The p21 mRNA half-life significantly changes as a result of RNase L modulation, indicating a post-transcriptional effect. Indeed, we found that RNase L promotes tristetraprolin (TTP/ZFP36) mRNA decay. This activity was not seen with dimerization- and nuclease-deficient RNase L mutants. Deficiency in TTP led to increases in p21 mRNA and protein. With induced ablation of RNase L, TTP mRNA and protein expressions were higher, while p21 expression became reduced. We further establish that TTP, but not C124R TTP mutant, binds to, and accelerates the decay of p21 mRNA. The p21 mRNA half-life was prolonged in TTP−/− MEFs. The TTP regulation of p21 mRNA decay required functional AU-rich elements. Thus, we demonstrate a novel mechanism of regulating G1 growth arrest by an RNase L-TTP-p21 axis. PMID:22718976

Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA*

PubMed Central

Woo, Ho-Hyung; Baker, Terri; Laszlo, Csaba; Chambers, Setsuko K.

2013-01-01

CSF-1 mRNA 3′UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3′UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3′UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of nucleolin's actions on CSF-1 mRNA and describe the dependence of miR-130a- and miR-301a-directed CSF-1 mRNA decay and inhibition of ovarian cancer cell motility on nucleolin. PMID:23471483

Universal Poisson Statistics of mRNAs with Complex Decay Pathways.

PubMed

Thattai, Mukund

2016-01-19

Messenger RNA (mRNA) dynamics in single cells are often modeled as a memoryless birth-death process with a constant probability per unit time that an mRNA molecule is synthesized or degraded. This predicts a Poisson steady-state distribution of mRNA number, in close agreement with experiments. This is surprising, since mRNA decay is known to be a complex process. The paradox is resolved by realizing that the Poisson steady state generalizes to arbitrary mRNA lifetime distributions. A mapping between mRNA dynamics and queueing theory highlights an identifiability problem: a measured Poisson steady state is consistent with a large variety of microscopic models. Here, I provide a rigorous and intuitive explanation for the universality of the Poisson steady state. I show that the mRNA birth-death process and its complex decay variants all take the form of the familiar Poisson law of rare events, under a nonlinear rescaling of time. As a corollary, not only steady-states but also transients are Poisson distributed. Deviations from the Poisson form occur only under two conditions, promoter fluctuations leading to transcriptional bursts or nonindependent degradation of mRNA molecules. These results place severe limits on the power of single-cell experiments to probe microscopic mechanisms, and they highlight the need for single-molecule measurements. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Staufen-mediated mRNA decay

PubMed Central

Park, Eonyoung; Maquat, Lynne E.

2013-01-01

Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base-pairing of 3'UTR sequences or by intermolecular base-pairing of 3'UTR sequences with a long noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Since both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1, SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms. PMID:23681777

Regulated mRNA Decay in Arabidopsis: A global analysis of differential control by hormones and the circadian clock

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

Green, Pamela J.

The long-term goal of this research was to better understand the influence of mRNA stability on gene regulation, particularly in response to hormones and the circadian clock. The primary aim of this project was to examine this using DNA microarrays, small RNA analysis and other approaches. We accomplished these objectives, although we were only able to detect small changes in mRNA stability in response to these stimuli. However, the work also contributed to a major breakthrough allowing the identification of small RNAs on a genomic scale in eukaryotes. Moreover, the project prompted us to develop a new way to analyzemore » mRNA decay genome wide. Thus, the research was hugely successful beyond our objectives.« less

Organizing Principles of Mammalian Nonsense-Mediated mRNA Decay

PubMed Central

Popp, Maximilian Wei-Lin; Maquat, Lynne E.

2014-01-01

Cells use messenger RNAs (mRNAs) to ensure the accurate dissemination of genetic information encoded by DNA. Given that mRNAs largely direct the synthesis of a critical effector of cellular phenotype, i.e., proteins, tight regulation of both the quality and quantity of mRNA is a prerequisite for effective cellular homeostasis. Here, we review nonsense-mediated mRNA decay (NMD), which is the best-characterized posttranscriptional quality control mechanism that cells have evolved in their cytoplasm to ensure transcriptome fidelity. We use protein quality control as a conceptual framework to organize what is known about NMD, highlighting overarching similarities between these two polymer quality control pathways, where the protein quality control and NMD pathways intersect, and how protein quality control can suggest new avenues for research into mRNA quality control. PMID:24274751

Staufen-mediated mRNA decay.

PubMed

Park, Eonyoung; Maquat, Lynne E

2013-01-01

Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'-UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base pairing of 3'-UTR sequences or by intermolecular base pairing of 3'-UTR sequences with a long-noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Because both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1; SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms. Copyright © 2013 John Wiley & Sons, Ltd.

Chromatoid Body Protein TDRD6 Supports Long 3’ UTR Triggered Nonsense Mediated mRNA Decay

PubMed Central

Fanourgakis, Grigorios; Akpinar, Müge; Dahl, Andreas; Jessberger, Rolf

2016-01-01

Chromatoid bodies (CBs) are spermiogenesis-specific organelles of largely unknown function. CBs harbor various RNA species, RNA-associated proteins and proteins of the tudor domain family like TDRD6, which is required for a proper CB architecture. Proteome analysis of purified CBs revealed components of the nonsense-mediated mRNA decay (NMD) machinery including UPF1. TDRD6 is essential for UPF1 localization to CBs, for UPF1-UPF2 and UPF1-MVH interactions. Upon removal of TDRD6, the association of several mRNAs with UPF1 and UPF2 is disturbed, and the long 3’ UTR-stimulated but not the downstream exon-exon junction triggered pathway of NMD is impaired. Reduced association of the long 3’ UTR mRNAs with UPF1 and UPF2 correlates with increased stability and enhanced translational activity. Thus, we identified TDRD6 within CBs as required for mRNA degradation, specifically the extended 3’ UTR-triggered NMD pathway, and provide evidence for the requirement of NMD in spermiogenesis. This function depends on TDRD6-promoted assembly of mRNA and decay enzymes in CBs. PMID:27149095

Somatic Mutation Patterns in Hemizygous Genomic Regions Unveil Purifying Selection during Tumor Evolution

PubMed Central

Basu, Swaraj; Larsson, Erik

2016-01-01

Identification of cancer driver genes using somatic mutation patterns indicative of positive selection has become a major goal in cancer genomics. However, cancer cells additionally depend on a large number of genes involved in basic cellular processes. While such genes should in theory be subject to strong purifying (negative) selection against damaging somatic mutations, these patterns have been elusive and purifying selection remains inadequately explored in cancer. Here, we hypothesized that purifying selection should be evident in hemizygous genomic regions, where damaging mutations cannot be compensated for by healthy alleles. Using a 7,781-sample pan-cancer dataset, we first confirmed this in POLR2A, an essential gene where hemizygous deletions are known to confer elevated sensitivity to pharmacological suppression. We next used this principle to identify several genes and pathways that show patterns indicative of purifying selection to avoid deleterious mutations. These include the POLR2A interacting protein INTS10 as well as genes involved in mRNA splicing, nonsense-mediated mRNA decay and other RNA processing pathways. Many of these genes belong to large protein complexes, and strong overlaps were observed with recent functional screens for gene essentiality in human cells. Our analysis supports that purifying selection acts to preserve the remaining function of many hemizygously deleted essential genes in tumors, indicating vulnerabilities that might be exploited by future therapeutic strategies. PMID:28027311

Heat Shock Response in Yeast Involves Changes in Both Transcription Rates and mRNA Stabilities

PubMed Central

Castells-Roca, Laia; García-Martínez, José; Moreno, Joaquín; Herrero, Enrique; Bellí, Gemma; Pérez-Ortín, José E.

2011-01-01

We have analyzed the heat stress response in the yeast Saccharomyces cerevisiae by determining mRNA levels and transcription rates for the whole transcriptome after a shift from 25°C to 37°C. Using an established mathematical algorithm, theoretical mRNA decay rates have also been calculated from the experimental data. We have verified the mathematical predictions for selected genes by determining their mRNA decay rates at different times during heat stress response using the regulatable tetO promoter. This study indicates that the yeast response to heat shock is not only due to changes in transcription rates, but also to changes in the mRNA stabilities. mRNA stability is affected in 62% of the yeast genes and it is particularly important in shaping the mRNA profile of the genes belonging to the environmental stress response. In most cases, changes in transcription rates and mRNA stabilities are homodirectional for both parameters, although some interesting cases of antagonist behavior are found. The statistical analysis of gene targets and sequence motifs within the clusters of genes with similar behaviors shows that both transcriptional and post-transcriptional regulons apparently contribute to the general heat stress response by means of transcriptional factors and RNA binding proteins. PMID:21364882

Stabilization and cytoskeletal-association of LDL receptor mRNA are mediated by distinct domains in its 3' untranslated region.

PubMed

Wilson, G M; Vasa, M Z; Deeley, R G

1998-05-01

The mRNA encoding the human low density lipoprotein (LDL) receptor is transiently stabilized after phorbol ester treatment of HepG2 cells and has been shown to associate with components of the cytoskeleton in this cell line (G. M. Wilson, E. A. Roberts, and R. G. Deeley, J. Lipid Res. 1997. 38: 437-446). Using an episomal expression system, fragments of the 3' untranslated region (3'UTR) of LDL receptor mRNA were transcribed in fusion with the coding region of beta-globin mRNA in HepG2 cells. Analyses of the decay kinetics of these beta-globin-LDL receptor fusion mRNA deletion mutants showed that sequences in the proximal 3'UTR of LDL receptor mRNA including several AU-rich elements (AREs) were sufficient to confer short constitutive mRNA half-life in the heterologous system. Stabilization of LDL receptor mRNA in the presence of PMA required sequences in the distal 3'UTR, at or near three Alu-like repetitive elements. Furthermore, the 3'UTR of LDL receptor mRNA conferred cytoskeletal association on the otherwise unassociated beta-globin mRNA, by a mechanism involving at least two distinct RNA elements. Comparisons of decay kinetics and subcellular localization of endogenous LDL receptor mRNA and beta-globin-LDL receptor mRNA fusions in HepG2 cells have demonstrated that several cis-acting elements in the receptor 3'UTR contribute to post-transcriptional regulation of receptor expression, and provide further support for involvement of the cytoskeleton in the regulation of LDL receptor mRNA turnover.

CYP3A5 mRNA degradation by nonsense-mediated mRNA decay.

PubMed

Busi, Florent; Cresteil, Thierry

2005-09-01

The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g.31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudoexon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.

Selenocysteine incorporation: A trump card in the game of mRNA decay

PubMed Central

Shetty, Sumangala P.; Copeland, Paul R.

2015-01-01

The incorporation of the 21st amino acid, selenocysteine (Sec), occurs on mRNAs that harbor in-frame stop codons because the Sec-tRNASec recognizes a UGA codon. This sets up an intriguing interplay between translation elongation, translation termination and the complex machinery that marks mRNAs that contain premature termination codons for degradation, leading to nonsense mediated mRNA decay (NMD). In this review we discuss the intricate and complex relationship between this key quality control mechanism and the process of Sec incorporation in mammals. PMID:25622574

Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome

PubMed Central

ORBAN, TAMAS I.; IZAURRALDE, ELISA

2005-01-01

RNA interference (RNAi) is a conserved RNA silencing pathway that leads to sequence-specific mRNA decay in response to the presence of double-stranded RNA (dsRNA). Long dsRNA molecules are first processed by Dicer into 21–22-nucleotide small interfering RNAs (siRNAs). The siRNAs are incorporated into a multimeric RNA-induced silencing complex (RISC) that cleaves mRNAs at a site determined by complementarity with the siRNAs. Following this initial endonucleolytic cleavage, the mRNA is degraded by a mechanism that is not completely understood. We investigated the decay pathway of mRNAs targeted by RISC in Drosophila cells. We show that 5′ mRNA fragments generated by RISC cleavage are rapidly degraded from their 3′ ends by the exosome, whereas the 3′ fragments are degraded from their 5′ ends by XRN1. Exosome-mediated decay of the 5′ fragments requires the Drosophila homologs of yeast Ski2p, Ski3p, and Ski8p, suggesting that their role as regulators of exosome activity is conserved. Our findings indicate that mRNAs targeted by siRNAs are degraded from the ends generated by RISC cleavage, without undergoing decapping or deadenylation. PMID:15703439

Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants

PubMed Central

Lattimore, Vanessa L.; Pearson, John F.; Currie, Margaret J.; Spurdle, Amanda B.; Robinson, Bridget A.; Walker, Logan C.

2018-01-01

PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates (n > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance. PMID:29774201

Investigation of Experimental Factors That Underlie BRCA1/2 mRNA Isoform Expression Variation: Recommendations for Utilizing Targeted RNA Sequencing to Evaluate Potential Spliceogenic Variants.

PubMed

Lattimore, Vanessa L; Pearson, John F; Currie, Margaret J; Spurdle, Amanda B; Robinson, Bridget A; Walker, Logan C

2018-01-01

PCR-based RNA splicing assays are commonly used in diagnostic and research settings to assess the potential effects of variants of uncertain clinical significance in BRCA1 and BRCA2 . The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium completed a multicentre investigation to evaluate differences in assay design and the integrity of published data, raising a number of methodological questions associated with cell culture conditions and PCR-based protocols. We utilized targeted RNA-seq to re-assess BRCA1 and BRCA2 mRNA isoform expression patterns in lymphoblastoid cell lines (LCLs) previously used in the multicentre ENIGMA study. Capture of the targeted cDNA sequences was carried out using 34 BRCA1 and 28 BRCA2 oligonucleotides from the Illumina Truseq Targeted RNA Expression platform. Our results show that targeted RNA-seq analysis of LCLs overcomes many of the methodology limitations associated with PCR-based assays leading us to make the following observations and recommendations: (1) technical replicates ( n  > 2) of variant carriers to capture methodology induced variability associated with RNA-seq assays, (2) LCLs can undergo multiple freeze/thaw cycles and can be cultured up to 2 weeks without noticeably influencing isoform expression levels, (3) nonsense-mediated decay inhibitors are essential prior to splicing assays for comprehensive mRNA isoform detection, (4) quantitative assessment of exon:exon junction levels across BRCA1 and BRCA2 can help distinguish between normal and aberrant isoform expression patterns. Experimentally derived recommendations from this study will facilitate the application of targeted RNA-seq platforms for the quantitation of BRCA1 and BRCA2 mRNA aberrations associated with sequence variants of uncertain clinical significance.

Trick or TREAT: A Scary-Good New Approach for Single-Molecule mRNA Decay Analysis.

PubMed

Russo, Joseph; Wilusz, Jeffrey

2017-11-02

In this issue of Molecular Cell, Horvathova et al. (2017) have developed a powerful approach to single-molecule assessment of RNA decay in living cells by exploiting the ability of flavivirus RNA structural elements to trap XRN1 decay intermediates in dual-labeled reporter constructs. Copyright © 2017 Elsevier Inc. All rights reserved.

Exaptive origins of regulated mRNA decay in eukaryotes

PubMed Central

Hamid, Fursham M.

2016-01-01

Eukaryotic gene expression is extensively controlled at the level of mRNA stability and the mechanisms underlying this regulation are markedly different from their archaeal and bacterial counterparts. We propose that two such mechanisms, nonsense‐mediated decay (NMD) and motif‐specific transcript destabilization by CCCH‐type zinc finger RNA‐binding proteins, originated as a part of cellular defense against RNA pathogens. These branches of the mRNA turnover pathway might have been used by primeval eukaryotes alongside RNA interference to distinguish their own messages from those of RNA viruses and retrotransposable elements. We further hypothesize that the subsequent advent of “professional” innate and adaptive immunity systems allowed NMD and the motif‐triggered mechanisms to be efficiently repurposed for regulation of endogenous cellular transcripts. This scenario explains the rapid emergence of archetypical mRNA destabilization pathways in eukaryotes and argues that other aspects of post‐transcriptional gene regulation in this lineage might have been derived through a similar exaptation route. PMID:27438915

Exaptive origins of regulated mRNA decay in eukaryotes.

PubMed

Hamid, Fursham M; Makeyev, Eugene V

2016-09-01

Eukaryotic gene expression is extensively controlled at the level of mRNA stability and the mechanisms underlying this regulation are markedly different from their archaeal and bacterial counterparts. We propose that two such mechanisms, nonsense-mediated decay (NMD) and motif-specific transcript destabilization by CCCH-type zinc finger RNA-binding proteins, originated as a part of cellular defense against RNA pathogens. These branches of the mRNA turnover pathway might have been used by primeval eukaryotes alongside RNA interference to distinguish their own messages from those of RNA viruses and retrotransposable elements. We further hypothesize that the subsequent advent of "professional" innate and adaptive immunity systems allowed NMD and the motif-triggered mechanisms to be efficiently repurposed for regulation of endogenous cellular transcripts. This scenario explains the rapid emergence of archetypical mRNA destabilization pathways in eukaryotes and argues that other aspects of post-transcriptional gene regulation in this lineage might have been derived through a similar exaptation route. © 2016 The Authors BioEssays Published by WILEY Periodicals, Inc.

A mechanism for exon skipping caused by nonsense or missense mutations in BRCA1 and other genes.

PubMed

Liu, H X; Cartegni, L; Zhang, M Q; Krainer, A R

2001-01-01

Point mutations can generate defective and sometimes harmful proteins. The nonsense-mediated mRNA decay (NMD) pathway minimizes the potential damage caused by nonsense mutations. In-frame nonsense codons located at a minimum distance upstream of the last exon-exon junction are recognized as premature termination codons (PTCs), targeting the mRNA for degradation. Some nonsense mutations cause skipping of one or more exons, presumably during pre-mRNA splicing in the nucleus; this phenomenon is termed nonsense-mediated altered splicing (NAS), and its underlying mechanism is unclear. By analyzing NAS in BRCA1, we show here that inappropriate exon skipping can be reproduced in vitro, and results from disruption of a splicing enhancer in the coding sequence. Enhancers can be disrupted by single nonsense, missense and translationally silent point mutations, without recognition of an open reading frame as such. These results argue against a nuclear reading-frame scanning mechanism for NAS. Coding-region single-nucleotide polymorphisms (cSNPs) within exonic splicing enhancers or silencers may affect the patterns or efficiency of mRNA splicing, which may in turn cause phenotypic variability and variable penetrance of mutations elsewhere in a gene.

The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing.

PubMed

Makeyev, Eugene V; Zhang, Jiangwen; Carrasco, Monica A; Maniatis, Tom

2007-08-03

Both microRNAs and alternative pre-mRNA splicing have been implicated in the development of the nervous system (NS), but functional interactions between these two pathways are poorly understood. We demonstrate that the neuron-specific microRNA miR-124 directly targets PTBP1 (PTB/hnRNP I) mRNA, which encodes a global repressor of alternative pre-mRNA splicing in nonneuronal cells. Among the targets of PTBP1 is a critical cassette exon in the pre-mRNA of PTBP2 (nPTB/brPTB/PTBLP), an NS-enriched PTBP1 homolog. When this exon is skipped, PTBP2 mRNA is subject to nonsense-mediated decay (NMD). During neuronal differentiation, miR-124 reduces PTBP1 levels, leading to the accumulation of correctly spliced PTBP2 mRNA and a dramatic increase in PTBP2 protein. These events culminate in the transition from non-NS to NS-specific alternative splicing patterns. We also present evidence that miR-124 plays a key role in the differentiation of progenitor cells to mature neurons. Thus, miR-124 promotes NS development, at least in part by regulating an intricate network of NS-specific alternative splicing.

Novel features of the XRN-family in Arabidopsis: Evidence that AtXRN4, one of several orthologs of nuclear Xrn2p/Rat1p, functions in the cytoplasm

PubMed Central

Kastenmayer, J. P.; Green, P. J.

2000-01-01

The 5′-3′ exoribonucleases Xrn1p and Xrn2p/Rat1p function in the degradation and processing of several classes of RNA in Saccharomyces cerevisiae. Xrn1p is the main enzyme catalyzing cytoplasmic mRNA degradation in multiple decay pathways, whereas Xrn2p/Rat1p functions in the processing of rRNAs and small nucleolar RNAs (snoRNAs) in the nucleus. Much less is known about the XRN-like proteins of multicellular eukaryotes; however, differences in their activities could explain differences in mRNA degradation between multicellular and unicellular eukaryotes. One such difference is the lack in plants and animals of mRNA decay intermediates like those generated in yeast when Xrn1p is blocked by poly(G) tracts that are inserted within mRNAs. We investigated the XRN-family in Arabidopsis thaliana and found it to have several novel features. First, the Arabidopsis genome contains three XRN-like genes (AtXRNs) that are structurally similar to Xrn2p/Rat1p, a characteristic unique to plants. Furthermore, our experimental results and sequence database searches indicate that Xrn1p orthologs may be absent from higher plants. Second, the lack of poly(G) mRNA decay intermediates in plants cannot be explained by the activity of the AtXRNs, because they are blocked by poly(G) tracts. Finally, complementation of yeast mutants and localization studies indicate that two of the AtXRNs likely function in the nucleus, whereas the third acts in the cytoplasm. Thus, the XRN-family in plants is more complex than in other eukaryotes, and, if an XRN-like enzyme plays a role in mRNA decay in plants, the likely participant is a cytoplasmic Xrn2p/Rat1p ortholog, rather than an Xrn1p ortholog. PMID:11106401

Posttranscriptional Control of Photosynthetic mRNA Decay under Stress Conditions Requires 3′ and 5′ Untranslated Regions and Correlates with Differential Polysome Association in Rice1[W][OA

PubMed Central

Park, Su-Hyun; Chung, Pil Joong; Juntawong, Piyada; Bailey-Serres, Julia; Kim, Youn Shic; Jung, Harin; Bang, Seung Woon; Kim, Yeon-Ki; Do Choi, Yang; Kim, Ju-Kon

2012-01-01

Abiotic stress, including drought, salinity, and temperature extremes, regulates gene expression at the transcriptional and posttranscriptional levels. Expression profiling of total messenger RNAs (mRNAs) from rice (Oryza sativa) leaves grown under stress conditions revealed that the transcript levels of photosynthetic genes are reduced more rapidly than others, a phenomenon referred to as stress-induced mRNA decay (SMD). By comparing RNA polymerase II engagement with the steady-state mRNA level, we show here that SMD is a posttranscriptional event. The SMD of photosynthetic genes was further verified by measuring the half-lives of the small subunit of Rubisco (RbcS1) and Chlorophyll a/b-Binding Protein1 (Cab1) mRNAs during stress conditions in the presence of the transcription inhibitor cordycepin. To discern any correlation between SMD and the process of translation, changes in total and polysome-associated mRNA levels after stress were measured. Total and polysome-associated mRNA levels of two photosynthetic (RbcS1 and Cab1) and two stress-inducible (Dehydration Stress-Inducible Protein1 and Salt-Induced Protein) genes were found to be markedly similar. This demonstrated the importance of polysome association for transcript stability under stress conditions. Microarray experiments performed on total and polysomal mRNAs indicate that approximately half of all mRNAs that undergo SMD remain polysome associated during stress treatments. To delineate the functional determinant(s) of mRNAs responsible for SMD, the RbcS1 and Cab1 transcripts were dissected into several components. The expressions of different combinations of the mRNA components were analyzed under stress conditions, revealing that both 3′ and 5′ untranslated regions are necessary for SMD. Our results, therefore, suggest that the posttranscriptional control of photosynthetic mRNA decay under stress conditions requires both 3′ and 5′ untranslated regions and correlates with differential polysome association. PMID:22566494

Overexpression of nucleolin in chronic lymphocytic leukemia cells induces stabilization of bcl2 mRNA

PubMed Central

Otake, Yoko; Soundararajan, Sridharan; Sengupta, Tapas K.; Kio, Ebenezer A.; Smith, James C.; Pineda-Roman, Mauricio; Stuart, Robert K.; Spicer, Eleanor K.

2007-01-01

B-cell chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal B cells that are resistant to apoptosis as a result of bcl2 oncogene overexpression. Studies were done to determine the mechanism for the up-regulation of bcl-2 protein observed in CD19+ CLL cells compared with CD19+ B cells from healthy volunteers. The 11-fold higher level of bcl-2 protein in CLL cells was positively correlated with a 26-fold elevation in the cytosolic level of nucleolin, a bcl2 mRNA–stabilizing protein. Measurements of the bcl2 heterogeneous nuclear/bcl2 mRNA (hnRNA)/mRNA ratios and the rates of bcl2 mRNA decay in cell extracts indicated that the 3-fold higher steady-state level of bcl2 mRNA in CLL cells was the result of increased bcl2 mRNA stability. Nucleolin was present throughout the nucleus and cytoplasm of CLL cells, whereas in normal B cells nucleolin was only detected in the nucleus. The addition of recombinant human nucleolin to extracts of normal B cells markedly slowed the rate of bcl2 mRNA decay. SiRNA knockdown of nucleolin in MCF-7 cells resulted in decreased levels of bcl2 mRNA and protein but no change in β-actin. These results indicate that bcl-2 overexpression in CLL cells is related to stabilization of bcl2 mRNA by nucleolin. PMID:17179226

The Rtr1p CTD phosphatase autoregulates its mRNA through a degradation pathway involving the REX exonucleases

PubMed Central

Hodko, Domagoj; Ward, Taylor; Chanfreau, Guillaume

2016-01-01

Rtr1p is a phosphatase that impacts gene expression by modulating the phosphorylation status of the C-terminal domain of the large subunit of RNA polymerase II. Here, we show that Rtr1p is a component of a novel mRNA degradation pathway that promotes its autoregulation through turnover of its own mRNA. We show that the 3′UTR of the RTR1 mRNA contains a cis element that destabilizes this mRNA. RTR1 mRNA turnover is achieved through binding of Rtr1p to the RTR1 mRNP in a manner that is dependent on this cis element. Genetic evidence shows that Rtr1p-mediated decay of the RTR1 mRNA involves the 5′-3′ DExD/H-box RNA helicase Dhh1p and the 3′-5′ exonucleases Rex2p and Rex3p. Rtr1p and Rex3p are found associated with Dhh1p, suggesting a model for recruiting the REX exonucleases to the RTR1 mRNA for degradation. Rtr1p-mediated decay potentially impacts additional transcripts, including the unspliced BMH2 pre-mRNA. We propose that Rtr1p may imprint its RNA targets cotranscriptionally and determine their downstream degradation mechanism by directing these transcripts to a novel turnover pathway that involves Rtr1p, Dhh1p, and the REX family of exonucleases. PMID:26843527

Wig1 prevents cellular senescence by regulating p21 mRNA decay through control of RISC recruitment

PubMed Central

Kim, Bong Cho; Lee, Hyung Chul; Lee, Je-Jung; Choi, Chang-Min; Kim, Dong-Kwan; Lee, Jae Cheol; Ko, Young-Gyu; Lee, Jae-Seon

2012-01-01

Premature senescence, a key strategy used to suppress carcinogenesis, can be driven by p53/p21 proteins in response to various stresses. Here, we demonstrate that Wig1 plays a critical role in this process through regulation of p21 mRNA stability. Wig1 controls the association of Argonaute2 (Ago2), a central component of the RNA-induced silencing complex (RISC), with target p21 mRNA via binding of the stem-loop structure near the microRNA (miRNA) target site. Depletion of Wig1 prohibited miRNA-mediated p21 mRNA decay and resulted in premature senescence. Wig1 plays an essential role in cell proliferation, as demonstrated in tumour xenografts in mice, and Wig1 and p21 mRNA levels are inversely correlated in human normal and cancer tissues. Together, our data indicate a novel role of Wig1 in RISC target accessibility, which is a key step in RNA-mediated gene silencing. In addition, these findings indicate that fine-tuning of p21 levels by Wig1 is essential for the prevention of cellular senescence. PMID:23085987

Wig1 prevents cellular senescence by regulating p21 mRNA decay through control of RISC recruitment.

PubMed

Kim, Bong Cho; Lee, Hyung Chul; Lee, Je-Jung; Choi, Chang-Min; Kim, Dong-Kwan; Lee, Jae Cheol; Ko, Young-Gyu; Lee, Jae-Seon

2012-11-14

Premature senescence, a key strategy used to suppress carcinogenesis, can be driven by p53/p21 proteins in response to various stresses. Here, we demonstrate that Wig1 plays a critical role in this process through regulation of p21 mRNA stability. Wig1 controls the association of Argonaute2 (Ago2), a central component of the RNA-induced silencing complex (RISC), with target p21 mRNA via binding of the stem-loop structure near the microRNA (miRNA) target site. Depletion of Wig1 prohibited miRNA-mediated p21 mRNA decay and resulted in premature senescence. Wig1 plays an essential role in cell proliferation, as demonstrated in tumour xenografts in mice, and Wig1 and p21 mRNA levels are inversely correlated in human normal and cancer tissues. Together, our data indicate a novel role of Wig1 in RISC target accessibility, which is a key step in RNA-mediated gene silencing. In addition, these findings indicate that fine-tuning of p21 levels by Wig1 is essential for the prevention of cellular senescence.

Timescales and bottlenecks in miRNA-dependent gene regulation.

PubMed

Hausser, Jean; Syed, Afzal Pasha; Selevsek, Nathalie; van Nimwegen, Erik; Jaskiewicz, Lukasz; Aebersold, Ruedi; Zavolan, Mihaela

2013-12-03

MiRNAs are post-transcriptional regulators that contribute to the establishment and maintenance of gene expression patterns. Although their biogenesis and decay appear to be under complex control, the implications of miRNA expression dynamics for the processes that they regulate are not well understood. We derived a mathematical model of miRNA-mediated gene regulation, inferred its parameters from experimental data sets, and found that the model describes well time-dependent changes in mRNA, protein and ribosome density levels measured upon miRNA transfection and induction. The inferred parameters indicate that the timescale of miRNA-dependent regulation is slower than initially thought. Delays in miRNA loading into Argonaute proteins and the slow decay of proteins relative to mRNAs can explain the typically small changes in protein levels observed upon miRNA transfection. For miRNAs to regulate protein expression on the timescale of a day, as miRNAs involved in cell-cycle regulation do, accelerated miRNA turnover is necessary.

Heat-induced ribosome pausing triggers mRNA co-translational decay in Arabidopsis thaliana

PubMed Central

Merret, Rémy; Nagarajan, Vinay K.; Carpentier, Marie-Christine; Park, Sunhee; Favory, Jean-Jacques; Descombin, Julie; Picart, Claire; Charng, Yee-yung; Green, Pamela J.; Deragon, Jean-Marc; Bousquet-Antonelli, Cécile

2015-01-01

The reprogramming of gene expression in heat stress is a key determinant to organism survival. Gene expression is downregulated through translation initiation inhibition and release of free mRNPs that are rapidly degraded or stored. In mammals, heat also triggers 5′-ribosome pausing preferentially on transcripts coding for HSC/HSP70 chaperone targets, but the impact of such phenomenon on mRNA fate remains unknown. Here, we provide evidence that, in Arabidopsis thaliana, heat provokes 5′-ribosome pausing leading to the XRN4-mediated 5′-directed decay of translating mRNAs. We also show that hindering HSC/HSP70 activity at 20°C recapitulates heat effects by inducing ribosome pausing and co-translational mRNA turnover. Strikingly, co-translational decay targets encode proteins with high HSC/HSP70 binding scores and hydrophobic N-termini, two characteristics that were previously observed for transcripts most prone to pausing in animals. This work suggests for the first time that stress-induced variation of translation elongation rate is an evolutionarily conserved process leading to the polysomal degradation of thousands of ‘non-aberrant’ mRNAs. PMID:25845591

Global survey of mRNA levels and decay rates of Chlamydia trachomatis trachoma and lymphogranuloma venereum biovars.

PubMed

Ferreira, Rita; Borges, Vítor; Borrego, Maria José; Gomes, João Paulo

2017-07-01

Interpreting the intricate bacterial transcriptomics implies understanding the dynamic relationship established between de novo transcription and the degradation of transcripts. Here, we performed a comparative overview of gene expression levels and mRNA decay rates for different-biovar (trachoma and lymphogranuloma venereum) strains of the obligate intracellular bacterium Chlamydia trachomatis . By using RNA-sequencing to measure gene expression levels at mid developmental stage and mRNA decay rates upon rifampicin-based transcription blockage, we observed that: i ) 60-70% of the top-50 expressed genes encode proteins with unknown function and proteins involved in "Translation, ribosomal structure and biogenesis" for all strains; ii ) the expression ranking by genes' functional categories was in general concordant among different-biovar strains; iii ) the median of the half-life time (t 1/2 ) values of transcripts were 15-17 min, indicating that the degree of transcripts' stability seems to correlate with the bacterial intracellular life-style, as these values are considerably higher than the ones observed in other studies for facultative intracellular and free-living bacteria; iv ) transcript decay rates were highly heterogeneous within each C. trachomatis strain and did not correlate with steady-state expression levels; v ) only at very few instances (essentially at gene functional category level) was possible to unveil dissimilarities potentially underlying phenotypic differences between biovars. In summary, the unveiled transcriptomic scenario, marked by a general lack of correlation between transcript production and degradation and a huge inter-transcript heterogeneity in decay rates, likely reflects the challenges underlying the unique biphasic developmental cycle of C. trachomatis and its intricate interactions with the human host, which probably exacerbate the complexity of the bacterial transcription regulation.

The MicroRNA miR-124 Promotes Neuronal Differentiation by Triggering Brain-Specific Alternative Pre-mRNA Splicing

PubMed Central

Makeyev, Eugene V.; Zhang, Jiangwen; Carrasco, Monica A.; Maniatis, Tom

2011-01-01

SUMMARY Both microRNAs and alternative pre-mRNA splicing have been implicated in the development of the nervous system (NS), but functional interactions between these two pathways are poorly understood. We demonstrate that the neuron-specific microRNA miR-124 directly targets PTBP1 (PTB/hnRNP I) mRNA, which encodes a global repressor of alternative pre-mRNA splicing in nonneuronal cells. Among the targets of PTBP1 is a critical cassette exon in the pre-mRNA of PTBP2 (nPTB/brPTB/PTBLP), an NS-enriched PTBP1 homolog. When this exon is skipped, PTBP2 mRNA is subject to nonsense-mediated decay (NMD). During neuronal differentiation, miR-124 reduces PTBP1 levels, leading to the accumulation of correctly spliced PTBP2 mRNA and a dramatic increase in PTBP2 protein. These events culminate in the transition from non-NS to NS-specific alternative splicing patterns. We also present evidence that miR-124 plays a key role in the differentiation of progenitor cells to mature neurons. Thus, miR-124 promotes NS development, at least in part by regulating an intricate network of NS-specific alternative splicing. PMID:17679093

Emerging functions of alternative splicing coupled with nonsense-mediated decay.

PubMed

Hamid, Fursham M; Makeyev, Eugene V

2014-08-01

Higher eukaryotes rely on AS (alternative splicing) of pre-mRNAs (mRNA precursors) to generate more than one protein product from a single gene and to regulate mRNA stability and translational activity. An important example of the latter function involves an interplay between AS and NMD (nonsense-mediated decay), a cytoplasmic quality control mechanism eliminating mRNAs containing PTCs (premature translation termination codons). Although originally identified as an error surveillance process, AS-NMD additionally provides an efficient strategy for deterministic regulation of gene expression outputs. In this review, we discuss recently published examples of AS-NMD and delineate functional contexts where recurrent use of this mechanism orchestrates expression of important genes.

Development of a time-resolved fluorometric method for observing hybridization in living cells using fluorescence resonance energy transfer.

PubMed Central

Tsuji, A; Sato, Y; Hirano, M; Suga, T; Koshimoto, H; Taguchi, T; Ohsuka, S

2001-01-01

We previously showed that a specific kind of mRNA (c-fos) was detected in a living cell under a microscope by introducing two fluorescently labeled oligodeoxynucleotides, each labeled with donor or acceptor, into the cytoplasm, making them hybridize to adjacent locations on c-fos mRNA, and taking images of fluorescence resonance energy transfer (FRET) (A. Tsuji, H. Koshimoto, Y. Sato, M. Hirano. Y. Sei-Iida, S. Kondo, and K. Ishibashi, 2000, Biophys. J. 78:3260-3274). On the formed hybrid, the distance between donor and acceptor becomes close and FRET occurs. To observe small numbers of mRNA in living cells using this method, it is required that FRET fluorescence of hybrid must be distinguished from fluorescence of excess amounts of non-hybridizing probes and from cell autofluorescence. To meet these requirements, we developed a time-resolved method using acceptor fluorescence decays. When a combination of a donor having longer fluorescence lifetime and an acceptor having shorter lifetime is used, the measured fluorescence decays of acceptors under FRET becomes slower than the acceptor fluorescence decay with direct excitation. A combination of Bodipy493/503 and Cy5 was selected as donor and acceptor. When the formed hybrid had a configuration where the target RNA has no single-strand part between the two fluorophores, the acceptor fluorescence of hybrid had a sufficiently longer delay to detect fluorescence of hybrid in the presence of excess amounts of non-hybridizing probes. Spatial separation of 10-12 bases between two fluorophores on the hybrid is also required. The decay is also much slower than cell autofluorescence, and smaller numbers of hybrid were detected with less interference of cell autofluorescence in the cytoplasm of living cells under a time-resolved fluorescence microscope with a time-gated function equipped camera. The present method will be useful when observing induced expressions of mRNA in living cells. PMID:11423432

The cytoplasmic mRNA degradation factor Pat1 is required for rRNA processing

PubMed Central

Muppavarapu, Mridula; Huch, Susanne; Nissan, Tracy

2016-01-01

ABSTRACT Pat1 is a key cytoplasmic mRNA degradation factor, the loss of which severely increases mRNA half-lives. Several recent studies have shown that Pat1 can enter the nucleus and can shuttle between the nucleus and the cytoplasm. As a result, many nuclear roles have been proposed for Pat1. In this study, we analyzed four previously suggested nuclear roles of Pat1 and show that Pat1 is not required for efficient pre-mRNA splicing or pre-mRNA decay in yeast. However, lack of Pat1 results in accumulation of pre-rRNA processing intermediates. Intriguingly, we identified a novel genetic relationship between Pat1 and the rRNA decay machinery, specifically the exosome and the TRAMP complex. While the pre-rRNA processing intermediates that accumulate in the pat1 deletion mutant are, at least to some extent, recognized as aberrant by the rRNA degradation machinery, it is unlikely that these accumulations are the cause of their synthetic sick relationship. Here, we show that the dysregulation of the levels of mRNAs related to ribosome biogenesis could be the cause of the accumulation of the pre-rRNA processing intermediates. Although our results support a role for Pat1 in transcription, they nevertheless suggest that the primary cause of the dysregulated mRNA levels is most likely due to Pat1's role in mRNA decapping and mRNA degradation. PMID:26918764

The cytoplasmic mRNA degradation factor Pat1 is required for rRNA processing.

PubMed

Muppavarapu, Mridula; Huch, Susanne; Nissan, Tracy

2016-01-01

Pat1 is a key cytoplasmic mRNA degradation factor, the loss of which severely increases mRNA half-lives. Several recent studies have shown that Pat1 can enter the nucleus and can shuttle between the nucleus and the cytoplasm. As a result, many nuclear roles have been proposed for Pat1. In this study, we analyzed four previously suggested nuclear roles of Pat1 and show that Pat1 is not required for efficient pre-mRNA splicing or pre-mRNA decay in yeast. However, lack of Pat1 results in accumulation of pre-rRNA processing intermediates. Intriguingly, we identified a novel genetic relationship between Pat1 and the rRNA decay machinery, specifically the exosome and the TRAMP complex. While the pre-rRNA processing intermediates that accumulate in the pat1 deletion mutant are, at least to some extent, recognized as aberrant by the rRNA degradation machinery, it is unlikely that these accumulations are the cause of their synthetic sick relationship. Here, we show that the dysregulation of the levels of mRNAs related to ribosome biogenesis could be the cause of the accumulation of the pre-rRNA processing intermediates. Although our results support a role for Pat1 in transcription, they nevertheless suggest that the primary cause of the dysregulated mRNA levels is most likely due to Pat1's role in mRNA decapping and mRNA degradation.

Serum-deprivation stimulates cap-binding by PARN at the expense of eIF4E, consistent with the observed decrease in mRNA stability

PubMed Central

Seal, Ruth; Temperley, Richard; Wilusz, Jeffrey; Lightowlers, Robert N.; Chrzanowska-Lightowlers, Zofia M. A.

2005-01-01

PARN, a poly(A)-specific ribonuclease, binds the 5′ cap-structure of mRNA and initiates deadenylation-dependent decay. Eukaryotic initiation factor 4E (eIF4E) also binds to the cap structure, an interaction that is critical for initiating cap-dependent translation. The stability of various mRNA transcripts in human cell lines is reduced under conditions of serum starvation as determined by both functional and chemical half-lives. Serum starvation also leads to enhanced cap association by PARN. In contrast, the 5′ cap occupancy by eIF4E decreases under serum-deprivation, as does the translation of reporter transcripts. Further, we show that PARN is a phosphoprotein and that this modification can be modulated by serum status. Taken together, these data are consistent with a natural competition existing at the 5′ cap structure between PARN and eIF4E that may be regulated by changes in post-translational modifications. These phosphorylation-induced changes in the interplay of PARN and eIF4E may determine whether the mRNA is translated or decayed. PMID:15653638

Insulin Signaling Augments eIF4E-Dependent Nonsense-Mediated mRNA Decay in Mammalian Cells.

PubMed

Park, Jungyun; Ahn, Seyoung; Jayabalan, Aravinth K; Ohn, Takbum; Koh, Hyun Chul; Hwang, Jungwook

2016-07-01

Nonsense-mediated mRNA decay (NMD) modulates the level of mRNA harboring a premature termination codon (PTC) in a translation-dependent manner. Inhibition of translation is known to impair NMD; however, few studies have investigated the correlation between enhanced translation and increased NMD. Here, we demonstrate that insulin signaling events increase translation, leading to an increase in NMD of eIF4E-bound transcripts. We provide evidence that (i) insulin-mediated enhancement of translation augments NMD and rapamycin abrogates this enhancement; (ii) an increase in AKT phosphorylation due to inhibition of PTEN facilitates NMD; (iii) insulin stimulation increases the binding of up-frameshift factor 1 (UPF1), most likely to eIF4E-bound PTC-containing transcripts; and (iv) insulin stimulation induces the colocalization of UPF1 and eIF4E in processing bodies. These results illustrate how extracellular signaling promotes the removal of eIF4E-bound NMD targets. Copyright © 2015 Elsevier B.V. All rights reserved.

Cytoplasmic Control of Sense-Antisense mRNA Pairs.

PubMed

Sinturel, Flore; Navickas, Albertas; Wery, Maxime; Descrimes, Marc; Morillon, Antonin; Torchet, Claire; Benard, Lionel

2015-09-22

Transcriptome analyses have revealed that convergent gene transcription can produce many 3'-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3'-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3'-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3'-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD) in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs) form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5'-3' cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Preharvest UV-C radiation influences physiological, biochemical, and transcriptional changes in strawberry cv. Camarosa.

PubMed

de Oliveira, Isadora Rubin; Crizel, Giseli Rodrigues; Severo, Joseana; Renard, Catherine M G C; Chaves, Fabio Clasen; Rombaldi, Cesar Valmor

2016-11-01

Ultraviolet C (UV-C) radiation is known for preventing fungal decay and enhancing phytochemical content in fruit when applied postharvest. However, limited knowledge is available regarding fruit responses to preharvest application of UV-C radiation. Thus, the effects of UV-C radiation on photosynthetic efficiency, dry matter accumulation and partitioning, fruit yield and decay, phytochemical content, and relative transcript accumulation of genes associated with these metabolic pathways were monitored in strawberry (Fragaria x ananassa Duch.) cv. Camarosa. A reduction in photosynthetic efficiency was followed by a decrease in light harvesting complex LhcIIb-1 mRNA accumulation as well as a decrease in yield per plant. Phenylalanine ammonia lyase activity, phenolic, anthocyanin, and L-ascorbic acid contents were higher in UV-C treated fruit. In addition, preharvest UV-C treatment reduced microorganism incidence in the greenhouse and on the fruit surface, increased the accumulation of β-1,3-Gluc and PR-1 mRNA, and prevented fruit decay. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Murine knockin model for progranulin-deficient frontotemporal dementia with nonsense-mediated mRNA decay

PubMed Central

Nguyen, Thi A.; Zhang, Jiasheng; Devireddy, Swathi; Zhou, Ping; Karydas, Anna M.; Xu, Xialian; Miller, Bruce L.; Rigo, Frank; Ferguson, Shawn M.; Walther, Tobias C.; Farese, Robert V.

2018-01-01

Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized GrnR493X knockin mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous GrnR493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels in GrnR493X mice and cell lines and in fibroblasts from patients containing the GRNR493X mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation. PMID:29511098

Regulation of c-Myc mRNA by L11 in Response to UV and Gamma Irradiation

DTIC Science & Technology

2014-12-01

function of miRNAs. For example, TTP promotes tumor ne- crosis factor alpha (TNF-") mRNA decay caused by miR-16 (29) and HuR facilitates the targeting of c...experiments would demonstrate an important function of L11 in regulating c-myc mRNA in response to DNA damage, offer useful information for developing...24 (Figure 1). We also tested an array of miRNAs possessing tumor suppressor functions for L11 binding miR-16 miR-1248 miR-3944 (-) miR-191 miR

Using complementary DNA from MyoD-transduced fibroblasts to sequence large muscle genes.

PubMed

Waddell, Leigh B; Monnier, Nicole; Cooper, Sandra T; North, Kathryn N; Clarke, Nigel F

2011-08-01

Large muscle genes are often sequenced using complementary DNA (cDNA) made from muscle messenger RNA (mRNA) to reduce the cost and workload associated with sequencing from genomic DNA. Two potential barriers are the availability of a frozen muscle biopsy, and difficulties in detecting nonsense mutations due to nonsense-mediated mRNA decay (NMD). We present patient examples showing that use of MyoD-transduced fibroblasts as a source of muscle-specific mRNA overcomes these potential difficulties in sequencing large muscle-related genes. Copyright © 2011 Wiley Periodicals, Inc.

Nitric Oxide Increases the Decay of Matrix Metalloproteinase 9 mRNA by Inhibiting the Expression of mRNA-Stabilizing Factor HuR

PubMed Central

Akool, El-Sayed; Kleinert, Hartmut; Hamada, Farid M. A.; Abdelwahab, Mohamed H.; Förstermann, Ulrich; Pfeilschifter, Josef; Eberhardt, Wolfgang

2003-01-01

Dysregulation of extracellular matrix turnover is an important feature of many inflammatory processes. Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin-1 beta. We demonstrate that NO does strongly destabilize MMP-9 mRNA, since different luciferase reporter gene constructs containing the MMP-9 3′ untranslated region (UTR) displayed significant reduced luciferase activity in response to the presence of NO. Moreover, by use of an in vitro degradation assay we found that the cytoplasmic fractions of NO-treated cells contained a higher capacity to degrade MMP-9 transcripts than those obtained from control cells. An RNA electrophoretic mobility shift assay demonstrated that three of four putative AU-rich elements present in the 3′ UTR of MMP-9 were constitutively occupied by the mRNA-stabilizing factor HuR and that the RNA binding was strongly attenuated by the presence of NO. The addition of recombinant glutathione transferase-HuR prevented the rapid decay of MMP-9 mRNA, whereas the addition of a neutralizing anti-HuR antibody caused an acceleration of MMP-9 mRNA degradation. Furthermore, the expression of HuR mRNA and protein was significantly reduced by exogenously and endogenously produced NO. These inhibitory effects were mimicked by the cGMP analog 8-bromo-cGMP and reversed by LY-83583, an inhibitor of soluble guanylyl cyclase. These results demonstrate that NO acts in a cGMP-dependent mechanism to inhibit the expression level of HuR, thereby reducing the stability of MMP-9 mRNA. PMID:12832476

Common pathological mutations in PQBP1 induce nonsense-mediated mRNA decay and enhance exclusion of the mutant exon.

PubMed

Musante, Luciana; Kunde, Stella-Amrei; Sulistio, Tina O; Fischer, Ute; Grimme, Astrid; Frints, Suzanna G M; Schwartz, Charles E; Martínez, Francisco; Romano, Corrado; Ropers, Hans-Hilger; Kalscheuer, Vera M

2010-01-01

The polyglutamine binding protein 1 (PQBP1) gene plays an important role in X-linked mental retardation (XLMR). Nine of the thirteen PQBP1 mutations known to date affect the AG hexamer in exon 4 and cause frameshifts introducing premature termination codons (PTCs). However, the phenotype in this group of patients is variable. To investigate the pathology of these PQBP1 mutations, we evaluated their consequences on mRNA and protein expression. RT-PCRs revealed mutation-specific reduction of PQBP1 mRNAs carrying the PTCs that can be partially restored by blocking translation, thus indicating a role for the nonsense-mediated mRNA decay pathway. In addition, these mutations resulted in altered levels of PQBP1 transcripts that skipped exon 4, probably as a result of altering important splicing motifs via nonsense-associated altered splicing (NAS). This hypothesis is supported by transfection experiments using wild-type and mutant PQBP1 minigenes. Moreover, we show that a truncated PQBP1 protein is indeed present in the patients. Remarkably, patients with insertion/deletion mutations in the AG hexamer express significantly increased levels of a PQBP1 isoform, which is very likely encoded by the transcripts without exon 4, confirming the findings at the mRNA level. Our study provides significant insight into the early events contributing to the pathogenesis of the PQBP1 related XLMR disease.

5′ to 3′ mRNA Decay Contributes to the Regulation of Arabidopsis Seed Germination by Dormancy1

PubMed Central

Basbouss-Serhal, Isabelle; Pateyron, Stéphanie; Cochet, Françoise

2017-01-01

The regulation of plant gene expression, necessary for development and adaptive responses, relies not only on RNA transcription but also on messenger RNA (mRNA) fate. To understand whether seed germination relies on the degradation of specific subsets of mRNA, we investigated whether the 5′ to 3′ RNA decay machinery participated in the regulation of this process. Arabidopsis (Arabidopsis thaliana) seeds of exoribonuclease4 (xrn4) and varicose (vcs) mutants displayed distinct dormancy phenotypes. Transcriptome analysis of xrn4-5 and vcs-8 mutant seeds allowed us to identify genes that are likely to play a role in the control of germination. Study of 5′ untranslated region features of these transcripts revealed that specific motifs, secondary energy, and GC content could play a role in their degradation by XRN4 and VCS, and Gene Ontology clustering revealed novel actors of seed dormancy and germination. Several specific transcripts identified as being putative targets of XRN4 and VCS in seeds (PECTIN LYASE-LIKE, ASPARTYL PROTEASE, DWD-HYPERSENSITIVE-TO-ABA3, and YELLOW STRIPE-LIKE5) were further studied by reverse genetics, and their functional roles in the germination process were confirmed by mutant analysis. These findings suggest that completion of germination and its regulation by dormancy also depend on the degradation of specific subsets of mRNA. PMID:28126845

Murine knockin model for progranulin-deficient frontotemporal dementia with nonsense-mediated mRNA decay.

PubMed

Nguyen, Andrew D; Nguyen, Thi A; Zhang, Jiasheng; Devireddy, Swathi; Zhou, Ping; Karydas, Anna M; Xu, Xialian; Miller, Bruce L; Rigo, Frank; Ferguson, Shawn M; Huang, Eric J; Walther, Tobias C; Farese, Robert V

2018-03-20

Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized Grn R493X knockin mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous Grn R493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels in Grn R493X mice and cell lines and in fibroblasts from patients containing the GRN R493X mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation. Copyright © 2018 the Author(s). Published by PNAS.

Demographic, socioeconomic, and behavioral factors affecting patterns of tooth decay in the permanent dentition: Principal components and factor analyses

PubMed Central

Shaffer, John R.; Polk, Deborah E.; Feingold, Eleanor; Wang, Xiaojing; Cuenco, Karen T.; Weeks, Daniel E.; DeSensi, Rebecca S.; Weyant, Robert J.; Crout, Richard; McNeil, Daniel W.; Marazita, Mary L.

2012-01-01

Objectives Dental caries of the permanent dentition is a multi-factorial disease resulting from the complex interplay of endogenous and environmental risk factors. The disease is not easily quantified due to the innumerable possible combinations of carious lesions across individual tooth surfaces of the permanent dentition. Global measures of decay, such as the DMFS index (which was developed for surveillance applications), may not be optimal for studying the epidemiology of dental caries because they ignore the distinct patterns of decay across the dentition. We hypothesize that specific risk factors may manifest their effects on specific tooth surfaces leading to patterns of decay that can be identified and studied. In this study we utilized two statistical methods of extracting patterns of decay from surface-level caries data in order to create novel phenotypes with which to study the risk factors affecting dental caries. Methods Intra-oral dental examinations were performed on 1,068 participants aged 18 to 75 years to assess dental caries. The 128 tooth surfaces of the permanent dentition were scored as carious or not and used as input for principal components analysis (PCA) and factor analysis (FA), two methods of identifying underlying patterns without a priori knowledge of the patterns. Demographic (age, sex, birth year, race/ethnicity, and educational attainment), anthropometric (height, body mass index, waist circumference), endogenous (saliva flow), and environmental (tooth brushing frequency, home water source, and home water fluoride) risk factors were tested for association with the caries patterns identified by PCA and FA, as well as DMFS, for comparison. The ten strongest patterns (i.e., those that explain the most variation in the data set) extracted by PCA and FA were considered. Results The three strongest patterns identified by PCA reflected (i) global extent of decay (i.e., comparable to DMFS index), (ii) pit and fissure surface caries, and (iii) smooth surface caries, respectively. The two strongest patterns identified by FA corresponded to (i) pit and fissure surface caries and (ii) maxillary incisor caries. Age and birth year were significantly associated with several patterns of decay, including global decay/DMFS index. Sex, race, educational attainment, and tooth brushing were each associated with specific patterns of decay, but not with global decay/DMFS index. Conclusions Taken together, these results support the notion that caries experience is separable into patterns attributable to distinct risk factors. This study demonstrates the utility of such novel caries patterns as new outcomes for exploring the complex, multifactorial nature of dental caries. PMID:23106439

Demographic, socioeconomic, and behavioral factors affecting patterns of tooth decay in the permanent dentition: principal components and factor analyses.

PubMed

Shaffer, John R; Polk, Deborah E; Feingold, Eleanor; Wang, Xiaojing; Cuenco, Karen T; Weeks, Daniel E; DeSensi, Rebecca S; Weyant, Robert J; Crout, Richard; McNeil, Daniel W; Marazita, Mary L

2013-08-01

Dental caries of the permanent dentition is a multifactorial disease resulting from the complex interplay of endogenous and environmental risk factors. The disease is not easily quantitated due to the innumerable possible combinations of carious lesions across individual tooth surfaces of the permanent dentition. Global measures of decay, such as the DMFS index (which was developed for surveillance applications), may not be optimal for studying the epidemiology of dental caries because they ignore the distinct patterns of decay across the dentition. We hypothesize that specific risk factors may manifest their effects on specific tooth surfaces leading to patterns of decay that can be identified and studied. In this study, we utilized two statistical methods of extracting patterns of decay from surface-level caries data to create novel phenotypes with which to study the risk factors affecting dental caries. Intra-oral dental examinations were performed on 1068 participants aged 18-75 years to assess dental caries. The 128 tooth surfaces of the permanent dentition were scored as carious or not and used as input for principal components analysis (PCA) and factor analysis (FA), two methods of identifying underlying patterns without a priori knowledge of the patterns. Demographic (age, sex, birth year, race/ethnicity, and educational attainment), anthropometric (height, body mass index, waist circumference), endogenous (saliva flow), and environmental (tooth brushing frequency, home water source, and home water fluoride) risk factors were tested for association with the caries patterns identified by PCA and FA, as well as DMFS, for comparison. The ten strongest patterns (i.e. those that explain the most variation in the data set) extracted by PCA and FA were considered. The three strongest patterns identified by PCA reflected (i) global extent of decay (i.e. comparable to DMFS index), (ii) pit and fissure surface caries and (iii) smooth surface caries, respectively. The two strongest patterns identified by FA corresponded to (i) pit and fissure surface caries and (ii) maxillary incisor caries. Age and birth year were significantly associated with several patterns of decay, including global decay/DMFS index. Sex, race, educational attainment, and tooth brushing were each associated with specific patterns of decay, but not with global decay/DMFS index. Taken together, these results support the notion that caries experience is separable into patterns attributable to distinct risk factors. This study demonstrates the utility of such novel caries patterns as new outcomes for exploring the complex, multifactorial nature of dental caries. © 2012 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Stress-dependent relocalization of translationally primed mRNPs to cytoplasmic granules that are kinetically and spatially distinct from P-bodies.

PubMed

Hoyle, Nathaniel P; Castelli, Lydia M; Campbell, Susan G; Holmes, Leah E A; Ashe, Mark P

2007-10-08

Cytoplasmic RNA granules serve key functions in the control of messenger RNA (mRNA) fate in eukaryotic cells. For instance, in yeast, severe stress induces mRNA relocalization to sites of degradation or storage called processing bodies (P-bodies). In this study, we show that the translation repression associated with glucose starvation causes the key translational mediators of mRNA recognition, eIF4E, eIF4G, and Pab1p, to resediment away from ribosomal fractions. These mediators then accumulate in P-bodies and in previously unrecognized cytoplasmic bodies, which we define as EGP-bodies. Our kinetic studies highlight the fundamental difference between EGP- and P-bodies and reflect the complex dynamics surrounding reconfiguration of the mRNA pool under stress conditions. An absence of key mRNA decay factors from EGP-bodies points toward an mRNA storage function for these bodies. Overall, this study highlights new potential control points in both the regulation of mRNA fate and the global control of translation initiation.

Decay patterns of brick wall in atmospheric environment: a possible analogue to rock weathering?

NASA Astrophysics Data System (ADS)

Prikryl, Richard; Weishauptová, Zuzana; Přikrylová, Jiřina; Jablonský, Jakub

2015-04-01

This study is focused on the decay of bricks exposed in enclosing wall of the Regional maternal hospital in Prague city centre (Czech Republic). The hospital, listed as a Czech architectural monument, has been constructed from locally produced bricks in neo-Gothic style in the period of 1867-1875. The bricks of the enclosing wall show sequence of decay patterns that resemble weathering forms observable on monuments built of natural stone. This study aims to study the observed decay patterns by means of in situ mapping and by analyses of decayed material (optical microscopy, SEM/EDS, X-ray diffraction, Hg-porosimetry, water soluble salts analysis) and to interpret them based on the phase composition and other properties of bricks. Finally, the decay patterns of studied brick wall are compared to known weathering sequences on porous rocks (both on natural outcrops and on artistic monuments).

Heritable patterns of tooth decay in the permanent dentition: principal components and factor analyses.

PubMed

Shaffer, John R; Feingold, Eleanor; Wang, Xiaojing; Tcuenco, Karen T; Weeks, Daniel E; DeSensi, Rebecca S; Polk, Deborah E; Wendell, Steve; Weyant, Robert J; Crout, Richard; McNeil, Daniel W; Marazita, Mary L

2012-03-09

Dental caries is the result of a complex interplay among environmental, behavioral, and genetic factors, with distinct patterns of decay likely due to specific etiologies. Therefore, global measures of decay, such as the DMFS index, may not be optimal for identifying risk factors that manifest as specific decay patterns, especially if the risk factors such as genetic susceptibility loci have small individual effects. We used two methods to extract patterns of decay from surface-level caries data in order to generate novel phenotypes with which to explore the genetic regulation of caries. The 128 tooth surfaces of the permanent dentition were scored as carious or not by intra-oral examination for 1,068 participants aged 18 to 75 years from 664 biological families. Principal components analysis (PCA) and factor analysis (FA), two methods of identifying underlying patterns without a priori surface classifications, were applied to our data. The three strongest caries patterns identified by PCA recaptured variation represented by DMFS index (correlation, r = 0.97), pit and fissure surface caries (r = 0.95), and smooth surface caries (r = 0.89). However, together, these three patterns explained only 37% of the variability in the data, indicating that a priori caries measures are insufficient for fully quantifying caries variation. In comparison, the first pattern identified by FA was strongly correlated with pit and fissure surface caries (r = 0.81), but other identified patterns, including a second pattern representing caries of the maxillary incisors, were not representative of any previously defined caries indices. Some patterns identified by PCA and FA were heritable (h(2) = 30-65%, p = 0.043-0.006), whereas other patterns were not, indicating both genetic and non-genetic etiologies of individual decay patterns. This study demonstrates the use of decay patterns as novel phenotypes to assist in understanding the multifactorial nature of dental caries.

HTLV-1 Tax plugs and freezes UPF1 helicase leading to nonsense-mediated mRNA decay inhibition.

PubMed

Fiorini, Francesca; Robin, Jean-Philippe; Kanaan, Joanne; Borowiak, Malgorzata; Croquette, Vincent; Le Hir, Hervé; Jalinot, Pierre; Mocquet, Vincent

2018-01-30

Up-Frameshift Suppressor 1 Homolog (UPF1) is a key factor for nonsense-mediated mRNA decay (NMD), a cellular process that can actively degrade mRNAs. Here, we study NMD inhibition during infection by human T-cell lymphotropic virus type I (HTLV-1) and characterise the influence of the retroviral Tax factor on UPF1 activity. Tax interacts with the central helicase core domain of UPF1 and might plug the RNA channel of UPF1, reducing its affinity for nucleic acids. Furthermore, using a single-molecule approach, we show that the sequential interaction of Tax with a RNA-bound UPF1 freezes UPF1: this latter is less sensitive to the presence of ATP and shows translocation defects, highlighting the importance of this feature for NMD. These mechanistic insights reveal how HTLV-1 hijacks the central component of NMD to ensure expression of its own genome.

Picornavirus Modification of a Host mRNA Decay Protein

PubMed Central

Rozovics, Janet M.; Chase, Amanda J.; Cathcart, Andrea L.; Chou, Wayne; Gershon, Paul D.; Palusa, Saiprasad; Wilusz, Jeffrey; Semler, Bert L.

2012-01-01

ABSTRACT Due to the limited coding capacity of picornavirus genomic RNAs, host RNA binding proteins play essential roles during viral translation and RNA replication. Here we describe experiments suggesting that AUF1, a host RNA binding protein involved in mRNA decay, plays a role in the infectious cycle of picornaviruses such as poliovirus and human rhinovirus. We observed cleavage of AUF1 during poliovirus or human rhinovirus infection, as well as interaction of this protein with the 5′ noncoding regions of these viral genomes. Additionally, the picornavirus proteinase 3CD, encoded by poliovirus or human rhinovirus genomic RNAs, was shown to cleave all four isoforms of recombinant AUF1 at a specific N-terminal site in vitro. Finally, endogenous AUF1 was found to relocalize from the nucleus to the cytoplasm in poliovirus-infected HeLa cells to sites adjacent to (but distinct from) putative viral RNA replication complexes. PMID:23131833

New insights into the interplay between the translation machinery and nonsense-mediated mRNA decay factors.

PubMed

Raimondeau, Etienne; Bufton, Joshua C; Schaffitzel, Christiane

2018-06-19

Faulty mRNAs with a premature stop codon (PTC) are recognized and degraded by nonsense-mediated mRNA decay (NMD). Recognition of a nonsense mRNA depends on translation and on the presence of NMD-enhancing or the absence of NMD-inhibiting factors in the 3'-untranslated region. Our review summarizes our current understanding of the molecular function of the conserved NMD factors UPF3B and UPF1, and of the anti-NMD factor Poly(A)-binding protein, and their interactions with ribosomes translating PTC-containing mRNAs. Our recent discovery that UPF3B interferes with human translation termination and enhances ribosome dissociation in vitro , whereas UPF1 is inactive in these assays, suggests a re-interpretation of previous experiments and modification of prevalent NMD models. Moreover, we discuss recent work suggesting new functions of the key NMD factor UPF1 in ribosome recycling, inhibition of translation re-initiation and nascent chain ubiquitylation. These new findings suggest that the interplay of UPF proteins with the translation machinery is more intricate than previously appreciated, and that this interplay quality-controls the efficiency of termination, ribosome recycling and translation re-initiation. © 2018 The Author(s).

Rous Sarcoma Virus RNA Stability Element Inhibits Deadenylation of mRNAs with Long 3′UTRs

PubMed Central

Balagopal, Vidya; Beemon, Karen L.

2017-01-01

All retroviruses use their full-length primary transcript as the major mRNA for Group-specific antigen (Gag) capsid proteins. This results in a long 3′ untranslated region (UTR) downstream of the termination codon. In the case of Rous sarcoma virus (RSV), there is a 7 kb 3′UTR downstream of the gag terminator, containing the pol, env, and src genes. mRNAs containing long 3′UTRs, like those with premature termination codons, are frequently recognized by the cellular nonsense-mediated mRNA decay (NMD) machinery and targeted for degradation. To prevent this, RSV has evolved an RNA stability element (RSE) in the RNA immediately downstream of the gag termination codon. This 400-nt RNA sequence stabilizes premature termination codons (PTCs) in gag. It also stabilizes globin mRNAs with long 3′UTRs, when placed downstream of the termination codon. It is not clear how the RSE stabilizes the mRNA and prevents decay. We show here that the presence of RSE inhibits deadenylation severely. In addition, the RSE also impairs decapping (DCP2) and 5′-3′ exonucleolytic (XRN1) function in knockdown experiments in human cells. PMID:28763028

Polypyrimidine tract binding protein 1 protects mRNAs from recognition by the nonsense-mediated mRNA decay pathway

PubMed Central

Ge, Zhiyun; Quek, Bao Lin; Beemon, Karen L; Hogg, J Robert

2016-01-01

The nonsense-mediated mRNA decay (NMD) pathway degrades mRNAs containing long 3'UTRs to perform dual roles in mRNA quality control and gene expression regulation. However, expansion of vertebrate 3'UTR functions has required a physical expansion of 3'UTR lengths, complicating the process of detecting nonsense mutations. We show that the polypyrimidine tract binding protein 1 (PTBP1) shields specific retroviral and cellular transcripts from NMD. When bound near a stop codon, PTBP1 blocks the NMD protein UPF1 from binding 3'UTRs. PTBP1 can thus mark specific stop codons as genuine, preserving both the ability of NMD to accurately detect aberrant mRNAs and the capacity of long 3'UTRs to regulate gene expression. Illustrating the wide scope of this mechanism, we use RNA-seq and transcriptome-wide analysis of PTBP1 binding sites to show that many human mRNAs are protected by PTBP1 and that PTBP1 enrichment near stop codons correlates with 3'UTR length and resistance to NMD. DOI: http://dx.doi.org/10.7554/eLife.11155.001 PMID:26744779

Plant RNA Regulatory Network and RNA Granules in Virus Infection.

PubMed

Mäkinen, Kristiina; Lõhmus, Andres; Pollari, Maija

2017-01-01

Regulation of post-transcriptional gene expression on mRNA level in eukaryotic cells includes translocation, translation, translational repression, storage, mRNA decay, RNA silencing, and nonsense-mediated decay. These processes are associated with various RNA-binding proteins and cytoplasmic ribonucleoprotein complexes many of which are conserved across eukaryotes. Microscopically visible aggregations formed by ribonucleoprotein complexes are termed RNA granules. Stress granules where the translationally inactive mRNAs are stored and processing bodies where mRNA decay may occur present the most studied RNA granule types. Diverse RNP-granules are increasingly being assigned important roles in viral infections. Although the majority of the molecular level studies on the role of RNA granules in viral translation and replication have been conducted in mammalian systems, some studies link also plant virus infection to RNA granules. An increasing body of evidence indicates that plant viruses require components of stress granules and processing bodies for their replication and translation, but how extensively the cellular mRNA regulatory network is utilized by plant viruses has remained largely enigmatic. Antiviral RNA silencing, which is an important regulator of viral RNA stability and expression in plants, is commonly counteracted by viral suppressors of RNA silencing. Some of the RNA silencing suppressors localize to cellular RNA granules and have been proposed to carry out their suppression functions there. Moreover, plant nucleotide-binding leucine-rich repeat protein-mediated virus resistance has been linked to enhanced processing body formation and translational repression of viral RNA. Many interesting questions relate to how the pathways of antiviral RNA silencing leading to viral RNA degradation and/or repression of translation, suppression of RNA silencing and viral RNA translation converge in plants and how different RNA granules and their individual components contribute to these processes. In this review we discuss the roles of cellular RNA regulatory mechanisms and RNA granules in plant virus infection in the light of current knowledge and compare the findings to those made in animal virus studies.

Regulation of mouse hepatic CYP2D9 mRNA expression by growth and adrenal hormones.

PubMed

Jarukamjorn, Kanokwan; Sakuma, Tsutomu; Jaruchotikamol, Atika; Oguro, Miki; Nemoto, Nobuo

2006-02-01

The constitutive expression of CYP2D9 is sexually dimorphic, namely, strong in males, but diminutive in females. Repetition of mimic growth hormone (GH) secretion pattern impressively returned the mRNA expression level to that in intact mice: the GH secretion pattern's regulation of CYP2D9 mRNA expression has been predominantly disrupted by exogenous GH-administration. The extensive decline of CYP2D9 mRNA expression becoming a sexually non-specific P450 in 9-week-old male mice exposed as neonates to monosodium L-glutamate (MSG) suggested that the male GH secretion pattern is a key to the regulation of male-specific CYP2D9 mRNA expression in adult mice. Dexamethasone (Dex) showed possibility to induce CYP2D9 mRNA expression in adult MSG-neonatally treated mice of either sex. However, the antagonism was observed by co-administration of Dex and GH in the males. Dex-administration in adrenalectomized mice significantly elevated CYP2D9 mRNA expression levels. These findings suggest that an adrenal hormone participates in the regulatory mechanism of CYP2D9 mRNA expression in association with GH.

Kinetics of hairpin ribozyme cleavage in yeast.

PubMed Central

Donahue, C P; Fedor, M J

1997-01-01

Hairpin ribozymes catalyze a self-cleavage reaction that provides a simple model for quantitative analyses of intracellular mechanisms of RNA catalysis. Decay rates of chimeric mRNAs containing self-cleaving ribozymes give a direct measure of intracellular cleavage kinetics in yeast. Intracellular ribozyme-mediated cleavage occurs at similar rates and shows similar inhibition by ribozyme mutations as ribozyme-mediated reactions in vitro, but only when ribozymes are located in a favorable mRNA sequence context. The impact of cleavage on mRNA abundance is shown to depend directly on intrinsic mRNA stability. Surprisingly, cleavage products are no more labile than uncleaved mRNAs despite the loss of terminal cap structures or poly (A). PMID:9292496

CCR4-NOT deadenylates mRNA associated with RNA-induced silencing complexes in human cells.

PubMed

Piao, Xianghua; Zhang, Xue; Wu, Ligang; Belasco, Joel G

2010-03-01

MicroRNAs (miRNAs) repress gene expression posttranscriptionally by inhibiting translation and by expediting deadenylation so as to trigger rapid mRNA decay. Their regulatory influence is mediated by the protein components of the RNA-induced silencing complex (RISC), which deliver miRNAs and siRNAs to their mRNA targets. Here, we present evidence that CCR4-NOT is the deadenylase that removes poly(A) from messages destabilized by miRNAs in human cells. Overproducing a mutationally inactivated form of either of the catalytic subunits of this deadenylase (CCR4 or CAF1/POP2) significantly impedes the deadenylation and decay of mRNA targeted by a partially complementary miRNA. The same deadenylase initiates the degradation of "off-target" mRNAs that are bound by an imperfectly complementary siRNA introduced by transfection. The greater inhibitory effect of inactive CAF1 or POP2 (versus inactive CCR4) suggests a predominant role for this catalytic subunit of CCR4-NOT in miRNA- or small interfering RNA (siRNA)-mediated deadenylation. These effects of mi/siRNAs and CCR4-NOT can be fully reproduced by directly tethering RISC to mRNA without the guidance of a small RNA, indicating that the ability of RISC to accelerate deadenylation is independent of RNA base pairing. Despite its importance for mi/siRNA-mediated deadenylation, CCR4-NOT appears not to associate significantly with RISC, as judged by the failure of CAF1 and POP2 to coimmunoprecipitate detectably with either the Ago or TNRC6 subunit of RISC, a finding at odds with deadenylase recruitment as the mechanism by which RISC accelerates poly(A) removal.

Protein functional features are reflected in the patterns of mRNA translation speed.

PubMed

López, Daniel; Pazos, Florencio

2015-07-09

The degeneracy of the genetic code makes it possible for the same amino acid string to be coded by different messenger RNA (mRNA) sequences. These "synonymous mRNAs" may differ largely in a number of aspects related to their overall translational efficiency, such as secondary structure content and availability of the encoded transfer RNAs (tRNAs). Consequently, they may render different yields of the translated polypeptides. These mRNA features related to translation efficiency are also playing a role locally, resulting in a non-uniform translation speed along the mRNA, which has been previously related to some protein structural features and also used to explain some dramatic effects of "silent" single-nucleotide-polymorphisms (SNPs). In this work we perform the first large scale analysis of the relationship between three experimental proxies of mRNA local translation efficiency and the local features of the corresponding encoded proteins. We found that a number of protein functional and structural features are reflected in the patterns of ribosome occupancy, secondary structure and tRNA availability along the mRNA. One or more of these proxies of translation speed have distinctive patterns around the mRNA regions coding for certain protein local features. In some cases the three patterns follow a similar trend. We also show specific examples where these patterns of translation speed point to the protein's important structural and functional features. This support the idea that the genome not only codes the protein functional features as sequences of amino acids, but also as subtle patterns of mRNA properties which, probably through local effects on the translation speed, have some consequence on the final polypeptide. These results open the possibility of predicting a protein's functional regions based on a single genomic sequence, and have implications for heterologous protein expression and fine-tuning protein function.

MicroRNA networks in mouse lung organogenesis.

PubMed

Dong, Jie; Jiang, Guoqian; Asmann, Yan W; Tomaszek, Sandra; Jen, Jin; Kislinger, Thomas; Wigle, Dennis A

2010-05-26

MicroRNAs (miRNAs) are known to be important regulators of both organ development and tumorigenesis. MiRNA networks and their regulation of messenger RNA (mRNA) translation and protein expression in specific biological processes are poorly understood. We explored the dynamic regulation of miRNAs in mouse lung organogenesis. Comprehensive miRNA and mRNA profiling was performed encompassing all recognized stages of lung development beginning at embryonic day 12 and continuing to adulthood. We analyzed the expression patterns of dynamically regulated miRNAs and mRNAs using a number of statistical and computational approaches, and in an integrated manner with protein levels from an existing mass-spectrometry derived protein database for lung development. In total, 117 statistically significant miRNAs were dynamically regulated during mouse lung organogenesis and clustered into distinct temporal expression patterns. 11,220 mRNA probes were also shown to be dynamically regulated and clustered into distinct temporal expression patterns, with 3 major patterns accounting for 75% of all probes. 3,067 direct miRNA-mRNA correlation pairs were identified involving 37 miRNAs. Two defined correlation patterns were observed upon integration with protein data: 1) increased levels of specific miRNAs directly correlating with downregulation of predicted mRNA targets; and 2) increased levels of specific miRNAs directly correlating with downregulation of translated target proteins without detectable changes in mRNA levels. Of 1345 proteins analyzed, 55% appeared to be regulated in this manner with a direct correlation between miRNA and protein level, but without detectable change in mRNA levels. Systematic analysis of microRNA, mRNA, and protein levels over the time course of lung organogenesis demonstrates dynamic regulation and reveals 2 distinct patterns of miRNA-mRNA interaction. The translation of target proteins affected by miRNAs independent of changes in mRNA level appears to be a prominent mechanism of developmental regulation in lung organogenesis.

A novel homozygous stop-codon mutation in human HFE responsible for nonsense-mediated mRNA decay.

PubMed

Padula, Maria Carmela; Martelli, Giuseppe; Larocca, Marilena; Rossano, Rocco; Olivieri, Attilio

2014-09-01

HFE-hemochromatosis (HH) is an autosomal disease characterized by excessive iron absorption. Homozygotes for H63D variant, and still less H63D heterozygotes, generally do not express HH phenotype. The data collected in our previous study in the province of Matera (Basilicata, Italy) underlined that some H63D carriers showed altered iron metabolism, without additional factors. In this study, we selected a cohort of 10/22 H63D carriers with severe biochemical iron overload (BIO). Additional analysis was performed for studying HFE exons, exon-intron boundaries, and untranslated regions (UTRs) by performing DNA extraction, PCR amplification and sequencing. The results showed a novel substitution (NM_000410.3:c.847C>T) in a patient exon 4 (GenBankJQ478433); it introduces a premature stop-codon (PTC). RNA extraction and reverse-transcription were also performed. Quantitative real-time PCR was carried out for verifying if our aberrant mRNA is targeted for nonsense-mediated mRNA decay (NMD); we observed that patient HFE mRNA was expressed much less than calibrator, suggesting that the mutated HFE protein cannot play its role in iron metabolism regulation, resulting in proband BIO. Our finding is the first evidence of a variation responsible for a PTC in iron cycle genes. The genotype-phenotype correlation observed in our cases could be related to the additional mutation. Copyright © 2014 Elsevier Inc. All rights reserved.

CUP promotes deadenylation and inhibits decapping of mRNA targets

PubMed Central

Igreja, Catia; Izaurralde, Elisa

2011-01-01

CUP is an eIF4E-binding protein (4E-BP) that represses the expression of specific maternal mRNAs prior to their posterior localization. Here, we show that CUP employs multiple mechanisms to repress the expression of target mRNAs. In addition to inducing translational repression, CUP maintains mRNA targets in a repressed state by promoting their deadenylation and protects deadenylated mRNAs from further degradation. Translational repression and deadenylation are independent of eIF4E binding and require both the middle and C-terminal regions of CUP, which collectively we termed the effector domain. This domain associates with the deadenylase complex CAF1–CCR4–NOT and decapping activators. Accordingly, in isolation, the effector domain is a potent trigger of mRNA degradation and promotes deadenylation, decapping and decay. However, in the context of the full-length CUP protein, the decapping and decay mediated by the effector domain are inhibited, and target mRNAs are maintained in a deadenylated, repressed form. Remarkably, an N-terminal regulatory domain containing a noncanonical eIF4E-binding motif is required to protect CUP-associated mRNAs from decapping and further degradation, suggesting that this domain counteracts the activity of the effector domain. Our findings indicate that the mode of action of CUP is more complex than previously thought and provide mechanistic insight into the regulation of mRNA expression by 4E-BPs. PMID:21937713

Genome-Wide Survey of Cold Stress Regulated Alternative Splicing in Arabidopsis thaliana with Tiling Microarray

PubMed Central

Leviatan, Noam; Alkan, Noam; Leshkowitz, Dena; Fluhr, Robert

2013-01-01

Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression. PMID:23776682

Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels

PubMed Central

Martins, Rute; Proença, Daniela; Silva, Bruno; Barbosa, Cristina; Silva, Ana Luísa; Faustino, Paula; Romão, Luísa

2012-01-01

Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 3′ untranslated region (UTR), along with exon seven. Therefore, this 3′ UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 3′-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites – two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its protein expression according to the physiological cellular requirements. PMID:22530027

Molecular evidence that the eukaryotic THO/TREX complex is required for efficient transcription elongation.

PubMed

Rondón, Ana G; Jimeno, Sonia; García-Rubio, María; Aguilera, Andrés

2003-10-03

THO/TREX is a conserved eukaryotic complex formed by the core THO complex plus proteins involved in mRNA metabolism and export such as Sub2 and Yra1. Mutations in any of the THO/TREX structural genes cause pleiotropic phenotypes such as transcription impairment, increased transcription-associated recombination, and mRNA export defects. To assay the relevance of THO/TREX complex in transcription, we performed in vitro transcription elongation assays in mutant cell extracts using supercoiled DNA templates containing two G-less cassettes. With these assays, we demonstrate that hpr1delta, tho2delta, and mft1delta mutants of the THO complex and sub2 mutants show significant reductions in the efficiency of transcription elongation. The mRNA expression defect of hpr1delta mutants was not due to an increase in mRNA decay, as determined by mRNA half-life measurements and mRNA time course accumulation experiments in the absence of Rrp6p exoribonuclease. This work demonstrates that THO and Sub2 are required for efficient transcription elongation, providing further evidence for the coupling between transcription and mRNA metabolism and export.

[MicroRNA in neurodegenerative disorders].

PubMed

Sobue, Gen

2013-01-01

MicroRNAs (miRNAs) bind to the 3'-untranslated region of mRNA, and thereby suppress the gene expression. Recent studies suggest that miRNAs modify the pathogenesis of cancer and neurodegeneration. Our study demonstrated that the expression levels of miR-196a is increased in a mouse model of spinal and bulbar muscular atrophy (SBMA), a neurodegenerative disease caused by the expansion of polyglutamine in androgen receptor (AR). In cultured neuronal cells, miR-196a decayed the mutant AR mRNA via silencing CUG triplet repeat RNA binding protein 2, a potent miR-196a targeting mRNA, which contributed to stabilize the mutant AR mRNA. Adeno-associated virus vector-mediated delivery of this miRNA attenuates the expression of the mutant AR, resulting in the mitigation of motor neuron degeneration in the SBMA mice. Introduction of miRNA appears to be a novel therapeutic strategy for devastating neurodegenerative diseases.

mRNA interactome capture in mammalian cells.

PubMed

Kastelic, Nicolai; Landthaler, Markus

2017-08-15

Throughout their entire life cycle, mRNAs are associated with RNA-binding proteins (RBPs), forming ribonucleoprotein (RNP) complexes with highly dynamic compositions. Their interplay is one key to control gene regulatory mechanisms from mRNA synthesis to decay. To assay the global scope of RNA-protein interactions, we and others have published a method combining crosslinking with highly stringent oligo(dT) affinity purification to enrich proteins associated with polyadenylated RNA (poly(A)+ RNA). Identification of the poly(A)+ RNA-bound proteome (also: mRNA interactome capture) has by now been applied to a diversity of cell lines and model organisms, uncovering comprehensive repertoires of RBPs and hundreds of novel RBP candidates. In addition to determining the RBP catalog in a given biological system, mRNA interactome capture allows the examination of changes in protein-mRNA interactions in response to internal and external stimuli, altered cellular programs and disease. Copyright © 2017. Published by Elsevier Inc.

Combined actions of multiple hairpin loop structures and sites of rate-limiting endonucleolytic cleavage determine differential degradation rates of individual segments within polycistronic puf operon mRNA.

PubMed Central

Klug, G; Cohen, S N

1990-01-01

Differential expression of the genes within the puf operon of Rhodobacter capsulatus is accomplished in part by differences in the rate of degradation of different segments of the puf transcript. We report here that decay of puf mRNA sequences specifying the light-harvesting I (LHI) and reaction center (RC) photosynthetic membrane peptides is initiated endoribonucleolytically within a discrete 1.4-kilobase segment of the RC-coding region. Deletion of this segment increased the half-life of the RC-coding region from 8 to 20 min while not affecting decay of LHI-coding sequences upstream from an intercistronic hairpin loop structure shown previously to impede 3'-to-5' degradation. Prolongation of RC segment half-life was dependent on the presence of other hairpin structures 3' to the RC region. Inserting the endonuclease-sensitive sites into the LHI-coding segment markedly accelerated its degradation. Our results suggest that differential degradation of the RC- and LHI-coding segments of puf mRNA is accomplished at least in part by the combined actions of RC region-specific endonuclease(s), one or more exonucleases, and several strategically located exonuclease-impeding hairpins. Images PMID:2394682

Nonsense-mediated mRNA decay: inter-individual variability and human disease

PubMed Central

Nguyen, Lam Son; Wilkinson, Miles; Gecz, Jozef

2013-01-01

Nonsense-Mediated mRNA Decay (NMD) is a regulatory pathway that functions to degrade transcripts containing premature termination codons (PTCs) and to maintain normal transcriptome homeostasis. Nonsense and frameshift mutations that generate PTCs cause approximately one-third of all known human genetic diseases and thus NMD has a potentially important role in human disease. In genetic disorders in which the affected genes carry PTC-generating mutations, NMD acts as a double-edge sword. While it can benefit the patient by degrading PTC-containing mRNAs that encode detrimental, dominant-negative truncated proteins, it can also make the disease worse when a PTC-containing mRNA is degraded that encodes a mutant but still functional protein. There is evidence that the magnitude of NMD varies between individuals, which, in turn, has been shown to correlate with both clinical presentations and the patients’ responses to drugs that promote read-through of PTCs. In this review, we examine the evidence supporting the existence of inter-individual variability in NMD efficiency and discuss the genetic factors that underlie this variability. We propose that inter-individual variability in NMD efficiency is a common phenomenon in human populations and that an individual’s NMD efficiency should be taken into consideration when testing, developing, and making therapeutic decisions for diseases caused by genes harboring PTCs. PMID:24239855

Membrane-association of mRNA decapping factors is independent of stress in budding yeast

PubMed Central

Huch, Susanne; Gommlich, Jessie; Muppavarapu, Mridula; Beckham, Carla; Nissan, Tracy

2016-01-01

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation. PMID:27146487

Membrane-association of mRNA decapping factors is independent of stress in budding yeast.

PubMed

Huch, Susanne; Gommlich, Jessie; Muppavarapu, Mridula; Beckham, Carla; Nissan, Tracy

2016-05-05

Recent evidence has suggested that the degradation of mRNA occurs on translating ribosomes or alternatively within RNA granules called P bodies, which are aggregates whose core constituents are mRNA decay proteins and RNA. In this study, we examined the mRNA decapping proteins, Dcp1, Dcp2, and Dhh1, using subcellular fractionation. We found that decapping factors co-sediment in the polysome fraction of a sucrose gradient and do not alter their behaviour with stress, inhibition of translation or inhibition of the P body formation. Importantly, their localisation to the polysome fraction is independent of the RNA, suggesting that these factors may be constitutively localised to the polysome. Conversely, polysomal and post-polysomal sedimentation of the decapping proteins was abolished with the addition of a detergent, which shifts the factors to the non-translating RNP fraction and is consistent with membrane association. Using a membrane flotation assay, we observed the mRNA decapping factors in the lower density fractions at the buoyant density of membrane-associated proteins. These observations provide further evidence that mRNA decapping factors interact with subcellular membranes, and we suggest a model in which the mRNA decapping factors interact with membranes to facilitate regulation of mRNA degradation.

Kaposi's Sarcoma-Associated Herpesvirus G-Protein-Coupled Receptor Prevents AU-Rich-Element-Mediated mRNA Decay

PubMed Central

Corcoran, Jennifer A.; Khaperskyy, Denys A.; Johnston, Benjamin P.; King, Christine A.; Cyr, David P.; Olsthoorn, Alisha V.

2012-01-01

During lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection, host gene expression is severely restricted by a process of global mRNA degradation known as host shutoff, which rededicates translational machinery to the expression of viral proteins. A subset of host mRNAs is spared from shutoff, and a number of these contain cis-acting AU-rich elements (AREs) in their 3′ untranslated regions. AREs are found in labile mRNAs encoding cytokines, growth factors, and proto-oncogenes. Activation of the p38/MK2 signal transduction pathway reverses constitutive decay of ARE-mRNAs, resulting in increased protein production. The viral G-protein-coupled receptor (vGPCR) is thought to play an important role in promoting the secretion of angiogenic molecules from KSHV-infected cells during lytic replication, but to date it has not been clear how vGPCR circumvents host shutoff. Here, we demonstrate that vGPCR activates the p38/MK2 pathway and stabilizes ARE-mRNAs, augmenting the levels of their protein products. Using MK2-deficient cells, we demonstrate that MK2 is essential for maximal vGPCR-mediated ARE-mRNA stabilization. ARE-mRNAs are normally delivered to cytoplasmic ribonucleoprotein granules known as processing bodies (PBs) for translational silencing and decay. We demonstrate that PB formation is prevented during KSHV lytic replication or in response to vGPCR-mediated activation of RhoA subfamily GTPases. Together, these data show for the first time that vGPCR impacts gene expression at the posttranscriptional level, coordinating an attack on the host mRNA degradation machinery. By suppressing ARE-mRNA turnover, vGPCR may facilitate escape of certain target mRNAs from host shutoff and allow secretion of angiogenic factors from lytically infected cells. PMID:22696654

Structure and Biological Function of the RNA Pyrophosphohydrolase BdRppH from Bdellovibrio bacteriovorus

PubMed Central

Messing, Simon A.J.; Gabelli, Sandra B.; Liu, Quansheng; Celesnik, Helena; Belasco, Joel G.; Piñeiro, Silvia A.; Amzel, L. Mario

2009-01-01

SUMMARY Until recently, the mechanism of mRNA decay in bacteria was thought to be different from that of eukaryotes. This paradigm changed with the discovery that RppH (ORF176/NudH/YgdP), an Escherichia coli enzyme that belongs to the Nudix superfamily, is an RNA resolution pyrophosphohydrolase that initiates mRNA decay by cleaving pyrophosphate from the 5′-triphosphate. Here we report the 1.9 Å structure of the Nudix hydrolase BdRppH from Bdellovibrio bacteriovorus, a bacterium that feeds on other Gram-negative bacteria. Based on the structure of the enzyme alone and in complex with GTP-Mg2+, we propose a mode of RNA binding similar to that of the nuclear decapping enzyme from Xenopus laevis, X29. In additional experiments, we show that BdRppH can indeed function in vitro and in vivo as an RNA pyrophosphohydrolase. These findings set the basis for the identification of possible decapping enzymes in other bacteria. PMID:19278661

Structure and Biological Function of the RNA Pyrophosphohydrolase BdRppH from Bdellovibrio bacteriovorus

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

Messing, S.; Gabelli, S; Liu, Q

2009-01-01

Until recently, the mechanism of mRNA decay in bacteria was thought to be different from that of eukaryotes. This paradigm changed with the discovery that RppH (ORF176/NudH/YgdP), an Escherichia coli enzyme that belongs to the Nudix superfamily, is an RNA pyrophosphohydrolase that initiates mRNA decay by cleaving pyrophosphate from the 5?-triphosphate. Here we report the 1.9 A resolution structure of the Nudix hydrolase BdRppH from Bdellovibrio bacteriovorus, a bacterium that feeds on other Gram-negative bacteria. Based on the structure of the enzyme alone and in complex with GTP-Mg2+, we propose a mode of RNA binding similar to that of themore » nuclear decapping enzyme from Xenopus laevis, X29. In additional experiments, we show that BdRppH can indeed function in vitro and in vivo as an RNA pyrophosphohydrolase. These findings set the basis for the identification of possible decapping enzymes in other bacteria.« less

Non-structural protein 1 of influenza viruses inhibits rapid mRNA degradation mediated by double-stranded RNA-binding protein, staufen1.

PubMed

Cho, Hana; Ahn, Sang Ho; Kim, Kyoung Mi; Kim, Yoon Ki

2013-07-11

Although non-structural protein 1 (NS1) of influenza viruses is not essential for virulence, this protein is involved in host-virus interactions, viral replication, and translation. In particular, NS1 is known to interact with the host protein, staufen1 (Stau1). This interaction is important for efficient viral replication. However, the underlying molecular mechanism by which NS1 influences the viral life cycle remains obscure. Here, we show using immunoprecipitation and artificial tethering that the N-terminus of NS1, NS1(1-73), interacts with Stau1, blocks the Stau1-Upf1 interaction, and consequently inhibits the efficiency of Stau1-mediated mRNA decay (SMD), but not nonsense-mediatedmRNA decay (NMD). The regulation of SMD efficiency by NS1 may contribute to building a more favorable cellular environment for viral replication. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

RNA-Binding Protein Dnd1 Promotes Breast Cancer Apoptosis by Stabilizing the Bim mRNA in a miR-221 Binding Site.

PubMed

Cheng, Feng; Pan, Ying; Lu, Yi-Min; Zhu, Lei; Chen, Shuzheng

2017-01-01

RNA-binding proteins (RBPs) and miRNAs are capable of controlling processes in normal development and cancer. Both of them could determine RNA transcripts fate from synthesis to decay. One such RBP, Dead end (Dnd1), is essential for regulating germ-cell viability and suppresses the germ-cell tumors development, yet how it exerts its functions in breast cancer has remained unresolved. The level of Dnd1 was detected in 21 cancerous tissues paired with neighboring normal tissues by qRT-PCR. We further annotated TCGA (The Cancer Genome Atlas) mRNA expression profiles and found that the expression of Dnd1 and Bim is positively correlated ( p = 0.04). Patients with higher Dnd1 expression level had longer overall survival ( p = 0.0014) by KM Plotter tool. Dnd1 knockdown in MCF-7 cells decreased Bim expression levels and inhibited apoptosis. While knockdown of Dnd1 promoted the decay of Bim mRNA 3'UTR, the stability of Bim-5'UTR was not affected. In addition, mutation of miR-221-binding site in Bim-3'UTR canceled the effect of Dnd1 on Bim mRNA. Knockdown of Dnd1 in MCF-7 cells confirmed that Dnd1 antagonized miR-221-inhibitory effects on Bim expression. Overall, our findings indicate that Dnd1 facilitates apoptosis by increasing the expression of Bim via its competitive combining with miR-221 in Bim-3'UTR. The new function of Dnd1 may contribute to a vital role in breast cancer development.

Post-transcriptional control by bacteriophage T4: mRNA decay and inhibition of translation initiation

PubMed Central

2010-01-01

Over 50 years of biological research with bacteriophage T4 includes notable discoveries in post-transcriptional control, including the genetic code, mRNA, and tRNA; the very foundations of molecular biology. In this review we compile the past 10 - 15 year literature on RNA-protein interactions with T4 and some of its related phages, with particular focus on advances in mRNA decay and processing, and on translational repression. Binding of T4 proteins RegB, RegA, gp32 and gp43 to their cognate target RNAs has been characterized. For several of these, further study is needed for an atomic-level perspective, where resolved structures of RNA-protein complexes are awaiting investigation. Other features of post-transcriptional control are also summarized. These include: RNA structure at translation initiation regions that either inhibit or promote translation initiation; programmed translational bypassing, where T4 orchestrates ribosome bypass of a 50 nucleotide mRNA sequence; phage exclusion systems that involve T4-mediated activation of a latent endoribonuclease (PrrC) and cofactor-assisted activation of EF-Tu proteolysis (Gol-Lit); and potentially important findings on ADP-ribosylation (by Alt and Mod enzymes) of ribosome-associated proteins that might broadly impact protein synthesis in the infected cell. Many of these problems can continue to be addressed with T4, whereas the growing database of T4-related phage genome sequences provides new resources and potentially new phage-host systems to extend the work into a broader biological, evolutionary context. PMID:21129205

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

Kim, Kyoung Mi; Cho, Hana; Kim, Yoon Ki, E-mail: yk-kim@korea.ac.kr

Highlights: Black-Right-Pointing-Pointer CDKN1A mRNA is a bona fide NMD substrate. Black-Right-Pointing-Pointer The uORF of CDKN1A mRNA is efficiently translated. Black-Right-Pointing-Pointer Translation of downstream main ORF is negatively regulated by translation of uORF in CDKN1A mRNA. -- Abstract: The first round of translation occurs on mRNAs bound by nuclear cap-binding complex (CBC), which is composed of nuclear cap-binding protein 80 and 20 (CBP80/20). During this round of translation, aberrant mRNAs are recognized and downregulated in abundance by nonsense-mediated mRNA decay (NMD), which is one of the mRNA quality control mechanisms. Here, our microarray analysis reveals that the level of cyclin-dependent kinasemore » inhibitor 1A (CDKN1A; also known as Waf1/p21) mRNAs increases in cells depleted of cellular NMD factors. Intriguingly, CDKN1A mRNA contains an upstream open reading frame (uORF), which is a NMD-inducing feature. Using chimeric reporter constructs, we find that the uORF of CDKN1A mRNA negatively modulates translation of the main downstream ORF. These findings provide biological insights into the possible role of NMD in diverse biological pathways mediated by CDKN1A.« less

Impairment of FOS mRNA stabilization following translation arrest in granulocytes from myelodysplastic syndrome patients.

PubMed

Feng, Xiaomin; Shikama, Yayoi; Shichishima, Tsutomu; Noji, Hideyoshi; Ikeda, Kazuhiko; Ogawa, Kazuei; Kimura, Hideo; Takeishi, Yasuchika; Kimura, Junko

2013-01-01

Although quantitative and qualitative granulocyte defects have been described in myelodysplastic syndromes (MDS), the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. We recently found that FOS mRNA elevation under translation-inhibiting stimuli was significantly smaller in granulocytes from MDS patients than in healthy individuals. The aim of this study is to clarify the cause of the impaired FOS induction in MDS. We first examined the mechanisms of FOS mRNA elevation using granulocytes from healthy donors cultured with the translation inhibitor emetine. Emetine increased both transcription and mRNA stability of FOS. p38 MAPK inhibition abolished the emetine-induced increase of FOS transcription but did not affect FOS mRNA stabilization. The binding of an AU-rich element (ARE)-binding protein HuR to FOS mRNA containing an ARE in 3'UTR was increased by emetine, and the knockdown of HuR reduced the FOS mRNA stabilizing effect of emetine. We next compared the emetine-induced transcription and mRNA stabilization of FOS between MDS patients and healthy controls. Increased rates of FOS transcription by emetine were similar in MDS and controls. In the absence of emetine, FOS mRNA decayed to nearly 17% of initial levels in 45 min in both groups. In the presence of emetine, however, 76.7±19.8% of FOS mRNA remained after 45 min in healthy controls, versus 37.9±25.5% in MDS (P<0.01). To our knowledge, this is the first report demonstrating attenuation of stress-induced FOS mRNA stabilization in MDS granulocytes.

Molecular events regulating messenger RNA stability in eukaryotes.

PubMed

Saini, K S; Summerhayes, I C; Thomas, P

1990-07-17

The regulation of mRNA turnover plays a major role in the overall control of gene expression. Transcriptional control of eukaryotic gene regulation by external and/or internal stimuli has received considerable attention and the purpose of this review is to highlight recent work elucidating the mechanisms underlying the steady-state levels of mRNAs in the cytoplasm. Protection of mRNA from the action of nucleases as it passes from the nucleus to the ribosomes for translation is achieved, at least in part, by its union with mRNA binding proteins and the presence of poly(A) tail. The half-life of a message represents a balance between the transcriptional activity and intracellular degradative processes. These properties can be modulated by the presence of specific nucleotide sequences in a mRNA along with cis- and trans-acting elements and accompanied by post-translational feed back mechanisms. Presently, various regulatory mechanisms involved in the mRNA decay process are ill-defined. The work described here illustrates the complexity of this emerging field of study and outlines its contribution to our understanding of gene regulation in eukaryotes.

Global Phosphoproteomics Identifies a Major Role for AKT and 14-3-3 in Regulating EDC3*

PubMed Central

Larance, Mark; Rowland, Alexander F.; Hoehn, Kyle L.; Humphreys, David T.; Preiss, Thomas; Guilhaus, Michael; James, David E.

2010-01-01

Insulin plays an essential role in metabolic homeostasis in mammals, and many of the underlying biochemical pathways are regulated via the canonical phosphatidylinositol 3-kinase/AKT pathway. To identify novel metabolic actions of insulin, we conducted a quantitative proteomics analysis of insulin-regulated 14-3-3-binding proteins in muscle cells. These studies revealed a novel role for insulin in the post-transcriptional regulation of mRNA expression. EDC3, a component of the mRNA decay and translation repression pathway associated with mRNA processing bodies, was shown to be phosphorylated by AKT downstream of insulin signaling. The major insulin-regulated site was mapped to Ser-161, and phosphorylation at this site led to increased 14-3-3 binding. Functional studies indicated that induction of 14-3-3 binding to EDC3 causes morphological changes in processing body structures, inhibition of microRNA-mediated mRNA post-transcriptional regulation, and alterations in the protein- protein interactions of EDC3. These data highlight an important new arm of the insulin signaling cascade in the regulation of mRNA utilization. PMID:20051463

Unproductively spliced ribosomal protein mRNAs are natural targets of mRNA surveillance in C. elegans

PubMed Central

Mitrovich, Quinn M.; Anderson, Philip

2000-01-01

Messenger RNA surveillance, the selective and rapid degradation of mRNAs containing premature stop codons, occurs in all eukaryotes tested. The biological role of this decay pathway, however, is not well understood. To identify natural substrates of mRNA surveillance, we used a cDNA-based representational difference analysis to identify mRNAs whose abundance increases in Caenorhabditis elegans smg(−) mutants, which are deficient for mRNA surveillance. Alternatively spliced mRNAs of genes encoding ribosomal proteins L3, L7a, L10a, and L12 are abundant natural targets of mRNA surveillance. Each of these genes expresses two distinct mRNAs. A productively spliced mRNA, whose abundance does not change in smg(−) mutants, encodes a normal, full-length, ribosomal protein. An unproductively spliced mRNA, whose abundance increases dramatically in smg(−) mutants, contains premature stop codons because of incomplete removal of an alternatively spliced intron. In transgenic animals expressing elevated quantities of RPL-12, a greater proportion of endogenous rpl-12 transcript is spliced unproductively. Thus, RPL-12 appears to autoregulate its own splicing, with unproductively spliced mRNAs being degraded by mRNA surveillance. We demonstrate further that alternative splicing of rpl introns is conserved among widely diverged nematodes. Our results suggest that one important role of mRNA surveillance is to eliminate unproductive by-products of gene regulation. PMID:10970881

hnRNP L binds to CA repeats in the 3'UTR of bcl-2 mRNA

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

Lee, Dong-Hyoung; Lim, Mi-Hyun; Youn, Dong-Ye

We previously reported that the CA-repeat sequence in the 3'-untranslated region (3'UTR) of bcl-2 mRNA is involved in the decay of bcl-2 mRNA. However, the trans-acting factor for the CA element in bcl-2 mRNA remains unidentified. The heterogeneous nuclear ribonucleoprotein L (hnRNP L), an intron splicing factor, has been reported to bind to CA repeats and CA clusters in the 3'UTR of several genes. We reported herein that the CA repeats of bcl-2 mRNA have the potential to form a distinct ribonuclear protein complex in cytoplasmic extracts of MCF-7 cells, as evidenced by RNA electrophoretic mobility shift assays (REMSA). Amore » super-shift assay using the hnRNP L antibody completely shifted the complex. Immunoprecipitation with the hnRNP L antibody and MCF-7 cells followed by RT-PCR revealed that hnRNP L interacts with endogenous bcl-2 mRNA in vivo. Furthermore, the suppression of hnRNP L in MCF-7 cells by the transfection of siRNA for hnRNP L resulted in a delay in the degradation of RNA transcripts including CA repeats of bcl-2 mRNA in vitro, suggesting that the interaction between hnRNPL and CA repeats of bcl-2 mRNA participates in destabilizing bcl-2 mRNA.« less

T-lymphocyte cytokine mRNA expression in cystic echinococcosis.

PubMed

Fauser, S; Kern, P

1997-04-01

In the present study we investigated cytokine mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with cystic echinococcosis (CE) after stimulation with different antigens. By using reverse transcriptase polymerase chain reaction (RT-PCR) we could demonstrate that restimulation with crude Echinococcus granulosus antigen (Eg-Ag) induced or enhanced Th2 cytokine mRNA expression, especially IL-5 (by using antigen from sheep cyst fluid) in 23 out of 26 investigated CE patients and IL-10 (by using antigen from camel cyst fluid) in 10 out of 10 investigated CE patients. In contrast, IL-5 mRNA expression was absent in PBMC of healthy controls after Eg-Ag stimulation. To determine the specificity of this reaction we stimulated PBMC from 11 CE patients with crude Echinococcus multilocularis antigen (Em-Ag) and PBMC from 8 CE patients with Toxocara canis antigen (Tc-Ag). We found that the PBMC of patients showed a similar mRNA cytokine pattern on stimulation with Em-Ag when compared with Eg-Ag stimulation. The cytokine mRNA pattern on stimulation with Tc-Ag, however, resembled the cytokine mRNA pattern of unstimulated PBMC. Furthermore, the stimulation of PBMC with crude Mycobacterium tuberculosis antigen (H37Ra) and purified protein derivative (PPD) of M. tuberculosis revealed distinct IL-5 mRNA expression in all investigated CE patients, whereas in healthy controls IL-5 mRNA expression was very weak or totally absent. Thus, our results indicate an induction of Th2 cytokine mRNA expression in CE patients, which is frequently observed in parasite infections. Interestingly, this response persists after stimulation with tuberculosis antigens, which normally induce Th1 response.

Nonsense-Mediated Decay in Genetic Disease: Friend or Foe?

PubMed Central

Miller, Jake N.; Pearce, David A.

2014-01-01

Eukaryotic cells utilize various RNA quality control mechanisms to ensure high fidelity of gene expression, thus protecting against the accumulation of nonfunctional RNA and the subsequent production of abnormal peptides. Messenger RNAs (mRNAs) are largely responsible for protein production, and mRNA quality control is particularly important for protecting the cell against the downstream effects of genetic mutations. Nonsense-mediated decay (NMD) is an evolutionarily conserved mRNA quality control system in all eukaryotes that degrades transcripts containing premature termination codons (PTCs). By degrading these aberrant transcripts, NMD acts to prevent the production of truncated proteins that could otherwise harm the cell through various insults, such as dominant negative effects or the ER stress response. Although NMD functions to protect the cell against the deleterious effects of aberrant mRNA, there is a growing body of evidence that mutation-, codon-, gene-, cell-, and tissue-specific differences in NMD efficiency can alter the underlying pathology of genetic disease. In addition, the protective role that NMD plays in genetic disease can undermine current therapeutic strategies aimed at increasing the production of full-length functional protein from genes harboring nonsense mutations. Here, we review the normal function of this RNA surveillance pathway and how it is regulated, provide current evidence for the role that it plays in modulating genetic disease phenotypes, and how NMD can be used as a therapeutic target. PMID:25485595

Evidence for convergent evolution of SINE-directed Staufen-mediated mRNA decay.

PubMed

Lucas, Bronwyn A; Lavi, Eitan; Shiue, Lily; Cho, Hana; Katzman, Sol; Miyoshi, Keita; Siomi, Mikiko C; Carmel, Liran; Ares, Manuel; Maquat, Lynne E

2018-01-30

Primate-specific Alu short interspersed elements (SINEs) as well as rodent-specific B and ID (B/ID) SINEs can promote Staufen-mediated decay (SMD) when present in mRNA 3'-untranslated regions (3'-UTRs). The transposable nature of SINEs, their presence in long noncoding RNAs, their interactions with Staufen, and their rapid divergence in different evolutionary lineages suggest they could have generated substantial modification of posttranscriptional gene-control networks during mammalian evolution. Some of the variation in SMD regulation produced by SINE insertion might have had a similar regulatory effect in separate mammalian lineages, leading to parallel evolution of the Staufen network by independent expansion of lineage-specific SINEs. To explore this possibility, we searched for orthologous gene pairs, each carrying a species-specific 3'-UTR SINE and each regulated by SMD, by measuring changes in mRNA abundance after individual depletion of two SMD factors, Staufen1 (STAU1) and UPF1, in both human and mouse myoblasts. We identified and confirmed orthologous gene pairs with 3'-UTR SINEs that independently function in SMD control of myoblast metabolism. Expanding to other species, we demonstrated that SINE-directed SMD likely emerged in both primate and rodent lineages >20-25 million years ago. Our work reveals a mechanism for the convergent evolution of posttranscriptional gene regulatory networks in mammals by species-specific SINE transposition and SMD.

Stability of gametocyte-specific Pfs25-mRNA in dried blood spots on filter paper subjected to different storage conditions

PubMed Central

2012-01-01

Background Real-time quantitative nucleic acid sequence-based amplification (QT-NASBA) is a sensitive method for detection of sub-microscopic gametocytaemia by measuring gametocyte-specific mRNA. Performing analysis on fresh whole blood samples is often not feasible in remote and resource-poor areas. Convenient methods for sample storage and transport are urgently needed. Methods Real-time QT-NASBA was performed on whole blood spiked with a dilution series of purified in-vitro cultivated gametocytes. The blood was either freshly processed or spotted on filter papers. Gametocyte detection sensitivity for QT-NASBA was determined and controlled by microscopy. Dried blood spot (DBS) samples were subjected to five different storage conditions and the loss of sensitivity over time was investigated. A formula to approximate the loss of Pfs25-mRNA due to different storage conditions and time was developed. Results Pfs25-mRNA was measured in time to positivity (TTP) and correlated well with the microscopic counts and the theoretical concentrations of the dilution series. TTP results constantly indicated higher amounts of RNA in filter paper samples extracted after 24 hours than in immediately extracted fresh blood. Among investigated storage conditions freezing at −20°C performed best with 98.7% of the Pfs25-mRNA still detectable at day 28 compared to fresh blood samples. After 92 days, the RNA detection rate was only slightly decreased to 92.9%. Samples stored at 37°C showed most decay with only 64.5% of Pfs25-mRNA detectable after one month. The calculated theoretical detection limit for 24 h-old DBS filter paper samples was 0.0095 (95% CI: 0.0025 to 0.0380) per μl. Conclusions The results suggest that the application of DBS filter papers for quantification of Plasmodium falciparum gametocytes with real-time QT-NASBA is practical and recommendable. This method proved sensitive enough for detection of sub-microscopic densities even after prolonged storage. Decay rates can be predicted for different storage conditions as well as durations. PMID:22545954

Rapid Recovery Gene Downregulation during Excess-Light Stress and Recovery in Arabidopsis.

PubMed

Crisp, Peter A; Ganguly, Diep R; Smith, Aaron B; Murray, Kevin D; Estavillo, Gonzalo M; Searle, Iain; Ford, Ethan; Bogdanović, Ozren; Lister, Ryan; Borevitz, Justin O; Eichten, Steven R; Pogson, Barry J

2017-08-01

Stress recovery may prove to be a promising approach to increase plant performance and, theoretically, mRNA instability may facilitate faster recovery. Transcriptome (RNA-seq, qPCR, sRNA-seq, and PARE) and methylome profiling during repeated excess-light stress and recovery was performed at intervals as short as 3 min. We demonstrate that 87% of the stress-upregulated mRNAs analyzed exhibit very rapid recovery. For instance, HSP101 abundance declined 2-fold every 5.1 min. We term this phenomenon rapid recovery gene downregulation (RRGD), whereby mRNA abundance rapidly decreases promoting transcriptome resetting. Decay constants ( k ) were modeled using two strategies, linear and nonlinear least squares regressions, with the latter accounting for both transcription and degradation. This revealed extremely short half-lives ranging from 2.7 to 60.0 min for 222 genes. Ribosome footprinting using degradome data demonstrated RRGD loci undergo cotranslational decay and identified changes in the ribosome stalling index during stress and recovery. However, small RNAs and 5'-3' RNA decay were not essential for recovery of the transcripts examined, nor were any of the six excess light-associated methylome changes. We observed recovery-specific gene expression networks upon return to favorable conditions and six transcriptional memory types. In summary, rapid transcriptome resetting is reported in the context of active recovery and cellular memory. © 2017 American Society of Plant Biologists. All rights reserved.

Rapid Recovery Gene Downregulation during Excess-Light Stress and Recovery in Arabidopsis[OPEN

PubMed Central

Estavillo, Gonzalo M.

2017-01-01

Stress recovery may prove to be a promising approach to increase plant performance and, theoretically, mRNA instability may facilitate faster recovery. Transcriptome (RNA-seq, qPCR, sRNA-seq, and PARE) and methylome profiling during repeated excess-light stress and recovery was performed at intervals as short as 3 min. We demonstrate that 87% of the stress-upregulated mRNAs analyzed exhibit very rapid recovery. For instance, HSP101 abundance declined 2-fold every 5.1 min. We term this phenomenon rapid recovery gene downregulation (RRGD), whereby mRNA abundance rapidly decreases promoting transcriptome resetting. Decay constants (k) were modeled using two strategies, linear and nonlinear least squares regressions, with the latter accounting for both transcription and degradation. This revealed extremely short half-lives ranging from 2.7 to 60.0 min for 222 genes. Ribosome footprinting using degradome data demonstrated RRGD loci undergo cotranslational decay and identified changes in the ribosome stalling index during stress and recovery. However, small RNAs and 5ʹ-3ʹ RNA decay were not essential for recovery of the transcripts examined, nor were any of the six excess light-associated methylome changes. We observed recovery-specific gene expression networks upon return to favorable conditions and six transcriptional memory types. In summary, rapid transcriptome resetting is reported in the context of active recovery and cellular memory. PMID:28705956

Translation initiation mediated by nuclear cap-binding protein complex.

PubMed

Ryu, Incheol; Kim, Yoon Ki

2017-04-01

In mammals, cap-dependent translation of mRNAs is initiated by two distinct mechanisms: cap-binding complex (CBC; a heterodimer of CBP80 and 20)-dependent translation (CT) and eIF4E-dependent translation (ET). Both translation initiation mechanisms share common features in driving cap- dependent translation; nevertheless, they can be distinguished from each other based on their molecular features and biological roles. CT is largely associated with mRNA surveillance such as nonsense-mediated mRNA decay (NMD), whereas ET is predominantly involved in the bulk of protein synthesis. However, several recent studies have demonstrated that CT and ET have similar roles in protein synthesis and mRNA surveillance. In a subset of mRNAs, CT preferentially drives the cap-dependent translation, as ET does, and ET is responsible for mRNA surveillance, as CT does. In this review, we summarize and compare the molecular features of CT and ET with a focus on the emerging roles of CT in translation. [BMB Reports 2017; 50(4): 186-193].

Role of transcription factor Sp1 and RNA binding protein HuR in the down-regulation of Dr+ Escherichia coli receptor protein Decay Accelerating Factor (DAF or CD55) by Nitric oxide

PubMed Central

Banadakoppa, Manu; Liebenthal, Daniel; Nowak, David E; Urvil, Petri; Yallampalli, Uma; Wilson, Gerald M; Kishor, Aparna; Yallampalli, Chandra

2012-01-01

We previously reported that nitric oxide (NO) reduces the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr Fimbria (Dr+). The epithelial invasion of Dr+ E. coli is dependent on the expression level of its cellular receptor decay accelerating factor (DAF). NO reduces the rate of bacteremia by down-regulating the expression of DAF. In this study, we elucidated the role of transcription factor Sp1 and RNA binding protein HuR in the down-regulation of human DAF by NO. We generated a series of deletion mutant constructs of DAF gene 5′-untranslated region and mapped NO-response region upstream to the core promoter region of the DAF gene. One of the several Sp1 binding sites in the DAF 5′-untranslated region was located within the NO-response region. The binding of Sp1 to this site was inhibited by NO. Furthermore, NO also promoted the degradation of DAF mRNA. The 3′-untranslated region of DAF harbors an AU-rich element and this element destabilized the mRNA transcript. The NO promoted the rapid degradation of DAF mRNA by inhibiting the binding of mRNA stabilizing protein HuR to this AU-rich region. The inhibition of binding of HuR to AU-rich region was due to the S-nitrosylation of one or more cysteine residues by NO. Thus, these data reveal the molecular mediators of transcriptional and post-transcriptional regulation of DAF by NO with implications in pathophysiology related to DAF. PMID:23176121

Role of transcription factor Sp1 and RNA binding protein HuR in the downregulation of Dr+ Escherichia coli receptor protein decay accelerating factor (DAF or CD55) by nitric oxide.

PubMed

Banadakoppa, Manu; Liebenthal, Daniel; Nowak, David E; Urvil, Petri; Yallampalli, Uma; Wilson, Gerald M; Kishor, Aparna; Yallampalli, Chandra

2013-02-01

We previously reported that nitric oxide (NO) reduces the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr Fimbria (Dr(+) ). The epithelial invasion of Dr(+) E. coli is dependent on the expression level of its cellular receptor decay accelerating factor (DAF). NO reduces the rate of bacteremia by downregulating the expression of DAF. In this study, we elucidated the role of transcription factor Sp1 and RNA binding protein HuR in the downregulation of human DAF by NO. We generated a series of deletion mutant constructs of DAF gene 5'-untranslated region and mapped the NO-response region upstream to the core promoter region of the DAF gene. One of the several Sp1 binding sites in the DAF 5'-untranslated region was located within the NO-response region. The binding of Sp1 to this site was inhibited by NO. Furthermore, NO also promoted the degradation of DAF mRNA. The 3'-untranslated region of DAF harbors an AU-rich element and this element destabilized the mRNA transcript. NO promoted the rapid degradation of DAF mRNA by inhibiting the binding of mRNA stabilizing protein HuR to this AU-rich region. The inhibition of binding of HuR to the AU-rich region was due to the S-nitrosylation of one or more cysteine residues by NO. Thus, these data reveal the molecular mediators of transcriptional and post-transcriptional regulation of DAF by NO with implications in pathophysiology related to DAF. © 2012 The Authors Journal compilation © 2012 FEBS.

Posttranscriptional regulation of retroviral gene expression: primary RNA transcripts play three roles as pre-mRNA, mRNA, and genomic RNA

PubMed Central

LeBlanc, Jason; Weil, Jason; Beemon, Karen

2013-01-01

After reverse transcription of the retroviral RNA genome and integration of the DNA provirus into the host genome, host machinery is used for viral gene expression along with viral proteins and RNA regulatory elements. Here, we discuss co-transcriptional and posttranscriptional regulation of retroviral gene expression, comparing simple and complex retroviruses. Cellular RNA polymerase II synthesizes full-length viral primary RNA transcripts that are capped and polyadenylated. All retroviruses generate a singly spliced env mRNA from this primary transcript, which encodes the viral glycoproteins. In addition, complex viral RNAs are alternatively spliced to generate accessory proteins, such as Rev, which is involved in posttranscriptional regulation of HIV-1 RNA. Importantly, the splicing of all retroviruses is incomplete; they must maintain and export a fraction of their primary RNA transcripts. This unspliced RNA functions both as the major mRNA for Gag and Pol proteins and as the packaged genomic RNA. Different retroviruses export their unspliced viral RNA from the nucleus to the cytoplasm by either Tap-dependent or Rev/CRM1-dependent routes. Translation of the unspliced mRNA involves frame-shifting or termination codon suppression so that the Gag proteins, which make up the capsid, are expressed more abundantly than the Pol proteins, which are the viral enzymes. After the viral polyproteins assemble into viral particles and bud from the cell membrane, a viral encoded protease cleaves them. Some retroviruses have evolved mechanisms to protect their unspliced RNA from decay by nonsense-mediated RNA decay and to prevent genome editing by the cellular APOBEC deaminases. PMID:23754689

7-methylguanosine diphosphate (m(7)GDP) is not hydrolyzed but strongly bound by decapping scavenger (DcpS) enzymes and potently inhibits their activity.

PubMed

Wypijewska, Anna; Bojarska, Elzbieta; Lukaszewicz, Maciej; Stepinski, Janusz; Jemielity, Jacek; Davis, Richard E; Darzynkiewicz, Edward

2012-10-09

Decapping scavenger (DcpS) enzymes catalyze the cleavage of a residual cap structure following 3' → 5' mRNA decay. Some previous studies suggested that both m(7)GpppG and m(7)GDP were substrates for DcpS hydrolysis. Herein, we show that mononucleoside diphosphates, m(7)GDP (7-methylguanosine diphosphate) and m(3)(2,2,7)GDP (2,2,7-trimethylguanosine diphosphate), resulting from mRNA decapping by the Dcp1/2 complex in the 5' → 3' mRNA decay, are not degraded by recombinant DcpS proteins (human, nematode, and yeast). Furthermore, whereas mononucleoside diphosphates (m(7)GDP and m(3)(2,2,7)GDP) are not hydrolyzed by DcpS, mononucleoside triphosphates (m(7)GTP and m(3)(2,2,7)GTP) are, demonstrating the importance of a triphosphate chain for DcpS hydrolytic activity. m(7)GTP and m(3)(2,2,7)GTP are cleaved at a slower rate than their corresponding dinucleotides (m(7)GpppG and m(3)(2,2,7)GpppG, respectively), indicating an involvement of the second nucleoside for efficient DcpS-mediated digestion. Although DcpS enzymes cannot hydrolyze m(7)GDP, they have a high binding affinity for m(7)GDP and m(7)GDP potently inhibits DcpS hydrolysis of m(7)GpppG, suggesting that m(7)GDP may function as an efficient DcpS inhibitor. Our data have important implications for the regulatory role of m(7)GDP in mRNA metabolic pathways due to its possible interactions with different cap-binding proteins, such as DcpS or eIF4E.

Upf1 senses 3′UTR length to potentiate mRNA decay

PubMed Central

Hogg, J. Robert; Goff, Stephen P.

2010-01-01

Summary The selective degradation of mRNAs by the nonsense-mediated decay pathway is a quality control process with important consequences for human disease. From initial studies using RNA hairpin-tagged mRNAs for purification of messenger ribonucleoproteins assembled on transcripts with HIV-1 3′ untranslated region (3′UTR) sequences, we uncover a two-step mechanism for Upf1-dependent degradation of mRNAs with long 3′UTRs. We demonstrate that Upf1 associates with mRNAs in a 3′UTR length-dependent manner and is highly enriched on transcripts containing 3′UTRs known to elicit NMD. Surprisingly, Upf1 recruitment and subsequent RNA decay can be antagonized by retroviral RNA elements that promote translational readthrough. By modulating the efficiency of translation termination, recognition of long 3′UTRs by Upf1 is uncoupled from the initiation of decay. We propose a model for 3′UTR length surveillance in which equilibrium binding of Upf1 to mRNAs precedes a kinetically distinct commitment to RNA decay. PMID:21029861

Staufen1 dimerizes via a conserved motif and a degenerate dsRNA-binding domain to promote mRNA decay

PubMed Central

Gleghorn, Michael L.; Gong, Chenguang; Kielkopf, Clara L.; Maquat, Lynne E.

2014-01-01

Staufen (STAU)1-mediated mRNA decay (SMD) degrades mammalian-cell mRNAs that bind the double-stranded (ds)RNA-binding protein STAU1 in their 3′-untranslated region. We report a new motif, which typifies STAU homologs from all vertebrate classes, that is responsible for human (h)STAU1 homodimerization. Our crystal structure and mutagenesis analyses reveal that this motif, now named the Staufen-swapping motif (SSM), and dsRNA-binding domain 5 (‘RBD’5) mediate protein dimerization: the two SSM α-helices of one molecule interact primarily through a hydrophobic patch with the two ‘RBD’5 α-helices of a second molecule. ‘RBD’5 adopts the canonical α-β-β-β-α fold of a functional RBD, but it lacks residues and features needed to bind duplex RNA. In cells, SSM-mediated hSTAU1 dimerization increases the efficiency of SMD by augmenting hSTAU1 binding to the ATP-dependent RNA helicase hUPF1. Dimerization regulates keratinocyte-mediated wound-healing and, undoubtedly, many other cellular processes. PMID:23524536

ICE1 promotes the link between splicing and nonsense-mediated mRNA decay

PubMed Central

Baird, Thomas D; Cheng, Ken Chih-Chien; Chen, Yu-Chi; Buehler, Eugen; Martin, Scott E; Inglese, James

2018-01-01

The nonsense-mediated mRNA decay (NMD) pathway detects aberrant transcripts containing premature termination codons (PTCs) and regulates expression of 5–10% of non-aberrant human mRNAs. To date, most proteins involved in NMD have been identified by genetic screens in model organisms; however, the increased complexity of gene expression regulation in human cells suggests that additional proteins may participate in the human NMD pathway. To identify proteins required for NMD, we performed a genome-wide RNAi screen against >21,000 genes. Canonical members of the NMD pathway were highly enriched as top hits in the siRNA screen, along with numerous candidate NMD factors, including the conserved ICE1/KIAA0947 protein. RNAseq studies reveal that depletion of ICE1 globally enhances accumulation and stability of NMD-target mRNAs. Further, our data suggest that ICE1 uses a putative MIF4G domain to interact with exon junction complex (EJC) proteins and promotes the association of the NMD protein UPF3B with the EJC. PMID:29528287

Effects of PTCs on nonsense-mediated mRNA decay are dependent on PTC location.

PubMed

Moon, Heegyum; Zheng, Xuexiu; Loh, Tiing Jen; Jang, Ha Na; Liu, Yongchao; Jung, Da-Woon; Williams, Darren R; Shen, Haihong

2017-03-01

The récepteur d'origine nantais (RON) gene is a proto-oncogene that is responsible for encoding the human macrophage-stimulating protein (MSP) 1 receptor. MSP activation induces RON-mediated cell dissociation, migration and matrix invasion. Isoforms of RON that exclude exons 5 and 6 encode the RONΔ160 protein, which promotes cell transformation in vitro and tumor metastasis in vivo . Premature termination codons (PTCs) in exons activate the nonsense-mediated mRNA decay (NMD) signaling pathway. The present study demonstrated that PTCs at various locations in the alternative exons 5 and 6 could induce NMD of the majority of the spliced, or partially spliced, isoforms. However, the isoforms that excluded exon 6 or exons 5 and 6 were markedly increased when produced from mutated minigenes with inserted PTCs. Furthermore, the unspliced isoform of intron 5 was not observed to be decreased by the presence of PTCs. Notably, these effects may be dependent on the location of the PTCs. The current study demonstrated a novel mechanism underlying the regulation of NMD in alternative splicing.

A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement*

PubMed Central

Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

2016-01-01

Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. PMID:26887951

A small RNA activates CFA synthase by isoform-specific mRNA stabilization

PubMed Central

Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

2013-01-01

Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5′ end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5′ untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability. PMID:24141880

A small RNA activates CFA synthase by isoform-specific mRNA stabilization.

PubMed

Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

2013-11-13

Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5' end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5' untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.

Effect of iron on accumulation of exotoxin A-specific mRNA in Pseudomonas aeruginosa.

PubMed Central

Lory, S

1986-01-01

A DNA probe from an internal fragment of the exotoxin A structural gene was used to study the effects of selected culture conditions on steady-state levels of exotoxin-specific mRNA in Pseudomonas aeruginosa. Cells grown under conditions of iron deprivation began to synthesize and excrete the exotoxin A polypeptide during the late exponential phase of growth and throughout the stationary phase of growth, concomitant with a sharp increase in exotoxin A mRNA pools in P. aeruginosa cells. The addition of iron to the medium resulted in the failure of these cells to synthesize exotoxin A mRNA, despite significantly enhanced growth. The inhibition of the production of exotoxin A and the accumulation of its mRNA by iron was dose dependent, with a half-maximal inhibitory concentration of FeSO4 of 5 to 10 microM. A blockade of the initiation of transcription by rifampin resulted in the decay of exotoxin A mRNA, with a half-life of approximately 8 to 10 min, depending on the media used for growth. The addition of iron to cells actively engaged in exotoxin A synthesis also resulted in a gradual decrease in the amount of this mRNA in bacteria. However, the rate of decline of mRNA induced by iron was relatively slow (half-life, 90 min), with a considerable lag time between the iron addition and the first detectable effect on mRNA. While iron clearly appears to influence the production of exotoxin A at the transcriptional level, the molecular basis of this effect may involve several interacting factors affecting the initiation of transcription and perhaps mRNA turnover. Images PMID:2430950

Uncovering Patterns of Inter-Urban Trip and Spatial Interaction from Social Media Check-In Data

PubMed Central

Liu, Yu; Sui, Zhengwei; Kang, Chaogui; Gao, Yong

2014-01-01

The article revisits spatial interaction and distance decay from the perspective of human mobility patterns and spatially-embedded networks based on an empirical data set. We extract nationwide inter-urban movements in China from a check-in data set that covers half a million individuals within 370 cities to analyze the underlying patterns of trips and spatial interactions. By fitting the gravity model, we find that the observed spatial interactions are governed by a power law distance decay effect. The obtained gravity model also closely reproduces the exponential trip displacement distribution. The movement of an individual, however, may not obey the same distance decay effect, leading to an ecological fallacy. We also construct a spatial network where the edge weights denote the interaction strengths. The communities detected from the network are spatially cohesive and roughly consistent with province boundaries. We attribute this pattern to different distance decay parameters between intra-province and inter-province trips. PMID:24465849

Uncovering patterns of inter-urban trip and spatial interaction from social media check-in data.

PubMed

Liu, Yu; Sui, Zhengwei; Kang, Chaogui; Gao, Yong

2014-01-01

The article revisits spatial interaction and distance decay from the perspective of human mobility patterns and spatially-embedded networks based on an empirical data set. We extract nationwide inter-urban movements in China from a check-in data set that covers half a million individuals within 370 cities to analyze the underlying patterns of trips and spatial interactions. By fitting the gravity model, we find that the observed spatial interactions are governed by a power law distance decay effect. The obtained gravity model also closely reproduces the exponential trip displacement distribution. The movement of an individual, however, may not obey the same distance decay effect, leading to an ecological fallacy. We also construct a spatial network where the edge weights denote the interaction strengths. The communities detected from the network are spatially cohesive and roughly consistent with province boundaries. We attribute this pattern to different distance decay parameters between intra-province and inter-province trips.

Tissue-specific mRNA expression profiling in grape berry tissues

PubMed Central

Grimplet, Jerome; Deluc, Laurent G; Tillett, Richard L; Wheatley, Matthew D; Schlauch, Karen A; Cramer, Grant R; Cushman, John C

2007-01-01

Background Berries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and transport processes. Seeds, which supply essential resources for embryo development, showed higher mRNA abundance of genes encoding phenylpropanoid biosynthetic enzymes, seed storage proteins, and late embryogenesis abundant proteins. Water-deficit stress affected the mRNA abundance of 13% of the genes with differential expression patterns occurring mainly in the pulp and skin. In pulp and seed tissues transcript abundance in most functional categories declined in water-deficit stressed vines relative to well-watered vines with transcripts for storage proteins and novel (no-hit) functional assignments being over represented. In the skin of berries from water-deficit stressed vines, however, transcripts from several functional categories including general phenypropanoid and ethylene metabolism, pathogenesis-related responses, energy, and interaction with the environment were significantly over-represented. Conclusion These results revealed novel insights into the tissue-specific expression mRNA expression patterns of an extensive repertoire of genes expressed in berry tissues. This work also establishes an extensive catalogue of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern tissue-specific expression patterns associated with tissue differentiation within berries. These results also confirmed that water-deficit stress has a profound effect on mRNA expression patterns particularly associated with the biosynthesis of aroma and color metabolites within skin and pulp tissues that ultimately impact wine quality. PMID:17584945

Regulation of gene expression in mammalian nervous system through alternative pre-mRNA splicing coupled with RNA quality control mechanisms.

PubMed

Yap, Karen; Makeyev, Eugene V

2013-09-01

Eukaryotic gene expression is orchestrated on a genome-wide scale through several post-transcriptional mechanisms. Of these, alternative pre-mRNA splicing expands the proteome diversity and modulates mRNA stability through downstream RNA quality control (QC) pathways including nonsense-mediated decay (NMD) of mRNAs containing premature termination codons and nuclear retention and elimination (NRE) of intron-containing transcripts. Although originally identified as mechanisms for eliminating aberrant transcripts, a growing body of evidence suggests that NMD and NRE coupled with deliberate changes in pre-mRNA splicing patterns are also used in a number of biological contexts for deterministic control of gene expression. Here we review recent studies elucidating molecular mechanisms and biological significance of these gene regulation strategies with a specific focus on their roles in nervous system development and physiology. This article is part of a Special Issue entitled 'RNA and splicing regulation in neurodegeneration'. Copyright © 2013 Elsevier Inc. All rights reserved.

Homozygous SALL1 Mutation Causes a Novel Multiple Congenital Anomaly—Mental Retardation Syndrome

PubMed Central

Vodopiutz, Julia; Zoller, Heinz; Fenwick, Aimée L.; Arnhold, Richard; Schmid, Max; Prayer, Daniela; Müller, Thomas; Repa, Andreas; Pollak, Arnold; Aufricht, Christoph; Wilkie, Andrew O.M.; Janecke, Andreas R.

2013-01-01

Objective To delineate a novel autosomal recessive multiple congenital anomaly-mental retardation (MCA-MR) syndrome in 2 female siblings of a consanguineous pedigree and to identify the disease-causing mutation. Study design Both siblings were clinically characterized and homozygosity mapping and sequencing of candidate genes were applied. The contribution of nonsense-mediated messenger RNA (mRNA) decay to the expression of mutant mRNA in fibroblasts of a healthy carrier and a control was studied by pyrosequencing. Results We identified the first homozygous SALL1 mutation, c.3160C > T (p.R1054*), in 2 female siblings presenting with multiple congenital anomalies, central nervous system defects, cortical blindness, and absence of psychomotor development (ie, a novel recognizable, autosomal recessive MCA-MR). The mutant SALL1 transcript partially undergoes nonsense-mediated mRNA decay and is present at 43% of the normal transcript level in the fibroblasts of a healthy carrier. Conclusion Previously heterozygous SALL1 mutations and deletions have been associated with dominantly inherited anal-renal-radial-ear developmental anomalies. We identified an allelic recessive SALL1-related MCA-MR. Our findings imply that quantity and quality of SALL1 transcript are important for SALL1 function and determine phenotype, and mode of inheritance, of allelic SALL1-related disorders. This novel MCA-MR emphasizes SALL1 function as critical for normal central nervous system development and warrants a detailed neurologic investigation in all individuals with SALL1 mutations. PMID:23069192

Unique Temporal Expression of Triplicated Long-Wavelength Opsins in Developing Butterfly Eyes

PubMed Central

Arikawa, Kentaro; Iwanaga, Tomoyuki; Wakakuwa, Motohiro; Kinoshita, Michiyo

2017-01-01

Following gene duplication events, the expression patterns of the resulting gene copies can often diverge both spatially and temporally. Here we report on gene duplicates that are expressed in distinct but overlapping patterns, and which exhibit temporally divergent expression. Butterflies have sophisticated color vision and spectrally complex eyes, typically with three types of heterogeneous ommatidia. The eyes of the butterfly Papilio xuthus express two green- and one red-absorbing visual pigment, which came about via gene duplication events, in addition to one ultraviolet (UV)- and one blue-absorbing visual pigment. We localized mRNAs encoding opsins of these visual pigments in developing eye disks throughout the pupal stage. The mRNAs of the UV and blue opsin are expressed early in pupal development (pd), specifying the type of the ommatidium in which they appear. Red sensitive photoreceptors first express a green opsin mRNA, which is replaced later by the red opsin mRNA. Broadband photoreceptors (that coexpress the green and red opsins) first express the green opsin mRNA, later change to red opsin mRNA and finally re-express the green opsin mRNA in addition to the red mRNA. Such a unique temporal and spatial expression pattern of opsin mRNAs may reflect the evolution of visual pigments and provide clues toward understanding how the spectrally complex eyes of butterflies evolved. PMID:29238294

Transterm—extended search facilities and improved integration with other databases

PubMed Central

Jacobs, Grant H.; Stockwell, Peter A.; Tate, Warren P.; Brown, Chris M.

2006-01-01

Transterm has now been publicly available for >10 years. Major changes have been made since its last description in this database issue in 2002. The current database provides data for key regions of mRNA sequences, a curated database of mRNA motifs and tools to allow users to investigate their own motifs or mRNA sequences. The key mRNA regions database is derived computationally from Genbank. It contains 3′ and 5′ flanking regions, the initiation and termination signal context and coding sequence for annotated CDS features from Genbank and RefSeq. The database is non-redundant, enabling summary files and statistics to be prepared for each species. Advances include providing extended search facilities, the database may now be searched by BLAST in addition to regular expressions (patterns) allowing users to search for motifs such as known miRNA sequences, and the inclusion of RefSeq data. The database contains >40 motifs or structural patterns important for translational control. In this release, patterns from UTRsite and Rfam are also incorporated with cross-referencing. Users may search their sequence data with Transterm or user-defined patterns. The system is accessible at . PMID:16381889

A comparison of postburn woodpecker foraging use of white fir (Abies concolor) and Jeffrey Pine (Pinus jeffreyi)

Treesearch

Kerry L. Farris; Steve Zack

2008-01-01

We examined the temporal patterns of the structural decay, insect infestation and woodpecker foraging patterns on white-fir and yellow pine following a prescribed burn in Lassen National Park, CA. Our objectives were to: 1) describe how pine and fir differ in their decay patterns and insect activity, and 2) determine how these differences reflect woodpecker foraging...

Predictive model for CO2 generation and decay in building envelopes

NASA Astrophysics Data System (ADS)

Aglan, Heshmat A.

2003-01-01

Understanding carbon dioxide generation and decay patterns in buildings with high occupancy levels is useful to identify their indoor air quality, air change rates, percent fresh air makeup, occupancy pattern, and how a variable air volume system to off-set undesirable CO2 level can be modulated. A mathematical model governing the generation and decay of CO2 in building envelopes with forced ventilation due to high occupancy is developed. The model has been verified experimentally in a newly constructed energy efficient healthy house. It was shown that the model accurately predicts the CO2 concentration at any time during the generation and decay processes.

Notch and Delta mRNAs in early-stage and mid-stage Drosophila embryos exhibit complementary patterns of protein producing potentials

PubMed Central

Shepherd, Andrew; Wesley, Uma; Wesley, Cedric

2010-01-01

Notch and Delta proteins generate Notch signaling that specifies cell fates during animal development. There is an intriguing phenomenon in Drosophila embryogenesis that has not received much attention and whose significance to embryogenesis is unknown. Notch and Delta mRNAs expressed in early-stage embryos are shorter than their counterparts in mid-stage embryos. We show here that the difference in sizes is due to mRNA 3′ processing at alternate polyadenylation sites. While the early-stage Notch mRNA has a lower protein-producing potential than the mid-stage Notch mRNA, the early-stage Delta mRNA has a higher protein-producing potential than the mid-stage Delta mRNA. Our data can explain the complementary patterns of Notch and Delta protein levels in early-stage and mid-stage embryos. Our data also raise the possibility that the manner and regulation of Notch signaling change in the course of embryogenesis and that this change is effected by 3′ UTR and mRNA 3′ processing factors. PMID:20201103

Modulation of transferrin receptor mRNA by transferrin-gallium in human myeloid HL60 and lymphoid CCRF-CEM leukaemic cells.

PubMed Central

Ul-Haq, R; Chitambar, C R

1993-01-01

Gallium binds to the iron transport protein transferrin (Tf), is incorporated into cells through transferrin receptors (TfR) and inhibits iron-dependent DNA synthesis. Since cellular TfR expression is tightly regulated by the availability of iron, we investigated the effects of transferrin-gallium (Tf-Ga) on TfR mRNA levels in myeloid HL60 and lymphoid CCRF-CEM cells. In HL60 cells, Tf-Ga increased TfR mRNA levels in a dose-dependent fashion. This increase in TfR mRNA was blocked by Tf-Fe and by cycloheximide. Analysis of the rate of mRNA decay in the presence of actinomycin D revealed that the half-life of TfR mRNA was increased in HL60 cells incubated with Tf-Ga. The rate of transcription of TfR mRNA was not increased by Tf-Ga. In contrast with HL60 cells, CCRF-CEM cells displayed a decrease in the level of TfR mRNA after incubation with Tf-Ga. Tf-Ga inhibited iron uptake in both HL60 and CCRF-CEM cells but increased the level of TfR mRNA only in HL60 cells, suggesting that the Tf-Ga induction of TfR mRNA was not solely due to inhibition of cellular iron uptake. At growth-inhibitory concentrations, Tf-Ga increased the TfR mRNA level in HL60 cells but decreased it in CCRF-CEM cells. Our studies suggest that in HL60 cells, gallium regulates TfR expression at the post-transcriptional level by mechanisms which require de novo protein synthesis and involve interaction with iron. The divergent effects of Tf-Ga on TfR mRNA in myeloid HL60 and lymphoid CCRF-CEM cells suggest that differences exist in the regulation of TfR expression between these two cell types. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8379943

Detection of active decay at groundline in utility poles

Treesearch

Alex L. Shigo; Walter C. Shortle; Julian Ochrymowych

1977-01-01

Active wood decay at groundline in in-service utility poles can be detected by a skilled inspector using: 1. A knowledge of basic patterns of decay. 2. Recognition of obvious signs of decay. 3. Proper interpretation of information obtained from a pulsed-current meter-Shigometer®-used with various probes and probing techniques.

Photographic monitoring of soiling and decay of roadside walls in central Oxford, England

NASA Astrophysics Data System (ADS)

Thornbush, Mary J.; Viles, Heather A.

2008-12-01

As part of the Environmental Monitoring of Integrated Transport Strategies (EMITS) project, which examined the impact of the Oxford Transport Strategy (OTS) on the soiling and decay of buildings and structures in central Oxford, England, a simple photographic survey of a sample of roadside walls was carried out in 1997, with re-surveys in 1999 and 2003. Thirty photographs were taken each time, covering an area of stonework approximately 30 × 30 cm in dimensions at 1-1.3 m above pavement level. The resulting images have been used to investigate, both qualitatively as well as quantitatively, the progression of soiling and decay. Comparison of images by eye reveals a number of minor changes in soiling and decay patterns, but generally indicates stability except at one site where dramatic, superficial damage occurred over 2 years. Quantitative analysis of decay features (concavities resulting from surface blistering, flaking, and scaling), using simple techniques in Adobe Photoshop, shows variable pixel-based size proportions of concavities across 6 years of survey. Colour images (in Lab Color) generally have a reduced proportion of pixels, representing decay features in comparison to black and white (Grayscale) images. The study conveys that colour images provide more information both for general observations of soiling and decay patterns and for segmentation of decay-produced concavities. The study indicates that simple repeat photography can reveal useful information about changing patterns of both soiling and decay, although unavoidable variation in external lighting conditions between re-surveys is a factor limiting the accuracy of change detection.

Expression of myosin heavy chain isoforms mRNA transcripts in the temporalis muscle of common chimpanzees (Pan troglodytes).

PubMed

Ciurana, Neus; Artells, Rosa; Muñoz, Carmen; Arias-Martorell, Júlia; Bello-Hellegouarch, Gaëlle; Casado, Aroa; Cuesta, Elisabeth; Pérez-Pérez, Alejandro; Pastor, Juan Francisco; Potau, Josep Maria

2017-11-01

The common chimpanzee (Pan troglodytes) is the primate that is phylogenetically most closely related to humans (Homo sapiens). In order to shed light on the anatomy and function of the temporalis muscle in the chimpanzee, we have analyzed the expression patterns of the mRNA transcripts of the myosin heavy chain (MyHC) isoforms in different parts of the muscle. We dissected the superficial, deep and sphenomandibularis portions of the temporalis muscle in five adult P. troglodytes and quantified the expression of the mRNA transcripts of the MyHC isoforms in each portion using real-time quantitative polymerase chain reaction. We observed significant differences in the patterns of expression of the mRNA transcripts of the MyHC-IIM isoform between the sphenomandibularis portion and the anterior superficial temporalis (33.6% vs 47.0%; P=0.032) and between the sphenomandibularis portion and the anterior deep temporalis (33.6% vs 43.0; P=0.016). We also observed non-significant differences between the patterns of expression in the anterior and posterior superficial temporalis. The differential expression patterns of the mRNA transcripts of the MyHC isoforms in the temporalis muscle in P. troglodytes may be related to the functional differences that have been observed in electromyographic studies in other species of primates. Our findings can be applicable to the fields of comparative anatomy, evolutionary anatomy, and anthropology. Copyright © 2017 Elsevier GmbH. All rights reserved.

A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement.

PubMed

Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

2016-04-15

Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Partial androgen insensitivity syndrome caused by a deep intronic mutation creating an alternative splice acceptor site of the AR gene.

PubMed

Ono, Hiroyuki; Saitsu, Hirotomo; Horikawa, Reiko; Nakashima, Shinichi; Ohkubo, Yumiko; Yanagi, Kumiko; Nakabayashi, Kazuhiko; Fukami, Maki; Fujisawa, Yasuko; Ogata, Tsutomu

2018-02-02

Although partial androgen insensitivity syndrome (PAIS) is caused by attenuated responsiveness to androgens, androgen receptor gene (AR) mutations on the coding regions and their splice sites have been identified only in <25% of patients with a diagnosis of PAIS. We performed extensive molecular studies including whole exome sequencing in a Japanese family with PAIS, identifying a deep intronic variant beyond the branch site at intron 6 of AR (NM_000044.4:c.2450-42 G > A). This variant created the splice acceptor motif that was accompanied by pyrimidine-rich sequence and two candidate branch sites. Consistent with this, reverse transcriptase (RT)-PCR experiments for cycloheximide-treated lymphoblastoid cell lines revealed a relatively large amount of aberrant mRNA produced by the newly created splice acceptor site and a relatively small amount of wildtype mRNA produced by the normal splice acceptor site. Furthermore, most of the aberrant mRNA was shown to undergo nonsense mediated decay (NMD) and, if a small amount of aberrant mRNA may have escaped NMD, such mRNA was predicted to generate a truncated AR protein missing some functional domains. These findings imply that the deep intronic mutation creating an alternative splice acceptor site resulted in the production of a relatively small amount of wildtype AR mRNA, leading to PAIS.

Differential expression of decorin and biglycan genes during mouse tooth development

NASA Technical Reports Server (NTRS)

Matsuura, T.; Duarte, W. R.; Cheng, H.; Uzawa, K.; Yamauchi, M.

2001-01-01

Small leucine-rich proteoglycans (SLRPs) have a number of biological functions and some of them are thought to regulate collagen mineralizaton in bone and tooth. We have previously identified and immunolocalized two members of the SLRPs family, decorin and biglycan, in bovine tooth/periodontium. To investigate their potential roles in tooth development, we examined the mRNA expression patterns of decorin, biglycan and type I collagen in newborn (day 19) mice tooth germs by in situ hybridization. At this developmental stage, the first maxillary and mandibular molars include stages before and after secretion of the predentin matrix, respectively. The expression of decorin mRNA coincided with that of type I collagen mRNA and was mostly observed in secretory odontoblasts, while the biglycan mRNA was expressed throughout the tooth germ, including pre-secretory odontoblasts/ameloblasts, dental papilla and stellate reticulum. However, its signal in secretory odontoblasts was not as evident as that of decorin. In mandibular incisors, where a significant amount of predentin matrix and a small amount of enamel matrix were already secreted, a similar differential expression pattern was observed. In secretory ameloblasts the biglycan mRNA expression was apparent, while that of decorin was not. These differential expression patterns suggest the distinct roles of biglycan and decorin in the process of tooth development.

Molecular signaling in intervertebral disk development.

PubMed

DiPaola, Christian P; Farmer, James C; Manova, Katia; Niswander, Lee A

2005-09-01

The purpose of this investigation is to identify and study the expression pattern of pertinent molecular factors involved in the differentiation of the intervertebral disk (IVD). It is likely that hedgehog genes and the BMP inhibitors are key factors involved in spinal joint formation. Radioactive in situ hybridization with mRNA probes for pax-1, SHH, IHH and Noggin gene was performed on mouse embryo and adult tissue. Immunohistochemistry was performed to localize hedgehog receptor, "patched" (ptc). From 14.5 dpc until birth pax-1 mRNA was expressed in the developing anulus fibrosus (AF). During the same developmental period Noggin mRNA is highly expressed throughout the spine, in the developing AF, while ptc protein and SHH mRNA were expressed in the developing nucleus pulposus (NP). IHH mRNA was expressed by condensing chondrocytes of the vertebral bodies and later becomes confined to the vertebral endplate. We show for the first time that pax-1 is expressed in the adult intervertebral disk. Ptc expression in the NP is an indicator of hedgehog protein signaling in the developing IVD. The expression pattern of the BMP inhibitor Noggin appears to be important for the normal formation of the IVD and may prove to play a role in its segmental pattern formation.

Differential IL-1β secretion by monocyte subsets is regulated by Hsp27 through modulating mRNA stability.

PubMed

Hadadi, Eva; Zhang, Biyan; Baidžajevas, Kajus; Yusof, Nurhashikin; Puan, Kia Joo; Ong, Siew Min; Yeap, Wei Hseun; Rotzschke, Olaf; Kiss-Toth, Endre; Wilson, Heather; Wong, Siew Cheng

2016-12-15

Monocytes play a central role in regulating inflammation in response to infection or injury, and during auto-inflammatory diseases. Human blood contains classical, intermediate and non-classical monocyte subsets that each express characteristic patterns of cell surface CD16 and CD14; each subset also has specific functional properties, but the mechanisms underlying many of their distinctive features are undefined. Of particular interest is how monocyte subsets regulate secretion of the apical pro-inflammatory cytokine IL-1β, which is central to the initiation of immune responses but is also implicated in the pathology of various auto-immune/auto-inflammatory conditions. Here we show that primary human non-classical monocytes, exposed to LPS or LPS + BzATP (3'-O-(4-benzoyl)benzyl-ATP, a P2X7R agonist), produce approx. 80% less IL-1β than intermediate or classical monocytes. Despite their low CD14 expression, LPS-sensing, caspase-1 activation and P2X7R activity were comparable in non-classical monocytes to other subsets: their diminished ability to produce IL-1β instead arose from 50% increased IL-1β mRNA decay rates, mediated by Hsp27. These findings identify the Hsp27 pathway as a novel therapeutic target for the management of conditions featuring dysregulated IL-1β production, and represent an advancement in understanding of both physiological inflammatory responses and the pathogenesis of inflammatory diseases involving monocyte-derived IL-1β.

Decay of S‐wave amplitudes with distance for earthquakes in the Charlevoix, Quebec, area: Effects of radiation pattern and directivity

USGS Publications Warehouse

Frankel, Arthur

2015-01-01

The decay of the Fourier spectral amplitudes of S waves over distances of 10–80 km near Charlevoix, Quebec, was determined using waveforms from seven earthquakes with MN 3.3–5.4. The S‐wave spectral amplitudes were corrected for site response and source amplitude by normalizing the coda‐wave spectrum at a fixed time after the origin time. The amplitude decay with distance was found to be less steep as the frequency increases from 1 to 14, contrary to what would be expected from anelastic and scattering attenuation for a point source with an isotropic radiation pattern. The decay at 14 Hz indicates that the geometrical spreading at distances less than 80 km is less steep than R−1.05. The steeper distance decay of the low‐frequency spectrum appears to be an artifact of the radiation pattern and rupture directivity, which affect the low‐frequency amplitude more than the high frequency. Synthetic seismograms were made for a horizontally layered crust for the Mw 4.6 Rivière du Loup earthquake and an Mw 3.3 event. The decay with distance of the 1 Hz spectral amplitudes of the synthetics is similar to that observed for the Rivière du Loup earthquake, indicating that radiation pattern and rupture directivity are important factors in determining the attenuation with distance at 1 Hz. For the Mw 3.3 earthquake, the distance decay of the 1 Hz spectral amplitudes was found to be sensitive to the focal mechanism. This study demonstrates that estimates of geometrical spreading made using 1 Hz amplitudes can be contaminated by radiation pattern and directivity effects and may not be applicable for constructing ground‐motion prediction equations for sources with other focal mechanisms and rupture behavior.

Late-onset spastic paraplegia: Aberrant SPG11 transcripts generated by a novel splice site donor mutation.

PubMed

Kawarai, Toshitaka; Miyamoto, Ryosuke; Mori, Atsuko; Oki, Ryosuke; Tsukamoto-Miyashiro, Ai; Matsui, Naoko; Miyazaki, Yoshimichi; Orlacchio, Antonio; Izumi, Yuishin; Nishida, Yoshihiko; Kaji, Ryuji

2015-12-15

We identified a novel homozygous mutation in the splice site donor (SSD) of intron 30 (c.5866+1G>A) in consanguineous Japanese SPG11 siblings showing late-onset spastic paraplegia using the whole-exome sequencing. Phenotypic variability was observed, including age-at-onset, dysarthria and pes cavus. Coding DNA sequencing revealed that the mutation affected the recognition of the constitutive SSD of intron 30, splicing upstream onto a nearby cryptic SSD in exon 30. The use of constitutive splice sites of intron 29 was confirmed by sequencing. The mutant transcripts are mostly subject to degradation by the nonsense-mediated mRNA decay system. SPG11 transcripts, escaping from the nonsense-mediated mRNA decay pathway, would generate a truncated protein (p.Tyr1900Phefs5X) containing the first 1899 amino acids and followed by 4 aberrant amino acids. This study showed a successful clinical application of whole-exome sequencing in spastic paraplegia and demonstrated a further evidence of allelic heterogeneity in SPG11. The confirmation of aberrant transcript by splice site mutation is a prerequisite for a more precise molecular diagnosis. Copyright © 2015 Elsevier B.V. All rights reserved.

DapF stabilizes the substrate-favoring conformation of RppH to stimulate its RNA-pyrophosphohydrolase activity in Escherichia coli.

PubMed

Wang, Qiang; Zhang, Delin; Guan, Zeyuan; Li, Dongqin; Pei, Kai; Liu, Jian; Zou, Tingting; Yin, Ping

2018-06-21

mRNA decay is an important strategy by which bacteria can rapidly adapt to their ever-changing surroundings. The 5'-terminus state of mRNA determines the velocity of decay of many types of RNA. In Escherichia coli, RNA pyrophosphohydrolase (RppH) is responsible for the removal of the 5'-terminal triphosphate from hundreds of mRNAs and triggers its rapid degradation by ribonucleases. A diaminopimelate epimerase, DapF, can directly interact with RppH and stimulate its hydrolysis activity in vivo and in vitro. However, the molecular mechanism remains to be elucidated. Here, we determined the complex structure of DapF-RppH as a heterotetramer in a 2:2 molar ratio. DapF-bound RppH exhibits an RNA-favorable conformation similar to the RNA-bound state, suggesting that association with DapF promotes and stabilizes RppH in a conformation that facilitates substrate RNA binding and thus stimulates the activity of RppH. To our knowledge, this is the first published structure of an RNA-pyrophosphohydrolysis complex in bacteria. Our study provides a framework for further investigation of the potential regulators involved in the RNA-pyrophosphohydrolysis process in prokaryotes.

Staufen2 functions in Staufen1-mediated mRNA decay by binding to itself and its paralog and promoting UPF1 helicase but not ATPase activity

PubMed Central

Park, Eonyoung; Gleghorn, Michael L.; Maquat, Lynne E.

2013-01-01

Staufen (STAU)1-mediated mRNA decay (SMD) is a posttranscriptional regulatory mechanism in mammals that degrades mRNAs harboring a STAU1-binding site (SBS) in their 3′-untranslated regions (3′ UTRs). We show that SMD involves not only STAU1 but also its paralog STAU2. STAU2, like STAU1, is a double-stranded RNA-binding protein that interacts directly with the ATP-dependent RNA helicase up-frameshift 1 (UPF1) to reduce the half-life of SMD targets that form an SBS by either intramolecular or intermolecular base-pairing. Compared with STAU1, STAU2 binds ∼10-fold more UPF1 and ∼two- to fivefold more of those SBS-containing mRNAs that were tested, and it comparably promotes UPF1 helicase activity, which is critical for SMD. STAU1- or STAU2-mediated augmentation of UPF1 helicase activity is not accompanied by enhanced ATP hydrolysis but does depend on ATP binding and a basal level of UPF1 ATPase activity. Studies of STAU2 demonstrate it changes the conformation of RNA-bound UPF1. These findings, and evidence for STAU1−STAU1, STAU2−STAU2, and STAU1−STAU2 formation in vitro and in cells, are consistent with results from tethering assays: the decrease in mRNA abundance brought about by tethering siRNA-resistant STAU2 or STAU1 to an mRNA 3′ UTR is inhibited by downregulating the abundance of cellular STAU2, STAU1, or UPF1. It follows that the efficiency of SMD in different cell types reflects the cumulative abundance of STAU1 and STAU2. We propose that STAU paralogs contribute to SMD by “greasing the wheels” of RNA-bound UPF1 so as to enhance its unwinding capacity per molecule of ATP hydrolyzed. PMID:23263869

Staufen2 functions in Staufen1-mediated mRNA decay by binding to itself and its paralog and promoting UPF1 helicase but not ATPase activity.

PubMed

Park, Eonyoung; Gleghorn, Michael L; Maquat, Lynne E

2013-01-08

Staufen (STAU)1-mediated mRNA decay (SMD) is a posttranscriptional regulatory mechanism in mammals that degrades mRNAs harboring a STAU1-binding site (SBS) in their 3'-untranslated regions (3' UTRs). We show that SMD involves not only STAU1 but also its paralog STAU2. STAU2, like STAU1, is a double-stranded RNA-binding protein that interacts directly with the ATP-dependent RNA helicase up-frameshift 1 (UPF1) to reduce the half-life of SMD targets that form an SBS by either intramolecular or intermolecular base-pairing. Compared with STAU1, STAU2 binds ~10-fold more UPF1 and ~two- to fivefold more of those SBS-containing mRNAs that were tested, and it comparably promotes UPF1 helicase activity, which is critical for SMD. STAU1- or STAU2-mediated augmentation of UPF1 helicase activity is not accompanied by enhanced ATP hydrolysis but does depend on ATP binding and a basal level of UPF1 ATPase activity. Studies of STAU2 demonstrate it changes the conformation of RNA-bound UPF1. These findings, and evidence for STAU1-STAU1, STAU2-STAU2, and STAU1-STAU2 formation in vitro and in cells, are consistent with results from tethering assays: the decrease in mRNA abundance brought about by tethering siRNA-resistant STAU2 or STAU1 to an mRNA 3' UTR is inhibited by downregulating the abundance of cellular STAU2, STAU1, or UPF1. It follows that the efficiency of SMD in different cell types reflects the cumulative abundance of STAU1 and STAU2. We propose that STAU paralogs contribute to SMD by "greasing the wheels" of RNA-bound UPF1 so as to enhance its unwinding capacity per molecule of ATP hydrolyzed.

Upregulation of estrogen receptor subtypes and vitellogenin mRNA in cinnamon clownfish Amphiprion melanopus during the sex change process: profiles on effects of 17beta-estradiol.

PubMed

Kim, Na Na; Jin, Deuk-Hee; Lee, Jehee; Kil, Gyung-Suk; Choi, Cheol Young

2010-10-01

In the present study, we investigated the expression pattern of estrogen receptors (esr) and vitellogenin (vtg) mRNA in the gonads and liver during sex change in cinnamon clownfish by using quantitative polymerase chain reaction. We divided gonadal development during the sex change from male to female into 3 stages (mature male, male at 90days after removing female, and mature female) and investigated esr and vtg mRNA expressions during the sex change. With female, the esr and vtg mRNA expressions increased. In western blot analysis, Esr1 protein was detected only in the ovaries of female cinnamon clownfish. Also, to understand the effect of 17beta-estradiol (E(2)), we investigated the esr and vtg mRNA expression patterns in the gonads and liver, and the changes in plasma E(2) level after E(2) injection. E(2) treatment increased both mRNA expression levels of esr and vtg and plasma E(2) levels. The present study describes the molecular characterization of esr subtypes and the interactions between esr and vtg after E(2) treatment in cinnamon clownfish. 2010 Elsevier Inc. All rights reserved.

Synthesis of compact patterns for NMR relaxation decay in intelligent "electronic tongue" for analyzing heavy oil composition

NASA Astrophysics Data System (ADS)

Lapshenkov, E. M.; Volkov, V. Y.; Kulagin, V. P.

2018-05-01

The article is devoted to the problem of pattern creation of the NMR sensor signal for subsequent recognition by the artificial neural network in the intelligent device "the electronic tongue". The specific problem of removing redundant data from the spin-spin relaxation signal pattern that is used as a source of information in analyzing the composition of oil and petroleum products is considered. The method is proposed that makes it possible to remove redundant data of the relaxation decay pattern but without introducing additional distortion. This method is based on combining some relaxation decay curve intervals that increment below the noise level such that the increment of the combined intervals is above the noise level. In this case, the relaxation decay curve samples that are located inside the combined intervals are removed from the pattern. This method was tested on the heavy-oil NMR signal patterns that were created by using the Carr-Purcell-Meibum-Gill (CPMG) sequence for recording the relaxation process. Parameters of CPMG sequence are: 100 μs - time interval between 180° pulses, 0.4s - duration of measurement. As a result, it was revealed that the proposed method allowed one to reduce the number of samples 15 times (from 4000 to 270), and the maximum detected root mean square error (RMS error) equals 0.00239 (equivalent to signal-to-noise ratio 418).

Genetic analyses of Per.C6 cell clones producing a therapeutic monoclonal antibody regarding productivity and long-term stability.

PubMed

Tsuruta, Lilian Rumi; Lopes Dos Santos, Mariana; Yeda, Fernanda Perez; Okamoto, Oswaldo Keith; Moro, Ana Maria

2016-12-01

Genetic characterization of protein-producing clones represents additional value to cell line development. In the present study, ten Per.C6 clones producing a Rebmab100 monoclonal antibody were selected using two cloning methods: six clones originated from limiting dilution cloning and four by the automated colony picker ClonePix FL. A stability program was performed for 50 generations, including 4 batches distributed along the timeframe to determine specific productivity (Qp) maintenance. Four stable clones (two from limiting dilution and two from ClonePix FL) were further evaluated. The relative mRNA expression levels of both heavy chain (HC) and light chain (LC) genes were verified at generations 0, 30-35, and 50-55 of the stability program. At generations 0 and 30-35, LC gene expression level was higher than HC gene, whereas at generation 50-55, the opposite prevailed. A high correlation was observed between Qp and HC or LC mRNA expression level for all clones at each generation analyzed along the continuous culture. The mRNA stability study was performed at steady-state culture. The LC gene displayed a higher half-life and lower decay constant than HC gene, accounting for the higher observed expression level of LC mRNA in comparison to HC mRNA. Clone R6 was highlighted due its high Qp, mRNA expression levels, and mRNA stability. Besides the benefits of applying genetic characterization for the selection of stable and high-producing clones, the present study shows for the first time the correlation between Qp and HC or LC expression levels and also mRNA stability in clones derived from human cell line Per.C6(®).

Topographical cone photopigment gene expression in deutan-type red-green color vision defects.

PubMed

Bollinger, Kathryn; Sjoberg, Stacy A; Neitz, Maureen; Neitz, Jay

2004-01-01

Eye donors were identified who had X-chromosome photopigment gene arrays like those of living deuteranomalous men; the arrays contained two genes encoding long-wavelength sensitive (L) pigments as well as genes to encode middle-wavelength sensitive (M) photopigment. Ultrasensitive methods failed to detect the presence of M photopigment mRNA in the retinas of these deutan donors. This provides direct evidence that deuteranomaly is caused by the complete absence of M pigment mRNA. Additionally, for those eyes with mRNA corresponding to two different L-type photopigments, the ratio of mRNA from the first vs. downstream L genes was analyzed across the retinal topography. Results show that the pattern of first relative to downstream L gene expression in the deuteranomalous retina is similar to the pattern of L vs. M gene expression found in normal retinas.

Use of sonic tomography to detect and quantify wood decay in living trees1

PubMed Central

Gilbert, Gregory S.; Ballesteros, Javier O.; Barrios-Rodriguez, Cesar A.; Bonadies, Ernesto F.; Cedeño-Sánchez, Marjorie L.; Fossatti-Caballero, Nohely J.; Trejos-Rodríguez, Mariam M.; Pérez-Suñiga, José Moises; Holub-Young, Katharine S.; Henn, Laura A. W.; Thompson, Jennifer B.; García-López, Cesar G.; Romo, Amanda C.; Johnston, Daniel C.; Barrick, Pablo P.; Jordan, Fulvia A.; Hershcovich, Shiran; Russo, Natalie; Sánchez, Juan David; Fábrega, Juan Pablo; Lumpkin, Raleigh; McWilliams, Hunter A.; Chester, Kathleen N.; Burgos, Alana C.; Wong, E. Beatriz; Diab, Jonathan H.; Renteria, Sonia A.; Harrower, Jennifer T.; Hooton, Douglas A.; Glenn, Travis C.; Faircloth, Brant C.; Hubbell, Stephen P.

2016-01-01

Premise of the study: Field methodology and image analysis protocols using acoustic tomography were developed and evaluated as a tool to estimate the amount of internal decay and damage of living trees, with special attention to tropical rainforest trees with irregular trunk shapes. Methods and Results: Living trunks of a diversity of tree species in tropical rainforests in the Republic of Panama were scanned using an Argus Electronic PiCUS 3 Sonic Tomograph and evaluated for the amount and patterns of internal decay. A protocol using ImageJ analysis software was used to quantify the proportions of intact and compromised wood. The protocols provide replicable estimates of internal decay and cavities for trees of varying shapes, wood density, and bark thickness. Conclusions: Sonic tomography, coupled with image analysis, provides an efficient, noninvasive approach to evaluate decay patterns and structural integrity of even irregularly shaped living trees. PMID:28101433

CODIT

Treesearch

Walter C. Shortle

2000-01-01

CODIT is an acronym for compartmentalization of decay in trees. It is a simple model system originally designed to help forest managers understand the patterns of discoloration and decay in living trees.

Detailed spectroscopy of 110Cd: Evidence for weak mixing and the emergence of γ-soft behavior

NASA Astrophysics Data System (ADS)

Garrett, P. E.; Bangay, J.; Diaz Varela, A.; Ball, G. C.; Cross, D. S.; Demand, G. A.; Finlay, P.; Garnsworthy, A. B.; Green, K. L.; Hackman, G.; Hannant, C. D.; Jigmeddorj, B.; Jolie, J.; Kulp, W. D.; Leach, K. G.; Orce, J. N.; Phillips, A. A.; Radich, A. J.; Rand, E. T.; Schumaker, M. A.; Svensson, C. E.; Sumithrarachchi, C.; Triambak, S.; Warr, N.; Wong, J.; Wood, J. L.; Yates, S. W.

2012-10-01

A study of the β+-electron capture decay of 110In into levels of 110Cd is combined with a reanalysis of data from a previous study of 110Cd with the (n,n'γ) reaction with monoenergetic neutrons. The γγ coincidences from the 110In decay leads to many new assignments of γ rays observed in the (n,n'γ) reaction, permitting the observation of weak low-energy transitions, and setting stringent upper limits on unobserved decay branches. The uncertainties on many of the lifetimes from the (n,n'γ) reaction are significantly reduced, and limits are established for the lifetimes of levels too long for a direct measurement. The absence of enhanced transitions between the previously assigned phonon states and the deformed intruder states strongly suggests that mixing between the configurations is generally weak, refuting the strong-mixing scenario as an explanation of the decay pattern of the excited 0+ states in 110Cd. The decay pattern of the nonintruder states is suggestive of a γ-soft rotor, or O(6) nucleus, rather than a vibrational, or U(5), pattern. The existence of a four-particle-six-hole proton excitation in 110Cd is also suggested.

WIG1 is crucial for AGO2-mediated ACOT7 mRNA silencing via miRNA-dependent and -independent mechanisms.

PubMed

Lee, Hyung Chul; Jung, Seung Hee; Hwang, Hyun Jung; Kang, Donghee; De, Supriyo; Dudekula, Dawood B; Martindale, Jennifer L; Park, Byungkyu; Park, Seung Kuk; Lee, Eun Kyung; Lee, Jeong-Hwa; Jeong, Sunjoo; Han, Kyungsook; Park, Heon Joo; Ko, Young-Gyu; Gorospe, Myriam; Lee, Jae-Seon

2017-06-20

RNA-binding proteins (RBPs) are involved in mRNA splicing, maturation, transport, translation, storage and turnover. Here, we identified ACOT7 mRNA as a novel target of human WIG1. ACOT7 mRNA decay was triggered by the microRNA miR-9 in a WIG1-dependent manner via classic recruitment of Argonaute 2 (AGO2). Interestingly, AGO2 was also recruited to ACOT7 mRNA in a WIG1-dependent manner in the absence of miR-9, which indicates an alternative model whereby WIG1 controls AGO2-mediated gene silencing. The WIG1-AGO2 complex attenuated translation initiation via an interaction with translation initiation factor 5B (eIF5B). These results were confirmed using a WIG1 tethering system based on the MS2 bacteriophage coat protein and a reporter construct containing an MS2-binding site, and by immunoprecipitation of WIG1 and detection of WIG1-associated proteins using liquid chromatography-tandem mass spectrometry. We also identified WIG1-binding motifs using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation analyses. Altogether, our data indicate that WIG1 governs the miRNA-dependent and the miRNA-independent recruitment of AGO2 to lower the stability of and suppress the translation of ACOT7 mRNA. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Type I Glanzmann thrombasthenia caused by an apparently silent beta3 mutation that results in aberrant splicing and reduced beta3 mRNA.

PubMed

Xie, Jingli; Pabón, Dina; Jayo, Asier; Butta, Nora; González-Manchón, Consuelo

2005-05-01

We report a novel genetic defect in a patient with type I Glanzmann thrombasthenia. Flow cytometry analysis revealed undetectable levels of platelet glycoproteins alphaIIb and beta3, although residual amounts of both proteins were detectable in immunoblotting analysis. Sequence analysis of reversely transcribed platelet beta3 mRNA showed a 100-base pair deletion in the 3'-boundary of exon 11, that results in a frame shift and appearance of a premature STOP codon. Analysis of the corresponding genomic DNA fragment revealed the presence of a homozygous C1815T transition in exon 11. The mutation does not change the amino acid residue but it creates an ectopic consensus splice donor site that is used preferentially, causing splicing out of part of exon 11. The parents of the proband, heterozygous for this mutation, were asymptomatic and had reduced platelet content of alphaIIbbeta3. PCR-based relative quantification of beta3 mRNA failed to detect the mutant transcript in the parents and showed a marked reduction in the patient. The results suggest that the thrombasthenic phenotype is, mainly, the result of the reduced availability of beta3-mRNA, most probably due to activation of the nonsense-mediated mRNA decay mechanism. They also show the convenience of analyzing both genomic DNA and mRNA, in order to ascertain the functional consequences of single nucleotide substitutions.

Tristetraprolin inhibits mitochondrial function through suppression of α-Synuclein expression in cancer cells

PubMed Central

Vo, Mai-Tram; Choi, Seong Hee; Lee, Ji-Heon; Hong, Chung Hwan; Kim, Jong Soo; Lee, Unn Hwa; Chung, Hyung-Min; Lee, Byung Ju; Park, Jeong Woo; Cho, Wha Ja

2017-01-01

Mitochondrial dynamics play critical roles in maintaining mitochondrial functions. Here, we report a novel mechanism for regulation of mitochondrial dynamics mediated by tristetraprolin (TTP), an AU-rich element (ARE)-binding protein. Overexpression of TTP resulted in elongated mitochondria, down-regulation of mitochondrial oxidative phosphorylation, reduced membrane potential, cytochrome c release, and increased apoptotic cell death in cancer cells. TTP overexpression inhibited the expression of α-Synuclein (α-Syn). TTP bound to the ARE within the mRNA 3′-untranslated regions (3′-UTRs) of α-Syn and enhanced the decay of α-Syn mRNA. Overexpression of α-Syn without the 3′-UTR restored TTP-induced defects in mitochondrial morphology, mitochondrial oxidative phosphorylation, membrane potential, and apoptotic cell death. Taken together, our data demonstrate that TTP acts as a regulator of mitochondrial dynamics through enhancing degradation of α-Syn mRNA in cancer cells. This finding will increase understanding of the molecular basis of mitochondrial dynamics. PMID:28410208

Effects of ethylene on gene expression in carrot roots

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

Nichols, S.E.

1984-01-01

To investigate ethylene effects on expression of genetic information, cDNA clones corresponding to ethylene-induced carrot root mRNAs were constructed and isolated. RNA dot blot analysis showed that for the three clones studied peak cytosolic mRNA prevalence occurred at 21 hours of treatment followed thereafter by rapid messenger decay. DNA filter excess hybridization to in vitro synthesized nuclear RNA showed that the ethylene-induced mRNA increase is engendered by transcription of previously quiescent genes. The kinetics and magnitude of changes in mRNA prevalence parallel changes in transcriptional activity; therefore, the ethylene effect is primarily at the level of the transcription. In vivomore » pulse labelling with (/sup 35/S)-methionine showed that between 18 and 27 hours of ethylene treatment a 2.5 fold increase in translational efficiency occurred for one message studied. The resulting protein is the predominant protein synthesized in carrots treated with ethylene for 27 hours. Thus, ethylene exerts multiple regulatory controls on the expression of genetic information.« less

Meth mouth severity in response to drug-use patterns and dental access in methamphetamine users.

PubMed

Brown, Ronni E; Morisky, Donald E; Silverstein, Steven J

2013-06-01

Meth mouth is the rapid development of tooth decay in methamphetamine users. Our study questioned whether drug-use patterns and dental care access are risk factors affecting the severity of meth mouth. Participants received dental examinations, and the number of decayed, missing and filled teeth (DMFT) were counted and used to measure meth mouth severity.

Alternative-splicing-mediated gene expression

NASA Astrophysics Data System (ADS)

Wang, Qianliang; Zhou, Tianshou

2014-01-01

Alternative splicing (AS) is a fundamental process during gene expression and has been found to be ubiquitous in eukaryotes. However, how AS impacts gene expression levels both quantitatively and qualitatively remains to be fully explored. Here, we analyze two common models of gene expression, each incorporating a simple splice mechanism that a pre-mRNA is spliced into two mature mRNA isoforms in a probabilistic manner. In the constitutive expression case, we show that the steady-state molecular numbers of two mature mRNA isoforms follow mutually independent Poisson distributions. In the bursting expression case, we demonstrate that the tail decay of the steady-state distribution for both mature mRNA isoforms that in general are not mutually independent can be characterized by the product of mean burst size and splicing probability. In both cases, we find that AS can efficiently modulate both the variability (measured by variance) and the noise level of the total mature mRNA, and in particular, the latter is always lower than the noise level of the pre-mRNA, implying that AS always reduces the noise. These results altogether reveal that AS is a mechanism of efficiently controlling the gene expression noise.

RNA splicing and splicing regulator changes in prostate cancer pathology.

PubMed

Munkley, Jennifer; Livermore, Karen; Rajan, Prabhakar; Elliott, David J

2017-09-01

Changes in mRNA splice patterns have been associated with key pathological mechanisms in prostate cancer progression. The androgen receptor (abbreviated AR) transcription factor is a major driver of prostate cancer pathology and activated by androgen steroid hormones. Selection of alternative promoters by the activated AR can critically alter gene function by switching mRNA isoform production, including creating a pro-oncogenic isoform of the normally tumour suppressor gene TSC2. A number of androgen-regulated genes generate alternatively spliced mRNA isoforms, including a prostate-specific splice isoform of ST6GALNAC1 mRNA. ST6GALNAC1 encodes a sialyltransferase that catalyses the synthesis of the cancer-associated sTn antigen important for cell mobility. Genetic rearrangements occurring early in prostate cancer development place ERG oncogene expression under the control of the androgen-regulated TMPRSS2 promoter to hijack cell behaviour. This TMPRSS2-ERG fusion gene shows different patterns of alternative splicing in invasive versus localised prostate cancer. Alternative AR mRNA isoforms play a key role in the generation of prostate cancer drug resistance, by providing a mechanism through which prostate cancer cells can grow in limited serum androgen concentrations. A number of splicing regulator proteins change expression patterns in prostate cancer and may help drive key stages of disease progression. Up-regulation of SRRM4 establishes neuronal splicing patterns in neuroendocrine prostate cancer. The splicing regulators Sam68 and Tra2β increase expression in prostate cancer. The SR protein kinase SRPK1 that modulates the activity of SR proteins is up-regulated in prostate cancer and has already given encouraging results as a potential therapeutic target in mouse models.

Differential ontogenetic patterns of levocabastine-sensitive neurotensin NT2 receptors and of NT1 receptors in the rat brain revealed by in situ hybridization.

PubMed

Lépée-Lorgeoux, I; Betancur, C; Rostène, W; Pélaprat, D

1999-03-12

The postnatal ontogeny of the levocabastine-sensitive neurotensin receptor (NT2) mRNA was studied by in situ hybridization in the rat brain and compared with the distribution of the levocabastine-insensitive NT1 receptor. NT2 receptor mRNA was absent at birth from all brain structures except the ependymal cell layer lining the ventricles. The development of NT2 receptor mRNA followed three ontogenetic patterns. The first pattern, involving the majority of the cerebral gray matter, was characterized by a continuous increase from postnatal day 5 (P5) to P30. The second one, involving regions rich in myelinated fibers such as the corpus callosum and lacunosum moleculare layer of the hippocampus, exhibited a pronounced increase between P5 and P10, peaked at P15 and was followed by a plateau or a slight decrease. The third pattern was observed in the ependymal cell layer lining the olfactory and lateral ventricles, where the high labeling already present at birth continued to increase during development. These different developmental patterns could reflect the variety of cells expressing NT2 receptor mRNA, including neurons, protoplasmic astrocytes in gray matter, fibrous astrocytes present in myelinated fibers tracts, and ependymal cells. In contrast, NT1 receptor mRNA, which seems to be associated only with neurons, was highly and transiently expressed during the perinatal period in the cerebral cortex, hippocampus and striatal neuroepithelium. Other regions, notably the ventral tegmental area and substantia nigra compacta, exhibited a gradual increase in NT1 receptor signal, reaching adult levels by P21. Both the differential localization and ontogenetic profiles of NT1 and NT2 receptor mRNAs suggest different involvement of these two receptors in brain functions and development. Copyright 1999 Elsevier Science B.V.

Mechanisms of ultraviolet disinfection and chlorination of Escherichia coli: Culturability, membrane permeability, metabolism, and genetic damage.

PubMed

Xu, Limei; Zhang, Chongmiao; Xu, Pengcheng; Wang, Xiaochang C

2018-03-01

Traditional culture methods may underestimate the tolerance of microorganisms to disinfectants because of the existence of viable but nonculturable or sublethally injured cells after disinfection. The selection of a strict method is crucial for the evaluation of disinfection performance. The actions of 2 typical disinfectants - ultraviolet (UV) and chlorine - on the fecal indicator Escherichia coli were investigated by the detection of culturability, membrane permeability, metabolic activity, deoxyribonucleic acid (DNA), and messenger ribonucleic acid (mRNA). During UV disinfection, the irreversible damages in the cell membrane and cellular adenosine triphosphate (ATP) were negligible at low UV doses (<80mJ/cm 2 ). However, membrane permeability was damaged at low doses of chlorine (<5mg/L), leading to leakage of cellular ATP. Our study showed that a slight lesion in DNA was detected even at high doses of UV (400mJ/cm 2 ) and chlorine (>5mg/L) treatments. The decay of mRNA was more rapid than that of DNA. The degradation level of mRNA depended on the choice of target genes. After exposure to 50mJ/cm 2 UV dose or 5mg/L chlorine for 30min, the DNA damage repair function (RecA mRNA) was inhibited. The mRNA involved in the DNA damage repair function can be a potential indicator of bacterial viability. Copyright © 2017. Published by Elsevier B.V.

Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

PubMed

Xie, Fang-Fei; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Hong; Wu, Jian; Guo, Yu-Fan; Zeng, Ke-Qin; Wang, Ming-Jun; Zhu, Xiao-Wei; Xia, Wei; Wang, Lan; He, Pei; Bing, Peng-Fei; Lu, Xin; Zhang, Yong-Hong; Lei, Shu-Feng

2018-01-01

DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1 ) between methylation level and mRNA is moderate (-0.63-0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P < 0.05 and more than 5.96% genes presented very strong correlation (R T4  > 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

The role of alternative splicing coupled to nonsense-mediated mRNA decay in human disease.

PubMed

da Costa, Paulo J; Menezes, Juliane; Romão, Luísa

2017-10-01

Alternative pre-mRNA splicing (AS) affects gene expression as it generates proteome diversity. Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature translation-termination codons (PTCs), preventing the production of truncated proteins that could result in disease. Several studies have also implicated NMD in the regulation of steady-state levels of physiological mRNAs. In addition, it is known that several regulated AS events do not lead to generation of protein products, as they lead to transcripts that carry PTCs and thus, they are committed to NMD. Indeed, an estimated one-third of naturally occurring, alternatively spliced mRNAs is targeted for NMD, being AS coupled to NMD (AS-NMD) an efficient strategy to regulate gene expression. In this review, we will focus on how AS mechanism operates and how can be coupled to NMD to fine-tune gene expression levels. Furthermore, we will demonstrate the physiological significance of the interplay among AS and NMD in human disease, such as cancer and neurological disorders. The understanding of how AS-NMD orchestrates expression of vital genes is of utmost importance for the advance in diagnosis, prognosis and treatment of many human disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

cry1Aa lacks stability elements at its 5'-UTR but integrity of its transcription terminator is critical to prevent decay of its transcript.

PubMed

Ramírez-Prado, Jorge Humberto; Martínez-Márquez, Eva Isabel; Olmedo-Alvarez, Gabriela

2006-07-01

We analyzed the influence of the 5' and 3' untranslated regions of the Bacillus thuringiensis cry1Aa on its mRNA stability. Although the cry1Aa gene has a stable transcript (8 min), its 5' UTR did not provide stability to the reporter gene uidA. Stability of cry1Aa could be increased to 40 min by addition of an SP82 stability element at the 5' UTR, suggesting that once the 5' and 3' ends were protected initiation of decay could be effectively blocked. We generated mutations in the transcription terminator and found that changes that reduced the stability of the stem, a larger loop, or elimination of the U-trail sharply decreased the half-life of the transcript. Therefore, unlike some stable bacterial transcripts, cry1Aa lacks special features at the end 5' to prevent decay, but its terminator is the main determinant of its stability.

Investigating microbial transformations of soil organic matter: synthesizing knowledge from disparate fields to guide new experimentation

NASA Astrophysics Data System (ADS)

Billings, S. A.; Tiemann, L. K.; Ballantyne, F., IV; Lehmeier, C. A.; Min, K.

2015-04-01

Discerning why some soil organic matter (SOM) leaves soil profiles relatively quickly while other compounds, especially at depth, can be retained for decades to millennia is challenging for a multitude of reasons. Simultaneous with soil-specific advances, multiple other disciplines have enhanced their knowledge bases in ways potentially useful for future investigations of SOM decay. In this article, we highlight observations highly relevant for those investigating SOM decay and retention but often emanating from disparate fields and residing in literature seldom cited in SOM research. We focus on recent work in two key areas. First, we turn to experimental approaches using natural and artificial aquatic environments to investigate patterns of microbially mediated OM transformations as environmental conditions change, and highlight how aquatic microbial responses to environmental change can reveal processes likely important to OM decay and retention in soils. Second, we emphasize the importance of establishing intrinsic patterns of decay kinetics for purified substrates commonly found in soils to develop baseline rates. These decay kinetics - which represent the upper limit of the reaction rates - can then be compared to substrate decay kinetics observed in natural samples, which integrate intrinsic decay reaction rates and edaphic factors essential to the site under study but absent in purified systems. That comparison permits the site-specific factors to be parsed from the fundamental decay kinetics, an important advance in our understanding of SOM decay (and thus persistence) in natural systems. We then suggest ways in which empirical observations from aquatic systems and purified substrate-enzyme reaction kinetics can be used to advance recent theoretical efforts in SOM-focused research. Finally, we suggest how the observations in aquatic and purified substrate-enzyme systems could be used to help unravel the puzzles presented by oft-observed patterns of SOM characteristics with depth, as one example of the many perplexing SOM-related problems.

Corticosteroids reduce IL-6 in ASM cells via up-regulation of MKP-1.

PubMed

Quante, Timo; Ng, Yee Ching; Ramsay, Emma E; Henness, Sheridan; Allen, Jodi C; Parmentier, Johannes; Ge, Qi; Ammit, Alaina J

2008-08-01

The mechanisms by which corticosteroids reduce airway inflammation are not completely understood. Traditionally, corticosteroids were thought to inhibit cytokines exclusively at the transcriptional level. Our recent evidence, obtained in airway smooth muscle (ASM), no longer supports this view. We have found that corticosteroids do not act at the transcriptional level to reduce TNF-alpha-induced IL-6 gene expression. Rather, corticosteroids inhibit TNF-alpha-induced IL-6 secretion by reducing the stability of the IL-6 mRNA transcript. TNF-alpha-induced IL-6 mRNA decays at a significantly faster rate in ASM cells pretreated with the corticosteroid dexamethasone (t(1/2) = 2.4 h), compared to vehicle (t(1/2) = 9.0 h; P < 0.05) (results are expressed as decay constants [k] [mean +/- SEM] and half-life [h]). Interestingly, the underlying mechanism of inhibition by corticosteroids is via the up-regulation of an endogenous mitogen-activated protein kinase (MAPK) inhibitor, MAPK phosphatase-1 (MKP-1). Corticosteroids rapidly up-regulate MKP-1 in a time-dependent manner (44.6 +/- 10.5-fold increase after 24 h treatment with dexamethasone; P < 0.05), and MKP-1 up-regulation was temporally related to the inhibition of TNF-alpha-induced p38 MAPK phosphorylation. Moreover, TNF-alpha acts via a p38 MAPK-dependent pathway to stabilize the IL-6 mRNA transcript (TNF-alpha, t(1/2) = 9.6 h; SB203580 + TNF-alpha, t(1/2) = 1.5 h), exogenous expression of MKP-1 significantly inhibits TNF-alpha-induced IL-6 secretion and MKP-1 siRNA reverses the inhibition of TNF-alpha-induced IL-6 secretion by dexamethasone. Taken together, these results suggest that corticosteroid-induced MKP-1 contributes to the repression of IL-6 secretion in ASM cells.

The Human T-Lymphotropic Virus Type 1 Tax Protein Inhibits Nonsense-Mediated mRNA Decay by Interacting with INT6/EIF3E and UPF1

PubMed Central

Mocquet, Vincent; Neusiedler, Julia; Rende, Francesca; Cluet, David; Robin, Jean-Philippe; Terme, Jean-Michel; Duc Dodon, Madeleine; Wittmann, Jürgen; Morris, Christelle; Le Hir, Hervé; Ciminale, Vincenzo

2012-01-01

In this report, we analyzed whether the degradation of mRNAs by the nonsense-mediated mRNA decay (NMD) pathway was affected in human T-lymphotropic virus type 1 (HTLV-1)-infected cells. This pathway was indeed strongly inhibited in C91PL, HUT102, and MT2 cells, and such an effect was also observed by the sole expression of the Tax protein in Jurkat and HeLa cells. In line with this activity, Tax binds INT6/EIF3E (here called INT6), which is a subunit of the translation initiation factor eukaryotic initiation factor 3 (eIF3) required for efficient NMD, as well as the NMD core factor upstream frameshift protein 1 (UPF1). It was also observed that Tax expression alters the morphology of processing bodies (P-bodies), the cytoplasmic structures which concentrate RNA degradation factors. The presence of UPF1 in these subcellular compartments was increased by Tax, whereas that of INT6 was decreased. In line with these effects, the level of the phosphorylated form of UPF1 was increased in the presence of Tax. Analysis of several mutants of the viral protein showed that the interaction with INT6 is necessary for NMD inhibition. The alteration of mRNA stability was observed to affect viral transcripts, such as that coding for the HTLV-1 basic leucine zipper factor (HBZ), and also several cellular mRNAs sensitive to the NMD pathway. Our data indicate that the effect of Tax on viral and cellular gene expression is not restricted to transcriptional control but can also involve posttranscriptional regulation. PMID:22553336

Molecular ontogenesis of digestive capability and associated endocrine control in Atlantic cod (Gadus morhua) larvae.

PubMed

Kortner, Trond M; Overrein, Ingrid; Oie, Gunvor; Kjørsvik, Elin; Bardal, Tora; Wold, Per-Arvid; Arukwe, Augustine

2011-10-01

We have profiled the expression of twelve genes, in order to provide an overview on the molecular ontogeny of digestive capability with the associated endocrine control during Atlantic cod (Gadus morhua) larval development. Enzyme activity levels for the key digestive enzyme, trypsin, was also measured. Specifically, transcripts for trypsin, amylase, lipolytic enzymes: bile salt activated lipase (BAL), phospholipase A2 (PLA2) and Acyl CoA dehydrogenase (ACADM), regulatory peptides: neuropeptide Y (NPY), orexin (OX) cholecystokinin (CCK) and cocaine and amphetamine-related transcript (CART), the somatotropic factors: growth hormone (GH), preprosomatostatin 1 (PPSS1) and thyroid hormone receptors (TRα and TRβ) were analyzed using quatitative (real-time) polymerase chain reaction (qPCR). Trypsin and BAL mRNA levels peaked at approximately day 17 and 25 post-hatch, respectively, and thereafter displayed a decreasing pattern until metamorphosis. GH mRNA levels decreased moderately from 3 to 33dph, and thereafter, an increase was observed until 46dph. TRα mRNA levels showed a fluctuating pattern peaking at day 39 post-hatch. TRβ mRNA levels were too low to obtain quantitative measurements. Amylase mRNA slightly increased from day 3 to 17 post-hatch, and thereafter showed a steady decrease until day 60. Interestingly, PLA2 mRNA expression showed a consistent increase throughout the study period, indicating an increasingly important role during larval development. Overall, data from this study indicate that cod larvae show differential developmental mode of expression patterns for key genes and endocrine factors that regulate digestive capability, growth and development. These data are discussed in relation to larval trypsin enzyme activity and previous reports for other teleost species. Copyright © 2011 Elsevier Inc. All rights reserved.

Analysis of Wave Velocity Patterns in Black Cherry Trees and its Effect on Internal Decay Detection

Treesearch

Guanghui Li; Xiping Wang; Jan Wiedenbeck; Robert J. Ross

2013-01-01

In this study, we examined stress wave velocity patterns in the cross sections of black cherry trees, developed analytical models of stress wave velocity in sound healthy trees, and then tested the effectiveness of the models as a tool for tree decay diagnosis. Acoustic tomography data of the tree cross sections were collected from 12 black cherry trees at a production...

Analysis of wave velocity patterns in black cherry trees and its effect on internal decay detection

Treesearch

Guanghui Li; Xiping Wang; Hailin Feng; Jan Wiedenbeck; Robert J. Ross

2014-01-01

In this study, we examined stress wave velocity patterns in the cross sections of black cherry trees, developed analytical models of stress wave velocity in sound healthy trees, and then tested the effectiveness of the models as a tool for tree decay diagnosis. Acoustic tomography data of the tree cross sections were collected from 12 black cherry trees at a production...

Complete ? -decay pattern for the high-priority decay-heat isotopes I 137 and Xe 137 determined using total absorption spectroscopy

DOE PAGES

Rasco, B. C.; Rykaczewski, K. P.; Fijalkowska, A.; ...

2017-05-31

We measured the complete -decay intensities of 137I and 137Xe with the Modular Total Absorption Spectrometer at Oak Ridge National Laboratory. We describe a novel technique for measuring the -delayed neutron energy spectrum, which also provides a measurement of the -neutron branching ratio, P n.

Altered skeletal pattern of gene expression in response to spaceflight and hindlimb elevation

NASA Technical Reports Server (NTRS)

Bikle, D. D.; Harris, J.; Halloran, B. P.; Morey-Holton, E.

1994-01-01

Spaceflight leads to osteopenia, in part by inhibiting bone formation. Using an animal model (hindlimb elevation) that simulates the weightlessness of spaceflight, we and others showed a reversible inhibition of bone formation and bone mineralization. In this study, we have measured the mRNA levels of insulin-like growth factor I (IGF-I), IGF-I receptor (IGF-IR), alkaline phosphatase, and osteocalcin in the tibiae of rats flown aboard National Aeronautics and Space Administration Shuttle Flight STS-54 and compared the results with those obtained from their ground-based controls and from the bones of hindlimb-elevated animals. Spaceflight and hindlimb elevation transiently increase the mRNA levels for IGF-I, IGF-IR, and alkaline phosphatase but decrease the mRNA levels for osteocalcin. The changes in osteocalcin and alkaline phosphatase mRNA levels are consistent with a shift toward decreased maturation, whereas the rise in IGF-I and IGF-IR mRNA levels may indicate a compensatory response to the fall in bone formation. We conclude that skeletal unloading during spaceflight or hindlimb elevation resets the pattern of gene expression in the osteoblast, giving it a less mature profile.

An evolutionary conserved pattern of 18S rRNA sequence complementarity to mRNA 5′ UTRs and its implications for eukaryotic gene translation regulation

PubMed Central

Pánek, Josef; Kolář, Michal; Vohradský, Jiří; Shivaya Valášek, Leoš

2013-01-01

There are several key mechanisms regulating eukaryotic gene expression at the level of protein synthesis. Interestingly, the least explored mechanisms of translational control are those that involve the translating ribosome per se, mediated for example via predicted interactions between the ribosomal RNAs (rRNAs) and mRNAs. Here, we took advantage of robustly growing large-scale data sets of mRNA sequences for numerous organisms, solved ribosomal structures and computational power to computationally explore the mRNA–rRNA complementarity that is statistically significant across the species. Our predictions reveal highly specific sequence complementarity of 18S rRNA sequences with mRNA 5′ untranslated regions (UTRs) forming a well-defined 3D pattern on the rRNA sequence of the 40S subunit. Broader evolutionary conservation of this pattern may imply that 5′ UTRs of eukaryotic mRNAs, which have already emerged from the mRNA-binding channel, may contact several complementary spots on 18S rRNA situated near the exit of the mRNA binding channel and on the middle-to-lower body of the solvent-exposed 40S ribosome including its left foot. We discuss physiological significance of this structurally conserved pattern and, in the context of previously published experimental results, propose that it modulates scanning of the 40S subunit through 5′ UTRs of mRNAs. PMID:23804757

Fatty acid binding proteins (FABPs) in prostate, bladder and kidney cancer cell lines and the use of IL-FABP as survival predictor in patients with renal cell carcinoma

PubMed Central

2011-01-01

Background Fatty acid binding proteins (FABP) play an important role in carcinogenesis. Modified FABP expression patterns were described for prostate, bladder and for renal cell carcinoma. Studies on metabolic relationships and interactions in permanent cell lines allow a deeper insight into molecular processes. The aim of this study is therefore a systematic overview on mRNA and protein expressions of seven FABPs in frequently used urological cell lines. Methods Nine cell lines of renal carcinomas, seven of urinary bladder carcinomas, and five of prostate carcinomas were investigated. Quantitative RT-qPCR and western blotting were used to determine different FABPs. In addition, 46 paired cancerous and noncancerous tissue samples from nephrectomy specimen with renal cell carcinomas were investigated regarding the ileum FABP mRNA expression level and associated with survival outcome. Results General characteristics of all urological carcinoma cell lines were the expression of E-and IL-FABP on mRNA and protein level, while the expressions differed between the cell lines. The protein expression was not always congruent with the mRNA expression. Renal cell carcinoma cell lines showed expressions of L-, H- and B-FABP mRNA in addition to the general FABP expression in five out of the eight investigated cell lines. In bladder cancer cell lines, we additionally found the expression of A-FABP mRNA in six cell lines, while H-FABP was present only in three cell lines. In prostate cancer cell lines, a strong reduction of A- and E- FABP mRNA was observed. The expression of B-FABP mRNA and protein was observed only in the 22 RV-1 cells. IL-FABP mRNA was over-expressed in renal tumour tissue. The IL-FABP ratio was identified as an independent indicator of survival outcome. Conclusions Distinctly different FABP expression patterns were observed not only between the cell lines derived from the three cancer types, but also between the cell lines from the same cancer. The FABP patterns in the cell lines do not always reflect the real situation in the tumours. These facts have to be considered in functional studies concerning the different FABPs. PMID:21767383

The fitness cost of mis-splicing is the main determinant of alternative splicing patterns.

PubMed

Saudemont, Baptiste; Popa, Alexandra; Parmley, Joanna L; Rocher, Vincent; Blugeon, Corinne; Necsulea, Anamaria; Meyer, Eric; Duret, Laurent

2017-10-30

Most eukaryotic genes are subject to alternative splicing (AS), which may contribute to the production of protein variants or to the regulation of gene expression via nonsense-mediated messenger RNA (mRNA) decay (NMD). However, a fraction of splice variants might correspond to spurious transcripts and the question of the relative proportion of splicing errors to functional splice variants remains highly debated. We propose a test to quantify the fraction of AS events corresponding to errors. This test is based on the fact that the fitness cost of splicing errors increases with the number of introns in a gene and with expression level. We analyzed the transcriptome of the intron-rich eukaryote Paramecium tetraurelia. We show that in both normal and in NMD-deficient cells, AS rates strongly decrease with increasing expression level and with increasing number of introns. This relationship is observed for AS events that are detectable by NMD as well as for those that are not, which invalidates the hypothesis of a link with the regulation of gene expression. Our results show that in genes with a median expression level, 92-98% of observed splice variants correspond to errors. We observed the same patterns in human transcriptomes and we further show that AS rates correlate with the fitness cost of splicing errors. These observations indicate that genes under weaker selective pressure accumulate more maladaptive substitutions and are more prone to splicing errors. Thus, to a large extent, patterns of gene expression variants simply reflect the balance between selection, mutation, and drift.

Drosophila Hephaestus/Polypyrimidine Tract Binding Protein Is Required for Dorso-Ventral Patterning and Regulation of Signalling between the Germline and Soma

PubMed Central

McDermott, Suzanne M.; Davis, Ilan

2013-01-01

In the Drosophila oocyte, gurken (grk) mRNA encodes a secreted TGF-α signal that specifies the future embryonic dorso-ventral axes by altering the fate of the surrounding epithelial follicle cells. We previously identified a number of RNA binding proteins that associate specifically with the 64 nucleotide grk localization signal, including the Drosophila orthologue of polypyrimidine tract-binding protein (PTB), Hephaestus (Heph). To test whether Heph is required for correct grk mRNA or protein function, we used immunoprecipitation to validate the association of Heph with grk mRNA and characterized the heph mutant phenotype. We found that Heph is a component of grk mRNP complexes but heph germline clones show that Heph is not required for grk mRNA localization. Instead, we identify a novel function for Heph in the germline and show that it is required for proper Grk protein localization. Furthermore, we show that Heph is required in the oocyte for the correct organization of the actin cytoskeleton and dorsal appendage morphogenesis. Our results highlight a requirement for an mRNA binding protein in the localization of Grk protein, which is independent of mRNA localization, and we propose that Heph is required in the germline for efficient Grk signalling to the somatic follicle cells during dorso-ventral patterning. PMID:23894566

Identification of a functionally distinct truncated BDNF mRNA splice variant and protein in Trachemys scripta elegans.

PubMed

Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

2013-01-01

Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein.

Identification of a Functionally Distinct Truncated BDNF mRNA Splice Variant and Protein in Trachemys scripta elegans

PubMed Central

Ambigapathy, Ganesh; Zheng, Zhaoqing; Li, Wei; Keifer, Joyce

2013-01-01

Brain-derived neurotrophic factor (BDNF) has a diverse functional role and complex pattern of gene expression. Alternative splicing of mRNA transcripts leads to further diversity of mRNAs and protein isoforms. Here, we describe the regulation of BDNF mRNA transcripts in an in vitro model of eyeblink classical conditioning and a unique transcript that forms a functionally distinct truncated BDNF protein isoform. Nine different mRNA transcripts from the BDNF gene of the pond turtle Trachemys scripta elegans (tBDNF) are selectively regulated during classical conditioning: exon I mRNA transcripts show no change, exon II transcripts are downregulated, while exon III transcripts are upregulated. One unique transcript that codes from exon II, tBDNF2a, contains a 40 base pair deletion in the protein coding exon that generates a truncated tBDNF protein. The truncated transcript and protein are expressed in the naïve untrained state and are fully repressed during conditioning when full-length mature tBDNF is expressed, thereby having an alternate pattern of expression in conditioning. Truncated BDNF is not restricted to turtles as a truncated mRNA splice variant has been described for the human BDNF gene. Further studies are required to determine the ubiquity of truncated BDNF alternative splice variants across species and the mechanisms of regulation and function of this newly recognized BDNF protein. PMID:23825634

Differential Expression Patterns of occ1-Related Genes in Adult Monkey Visual Cortex

PubMed Central

Takahata, Toru; Komatsu, Yusuke; Watakabe, Akiya; Hashikawa, Tsutomu; Tochitani, Shiro

2009-01-01

We have previously revealed that occ1 is preferentially expressed in the primary visual area (V1) of the monkey neocortex. In our attempt to identify more area-selective genes in the macaque neocortex, we found that testican-1, an occ1-related gene, and its family members also exhibit characteristic expression patterns along the visual pathway. The expression levels of testican-1 and testican-2 mRNAs as well as that of occ1 mRNA start of high in V1, progressively decrease along the ventral visual pathway, and end of low in the temporal areas. Complementary to them, the neuronal expression of SPARC mRNA is abundant in the association areas and scarce in V1. Whereas occ1, testican-1, and testican-2 mRNAs are preferentially distributed in thalamorecipient layers including “blobs,” SPARC mRNA expression avoids these layers. Neither SC1 nor testican-3 mRNA expression is selective to particular areas, but SC1 mRNA is abundantly observed in blobs. The expressions of occ1, testican-1, testican-2, and SC1 mRNA were downregulated after monocular tetrodotoxin injection. These results resonate with previous works on chemical and functional gradients along the primate occipitotemporal visual pathway and raise the possibility that these gradients and functional architecture may be related to the visual activity–dependent expression of these extracellular matrix glycoproteins. PMID:19073625

Controlling nuclear RNA levels.

PubMed

Schmid, Manfred; Jensen, Torben Heick

2018-05-10

RNA turnover is an integral part of cellular RNA homeostasis and gene expression regulation. Whereas the cytoplasmic control of protein-coding mRNA is often the focus of study, we discuss here the less appreciated role of nuclear RNA decay systems in controlling RNA polymerase II (RNAPII)-derived transcripts. Historically, nuclear RNA degradation was found to be essential for the functionalization of transcripts through their proper maturation. Later, it was discovered to also be an important caretaker of nuclear hygiene by removing aberrant and unwanted transcripts. Recent years have now seen a set of new protein complexes handling a variety of new substrates, revealing functions beyond RNA processing and the decay of non-functional transcripts. This includes an active contribution of nuclear RNA metabolism to the overall cellular control of RNA levels, with mechanistic implications during cellular transitions.

Evidence that Poly(A) Binding Protein C1 Binds Nuclear Pre-mRNA Poly(A) Tails

PubMed Central

Hosoda, Nao; Lejeune, Fabrice; Maquat, Lynne E.

2006-01-01

In mammalian cells, poly(A) binding protein C1 (PABP C1) has well-known roles in mRNA translation and decay in the cytoplasm. However, PABPC1 also shuttles in and out of the nucleus, and its nuclear function is unknown. Here, we show that PABPC1, like the major nuclear poly(A) binding protein PABPN1, associates with nuclear pre-mRNAs that are polyadenylated and intron containing. PABPC1 does not bind nonpolyadenylated histone mRNA, indicating that the interaction of PABPC1 with pre-mRNA requires a poly(A) tail. Consistent with this conclusion, UV cross-linking results obtained using intact cells reveal that PABPC1 binds directly to pre-mRNA poly(A) tails in vivo. We also show that PABPC1 immunopurifies with poly(A) polymerase, suggesting that PABPC1 is acquired by polyadenylated transcripts during poly(A) tail synthesis. Our findings demonstrate that PABPC1 associates with polyadenylated transcripts earlier in mammalian mRNA biogenesis than previously thought and offer insights into the mechanism by which PABPC1 is recruited to newly synthesized poly(A). Our results are discussed in the context of pre-mRNA processing and stability and mRNA trafficking and the pioneer round of translation. PMID:16581783

Quality control of mRNP biogenesis: networking at the transcription site.

PubMed

Eberle, Andrea B; Visa, Neus

2014-08-01

Eukaryotic cells carry out quality control (QC) over the processes of RNA biogenesis to inactivate or eliminate defective transcripts, and to avoid their production. In the case of protein-coding transcripts, the quality controls can sense defects in the assembly of mRNA-protein complexes, in the processing of the precursor mRNAs, and in the sequence of open reading frames. Different types of defect are monitored by different specialized mechanisms. Some of them involve dedicated factors whose function is to identify faulty molecules and target them for degradation. Others are the result of a more subtle balance in the kinetics of opposing activities in the mRNA biogenesis pathway. One way or another, all such mechanisms hinder the expression of the defective mRNAs through processes as diverse as rapid degradation, nuclear retention and transcriptional silencing. Three major degradation systems are responsible for the destruction of the defective transcripts: the exosome, the 5'-3' exoribonucleases, and the nonsense-mediated mRNA decay (NMD) machinery. This review summarizes recent findings on the cotranscriptional quality control of mRNA biogenesis, and speculates that a protein-protein interaction network integrates multiple mRNA degradation systems with the transcription machinery. Copyright © 2014 Elsevier Ltd. All rights reserved.

Exon 2-mediated c-myc mRNA decay in vivo is independent of its translation.

PubMed Central

Pistoi, S; Roland, J; Babinet, C; Morello, D

1996-01-01

We have previously shown that the steady-state level of c-myc mRNA in vivo is primarily controlled by posttranscriptional regulatory mechanisms. To identify the sequences involved in this process, we constructed a series of H-2/myc transgenic lines in which various regions of the human c-MYC gene were placed under the control of the quasi-ubiquitous H-2K class I regulatory sequences. We demonstrated that the presence of one of the two coding exons, exon 2 or exon 3, is sufficient to confer a level of expression of transgene mRNA similar to that of endogenous c-myc in various adult tissues as well as after partial hepatectomy or after protein synthesis inhibition. We now focus on the molecular mechanisms involved in modulation of expression of mRNAs containing c-myc exon 2 sequences, with special emphasis on the coupling between translation and c-myc mRNA turnover. We have undertaken an analysis of expression, both at the mRNA level and at the protein level, of new transgenic constructs in which the translation is impaired either by disruption of the initiation codon or by addition of stop codons upstream of exon 2. Our results show that the translation of c-myc exon 2 is not required for regulated expression of the transgene in the different situations analyzed, and therefore they indicate that the mRNA destabilizing function of exon 2 is independent of translation by ribosomes. Our investigations also reveal that, in the thymus, some H-2/myc transgenes express high levels of mRNA but low levels of protein. Besides the fact that these results suggest the existence of tissue-specific mechanisms that control c-myc translatability in vivo, they also bring another indication of the uncoupling of c-myc mRNA translation and degradation. PMID:8756668

G protein-coupled estrogen receptor 1 (GPER, GPR 30) in normal human endometrium and early pregnancy decidua.

PubMed

Kolkova, Z; Noskova, V; Ehinger, A; Hansson, S; Casslén, B

2010-10-01

The recently identified trans-membrane G protein-coupled estrogen receptor 1 (GPER, GPR30) has been implicated in rapid non-genomic effects of estrogens. This focuses on expression and localization of GPER mRNA and protein in normal cyclic endometrium and early pregnancy decidua. Real-time PCR, western blotting, in situ hybridization and immuno-histochemistry were used. Endometrial expression of GPER mRNA was lower in the secretory phase than in the proliferative phase, and even lower in the decidua. The expression pattern was similar to that of ERα mRNA, but different from that of ERβ mRNA. Western blot detected GPER protein as a 54 kDa band in all endometrial and decidual samples. In contrast to the mRNA, GPER protein did not show cyclic variations. Apparently, a lower amount of mRNA is sufficient to maintain protein levels in the secretory phase. GPER mRNA was predominantly localized in the epithelium of mid- and late-proliferative phase endometrium, whereas expression in early proliferative and secretory glands could not be distinguished from the diffuse stromal signal, which was present throughout the cycle. Immuno-staining for GPER was stronger in glandular and luminal epithelium than in the stroma throughout the cycle. The cyclic variations of GPER mRNA obviously relate to strong epithelial expression in the proliferative phase, and the expression pattern suggests regulation by ovarian steroids. GPER protein is present in endometrial tissue throughout the cycle, and the epithelial localization suggests potential functions during sperm migration at mid-cycle, as well as decidualization and blastocyst implantation in the mid-secretory phase.

In the right place at the right time: visualizing and understanding mRNA localization

PubMed Central

Buxbaum, Adina R.; Haimovich, Gal

2015-01-01

The spatial regulation of protein translation is an efficient way to create functional and structural asymmetries in cells. Recent research has furthered our understanding of how individual cells spatially organize protein synthesis, by applying innovative technology to characterize the relationship between mRNAs and their regulatory proteins, single-mRNA trafficking dynamics, physiological effects of abrogating mRNA localization in vivo and for endogenous mRNA labelling. The implementation of new imaging technologies has yielded valuable information on mRNA localization, for example, by observing single molecules in tissues. The emerging movements and localization patterns of mRNAs in morphologically distinct unicellular organisms and in neurons have illuminated shared and specialized mechanisms of mRNA localization, and this information is complemented by transgenic and biochemical techniques that reveal the biological consequences of mRNA mislocalization. PMID:25549890

A 3' UTR-Derived Small RNA Provides the Regulatory Noncoding Arm of the Inner Membrane Stress Response.

PubMed

Chao, Yanjie; Vogel, Jörg

2016-02-04

Small RNAs (sRNAs) from conserved noncoding genes are crucial regulators in bacterial signaling pathways but have remained elusive in the Cpx response to inner membrane stress. Here we report that an alternative biogenesis pathway releasing the conserved mRNA 3' UTR of stress chaperone CpxP as an ∼60-nt sRNA provides the noncoding arm of the Cpx response. This so-called CpxQ sRNA, generated by general mRNA decay through RNase E, acts as an Hfq-dependent repressor of multiple mRNAs encoding extracytoplasmic proteins. Both CpxQ and the Cpx pathway are required for cell survival under conditions of dissipation of membrane potential. Our discovery of CpxQ illustrates how the conversion of a transcribed 3' UTR into an sRNA doubles the output of a single mRNA to produce two factors with spatially segregated functions during inner membrane stress: a chaperone that targets problematic proteins in the periplasm and a regulatory RNA that dampens their synthesis in the cytosol. Copyright © 2016 Elsevier Inc. All rights reserved.

The Structure and Function of the Rous Sarcoma virus RNA Stability Element

PubMed Central

Withers, Johanna B.; Beemon, Karen L.

2013-01-01

For simple retroviruses, such as the Rous sarcoma virus (RSV), post-transcriptional control elements regulate viral RNA splicing, export, stability, and packaging into virions. These RNA sequences interact with cellular host proteins to regulate and facilitate productive viral infections. One such element, known as the RSV stability element (RSE), is required for maintaining stability of the full-length unspliced RNA. This viral RNA serves as the mRNA for the Gag and Pol proteins and also as the genome packaged in progeny virions. When the RSE is deleted from the viral RNA, the unspliced RNA becomes unstable and is degraded in a Upf1-dependent manner. Current evidence suggests that the RSE inhibits recognition of the viral gag termination codon by the nonsense-mediated mRNA decay (NMD) pathway. We believe that the RSE acts as an insulator to NMD, thereby preventing at least one of the required functional steps that target an mRNA for degradation. Here, we discuss the history of the RSE and the current model of how the RSE is interacting with cellular NMD factors. PMID:21769913

Experimental reconstitution of chronic ER stress in the liver reveals feedback suppression of BiP mRNA expression

PubMed Central

Gomez, Javier A; Rutkowski, D Thomas

2016-01-01

Endoplasmic reticulum (ER) stress is implicated in many chronic diseases, but very little is known about how the unfolded protein response (UPR) responds to persistent ER stress in vivo. Here, we experimentally reconstituted chronic ER stress in the mouse liver, using repeated injection of a low dose of the ER stressor tunicamycin. Paradoxically, this treatment led to feedback-mediated suppression of a select group of mRNAs, including those encoding the ER chaperones BiP and GRP94. This suppression was due to both silencing of the ATF6α pathway of UPR-dependent transcription and enhancement of mRNA degradation, possibly via regulated IRE1-dependent decay (RIDD). The suppression of mRNA encoding BiP was phenocopied by ectopic overexpression of BiP protein, and was also observed in obese mice. Our findings suggest that persistent cycles of UPR activation and deactivation create an altered, quasi-stable setpoint for UPR-dependent transcriptional regulation—an outcome that could be relevant to conditions such as metabolic syndrome. DOI: http://dx.doi.org/10.7554/eLife.20390.001 PMID:27938665

Decapping activators in Saccharomyces cerevisiae act by multiple mechanisms.

PubMed

Nissan, Tracy; Rajyaguru, Purusharth; She, Meipei; Song, Haiwei; Parker, Roy

2010-09-10

Eukaryotic mRNA degradation often occurs in a process whereby translation initiation is inhibited and the mRNA is targeted for decapping. In yeast cells, Pat1, Scd6, Edc3, and Dhh1 all function to promote decapping by an unknown mechanism(s). We demonstrate that purified Scd6 and a region of Pat1 directly repress translation in vitro by limiting the formation of a stable 48S preinitiation complex. Moreover, while Pat1, Edc3, Dhh1, and Scd6 all bind the decapping enzyme, only Pat1 and Edc3 enhance its activity. We also identify numerous direct interactions between Pat1, Dcp1, Dcp2, Dhh1, Scd6, Edc3, Xrn1, and the Lsm1-7 complex. These observations identify three classes of decapping activators that function to directly repress translation initiation and/or stimulate Dcp1/2. Moreover, Pat1 is identified as critical in mRNA decay by first inhibiting translation initiation, then serving as a scaffold to recruit components of the decapping complex, and finally activating Dcp2. Copyright © 2010 Elsevier Inc. All rights reserved.

Conceptual Modeling in Systems Biology Fosters Empirical Findings: The mRNA Lifecycle

PubMed Central

Dori, Dov; Choder, Mordechai

2007-01-01

One of the main obstacles to understanding complex biological systems is the extent and rapid evolution of information, way beyond the capacity individuals to manage and comprehend. Current modeling approaches and tools lack adequate capacity to model concurrently structure and behavior of biological systems. Here we propose Object-Process Methodology (OPM), a holistic conceptual modeling paradigm, as a means to model both diagrammatically and textually biological systems formally and intuitively at any desired number of levels of detail. OPM combines objects, e.g., proteins, and processes, e.g., transcription, in a way that is simple and easily comprehensible to researchers and scholars. As a case in point, we modeled the yeast mRNA lifecycle. The mRNA lifecycle involves mRNA synthesis in the nucleus, mRNA transport to the cytoplasm, and its subsequent translation and degradation therein. Recent studies have identified specific cytoplasmic foci, termed processing bodies that contain large complexes of mRNAs and decay factors. Our OPM model of this cellular subsystem, presented here, led to the discovery of a new constituent of these complexes, the translation termination factor eRF3. Association of eRF3 with processing bodies is observed after a long-term starvation period. We suggest that OPM can eventually serve as a comprehensive evolvable model of the entire living cell system. The model would serve as a research and communication platform, highlighting unknown and uncertain aspects that can be addressed empirically and updated consequently while maintaining consistency. PMID:17849002

β decay studies of n-rich Cs isotopes with the ISOLDE Decay Station

NASA Astrophysics Data System (ADS)

Lică, R.; Benzoni, G.; Morales, A. I.; Borge, M. J. G.; Fraile, L. M.; Mach, H.; Madurga, M.; Sotty, C.; Vedia, V.; De Witte, H.; Benito, J.; Berry, T.; Blasi, N.; Bracco, A.; Camera, F.; Ceruti, S.; Charviakova, V.; Cieplicka-Oryńczak, N.; Costache, C.; Crespi, F. C. L.; Creswell, J.; Fernández-Martínez, G.; Fynbo, H.; Greenlees, P.; Homm, I.; Huyse, M.; Jolie, J.; Karayonchev, V.; Köster, U.; Konki, J.; Kröll, T.; Kurcewicz, J.; Kurtukian-Nieto, T.; Lazarus, I.; Leoni, S.; Lund, M.; Marginean, N.; Marginean, R.; Mihai, C.; Mihai, R.; Negret, A.; Orduz, A.; Patyk, Z.; Pascu, S.; Pucknell, V.; Rahkila, P.; Regis, J. M.; Rotaru, F.; Saed-Sami, N.; Sánchez-Tembleque, V.; Stanoiu, M.; Tengblad, O.; Thuerauf, M.; Turturica, A.; Van Duppen, P.; Warr, N.

2017-05-01

Neutron-rich Ba isotopes are expected to exhibit octupolar correlations, reaching their maximum in isotopes around mass A = 146. The odd-A neutron-rich members of this isotopic chain show typical patterns related to non-axially symmetric shapes, which are however less marked compared to even-A ones, pointing to a major contribution from vibrations. In the present paper we present results from a recent study focused on 148-150Cs β-decay performed at the ISOLDE Decay Station equipped with fast-timing detectors. A detailed analysis of the measured decay half-lives and decay scheme of 149Ba is presented, giving a first insight in the structure of this neutron-rich nucleus.

Diversity in mRNA expression of the serine-type carboxypeptidase ocpG in Aspergillus oryzae through intron retention.

PubMed

Ishida, Ken; Kuboshima, Megumi; Morita, Hiroto; Maeda, Hiroshi; Okamoto, Ayako; Takeuchi, Michio; Yamagata, Youhei

2014-01-01

Alternative splicing is thought to be a means for diversification of products by mRNA modification. Although some intron retentions are predicted by transcriptome analysis in Aspergillus oryzae, its physiological significance remains unknown. We found that intron retention occurred occasionally in the serine-type carboxypeptidase gene, ocpG. Analysis under various culture conditions revealed that extracellular nitrogen conditions influence splicing patterns; this suggested that there might be a correlation between splicing efficiency and the necessity of OcpG activity for obtaining a nitrogen source. Since further analysis showed that splicing occurred independently in each intron, we constructed ocpG intron-exchanging strain by interchanging the positions of intron-1 and intron-2. The splicing pattern indicated the probability that ocpG intron retention was affected by the secondary structures of intronic mRNA.

The synthesis of polyadenylated messenger RNA in herpes simplex type I virus infected BHK cells.

PubMed

Harris, T J; Wildy, P

1975-09-01

The pattern of polyadenylated messenger RNA (mRNA) synthesis in BHK cell monolayers, infected under defined conditions with herpes simplex type I virus has been investigated by polyacrylamide gel electrophoresis or pulse-labelled RNA isolated by oligo dT-cellulose chromatography. Two classes of mRNA molecules were synthesized in infected cells; these were not detected in uninfected cells. The rate of synthesis of the larger, 18 to 30S RNA class reached a maximum soon after injection and then declined, whereas the rate of synthesis of the 7 to 11 S RNA class did not reach a maximum until much later and did not decline. In the presence of cytosine arabinoside, the rate of mRNA synthesis in infected cells was reduced but the electrophoretic pattern remained the same.

Evaluation of the effect of different stretching patterns on force decay and tensile properties of elastomeric ligatures

PubMed Central

Aminian, Amin; Nakhaei, Samaneh; Agahi, Raha Habib; Rezaeizade, Masoud; Aliabadi, Hamed Mirzazadeh; Heidarpour, Majid

2015-01-01

Background: There have been numerous researches on elastomeric ligatures, but clinical conditions in different stages of treatment are not exactly similar to laboratory conditions. The aim of this in vitro study was to simulate clinical conditions and evaluate the effect of three stretching patterns on the amount of force, tensile strength (TS) and extension to TS of the elastomers during 8 weeks. Materials and Methods: Forces, TS and extension to TS of two different brands of elastomers were measured at initial, 24 h and 2, 4, and 8-week intervals using a testing machine. During the study period, the elastomers were stored in three different types of jig (uniform stretching, 1 and 3 mm point stretching) designed by the computer-aided design and computer-aided manufacturing technique in order to simulate the different stages of orthodontic treatment. Results: The elastomeric ligatures under study exhibited a similar force decay pattern. The maximum force decay occurred during the first 24 h (49.9% ± 15%) and the amount of force decay was 75.7% ± 8% after 8 weeks. In general, the TS decreased during the study period, and the amount of extension to TS increased. Conclusion: Although the elastic behavior of all ligatures under study was similar, the amount of residual force, TS and extension to TS increased in elastomers under point stretching pattern. PMID:26759597

Evaluation of the effect of different stretching patterns on force decay and tensile properties of elastomeric ligatures.

PubMed

Aminian, Amin; Nakhaei, Samaneh; Agahi, Raha Habib; Rezaeizade, Masoud; Aliabadi, Hamed Mirzazadeh; Heidarpour, Majid

2015-01-01

There have been numerous researches on elastomeric ligatures, but clinical conditions in different stages of treatment are not exactly similar to laboratory conditions. The aim of this in vitro study was to simulate clinical conditions and evaluate the effect of three stretching patterns on the amount of force, tensile strength (TS) and extension to TS of the elastomers during 8 weeks. Forces, TS and extension to TS of two different brands of elastomers were measured at initial, 24 h and 2, 4, and 8-week intervals using a testing machine. During the study period, the elastomers were stored in three different types of jig (uniform stretching, 1 and 3 mm point stretching) designed by the computer-aided design and computer-aided manufacturing technique in order to simulate the different stages of orthodontic treatment. The elastomeric ligatures under study exhibited a similar force decay pattern. The maximum force decay occurred during the first 24 h (49.9% ± 15%) and the amount of force decay was 75.7% ± 8% after 8 weeks. In general, the TS decreased during the study period, and the amount of extension to TS increased. Although the elastic behavior of all ligatures under study was similar, the amount of residual force, TS and extension to TS increased in elastomers under point stretching pattern.

Wood and humus decay strategies by white-rot basidiomycetes correlate with two different dye decolorization and enzyme secretion patterns on agar plates.

PubMed

Barrasa, José M; Blanco, María N; Esteve-Raventós, Fernando; Altés, Alberto; Checa, Julia; Martínez, Angel T; Ruiz-Dueñas, Francisco J

2014-11-01

During several forays for ligninolytic fungi in different Spanish native forests, 35 white-rot basidiomycetes growing on dead wood (16 species from 12 genera) and leaf litter (19 species from 10 genera) were selected for their ability to decolorize two recalcitrant aromatic dyes (Reactive Blue 38 and Reactive Black 5) added to malt extract agar medium. In this study, two dye decolorization patterns were observed and correlated with two ecophysiological groups (wood and humus white-rot basidiomycetes) and three taxonomical groups (orders Polyporales, Hymenochaetales and Agaricales). Depending on the above groups, different decolorization zones were observed on the dye-containing plates, being restricted to the colony area or extending to the surrounding medium, which suggested two different decay strategies. These two strategies were related to the ability to secrete peroxidases and laccases inside (white-rot wood Polyporales, Hymenochaetales and Agaricales) and outside (white-rot humus Agaricales) of the fungal colony, as revealed by enzymatic tests performed directly on the agar plates. Similar oxidoreductases production patterns were observed when fungi were grown in the absence of dyes, although the set of enzyme released was different. All these results suggest that the decolorization patterns observed could be related with the existence of two decay strategies developed by white-rot basidiomycetes adapted to wood and leaf litter decay in the field. Published by Elsevier Inc.

Investigating microbial transformations of soil organic matter: synthesizing knowledge from disparate fields to guide new experimentation

NASA Astrophysics Data System (ADS)

Billings, S. A.; Tiemann, L. K.; Ballantyne, F., IV; Lehmeier, C.; Min, K.

2014-12-01

Investigators of soil organic matter (SOM) transformations struggle with a deceptively simple-sounding question: "Why does some SOM leave the soil profile relatively quickly, while other compounds, especially those at depth, appear to be retained on timescales ranging from the decadal to the millennial?" This question is important on both practical and academic levels, but addressing it is challenging for a multitude of reasons. Simultaneous with soil-specific advances, multiple other disciplines have enhanced their knowledge bases in ways potentially useful for future investigations of SOM decay. In this article, we highlight observations highly relevant for those investigating SOM decay and retention but often emanating from disparate fields and residing in literature seldom cited in SOM research. We focus on recent work in two key areas. First, we turn to experimental approaches using natural and artificial aquatic environments to investigate patterns of microbially-mediated OM transformations as environmental conditions change, and highlight how aquatic microbial responses to environmental change can reveal processes likely important to OM decay and retention in soils. Second, we emphasize the importance of establishing intrinsic patterns of decay kinetics for purified substrates commonly found in soils to develop baseline rates. These decay kinetics - which represent the upper limit of the reaction rates - can then be compared to substrate decay kinetics observed in natural samples, which integrate intrinsic decay reaction rates and edaphic factors essential to the site under study but absent in purified systems. That comparison permits the site-specific factors to be parsed from the fundamental decay kinetics, an important advance in our understanding of SOM decay (and thus persistence) in natural systems. We then suggest ways in which empirical observations from aquatic systems and purified enzyme-substrate reaction kinetics can be used to advance recent theoretical efforts in SOM-focused research. Finally, we suggest how the observations in aquatic and purified enzyme-substrate systems could be used to help unravel the puzzles presented by oft-observed patterns of SOM characteristics with depth, as one example of the many perplexing SOM-related problems.

Imaging mRNA In Vivo, from Birth to Death.

PubMed

Tutucci, Evelina; Livingston, Nathan M; Singer, Robert H; Wu, Bin

2018-05-20

RNA is the fundamental information transfer system in the cell. The ability to follow single messenger RNAs (mRNAs) from transcription to degradation with fluorescent probes gives quantitative information about how the information is transferred from DNA to proteins. This review focuses on the latest technological developments in the field of single-mRNA detection and their usage to study gene expression in both fixed and live cells. By describing the application of these imaging tools, we follow the journey of mRNA from transcription to decay in single cells, with single-molecule resolution. We review current theoretical models for describing transcription and translation that were generated by single-molecule and single-cell studies. These methods provide a basis to study how single-molecule interactions generate phenotypes, fundamentally changing our understating of gene expression regulation.

Aromatase, steroid-5-alpha-reductase type 1 and type 2 mRNA expression in gonads and in brain of Xenopus laevis during ontogeny.

PubMed

Urbatzka, R; Lutz, I; Kloas, W

2007-01-01

The key enzymes involved in the production of endogenous sex steroids are steroid-5-alpha-reductase and aromatase converting testosterone (T) into dihydrotestosterone (DHT) and into estradiol (E2), respectively. To gain more insights into the molecular mechanisms of sexual differentiation of amphibians, we determined the mRNA expression of steroid-5-alpha-reductase type1 (Srd5a1), type2 (Srd5a2) and aromatase (Aro) during ontogeny starting from the egg and ending after completion of metamorphosis in Xenopus laevis. Expression of all three enzymes was measured by means of semi-quantitative RT-PCR, determining for the first time Srd5a1 and Srd5a2 mRNA expression in amphibians. mRNA was analyzed in whole body homogenates from stage 12 to 48, while brain and gonads with kidney were studied separately from stage 48 to 66. Different ontogenetic mRNA expression patterns were observed for all genes analyzed, revealing early mRNA expression of Srd5a1 already in the egg at stage 12 whereas Srd5a2 and Aro was detected at stage 39. Sex-specific mRNA expressions of Srd5a2 and of Aro were determined in the gonads with kidney but not in brain. Srd5a2 was two-fold higher expressed in testes than in ovaries while Aro mRNA was ten-fold higher in ovaries. No gender-specific mRNA expression was observed for Srd5a1 in gonads and in brain. The ontogenetic patterns of Aro, Srd5a1 and Srd5a2 suggest that these genes are involved in sexual differentiation of gonads and brain already in early developmental stages. Especially in gonads Srd5a2 seems to be important for physiological regulation of testis development while Aro is associated with the development of ovaries.

Changes in cholecystokinin and peptide Y gene expression with feeding in yellowtail (Seriola quinqueradiata): relation to pancreatic exocrine regulation.

PubMed

Murashita, Koji; Fukada, Haruhisa; Hosokawa, Hidetsuyo; Masumoto, Toshiro

2007-03-01

In fish, the regulation of digestive enzyme secretion by hormonal control such as cholecystokinin (CCK) and neuropeptide Y (NPY)-related peptide is not well understood. To investigate the roles of fish CCK and peptide Y (PY) in digestive enzyme secretion, mRNA levels of CCK and PY, pyloric caeca enzyme activities and mRNA levels of pancreatic digestive enzymes (lipase, trypsin and amylase) were measured at pre- and post-prandial stages in yellowtail. Pyloric caeca were sampled at 0, 0.5, 1.5, 3, 6, 12, 24 and 48 h after feeding. The mRNA levels of trypsin and amylase increased after feeding, suggesting that transcription was induced by feed ingestion. Digestive enzyme activities decreased in exocrine pancreas after feeding, suggesting the stored enzyme was secreted from pancreas post-prandially. mRNA levels for CCK displayed a time-dependent increase, peaking between 1.5 and 3 h after-feeding followed by a rapid decrease 3 to 6 h after feeding. The mRNA expression pattern of PY was inverse to the pattern of CCK, decreasing until 1.5 h after feeding and then rising to initial levels by 12 h after feeding. These results suggest that CCK and PY work antagonistically in the exocrine pancreas of yellowtail.

The loss of SMG1 causes defects in quality control pathways in Physcomitrella patens

PubMed Central

Lang, Daniel; Zimmer, Andreas D; Causier, Barry

2018-01-01

Abstract Nonsense-mediated mRNA decay (NMD) is important for RNA quality control and gene regulation in eukaryotes. NMD targets aberrant transcripts for decay and also directly influences the abundance of non-aberrant transcripts. In animals, the SMG1 kinase plays an essential role in NMD by phosphorylating the core NMD factor UPF1. Despite SMG1 being ubiquitous throughout the plant kingdom, little is known about its function, probably because SMG1 is atypically absent from the genome of the model plant, Arabidopsis thaliana. By combining our previously established SMG1 knockout in moss with transcriptome-wide analysis, we reveal the range of processes involving SMG1 in plants. Machine learning assisted analysis suggests that 32% of multi-isoform genes produce NMD-targeted transcripts and that splice junctions downstream of a stop codon act as the major determinant of NMD targeting. Furthermore, we suggest that SMG1 is involved in other quality control pathways, affecting DNA repair and the unfolded protein response, in addition to its role in mRNA quality control. Consistent with this, smg1 plants have increased susceptibility to DNA damage, but increased tolerance to unfolded protein inducing agents. The potential involvement of SMG1 in RNA, DNA and protein quality control has major implications for the study of these processes in plants. PMID:29596649

A Novel de novo CDH1 Germline Variant Aids in the Classification of C-terminal E-cadherin Alterations Predicted to Escape Nonsense-Mediated mRNA Decay.

PubMed

Krempely, Kate; Karam, Rachid

2018-05-24

Most truncating CDH1 pathogenic alterations confer an elevated lifetime risk of diffuse gastric cancer and lobular breast cancer. However, transcripts containing carboxyl-terminal (C-terminal) premature stop codons have been demonstrated to escape the nonsense-mediated mRNA decay (NMD) pathway, and gastric and breast cancer risks associated with these truncations should be carefully evaluated. A female patient underwent multigene panel testing due to a personal history of invasive lobular breast cancer diagnosed at age 54, which identified the germline CDH1 nonsense alteration, c.2506G>T (p.E836*), in the last exon of the gene. Subsequent parental testing for the alteration was negative and additional short tandem repeat analysis confirmed the familial relationships and the de novo occurrence in the proband. Based on the de novo occurrence, clinical history, and rarity in general population databases, this alteration was classified as a likely pathogenic variant. This is the most C-terminal pathogenic alteration reported to date. Additionally, this alteration contributed to the classification of six other upstream CDH1 C-terminal truncating variants as pathogenic or likely pathogenic. Identifying the most distal pathogenic alteration provides evidence to classify other C-terminal truncating variants as either pathogenic or benign, a fundamental step to offering pre-symptomatic screening and prophylactic procedures to the appropriate patients. Cold Spring Harbor Laboratory Press.

bicoid mRNA localises to the Drosophila oocyte anterior by random Dynein-mediated transport and anchoring

PubMed Central

Trovisco, Vítor; Belaya, Katsiaryna; Nashchekin, Dmitry; Irion, Uwe; Sirinakis, George; Butler, Richard; Lee, Jack J; Gavis, Elizabeth R; St Johnston, Daniel

2016-01-01

bicoid mRNA localises to the Drosophila oocyte anterior from stage 9 of oogenesis onwards to provide a local source for Bicoid protein for embryonic patterning. Live imaging at stage 9 reveals that bicoid mRNA particles undergo rapid Dynein-dependent movements near the oocyte anterior, but with no directional bias. Furthermore, bicoid mRNA localises normally in shot2A2, which abolishes the polarised microtubule organisation. FRAP and photo-conversion experiments demonstrate that the RNA is stably anchored at the anterior, independently of microtubules. Thus, bicoid mRNA is localised by random active transport and anterior anchoring. Super-resolution imaging reveals that bicoid mRNA forms 110–120 nm particles with variable RNA content, but constant size. These particles appear to be well-defined structures that package the RNA for transport and anchoring. DOI: http://dx.doi.org/10.7554/eLife.17537.001 PMID:27791980

Antisense RNA: effect of ribosome binding sites, target location, size, and concentration on the translation of specific mRNA molecules.

PubMed

Daugherty, B L; Hotta, K; Kumar, C; Ahn, Y H; Zhu, J D; Pestka, S

1989-01-01

A series of plasmids were constructed to generate RNA complementary to the beta-galactosidase messenger RNA under control of the phage lambda PL promoter. These plasmids generate anti-lacZ mRNA bearing or lacking a synthetic ribosome binding site adjacent to the lambda PL promoter and/or the lacZ ribosome binding site in reverse orientation. Fragments of lacZ DNA from the 5' and/or the 3' region were used in these constructions. When these anti-mRNA molecules were produced in Escherichia coli 294, maximal inhibition of beta-galactosidase synthesis occurred when a functional ribosome binding site was present near the 5' end of the anti-mRNA and the anti-mRNA synthesized was complementary to the 5' region of the mRNA corresponding to the lacZ ribosome binding site and/or the 5'-coding sequence. Anti-mRNAs producing maximal inhibition of beta-galactosidase synthesis exhibited an anti-lacZ mRNA:normal lacZ mRNA ratio of 100:1 or higher. Those showing lower levels of inhibition exhibited much lower anti-lacZ mRNA:normal lacZ mRNA ratios. A functional ribosome binding site at the 5'-end was found to decrease the decay rate of the anti-lacZ mRNAs. In addition, the incorporation of a transcription terminator just downstream of the antisense segment provided for more efficient inhibition of lacZ mRNA translation due to synthesis of smaller and more abundant anti-lacZ mRNAs. The optimal constructions produced undetectable levels of beta-galactosidase synthesis.

KSRP Modulation of GAP-43 mRNA Stability Restricts Axonal Outgrowth in Embryonic Hippocampal Neurons

PubMed Central

Bird, Clark W.; Gardiner, Amy S.; Bolognani, Federico; Tanner, Daniel C.; Chen, Ching-Yi; Lin, Wei-Jye; Yoo, Soonmoon; Twiss, Jeffery L.; Perrone- Bizzozero, Nora

2013-01-01

The KH-type splicing regulatory protein (KSRP) promotes the decay of AU-rich element (ARE)-containing mRNAs. Although KSRP is expressed in the nervous system, very little is known about its role in neurons. In this study, we examined whether KSRP regulates the stability of the ARE-containing GAP-43 mRNA. We found that KSRP destabilizes this mRNA by binding to its ARE, a process that requires the presence of its fourth KH domain (KH4). Furthermore, KSRP competed with the stabilizing factor HuD for binding to these sequences. We also examined the functional consequences of KSRP overexpression and knockdown on the differentiation of primary hippocampal neurons in culture. Overexpression of full length KSRP or KSRP without its nuclear localization signal hindered axonal outgrowth in these cultures, while overexpression of a mutant protein without the KH4 domain that has less affinity for binding to GAP-43′s ARE had no effect. In contrast, depletion of KSRP led to a rise in GAP-43 mRNA levels and a dramatic increase in axonal length, both in KSRP shRNA transfected cells and neurons cultured from Ksrp+/− and Ksrp −/−embryos. Finally we found that overexpression of GAP-43 rescued the axonal outgrowth deficits seen with KSRP overexpression, but only when cells were transfected with GAP-43 constructs containing 3′ UTR sequences targeting the transport of this mRNA to axons. Together, our results suggest that KSRP is an important regulator of mRNA stability and axonal length that works in direct opposition to HuD to regulate the levels of GAP-43 and other ARE-containing neuronal mRNAs. PMID:24244461

v-Src oncogene product increases sphingosine kinase 1 expression through mRNA stabilization: alteration of AU-rich element-binding proteins.

PubMed

Sobue, S; Murakami, M; Banno, Y; Ito, H; Kimura, A; Gao, S; Furuhata, A; Takagi, A; Kojima, T; Suzuki, M; Nozawa, Y; Murate, T

2008-10-09

Sphingosine kinase 1 (SPHK1) is overexpressed in solid tumors and leukemia. However, the mechanism of SPHK1 overexpression by oncogenes has not been defined. We found that v-Src-transformed NIH3T3 cells showed a high SPHK1 mRNA, SPHK1 protein and SPHK enzyme activity. siRNA of SPHK1 inhibited the growth of v-Src-NIH3T3, suggesting the involvement of SPHK1 in v-Src-induced oncogenesis. v-Src-NIH3T3 showed activations of protein kinase C-alpha, signal transducers and activators of transcription 3 and c-Jun NH(2)-terminal kinase. Their inhibition suppressed SPHK1 expression in v-Src-NIH3T3, whereas their overexpression increased SPHK1 mRNA in NIH3T3. Unexpectedly, the nuclear run-on assay and the promoter analysis using 5'-promoter region of mouse SPHK1 did not show any significant difference between mock- and v-Src-NIH3T3. Furthermore, the half-life of SPHK1 mRNA in mock-NIH3T3 was nearly 15 min, whereas that of v-Src-NIH3T3 was much longer. Examination of two AU-rich region-binding proteins, AUF1 and HuR, that regulate mRNA decay reciprocally, showed decreased total AUF1 protein associated with increased tyrosine-phosphorylated form and increased serine-phosphorylated HuR protein in v-Src-NIH3T3. Modulation of AUF1 and HuR by their overexpression or siRNA revealed that SPHK1 mRNA in v-Src- and mock-NIH3T3 was regulated reciprocally by these factors. Our results showed, for the first time, a novel mechanism of v-Src-induced SPHK1 overexpression.

Discovering mutated driver genes through a robust and sparse co-regularized matrix factorization framework with prior information from mRNA expression patterns and interaction network.

PubMed

Xi, Jianing; Wang, Minghui; Li, Ao

2018-06-05

Discovery of mutated driver genes is one of the primary objective for studying tumorigenesis. To discover some relatively low frequently mutated driver genes from somatic mutation data, many existing methods incorporate interaction network as prior information. However, the prior information of mRNA expression patterns are not exploited by these existing network-based methods, which is also proven to be highly informative of cancer progressions. To incorporate prior information from both interaction network and mRNA expressions, we propose a robust and sparse co-regularized nonnegative matrix factorization to discover driver genes from mutation data. Furthermore, our framework also conducts Frobenius norm regularization to overcome overfitting issue. Sparsity-inducing penalty is employed to obtain sparse scores in gene representations, of which the top scored genes are selected as driver candidates. Evaluation experiments by known benchmarking genes indicate that the performance of our method benefits from the two type of prior information. Our method also outperforms the existing network-based methods, and detect some driver genes that are not predicted by the competing methods. In summary, our proposed method can improve the performance of driver gene discovery by effectively incorporating prior information from interaction network and mRNA expression patterns into a robust and sparse co-regularized matrix factorization framework.

Brain Activation Patterns at Exhaustion in Rats That Differ in Inherent Exercise Capacity

PubMed Central

Foley, Teresa E.; Brooks, Leah R.; Gilligan, Lori J.; Burghardt, Paul R.; Koch, Lauren G.; Britton, Steven L.; Fleshner, Monika

2012-01-01

In order to further understand the genetic basis for variation in inherent (untrained) exercise capacity, we examined the brains of 32 male rats selectively bred for high or low running capacity (HCR and LCR, respectively). The aim was to characterize the activation patterns of brain regions potentially involved in differences in inherent running capacity between HCR and LCR. Using quantitative in situ hybridization techniques, we measured messenger ribonuclease (mRNA) levels of c-Fos, a marker of neuronal activation, in the brains of HCR and LCR rats after a single bout of acute treadmill running (7.5–15 minutes, 15° slope, 10 m/min) or after treadmill running to exhaustion (15–51 minutes, 15° slope, initial velocity 10 m/min). During verification of trait differences, HCR rats ran six times farther and three times longer prior to exhaustion than LCR rats. Running to exhaustion significantly increased c-Fos mRNA activation of several brain areas in HCR, but LCR failed to show significant elevations of c-Fos mRNA at exhaustion in the majority of areas examined compared to acutely run controls. Results from these studies suggest that there are differences in central c-Fos mRNA expression, and potential brain activation patterns, between HCR and LCR rats during treadmill running to exhaustion and these differences could be involved in the variation in inherent running capacity between lines. PMID:23028992

Comprehensive Study On The Metastable Negative Ion Fragmentation Of Individual Dna Components And Larger Oligonucleotides

NASA Astrophysics Data System (ADS)

Ingolfsson, O.; Flosadottir, H. D.; Omarsson, B.; Ilko, B.

2010-07-01

Here we present a systematic study on the unimolecular decay pathways of the deprotonated building blocks of DNA and RNA to address the following questions: 1. Are the negative ion fragmentation patterns observed in the metastable decay of individual DNA components still evident when these are combined to larger oligonucleotides? 2. What is the significance of the charge location in determining the fragmentation pathways in the metastable decay process? 3. Are those metastable decay channels relevant in dissociative electron attachment to DNA components? To address these questions we have studied the fragmentation patterns of the deprotonated ribose and ribose 5'-monophosphate, the fragmentation patterns of the individual bases, all nucleosides and all 2'-deoxynucleosides as well as the individual nucleotides and several combinations of hexameric oligonucleotides. Furthermore, to understand the significance of the charge location in determining the fragmentation path in the metastable decay process of these deprotonated ions we have also studied modified uridine and guanosine. These have been modified to block different deprotonation sites and thus to control the initial step in the in the fragmentation process i.e. the site of deprotonation. In addition to our experimental approach we have also simulated the metastable fragmentation of the deprotonated uridine and 2'-deoxyguanosine to clarify the mechanisms and fragmentation patterns observed. Where data is available, the results are compared to dissociative electron attachment to DNA components and discussed in context to the underlying mechanism. Experiments on modified nucleosides where selected deprotonation sites have been blocked are used to verify the predicted reaction paths and imulations on uridine and 2'-deoxyguanosine are compared to the experimental results and used to shed light on the mechanisms involved.

Optical proximity correction (OPC) in near-field lithography with pixel-based field sectioning time modulation

NASA Astrophysics Data System (ADS)

Oh, Seonghyeon; Han, Dandan; Shim, Hyeon Bo; Hahn, Jae W.

2018-01-01

Subwavelength features have been successfully demonstrated in near-field lithography. In this study, the point spread function (PSF) of a near-field beam spot from a plasmonic ridge nanoaperture is discussed with regard to the complex decaying characteristic of a non-propagating wave and the asymmetry of the field distribution for pattern design. We relaxed the shape complexity of the field distribution with pixel-based optical proximity correction (OPC) for simplifying the pattern image distortion. To enhance the pattern fidelity for a variety of arbitrary patterns, field-sectioning structures are formulated via convolutions with a time-modulation function and a transient PSF along the near-field dominant direction. The sharpness of corners and edges, and line shortening can be improved by modifying the original target pattern shape using the proposed approach by considering both the pattern geometry and directionality of the field decay for OPC in near-field lithography.

Optical proximity correction (OPC) in near-field lithography with pixel-based field sectioning time modulation.

PubMed

Oh, Seonghyeon; Han, Dandan; Shim, Hyeon Bo; Hahn, Jae W

2018-01-26

Subwavelength features have been successfully demonstrated in near-field lithography. In this study, the point spread function (PSF) of a near-field beam spot from a plasmonic ridge nanoaperture is discussed with regard to the complex decaying characteristic of a non-propagating wave and the asymmetry of the field distribution for pattern design. We relaxed the shape complexity of the field distribution with pixel-based optical proximity correction (OPC) for simplifying the pattern image distortion. To enhance the pattern fidelity for a variety of arbitrary patterns, field-sectioning structures are formulated via convolutions with a time-modulation function and a transient PSF along the near-field dominant direction. The sharpness of corners and edges, and line shortening can be improved by modifying the original target pattern shape using the proposed approach by considering both the pattern geometry and directionality of the field decay for OPC in near-field lithography.

Minimal 2'-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity

PubMed Central

Basila, Megan; Kelley, Melissa L.

2017-01-01

Since its initial application in mammalian cells, CRISPR-Cas9 has rapidly become a preferred method for genome engineering experiments. The Cas9 nuclease is targeted to genomic DNA using guide RNAs (gRNA), either as the native dual RNA system consisting of a DNA-targeting CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), or as a chimeric single guide RNA (sgRNA). Entirely DNA-free CRISPR-Cas9 systems using either Cas9 protein or Cas9 mRNA and chemically synthesized gRNAs allow for transient expression of CRISPR-Cas9 components, thereby reducing the potential for off-targeting, which is a significant advantage in therapeutic applications. In addition, the use of synthetic gRNA allows for the incorporation of chemical modifications for enhanced properties including improved stability. Previous studies have demonstrated the utility of chemically modified gRNAs, but have focused on one pattern with multiple modifications in co-electroporation with Cas9 mRNA or multiple modifications and patterns with Cas9 plasmid lipid co-transfections. Here we present gene editing results using a series of chemically modified synthetic sgRNA molecules and chemically modified crRNA:tracrRNA molecules in both electroporation and lipid transfection assessing indel formation and/or phenotypic gene knockout. We show that while modifications are required for co-electroporation with Cas9 mRNA, some modification patterns of the gRNA are toxic to cells compared to the unmodified gRNA and most modification patterns do not significantly improve gene editing efficiency. We also present modification patterns of the gRNA that can modestly improve Cas9 gene editing efficiency when co-transfected with Cas9 mRNA or Cas9 protein (> 1.5-fold difference). These results indicate that for certain applications, including those relevant to primary cells, the incorporation of some, but not all chemical modification patterns on synthetic crRNA:tracrRNA or sgRNA can be beneficial to CRISPR-Cas9 gene editing. PMID:29176845

Minimal 2'-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity.

PubMed

Basila, Megan; Kelley, Melissa L; Smith, Anja van Brabant

2017-01-01

Since its initial application in mammalian cells, CRISPR-Cas9 has rapidly become a preferred method for genome engineering experiments. The Cas9 nuclease is targeted to genomic DNA using guide RNAs (gRNA), either as the native dual RNA system consisting of a DNA-targeting CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), or as a chimeric single guide RNA (sgRNA). Entirely DNA-free CRISPR-Cas9 systems using either Cas9 protein or Cas9 mRNA and chemically synthesized gRNAs allow for transient expression of CRISPR-Cas9 components, thereby reducing the potential for off-targeting, which is a significant advantage in therapeutic applications. In addition, the use of synthetic gRNA allows for the incorporation of chemical modifications for enhanced properties including improved stability. Previous studies have demonstrated the utility of chemically modified gRNAs, but have focused on one pattern with multiple modifications in co-electroporation with Cas9 mRNA or multiple modifications and patterns with Cas9 plasmid lipid co-transfections. Here we present gene editing results using a series of chemically modified synthetic sgRNA molecules and chemically modified crRNA:tracrRNA molecules in both electroporation and lipid transfection assessing indel formation and/or phenotypic gene knockout. We show that while modifications are required for co-electroporation with Cas9 mRNA, some modification patterns of the gRNA are toxic to cells compared to the unmodified gRNA and most modification patterns do not significantly improve gene editing efficiency. We also present modification patterns of the gRNA that can modestly improve Cas9 gene editing efficiency when co-transfected with Cas9 mRNA or Cas9 protein (> 1.5-fold difference). These results indicate that for certain applications, including those relevant to primary cells, the incorporation of some, but not all chemical modification patterns on synthetic crRNA:tracrRNA or sgRNA can be beneficial to CRISPR-Cas9 gene editing.

Local Context Finder (LCF) reveals multidimensional relationships among mRNA expression profiles of Arabidopsis responding to pathogen infection

PubMed Central

Katagiri, Fumiaki; Glazebrook, Jane

2003-01-01

A major task in computational analysis of mRNA expression profiles is definition of relationships among profiles on the basis of similarities among them. This is generally achieved by pattern recognition in the distribution of data points representing each profile in a high-dimensional space. Some drawbacks of commonly used pattern recognition algorithms stem from their use of a globally linear space and/or limited degrees of freedom. A pattern recognition method called Local Context Finder (LCF) is described here. LCF uses nonlinear dimensionality reduction for pattern recognition. Then it builds a network of profiles based on the nonlinear dimensionality reduction results. LCF was used to analyze mRNA expression profiles of the plant host Arabidopsis interacting with the bacterial pathogen Pseudomonas syringae. In one case, LCF revealed two dimensions essential to explain the effects of the NahG transgene and the ndr1 mutation on resistant and susceptible responses. In another case, plant mutants deficient in responses to pathogen infection were classified on the basis of LCF analysis of their profiles. The classification by LCF was consistent with the results of biological characterization of the mutants. Thus, LCF is a powerful method for extracting information from expression profile data. PMID:12960373

The relative expression levels of insulin-like growth factor 1 and myostatin mRNA in the asynchronous development of skeletal muscle in ducks during early development.

PubMed

Hu, Yan; Liu, Hongxiang; Shan, Yanju; Ji, Gaige; Xu, Wenjuan; Shu, Jingting; Li, Huifang

2015-08-10

Genetic selection is a powerful tool for modifying poultry muscle yield. Insulin-like growth factor I (IGF-I) and myostatin (MSTN) are important regulators of muscle growth, especially in the myogenesis stage. This study compared the developmental pattern of the pectoralis major (PM) and lateral gastrocnemius (LM) muscles, mRNA expression characterization of IGF-I and MSTN-A and their correlation between 14 days in ovo and 1 week post-hatch in two Chinese local duck breeds. During early development, the growth of duck PM and LM followed an asynchronous pattern. Variations in PM growth rate observed with development followed the relative variations of MSTN and IGF-I expression; however, the same behavior was not observed in LM. Moreover, the profile of IGF-I expression in duck skeletal muscles indicated that genetic selection for high meat-yield poultry has altered the temporal expression of IGF-I and affected cellular characteristics and mass by hatch in a PM-specific manner. The MSTN-A expression profile showed synchronization with the growth of skeletal muscle and peaks of myofiber proliferation. The expression patterns of IGF-I and MSTN suggest that duck pectoralis fibers are prioritized for proliferation in embryogenesis. The IGF-1/MSTN-A mRNA ratios in PM and LM presented very similar trends in the changes of myofiber characteristics, and differences in the IGF-1/MSTN-A mRNA ratio in PM between the two breeds corresponded to the timing of differences in PM mass between the varieties. Our results support the hypothesis that relative levels of IGF-I and MSTN mRNA may participate in ordering muscle growth rates with selected development. Copyright © 2015 Elsevier B.V. All rights reserved.

Identification and Migration of Primordial Germ Cells in Atlantic Salmon, Salmo salar: Characterization of Vasa, Dead End, and Lymphocyte Antigen 75 Genes

PubMed Central

Nagasawa, Kazue; Fernandes, Jorge MO; Yoshizaki, Goro; Miwa, Misako; Babiak, Igor

2013-01-01

No information exists on the identification of primordial germ cells (PGCs) in the super-order Protacanthopterygii, which includes the Salmonidae family and Atlantic salmon (Salmo salar L.), one of the most commercially important aquatic animals worldwide. In order to identify salmon PGCs, we cloned the full-length cDNA of vasa, dead end (dnd), and lymphocyte antigen 75 (ly75/CD205) genes as germ cell marker candidates, and analyzed their expression patterns in both adult and embryonic stages of Atlantic salmon. Semi-quantitative RT-PCR results showed that salmon vasa and dnd were specifically expressed in testis and ovary, and vasa, dnd, and ly75 mRNA were maternally deposited in the egg. vasa mRNA was consistently detected throughout embryogenesis while dnd and ly75 mRNA were gradually degraded during cleavages. In situ analysis revealed the localization of vasa and dnd mRNA and Ly75 protein in PGCs of hatched larvae. Whole-mount in situ hybridization detected vasa mRNA during embryogenesis, showing a distribution pattern somewhat different to that of zebrafish; specifically, at mid-blastula stage, vasa-expressing cells were randomly distributed at the central part of blastodisc, and then they migrated to the presumptive region of embryonic shield. Therefore, the typical vasa localization pattern of four clusters during blastulation, as found in zebrafish, was not present in Atlantic salmon. In addition, salmon PGCs could be specifically labeled with a green fluorescence protein (GFP) using gfp-rt-vasa 3′-UTR RNA microinjection for further applications. These findings may assist in understanding PGC development not only in Atlantic salmon but also in other salmonids. PMID:23239145

Localization and expression of messenger RNAs for the peroxisome proliferator-activated receptors in ovarian tissue from naturally cycling and pseudopregnant rats.

PubMed

Komar, Carolyn M; Curry, Thomas E

2002-05-01

Structural and functional development of the corpus luteum (CL) involves tissue remodeling, angiogenesis, lipid metabolism, and steroid production. The peroxisome proliferator-activated receptors (PPARs) have been shown to play a role in these as well as in a multitude of other cellular processes. To examine the expression of mRNA corresponding to the PPAR family members (alpha, delta, and gamma) in luteal tissue, ovaries were collected from gonadotropin-treated, immature rats on Days 1, 4, 8, and 14 of pseudopregnancy and from adult, cycling animals on each day of the estrous cycle. Ovaries were processed for in situ hybridization or RNA isolation for analysis by RNase protection assay. The expression of PPARgamma mRNA was abundant in granulosa cells of developing follicles during both pseudopregnancy and the estrous cycle and was low to undetectable in CL from pseudopregnant rats. However, luteal tissue in cycling animals, especially CL remaining from previous cycles, had high levels of PPARgamma mRNA. The PPARalpha mRNA was localized mainly in the theca and stroma, and PPARdelta mRNA was expressed throughout the ovary. Levels of mRNA for PPARgamma decreased between Days 1 and 4 of pseudopregnancy, and PPARalpha mRNA levels were lower on the day of estrus compared to pro- and metestrus (P < 0.05). The PPARdelta mRNA levels remained steady throughout the estrous cycle and pseudopregnancy. These data illustrate a difference in the luteal expression of mRNA for PPARgamma between the adult, cycling rat and the immature, gonadotropin-treated rat. This differential pattern of expression may be related to the difference in timing of the preovulatory prolactin surge, because the gonadotropin-primed animals would not experience a prolactin surge coincident with the LH surge, as occurs in adult, cycling animals. Additionally, the expression pattern of PPARdelta mRNA indicates that it may be involved in cellular functions involved with maintaining basal ovarian function, whereas PPARalpha may play a role in lipid metabolism in the theca and stroma.

Distance Decay of Similarity in Neotropical Diatom Communities

PubMed Central

Wetzel, Carlos E.; Bicudo, Denise de C.; Ector, Luc; Lobo, Eduardo A.; Soininen, Janne; Landeiro, Victor L.; Bini, Luis M.

2012-01-01

Background The regression of similarity against distance unites several ecological phenomena, and thus provides a highly useful approach for illustrating the spatial turnover across sites. Our aim was to test whether the rates of decay in community similarity differ between diatom growth forms suggested to show different dispersal ability. We hypothesized that the diatom group with lower dispersal ability (i.e. periphyton) would show higher distance decay rates than a group with higher dispersal ability (i.e. plankton). Methods/Principal findings Periphyton and phytoplankton samples were gathered at sites distributed over an area of approximately 800 km length in the Negro River, Amazon basin, Brazil, South America (3°08′00″S; 59°54′30″W). Distance decay relationships were then estimated using distance-based regressions, and the coefficients of these regressions were compared among the groups with different dispersal abilities to assess our predictions. We found evidence that different tributaries and reaches of the Negro River harbor different diatom communities. As expected, the rates of distance decay in community similarity were higher for periphyton than for phytoplankton indicating the lower dispersal ability of periphytic taxa. Conclusions/Significance Our study demonstrates that the comparison of distance decay relationships among taxa with similar ecological requirements, but with different growth form and thus dispersal ability provides a sound approach to evaluate the effects of dispersal ability on beta diversity patterns. Our results are also in line with the growing body of evidence indicating that microorganisms exhibit biogeographic patterns. Finally, we underscore that clumbing all microbial taxa into one group may be a flawed approach to test whether microbes exhibit biogeographic patterns. PMID:23028767

Ovarian Expression and Regulation of the Stromelysins During the Periovulatory Period in the Human and the Rat1

PubMed Central

McCord, Lauren A.; Li, Feixue; Rosewell, Katherine L.; Brännström, Mats; Curry, Thomas E.

2011-01-01

ABSTRACT The matrix metalloproteinases (MMPs) are postulated to facilitate follicular rupture. In the present study, expression of the stromelysins (MMP3, MMP10, MMP11) was analyzed in the periovulatory human and rat ovary. Human granulosa and theca cells were collected from the dominant follicle at various times after human chorionic gonadotropin (hCG). Intact rat ovaries, granulosa cells, and residual tissue (tissue remaining after granulosa cell collection) were isolated from equine CG (eCG)-hCG-primed animals. Mmp10 mRNA was highly induced in human granulosa and theca cells and intact rat ovaries, granulosa cells, and residual tissue. Localization of MMP10 to granulosa and theca cells in both human and rat ovarian follicles was confirmed by immunohistochemistry. Mmp3 mRNA was unchanged in human cells and rat granulosa cells, but increased in intact rat ovaries and residual tissue. Mmp11 mRNA decreased following hCG treatment in human granulosa and theca cells as well as rat granulosa cells. Regulation of Mmp10 in cultured rat granulosa cells revealed that the EGF inhibitor AG1478 and the progesterone receptor antagonist RU486 suppressed the induction of Mmp10 mRNA, whereas the prostaglandin inhibitor NS398 had no effect. Studies on the Mmp10 promoter demonstrated that forskolin plus PMA stimulated promoter activity, which was dependent upon a proximal AP1 site. In conclusion, there are divergent patterns of stromelysin expression associated with ovulation, with a marked induction of Mmp10 mRNA and a decrease in Mmp11 mRNA, yet a species-dependent pattern on Mmp3 mRNA expression. The induction of Mmp10 expression suggests an important role for this MMP in the follicular changes associated with ovulation and subsequent luteinization. PMID:22116802

Ovarian expression and regulation of the stromelysins during the periovulatory period in the human and the rat.

PubMed

McCord, Lauren A; Li, Feixue; Rosewell, Katherine L; Brännström, Mats; Curry, Thomas E

2012-03-01

The matrix metalloproteinases (MMPs) are postulated to facilitate follicular rupture. In the present study, expression of the stromelysins (MMP3, MMP10, MMP11) was analyzed in the periovulatory human and rat ovary. Human granulosa and theca cells were collected from the dominant follicle at various times after human chorionic gonadotropin (hCG). Intact rat ovaries, granulosa cells, and residual tissue (tissue remaining after granulosa cell collection) were isolated from equine CG (eCG)-hCG-primed animals. Mmp10 mRNA was highly induced in human granulosa and theca cells and intact rat ovaries, granulosa cells, and residual tissue. Localization of MMP10 to granulosa and theca cells in both human and rat ovarian follicles was confirmed by immunohistochemistry. Mmp3 mRNA was unchanged in human cells and rat granulosa cells, but increased in intact rat ovaries and residual tissue. Mmp11 mRNA decreased following hCG treatment in human granulosa and theca cells as well as rat granulosa cells. Regulation of Mmp10 in cultured rat granulosa cells revealed that the EGF inhibitor AG1478 and the progesterone receptor antagonist RU486 suppressed the induction of Mmp10 mRNA, whereas the prostaglandin inhibitor NS398 had no effect. Studies on the Mmp10 promoter demonstrated that forskolin plus PMA stimulated promoter activity, which was dependent upon a proximal AP1 site. In conclusion, there are divergent patterns of stromelysin expression associated with ovulation, with a marked induction of Mmp10 mRNA and a decrease in Mmp11 mRNA, yet a species-dependent pattern on Mmp3 mRNA expression. The induction of Mmp10 expression suggests an important role for this MMP in the follicular changes associated with ovulation and subsequent luteinization.

Regulation of DM-20 mRNA expression and intracellular translocation of glutathione-S-transferase pi isoform during oligodendrocyte differentiation in the adult rat spinal cord.

PubMed

Kitada, Masaaki; Takeda, Kazuya; Dezawa, Mari

2016-07-01

We previously demonstrated that NG2-positive oligodendrocyte precursor cells (OPCs) do not express DM-20 mRNA and identified a distinct DM-20 mRNA-positive cell population expressing glutathione-S-transferase pi isoform (GST-pi) in the nucleus (GST-pi(Nuc)) of the adult rat spinal cord. As GST-pi intranuclear localization correlates with progenitor cell properties, we examined the differentiation status of this cell population under the intensive 5-bromo-2'-deoxyuridine (BrdU) administration method, consisting of intraperitoneal BrdU injections every 2 h for 48 h. We observed that a certain population of proliferating/proliferated cells expressed DM-20 mRNA, and sometimes two proliferating/proliferated cells were observed still attached to each other. We performed triple staining for BrdU, DM-20 mRNA, and NG2 and found pairs of neighboring BrdU-positive cells, which were considered to originate from the same progenitor cells and where both cells expressed DM-20 mRNA. Triple staining for BrdU, DM-20 mRNA, and GST-pi detected proliferating/proliferated cells exhibiting the GST-pi(Nuc)/DM-20 mRNA-positive expression pattern. These findings suggested the presence of a GST-pi(Nuc)/DM-20 mRNA-positive oligodendrocyte-lineage progenitor cell population in the adult rat spinal cord. However, we did not find any pair of neighboring BrdU-positive cells with this expression pattern. These observations collectively support the idea that GST-pi(Nuc)/DM-20 mRNA-expressing cells are the progeny of NG2-positive OPCs rather than a novel type of oligodendrocyte-lineage progenitor cells and that DM-20 mRNA expression is dynamically regulated during differentiation of OPCs into oligodendrocytes.

Magnetic pattern at supergranulation scale: the void size distribution

NASA Astrophysics Data System (ADS)

Berrilli, F.; Scardigli, S.; Del Moro, D.

2014-08-01

The large-scale magnetic pattern observed in the photosphere of the quiet Sun is dominated by the magnetic network. This network, created by photospheric magnetic fields swept into convective downflows, delineates the boundaries of large-scale cells of overturning plasma and exhibits "voids" in magnetic organization. These voids include internetwork fields, which are mixed-polarity sparse magnetic fields that populate the inner part of network cells. To single out voids and to quantify their intrinsic pattern we applied a fast circle-packing-based algorithm to 511 SOHO/MDI high-resolution magnetograms acquired during the unusually long solar activity minimum between cycles 23 and 24. The computed void distribution function shows a quasi-exponential decay behavior in the range 10-60 Mm. The lack of distinct flow scales in this range corroborates the hypothesis of multi-scale motion flows at the solar surface. In addition to the quasi-exponential decay, we have found that the voids depart from a simple exponential decay at about 35 Mm.

Patterns of particle distribution in multiparticle systems by random walks with memory enhancement and decay

NASA Astrophysics Data System (ADS)

Tan, Zhi-Jie; Zou, Xian-Wu; Huang, Sheng-You; Zhang, Wei; Jin, Zhun-Zhi

2002-07-01

We investigate the pattern of particle distribution and its evolution with time in multiparticle systems using the model of random walks with memory enhancement and decay. This model describes some biological intelligent walks. With decrease in the memory decay exponent α, the distribution of particles changes from a random dispersive pattern to a locally dense one, and then returns to the random one. Correspondingly, the fractal dimension Df,p characterizing the distribution of particle positions increases from a low value to a maximum and then decreases to the low one again. This is determined by the degree of overlap of regions consisting of sites with remanent information. The second moment of the density ρ(2) was introduced to investigate the inhomogeneity of the particle distribution. The dependence of ρ(2) on α is similar to that of Df,p on α. ρ(2) increases with time as a power law in the process of adjusting the particle distribution, and then ρ(2) tends to a stable equilibrium value.

Decay Pattern of Pygmy States Observed in Neutron-Rich Ne26

NASA Astrophysics Data System (ADS)

Gibelin, J.; Beaumel, D.; Motobayashi, T.; Blumenfeld, Y.; Aoi, N.; Baba, H.; Elekes, Z.; Fortier, S.; Frascaria, N.; Fukuda, N.; Gomi, T.; Ishikawa, K.; Kondo, Y.; Kubo, T.; Lima, V.; Nakamura, T.; Saito, A.; Satou, Y.; Scarpaci, J.-A.; Takeshita, E.; Takeuchi, S.; Teranishi, T.; Togano, Y.; Vinodkumar, A. M.; Yanagisawa, Y.; Yoshida, K.

2008-11-01

Coulomb excitation of the exotic neutron-rich nucleus Ne26 on a Pb208 target was measured at 58MeV/u in order to search for low-lying E1 strength above the neutron emission threshold. This radioactive beam experiment was carried out at the RIKEN Accelerator Research Facility. Using the invariant mass method in the Ne25+n channel, we observe a sizable amount of E1 strength between 6 and 10 MeV excitation energy. By performing a multipole decomposition of the differential cross section, a reduced dipole transition probability of B(E1)=0.49±0.16e2fm2 is deduced, corresponding to 4.9±1.6% of the Thomas-Reiche-Kuhn sum rule. For the first time, the decay pattern of low-lying strength in a neutron-rich nucleus is measured. The extracted decay pattern is not consistent with several mean-field theory descriptions of the pygmy states.

AKT phosphorylates H3-threonine 45 to facilitate termination of gene transcription in response to DNA damage.

PubMed

Lee, Jong-Hyuk; Kang, Byung-Hee; Jang, Hyonchol; Kim, Tae Wan; Choi, Jinmi; Kwak, Sojung; Han, Jungwon; Cho, Eun-Jung; Youn, Hong-Duk

2015-05-19

Post-translational modifications of core histones affect various cellular processes, primarily through transcription. However, their relationship with the termination of transcription has remained largely unknown. In this study, we show that DNA damage-activated AKT phosphorylates threonine 45 of core histone H3 (H3-T45). By genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis, H3-T45 phosphorylation was distributed throughout DNA damage-responsive gene loci, particularly immediately after the transcription termination site. H3-T45 phosphorylation pattern showed close-resemblance to that of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation, which establishes the transcription termination signal. AKT1 was more effective than AKT2 in phosphorylating H3-T45. Blocking H3-T45 phosphorylation by inhibiting AKT or through amino acid substitution limited RNA decay downstream of mRNA cleavage sites and decreased RNA polymerase II release from chromatin. Our findings suggest that AKT-mediated phosphorylation of H3-T45 regulates the processing of the 3' end of DNA damage-activated genes to facilitate transcriptional termination. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

Brünner, F.; Parganlija, D.; Rebhan, A.

We present new results on the decay patterns of scalar and tensor glueballs in the top-down holographic Witten-Sakai-Sugimoto model. This model, which has only one free dimensionless parameter, gives semi-quantitative predictions for the vector meson spectrum, their decay widths, and also a gluon condensate in agreement with SVZ sum rules. The holographic predictions for scalar glueball decay rates are compared with experimental data for the widely discussed gluon candidates f{sub 0}(1500) and f{sub 0}(1710)

Preservation and phylogeny of Cambrian ecdysozoans tested by experimental decay of Priapulus

NASA Astrophysics Data System (ADS)

Sansom, Robert S.

2016-09-01

The exceptionally preserved Cambrian fossil record provides unique insight into the early evolutionary history of animals. Understanding of the mechanisms of exceptional soft tissue preservation frames all interpretations of the fauna and its evolutionary significance. This is especially true for recent interpretations of preserved nervous tissues in fossil ecdysozoans. However, models of soft tissue preservation lack empirical support from actualistic studies. Here experimental decay of the priapulid Priapulus reveal consistent bias towards rapid loss of internal non-cuticular anatomy compared with recalcitrant cuticular anatomy. This is consistent with models of Burgess Shale-type preservation and indicates that internal tissues are unlikely to be preserved with fidelity if organically preserved. This pattern, along with extreme body margin distortion, is consistent with onychophoran decay, and is therefore resolved as general for early ecdysozoans. Application of these patterns to phylogenetic data finds scalidophoran taxa to be very sensitive to taphonomically informed character coding, but not panarthropodan taxa. Priapulid decay also have unexpected relevance for interpretation of myomeres in fossil chordates. The decay data presented serve not only as a test of models of preservation but also a framework with which to interpret ecdysozoan fossil anatomies, and the subsequent evolutionary inferences drawn from them.

Preservation and phylogeny of Cambrian ecdysozoans tested by experimental decay of Priapulus

PubMed Central

Sansom, Robert S.

2016-01-01

The exceptionally preserved Cambrian fossil record provides unique insight into the early evolutionary history of animals. Understanding of the mechanisms of exceptional soft tissue preservation frames all interpretations of the fauna and its evolutionary significance. This is especially true for recent interpretations of preserved nervous tissues in fossil ecdysozoans. However, models of soft tissue preservation lack empirical support from actualistic studies. Here experimental decay of the priapulid Priapulus reveal consistent bias towards rapid loss of internal non-cuticular anatomy compared with recalcitrant cuticular anatomy. This is consistent with models of Burgess Shale-type preservation and indicates that internal tissues are unlikely to be preserved with fidelity if organically preserved. This pattern, along with extreme body margin distortion, is consistent with onychophoran decay, and is therefore resolved as general for early ecdysozoans. Application of these patterns to phylogenetic data finds scalidophoran taxa to be very sensitive to taphonomically informed character coding, but not panarthropodan taxa. Priapulid decay also have unexpected relevance for interpretation of myomeres in fossil chordates. The decay data presented serve not only as a test of models of preservation but also a framework with which to interpret ecdysozoan fossil anatomies, and the subsequent evolutionary inferences drawn from them. PMID:27595908

VDR regulation of microRNA differs across prostate cell models suggesting extremely flexible control of transcription.

PubMed

Singh, Prashant K; Long, Mark D; Battaglia, Sebastiano; Hu, Qiang; Liu, Song; Sucheston-Campbell, Lara E; Campbell, Moray J

2015-01-01

The Vitamin D Receptor (VDR) is a member of the nuclear receptor superfamily and is of therapeutic interest in cancer and other settings. Regulation of microRNA (miRNA) by the VDR appears to be important to mediate its actions, for example, to control cell growth. To identify if and to what extent VDR-regulated miRNA patterns change in prostate cancer progression, we undertook miRNA microarray analyses in 7 cell models representing non-malignant and malignant prostate cells (RWPE-1, RWPE-2, HPr1, HPr1AR, LNCaP, LNCaP-C4-2, and PC-3). To focus on primary VDR regulatory events, we undertook expression analyses after 30 minutes treatment with 1α,25(OH)2D3. Across all models, 111 miRNAs were significantly modulated by 1α,25(OH)2D3 treatment. Of these, only 5 miRNAs were modulated in more than one cell model, and of these, only 3 miRNAs were modulated in the same direction. The patterns of miRNA regulation, and the networks they targeted, significantly distinguished the different cell types. Integration of 1α,25(OH)2D3-regulated miRNAs with published VDR ChIP-seq data showed significant enrichment of VDR peaks in flanking regions of miRNAs. Furthermore, mRNA and miRNA expression analyses in non-malignant RWPE-1 cells revealed patterns of miRNA and mRNA co-regulation; specifically, 13 significant reciprocal patterns were identified and these patterns were also observed in TCGA prostate cancer data. Lastly, motif search analysis revealed differential motif enrichment within VDR peaks flanking mRNA compared to miRNA genes. Together, this study revealed that miRNAs are rapidly regulated in a highly cell-type specific manner, and are significantly co-integrated with mRNA regulation.

Viral Evasion and Manipulation of Host RNA Quality Control Pathways

PubMed Central

2016-01-01

Viruses have evolved diverse strategies to maximize the functional and coding capacities of their genetic material. Individual viral RNAs are often used as substrates for both replication and translation and can contain multiple, sometimes overlapping open reading frames. Further, viral RNAs engage in a wide variety of interactions with both host and viral proteins to modify the activities of important cellular factors and direct their own trafficking, packaging, localization, stability, and translation. However, adaptations increasing the information density of small viral genomes can have unintended consequences. In particular, viral RNAs have developed features that mark them as potential targets of host RNA quality control pathways. This minireview focuses on ways in which viral RNAs run afoul of the cellular mRNA quality control and decay machinery, as well as on strategies developed by viruses to circumvent or exploit cellular mRNA surveillance. PMID:27226372

Viral Evasion and Manipulation of Host RNA Quality Control Pathways.

PubMed

Hogg, J Robert

2016-08-15

Viruses have evolved diverse strategies to maximize the functional and coding capacities of their genetic material. Individual viral RNAs are often used as substrates for both replication and translation and can contain multiple, sometimes overlapping open reading frames. Further, viral RNAs engage in a wide variety of interactions with both host and viral proteins to modify the activities of important cellular factors and direct their own trafficking, packaging, localization, stability, and translation. However, adaptations increasing the information density of small viral genomes can have unintended consequences. In particular, viral RNAs have developed features that mark them as potential targets of host RNA quality control pathways. This minireview focuses on ways in which viral RNAs run afoul of the cellular mRNA quality control and decay machinery, as well as on strategies developed by viruses to circumvent or exploit cellular mRNA surveillance. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

IL-17A acts via p38 MAPK to increase stability of TNF-alpha-induced IL-8 mRNA in human ASM.

PubMed

Henness, Sheridan; van Thoor, Eveline; Ge, Qi; Armour, Carol L; Hughes, J Margaret; Ammit, Alaina J

2006-06-01

Human airway smooth muscle (ASM) plays an immunomodulatory role in asthma. Recently, IL-17A has become of increasing interest in asthma, being found at elevated levels in asthmatic airways and emerging as playing an important role in airway neutrophilia. IL-17A predominantly exerts its neutrophil orchestrating role indirectly via the induction of cytokines by resident airway structural cells. Here, we perform an in vitro study to show that although IL-17A did not induce secretion of the CXC chemokine IL-8 from ASM cells, IL-17A significantly potentiates TNF-alpha-induced IL-8 protein secretion and gene expression in a concentration- and time-dependent manner (P < 0.05). Levels of IL-8 protein produced after 24 h of incubation with TNF-alpha were enhanced 2.7-fold in the presence of IL-17A, and conditioned media significantly enhanced neutrophil chemotaxis in vitro. As IL-17A had no effect on the activity of NF-kappaB, a key transcriptional regulator of IL-8 gene expression, we then examined whether IL-17A acts at the posttranscriptional level. We found that IL-17A significantly augmented TNF-alpha-induced IL-8 mRNA stability. Interestingly, this enhanced stability occurred via a p38 MAPK-dependent pathway. The decay of IL-8 mRNA transcripts proceeded at a significantly faster rate when cells were pretreated with the p38 MAPK inhibitor SB-203580 (-0.05763 +/- 0.01964, t(1/2) = 12.0 h), compared with vehicle (-0.01030 +/- 0.007963, t(1/2) = 67.3 h) [results are expressed as decay constant (means +/- SE) and half-life (t(1/2) in h): P < 0.05]. Collectively, these results demonstrate that IL-17A amplifies the synthetic function of ASM cells, acting via a p38 MAPK-dependent posttranscriptional pathway to augment TNF-alpha-induced secretion of the potent neutrophil chemoattractant IL-8 from ASM cells.

Pou1f1, the key transcription factor related to somatic growth in tilapia (Orechromis niloticus), is regulated by two independent post-transcriptional regulation mechanisms.

PubMed

Wang, Dongfang; Qin, Jingkai; Jia, Jirong; Yan, Peipei; Li, Wensheng

2017-01-29

This study aims to determine the post-transcriptional regulation mechanism of the transcription factor pou1f1 (pou class 1 homeobox 1), which is the key gene for pituitary development, somatic growth in vertebrates, and transcription of several hormone genes in teleost fish. MicroRNA miR-223-3p was identified as a bona fide target of pou1f; overexpression of miR-223-3p in primary pituitary cells led to the down-regulation of pou1f1 and downstream genes, and inhibition of miR-223-3p led to the up-regulation of pou1f1 in Nile tilapia dispersed primary pituitary cells. An adenylate-uridylate-rich element (AU-Rich element) was found in the 3'UTR of pou1f1 mRNA, and deletion of the AU-Rich element led to slower mRNA decay and therefore more protein output. A potential mutual relationship between miR-223-3p and the AU-rich element was also investigated, and the results demonstrated that with or without the AU-Rich element, miR-223-3p induced the up-regulation of a reporter system under serum starvation conditions, indicating that miR-223-3p and the AU-Rich element function independent of each other. This study is the first to investigate the post-transcriptional mechanism of pou1f1, which revealed that miR-223-3p down-regulated pou1f1 and downstream gene expressions, and the AU-Rich element led to rapid decay of pou1f1 mRNA. MicroRNA miR-223-3p and the AU-Rich element co-regulated the post-transcriptional expression of pou1f1 independently in Nile tilapia, demonstrating that pou1f1 is under the control of a dual post-transcription regulation mechanism. Copyright © 2016 Elsevier Inc. All rights reserved.

Sodium lauryl sulphate alters the mRNA expression of lipid-metabolizing enzymes and PPAR signalling in normal human skin in vivo.

PubMed

Törmä, Hans; Berne, Berit

2009-12-01

Detergents irritate skin and affect skin barrier homeostasis. In this study, healthy skin was exposed to 1% sodium lauryl sulphate (SLS) in water for 24 h. Biopsies were taken 6 h to 8 days post exposure. Lipid patterns were stained in situ and real-time polymerase chain reaction (PCR) was used to examine mRNA expression of enzymes synthesizing barrier lipids, peroxisome proliferator-activated receptors (PPAR) and lipoxygenases. The lipid pattern was disorganized from 6 h to 3 days after SLS exposure. Concomitant changes in mRNA expression included: (i) reduction, followed by induction, of ceramide-generating beta-glucocerebrosidase, (ii) increase on day 1 of two other enzymes for ceramide biosynthesis and (iii) persistent reduction of acetyl-CoA carboxylase-B, a key enzyme in fatty acid synthesis. Surprisingly, the rate-limiting enzyme in cholesterol synthesis, HMG-CoA reductase, was unaltered. Among putative regulators of barrier lipids synthesis, PPARalpha and PPARgamma exhibited reduced mRNA expression, while PPARbeta/delta and LXRbeta were unaltered. Epidermal lipoxygenase-3, which may generate PPARalpha agonists, exhibited reduced expression. In conclusion, SLS induces reorganization of lipids in the stratum corneum, which play a role in detergents' destruction of the barrier. The changes in mRNA expression of enzymes involved in synthesizing barrier lipids are probably important for the restoration of the barrier.

Detailed analysis of the δ-crystallin mRNA-expressing region in early development of the chick pituitary gland.

PubMed

Inoue, Makiko; Shiina, Tomoya; Aizawa, Sayaka; Sakata, Ichiro; Takagi, Hiroyasu; Sakai, Takafumi

2012-06-01

Although δ-crystallin (δ-crys), also known as lens protein, is transiently expressed in Rathke's pouch (RP) of the chick embryo, detailed temporal and spatial expression patterns have been obscure. In this study, to understand the relationship between the δ-crys mRNA-expressing region and RP formation, we examined the embryonic expression pattern of δ-crys mRNA in the primordium of the adenohypophysis. δ-crys mRNA expression was initially found at stage 15 anterior to the foregut and posterior to the invaginated oral ectoderm. After RP formation, the δ-crys mRNA was expressed in the post-ventral region of RP and the anterior region of RP. δ-crys mRNA expression was then restricted to the cephalic lobe of the pituitary gland. From stage 20, the δ-crys and alpha-glycoprotein subunit (αGSU) mRNA-expressing regions were almost completely overlapping. The αGSU mRNA-expressing region is thought to be the primordium of the pars tuberalis, and these regions were overlapped with the Lhx3 mRNA-expressing region. The intensity of δ-crys mRNA expression gradually decreased with development and completely disappeared by stage 34. These results suggest that the embryonic chick pituitary gland consists of two different regions labeled with δ-crys and Lhx3.

Structural and kinetic insights into stimulation of RppH-dependent RNA degradation by the metabolic enzyme DapF.

PubMed

Gao, Ang; Vasilyev, Nikita; Luciano, Daniel J; Levenson-Palmer, Rose; Richards, Jamie; Marsiglia, William M; Traaseth, Nathaniel J; Belasco, Joel G; Serganov, Alexander

2018-05-04

Vitally important for controlling gene expression in eukaryotes and prokaryotes, the deprotection of mRNA 5' termini is governed by enzymes whose activity is modulated by interactions with ancillary factors. In Escherichia coli, 5'-end-dependent mRNA degradation begins with the generation of monophosphorylated 5' termini by the RNA pyrophosphohydrolase RppH, which can be stimulated by DapF, a diaminopimelate epimerase involved in amino acid and cell wall biosynthesis. We have determined crystal structures of RppH-DapF complexes and measured rates of RNA deprotection. These studies show that DapF potentiates RppH activity in two ways, depending on the nature of the substrate. Its stimulatory effect on the reactivity of diphosphorylated RNAs, the predominant natural substrates of RppH, requires a substrate long enough to reach DapF in the complex, while the enhanced reactivity of triphosphorylated RNAs appears to involve DapF-induced changes in RppH itself and likewise increases with substrate length. This study provides a basis for understanding the intricate relationship between cellular metabolism and mRNA decay and reveals striking parallels with the stimulation of decapping activity in eukaryotes.

Interleukin-1 homologues IL-1F7b and IL-18 contain functional mRNA instability elements within the coding region responsive to lipopolysaccharide

PubMed Central

2004-01-01

IL-1F7b, a novel homologue of the IL-1 (interleukin 1) family, was discovered by computational cloning. We demonstrated that IL-1F7b shares critical amino acid residues with IL-18 and binds to the IL-18-binding protein enhancing its ability to inhibit IL-18-induced interferon-γ. We also showed that low levels of IL-1F7b are constitutively present intracellularly in human blood monocytes. In this study, we demonstrate that similar to IL-18, both mRNA and intracellular protein expression of IL-1F7b are up-regulated by LPS (lipopolysaccharide) in human monocytes. In stable transfectants of murine RAW264.7 macrophage cells, there was no IL-1F7b protein expression despite a highly active CMV promoter. We found that IL-1F7b-specific mRNA was rapidly degraded in transfected cells, via a 3′-UTR (untranslated region)-independent control of IL-1F7b transcript stability. After LPS stimulation, there was a rapid transient increase in IL-1F7b-specific mRNA and concomitant protein levels. Using sequence alignment, we found a conserved ten-nucleotide homology box within the open reading frame of IL-F7b, which is flanking the coding region instability elements of some selective genes. In-frame deletion of downstream exon 5 from the full-length IL-1F7b cDNA markedly increased the levels of IL-1F7b mRNA. A similar coding region element is located in IL-18. When transfected into RAW264.7 macrophages, IL-18 mRNA was also unstable unless treated with LPS. These results indicate that both IL-1F7b and IL-18 mRNA contain functional instability determinants within their coding region, which influence mRNA decay as a novel mechanism to regulate the expression of IL-1 family members. PMID:15046617

Five Potential Crises.

ERIC Educational Resources Information Center

Futurist, 1984

1984-01-01

Five areas that have great potential for becoming crises in the future are described: a warming of the earth's climate, changing weather patterns and growing seasons; water shortage; the decay of the physical infrastructure, e.g., decay of roads, bridges; breakdown of the international monetary and trading system; and nuclear warfare. (Author/RM)

Recurrence Methods for the Identification of Morphogenetic Patterns

PubMed Central

Facchini, Angelo; Mocenni, Chiara

2013-01-01

This paper addresses the problem of identifying the parameters involved in the formation of spatial patterns in nonlinear two dimensional systems. To this aim, we perform numerical experiments on a prototypical model generating morphogenetic Turing patterns, by changing both the spatial frequency and shape of the patterns. The features of the patterns and their relationship with the model parameters are characterized by means of the Generalized Recurrence Quantification measures. We show that the recurrence measures Determinism and Recurrence Entropy, as well as the distribution of the line lengths, allow for a full characterization of the patterns in terms of power law decay with respect to the parameters involved in the determination of their spatial frequency and shape. A comparison with the standard two dimensional Fourier transform is performed and the results show a better performance of the recurrence indicators in identifying a reliable connection with the spatial frequency of the patterns. Finally, in order to evaluate the robustness of the estimation of the power low decay, extensive simulations have been performed by adding different levels of noise to the patterns. PMID:24066062

The expression patterns of the clock genes period and timeless are affected by photoperiod in the Mediterranean corn stalk borer, Sesamia nonagrioides.

PubMed

Kontogiannatos, Dimitrios; Gkouvitsas, Theodoros; Kourti, Anna

2017-01-01

To obtain clues to the link between the molecular mechanism of circadian and photoperiod clocks, we cloned two circadian clock genes, period (per) and timeless (tim) from the moth Sesamia nonagrioides, which undergoes facultative diapause controlled by photoperiod. Sequence analysis revealed a high degree of conservation among the compared insects fοr both genes. We also investigated the expression patterns of per and tim in brains of larvae growing under 16L:8D (long days), constant darkness (DD) and 10L:14D (short days) conditions by qPCR assays. The results showed that mRNA accumulations encoding both genes exhibited diel oscillations under different photoperiods. The oscillation of per and tim mRNA, under short-day photoperiod differed from long-day. The difference between long-day and short-day conditions in the pattern of mRNA levels of per and tim appears to distinguish photoperiodic conditions clearly and both genes were influenced by photoperiod in different ways. We infer that not all photoperiodic clocks of insects interact with circadian clocks in the same fashion. Our results suggest that transcriptional regulations of the both clock genes act in the diapause programing in S. nonagrioides. The expression patterns of these genes are affected by photoperiod but runs with 24 h by entrainment to daily environmental cues. © 2016 Wiley Periodicals, Inc.

Polygalacturonase Gene Expression in Rutgers, rin, nor, and Nr Tomato Fruits 1

PubMed Central

DellaPenna, Dean; Kates, David S.; Bennett, Alan B.

1987-01-01

Polygalacturonase (PG) gene expression was studied in normally ripening tomato fruit (Lycopersicon esculentum Mill, cv Rutgers) and in three ripening-impaired mutants, rin, nor, and Nr. Normal and mutant fruit of identical chronological age were analyzed at 41, 49, and 62 days after pollination. These stages corresponded to mature-green, ripe, and overripe, respectively, for Rutgers. The amount of PG mRNA in Rutgers was highest at 49 days and accounted for 2.3% of the total mRNA mass but at 62 days had decreased to 0.004% of the total mRNA mass. In Nr, the amount of PG mRNA steadily increased between 41 and 62 days after pollination, reaching a maximum level of 0.5% of the total mRNA mass. The mutant nor exhibited barely detectable levels of PG mRNA at all stages tested. Surprisingly, PG mRNA, comprising approximately 0.06% of the mRNA mass, was detected in 49 day rin fruit. This mRNA accumulation occurred in the absence of elevated ethylene production by the fruit and resulted in the synthesis of enzymically active PG I. The different patterns of PG mRNA accumulation in the three mutants suggests that distinct molecular mechanisms contribute to reduced PG expression in each ripening-impaired mutant. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 PMID:16665727

Specificity and Evolutionary Conservation of the Escherichia coli RNA Pyrophosphohydrolase RppH*

PubMed Central

Foley, Patricia L.; Hsieh, Ping-kun; Luciano, Daniel J.; Belasco, Joel G.

2015-01-01

Bacterial RNA degradation often begins with conversion of the 5′-terminal triphosphate to a monophosphate by the RNA pyrophosphohydrolase RppH, an event that triggers rapid ribonucleolytic attack. Besides its role as the master regulator of 5′-end-dependent mRNA decay, RppH is important for the ability of pathogenic bacteria to invade host cells, yet little is known about how it chooses its targets. Here, we show that Escherichia coli RppH (EcRppH) requires at least two unpaired nucleotides at the RNA 5′ end and prefers three or more such nucleotides. It can tolerate any nucleotide at the first three positions but has a modest preference for A at the 5′ terminus and either a G or A at the second position. Mutational analysis has identified EcRppH residues crucial for substrate recognition or catalysis. The promiscuity of EcRppH differentiates it from its Bacillus subtilis counterpart, which has a strict RNA sequence requirement. EcRppH orthologs likely to share its relaxed sequence specificity are widespread in all classes of Proteobacteria, except Deltaproteobacteria, and in flowering plants. By contrast, the phylogenetic range of recognizable B. subtilis RppH orthologs appears to be restricted to the order Bacillales. These findings help to explain the selective influence of RppH on bacterial mRNA decay and show that RppH-dependent degradation has diversified significantly during the course of evolution. PMID:25657006

Specificity and evolutionary conservation of the Escherichia coli RNA pyrophosphohydrolase RppH.

PubMed

Foley, Patricia L; Hsieh, Ping-kun; Luciano, Daniel J; Belasco, Joel G

2015-04-10

Bacterial RNA degradation often begins with conversion of the 5'-terminal triphosphate to a monophosphate by the RNA pyrophosphohydrolase RppH, an event that triggers rapid ribonucleolytic attack. Besides its role as the master regulator of 5'-end-dependent mRNA decay, RppH is important for the ability of pathogenic bacteria to invade host cells, yet little is known about how it chooses its targets. Here, we show that Escherichia coli RppH (EcRppH) requires at least two unpaired nucleotides at the RNA 5' end and prefers three or more such nucleotides. It can tolerate any nucleotide at the first three positions but has a modest preference for A at the 5' terminus and either a G or A at the second position. Mutational analysis has identified EcRppH residues crucial for substrate recognition or catalysis. The promiscuity of EcRppH differentiates it from its Bacillus subtilis counterpart, which has a strict RNA sequence requirement. EcRppH orthologs likely to share its relaxed sequence specificity are widespread in all classes of Proteobacteria, except Deltaproteobacteria, and in flowering plants. By contrast, the phylogenetic range of recognizable B. subtilis RppH orthologs appears to be restricted to the order Bacillales. These findings help to explain the selective influence of RppH on bacterial mRNA decay and show that RppH-dependent degradation has diversified significantly during the course of evolution. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

mRNA expression pattern of selected candidate genes differs in bovine oviductal epithelial cells in vitro compared with the in vivo state and during cell culture passages.

PubMed

Danesh Mesgaran, Sadjad; Sharbati, Jutta; Einspanier, Ralf; Gabler, Christoph

2016-08-15

The mammalian oviduct provides the optimal environment for gamete maturation including sperm capacitation, fertilization, and development of the early embryo. Various cell culture models for primary bovine oviductal epithelial cells (BOEC) were established to reveal such physiological events. The aim of this study was to evaluate 17 candidate mRNA expression patterns in oviductal epithelial cells (1) in transition from in vivo cells to in vitro cells; (2) during three consecutive cell culture passages; (3) affected by the impact of LOW or HIGH glucose content media; and (4) influenced by different phases of the estrous cycle in vivo and in vitro. In addition, the release of a metabolite and proteins from BOEC at two distinct cell culture passage numbers was estimated to monitor the functionality. BOEC from 8 animals were isolated and cultured for three consecutive passages. Total RNA was extracted from in vivo and in vitro samples and subjected to reverse transcription quantitative polymerase chain reaction to reveal mRNA expression of selected candidate genes. The release of prostaglandin E2 (PGE2), oviduct-specific glycoprotein 1 (OVGP1) and interleukin 8 (IL8) by BOEC was measured by EIA or ELISA after 24 h. Almost all candidate genes (prostaglandin synthases, enzymes of cellular metabolism and mucins) mRNA expression pattern differed compared in vivo with in vitro state. In addition, transcription of most candidate genes was influenced by the number of cell culture passages. Different glucose medium content did not affect mRNA expression of most candidate genes. The phase of the estrous cycle altered some candidate mRNA expression in BOEC in vitro at later passages. The release of PGE2 and OVGP1 between passages did not differ. However, BOEC in passage 3 released significantly higher amount of IL8 compared with cells in passage 0. This study supports the hypothesis that candidate mRNA expression in BOEC was influenced by transition from the in vivo situation to the new in vitro environment and during consecutive passages. The consequence of cell culture passaging on BOEC ability to release bioactive compounds should be considered.

Widespread promoter-mediated coordination of transcription and mRNA degradation

PubMed Central

2012-01-01

Background Previous work showed that mRNA degradation is coordinated with transcription in yeast, and in several genes the control of mRNA degradation was linked to promoter elements through two different mechanisms. Here we show at the genomic scale that the coordination of transcription and mRNA degradation is promoter-dependent in yeast and is also observed in humans. Results We first demonstrate that swapping upstream cis-regulatory sequences between two yeast species affects both transcription and mRNA degradation and suggest that while some cis-regulatory elements control either transcription or degradation, multiple other elements enhance both processes. Second, we show that adjacent yeast genes that share a promoter (through divergent orientation) have increased similarity in their patterns of mRNA degradation, providing independent evidence for the promoter-mediated coupling of transcription to mRNA degradation. Finally, analysis of the differences in mRNA degradation rates between mammalian cell types or mammalian species suggests a similar coordination between transcription and mRNA degradation in humans. Conclusions Our results extend previous studies and suggest a pervasive promoter-mediated coordination between transcription and mRNA degradation in yeast. The diverse genes and regulatory elements associated with this coordination suggest that it is generated by a global mechanism of gene regulation and modulated by gene-specific mechanisms. The observation of a similar coupling in mammals raises the possibility that coupling of transcription and mRNA degradation may reflect an evolutionarily conserved phenomenon in gene regulation. PMID:23237624

Estrogen receptor mRNA expression patterns in the liver and ovary of female rainbow trout over a complete reproductive cycle

PubMed Central

Nagler, James J.; Cavileer, Timothy D.; Verducci, Joseph S.; Schultz, Irvin R.; Hook, Sharon E.; Hayton, William L.

2012-01-01

Estrogens are critical hormones involved in reproduction and need to bind to estrogen receptors in target organs for biological activity. Fishes have two distinct estrogen receptor subtypes, alpha (α) and beta (β), with variable combinations of additional isoforms of each subtype dependent on the history of genome duplication within a taxon. The comparative expression patterns of estrogen receptor isoforms during the female reproductive cycle will provide important insights into the unique function and importance of each. The purpose of this study was to measure the mRNAs for the four estrogen receptor isoforms (erα1, erα2, erβ1, erβ2) in the liver and ovary of adult, female rainbow trout over the course of an annual reproductive cycle. The expression of estrogen receptor mRNA isoforms was measured by quantitative real-time RT-PCR. Several reproductive indices (gonadosomatic index, maximum oocyte diameter, plasma estradiol-17β, plasma vitellogenin, and ovulation) were also quantified for comparison and used in a correlation analysis to examine any inter-relationships. Of the four isoforms, the expression of erα1 was highest in the liver, and had a significant positive correlation with liver erβ1 expression. Liver expression of erα2 mRNA was the lowest, but showed a significant positive correlation with maximum oocyte diameter in the ovary. The pattern of the erβ isoforms in liver was one of initially elevated mRNA expression followed by a gradual decrease as reproductive development proceeded. In the ovary the erβ1 isoform had the highest mRNA expression of all estrogen receptor isoforms, at the beginning of the reproductive cycle, but then decreased afterward. Both ovarian erβ isoforms had a significant positive correlation with one another. In contrast, erα2 mRNA expression showed a high maximum level in the ovary near the end of the cycle along with a significant positive correlation with plasma estradiol-17β levels; the highest gonadosomatic indices, maximum oocyte diameter, and vitellogenin levels occurred then too. PMID:22732076

Differential expression of decorin and biglycan genes during palatogenesis in normal and retinoic acid-treated mice.

PubMed

Zhang, Yuxiang; Mori, Tetsuji; Iseki, Ken; Hagino, Seita; Takaki, Hiromi; Takeuchi, Mayumi; Hikake, Tsuyoshi; Tase, Choichiro; Murakawa, Masahiro; Yokoya, Sachihiko; Wanaka, Akio

2003-04-01

Proteoglycans are involved in secondary palate formation. In the present study, we focused on two small leucine-rich proteoglycans, decorin and biglycan, because they assembled extracellular matrix molecules such as collagens and modulated signaling pathway of transforming growth factor-beta. To investigate the functions of decorin and biglycan in palatogenesis, we compared their mRNA expression patterns between normal palate and retinoic acid-induced cleft palate in mice by using in situ hybridization analysis during the period of embryonic day 13.5 (E13.5) to E15.5. On E13.5, decorin mRNA was expressed in the epithelia and mesenchyme on the nasal side of the developing secondary palate. During the period the palate shelves were fusing (E14.5), decorin mRNA was strongly expressed in the mesenchyme but its expression pattern was asymmetric; decorin mRNA expression area in the nasal side was broader than that in the oral side. The expression of decorin mRNA was hardly detected in the mesenchyme on either side of the medial edge epithelium. After fusion (E15.5), its expression converged to the mesenchyme just around the palatine bone. Biglycan mRNA was ubiquitously distributed throughout the palatal mesenchyme for the mid-gestation period. Its expression area became limited to the ossification area within the palate after the late gestation period. In the retinoic acid-treated mice, the area of the decorin gene expression expanded to the core region of the palate primordium where little signal was observed in control mice. On the other hand, biglycan in the retinoic acid-treated mice did not show remarkable change in its distribution patterns compared with that in the control mice. These findings suggest that decorin and biglycan play distinct roles in palatogenesis, and decorin was more actively involved in the process of secondary palate formation than biglycan. Up-regulation of decorin gene expression in the retinoic acid-treated mice might influence the pathogenesis of cleft palate. Copyright 2003 Wiley-Liss, Inc.

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. A comparison of fragmentation patterns of linear dextran obtained by in-source decay, post-source decay and collision-induced dissociation and the stability of linear and cyclic glucans studied by in-source decay.

PubMed

Bashir, Sajid; Giannakopulos, Anastassios E; Derrick, Peter J; Critchley, Peter; Bottrill, Andrew; Padley, Henry J

2004-01-01

In the first part of this study fragmentation patterns from a range of dextran oligomers (containing 4-20 anhydroglucose units) were compared in three different methods of analysis coupled with matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry. Collision-induced-dissociation (CID), prompt in-source decay (ISD) and post-source decay (PSD) all caused cleavage of the glycosidic bonds. Both CID and to a lesser extent ISD caused further cleavage of pyranose rings of the individual sugar residues. There was very little cleavage of pyranose rings detected in the PSD spectrum. Derivatisation of the reducing end-groups of the oligodextrans with 1-phenyl-3-methyl-5-pyrazolone (PMP) restricted cleavage in the MALDI mass spectrometer to the non-reducing end, and further it enabled the saccharides to be separated by HPLC so that a single chain length could be examined as a standard. Maltoheptaose was also used as a standard. In the second part of the study prompt ISD-MALDI mass spectrometry was used to compare the fragmentation of three oligoglucans, dextran, maltodextrin and gamma cyclodextrin, that have different linkages and different secondary structure. The results showed that the degree of fragmentation correlated with the degree of freedom in the saccharide chains in solution determined by NMR. Dextran the most random conformation was fragmented most whereas there was little evidence of any fragments, not even glycosidic bond breakage from cyclodextrin, even when the laser power was increased considerably. The fragmentation pattern of maltodextrin was intermediate. The patterns of fragmentation produced by MALDI mass spectrometry, particularly where standards are available to calibrate the spectrum and the energy of the laser is controlled, can be used to predict the type of linkage present.

An NXF1 mRNA with a retained intron is expressed in hippocampal and neocortical neurons and is translated into a protein that functions as an Nxf1 cofactor.

PubMed

Li, Ying; Bor, Yeou-Cherng; Fitzgerald, Mark P; Lee, Kevin S; Rekosh, David; Hammarskjold, Marie-Louise

2016-12-01

The Nxf1 protein is a major nuclear export receptor for the transport of mRNA, and it also is essential for export of retroviral mRNAs with retained introns. In the latter case, it binds to RNA elements known as constitutive transport elements (CTEs) and functions in conjunction with a cofactor known as Nxt1. The NXF1 gene also regulates expression of its own intron-containing RNA through the use of a functional CTE within intron 10. mRNA containing this intron is exported to the cytoplasm, where it can be translated into the 356-amino acid short Nxf1(sNxf1) protein, despite the fact that it is a prime candidate for nonsense-mediated decay (NMD). Here we demonstrate that sNxf1 is highly expressed in nuclei and dendrites of hippocampal and neocortical neurons in rodent brain. Additionally, we show that sNxf1 localizes in RNA granules in neurites of differentiated N2a mouse neuroblastoma cells, where it shows partial colocalization with Staufen2 isoform SS, a protein known to play a role in dendritic mRNA trafficking. We also show that sNxf1 forms heterodimers in conjunction with the full-length Nxf1 and that sNxf1 can replace Nxt1 to enhance the expression of CTE-containing mRNA and promote its association with polyribosomes. © 2016 Li et al. 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).

Remodeling of the pioneer translation initiation complex involves translation and the karyopherin importin β

PubMed Central

Sato, Hanae; Maquat, Lynne E.

2009-01-01

Mammalian mRNAs lose and acquire proteins throughout their life span while undergoing processing, transport, translation, and decay. How translation affects messenger RNA (mRNA)–protein interactions is largely unknown. The pioneer round of translation uses newly synthesized mRNA that is bound by cap-binding protein 80 (CBP80)–CBP20 (also known as the cap-binding complex [CBC]) at the cap, poly(A)-binding protein N1 (PABPN1) and PABPC1 at the poly(A) tail, and, provided biogenesis involves pre-mRNA splicing, exon junction complexes (EJCs) at exon–exon junctions. Subsequent rounds of translation engage mRNA that is bound by eukaryotic translation initiation factor 4E (eIF4E) at the cap and PABPC1 at the poly(A) tail, but that lacks detectable EJCs and PABPN1. Using the level of intracellular iron to regulate the translation of specific mRNAs, we show that translation promotes not only removal of EJC constituents, including the eIF4AIII anchor, but also replacement of PABPN1 by PABPC1. Remarkably, translation does not affect replacement of CBC by eIF4E. Instead, replacement of CBC by eIF4E is promoted by importin β (IMPβ): Inhibiting the binding of IMPβ to the complex of CBC–IMPα at an mRNA cap using the IMPα IBB (IMPβ-binding) domain or a RAN variant increases the amount of CBC-bound mRNA and decreases the amount of eIF4E-bound mRNA. Our studies uncover a previously unappreciated role for IMPβ and a novel paradigm for how newly synthesized messenger ribonucleoproteins (mRNPs) are matured. PMID:19884259

Pattern-Recognition Receptor Signaling Regulator mRNA Expression in Humans and Mice, and in Transient Inflammation or Progressive Fibrosis

PubMed Central

Günthner, Roman; Kumar, Vankayala Ramaiah Santhosh; Lorenz, Georg; Anders, Hans-Joachim; Lech, Maciej

2013-01-01

The cell type-, organ-, and species-specific expression of the pattern-recognition receptors (PRRs) are well described but little is known about the respective expression profiles of their negative regulators. We therefore determined the mRNA expression levels of A20, CYLD, DUBA, ST2, CD180, SIGIRR, TANK, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, SHP1, SHP2, TOLLIP, IRF4, SIKE, NLRX1, ERBIN, CENTB1, and Clec4a2 in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. Additionally, we characterized their expression profiles in mononuclear blood cells upon bacterial endotoxin, which showed a consistent induction of A20, SOCS3, IRAK-M, and Clec4a2 in human and murine cells. Furthermore, we studied the expression pattern in transient kidney ischemia-reperfusion injury versus post-ischemic atrophy and fibrosis in mice. A20, CD180, ST2, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, IRF4, CENTB1, and Clec4a2 were all induced, albeit at different times of injury and repair. Progressive fibrosis was associated with a persistent induction of these factors. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to PRR-mediated innate immunity, which seems to be involved in tissue injury, tissue regeneration and in progressive tissue scarring. PMID:24009023

Transcription mapping and expression patterns of genes in the major immediate-early region of Kaposi's sarcoma-associated herpesvirus.

PubMed

Saveliev, Alexei; Zhu, Fan; Yuan, Yan

2002-08-01

Viral immediate-early (IE) genes are the first class of viral genes expressed during primary infection or reactivation from latency. They usually encode regulatory proteins that play crucial roles in viral life cycle. In a previous study, four regions in the KSHV genome were found to be actively transcribed in the immediate-early stage of viral reactivation in primary effusion lymphoma cells. Three immediate-early transcripts were characterized in these regions, as follows: mRNAs for ORF50 (KIE-1), ORF-45 (KIE-2), and ORF K4.2 (KIE-3) (F. X. Zhu, T. Cusano, and Y. Yuan, 1999, J. Virol. 73, 5556-5567). In the present study, we further analyzed the expression of genes in these IE regions in BC-1 and BCBL-1 cells. One of the immediate-early regions (KIE-1) that encompasses ORF50 and other genes was intensively studied to establish a detailed transcription map and expression patterns of genes in this region. This study led to identification of several novel IE transcripts in this region. They include a 2.6-kb mRNA which encodes ORF48/ORF29b, a family of transcripts that are complementary to ORF50 mRNA and a novel K8 IE mRNA of 1.5 kb. Together with the IE mRNA for ORF50 which was identified previously, four immediate-early genes have been mapped to KIE-1 region. Therefore, we would designate KIE-1 the major immediate-early region of KSHV. In addition, we showed that transcription of K8 gene is controlled by two promoters, yielding two transcripts, an immediate-early mRNA of 1.5 kb and a delayed-early mRNA of 1.3 kb.

Nonsense-mediated mRNA decay factor Upf2 exists in both the nucleoplasm and the cytoplasm

PubMed Central

TATSUNO, TAKANORI; NAKAMURA, YUKA; MA, SHAOFU; TOMOSUGI, NAOHISA; ISHIGAKI, YASUHITO

2016-01-01

Upf2 protein predominantly localizes to the cytoplasmic fraction, and binds to the exon junction complex (EJC) on spliced mRNA. The present study aimed to determine the cellular site where the interaction between Upf2 and EJC occurs. First, the cell lysate was fractionated into the cytoplasm and nucleoplasm, and western blotting to detect levels of Upf2 protein was performed. Upf2 was clearly detected in the cytoplasm and in the nucleoplasm. Secondly, immunostaining was performed, and the majority of Upf2 was detected in the cytoplasmic perinuclear region; a small quantity of Upf2 was detected in the intranuclear region. RNase treatment of the cells reduced the Upf2 immunostained signal. The immune-purified fractions containing nuclear and cytoplasmic Upf2 also contained one of the EJC core factors, RBM8A. These results implied the existence of Upf2 in the nucleoplasm and the cytoplasm, and it appeared to be involved in the construction of the mRNA complex. In order to verify the construction of Upf2-binding EJC in the nucleoplasm, an in situ proximity ligation assay was performed with anti-Upf2 and anti-RBM8A antibodies. These results demonstrated that their interaction occurred not only in the cytoplasmic region, but also in the intranuclear region. Taken together, these results suggested that Upf2 combines with EJC in both the cytoplasmic and the intranuclear fractions, and that it is involved in mRNA metabolism in human cells. PMID:27221324

Unfolded protein response transducer IRE1-mediated signaling independent of XBP1 mRNA splicing is not required for growth and development of medaka fish

PubMed Central

Ishikawa, Tokiro; Kashima, Makoto; Nagano, Atsushi J; Ishikawa-Fujiwara, Tomoko; Kamei, Yasuhiro; Todo, Takeshi

2017-01-01

When activated by the accumulation of unfolded proteins in the endoplasmic reticulum, metazoan IRE1, the most evolutionarily conserved unfolded protein response (UPR) transducer, initiates unconventional splicing of XBP1 mRNA. Unspliced and spliced mRNA are translated to produce pXBP1(U) and pXBP1(S), respectively. pXBP1(S) functions as a potent transcription factor, whereas pXBP1(U) targets pXBP1(S) to degradation. In addition, activated IRE1 transmits two signaling outputs independent of XBP1, namely activation of the JNK pathway, which is initiated by binding of the adaptor TRAF2 to phosphorylated IRE1, and regulated IRE1-dependent decay (RIDD) of various mRNAs in a relatively nonspecific manner. Here, we conducted comprehensive and systematic genetic analyses of the IRE1-XBP1 branch of the UPR using medaka fish and found that the defects observed in XBP1-knockout or IRE1-knockout medaka were fully rescued by constitutive expression of pXBP1(S). Thus, the JNK and RIDD pathways are not required for the normal growth and development of medaka. The unfolded protein response sensor/transducer IRE1-mediated splicing of XBP1 mRNA encoding its active downstream transcription factor to maintain the homeostasis of the endoplasmic reticulum is sufficient for growth and development of medaka fish. PMID:28952924

Sequencing of mRNA identifies re-expression of fetal splice variants in cardiac hypertrophy

PubMed Central

Ames, EG; Lawson, MJ; Mackey, AJ; Holmes, JW

2013-01-01

Cardiac hypertrophy has been well-characterized at the level of transcription. During cardiac hypertrophy, genes normally expressed primarily during fetal heart development are reexpressed, and this fetal gene program is believed to be a critical component of the hypertrophic process. Recently, alternative splicing of mRNA transcripts has been shown to be temporally regulated during heart development, leading us to consider whether fetal patterns of splicing also reappear during hypertrophy. We hypothesized that patterns of alternative splicing occurring during heart development are recapitulated during cardiac hypertrophy. Here we present a study of isoform expression during pressure-overload cardiac hypertrophy induced by 10 days of transverse aortic constriction (TAC) in rats and in developing fetal rat hearts compared to sham-operated adult rat hearts, using high-throughput sequencing of poly(A) tail mRNA. We find a striking degree of overlap between the isoforms expressed differentially in fetal and pressure-overloaded hearts compared to control: forty-four percent of the isoforms with significantly altered expression in TAC hearts are also expressed at significantly different levels in fetal hearts compared to control (P < 0.001). The isoforms that are shared between hypertrophy and fetal heart development are significantly enriched for genes involved in cytoskeletal organization, RNA processing, developmental processes, and metabolic enzymes. Our data strongly support the concept that mRNA splicing patterns normally associated with heart development recur as part of the hypertrophic response to pressure overload. These findings suggest that cardiac hypertrophy shares post-transcriptional as well as transcriptional regulatory mechanisms with fetal heart development. PMID:23688780

3′UTR AU-rich elements (AREs) and the RNA-binding protein Tristetraprolin (TTP) are not required for the LPS-mediated destabilization of phospholipase-Cβ-2 mRNA in murine macrophages

PubMed Central

Shukla, Smita; Elson, Genie; Blackshear, Perry J.; Lutz, Carol S.; Leibovich, S. Joseph

2017-01-01

We have shown previously that bacterial lipopolysaccharide (LPS)-mediated suppression of Phospholipase-Cβ-2 (PLCβ-2) expression is involved in M1 (inflammatory) to M2-like (wound healing) phenotypic switching of macrophages triggered by adenosine. This suppression is mediated post-transcriptionally by destabilization of PLCβ-2 mRNA. To investigate the mechanism of this LPS-mediated destabilization, we examined the roles of RNA-binding agents including microRNAs and RNA-binding proteins that are involved in regulating stability of mRNAs encoding growth factors, inflammatory mediators and proto-oncogenes. Adenylate and Uridylate (AU)-rich elements (AREs) in 3′UTRs are specific recognition sites for RNA-binding proteins including Tristetraprolin (TTP), HuR and AUF1, and for microRNAs that are involved in regulating mRNA stability. In this study, we investigated the role of TTP and AREs in regulating PLCβ-2 mRNA stability. The 3′UTR of the PLCβ-2 gene was inserted into the pLightswitch luciferase reporter plasmid and transfected into RAW264.7 cells. LPS suppressed Luciferase expression from this reporter. Luciferase expression from mutant 3′UTR constructs lacking AREs was similarly down-regulated, suggesting that these regions are not required for LPS-mediated suppression of PLCβ-2. TTP was rapidly upregulated in both primary murine macrophages and RAW264.7 cells in response to LPS. Suppression of PLCβ-2 by LPS was examined using macrophages from mice lacking TTP. LPS suppressed PLCβ-2 expression to the same extent in wild type and TTP−/− macrophages. Also, the rate of decay of PLCβ-2 mRNA in LPS-treated macrophages following transcriptional blockade was similar in wild type and TTP−/− macrophages, clearly indicating that TTP is not involved in LPS-mediated destabilization of PLCβ-2 mRNA in macrophages. PMID:28124257

Sequence Divergence in the 3′ Untranslated Regions of Human ζ- and α-Globin mRNAs Mediates a Difference in Their Stabilities and Contributes to Efficient α-to-ζ Gene Developmental Switching

PubMed Central

Russell, J. Eric; Morales, Julia; Makeyev, Aleksandr V.; Liebhaber, Stephen A.

1998-01-01

The developmental stage-specific expression of human globin proteins is characterized by a switch from the coexpression of ζ- and α-globin in the embryonic yolk sac to exclusive expression of α-globin during fetal and adult life. Recent studies with transgenic mice demonstrate that in addition to transcriptional control elements, full developmental silencing of the human ζ-globin gene requires elements encoded within the transcribed region. In the current work, we establish that these latter elements operate posttranscriptionally by reducing the relative stability of ζ-globin mRNA. Using a transgenic mouse model system, we demonstrate that human ζ-globin mRNA is unstable in adult erythroid cells relative to the highly stable human α-globin mRNA. A critical determinant of the difference between α- and ζ-globin mRNA stability is mapped by in vivo expression studies to their respective 3′ untranslated regions (3′UTRs). In vitro messenger ribonucleoprotein (mRNP) assembly assays demonstrate that the α- and ζ-globin 3′UTRs assemble a previously described mRNP stability-determining complex, the α-complex, with distinctly different affinities. The diminished efficiency of α-complex assembly on the ζ 3′UTR results from a single C→G nucleotide substitution in a crucial polypyrimidine tract contained by both the human α- and ζ-globin mRNA 3′UTRs. A potential pathway for accelerated ζ-globin mRNA decay is suggested by the observation that its 3′UTR encodes a shortened poly(A) tail. Based upon these data, we propose a model for ζ-globin gene silencing in fetal and adult erythroid cells in which posttranscriptional controls play a central role by providing for accelerated clearance of ζ-globin transcripts. PMID:9528789

3'UTR AU-Rich Elements (AREs) and the RNA-Binding Protein Tristetraprolin (TTP) Are Not Required for the LPS-Mediated Destabilization of Phospholipase-Cβ-2 mRNA in Murine Macrophages.

PubMed

Shukla, Smita; Elson, Genie; Blackshear, Perry J; Lutz, Carol S; Leibovich, S Joseph

2017-04-01

We have shown previously that bacterial lipopolysaccharide (LPS)-mediated suppression of phospholipase-Cβ-2 (PLCβ-2) expression is involved in M1 (inflammatory) to M2-like (wound healing) phenotypic switching of macrophages triggered by adenosine. This suppression is mediated post-transcriptionally by destabilization of PLCβ-2 mRNA (messenger ribonucleic acid). To investigate the mechanism of this LPS-mediated destabilization, we examined the roles of RNA-binding agents including microRNAs and RNA-binding proteins that are involved in regulating stability of mRNAs encoding growth factors, inflammatory mediators, and proto-oncogenes. Adenylate and uridylate (AU)-rich elements (AREs) in 3'UTRs are specific recognition sites for RNA-binding proteins including tristetraprolin (TTP), HuR, and AUF1 and for microRNAs that are involved in regulating mRNA stability. In this study, we investigated the role of TTP and AREs in regulating PLCβ-2 mRNA stability. The 3'UTR of the PLCβ-2 gene was inserted into the pLightswitch luciferase reporter plasmid and transfected into RAW264.7 cells. LPS suppressed luciferase expression from this reporter. Luciferase expression from mutant 3'UTR constructs lacking AREs was similarly downregulated, suggesting that these regions are not required for LPS-mediated suppression of PLCβ-2. TTP was rapidly upregulated in both primary murine macrophages and RAW264.7 cells in response to LPS. Suppression of PLCβ-2 by LPS was examined using macrophages from mice lacking TTP (TTP -/- ). LPS suppressed PLCβ-2 expression to the same extent in wild type (WT) and TTP -/- macrophages. Also, the rate of decay of PLCβ-2 mRNA in LPS-treated macrophages following transcriptional blockade was similar in WT and TTP -/- macrophages, clearly indicating that TTP is not involved in LPS-mediated destabilization of PLCβ-2 mRNA in macrophages.

Developmental expression of the neuroligins and neurexins in fragile X mice.

PubMed

Lai, Jonathan K Y; Doering, Laurie C; Foster, Jane A

2016-03-01

Neuroligins and neurexins are transsynaptic proteins involved in the maturation of glutamatergic and GABAergic synapses. Research has identified synaptic proteins and function as primary contributors to the development of fragile X syndrome. Fragile X mental retardation protein (FMRP), the protein that is lacking in fragile X syndrome, binds neuroligin-1 and -3 mRNA. Using in situ hybridization, we examined temporal and spatial expression patterns of neuroligin (NLGN) and neurexin (NRXN) mRNAs in the somatosensory (S1) cortex and hippocampus in wild-type (WT) and fragile X knockout (FMR1-KO) mice during the first 5 weeks of postnatal life. Genotype-based differences in expression included increased NLGN1 mRNA in CA1 and S1 cortex, decreased NLGN2 mRNA in CA1 and dentate gyrus (DG) regions of the hippocampus, and increased NRXN3 mRNA in CA1, DG, and S1 cortex between female WT and FMR1-KO mice. In male mice, decreased expression of NRXN3 mRNA was observed in CA1 and DG regions of FMR1-KO mice. Sex differences in hippocampal expression of NLGN2, NRXN1, NRXN2, and NRXN3 mRNAs and in S1 cortex expression of NRXN3 mRNAs were observed WT mice, whereas sex differences in NLGN3, NRXN1, NRXN2, and NRXN3 mRNA expression in the hippocampus and in NLGN1, NRXN2 and NRXN3 mRNA expression in S1 cortex were detected in FMR1-KO mice. These results provide a neuroanatomical map of NLGN and NRXN expression patterns over postnatal development in WT and FMR1-KO mice. The differences in developmental trajectory of these synaptic proteins could contribute to long-term differences in CNS wiring and synaptic function. © 2015 Wiley Periodicals, Inc.

Altered Gastrointestinal Function in the Neuroligin-3 Mouse Model of Autism

DTIC Science & Technology

2013-10-01

GABA neurotransmission in the brain. This work aims to examine the spatiotemporal distribution patterns of NL3 and related proteins and mRNA in gut ...implicated in ASD are upregulated during gut development presynaptic localization of the neuroligin-3 protein 16. SECURITY CLASSIFICATION OF: U...related proteins and mRNA in gut tissue from these mice. This project aims to determine biological mechanisms contributing to gastrointestinal dysfunction

The history of infrastructures and the future of cyberinfrastructure in the Earth system sciences

NASA Astrophysics Data System (ADS)

Edwards, P. N.

2012-12-01

Infrastructures display similar historical patterns of inception, development, growth and decay. They typically begin as centralized systems which later proliferate into competing variants. Users' desire for seamless functionality tends eventually to push these variants toward interoperability, usually through "gateway" technologies that link incompatible systems into networks. Another stage is reached when these networks are linked to others, as in the cases of container transport (connecting trucking, rail, and shipping) or the Internet. End stages of infrastructure development include "splintering" (specialized service tiering) and decay, as newer infrastructures displace older ones. Temporal patterns are also visible in historical infrastructure development. This presentation, by a historian of science and technology, describes these patterns through examples of both physical and digital infrastructures, focusing on the global weather forecast infrastructure since the 19th century. It then investigates how some of these patterns might apply to the future of cyberinfrastructure for the Earth system sciences.

Proper accounting for time increases crop-based biofuels' greenhouse gas deficit versus petroleum

NASA Astrophysics Data System (ADS)

O'Hare, M.; Plevin, R. J.; Martin, J. I.; Jones, A. D.; Kendall, A.; Hopson, E.

2009-04-01

The global warming intensities of crop-based biofuels and fossil fuels differ not only in amount but also in their discharge patterns over time. Early discharges, for example, from market-mediated land use change, will have created more global warming by any time in the future than later discharges, owing to the slow decay of atmospheric CO2. A spreadsheet model of this process, BTIME, captures this important time pattern effect using the Bern CO2 decay model to allow fuels to be compared for policy decisions on the basis of their real warming effects with a variety of user-supplied parameter values. The model also allows economic discounting of climate effects extended far into the future. Compared to approaches that simply sum greenhouse gas emissions over time, recognizing the physics of atmospheric CO2 decay significantly increases the deficit relative to fossil fuel of any biofuel causing land use change.

Evaluation of Force Degradation Pattern of Elastomeric Ligatures and Elastomeric Separators in Active Tieback State.

PubMed

Mohammadi, Amir; Mahmoodi, Farhang

2015-01-01

Background and aims. The purpose of this study was to evaluate initial force and force decay of commercially available elastomeric ligatures and elastomeric separators in active tieback state in a simulated oral environment. Materials and methods. A total of 288 elastomeric ligatures and elastomeric separators from three manufacturers (Dentaurum, RMO, 3M Unitek) were stretched to 100% and 150% of their original inner diameter. Force levels were measured initially and at 3-minute, 24-hour, and 1-, 2-, 3- and 4-week intervals. Data were analyzed by univariate analysis of variance and a post hoc Tukey test. Results. The means of initial forces of elastomeric ligatures and separators from three above-mentioned companies, when stretched to 100% of their inner diameters, were 199, 305 and 284 g, and 330, 416, 330 g; when they were stretched to 150% of their inner diameters the values were 286, 422 and 375 g, and 433, 540 and 504 g, respectively. In active tieback state, 11-18% of the initial force of the specimens was lost within the first 3 minutes and 29-63% of the force decay occurred in the first 24 hours; then force decay rate decreased. 62-81% of the initial force was lost in 4 weeks. Although force decay pattern was identical in all the products, the initial force and force decay of Dentaurum elastomeric products were less than the similar products of other companies (P<0.05). Under the same conditions, the force of elastomeric separators was greater than elastomeric ligatures of the same company. Conclusion. Regarding the force pattern of elastomeric ligatures and separators and optimal force for tooth movement, many of these products can be selected for applying orthodontic forces in active tieback state.

Acidic digestion in a teleost: postprandial and circadian pattern of gastric pH, pepsin activity, and pepsinogen and proton pump mRNAs expression.

PubMed

Yúfera, Manuel; Moyano, Francisco J; Astola, Antonio; Pousão-Ferreira, Pedro; Martínez-Rodríguez, Gonzalo

2012-01-01

Two different modes for regulation of stomach acid secretion have been described in vertebrates. Some species exhibit a continuous acid secretion maintaining a low gastric pH during fasting. Others, as some teleosts, maintain a neutral gastric pH during fasting while the hydrochloric acid is released only after the ingestion of a meal. Those different patterns seem to be closely related to specific feeding habits. However, our recent observations suggest that this acidification pattern could be modified by changes in daily feeding frequency and time schedule. The aim of this study was to advance in understanding the regulation mechanisms of stomach digestion and pattern of acid secretion in teleost fish. We have examined the postprandial pattern of gastric pH, pepsin activity, and mRNA expression for pepsinogen and proton pump in white seabream juveniles maintained under a light/dark 12/12 hours cycle and receiving only one morning meal. The pepsin activity was analyzed according to the standard protocol buffering at pH 2 and using the actual pH measured in the stomach. The results show how the enzyme precursor is permanently available while the hydrochloric acid, which activates the zymogen fraction, is secreted just after the ingestion of food. Results also reveal that analytical protocol at pH 2 notably overestimates true pepsin activity in fish stomach. The expression of the mRNA encoding pepsinogen and proton pump exhibited almost parallel patterns, with notable increases during the darkness period and sharp decreases just before the morning meal. These results indicate that white seabream uses the resting hours for recovering the mRNA stock that will be quickly used during the feeding process. Our data clearly shows that both daily illumination pattern and feeding time are involved at different level in the regulation of the secretion of digestive juices.

Environmentally Persistent Free Radicals and Their Lifetimes in PM2.5

PubMed Central

Gehling, William; Dellinger, Barry

2015-01-01

For the first time, an expansive study into the concentration and extended decay behavior of environmentally persistent free radicals in PM2.5 was performed. Results from this study revealed three types of radical decay—a fast decay, slow decay, and no decay—following one of four decay patterns: a relatively fast decay exhibiting a 1/e lifetime of 1–21 days accompanied by a slow decay with a 1/e lifetime of 21–5028 days (47% of samples); a single slow decay including a 1/e lifetime of 4–2083 days (24% of samples); no decay (18% of samples); and a relatively fast decay displaying an average 1/e lifetime of 0.25–21 days followed by no decay (11% of samples). Phenol correlated well with the initial radical concentration and fast decay rate. Other correlations for common atmospheric pollutants (ozone, NOx, SO2, etc.) as well as meteorological conditions suggested photochemical processes impact the initial radical concentration and fast decay rate. The radical signal in PM2.5 was remarkably similar to semiquinones in cigarette smoke. Accordingly, radicals inhaled from PM2.5 were related to the radicals inhaled from smoking cigarettes, expressed as the number of equivalent cigarettes smoked. This calculated to 0.4–0.9 cigarettes per day for nonextreme air quality in the United States. PMID:23844657

Transcriptional Response of the Archaeal Ammonia Oxidizer Nitrosopumilus maritimus to Low and Environmentally Relevant Ammonia Concentrations

PubMed Central

Stahl, David A.

2013-01-01

The ability of chemoautotrophic ammonia-oxidizing archaea to compete for ammonia among marine microorganisms at low ambient concentrations has been in part attributed to their extremely high affinity for ammonia, but as yet there is no mechanistic understanding of supporting metabolism. We examined transcription of selected genes for anabolic functions (CO2 fixation, ammonia transport, and cell wall synthesis) and a central catabolic function (ammonia oxidation) in the thaumarchaeon Nitrosopumilus maritimus SCM1 growing at two ammonia concentrations, as measured by combined ammonia and ammonium, one well above the Km for ammonia oxidation (∼500 μM) and the other well below the Km (<10 nM). Transcript levels were generally immediately and differentially repressed when cells transitioned from ammonia-replete to ammonia-limiting conditions. Transcript levels for ammonia oxidation, CO2 fixation, and one of the ammonia transport genes were approximately the same at high and low ammonia availability. Transcripts for all analyzed genes decreased with time in the complete absence of ammonia, but with various rates of decay. The new steady-state mRNA levels established are presumably more reflective of the natural physiological state of ammonia-oxidizing archaea and offer a reference for interpreting message abundance patterns in the natural environment. PMID:23995944

Contribution of copy number variants involving nonsense-mediated mRNA decay pathway genes to neuro-developmental disorders.

PubMed

Nguyen, Lam S; Kim, Hyung-Goo; Rosenfeld, Jill A; Shen, Yiping; Gusella, James F; Lacassie, Yves; Layman, Lawrence C; Shaffer, Lisa G; Gécz, Jozef

2013-05-01

The nonsense-mediated mRNA decay (NMD) pathway functions not only to degrade transcripts containing premature termination codons (PTC), but also to regulate the transcriptome. UPF3B and RBM8A, important components of NMD, have been implicated in various forms of intellectual disability (ID) and Thrombocytopenia with Absent Radius (TAR) syndrome, which is also associated with ID. To gauge the contribution of other NMD factors to ID, we performed a comprehensive search for copy number variants (CNVs) of 18 NMD genes among individuals with ID and/or congenital anomalies. We identified 11 cases with heterozygous deletions of the genomic region encompassing UPF2, which encodes for a direct interacting protein of UPF3B. Using RNA-Seq, we showed that the genome-wide consequence of reduced expression of UPF2 is similar to that seen in patients with UPF3B mutations. Out of the 1009 genes found deregulated in patients with UPF2 deletions by at least 2-fold, majority (95%) were deregulated similarly in patients with UPF3B mutations. This supports the major role of deletion of UPF2 in ID. Furthermore, we found that four other NMD genes, UPF3A, SMG6, EIF4A3 and RNPS1 are frequently deleted and/or duplicated in the patients. We postulate that dosage imbalances of these NMD genes are likely to be the causes or act as predisposing factors for neuro-developmental disorders. Our findings further emphasize the importance of NMD pathway(s) in learning and memory.

Granules harboring translationally active mRNAs provide a platform for P-body formation following stress.

PubMed

Lui, Jennifer; Castelli, Lydia M; Pizzinga, Mariavittoria; Simpson, Clare E; Hoyle, Nathaniel P; Bailey, Kathryn L; Campbell, Susan G; Ashe, Mark P

2014-11-06

The localization of mRNA to defined cytoplasmic sites in eukaryotic cells not only allows localized protein production but also determines the fate of mRNAs. For instance, translationally repressed mRNAs localize to P-bodies and stress granules where their decay and storage, respectively, are directed. Here, we find that several mRNAs are localized to granules in unstressed, actively growing cells. These granules play a key role in the stress-dependent formation of P-bodies. Specific glycolytic mRNAs are colocalized in multiple granules per cell, which aggregate during P-body formation. Such aggregation is still observed under conditions or in mutants where P-bodies do not form. In unstressed cells, the mRNA granules appear associated with active translation; this might enable a coregulation of protein expression from the same pathways or complexes. Parallels can be drawn between this coregulation and the advantage of operons in prokaryotic systems. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

ME31B globally represses maternal mRNAs by two distinct mechanisms during the Drosophila maternal-to-zygotic transition.

PubMed

Wang, Miranda; Ly, Michael; Lugowski, Andrew; Laver, John D; Lipshitz, Howard D; Smibert, Craig A; Rissland, Olivia S

2017-09-06

In animal embryos, control of development is passed from exclusively maternal gene products to those encoded by the embryonic genome in a process referred to as the maternal-to-zygotic transition (MZT). We show that the RNA-binding protein, ME31B, binds to and represses the expression of thousands of maternal mRNAs during the Drosophila MZT. However, ME31B carries out repression in different ways during different phases of the MZT. Early, it represses translation while, later, its binding leads to mRNA destruction, most likely as a consequence of translational repression in the context of robust mRNA decay. In a process dependent on the PNG kinase, levels of ME31B and its partners, Cup and Trailer Hitch (TRAL), decrease by over 10-fold during the MZT, leading to a change in the composition of mRNA-protein complexes. We propose that ME31B is a global repressor whose regulatory impact changes based on its biological context.

Beyond the known functions of the CCR4-NOT complex in gene expression regulatory mechanisms: New structural insights to unravel CCR4-NOT mRNA processing machinery.

PubMed

Ukleja, Marta; Valpuesta, José María; Dziembowski, Andrzej; Cuellar, Jorge

2016-10-01

Large protein assemblies are usually the effectors of major cellular processes. The intricate cell homeostasis network is divided into numerous interconnected pathways, each controlled by a set of protein machines. One of these master regulators is the CCR4-NOT complex, which ultimately controls protein expression levels. This multisubunit complex assembles around a scaffold platform, which enables a wide variety of well-studied functions from mRNA synthesis to transcript decay, as well as other tasks still being identified. Solving the structure of the entire CCR4-NOT complex will help to define the distribution of its functions. The recently published three-dimensional reconstruction of the complex, in combination with the known crystal structures of some of the components, has begun to address this. Methodological improvements in structural biology, especially in cryoelectron microscopy, encourage further structural and protein-protein interaction studies, which will advance our comprehension of the gene expression machinery. © 2016 WILEY Periodicals, Inc.

Control of myogenesis by rodent SINE-containing lncRNAs

PubMed Central

Wang, Jiashi; Gong, Chenguang; Maquat, Lynne E.

2013-01-01

Staufen1-mediated mRNA decay (SMD) degrades mRNAs that harbor a Staufen1-binding site (SBS) in their 3′ untranslated regions (UTRs). Human SBSs can form by intermolecular base-pairing between a 3′ UTR Alu element and an Alu element within a long noncoding RNA (lncRNA) called a ½-sbsRNA. Since Alu elements are confined to primates, it was unclear how SMD occurs in rodents. Here we identify mouse mRNA 3′ UTRs and lncRNAs that contain a B1, B2, B4, or identifier (ID) element. We show that SMD occurs in mouse cells via mRNA–lncRNA base-pairing of partially complementary elements and that mouse ½-sbsRNA (m½-sbsRNA)-triggered SMD regulates C2C12 cell myogenesis. Our findings define new roles for lncRNAs as well as B and ID short interspersed elements (SINEs) in mice that undoubtedly influence many developmental and homeostatic pathways. PMID:23558772

Phenotypic Evolution of UNC80 Loss of Function

PubMed Central

Valkanas, Elise; Schaffer, Katherine; Dunham, Christopher; Maduro, Valerie; du Souich, Christèle; Rupps, Rosemarie; Adams, David R.; Baradaran-Heravi, Alireza; Flynn, Elise; Malicdan, May C.; Gahl, William A.; Toro, Camilo; Boerkoel, Cornelius F.

2017-01-01

Failure to thrive arises as a complication of a heterogeneous group of disorders. We describe two female siblings with spastic paraplegia and global developmental delay but also, atypically for the HSPs, poor weight gain classified as failure to thrive. After extensive clinical and biochemical investigations failed to identify the etiology, we used exome sequencing to identify biallelic UNC80 mutations (NM_032504.1:c.[3983-3_3994delinsA];[2431C>T]. The paternally inherited NM_032504.1:c.3983-3_3994delinsA is predicted to encode p.Ser1328Argfs*19 and the maternally inherited NM_032504.1:c.2431C>T is predicted to encode p.Arg811*. No UNC80 mRNA was detectable in patient cultured skin fibroblasts, suggesting UNC80 loss of function by nonsense mediated mRNA decay. Further supporting the UNC80 mutations as causative of these siblings disorder, biallelic mutations in UNC80 have recently been described among individuals with an overlapping phenotype. This report expands the disease spectrum associated with UNC80 mutations. PMID:27513830

Conservation of miRNA-mediated silencing mechanisms across 600 million years of animal evolution.

PubMed

Mauri, Marta; Kirchner, Marieluise; Aharoni, Reuven; Ciolli Mattioli, Camilla; van den Bruck, David; Gutkovitch, Nadya; Modepalli, Vengamanaidu; Selbach, Matthias; Moran, Yehu; Chekulaeva, Marina

2017-01-25

Our current knowledge about the mechanisms of miRNA silencing is restricted to few lineages such as vertebrates, arthropods, nematodes and land plants. miRNA-mediated silencing in bilaterian animals is dependent on the proteins of the GW182 family. Here, we dissect the function of GW182 protein in the cnidarian Nematostella, separated by 600 million years from other Metazoa. Using cultured human cells, we show that Nematostella GW182 recruits the CCR4-NOT deadenylation complexes via its tryptophan-containing motifs, thereby inhibiting translation and promoting mRNA decay. Further, similarly to bilaterians, GW182 in Nematostella is recruited to the miRNA repression complex via interaction with Argonaute proteins, and functions downstream to repress mRNA. Thus, our work suggests that this mechanism of miRNA-mediated silencing was already active in the last common ancestor of Cnidaria and Bilateria. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Proteomic Analysis of the Mediator Complex Interactome in Saccharomyces cerevisiae.

PubMed

Uthe, Henriette; Vanselow, Jens T; Schlosser, Andreas

2017-02-27

Here we present the most comprehensive analysis of the yeast Mediator complex interactome to date. Particularly gentle cell lysis and co-immunopurification conditions allowed us to preserve even transient protein-protein interactions and to comprehensively probe the molecular environment of the Mediator complex in the cell. Metabolic 15 N-labeling thereby enabled stringent discrimination between bona fide interaction partners and nonspecifically captured proteins. Our data indicates a functional role for Mediator beyond transcription initiation. We identified a large number of Mediator-interacting proteins and protein complexes, such as RNA polymerase II, general transcription factors, a large number of transcriptional activators, the SAGA complex, chromatin remodeling complexes, histone chaperones, highly acetylated histones, as well as proteins playing a role in co-transcriptional processes, such as splicing, mRNA decapping and mRNA decay. Moreover, our data provides clear evidence, that the Mediator complex interacts not only with RNA polymerase II, but also with RNA polymerases I and III, and indicates a functional role of the Mediator complex in rRNA processing and ribosome biogenesis.

SKIV2L Mutations Cause Syndromic Diarrhea, or Trichohepatoenteric Syndrome

PubMed Central

Fabre, Alexandre; Charroux, Bernard; Martinez-Vinson, Christine; Roquelaure, Bertrand; Odul, Egritas; Sayar, Ersin; Smith, Hilary; Colomb, Virginie; Andre, Nicolas; Hugot, Jean-Pierre; Goulet, Olivier; Lacoste, Caroline; Sarles, Jacques; Royet, Julien; Levy, Nicolas; Badens, Catherine

2012-01-01

Syndromic diarrhea (or trichohepatoenteric syndrome) is a rare congenital bowel disorder characterized by intractable diarrhea and woolly hair, and it has recently been associated with mutations in TTC37. Although databases report TTC37 as being the human ortholog of Ski3p, one of the yeast Ski-complex cofactors, this lead was not investigated in initial studies. The Ski complex is a multiprotein complex required for exosome-mediated RNA surveillance, including the regulation of normal mRNA and the decay of nonfunctional mRNA. Considering the fact that TTC37 is homologous to Ski3p, we explored a gene encoding another Ski-complex cofactor, SKIV2L, in six individuals presenting with typical syndromic diarrhea without variation in TTC37. We identified mutations in all six individuals. Our results show that mutations in genes encoding cofactors of the human Ski complex cause syndromic diarrhea, establishing a link between defects of the human exosome complex and a Mendelian disease. PMID:22444670

Upregulation of Ryk expression in rat dorsal root ganglia after peripheral nerve injury.

PubMed

Li, Xin; Li, Yao-hua; Yu, Shun; Liu, Yaobo

2008-10-22

To study changes of Ryk expression in dorsal root ganglia (DRG) after peripheral nerve injury, we set up an animal model of unilateral sciatic nerve lesioned rats. Changes of Ryk protein expression in DRG neurons after unilateral sciatic nerve injury were investigated by immunostaining. Changes of Ryk mRNA were also tested by semi-quantitative PCR concurrently. We found, both at the level of protein and mRNA, that Ryk could be induced in cells of ipsilateral DRG after unilateral sciatic nerve lesion. Further investigation by co-immunostaining confirmed that the Ryk-immunoreactive (Ryk-IR) cells were NeuN-immunoreactive (NeuN-IR) neurons of DRG. We also showed the pattern of Ryk induction in DRG neurons after sciatic nerve injury: the number of Ryk IR neurons peaked at 2 weeks post-lesion and decreased gradually by 3 weeks post-lesion. The proportions of different sized Ryk IR neurons were also observed and counted at various stages after nerve lesion. Analysis of Ryk mRNA by RT-PCR showed the same induction pattern as by immunostaining. Ryk mRNA was not expressed in normal or contralateral DRG, but was expressed 1, 2 and 3 weeks post-lesion in the ipsilateral DRG. Ryk mRNA levels increased slightly from 1 to 2 weeks, decreased then by 3 weeks post-lesion. These results indicate that Ryk might be involved in peripheral nerve plasticity after injury. This is a novel function apart from its well-known fundamental activity as a receptor mediating axon guidance and outgrowth.

Effect of ration size on fillet fatty acid composition, phospholipid allostasis and mRNA expression patterns of lipid regulatory genes in gilthead sea bream (Sparus aurata).

PubMed

Benedito-Palos, Laura; Calduch-Giner, Josep A; Ballester-Lozano, Gabriel F; Pérez-Sánchez, Jaume

2013-04-14

The effect of ration size on muscle fatty acid (FA) composition and mRNA expression levels of key regulatory enzymes of lipid and lipoprotein metabolism have been addressed in juveniles of gilthead sea bream fed a practical diet over the course of an 11-week trial. The experimental setup included three feeding levels: (i) full ration until visual satiety, (ii) 70 % of satiation and (iii) 70 % of satiation with the last 2 weeks at the maintenance ration. Feed restriction reduced lipid content of whole body by 30 % and that of fillet by 50 %. In this scenario, the FA composition of fillet TAG was not altered by ration size, whereas that of phospholipids was largely modified with a higher retention of arachidonic acid and DHA. The mRNA transcript levels of lysophosphatidylcholine acyltransferases, phosphatidylethanolamine N-methyltransferase and FA desaturase 2 were not regulated by ration size in the present experimental model. In contrast, mRNA levels of stearoyl-CoA desaturases were markedly down-regulated by feed restriction. An opposite trend was found for a muscle-specific lipoprotein lipase, which is exclusive of fish lineage. Several upstream regulatory transcriptions were also assessed, although nutritionally mediated changes in mRNA transcripts were almost reduced to PPARα and β, which might act in a counter-regulatory way on lipolysis and lipogenic pathways. This gene expression pattern contributes to the construction of a panel of biomarkers to direct marine fish production towards muscle lean phenotypes with increased retentions of long-chain PUFA.

Platinum sensitivity and DNA repair in a recently established panel of patient-derived ovarian carcinoma xenografts

PubMed Central

Guffanti, Federica; Fratelli, Maddalena; Ganzinelli, Monica; Bolis, Marco; Ricci, Francesca; Bizzaro, Francesca; Chilà, Rosaria; Sina, Federica Paola; Fruscio, Robert; Lupia, Michela; Cavallaro, Ugo; Cappelletti, Maria Rosa; Generali, Daniele; Giavazzi, Raffaella; Damia, Giovanna

2018-01-01

A xenobank of patient-derived (PDX) ovarian tumor samples has been established consisting of tumors with different sensitivity to cisplatin (DDP), from very responsive to resistant. As the DNA repair pathway is an important driver in tumor response to DDP, we analyzed the mRNA expression of 20 genes involved in the nucleotide excision repair, fanconi anemia, homologous recombination, base excision repair, mismatch repair and translesion repair pathways and the methylation patterns of some of these genes. We also investigated the correlation with the response to platinum-based therapy. The mRNA levels of the selected genes were evaluated by Real Time-PCR (RT-PCR) with ad hoc validated primers and gene promoter methylation by pyrosequencing. All the DNA repair genes were variably expressed in all 42 PDX samples analyzed, with no particular histotype-specific pattern of expression. In high-grade serous/endometrioid PDXs, the CDK12 mRNA expression levels positively correlated with the expression of TP53BP1, PALB2, XPF and POLB. High-grade serous/endometrioid PDXs with TP53 mutations had significantly higher levels of POLQ, FANCD2, RAD51 and POLB than high-grade TP53 wild type PDXs. The mRNA levels of CDK12, PALB2 and XPF inversely associated with the in vivo DDP antitumor activity; higher CDK12 mRNA levels were associated with a higher recurrence rate in ovarian patients with low residual tumor. These data support the important role of CDK12 in the response to a platinum based therapy in ovarian patients. PMID:29872499

Effects of gonadoliberin analogue triptorelin on the pituitary-testicular complex in neonatal rats.

PubMed

Dygalo, N N; Shemenkova, T V; Kalinina, T S; Shishkina, G T

2014-02-01

Triptorelin, a synthetic analogue of neurohormone gonadoliberin (gonadotropin-releasing hormone, GnRH) administered daily to rats on postnatal days 5-7 suppressed the expression of GnRH receptor in the pituitary gland, but did not change functioning of the pituitary-testicular complex. Administration of triptorelin on postnatal days 12-14 (i.e. during the formation of pulsatile pattern of GnRH secretion and increasing levels of its mRNA receptor in the pituitary gland) had no effect on receptor expression, but increased the levels of luteinizing hormone mRNA in the pituitary gland and the weight of testes. At that time, blood levels of testosterone were lowered, which indicated disturbed pulsatile pattern of GnRH secretion.

Wood decay in living and dead trees: A pictorial overview

Treesearch

Walter C. Shortle; Kenneth R. Dudzik

2012-01-01

Pioneering research by Alex L. Shigo and his associates has produced a series of pictorial guidelines to provide a better understanding of how trees respond to wounding and subsequent microbial infections that lead to wood decay. The purpose of this paper is to visually summarize through the use of 96 color photographs and illustrations, the varied patterns of wood...

Experimental observations on the decay of environmental DNA from bighead and silver carps

USGS Publications Warehouse

Lance, Richard F.; Klymus, Katy E.; Richter, Cathy; Guan, Xin; Farrington, Heather L.; Carr, Matthew R.; Thompson, Nathan; Chapman, Duane C.; Baerwaldt, Kelly L.

2017-01-01

Interest in the field of environmental DNA (eDNA) is growing rapidly and eDNA surveys are becoming an important consideration for aquatic resource managers dealing with invasive species. However, in order for eDNA monitoring to mature as a research and management tool, there are several critical knowledge gaps that must be filled. One such gap is the fate of eDNA materials in the aquatic environment. Understanding the environmental factors that influence the decay of eDNA and how these factors impact detection probabilities over time and space could have significant implications for eDNA survey design and data interpretation. Here we experimentally explore decay of eDNA associated with bighead carp (Hypophthalmichthys nobilis) biological waste collected from an aquaculture filtration system and with sperm collected from captive silver carp (H. molitrix), and how decay may be influenced by differing levels of water turbulence, temperature, microbial load, and pH. We found that the decay patterns of eDNA associated with both H. nobilis biological waste and H. molitrix milt significantly fit monophasic exponential decay curves. Secondly, we observed that the highest temperature we tested resulted in a decay half-life as much as 5.5× more rapid than the lowest temperature we tested. When we suppressed microbial loads in eDNA samples, we observed that overall losses of eDNA were reduced by about 2.5×. When we amended eDNA samples with pond water the half-life of eDNA was reduced by about 2.25×, despite relatively little apparent increase in the overall microbial load. This pattern indicated that species constituency of the microbial community, in addition to microbial load, might play a critical role in eDNA degradation. A shift in pH from 6.5 to 8.0 in the samples resulted in a 1.6× reduction in eDNA halflife. Water turbulence in our study had no apparent effect on eDNA decay. When we combined different temperature, pH, and microbial load treatments to create a rapid decay condition and a slow decay condition, and tracked eDNA decay over 91 days, we observed a 5.0× greater loss of eDNA by Day 5 under rapid decay conditions than under slow decay conditions. At the end of the trials, the differences in eDNA loss between the rapid decay and baseline and slow decay conditions were 0.1× and 3.3×, respectively. Our results strongly demonstrate the potential for environmental factors to influence eDNA fate and, thus, the interpretation of eDNA survey results.

TCSPC based approaches for multiparameter detection in living cells

NASA Astrophysics Data System (ADS)

Jahn, Karolina; Buschmann, Volker; Koberling, Felix; Hille, Carsten

2014-03-01

In living cells a manifold of processes take place simultaneously. This implies a precise regulation of intracellular ion homeostasis. In order to understand their spatio-temporal pattern comprehensively, the development of multiplexing concepts is essential. Due to the multidimensional characteristics of fluorescence dyes (absorption and emission spectra, decay time, anisotropy), the highly sensitive and non-invasive fluorescence microscopy is a versatile tool for realising multiplexing concepts. A prerequisite are analyte-specific fluorescence dyes with low cross-sensitivity to other dyes and analytes, respectively. Here, two approaches for multiparameter detection in living cells are presented. Insect salivary glands are well characterised secretory active tissues which were used as model systems to evaluate multiplexing concepts. Salivary glands secrete a KCl-rich or NaCl-rich fluid upon stimulation which is mainly regulated by intracellular Ca2+ as second messenger. Thus, pairwise detection of intracellular Na+, Cl- and Ca2+ with the fluorescent dyes ANG2, MQAE and ACR were tested. Therefore, the dyes were excited simultaneously (2-photon excitation) and their corresponding fluorescence decay times were recorded within two spectral ranges using time-correlated singlephoton counting (TCSPC). A second approach presented here is based on a new TCSPC-platform covering decay time detection from picoseconds to milliseconds. Thereby, nanosecond decaying cellular fluorescence and microsecond decaying phosphorescence of Ruthenium-complexes, which is quenched by oxygen, were recorded simultaneously. In both cases changes in luminescence decay times can be linked to changes in analyte concentrations. In consequence of simultaneous excitation as well as detection, it is possible to get a deeper insight into spatio-temporal pattern in living tissues.

Sodium fluoride affects zebrafish behaviour and alters mRNA expressions of biomarker genes in the brain: Role of Nrf2/Keap1.

PubMed

Mukhopadhyay, Debdip; Priya, Pooja; Chattopadhyay, Ansuman

2015-09-01

Sodium fluoride (NaF), used as pesticides and for industrial purposes are deposited in the water bodies and therefore affects its biota. Zebrafish exposed to NaF in laboratory condition showed hyperactivity and frequent surfacing activity, somersaulting and vertical swimming pattern as compared to the control group. Reactive oxygen species level was elevated and glutathione level was depleted along with increased malondialdehyde content in the brain. Levels of glutathione-s-transferase (GST), catalase (CAT) and superoxide dismutase were also elevated in the treatment groups. Expression of mRNA of nuclear factor erythroid 2 related factor 2 (Nrf2) and its inhibitor Kelch-like ECH-associated protein 1 (Keap1) during stress condition were observed along with Gst, Cat, NADPH: quinone oxidoreductase 1(Nqo1) and p38. Except Keap1, all other genes exhibited elevated expression. Nrf2/Keap1 proteins had similar expression pattern as their corresponding mRNA. The findings in this study might help to understand the molecular mechanism of fluoride induced neurotoxicity in fish. Copyright © 2015 Elsevier B.V. All rights reserved.

Deep Sequencing Reveals Direct Targets of Gammaherpesvirus-Induced mRNA Decay and Suggests That Multiple Mechanisms Govern Cellular Transcript Escape

PubMed Central

Clyde, Karen; Glaunsinger, Britt A.

2011-01-01

One characteristic of lytic infection with gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and murine herpesvirus 68 (MHV68), is the dramatic suppression of cellular gene expression in a process known as host shutoff. The alkaline exonuclease proteins (KSHV SOX, MHV-68 muSOX and EBV BGLF5) have been shown to induce shutoff by destabilizing cellular mRNAs. Here we extend previous analyses of cellular mRNA abundance during lytic infection to characterize the effects of SOX and muSOX, in the absence of other viral genes, utilizing deep sequencing technology (RNA-seq). Consistent with previous observations during lytic infection, the majority of transcripts are downregulated in cells expressing either SOX or muSOX, with muSOX acting as a more potent shutoff factor than SOX. Moreover, most cellular messages fall into the same expression class in both SOX- and muSOX-expressing cells, indicating that both factors target similar pools of mRNAs. More abundant mRNAs are more efficiently downregulated, suggesting a concentration effect in transcript targeting. However, even among highly expressed genes there are mRNAs that escape host shutoff. Further characterization of select escapees reveals multiple mechanisms by which cellular genes can evade downregulation. While some mRNAs are directly refractory to SOX, the steady state levels of others remain unchanged, presumably as a consequence of downstream effects on mRNA biogenesis. Collectively, these studies lay the framework for dissecting the mechanisms underlying the susceptibility of mRNA to destruction during lytic gammaherpesvirus infection. PMID:21573023

Autotaxin Expression Is Regulated at the Post-transcriptional Level by the RNA-binding Proteins HuR and AUF1.

PubMed

Sun, Shuhong; Zhang, Xiaotian; Lyu, Lin; Li, Xixi; Yao, Siliang; Zhang, Junjie

2016-12-09

Autotaxin (ATX) is a key enzyme that converts lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA), a lysophospholipid mediator that regulates cellular activities through its specific G protein-coupled receptors. The ATX-LPA axis plays an important role in various physiological and pathological processes, especially in inflammation and cancer development. Although the transcriptional regulation of ATX has been widely studied, the post-transcriptional regulation of ATX is largely unknown. In this study, we identified conserved adenylate-uridylate (AU)-rich elements in the ATX mRNA 3'-untranslated region (3'UTR). The RNA-binding proteins HuR and AUF1 directly bound to the ATX mRNA 3'UTR and had antagonistic functions in ATX expression. HuR enhanced ATX expression by increasing ATX mRNA stability, whereas AUF1 suppressed ATX expression by promoting ATX mRNA decay. HuR and AUF1 were involved in ATX regulation in Colo320 human colon cancer cells and the LPS-stimulated human monocytic THP-1 cells. HuR knockdown suppressed ATX expression in B16 mouse melanoma cells, leading to inhibition of cell migration. This effect was reversed by AUF1 knockdown to recover ATX expression or by the addition of LPA. These results suggest that the post-transcriptional regulation of ATX expression by HuR and AUF1 modulates cancer cell migration. In summary, we identified HuR and AUF1 as novel post-transcriptional regulators of ATX expression, thereby elucidating a novel mechanism regulating the ATX-LPA axis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Programming mRNA decay to modulate synthetic circuit resource allocation

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

Venturelli, Ophelia S.; Tei, Mika; Bauer, Stefan

Synthetic circuits embedded in host cells compete with cellular processes for limited intracellular resources. Here we show how funnelling of cellular resources, after global transcriptome degradation by the sequence-dependent endoribonuclease MazF, to a synthetic circuit can increase production. Target genes are protected from MazF activity by recoding the gene sequence to eliminate recognition sites, while preserving the amino acid sequence. The expression of a protected fluorescent reporter and flux of a high-value metabolite are significantly enhanced using this genome-scale control strategy. Proteomics measurements discover a host factor in need of protection to improve resource redistribution activity. A computational model demonstratesmore » that the MazF mRNA-decay feedback loop enables proportional control of MazF in an optimal operating regime. Transcriptional profiling of MazF-induced cells elucidates the dynamic shifts in transcript abundance and discovers regulatory design elements. Altogether, our results suggest that manipulation of cellular resource allocation is a key control parameter for synthetic circuit design.« less

Programming mRNA decay to modulate synthetic circuit resource allocation

DOE PAGES

Venturelli, Ophelia S.; Tei, Mika; Bauer, Stefan; ...

2017-04-26

Synthetic circuits embedded in host cells compete with cellular processes for limited intracellular resources. Here we show how funnelling of cellular resources, after global transcriptome degradation by the sequence-dependent endoribonuclease MazF, to a synthetic circuit can increase production. Target genes are protected from MazF activity by recoding the gene sequence to eliminate recognition sites, while preserving the amino acid sequence. The expression of a protected fluorescent reporter and flux of a high-value metabolite are significantly enhanced using this genome-scale control strategy. Proteomics measurements discover a host factor in need of protection to improve resource redistribution activity. A computational model demonstratesmore » that the MazF mRNA-decay feedback loop enables proportional control of MazF in an optimal operating regime. Transcriptional profiling of MazF-induced cells elucidates the dynamic shifts in transcript abundance and discovers regulatory design elements. Altogether, our results suggest that manipulation of cellular resource allocation is a key control parameter for synthetic circuit design.« less

Diminished nuclear RNA decay upon Salmonella infection upregulates antibacterial noncoding RNAs.

PubMed

Imamura, Katsutoshi; Takaya, Akiko; Ishida, Yo-Ichi; Fukuoka, Yayoi; Taya, Toshiki; Nakaki, Ryo; Kakeda, Miho; Imamachi, Naoto; Sato, Aiko; Yamada, Toshimichi; Onoguchi-Mizutani, Rena; Akizuki, Gen; Tanu, Tanzina; Tao, Kazuyuki; Miyao, Sotaro; Suzuki, Yutaka; Nagahama, Masami; Yamamoto, Tomoko; Jensen, Torben Heick; Akimitsu, Nobuyoshi

2018-06-07

Cytoplasmic mRNA degradation controls gene expression to help eliminate pathogens during infection. However, it has remained unclear whether such regulation also extends to nuclear RNA decay. Here, we show that 145 unstable nuclear RNAs, including enhancer RNAs (eRNAs) and long noncoding RNAs (lncRNAs) such as NEAT1v2, are stabilized upon Salmonella infection in HeLa cells. In uninfected cells, the RNA exosome, aided by the Nuclear EXosome Targeting (NEXT) complex, degrades these labile transcripts. Upon infection, the levels of the exosome/NEXT components, RRP6 and MTR4, dramatically decrease, resulting in transcript stabilization. Depletion of lncRNAs, NEAT1v2, or eRNA07573 in HeLa cells triggers increased susceptibility to Salmonella infection concomitant with the deregulated expression of a distinct class of immunity-related genes, indicating that the accumulation of unstable nuclear RNAs contributes to antibacterial defense. Our results highlight a fundamental role for regulated degradation of nuclear RNA in the response to pathogenic infection. © 2018 The Authors.

Macromolecular Crowding Induces Spatial Correlations That Control Gene Expression Bursting Patterns.

PubMed

Norred, S Elizabeth; Caveney, Patrick M; Chauhan, Gaurav; Collier, Lauren K; Collier, C Patrick; Abel, Steven M; Simpson, Michael L

2018-05-18

Recent superresolution microscopy studies in E. coli demonstrate that the cytoplasm has highly variable local concentrations where macromolecular crowding plays a central role in establishing membrane-less compartmentalization. This spatial inhomogeneity significantly influences molecular transport and association processes central to gene expression. Yet, little is known about how macromolecular crowding influences gene expression bursting-the episodic process where mRNA and proteins are produced in bursts. Here, we simultaneously measured mRNA and protein reporters in cell-free systems, showing that macromolecular crowding decoupled the well-known relationship between fluctuations in the protein population (noise) and mRNA population statistics. Crowded environments led to a 10-fold increase in protein noise even though there were only modest changes in the mRNA population and fluctuations. Instead, cell-like macromolecular crowding created an inhomogeneous spatial distribution of mRNA ("spatial noise") that led to large variability in the protein production burst size. As a result, the mRNA spatial noise created large temporal fluctuations in the protein population. These results highlight the interplay between macromolecular crowding, spatial inhomogeneities, and the resulting dynamics of gene expression, and provide insights into using these organizational principles in both cell-based and cell-free synthetic biology.

Techniques for the treatment of IR divergences in decay processes at NLO and application to the top-quark decay.

PubMed

Basso, Lorenzo; Dittmaier, Stefan; Huss, Alexander; Oggero, Luisa

We present the extension of two general algorithms for the treatment of infrared singularities arising in electroweak corrections to decay processes at next-to-leading order: the dipole subtraction formalism and the one-cutoff slicing method. The former is extended to the case of decay kinematics which has not been considered in the literature so far. The latter is generalised to production and decay processes with more than two charged particles, where new "surface" terms arise. Arbitrary patterns of massive and massless external particles are considered, including the treatment of infrared singularities in dimensional or mass regularisation. As an application of the two techniques we present the calculation of the next-to-leading order QCD and electroweak corrections to the top-quark decay width including all off-shell and decay effects of intermediate [Formula: see text] bosons. The result, e.g., represents a building block of a future calculation of NLO electroweak effects to off-shell top-quark pair ([Formula: see text]) production. Moreover, this calculation can serve as the first step towards an event generator for top-quark decays at next-to-leading order accuracy, which can be used to attach top-quark decays to complicated many-particle top-quark processes, such as for [Formula: see text] or [Formula: see text].

SEM study of the effects of bacteria and yeasts on wood decay by brown and white-rot fungi. [Enterobacter, Cryptococcus Pichia, and Saccharomyces

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

Blanchette, R.A.; Shaw, C.G.; Cohen, A.L.

The scanning electron microscope was used to 1) examine the associations among microorganisms during wood decay and 2) observe the effect of these organisms on degradation of cell wall components. Bacteria (Enterobacter) and yeasts (Cryptococcus Pichia, and Saccharomyces) were found to have a mutualistic association with a white-rot fungus during decay of coniferous wood. Coriolus (Polyporus versicolar) degraded cell wall components in a typical ''erosion trough'' manner (i.e., by lysing zones around fungal hyphae). Bacteria and yeasts were seen only in these lysed zones. Typical gross decay patterns caused by the white-rot fungus were unaltered by bacteria and yeasts. Themore » SEM study suggests that the decay process is enhanced when these organisms are associated. In contrast, the same bacteria and yeasts were inhibitory when combined with a brown-rot fungus.« less

Claudin gene expression patterns do not associate with interspecific differences in paracellular nutrient absorption.

PubMed

Price, Edwin R; Rott, Katherine H; Caviedes-Vidal, Enrique; Karasov, William H

2016-01-01

Bats exhibit higher paracellular absorption of glucose-sized molecules than non-flying mammals, a phenomenon that may be driven by higher permeability of the intestinal tight junctions. The various claudins, occludin, and other proteins making up the tight junctions are thought to determine their permeability properties. Here we show that absorption of the paracellular probe l-arabinose is higher in a bat (Eptesicus fuscus) than in a vole (Microtus pennsylvanicus) or a hedgehog (Atelerix albiventris). Furthermore, histological measurements demonstrated that hedgehogs have many more enterocytes in their intestines, suggesting that bats cannot have higher absorption of arabinose simply by having more tight junctions. We therefore investigated the mRNA levels of several claudins and occludin, because these proteins may affect permeability of tight junctions to macronutrients. To assess the expression levels of claudins per tight junction, we normalized the mRNA levels of the claudins to the constitutively expressed tight junction protein ZO-1, and combined these with measurements previously made in a bat and a rodent to determine if there were among-species differences. Although expression ratios of several genes varied among species, there was not a consistent difference between bats and non-flyers in the expression ratio of any particular gene. Protein expression patterns may differ from mRNA expression patterns, and might better explain differences among species in arabinose absorption. Copyright © 2015 Elsevier Inc. All rights reserved.

Thyroid hormone receptor beta2 is strongly up-regulated at all levels of the hypothalamo-pituitary-thyroidal axis during late embryogenesis in chicken.

PubMed

Grommen, Sylvia V H; Arckens, Lutgarde; Theuwissen, Tim; Darras, Veerle M; De Groef, Bert

2008-03-01

In this study, we tried to elucidate the changes in thyroid hormone (TH) receptor beta2 (TRbeta2) expression at the different levels of the hypothalamo-pituitary-thyroidal (HPT) axis during the last week of chicken embryonic development and hatching, a period characterized by an augmented activity of the HPT axis. We quantified TRbeta2 mRNA in retina, pineal gland, and the major control levels of the HPT axis - brain, pituitary, and thyroid gland - at day 18 of incubation, and found the most abundant mRNA content in retina and pituitary. Thyroidal TRbeta2 mRNA content increased dramatically between embryonic day 14 and 1 day post-hatch. In pituitary and hypothalamus, TRbeta2 mRNA expression rose gradually, in parallel with increases in plasma thyroxine concentrations. Using in situ hybridization, we have demonstrated the presence of TRbeta2 mRNA throughout the diencephalon and confirmed the elevation in TRbeta2 mRNA expression in the hypophyseal thyrotropes. In vitro incubation with THs caused a down-regulation of TRbeta2 mRNA levels in embryonic but not in post-hatch pituitaries. The observed expression patterns in pituitary and diencephalon may point to substantial changes in TRbeta2-mediated TH feedback active during the perinatal period. The strong rise in thyroidal TRbeta2 mRNA content could be indicative of an augmented modulation of thyroid development and/or function by THs toward and after hatching. Finally, THs proved to exert an age-dependent effect on pituitary TRbeta2 mRNA expression.

Combination of Fluorescent in situ Hybridization (FISH) and Immunofluorescence Imaging for Detection of Cytokine Expression in Microglia/Macrophage Cells

PubMed Central

Fe Lanfranco, Maria; Loane, David J.; Mocchetti, Italo; Burns, Mark P.; Villapol, Sonia

2017-01-01

Microglia and macrophage cells are the primary producers of cytokines in response to neuroinflammatory processes. But these cytokines are also produced by other glial cells, endothelial cells, and neurons. It is essential to identify the cells that produce these cytokines to target their different levels of activation. We used dual RNAscope® fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) techniques to visualize the mRNA expression pattern of pro- and anti-inflammatory cytokines in microglia/macrophages cells. Using these methods, we can associate one mRNA to specific cell types when combining with different cellular markers by immunofluorescence. Results from RNAscope® probes IL-1β, TNFα, TGFβ, IL-10 or Arg1, showed colocalization with antibodies for microglia/macrophage cells. These target probes showed adequate sensitivity and specificity to detect mRNA expression. New FISH detection techniques combined with immunohistochemical techniques will help to jointly determine the protein and mRNA localization, as well as provide reliable quantification of the mRNA expression levels. PMID:29238736

Expression of inflammation-related genes in aldosterone-producing adenomas with KCNJ5 mutation.

PubMed

Murakami, Masanori; Yoshimoto, Takanobu; Nakano, Yujiro; Tsuchiya, Kyoichiro; Minami, Isao; Bouchi, Ryotaro; Fujii, Yasuhisa; Nakabayashi, Kazuhiko; Hashimoto, Koshi; Hata, Ken-Ichiro; Kihara, Kazunori; Ogawa, Yoshihiro

2016-08-05

The adrenocortical cells have been shown to produce various inflammatory cytokines such as TNFα and IL-6, which could modulate steroidogenesis. However, the role of inflammatory cytokines in aldosterone-producing adenomas (APAs) is not fully understood. In the present study, we examined the relationships between mRNA expression levels of the inflammation-related genes and somatic mutations in APA tissues. We evaluated mRNA expression levels of TNFA, IL6, and NFKB1 in APA tissues obtained from 44 Japanese APA patients. We revealed that mRNA expression patterns of the inflammation-related genes depended on a KCNJ5 somatic mutation. In addition, we showed that mRNA expression levels of the inflammation-related genes correlated with those of the steroidogenic enzyme CYP11B1 in the patients with APAs. The present study documented for the first time the expression of inflammation-related genes in APAs and the correlation of their expression levels with the KCNJ5 mutation status and mRNA expression levels of steroidogenic enzymes, indicating the pathophysiological relevance of inflammation-related genes in APAs. Copyright © 2016 Elsevier Inc. All rights reserved.

Epidermal growth factor (EGF) receptor-ligand based molecular staging predicts prognosis in head and neck squamous cell carcinoma partly due to deregulated EGF- induced amphiregulin expression.

PubMed

Gao, Jian; Ulekleiv, Camilla H; Halstensen, Trond S

2016-09-26

Increased expression of epidermal growth factor receptor (EGFR) and its ligands is associated with poor prognosis and chemoresistance in many carcinoma types, but its role in head and neck squamous cell carcinoma (HNSCC) is unclear. Our aim was to clarify whether mRNA expression of EGFR-ligands was linked to prognosis and cisplatin resistance, and if so, which ligand was most important and how was the expression regulated. To examine the prognostic effect of EGFR-ligand expression, we analyzed tumorous mRNA expression in 399 HNSCC patients. The intracellular signaling pathways controlling epidermal growth factor (EGF)-induced amphiregulin (AREG) expression were examined in three oral squamous cell carcinoma (OSCC) cell lines. Effect of AREG on cisplatin resistance was examined by viability assays in four-, and by association in 11 OSCC cell lines. The patients were divided into five groups according to the median mRNA expression levels of four EGFR ligands, i.e. AREG, EGF, heparin-binding EGF-like growth factor (HBEGF) and beta-cellulin (BTC). The number of increased-expressed EGFR-ligands were progressively correlated to five-year survival, even in advanced TNM-stage IV patients, where five-year mortality increased from 26 % if tumor expressed none to one EGFR-ligand, to 45 % in three to four ligand expressing tumors. Thus, staging the tumor according to these EGFR-ligand mRNA expression pattern completely out performed TNM staging in predicting prognosis. Multivariate analysis identified AREG as the dominating predictor, and AREG was overexpressed in OSCC compared to tumors from other sites. Both EGF and HBEGF stimulation induced strong AREG increase in OSCC cell lines, which was partially mediated by the extracellular signal-regulated kinase 1/2 pathway, and negatively regulated by p38, c-Jun N-terminal kinase, and phosphoinositide-3 kinase. Although increased AREG mRNA expression predicted unfavorable prognosis in platinum treated HNSCC patients, AREG did not mediate cisplatin resistance in the OSCC cell lines. Increased tumorous mRNA expression of four EGFR ligands was progressively associated with poor prognosis in HNSCC. Thus, EGFR-ligands mRNA expression pattern may be a new prognostic biomarker. The tightly regulated EGF-induced AREG mRNA expression was partly lost in the OSCC cell lines and restoring its regulation may be a new target in cancer treatment. Not applicable as the clinical data of the 498 HNSCC patients and their mRNA expression profiles were collected from the open TCGA database: http://cancergenome.nih.gov/cancersselected/headandneck .

Compartmentalization today

Treesearch

Kevin T. Smith

2006-01-01

For more than 30 years, the compartmentdization concept has helped tree care practitioners and land managers interpret patterns of decay in living trees. Understanding these patterns can help guide the selection of treatments that meet the needs of people and communities while respecting the underlying tree biology. At its simplest, compartmentalization resists the...

Mitochondrial Lysates Induce Inflammation and Alzheimer’s Disease-Relevant Changes in Microglial and Neuronal Cells

PubMed Central

Wilkins, Heather M.; Carl, Steven M.; Weber, Sam G.; Ramanujan, Suruchi A.; Festoff, Barry W.; Linseman, Daniel A.; Swerdlow, Russell H.

2015-01-01

Neuroinflammation occurs in AD. While AD genetic studies implicate inflammation-relevant genes and fibrillar amyloid β protein promotes inflammation, our understanding of AD neuroinflammation nevertheless remains incomplete. In this study we hypothesized damage-associated molecular pattern (DAMP) molecules arising from mitochondria, intracellular organelles that resemble bacteria, could contribute to AD neuroinflammation. To preliminarily test this possibility, we exposed neuronal and microglial cell lines to enriched mitochondrial lysates. BV2 microglial cells treated with mitochondrial lysates showed decreased TREM2 mRNA, increased TNFα mRNA, increased MMP-8 mRNA, increased IL-8 mRNA, redistribution of NFκB to the nucleus, and increased p38 MAPK phosphorylation. SH-SY5Y neuronal cells treated with mitochondrial lysates showed increased TNFα mRNA, increased NFκB protein, decreased IκBα protein, increased AβPP mRNA, and increased AβPP protein. Enriched mitochondrial lysates from SH-SY5Y cells lacking detectable mitochondrial DNA (ρ0 cells) failed to induce any of these changes, while mtDNA obtained directly from mitochondria (but not PCR-amplified mtDNA) increased BV2 cell TNFα mRNA. These results indicate at least one mitochondrial-derived DAMP molecule, mtDNA, can induce inflammatory changes in microglial and neuronal cell lines. Our data are consistent with the hypothesis that a mitochondrial-derived DAMP molecule or molecules could contribute to AD neuroinflammation. PMID:25537010

Stool metatranscriptomics: A technical guideline for mRNA stabilisation and isolation.

PubMed

Reck, Michael; Tomasch, Jürgen; Deng, Zhiluo; Jarek, Michael; Husemann, Peter; Wagner-Döbler, Irene

2015-07-04

The complex microbiome of the gut has an enormous impact on human health. Analysis of the transcriptional activity of microorganisms through mRNA sequencing (metatranscriptomics) opens a completely new window into their activity in vivo, but it is highly challenging due to numerous technical and bioinformatical obstacles. Here we present an optimized pipeline for extraction of high quality mRNA from stool samples. Comparison of three commercially available RNA extraction kits with the method of Zoetendal revealed that the Powermicrobiome Kit (MoBio) performed best with respect to RNA yield and purity. Next, the influence of the stabilization reagent during sample storage for up to 15 days was studied. RIN analysis and qRT-PCR of spiked-in and indigenous genes revealed that RNA Later preserved mRNA integrity most efficiently, while samples conserved in RNA Protect showed substantial mRNA decay. Using the optimized pipeline developed here, recovery rates for spiked-in E.coli cells expressing fluorescing proteins were 8.7-9.7% for SuperfolderGFP and 14.7-17.8% for mCherry. The mRNA of stabilized stool samples as well as of snap-frozen controls was sequenced with Illumina Hiseq, yielding on average 74 million reads per sample. PCoA analysis, taxonomic classification using Kraken and functional classification using bwa showed that the transcriptomes of samples conserved in RNA Later were unchanged for up to 6 days even at room temperature, while RNA Protect was inefficient for storage durations exceeding 24 h. However, our data indicate that RNA Later introduces a bias which is then maintained throughout storage, while RNA Protect conserved samples are initially more similar to the snap frozen controls. RNA Later conserved samples had a reduced abundance of e.g. Prevotellaceae transcripts and were depleted for e.g. COG category "Carbohydrate transport and metabolism". Since the overall similarity between all stool transcriptional profiles studied here was >0.92, these differences are unlikely to affect global comparisons, but should be taken into account when rare but critically important members of the stool microbiome are being studied.

HuR binding to cytoplasmic mRNA is perturbed by heat shock

PubMed Central

Gallouzi, Imed-Eddine; Brennan, Christopher M.; Stenberg, Myrna G.; Swanson, Maurice S.; Eversole, Ashley; Maizels, Nancy; Steitz, Joan A.

2000-01-01

AU-rich elements (AREs) located in the 3′ untranslated region target the mRNAs encoding many protooncoproteins, cytokines, and lymphokines for rapid degradation. HuR, a ubiquitously expressed member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins, binds ARE sequences and selectively stabilizes ARE-containing reporter mRNAs when overexpressed in transiently transfected cells. HuR appears predominantly nucleoplasmic but has been shown to shuttle between the nucleus and cytoplasm via a novel shuttling sequence HNS. We report generation of a mouse monoclonal antibody 3A2 that both immunoblots and immunoprecipitates HuR protein; it recognizes an epitope located in the first of HuR's three RNA recognition motifs. This antibody was used to probe HuR interactions with mRNA before and after heat shock, a condition that has been reported to stabilize ARE-containing mRNAs. At 37°C, approximately one-third of the cytoplasmic HuR appears polysome associated, and in vivo UV crosslinking reveals that HuR interactions with poly(A)+ RNA are predominantly cytoplasmic rather than nuclear. This comprises evidence that HuR directly interacts with mRNA in vivo. After heat shock, 12–15% of HuR accumulates in discrete foci in the cytoplasm, but surprisingly the majority of HuR crosslinks instead to nuclear poly(A)+ RNA, whose levels are dramatically increased in the stressed cells. This behavior of HuR differs from that of another ARE-binding protein, hnRNP D, which has been implicated as an effector of mRNA decay rather than mRNA stabilization and of the general pre-RNA-binding protein hnRNP A1. We interpret these differences to mean that the temporal association of HuR with ARE-containing mRNAs is different from that of these other two proteins. PMID:10737787

The orientation distribution of tunneling-related quantities

NASA Astrophysics Data System (ADS)

Seif, W. M.; Refaie, A. I.; Botros, M. M.

2018-03-01

In the nuclear tunneling processes involving deformed nuclei, most of the tunneling-related quantities depend on the relative orientations of the participating nuclei. In the presence of different multipole deformations, we study the variation of a few relevant quantities for the α-decay and the sub-barrier fusion processes, in an orientation degree of freedom. The knocking frequency and the penetration probability are evaluated within the Wentzel-Kramers-Brillouin approximation. The interaction potential is calculated with Skyrme-type nucleon-nucleon interaction. We found that the width of the potential pocket, the Coulomb barrier radius, the penetration probability, the α-decay width, and the fusion cross-section follow consistently the orientation-angle variation of the radius of the deformed nucleus. The orientation distribution patterns of the pocket width, the barrier radius, the logarithms of the penetrability, the decay width, and the fusion cross-section are found to be highly analogous to pattern of the deformed-nucleus radius. The curve patterns of the orientation angle distributions of the internal pocket depth, the Coulomb barrier height and width, as well as the knocking frequency simulate inversely the variation of the deformed nucleus radius. The predicted orientation behaviors will be of a special interest in predicting the optimum orientations for the tunneling processes.

Regional co-location pattern scoping on a street network considering distance decay effects of spatial interaction

PubMed Central

Yu, Wenhao

2017-01-01

Regional co-location scoping intends to identify local regions where spatial features of interest are frequently located together. Most of the previous researches in this domain are conducted on a global scale and they assume that spatial objects are embedded in a 2-D space, but the movement in urban space is actually constrained by the street network. In this paper we refine the scope of co-location patterns to 1-D paths consisting of nodes and segments. Furthermore, since the relations between spatial events are usually inversely proportional to their separation distance, the proposed method introduces the “Distance Decay Effects” to improve the result. Specifically, our approach first subdivides the street edges into continuous small linear segments. Then a value representing the local distribution intensity of events is estimated for each linear segment using the distance-decay function. Each kind of geographic feature can lead to a tessellated network with density attribute, and the generated multiple networks for the pattern of interest will be finally combined into a composite network by calculating the co-location prevalence measure values, which are based on the density variation between different features. Our experiments verify that the proposed approach is effective in urban analysis. PMID:28763496

A photo guide to the patterns of discoloration and decay in living northern hardwood trees

Treesearch

Alex L. Shigo; Edwin vH. Larson

1969-01-01

This guide has been prepared to help you estimate the extent and severity of discoloration and decay in northern hardwood trees. Though this certainly is not the last word on the subject, our research in this has progressed to the point where our findings, combined with the findings of others, can be put to practical use. By dissecting living trees and studying the...

Formation of a three-dimensional plasma boundary after decay of the plasma response to resonant magnetic perturbation fields

NASA Astrophysics Data System (ADS)

Schmitz, O.; Evans, T. E.; Fenstermacher, M. E.; Lanctot, M. J.; Lasnier, C. L.; Mordijck, S.; Moyer, R. A.; Reimerdes, H.; the DIII-D Team

2014-01-01

First time experimental evidence is presented for a direct link between the decay of a n = 3 plasma response and the formation of a three-dimensional (3D) plasma boundary. We inspect a lower single-null L-mode plasma which first reacts at sufficiently high rotation with an ideal resonant screening response to an external toroidal mode number n = 3 resonant magnetic perturbation field. Decay of this response due to reduced bulk plasma rotation changes the plasma state considerably. Signatures such as density pump out and a spin up of the edge rotation—which are usually connected to formation of a stochastic boundary—are detected. Coincident, striation of the divertor single ionized carbon emission and a 3D emission structure in double ionized carbon at the separatrix is seen. The striated C II pattern follows in this stage the perturbed magnetic footprint modelled without a plasma response (vacuum approach). This provides for the first time substantial experimental evidence, that a 3D plasma boundary with direct impact on the divertor particle flux pattern is formed as soon as the internal plasma response decays. The resulting divertor structure follows the vacuum modelled magnetic field topology. However, the inward extension of the perturbed boundary layer can still not directly be determined from these measurements.

Cell type-dependent gene regulation by Staufen2 in conjunction with Upf1.

PubMed

Miki, Takashi; Kamikawa, Yasunao; Kurono, Sadamu; Kaneko, Yuka; Katahira, Jun; Yoneda, Yoshihiro

2011-11-16

dendritic mRNA transport machines. Although Stau2 is thought to be involved in the dendritic targeting of several mRNAs in neurons, the mechanism whereby Stau2 regulates these mRNAs is unknown. To elucidate the functions of Stau2, we screened for novel binding partners by affinity purification of GST-tagged Stau2 from 293F cells. Three RNA helicases, RNA helicase A, Upf1 and Mov10, were identified in Stau2-containing complexes. We focused our studies on Upf1, a key player in nonsense-mediated mRNA decay. Stau2 was found to bind directly to Upf1 in an RNA-independent manner in vitro. Tethering Stau2 to the 3'-untranslated region (UTR) of a reporter gene had little effect on its expression in HeLa cells. In contrast, when the same tethering assay was performed in 293F cells, we observed an increase in reporter protein levels. This upregulation of protein expression by Stau2 turned out to be dependent on Upf1. Moreover, we found that in 293F cells, Stau2 upregulates the reporter mRNA level in an Upf1-independent manner. These results indicate that the recruitment of Stau2 alone or in combination with Upf1 differentially affects the fate of mRNAs. Moreover, the results suggest that Stau2-mediated fate determination could be executed in a cell type-specific manner.

Interaction of RNA-binding protein HuR and miR-466i regulates GM-CSF expression.

PubMed

Chen, Jing; Adamiak, William; Huang, Ganlei; Atasoy, Ulus; Rostami, Abdolmohamad; Yu, Shiguang

2017-12-08

Granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by T helper 17 (Th17) cells plays an essential role in autoimmune diseases. Transcriptional regulation of Th17 cell differentiation has been extensively studied, but post-transcriptional regulation of Th17 cell differentiation has remained less well characterized. The RNA-binding protein HuR functions to promote the stability of target mRNAs via binding the AU-rich elements of the 3' untranslated region (3'UTR) of numerous pro-inflammatory cytokines including IL-4, IL-13, IL-17 and TNF-α. However, whether HuR regulates GM-CSF expression in Th17 cells has not been fully investigated. Here we showed that HuR conditional knockout (KO) Th17 cells have decreased GM-CSF mRNA in comparison with wild-type (WT) Th17 cells, and that HuR binds directly to GM-CSF mRNA 3'UTR. Interestingly, HuR deficiency increased the levels of certain microRNA expression in Th17 cells; for example, miR-466i functioned to mediate GM-CSF and IL-17 mRNA decay, which was confirmed by in vitro luciferase assay. Furthermore, we found that HuR promoted Mxi1 expression to inhibit certain miRNA expression. Taken together, these findings indicate that interaction of HuR and miR-466i orchestrates GM-CSF expression in Th17 cells.

Detection of novel NF1 mutations and rapid mutation prescreening with Pyrosequencing.

PubMed

Brinckmann, Anja; Mischung, Claudia; Bässmann, Ingelore; Kühnisch, Jirko; Schuelke, Markus; Tinschert, Sigrid; Nürnberg, Peter

2007-12-01

Neurofibromatosis type 1 (NF1) is caused by mutations in the neurofibromin (NF1) gene. Mutation analysis of NF1 is complicated by its large size, the lack of mutation hotspots, pseudogenes and frequent de novo mutations. Additionally, the search for NF1 mutations on the mRNA level is often hampered by nonsense-mediated mRNA decay (NMD) of the mutant allele. In this study we searched for mutations in a cohort of 38 patients and investigated the relationship between mutation type and allele-specific transcription from the wild-type versus mutant alleles. Quantification of relative mRNA transcript numbers was done by Pyrosequencing, a novel real-time sequencing method whose signals can be quantified very accurately. We identified 21 novel mutations comprising various mutation types. Pyrosequencing detected a definite relationship between allelic NF1 transcript imbalance due to NMD and mutation type in 24 of 29 patients who all carried frame-shift or nonsense mutations. NMD was absent in 5 patients with missense and silent mutations, as well as in 4 patients with splice-site mutations that did not disrupt the reading frame. Pyrosequencing was capable of detecting NMD even when the effects were only moderate. Diagnostic laboratories could thus exploit this effect for rapid prescreening for NF1 mutations as more than 60% of the mutations in this gene disrupt the reading frame and are prone to NMD.

Functional Analyses of c.2268dup in Thyroid Peroxidase Gene Associated with Goitrous Congenital Hypothyroidism

PubMed Central

Harun, Fatimah; Jalaludin, Muhammad Yazid; Lim, Chor Yin; Ng, Khoon Leong

2014-01-01

The c.2268dup mutation in thyroid peroxidase (TPO) gene was reported to be a founder mutation in Taiwanese patients with dyshormonogenetic congenital hypothyroidism (CH). The functional impact of the mutation is not well documented. In this study, homozygous c.2268dup mutation was detected in two Malaysian-Chinese sisters with goitrous CH. Normal and alternatively spliced TPO mRNA transcripts were present in thyroid tissues of the two sisters. The abnormal transcript contained 34 nucleotides originating from intron 12. The c.2268dup is predicted to generate a premature termination codon (PTC) at position 757 (p.Glu757X). Instead of restoring the normal reading frame, the alternatively spliced transcript has led to another stop codon at position 740 (p.Asp739ValfsX740). The two PTCs are located at 116 and 201 nucleotides upstream of the exons 13/14 junction fulfilling the requirement for a nonsense-mediated mRNA decay (NMD). Quantitative RT-PCR revealed an abundance of unidentified transcripts believed to be associated with the NMD. TPO enzyme activity was not detected in both patients, even though a faint TPO band of about 80 kD was present. In conclusion, the c.2268dup mutation leads to the formation of normal and alternatively spliced TPO mRNA transcripts with a consequential loss of TPO enzymatic activity in Malaysian-Chinese patients with goitrous CH. PMID:24745015

Insulin-like growth factors I and II in starry flounder (Platichthys stellatus): molecular cloning and differential expression during embryonic development.

PubMed

Xu, Yongjiang; Zang, Kun; Liu, Xuezhou; Shi, Bao; Li, Cunyu; Shi, Xueying

2015-02-01

In order to elucidate the possible roles of insulin-like growth factors I and II (IGF-I and IGF-II) in the embryonic development of Platichthys stellatus, their cDNAs were isolated and their spatial expression pattern in adult organs and temporal expression pattern throughout embryonic development were examined by quantitative real-time PCR assay. The IGF-I cDNA sequence was 1,268 bp in length and contained an open reading frame (ORF) of 558 bp, which encoded 185 amino acid residues. With respect to IGF-II, the full-length cDNA was 899 bp in length and contained a 648-bp ORF, which encoded 215 amino acid residues. The amino acid sequences of IGF-I and IGF-II exhibited high identities with their fish counterparts. The highest IGF-I mRNA level was found in the liver for both sexes, whereas the IGF-II gene was most abundantly expressed in female liver and male liver, gill, and brain. The sex-specific and spatial expression patterns of IGF-I and IGF-II mRNAs are thought to be related to the sexually dimorphic growth and development of starry flounder. Both IGF-I and IGF-II mRNAs were detected in unfertilized eggs, which indicated that IGF-I and IGF-II were parentally transmitted. Nineteen embryonic development stages were tested. IGF-I mRNA level remained high from unfertilized eggs to low blastula followed by a significant decrease at early gastrula and then maintained a lower level. In contrast, IGF-II mRNA level was low from unfertilized eggs to high blastula and peaked at low blastula followed by a gradual decrease. Moreover, higher levels of IGF-I mRNA than that of IGF-II were found from unfertilized eggs to high blastula, vice versa from low blastula to newly hatched larva, and the different expression pattern verified the differential roles of IGF-I and IGF-II in starry flounder embryonic development. These results could help in understanding the endocrine mechanism involved in the early development and growth of starry flounder.

UPF1 Governs Synaptic Plasticity through Association with a STAU2 RNA Granule.

PubMed

Graber, Tyson E; Freemantle, Erika; Anadolu, Mina N; Hébert-Seropian, Sarah; MacAdam, Robyn L; Shin, Unkyung; Hoang, Huy-Dung; Alain, Tommy; Lacaille, Jean-Claude; Sossin, Wayne S

2017-09-20

Neuronal mRNAs can be packaged in reversibly stalled polysome granules before their transport to distant synaptic locales. Stimulation of synaptic metabotropic glutamate receptors (mGluRs) reactivates translation of these particular mRNAs to produce plasticity-related protein; a phenomenon exhibited during mGluR-mediated LTD. This form of plasticity is deregulated in Fragile X Syndrome, a monogenic form of autism in humans, and understanding the stalling and reactivation mechanism could reveal new approaches to therapies. Here, we demonstrate that UPF1, known to stall peptide release during nonsense-mediated RNA decay, is critical for assembly of stalled polysomes in rat hippocampal neurons derived from embryos of either sex. Moreover, UPF1 and its interaction with the RNA binding protein STAU2 are necessary for proper transport and local translation from a prototypical RNA granule substrate and for mGluR-LTD in hippocampal neurons. These data highlight a new, neuronal role for UPF1, distinct from its RNA decay functions, in regulating transport and/or translation of mRNAs that are critical for synaptic plasticity. SIGNIFICANCE STATEMENT The elongation and/or termination steps of mRNA translation are emerging as important control points in mGluR-LTD, a form of synaptic plasticity that is compromised in a severe monogenic form of autism, Fragile X Syndrome. Deciphering the molecular mechanisms controlling this type of plasticity may thus open new therapeutic opportunities. Here, we describe a new role for the ATP-dependent helicase UPF1 and its interaction with the RNA localization protein STAU2 in mediating mGluR-LTD through the regulation of mRNA translation complexes stalled at the level of elongation and/or termination. Copyright © 2017 the authors 0270-6474/17/379116-16$15.00/0.

Classification and machine recognition of severe weather patterns

NASA Technical Reports Server (NTRS)

Wang, P. P.; Burns, R. C.

1976-01-01

Forecasting and warning of severe weather conditions are treated from the vantage point of pattern recognition by machine. Pictorial patterns and waveform patterns are distinguished. Time series data on sferics are dealt with by considering waveform patterns. A severe storm patterns recognition machine is described, along with schemes for detection via cross-correlation of time series (same channel or different channels). Syntactic and decision-theoretic approaches to feature extraction are discussed. Active and decayed tornados and thunderstorms, lightning discharges, and funnels and their related time series data are studied.

Spectroscopy of excited states of unbound nuclei 30Ar and 29Cl

NASA Astrophysics Data System (ADS)

Xu, X.-D.; Mukha, I.; Grigorenko, L. V.; Scheidenberger, C.; Acosta, L.; Casarejos, E.; Chudoba, V.; Ciemny, A. A.; Dominik, W.; Duénas-Díaz, J.; Dunin, V.; Espino, J. M.; Estradé, A.; Farinon, F.; Fomichev, A.; Geissel, H.; Golubkova, T. A.; Gorshkov, A.; Janas, Z.; Kamiński, G.; Kiselev, O.; Knöbel, R.; Krupko, S.; Kuich, M.; Litvinov, Yu. A.; Marquinez-Durán, G.; Martel, I.; Mazzocchi, C.; Nociforo, C.; Ordúz, A. K.; Pfützner, M.; Pietri, S.; Pomorski, M.; Prochazka, A.; Rymzhanova, S.; Sánchez-Benítez, A. M.; Sharov, P.; Simon, H.; Sitar, B.; Slepnev, R.; Stanoiu, M.; Strmen, P.; Szarka, I.; Takechi, M.; Tanaka, Y. K.; Weick, H.; Winkler, M.; Winfield, J. S.

2018-03-01

Several states of proton-unbound isotopes 30Ar and 29Cl were investigated by measuring their in-flight decay products, 28S + proton + proton and 28S + proton, respectively. A refined analysis of 28S-proton angular correlations indicates that the ground state of 30Ar is located at 2 .45-0.10+0.05 MeV above the two-proton emission threshold. The investigation of the decay mechanism of the 30Ar ground state demonstrates that it has the transition dynamics. In the "transitional" region, the correlation patterns of the decay products present a surprisingly strong sensitivity to the two-proton decay energy of the 30Ar ground state and the one-proton decay energy as well as the one-proton decay width of the 29Cl ground state. The comparison of the experimental 28S-proton angular correlations with those resulting from Monte Carlo simulations of the detector response illustrates that other observed 30Ar excited states decay by sequential emission of protons via intermediate resonances in 29Cl. Based on the findings, the decay schemes of the observed states in 30Ar and 29Cl were constructed. For calibration purposes and for checking the performance of the experimental setup, decays of the previously known states of a two-proton emitter 19Mg were remeasured. Evidences for one new excited state in 19Mg and two unknown states in 18Na were found.

CsrA Participates in a PNPase Autoregulatory Mechanism by Selectively Repressing Translation of pnp Transcripts That Have Been Previously Processed by RNase III and PNPase

PubMed Central

Park, Hongmarn; Yakhnin, Helen; Connolly, Michael; Romeo, Tony

2015-01-01

ABSTRACT Csr is a conserved global regulatory system that represses or activates gene expression posttranscriptionally. CsrA of Escherichia coli is a homodimeric RNA binding protein that regulates transcription elongation, translation initiation, and mRNA stability by binding to the 5′ untranslated leader or initial coding sequence of target transcripts. pnp mRNA, encoding the 3′ to 5′ exoribonuclease polynucleotide phosphorylase (PNPase), was previously identified as a CsrA target by transcriptome sequencing (RNA-seq). Previous studies also showed that RNase III and PNPase participate in a pnp autoregulatory mechanism in which RNase III cleavage of the untranslated leader, followed by PNPase degradation of the resulting 5′ fragment, leads to pnp repression by an undefined translational repression mechanism. Here we demonstrate that CsrA binds to two sites in pnp leader RNA but only after the transcript is fully processed by RNase III and PNPase. In the absence of processing, both of the binding sites are sequestered in an RNA secondary structure, which prevents CsrA binding. The CsrA dimer bridges the upstream high-affinity site to the downstream site that overlaps the pnp Shine-Dalgarno sequence such that bound CsrA causes strong repression of pnp translation. CsrA-mediated translational repression also leads to a small increase in the pnp mRNA decay rate. Although CsrA has been shown to regulate translation and mRNA stability of numerous genes in a variety of organisms, this is the first example in which prior mRNA processing is required for CsrA-mediated regulation. IMPORTANCE CsrA protein represses translation of numerous mRNA targets, typically by binding to multiple sites in the untranslated leader region preceding the coding sequence. We found that CsrA represses translation of pnp by binding to two sites in the pnp leader transcript but only after it is processed by RNase III and PNPase. Processing by these two ribonucleases alters the mRNA secondary structure such that it becomes accessible to the ribosome for translation as well as to CsrA. As one of the CsrA binding sites overlaps the pnp ribosome binding site, bound CsrA prevents ribosome binding. This is the first example in which regulation by CsrA requires prior mRNA processing and should link pnp expression to conditions affecting CsrA activity. PMID:26438818

Macromolecular Crowding Induces Spatial Correlations That Control Gene Expression Bursting Patterns

DOE PAGES

Norred, Sarah Elizabeth; Caveney, Patrick M.; Chauhan, Gaurav; ...

2018-04-24

Recent superresolution microscopy studies in E. coli demonstrate that the cytoplasm has highly variable local concentrations where macromolecular crowding plays a central role in establishing membrane-less compartmentalization. This spatial inhomogeneity significantly influences molecular transport and association processes central to gene expression. Yet, little is known about how macromolecular crowding influences gene expression bursting—the episodic process where mRNA and proteins are produced in bursts. Here, we simultaneously measured mRNA and protein reporters in cell-free systems, showing that macromolecular crowding decoupled the well-known relationship between fluctuations in the protein population (noise) and mRNA population statistics. Crowded environments led to a 10-fold increasemore » in protein noise even though there were only modest changes in the mRNA population and fluctuations. Instead, cell-like macromolecular crowding created an inhomogeneous spatial distribution of mRNA (“spatial noise”) that led to large variability in the protein production burst size. As a result, the mRNA spatial noise created large temporal fluctuations in the protein population. Furthermore, these results highlight the interplay between macromolecular crowding, spatial inhomogeneities, and the resulting dynamics of gene expression, and provide insights into using these organizational principles in both cell-based and cell-free synthetic biology.« less

Macromolecular Crowding Induces Spatial Correlations That Control Gene Expression Bursting Patterns

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

Norred, Sarah Elizabeth; Caveney, Patrick M.; Chauhan, Gaurav

Recent superresolution microscopy studies in E. coli demonstrate that the cytoplasm has highly variable local concentrations where macromolecular crowding plays a central role in establishing membrane-less compartmentalization. This spatial inhomogeneity significantly influences molecular transport and association processes central to gene expression. Yet, little is known about how macromolecular crowding influences gene expression bursting—the episodic process where mRNA and proteins are produced in bursts. Here, we simultaneously measured mRNA and protein reporters in cell-free systems, showing that macromolecular crowding decoupled the well-known relationship between fluctuations in the protein population (noise) and mRNA population statistics. Crowded environments led to a 10-fold increasemore » in protein noise even though there were only modest changes in the mRNA population and fluctuations. Instead, cell-like macromolecular crowding created an inhomogeneous spatial distribution of mRNA (“spatial noise”) that led to large variability in the protein production burst size. As a result, the mRNA spatial noise created large temporal fluctuations in the protein population. Furthermore, these results highlight the interplay between macromolecular crowding, spatial inhomogeneities, and the resulting dynamics of gene expression, and provide insights into using these organizational principles in both cell-based and cell-free synthetic biology.« less

Exotic decays of the 125 GeV Higgs boson

DOE PAGES

Curtin, David; Essig, Rouven; Gori, Stefania; ...

2014-10-13

We perform an extensive survey of nonstandard Higgs decays that are consistent with the 125 GeV Higgs-like resonance. Our aim is to motivate a large set of new experimental analyses on the existing and forthcoming data from the Large Hadron Collider (LHC). The explicit search for exotic Higgs decays presents a largely untapped discovery opportunity for the LHC collaborations, as such decays may be easily missed by other searches. We emphasize that the Higgs is uniquely sensitive to the potential existence of new weakly coupled particles and provide a unified discussion of a large class of both simplified and completemore » models that give rise to characteristic patterns of exotic Higgs decays. We assess the status of exotic Higgs decays after LHC run I. In many cases we are able to set new nontrivial constraints by reinterpreting existing experimental analyses. We point out that improvements are possible with dedicated analyses and perform some preliminary collider studies. As a result, we prioritize the analyses according to their theoretical motivation and their experimental feasibility.« less

Patterns of gene expression in atrophying skeletal muscles: response to food deprivation

NASA Technical Reports Server (NTRS)

Jagoe, R. Thomas; Lecker, Stewart H.; Gomes, Marcelo; Goldberg, Alfred L.

2002-01-01

During fasting and many systemic diseases, muscle undergoes rapid loss of protein and functional capacity. To define the transcriptional changes triggering muscle atrophy and energy conservation in fasting, we used cDNA microarrays to compare mRNAs from muscles of control and food-deprived mice. Expression of >94% of genes did not change, but interesting patterns emerged among genes that were differentially expressed: 1) mRNAs encoding polyubiquitin, ubiquitin extension proteins, and many (but not all) proteasome subunits increased, which presumably contributes to accelerated protein breakdown; 2) a dramatic increase in mRNA for the ubiquitin ligase, atrogin-1, but not most E3s; 3) a significant suppression of mRNA for myosin binding protein H (but not other myofibrillar proteins) and IGF binding protein 5, which may favor cell protein loss; 4) decreases in mRNAs for several glycolytic enzymes and phosphorylase kinase subunits, and dramatic increases in mRNAs for pyruvate dehydrogenase kinase 4 and glutamine synthase, which should promote glucose sparing and gluconeogenesis. During fasting, metallothionein mRNA increased dramatically, mRNAs for extracellular matrix components fell, and mRNAs that may favor cap-independent mRNA translation rose. Significant changes occurred in mRNAs for many growth-related proteins and transcriptional regulators. These transcriptional changes indicate a complex adaptive program that should favor protein degradation and suppress glucose oxidation in muscle. Similar analysis of muscles atrophying for other causes is allowing us to identify a set of atrophy-specific changes in gene expression.

Differential Accumulation of Sunflower Tetraubiquitin mRNAs during Zygotic Embryogenesis and Developmental Regulation of Their Heat-Shock Response.

PubMed Central

Almoguera, C.; Coca, M. A.; Jordano, J.

1995-01-01

We have isolated and sequenced Ha UbiS, a cDNA for a dry-seed-stored mRNA that encodes tetraubiquitin. We have observed differential accumulation of tetraubiquitin mRNAs during sunflower (Helianthus annuus L.) zygotic embryogenesis. These mRNAs were up-regulated during late embryogenesis and reached higher prevalence in the dry seed, where they were found to be associated mainly with provascular tissue. UbiS mRNA, as confirmed by Rnase A protection experiments, accumulated also in response to heat shock, but only in leaves and later during postgerminative development. These novel observations demonstrate expression during seed maturation of specific plant polyubiquitin transcripts and developmental regulation of their heat-shock response. Using ubiquitin antibodies we also detected discrete, seed-specific proteins with distinct temporal expression patterns during zygotic embryogenesis. Some of these patterns were concurrent with UbiS mRNA accumulation in seeds. The most abundant ubiquitin-reacting proteins found in mature seeds were small (16-22 kD) and acidic (isoelectric points of 6.1-7.4). Possible functional implications for UbiS expression elicited from these observations are discussed. PMID:12228401

The G protein-coupled estrogen receptor 1 (GPER/GPR30) does not predict survival in patients with ovarian cancer

PubMed Central

2012-01-01

Background Even though ovarian tumors are not generally considered estrogen-sensitive, estrogens may still have an impact on ovarian tumor progression. The recently identified trans-membrane estrogen receptor GPER is involved in rapid estrogen signaling. Furthermore, it binds selective estrogen receptor modulators with agonistic effect, which could explain tamoxifen controversies. Methods GPER mRNA was assayed with quantitative real-time PCR (qPCR) in 42 primary ovarian tumors and 7 ovarian cancer cell lines. ERα and ERβ mRNA were analyzed for comparison. GPER protein was semi-quantified with densitometric scanning of Western blots and its tissue distribution analyzed with immunohistochemistry (IHC) in 40 ovarian tumors. In addition, IHC was evaluated in a tissue microarray (TMA) of 150 primary malignant ovarian tumors. Results All tumor samples contained GPER mRNA. The content of mRNA was not different between benign and malignant tumors, but one third of malignant samples over-expressed GPER mRNA. The content of ERα mRNA was higher in malignant than in benign tumors, whereas ERβ mRNA was higher in benign than in malignant tumors. GPER mRNA was detected in all seven ovarian cancer cell lines with highest levels in TOV21G and TOV112D cells. Similar expression pattern was seen for ERβ mRNA. Western blot demonstrated GPER protein in all tumor samples. Semi-quantification showed no difference between benign and malignant tumors, but about one third of malignant samples over-expressed GPER protein. GPER staining was localized mainly in epithelial cells. In the TMA study we found no correlation between GPER staining and clinical stage, histological grade or patient survival. Conclusions GPER mRNA as well as GPER protein is present in both benign and malignant ovarian tumor tissue. About one third of malignant tumors over-expressed both GPER mRNA and protein. This, however, correlated neither with histological or clinical parameters nor with patient survival. PMID:22424333

Multiple lipopolysaccharide (LPS) injections alter interleukin 6 (IL-6), IL-7, IL-10 and IL-6 and IL-7 receptor mRNA in CNS and spleen.

PubMed

Szot, Patricia; Franklin, Allyn; Figlewicz, Dianne P; Beuca, Timothy Petru; Bullock, Kristin; Hansen, Kim; Banks, William A; Raskind, Murray A; Peskind, Elaine R

2017-07-04

Neuroinflammation is proposed to be an important component in the development of several central nervous system (CNS) disorders including depression, Alzheimer's disease, Parkinson's disease, and traumatic brain injury. However, exactly how neuroinflammation leads to, or contributes to, these central disorders is unclear. The objective of the study was to examine and compare the expression of mRNAs for interleukin-6 (IL-6), IL-7, IL-10 and the receptors for IL-6 (IL-6R) and IL-7 (IL-7R) using in situ hybridization in discrete brain regions and in the spleen after multiple injections of 3mg/kg lipopolysaccharide (LPS), a model of neuroinflammation. In the spleen, LPS significantly elevated IL-6 mRNA expression, then IL-10 mRNA, with no effect on IL-7 or IL-7R mRNA, while significantly decreasing IL-6R mRNA expression. In the CNS, LPS administration had the greatest effect on IL-6 and IL-6R mRNA. LPS increased IL-6 mRNA expression only in non-neuronal cells throughout the brain, but significantly elevated IL-6R mRNA in neuronal populations, where observed, except the cerebellum. LPS resulted in variable effects on IL-10 mRNA, and had no effect on IL-7 or IL-7R mRNA expression. These studies indicate that LPS-induced neuroinflammation has substantial but variable effects on the regional and cellular patterns of CNS IL-6, IL-7 and IL-10, and for IL-6R and IL-7R mRNA expression. It is apparent that administration of LPS can affect non-neuronal and neuronal cells in the brain. Further research is required to determine how CNS inflammatory changes associated with IL-6, IL-10 and IL-6R could in turn contribute to the development of CNS neurological disorders. Published by Elsevier Ltd.

Hypoxia inducible factor 1 links fast-patterned muscle activity and fast muscle phenotype in rats.

PubMed

Lunde, Ida G; Anton, Siobhan L; Bruusgaard, Jo C; Rana, Zaheer A; Ellefsen, Stian; Gundersen, Kristian

2011-03-15

Exercise influences muscle phenotype by the specific pattern of action potentials delivered to the muscle, triggering intracellular signalling pathways. PO2 can be reduced by an order of magnitude in working muscle. In humans, carriers of a hyperactive polymorphism of the transcription factor hypoxia inducible factor 1α (HIF-1α) have 50% more fast fibres, and this polymorphism is prevalent among strength athletes. We have investigated the putative role of HIF-1α in mediating activity changes in muscle.When rat muscles were stimulated with short high frequency bursts of action potentials known to induce a fast muscle phenotype, HIF-1α increased by about 80%. In contrast, a pattern consisting of long low frequency trains known to make fast muscles slow reduced the HIF-1α level of the fast extensor digitorum longus (EDL) muscle by 44%. Nuclear protein extracts from normal EDL contained 2.3-fold more HIF-1α and 4-fold more HIF-1β than the slow soleus muscle, while von-Hippel-Lindau protein was 4.8-fold higher in slow muscles. mRNA displayed a reciprocal pattern; thus FIH-1 mRNA was almost 2-fold higher in fast muscle, while the HIF-1α level was half, and consequently protein/mRNA ratio for HIF-1α was more than 4-fold higher in the fast muscle, suggesting that HIF-1α is strongly suppressed post-transcriptionally in slow muscles.When HIF-1α was overexpressed for 14 days after somatic gene transfer in adult rats, a slow-to-fast transformation was observed, encompassing an increase in fibre cross sectional area, oxidative enzyme activity and myosin heavy chain. The latter was shown to be regulated at the mRNA level in C2C12 myotubes.

A 17-month time course study of human RNA and DNA degradation in body fluids under dry and humid environmental conditions.

PubMed

Sirker, Miriam; Schneider, Peter M; Gomes, Iva

2016-11-01

Blood, saliva, and semen are some of the forensically most relevant biological stains commonly found at crime scenes, which can often be of small size or challenging due to advanced decay. In this context, it is of great importance to possess reliable knowledge about the effects of degradation under different environmental conditions and to use appropriate methods for retrieving maximal information from limited sample amount. In the last decade, RNA analysis has been demonstrated to be a reliable approach identifying the cell or tissue type of an evidentiary body fluid trace. Hence, messenger RNA (mRNA) profiling is going to be implemented into forensic casework to supplement the routinely performed short tandem repeat (STR) analysis, and therefore, the ability to co-isolate RNA and DNA from the same sample is a prerequisite. The objective of this work was to monitor and compare the degradation process of both nucleic acids for human blood, saliva, and semen stains at three different concentrations, exposed to dry and humid conditions during a 17-month time period. This study also addressed the question whether there are relevant differences in the efficiency of automated, magnetic bead-based single DNA or RNA extraction methods compared to a manually performed co-extraction method using silica columns. Our data show that mRNA, especially from blood and semen, can be recovered over the entire time period surveyed without compromising the success of DNA profiling; mRNA analysis indicates to be a robust and reliable technique to identify the biological source of aged stain material. The co-extraction method appears to provide mRNA and DNA of sufficient quantity and quality for all different forensic investigation procedures. Humidity and accompanied mold formation are detrimental to both nucleic acids.

Functional characterization of the novel intronic nucleotide change c.288+9C>T within the BCKDHA gene: understanding a variant presentation of maple syrup urine disease.

PubMed

Fernández-Guerra, Paula; Navarrete, Rosa; Weisiger, Kara; Desviat, Lourdes R; Packman, Seymour; Ugarte, Magdalena; Rodríguez-Pombo, Pilar

2010-12-01

Mutations in any of the three different genes--BCKDHA, BCKDHB, and DBT--encoding for the E1α, E1β, and E2 catalytic components of the branched-chain α-ketoacid dehydrogenase complex can cause maple syrup urine disease (MSUD). Disease severity ranges from the classic to the mildest variant types and precise genotypes, mostly based on missense mutations, have been associated to the less severe presentations of the disease. Herein, we examine the consequences at the messenger RNA (mRNA) level of the novel intronic alteration c.288+9C>T found in heterozygous fashion in a BCKDHA variant MSUD patient who also carries the nucleotide change c.745G>A (p.Gly249Ser), previously described as a severe change. Direct analysis of the processed transcripts from the patient showed--in addition to a low but measurable level of normal mRNA product--an aberrantly spliced mRNA containing a 7-bp fragment of intron 2, which could be rescued when the patient's cells were treated with emetine. This aberrant transcript with a premature stop codon would be unstable, supporting the possible activation of nonsense-mediated mRNA decay pathway. Consistent with this finding, minigene splicing assays demonstrated that the point mutation c.288+9C>T is sufficient to create a cryptic splice site and cause the observed 7-bp insertion. Furthermore, our results strongly suggest that the c.288+9C>T allele in the patient generates both normal and aberrant transcripts that could sustain the variant presentation of the disease, highlighting the importance of correct genotyping to establish genotype-phenotype correlations and as basis for the development of therapeutic interventions.

Preprotachykinin A mRNA expression in the rat brain during development.

PubMed

Brené, S; Lindefors, N; Friedman, W J; Persson, H

1990-12-15

Expression of preprotachykinin A (PPT-A) mRNA was analyzed by northern blots using mRNA prepared from rat brain at 12 different developmental stages ranging from embryonic day 15 (E15) to adult. A single PPT-A mRNA of 1.3 kb was detected throughout development. PPT-A mRNA was detected as early as E15 and an approximately 3-fold increase occurred at birth. This amount remained until 3 weeks of age when the level increased, reaching a peak at 5 weeks of age. Adult amounts were approximately 3-fold higher than the levels at birth. The distribution of PPT-A mRNA-expressing cells in rat brain was studied by in situ hybridization on sections from embryonic day 20, postnatal days 4 and 7 as well as adult. Cells expressing PPT-A mRNA were detected in the forebrain at all 4 ages analyzed. However, the hybridization pattern and the labeling intensity varied in different brain regions during development. In cingulate cortex, intense labeling was seen in numerous cells at embryonic day 20 and postnatal days 4 and 7, whereas in the adult cingulate cortex only a few scattered labeled cells were observed. In frontoparietal cortex labeled cells were found from postnatal day 4 to adult, with the highest density of labeled cells at P7. Developmental differences in both the distribution of PPT-A mRNA-expressing cells and the level of PPT-A mRNA expression were also found in caudate-putamen, lateral hypothalamus and amygdala. Thus, our results show several changes in PPT-A mRNA expression during ontogeny, indicating a region and time-specific regulation of PPT-A mRNA expression during brain maturation.

Expression of a GALACTINOL SYNTHASE Gene in Tomato Seeds Is Up-Regulated before Maturation Desiccation and Again after Imbibition whenever Radicle Protrusion Is Prevented1

PubMed Central

Downie, Bruce; Gurusinghe, Sunitha; Dahal, Petambar; Thacker, Richard R.; Snyder, John C.; Nonogaki, Hiroyuki; Yim, Kyuock; Fukanaga, Keith; Alvarado, Veria; Bradford, Kent J.

2003-01-01

Raffinose family oligosaccharides (RFOs) have been implicated in mitigating the effects of environmental stresses on plants. In seeds, proposed roles for RFOs include protecting cellular integrity during desiccation and/or imbibition, extending longevity in the dehydrated state, and providing substrates for energy generation during germination. A gene encoding galactinol synthase (GOLS), the first committed enzyme in the biosynthesis of RFOs, was cloned from tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds, and its expression was characterized in tomato seeds and seedlings. GOLS (LeGOLS-1) mRNA accumulated in developing tomato seeds concomitant with maximum dry weight deposition and the acquisition of desiccation tolerance. LeGOLS-1 mRNA was present in mature, desiccated seeds but declined within 8 h of imbibition in wild-type seeds. However, LeGOLS-1 mRNA accumulated again in imbibed seeds prevented from completing germination by dormancy or water deficit. Gibberellin-deficient (gib-1) seeds maintained LeGOLS-1 mRNA amounts after imbibition unless supplied with gibberellin, whereas abscisic acid (ABA) did not prevent the loss of LeGOLS-1 mRNA from wild-type seeds. The presence of LeGOLS-1 mRNA in ABA-deficient (sitiens) tomato seeds indicated that wild-type amounts of ABA are not necessary for its accumulation during seed development. In all cases, LeGOLS-1 mRNA was most prevalent in the radicle tip. LeGOLS-1 mRNA accumulation was induced by dehydration but not by cold in germinating seeds, whereas both stresses induced LeGOLS-1 mRNA accumulation in seedling leaves. The physiological implications of LeGOLS-1 expression patterns in seeds and leaves are discussed in light of the hypothesized role of RFOs in plant stress tolerance. PMID:12644684

Profiles of mRNA expression of related genes in the duck hypothalamus-pituitary growth axis during embryonic and early post-hatch development.

PubMed

Hu, Yan; Liu, Hongxiang; Song, Chi; Xu, Wenjuan; Ji, Gaige; Zhu, Chunhong; Shu, Jingting; Li, Huifang

2015-03-15

In this study, the ontogeny of body and liver weight and the pattern of related gene mRNA expression in the hypothalamus-pituitary growth axis (HPGA) of two different duck breeds (Anas platyrhynchos domestica) were compared during embryonic and post-hatch development. Duck hypothalamic growth hormone release hormone (GHRH), somatostatin (SS), pituitary growth hormone (GH), liver growth hormone receptor (GHR) and insulin-like growth factor-I (IGF-1) mRNA were first detected on the 13th embryonic day. During early duck development, SS maintained a lower expression status, whereas the other four genes exhibited highly significant variations in an age-specific manner. Highly significant breed specificity was observed with respect to hepatic IGF-1 mRNA expression, which showed a significant breed-age interaction effect. Compared with previous studies on chickens, significant species differences were observed regarding the mRNA expression of bird embryonic HPGA-related genes. During early development, highly significant breed and age specificity were observed with respect to developmental changes in body and liver weight, and varying degrees of significant linear correlation were found between these performances and the mRNA expression of HPGA-related genes in the duck HPGA. These results suggest that different genetic backgrounds may lead to differences in duck growth and HPGA-related gene mRNA expression, and the differential mRNA expression of related genes in the duck HPGA may be particularly important in the early growth of ducks. Furthermore, hepatic IGF-1 mRNA expression presented highly significant breed specificity, and evidence suggests the involvement of hepatic IGF-1 in mediating genetic effects on embryo and offspring growth in ducks. Copyright © 2015 Elsevier B.V. All rights reserved.

Overlapping regional distribution of CCK and TPPII mRNAs in Cynomolgus monkey brain and correlated levels in human cerebral cortex (BA 10).

PubMed

Radu, Diana; Tomkinson, Birgitta; Zachrisson, Olof; Weber, Günther; de Belleroche, Jacqueline; Hirsch, Steven; Lindefors, Nils

2006-08-09

Tripeptidyl peptidase II (TPPII) is a high molecular weight exopeptidase important in inactivating extracellular cholecystokinin (CCK). Our aims were to study the anatomical localization of TPPII and CCK mRNA in the Cynomolgus monkey brain as a basis for a possible functional anatomical connection between enzyme (TPPII) and substrate (CCK) and examine if indications of changes in substrate availability in the human brain might be reflected in changes of levels of TPPII mRNA. mRNA in situ hybridization on postmortem brain from patients having had a schizophrenia diagnosis as compared to controls and on monkey and rat brain slices. overlapping distribution patterns of mRNAs for TPPII and CCK in rat and monkey. High amounts of TPPII mRNA are seen in the neocortex, especially in the frontal region and the hippocampus. TPPII mRNA is also present in the basal ganglia and cerebellum where CCK immunoreactivity and/or CCK B receptors have been found in earlier studies, suggesting presence of CCK-ergic afferents from other brain regions. Levels of mRNAs for CCK and TPPII show a positive correlation in postmortem human cerebral cortex Brodmann area (BA) 10. TPPII mRNA might be affected following schizophrenia. overall TPPII and CCK mRNA show a similar distribution in rat and monkey brain, confirming and extending earlier studies in rodents. In addition, correlated levels of TPPII and CCK mRNA in human BA 10 corroborate a functional link between CCK and TPPII in the human brain.

Effect of body mass and clothing on decomposition of pig carcasses.

PubMed

Matuszewski, Szymon; Konwerski, Szymon; Frątczak, Katarzyna; Szafałowicz, Michał

2014-11-01

Carcass mass and carcass clothing are factors of potential high forensic importance. In casework, corpses differ in mass and kind or extent of clothing; hence, a question arises whether methods for post-mortem interval estimation should take these differences into account. Unfortunately, effects of carcass mass and clothing on specific processes in decomposition and related entomological phenomena are unclear. In this article, simultaneous effects of these factors are analysed. The experiment followed a complete factorial block design with four levels of carcass mass (small carcasses 5-15 kg, medium carcasses 15.1-30 kg, medium/large carcasses 35-50 kg, large carcasses 55-70 kg) and two levels of carcass clothing (clothed and unclothed). Pig carcasses (N = 24) were grouped into three blocks, which were separated in time. Generally, carcass mass revealed significant and frequently large effects in almost all analyses, whereas carcass clothing had only minor influence on some phenomena related to the advanced decay. Carcass mass differently affected particular gross processes in decomposition. Putrefaction was more efficient in larger carcasses, which manifested itself through earlier onset and longer duration of bloating. On the other hand, active decay was less efficient in these carcasses, with relatively low average rate, resulting in slower mass loss and later onset of advanced decay. The average rate of active decay showed a significant, logarithmic increase with an increase in carcass mass, but only in these carcasses on which active decay was driven solely by larval blowflies. If a blowfly-driven active decay was followed by active decay driven by larval Necrodes littoralis (Coleoptera: Silphidae), which was regularly found in medium/large and large carcasses, the average rate showed only a slight and insignificant increase with an increase in carcass mass. These results indicate that lower efficiency of active decay in larger carcasses is a consequence of a multi-guild and competition-related pattern of this process. Pattern of mass loss in large and medium/large carcasses was not sigmoidal, but rather exponential. The overall rate of decomposition was strongly, but not linearly, related to carcass mass. In a range of low mass decomposition rate increased with an increase in mass, then at about 30 kg, there was a distinct decrease in rate, and again at about 50 kg, the rate slightly increased. Until about 100 accumulated degree-days larger carcasses gained higher total body scores than smaller carcasses. Afterwards, the pattern was reversed; moreover, differences between classes of carcasses enlarged with the progress of decomposition. In conclusion, current results demonstrate that cadaver mass is a factor of key importance for decomposition, and as such, it should be taken into account by decomposition-related methods for post-mortem interval estimation.

Wood decay: a submicroscopic view

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

Blanchette, R.A.

1980-01-01

Typical patterns of decay in softwoods are shown by ultrastructural differences revealed by SEM. Illustrative micrographs are reproduced showing fungi and their effects. Brown rot fungi (e.g. Fomitopsis pinicolor) degrade cellulose leaving a lignin skeleton. White rot fungi (e.g. Coriolus versicolor and Hirschioporus abietinus) degrade both lignin and cellulose. White pocket rots (e.g. Phellinus pini) primarily degrade lignin; they have potential for use in paper making, or the production of animal feed.

Expression Profile of Cytokines and Enzymes mRNA in Blood Leukocytes of Dogs with Leptospirosis and Its Associated Pulmonary Hemorrhage Syndrome.

PubMed

Maissen-Villiger, Carla A; Schweighauser, Ariane; van Dorland, H Anette; Morel, Claudine; Bruckmaier, Rupert M; Zurbriggen, Andreas; Francey, Thierry

2016-01-01

Dogs with leptospirosis show similar organ manifestations and disease course as human patients, including acute kidney injury and pulmonary hemorrhage, making this naturally-occurring infection a good animal model for human leptospirosis. Expression patterns of cytokines and enzymes have been correlated with disease manifestations and clinical outcome in humans and animals. The aim of this study was to describe mRNA expression of pro- and anti-inflammatory mediators in canine leptospirosis and to compare it with other renal diseases to identify patterns characterizing the disease and especially its pulmonary form. The mRNA abundance of cytokines (IL-1α, IL-1β, IL-8, IL-10, TNF-α, TGF-β) and enzymes (5-LO, iNOS) was measured prospectively in blood leukocytes from 34 dogs with severe leptospirosis and acute kidney injury, including 22 dogs with leptospirosis-associated pulmonary hemorrhages. Dogs with leptospirosis were compared to 14 dogs with acute kidney injury of other origin than leptospirosis, 8 dogs with chronic kidney disease, and 10 healthy control dogs. Canine leptospirosis was characterized by high 5-LO and low TNF-α expression compared to other causes of acute kidney injury, although the decreased TNF-α expression was also seen in chronic kidney disease. Leptospirosis-associated pulmonary hemorrhage was not characterized by a specific pattern, with only mild changes noted, including increased IL-10 and decreased 5-LO expression on some days in affected dogs. Fatal outcome from pulmonary hemorrhages was associated with low TNF-α, high IL-1β, and high iNOS expression, a pattern possibly expressed also in dogs with other forms of acute kidney injury. The patterns of cytokine and enzyme expression observed in the present study indicate a complex pro- and anti-inflammatory response to the infection with leptospires. The recognition of these signatures may be of diagnostic and prognostic relevance for affected individuals and they may indicate options for newer therapies targeting the identified pathways.

Evidence for decay in verbal short-term memory: a commentary on Berman, Jonides, and Lewis (2009).

PubMed

Campoy, Guillermo

2012-07-01

M. G. Berman, J. Jonides, and R. L. Lewis (2009) adapted the recent-probes task to investigate the causes of forgetting in short-term memory. In 7 experiments, they studied the persistence of memory traces by assessing the level of proactive interference generated by previous-trial items over a range of intertrial intervals. None of the experiments found a reduction in proactive interference over time, which they interpreted as evidence against time-based decay. However, it is possible that decay actually occurred over a shorter time period than was tested in this study, wherein the shortest decay interval was 3,300 ms. By reducing the time scale, the 2 experiments reported in the current commentary revealed a sharp decrease in proactive interference over time, with this reduction reaching a plateau in less than 3 s. This pattern suggests that decay operates in the early stages, whereas subsequent forgetting is likely to be due to interference. 2012 APA, all rights reserved

Obesity- and aging-induced excess of central transforming growth factor-β potentiates diabetic development via an RNA stress response

PubMed Central

Yan, Jingqi; Zhang, Hai; Yin, Ye; Li, Juxue; Tang, Yizhe; Purkayastha, Sudarshana; Li, Lianxi; Cai, Dongsheng

2014-01-01

The brain, in particular the hypothalamus, plays a role in regulating glucose homeostasis; however, it remains unclear if the brain is causally involved in diabetic development. Here, we identified that hypothalamic TGF-β is excessive under conditions of not only obesity but aging, which are two general etiological factors of diabetes. Pharmacological and genetic approaches consistently revealed that brain TGF-β excess caused hyperglycemia and glucose intolerance in a body weight-independent manner. Cell-specific genetic models demonstrated that astrocytes are responsible for brain TGF-β excess, and POMC neurons are crucial for the pro-diabetic effect of TGF-β excess. Mechanistically, TGF-β excess induced hypothalamic RNA stress response to accelerate IκBα mRNA decay, leading to an atypical, mRNA metabolism-driven hypothalamic NF-κB activation which links obesity as well as aging to hypothalamic inflammation. In conclusion, brain TGF-β excess and induction of RNA stress response and hypothalamic inflammation are important for the pro-diabetic effects of obesity or aging. PMID:25086906

Proteomic Analysis of the Arabidopsis Nucleolus Suggests Novel Nucleolar FunctionsD⃞

PubMed Central

Pendle, Alison F.; Clark, Gillian P.; Boon, Reinier; Lewandowska, Dominika; Lam, Yun Wah; Andersen, Jens; Mann, Matthias; Lamond, Angus I.; Brown, John W. S.; Shaw, Peter J.

2005-01-01

The eukaryotic nucleolus is involved in ribosome biogenesis and a wide range of other RNA metabolism and cellular functions. An important step in the functional analysis of the nucleolus is to determine the complement of proteins of this nuclear compartment. Here, we describe the first proteomic analysis of plant (Arabidopsis thaliana) nucleoli, in which we have identified 217 proteins. This allows a direct comparison of the proteomes of an important nuclear structure between two widely divergent species: human and Arabidopsis. The comparison identified many common proteins, plant-specific proteins, proteins of unknown function found in both proteomes, and proteins that were nucleolar in plants but nonnucleolar in human. Seventy-two proteins were expressed as GFP fusions and 87% showed nucleolar or nucleolar-associated localization. In a striking and unexpected finding, we have identified six components of the postsplicing exon-junction complex (EJC) involved in mRNA export and nonsense-mediated decay (NMD)/mRNA surveillance. This association was confirmed by GFP-fusion protein localization. These results raise the possibility that in plants, nucleoli may have additional functions in mRNA export or surveillance. PMID:15496452

Opposing PKA and Hog1 signals control the post-transcriptional response to glucose availability in Cryptococcus neoformans.

PubMed

Banerjee, Dithi; Bloom, Amanda L M; Panepinto, John C

2016-10-01

The pathogenic fungus Cryptococcus neoformans must adapt to glucose-limited conditions in the lung and glucose replete conditions upon dissemination to the brain. We report that glucose controls ribosome biogenesis and translation by modulating mRNA decay through a balance of PKA and Hog1 signalling. Glucose signalling through PKA stabilized ribosomal protein (RP) mRNAs whereas glucose starvation destabilized RP transcripts through Hog1. Glucose starvation-induced oxidative stress response genes, and treatment of glucose-fed cells with reactive oxygen species (ROS) generating compounds repressed RP transcripts, both of which were dependent on Hog1. Stabilization of RP transcripts led to retention of polysomes in a hog1Δ mutant, whereas stabilization of RP transcripts by cyclic AMP did not affect translation repression, suggesting that Hog1 alone signals translation repression. In sum, this work describes a novel antagonism between PKA and Hog1 controlling ribosome biogenesis via mRNA stability in response to glucose availability in this important human pathogen. © 2016 John Wiley & Sons Ltd.

Proteomic Analysis of the Mediator Complex Interactome in Saccharomyces cerevisiae

PubMed Central

Uthe, Henriette; Vanselow, Jens T.; Schlosser, Andreas

2017-01-01

Here we present the most comprehensive analysis of the yeast Mediator complex interactome to date. Particularly gentle cell lysis and co-immunopurification conditions allowed us to preserve even transient protein-protein interactions and to comprehensively probe the molecular environment of the Mediator complex in the cell. Metabolic 15N-labeling thereby enabled stringent discrimination between bona fide interaction partners and nonspecifically captured proteins. Our data indicates a functional role for Mediator beyond transcription initiation. We identified a large number of Mediator-interacting proteins and protein complexes, such as RNA polymerase II, general transcription factors, a large number of transcriptional activators, the SAGA complex, chromatin remodeling complexes, histone chaperones, highly acetylated histones, as well as proteins playing a role in co-transcriptional processes, such as splicing, mRNA decapping and mRNA decay. Moreover, our data provides clear evidence, that the Mediator complex interacts not only with RNA polymerase II, but also with RNA polymerases I and III, and indicates a functional role of the Mediator complex in rRNA processing and ribosome biogenesis. PMID:28240253

Genome-wide screen identifies novel machineries required for both ciliogenesis and cell cycle arrest upon serum starvation

PubMed Central

Kim, Ji Hyun; Ki, Soo Mi; Joung, Je-Gun; Scott, Eric; Heynen-Genel, Susanne; Aza-Blanc, Pedro; Kwon, Chang Hyuk; Kim, Joon; Gleeson, Joseph G.; Lee, Ji Eun

2016-01-01

Biogenesis of the primary cilium, a cellular organelle mediating various signaling pathways, is generally coordinated with cell cycle exit/re-entry. Although the dynamic cell cycle-associated profile of the primary cilium has been largely accepted, the mechanism governing the link between ciliogenesis and cell cycle progression has been poorly understood. Using a human genome-wide RNAi screen, we identify genes encoding subunits of the spliceosome and proteasome as novel regulators of ciliogenesis. We demonstrate that 1) the mRNA processing-related hits are essential for RNA expression of molecules acting in cilia disassembly, such as AURKA and PLK1, and 2) the ubiquitin-proteasome systems (UPS)-involved hits are necessary for proteolysis of molecules acting in cilia assembly, such as IFT88 and CPAP. In particular, we show that these screen hit-associated mechanisms are crucial for both cilia assembly and cell cycle arrest in response to serum withdrawal. Finally, our data suggest that the mRNA processing mechanism may modulate the UPS-dependent decay of cilia assembly regulators to control ciliary resorption-coupled cell cycle re-entry. PMID:27033521

Tilmicosin does not inhibit interleukin-8 gene expression in the bovine lung experimentally infected with Mannheimia (Pasteurella) haemolytica.

PubMed Central

Goubau, S; Morck, D W; Buret, A

2000-01-01

The expression of the interleukin-8 (IL-8) gene was examined by in situ hybridization in lung tissues from calves experimentally infected with Mannheimia (Pasteurella) haemolytica and treated with tilmicosin. Interleukin-8 mRNA expression was detected in alveolar areas, particularly along interlobular septa, in the lumen, and in the epithelial cells of some bronchioles. In lesional lung tissues from animals that had received tilmicosin, we found large areas with limited inflammation. There was no staining for IL-8 mRNA in these areas. In contrast, in strongly inflamed areas, the same patterns and intensities of staining for IL-8 mRNA were detected in tilmicosin- and sham-treated animals. We conclude that tilmicosin does not affect the expression of IL-8 mRNA in tissue showing microscopic signs of inflammation. Together with previous reports, this supports the view that the pro-apoptotic properties of tilmicosin on neutrophils do not compromise the host defense mechanisms required to control the infection. Images Figure 1. PMID:11041503

Influence of culture medium composition on relative mRNA abundances in domestic cat embryos.

PubMed

Hribal, R; Jewgenow, K; Braun, B C; Comizzoli, P

2013-04-01

Different culture conditions have been used to produce domestic cat embryos. As part of the in vitro procedures, the medium composition significantly affects the quality of the embryo development also. Quality assessments based on cleavage kinetics and blastomere symmetry are useful, but embryos also can differ in their relative gene expression patterns despite similar morphological characteristics. The aim of this study was to compare cat embryos produced with two different in vitro culture systems routinely used in two different laboratories [Smithsonian Conservation Biology Institute, Washington D.C., USA (SCBI) and Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany (IZW)]. Specifically, relative mRNA expression patterns of critical genes for pre-implantation embryo development were assessed in both conditions. Embryos were produced in parallel in both culture systems by IVF using frozen-thawed ejaculated semen in the United States and fresh epididymal sperm in Germany. Success of embryo development in vitro was recorded as well as relative mRNA abundances [DNA methyltransferases 1 and 3A (DNMT1, DNMT3A), gap junction protein alpha 1 (GJA1), octamer-binding transcription factor 4 [OCT4], insulin-like growth factors 1 and 2 receptors (IGF1R, IGF2R), beta-actin (ACTB)] in pools of days 4-5 morulae by semi-quantitative RT-PCR assay. Percentages of cleaved embryos were similar (p > 0.05) between both culture systems, regardless of the location. OCT4 mRNA abundance was higher (p < 0.05) in embryos derived in the SCBI culture system compared with those from the IZW system when epididymal sperm was used for IVF. No clear correlation between the expression pattern and the culture system could be found for all other genes. It is suggested that OCT4 expression might be affected by the media composition in some conditions and can be the indicator of a better embryo quality. © 2012 Blackwell Verlag GmbH.

The assembly of ecological communities inferred from taxonomic and functional composition

Treesearch

Eric R. Sokol; E.F. Benfield; Lisa K. Belden; H. Maurice. Valett

2011-01-01

Among-site variation in metacommunities (beta diversity) is typically correlated with the distance separating the sites (spatial lag). This distance decay in similarity pattern has been linked to both niche-based and dispersal-based community assembly hypotheses. Here we show that beta diversity patterns in community composition, when supplemented with functional-trait...

The Growth and Decay of Equatorial Backscatter Plumes.

DTIC Science & Technology

1980-02-01

spatially connected to bottomside backscatter, a feature noted in Jica- marca radar observations that led Woodman and La Hoz (1976) to speculate that...described in Section Ill-B, this pattern of plume growth resembles the "C-shaped" and "fishtail" patterns found in Jica- marca radar RTI displays of 50-MHz

Auditory Pattern Memory: Mechanisms of Tonal Sequence Discrimination by Human Observers

DTIC Science & Technology

1988-10-30

and Creelman (1977) in a study of categorical perception. Tanner’s model included a short-term decaying memory for the acoustic input to the system plus...auditory pattern components, J. &Coust. Soc. 91 Am., 76, 1037- 1044. Macmillan, N. A., Kaplan H. L., & Creelman , C. D. (1977). The psychophysics of

Recognizing the tenascin-X deficient type of Ehlers-Danlos syndrome: a cross-sectional study in 17 patients.

PubMed

Demirdas, S; Dulfer, E; Robert, L; Kempers, M; van Beek, D; Micha, D; van Engelen, B G; Hamel, B; Schalkwijk, J; Loeys, B; Maugeri, A; Voermans, N C

2017-03-01

The tenascin-X (TNX) deficient type Ehlers-Danlos syndrome (EDS) is similar to the classical type of EDS. Because of the limited awareness among geneticists and the challenge of the molecular analysis of the TNXB gene, the TNX-deficient type EDS is probably to be under diagnosed. We therefore performed an observational, cross-sectional study. History and physical examination were performed. Results of serum TNX measurements were collected and mutation analysis was performed by a combination of next-generation sequencing (NGS), Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Included were 17 patients of 11 families with autosomal recessive inheritance and childhood onset. All patients had hyperextensible skin without atrophic scarring. Hypermobility of the joints was observed in 16 of 17 patients. Deformities of the hands and feet were observed frequently. TNX serum level was tested and absent in 11 patients (seven families). Genetic testing was performed in all families; 12 different mutations were detected, most of which are suspected to lead to non-sense mRNA mediated decay. In short, patients with the TNX-deficient type EDS typically have generalized joint hypermobility, skin hyperextensibility and easy bruising. In contrast to the classical type, the inheritance pattern is autosomal recessive and atrophic scarring is absent. Molecular analysis of TNXB in a diagnostic setting is challenging. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

Use of mRNA expression signatures to discover small molecule inhibitors of skeletal muscle atrophy

PubMed Central

Adams, Christopher M.; Ebert, Scott M.; Dyle, Michael C.

2017-01-01

Purpose of review Here, we discuss a recently developed experimental strategy for discovering small molecules with potential to prevent and treat skeletal muscle atrophy. Recent findings Muscle atrophy involves and requires widespread changes in skeletal muscle gene expression, which generate complex but measurable patterns of positive and negative changes in skeletal muscle mRNA levels (a.k.a. mRNA expression signatures of muscle atrophy). Many bioactive small molecules generate their own characteristic mRNA expression signatures, and by identifying small molecules whose signatures approximate mirror images of muscle atrophy signatures, one may identify small molecules with potential to prevent and/or reverse muscle atrophy. Unlike a conventional drug discovery approach, this strategy does not rely on a predefined molecular target but rather exploits the complexity of muscle atrophy to identify small molecules that counter the entire spectrum of pathological changes in atrophic muscle. We discuss how this strategy has been used to identify two natural compounds, ursolic acid and tomatidine, that reduce muscle atrophy and improve skeletal muscle function. Summary Discovery strategies based on mRNA expression signatures can elucidate new approaches for preserving and restoring muscle mass and function. PMID:25807353

Use of mRNA expression signatures to discover small molecule inhibitors of skeletal muscle atrophy.

PubMed

Adams, Christopher M; Ebert, Scott M; Dyle, Michael C

2015-05-01

Here, we discuss a recently developed experimental strategy for discovering small molecules with potential to prevent and treat skeletal muscle atrophy. Muscle atrophy involves and requires widespread changes in skeletal muscle gene expression, which generate complex but measurable patterns of positive and negative changes in skeletal muscle mRNA levels (a.k.a. mRNA expression signatures of muscle atrophy). Many bioactive small molecules generate their own characteristic mRNA expression signatures, and by identifying small molecules whose signatures approximate mirror images of muscle atrophy signatures, one may identify small molecules with potential to prevent and/or reverse muscle atrophy. Unlike a conventional drug discovery approach, this strategy does not rely on a predefined molecular target but rather exploits the complexity of muscle atrophy to identify small molecules that counter the entire spectrum of pathological changes in atrophic muscle. We discuss how this strategy has been used to identify two natural compounds, ursolic acid and tomatidine, that reduce muscle atrophy and improve skeletal muscle function. Discovery strategies based on mRNA expression signatures can elucidate new approaches for preserving and restoring muscle mass and function.

Allocation of Interferon Gamma mRNA Positive Cells in Caecum Hallmarks a Protective Trait Against Histomonosis.

PubMed

Kidane, Fana Alem; Mitra, Taniya; Wernsdorf, Patricia; Hess, Michael; Liebhart, Dieter

2018-01-01

Histomonosis is a parasitic disease of gallinaceous birds characterized by necrotic lesions in cacum and liver that usually turns fatal in turkeys while it is less severe in chickens. Vaccination using in vitro attenuated Histomonas meleagridis has been experimentally shown to confer protection against histomonosis. The protective mechanisms that underpin the vaccine-induced immune response are not resolved so far. Therefore, the actual study aimed to evaluate the location and quantitative distribution patterns of signature cytokines of type 1 [interferon gamma (IFN-γ)] or type 2 [interleukin (IL)-13] immune responses in vaccinated or infected hosts. An intergroup and interspecies difference in the spatial and temporal distribution patterns of cytokine mRNA positive cells was evident. Quantification of cells showed a significantly decreased percentage of IFN-γ mRNA positive cells at 4 days post-inoculation (DPI) in caeca of turkeys inoculated exclusively with the attenuated or the virulent inocula, compared to control birds. The decrement was followed by a surge of cells expressing mRNA for IFN-γ or IL-13, reaching a peak of increment at 10 DPI. By contrast, turkeys challenged following vaccination showed a slight increment of cecal IFN-γ mRNA positive cells at 4 DPI after which positive cell counts became comparable to control birds. The increase in infected birds was accompanied by an extensive distribution of positively stained cells up to the muscularis layer of cecal tissue whereas the vaccine group maintained an intact mucosal structure. In chickens, the level of changes of positive cells was generally lower compared to turkeys. However, control chickens were found with a higher percentage of IFN-γ mRNA positive cells in cecum compared to their turkey counterparts indicating a higher resistance to histomonosis, similar to the observation in immunized turkeys. In chickens, it could be shown that the changes of cytokine-positive cells were related to variations of mononuclear cells quantified by immunofluorescence. Furthermore, gene expression measured by reverse transcription quantitative real time PCR confirmed variations in organs between the different groups of both bird species. Overall, it can be concluded that a proportionally increased, yet controlled, allocation of IFN-γ mRNA positive cells in caeca hallmarks a protective trait against histomonosis.

L-leucine, beta-hydroxy-beta-methylbutyric acid (HMB) and creatine monohydrate prevent myostatin-induced Akirin-1/Mighty mRNA down-regulation and myotube atrophy.

PubMed

Mobley, Christopher Brooks; Fox, Carlton D; Ferguson, Brian S; Amin, Rajesh H; Dalbo, Vincent J; Baier, Shawn; Rathmacher, John A; Wilson, Jacob M; Roberts, Michael D

2014-01-01

The purpose of this study was to examine if L-leucine (Leu), β-hydroxy-β-methylbutyrate (HMB), or creatine monohydrate (Crea) prevented potential atrophic effects of myostatin (MSTN) on differentiated C2C12 myotubes. After four days of differentiation, myotubes were treated with MSTN (10 ng/ml) for two additional days and four treatment groups were studied: 1) 3x per day 10 mM Leu, 2) 3x per day 10 mM HMB, 3) 3x per day 10 mM Crea, 4) DM only. Myotubes treated with DM without MSTN were analyzed as the control condition (DM/CTL). Following treatment, cells were analyzed for total protein, DNA content, RNA content, muscle protein synthesis (MPS, SUnSET method), and fiber diameter. Separate batch treatments were analyzed for mRNA expression patterns of myostatin-related genes (Akirin-1/Mighty, Notch-1, Ski, MyoD) as well as atrogenes (MuRF-1, and MAFbx/Atrogin-1). MSTN decreased fiber diameter approximately 30% compared to DM/CTL myotubes (p < 0.001). Leu, HMB and Crea prevented MSTN-induced atrophy. MSTN did not decrease MPS levels compared to DM/CTL myotubes, but MSTN treatment decreased the mRNA expression of Akirin-1/Mighty by 27% (p < 0.001) and MyoD by 26% (p < 0.01) compared to DM/CTL myotubes. shRNA experiments confirmed that Mighty mRNA knockdown reduced myotube size, linking MSTN treatment to atrophy independent of MPS. Remarkably, MSTN + Leu and MSTN + HMB myotubes had similar Akirin-1/Mighty and MyoD mRNA levels compared to DM/CTL myotubes. Furthermore, MSTN + Crea myotubes exhibited a 36% (p < 0.05) and 86% (p < 0.001) increase in Akirin-1/Mighty mRNA compared to DM/CTL and MSTN-only treated myotubes, respectively. Leu, HMB and Crea may reduce MSTN-induced muscle fiber atrophy by influencing Akirin-1/Mighty mRNA expression patterns. Future studies are needed to examine if Leu, HMB and Crea independently or synergistically affect Akirin-1/Mighty expression, and how Akirin-1/Mighty expression mechanistically relates to skeletal muscle hypertrophy in vivo.

L-leucine, beta-hydroxy-beta-methylbutyric acid (HMB) and creatine monohydrate prevent myostatin-induced Akirin-1/Mighty mRNA down-regulation and myotube atrophy

PubMed Central

2014-01-01

Background The purpose of this study was to examine if L-leucine (Leu), β-hydroxy-β-methylbutyrate (HMB), or creatine monohydrate (Crea) prevented potential atrophic effects of myostatin (MSTN) on differentiated C2C12 myotubes. Methods After four days of differentiation, myotubes were treated with MSTN (10 ng/ml) for two additional days and four treatment groups were studied: 1) 3x per day 10 mM Leu, 2) 3x per day 10 mM HMB, 3) 3x per day 10 mM Crea, 4) DM only. Myotubes treated with DM without MSTN were analyzed as the control condition (DM/CTL). Following treatment, cells were analyzed for total protein, DNA content, RNA content, muscle protein synthesis (MPS, SUnSET method), and fiber diameter. Separate batch treatments were analyzed for mRNA expression patterns of myostatin-related genes (Akirin-1/Mighty, Notch-1, Ski, MyoD) as well as atrogenes (MuRF-1, and MAFbx/Atrogin-1). Results MSTN decreased fiber diameter approximately 30% compared to DM/CTL myotubes (p < 0.001). Leu, HMB and Crea prevented MSTN-induced atrophy. MSTN did not decrease MPS levels compared to DM/CTL myotubes, but MSTN treatment decreased the mRNA expression of Akirin-1/Mighty by 27% (p < 0.001) and MyoD by 26% (p < 0.01) compared to DM/CTL myotubes. shRNA experiments confirmed that Mighty mRNA knockdown reduced myotube size, linking MSTN treatment to atrophy independent of MPS. Remarkably, MSTN + Leu and MSTN + HMB myotubes had similar Akirin-1/Mighty and MyoD mRNA levels compared to DM/CTL myotubes. Furthermore, MSTN + Crea myotubes exhibited a 36% (p < 0.05) and 86% (p < 0.001) increase in Akirin-1/Mighty mRNA compared to DM/CTL and MSTN-only treated myotubes, respectively. Conclusions Leu, HMB and Crea may reduce MSTN-induced muscle fiber atrophy by influencing Akirin-1/Mighty mRNA expression patterns. Future studies are needed to examine if Leu, HMB and Crea independently or synergistically affect Akirin-1/Mighty expression, and how Akirin-1/Mighty expression mechanistically relates to skeletal muscle hypertrophy in vivo. PMID:25132809

Differential neonatal imprinting and regulation by estrogen of estrogen receptor subtypes alpha and beta and of the truncated estrogen receptor product (TERP-1) mRNA expression in the male rat pituitary.

PubMed

Tena-Sempere, M; Barreiro, M L; González, L C; Pinilla, L; Aguilar, E

2001-11-01

Two distinct nuclear estrogen receptors (ERs) have been identified, the classical one, renamed ERalpha, and the more recently cloned ERbeta. In a variety of tissues, gene expression of both receptor subtypes results in the generation of multiple transcripts encoding the full-length as well as several alternately spliced isoforms. In the rat pituitary, a truncated, tissue-specific variant of ERalpha, called TERP-1, has been identified and found able to modulate ERalpha and ERbeta activity. So far, its pattern of expression and hormonal regulation have been mostly studied in females. The present study was designed to analyze the pattern of expression of TERP-1 mRNA in the male rat pituitary at different stages of postnatal development, and to evaluate the impact of neonatal imprinting and estrogen treatment upon TERP-1 expression in the male pituitary. Assessment of TERP-1 mRNA levels by semi-quantitative RT-PCR, using a variant-specific primer pair, revealed that TERP-1 is also expressed in the male rat pituitary. Relative mRNA expression levels changed markedly during postnatal development, with moderate expression of the TERP-1 transcript at birth, barely detectable levels during the infantile-prepubertal period, and maximal values in adulthood. Expression of TERP-1 was sensitive to neonatal estrogen exposure, which resulted in a significant, persistent increase in mRNA levels from the infantile period until puberty. This phenomenon was not mimicked by neonatal blockade of endogenous GnRH. In addition, estrogen was able to acutely up-regulate pituitary TERP-1 mRNA expression levels in prepubertal (30-day-old) and adult (75-day-old) males. Interestingly, neonatal imprinting as well as acute estrogen treatment resulted in opposite effects on TERP-1 and full-length ERalpha and ERbeta transcripts, the latter being decreased under both conditions. In conclusion, our data indicate that TERP-1 mRNA is expressed in a developmentally regulated manner in the male rat pituitary, and is affected by neonatal estrogen imprinting and acute estrogen treatment. Regulation of TERP-1 expression by neonatal or acute estrogen treatment may thus represent an additional tuning mechanism for estrogen actions in the male rat pituitary. Copyright 2001 S. Karger AG, Basel

[Positioning of mRNA 3' of the a site bound codon on the human 80S ribosome].

PubMed

Molotkov, M V; Graĭfer, D M; Demeshkina, N A; Repkova, M N; Ven'iaminova, A G; Karpova, G G

2005-01-01

Short mRNA analogues carrying a UUU triplet at the 5'-termini and a perfluorophenylazide group at either the N7 atom of the guanosine or the C5 atom of the uridine 3' of the triplet were applied to study positioning of mRNA 3' of the A site codon. Complexes of 80S ribosomes with the mRNA analogues were obtained in the presence of tRNAPhe that directed UUU codon to the P site and consequently provided placement of the nucleotide with cross-linker in positions +9 or +12 with respect to the first nucleotide of the P site bound codon. Both types mRNA analogues cross-linked to the 18S rRNA and 40S proteins under mild UV-irradiation. Cross-linking patterns in the complexes where modified nucleotides of the mRNA analogues were in position +7 were analyzed for comparison (cross-linking to the 18S rRNA in such complexes has been studied previously). The efficiency of cross-linking to the ribosomal components depended on the nature of the modified nucleotide in the mRNA analogue and its position on the ribosome, extent of cross-linking to the 18S rRNA being decreased drastically when the modified nucleotide was moved from position +7 to position +12. The nucleotides of 18S rRNA cross-linked to mRNA analogues were determined. Modified nucleotides in positions +9 and +12 cross-linked to the invariant dinucleotide A1824/A1825 and to variable A1823 in the 3'-minidomain of 18S rRNA as well as to protein S15. The same ribosomal components have been found earlier to cross-link to modified mRNA nucleotides in positions from +4 to +7. Besides, all mRNA analogues cross-linked to the invariant nucleotide c1698 in the 3'-minidomain and to and the conserved region 605-620 closing helix 18 in the 5'-domain.

Mir-17-3p Controls Spinal Neural Progenitor Patterning by Regulating Olig2/Irx3 Cross-repressive Loop

PubMed Central

Chen, Jun-An; Huang, Yuan-Ping; Mazzoni, Esteban O.; Tan, G. Christopher; Zavadil, Jiri; Wichterle, Hynek

2011-01-01

SUMMARY Neural patterning relies on transcriptional cross-repressive interactions that ensure unequivocal assignment of neural progenitor identity to proliferating cells. Progenitors of spinal motor neurons (pMN) and V2 interneurons (p2) are specified by a pair of cross-repressive transcription factors Olig2 and Irx3. Lineage tracing revealed that many p2 progenitors transiently express the pMN marker Olig2 during spinal cord development. Here we demonstrate that the repression of Olig2 in p2 domain is controlled by mir-17-3p microRNA-mediated silencing of Olig2 mRNA. Mice lacking all microRNAs or just the mir-17~92 cluster manifest a dorsal shift in pMN/p2 boundary and impairment in the production of V2 interneurons. Our findings suggest that microRNA-mediated repression of Olig2 mRNA plays a critical role during the patterning of ventral spinal progenitor domains by shifting the balance of cross-repressive interactions between Olig2 and Irx3 transcription factors. PMID:21338882

Non-Smad TGF-β signaling components are possible biomarkers of tamoxifen resistance

NASA Astrophysics Data System (ADS)

Babyshkina, N.; Zavyalova, M.; Patalyak, S.; Dronova, T.; Slonimskaya, E.; Cherdyntseva, N.

2017-09-01

A crosstalk between the estrogen receptor alpha (ERα) and tyrosine kinase receptors contribute to endocrine resistance. We investigated the effect of the four Smad-independent TGF-β signaling components and the distribution pattern of ERα expression on the response to adjuvant tamoxifen treatment in 122 estrogen positive breast cancer patients. We identified a low mRNA expression of TGF-βR1 in tamoxifen resistant group patients (TR) in contrast to tamoxifen sensitive group (TS). Similarly, negative TGF-βR1 expression was significantly higher in TR patients than in TS patients. The expression of TGF-βR1 was strongly correlated with the distribution pattern of ERα expression, level of CD44+/CD24-/low cells and Akt (pS473) expression. The patients with a low mRNA expression of TGF-βR1 as well as with a negative TGF-βR1 expression had an unfavorable prognosis concerning progression-free survival. The expression of TGF-βR1 and the distribution pattern of ERα expression can be considered as additional molecular predictive markers for estrogen positive breast cancer patients treated with adjuvant tamoxifen.

Biodegradation kinetics of dissolved organic matter chromatographic fractions in an intermittent river

NASA Astrophysics Data System (ADS)

Catalán, N.; Casas-Ruiz, J. P.; von Schiller, D.; Proia, L.; Obrador, B.; Zwirnmann, E.; Marcé, R.

2017-01-01

Controls on the degradation of dissolved organic matter (DOM) are complex but key to understand the role of freshwaters in the carbon cycle. Both the origin and previous degradation history have been suggested to determine DOM reactivity, but it is still a major challenge to understand the links between DOM composition and biodegradation kinetics. An appropriate context to study these links are intermittent rivers, as summer drought naturally diversifies DOM sources and sinks. Here we investigated the biodegradation kinetics of DOM in the main aquatic environments present in a temporary river. During dark incubations we traced the dynamics of bulk DOM and its main chromatographic fractions defined using LC-OCD: high molecular weight substances (HMWS), low molecular weight substances (LMWS), and humic substances and building blocks. Bulk DOM decay patterns were successfully fitted to the reactivity continuum (RC) biodegradation model. The RC parameters depicted running waters as the sites presenting a more reactive DOM, and temporary pools, enriched in leaf litter, as the ones with slowest DOM decay. The decay patterns of each DOM fraction were consistent throughout sites. LMWS and HMWS decayed in all cases and could be modeled using the RC model. Notably, the dynamics of LMWS controlled the bulk DOM kinetics. We discuss the mechanistic basis for the chromatographic fractions' kinetics during biodegradation and the implications that preconditioning and summer drought can have for DOM biodegradation in intermittent rivers.

The SMN1 common variant c.22 dupA in Chinese patients causes spinal muscular atrophy by nonsense-mediated mRNA decay in humans.

PubMed

Bai, JinLi; Qu, YuJin; Cao, YanYan; Yang, Lan; Ge, Lin; Jin, YuWei; Wang, Hong; Song, Fang

2018-02-20

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is mostly caused by homozygous deletion of the SMN1 gene. Approximately 5%-10% of SMA patients are believed to have SMN1 variants. c.22 dupA (p.Ser8lysfs*23) has been identified as the most frequent variant in the Chinese SMA population and to be associated with a severe phenotype. However, the exact molecular mechanism of the variant on the pathogenesis of SMA is unclear. We observed that SMN1 mRNA and the SMN protein in the peripheral blood cells of a patient with c.22 dupA were lower than those of controls. The aim of this study is to investigate whether nonsense-mediated mRNA decay (NMD) plays a role in the mechanism of the c.22 dupA variant of the SMN1 gene as it causes SMA. Two lymphoblasts cell lines from two patients (patient 1 and 2) with the c.22 dupA, and one dermal fibroblasts cell line from patient 2 were included in our study. Two-stage validation of the NMD mechanism was supplied. We first measured the changes in the transcript levels of the SMN1 gene by real-time quantitative PCR after immortalized B-lymphoblasts and dermal fibroblasts cells of the SMA patients were treated with inhibitors of the NMD pathway, including puromycin and cyclohemide. Next, lentivirus-mediated knockdown of the key NMD factor-Up-frameshift protein 1 (UPF1)-was performed in the fibroblasts cell line to further clarify whether the variant led to NMD, as UPF1 recognizes abnormally terminated transcripts as NMD substrates during translation. SC35 1.7-kb transcripts, a physiological NMD substrate was determined to be a NMD positive gene in our experiments. The two inhibitors resulted in a dramatic escalation of the levels of the full-length SMN1 (fl-SMN1) transcripts. Additionally, the SC35 1.7-kb mRNA levels were also increased, suggesting that NMD pathway is suppressed by the two inhibitors. For the 3 cell lines, the fold increase of the SMN1 transcript levels of cycloheximide ranged from 2.5±0.4 to 8.3±0.1, 1.9±0.2 to 5.0±0.7 and 2.2±0.1 to 4.9±0.2 for two lymphoblastoid cell lines and one fibroblasts cell line, respectively. For these cell lines, the fold increases of the SMN1 transcript levels of puromycin were as follows: 5.5±0.2 to 19.5±4.0, 3.1±0.3 to 9.9±1.8 and 1.5±0.2 to 6.5±0.5. Meanwhile, the SC35 1.7-kb transcript levels were markedly increased in all 3 cell lines. In addition, lentivirus-mediated UPF1 knockdown lead to a reduction of the UPF1 protein level to 22.5% compared to the negative control lentivirus. Additionally, knockdown of the UPF1 gene also promoted mRNA expression of the SC35 1.7kb and fl-SMN1 genes. The increases of the SMN1 and SC35 1.7-kb mRNA levels reached about 4- and 6.5-fold in fibroblasts derived from the patient 2, respectively. Altogether, our study provides the first evidence that the c.22 dupA variant in the SMN1 gene triggers NMD. SMA pathogenesis in the patient is associated with mRNA degradation of SMN1, but not the truncated SMN protein. Copyright © 2017. Published by Elsevier B.V.

Differential regulation of duplicate light-dependent protochlorophyllide oxidoreductases in the diatom Phaeodactylum tricornutum

DOE PAGES

Hunsperger, Heather M.; Ford, Christopher J.; Miller, James S.; ...

2016-07-01

Diatoms (Bacilliariophyceae) encode two light-dependent protochlorophyllide oxidoreductases (POR1 and POR2) that catalyze the penultimate step of chlorophyll biosynthesis in the light. Algae live in dynamic environments whose changing light levels induce photoacclimative metabolic shifts, including altered cellular chlorophyll levels. We hypothesized that the two POR proteins may be differentially adaptive under varying light conditions. Using the diatom Phaeodactylum tricornutum as a test system, differences in POR protein abundance and por gene expression were examined when this organism was grown on an alternating light:dark cycles at different irradiances; exposed to continuous light; and challenged by a significant decrease in light availability.more » As a result, for cultures maintained on a 12h light: 12h dark photoperiod at 200μEm –2 s –1 ( 200L/D), both por genes were up-regulated during the light and down-regulated in the dark, though por1 transcript abundance rose and fell earlier than that of por2. Little concordance occurred between por1 mRNA and POR1 protein abundance. In contrast, por2 mRNA and POR2 protein abundances followed similar diurnal patterns. When 200L/D P. tricornutum cultures were transferred to continuous light ( 200L/L), the diurnal regulatory pattern of por1 mRNA abundance but not of por2 was disrupted, and POR1 but not POR2 protein abundance dropped steeply. Under 1200μEm –2 s –1 ( 1200L/D), both por1 mRNA and POR1 protein abundance displayed diurnal oscillations. A compromised diel por2 mRNA response under 1200L/D did not impact the oscillation in POR2 abundance. When cells grown at 1200L/D were then shifted to 50μEm –2 s –1 (50L/D), por1 and por2 mRNA levels decreased swiftly but briefly upon light reduction. Thereafter, POR1 but not POR2 protein levels rose significantly in response to this light stepdown.« less

Influenza A viruses suppress cyclooxygenase-2 expression by affecting its mRNA stability.

PubMed

Dudek, Sabine Eva; Nitzsche, Katja; Ludwig, Stephan; Ehrhardt, Christina

2016-06-06

Infection with influenza A viruses (IAV) provokes activation of cellular defence mechanisms contributing to the innate immune and inflammatory response. In this process the cyclooxygenase-2 (COX-2) plays an important role in the induction of prostaglandin-dependent inflammation. While it has been reported that COX-2 is induced upon IAV infection, in the present study we observed a down-regulation at later stages of infection suggesting a tight regulation of COX-2 by IAV. Our data indicate the pattern-recognition receptor RIG-I as mediator of the initial IAV-induced COX-2 synthesis. Nonetheless, during on-going IAV replication substantial suppression of COX-2 mRNA and protein synthesis could be detected, accompanied by a decrease in mRNA half-life. Interestingly, COX-2 mRNA stability was not only imbalanced by IAV replication but also by stimulation of cells with viral RNA. Our results reveal tristetraprolin (TTP), which is known to bind COX-2 mRNA and promote its rapid degradation, as regulator of COX-2 expression in IAV infection. During IAV replication and viral RNA accumulation TTP mRNA synthesis was induced, resulting in reduced COX-2 levels. Accordingly, the down-regulation of TTP resulted in increased COX-2 protein expression after IAV infection. These findings indicate a novel IAV-regulated cellular mechanism, contributing to the repression of host defence and therefore facilitating viral replication.

Expression of fructose-1,6-bisphosphatase mRNA isoforms in normal and basal forebrain cholinergic lesioned rat brain.

PubMed

Löffler, T; Al-Robaiy, S; Bigl, M; Eschrich, K; Schliebs, R

2001-06-01

Fructose-1,6-bisphosphatase is one of the key enzymes in the gluconeogenic pathway predominantly occurring in liver, kidney and muscle. In the brain, fructose-1,6-bisphosphatase has been suggested to be an astrocyte-specific enzyme but the functional importance of glyconeogenesis in the brain is still unclear. To further elucidate the cellular source of fructose-1,6-bisphosphatase in the brain, non-radioactive in situ hybridizations were performed using digoxigenin-labeled RNA probes based on the sequence of recently cloned rat liver and muscle fructose-1,6-bisphosphatase cDNAs. In situ hybridization using a riboprobe for the liver isoform revealed a location of the hybridization signal mainly in neurons, while rat muscle fructose-1,6-bisphosphatase mRNA was detected in both neurons and astrocytes in the hippocampal formation and in layer I of the cerebral cortex.RT-PCR using RNA preparations of rat astrocytes, neurons, and adult whole brain demonstrated a localization of liver fructose-1,6-bisphosphatase mRNA isoform in neurons but not in astrocytes. The muscle fructose-1,6-bisphosphatase mRNA isoform could be detected by RT-PCR in total rat brain, astrocytic, and neuronal mRNA preparations. The isoforms of fructose-1,6-bisphosphatase mRNA seemingly demonstrate a distinct cellular expression pattern in rat brain suggesting a role of glyconeogenesis in both neurons and glial cells.

Cytokine mRNA expression in normal skin of various age populations before and after engraftment onto nude mice.

PubMed

Gilhar, A; Ullmann, Y; Shalagino, R; Weisinger, G

1998-01-01

Whether the impact of skin biological age on cytokine expression is a result of this tissue's proliferation potential or not is an important issue in dermatology. We investigated these questions by monitoring cytokine marker mRNA expression from human skin samples from healthy groups of individuals. The skin samples studied represented three age groups: fetal (17-21 weeks), young (18-35 years) and aged (76-88 years). Furthermore, upon skin transplantation of tissue from different age groups onto nude mice, we investigated whether cytokine marker RNA levels would change or normalize. Interestingly, both TNF-alpha and P53 mRNA showed a similar pattern of expression. Both were significantly higher in fetal skin (p < 0.0001 and p < 0.05, respectively), and no difference was noted between aged versus young skin. In contrast to this, IL1-alpha mRNA was expressed at its lowest and highest levels in fetal and young skin, respectively. Following skin transplantation, cytokines and P53 mRNA expression were normalized to similar levels in all age groups. This study implies that when cytokine expression was determined directly at the mRNA level, post-natal expression was not significantly different at either age group. Furthermore, it seems that the environmental conditions surrounding the grafted human skin found on nude mice encouraged normalization of donor cytokine expression.

Differential Gene Expression Associated with Honey Bee Grooming Behavior in Response to Varroa Mites.

PubMed

Hamiduzzaman, Mollah Md; Emsen, Berna; Hunt, Greg J; Subramanyam, Subhashree; Williams, Christie E; Tsuruda, Jennifer M; Guzman-Novoa, Ernesto

2017-05-01

Honey bee (Apis mellifera) grooming behavior is an important mechanism of resistance against the parasitic mite Varroa destructor. This research was conducted to study associations between grooming behavior and the expression of selected immune, neural, detoxification, developmental and health-related genes. Individual bees tested in a laboratory assay for various levels of grooming behavior in response to V. destructor were also analyzed for gene expression. Intense groomers (IG) were most efficient in that they needed significantly less time to start grooming and fewer grooming attempts to successfully remove mites from their bodies than did light groomers (LG). In addition, the relative abundance of the neurexin-1 mRNA, was significantly higher in IG than in LG, no groomers (NG) or control (bees without mite). The abundance of poly U binding factor kd 68 and cytochrome p450 mRNAs were significantly higher in IG than in control bees. The abundance of hymenoptaecin mRNA was significantly higher in IG than in NG, but it was not different from that of control bees. The abundance of vitellogenin mRNA was not changed by grooming activity. However, the abundance of blue cheese mRNA was significantly reduced in IG compared to LG or NG, but not to control bees. Efficient removal of mites by IG correlated with different gene expression patterns in bees. These results suggest that the level of grooming behavior may be related to the expression pattern of vital honey bee genes. Neurexin-1, in particular, might be useful as a bio-marker for behavioral traits in bees.

Heart failure-associated changes in RNA splicing of sarcomere genes.

PubMed

Kong, Sek Won; Hu, Yong Wu; Ho, Joshua W K; Ikeda, Sadakatsu; Polster, Sean; John, Ranjit; Hall, Jennifer L; Bisping, Egbert; Pieske, Burkert; dos Remedios, Cristobal G; Pu, William T

2010-04-01

Alternative mRNA splicing is an important mechanism for regulation of gene expression. Altered mRNA splicing occurs in association with several types of cancer, and a small number of disease-associated changes in splicing have been reported in heart disease. However, genome-wide approaches have not been used to study splicing changes in heart disease. We hypothesized that mRNA splicing is different in diseased hearts compared with control hearts. We used the Affymetrix Exon array to globally evaluate mRNA splicing in left ventricular myocardial RNA from controls (n=15) and patients with ischemic cardiomyopathy (n=15). We observed a broad and significant decrease in mRNA splicing efficiency in heart failure, which affected some introns to a greater extent than others. The profile of mRNA splicing separately clustered ischemic cardiomyopathy and control samples, suggesting distinct changes in mRNA splicing between groups. Reverse transcription-polymerase chain reaction validated 9 previously unreported alternative splicing events. Furthermore, we demonstrated that splicing of 4 key sarcomere genes, cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), myosin heavy chain 7 (MYH7), and filamin C, gamma (FLNC), was significantly altered in ischemic cardiomyopathy and in dilated cardiomyopathy and aortic stenosis. In aortic stenosis samples, these differences preceded the onset of heart failure. Remarkably, the ratio of minor to major splice variants of TNNT2, MYH7, and FLNC classified independent test samples as control or disease with >98% accuracy. Our data indicate that mRNA splicing is broadly altered in human heart disease and that patterns of aberrant RNA splicing accurately assign samples to control or disease classes.

Neonatal hyper- and hypothyroidism alter the myoglobin gene expression program in adulthood.

PubMed

Souza, K de Picoli; Nunes, M T

2014-08-01

Myoglobin acts as an oxygen store and a reactive oxygen species acceptor in muscles. We examined myoglobin mRNA in rat cardiac ventricle and skeletal muscles during the first 42 days of life and the impact of transient neonatal hypo- and hyperthyroidism on the myoglobin gene expression pattern. Cardiac ventricle and skeletal muscles of Wistar rats at 7-42 days of life were quickly removed, and myoglobin mRNA was determined by Northern blot analysis. Rats were treated with propylthiouracil (5-10 mg/100 g) and triiodothyronine (0.5-50 µg/100 g) for 5, 15, or 30 days after birth to induce hypo- and hyperthyroidism and euthanized either just after treatment or at 90 days. During postnatal (P) days 7-28, the ventricle myoglobin mRNA remained unchanged, but it gradually increased in skeletal muscle (12-fold). Triiodothyronine treatment, from days P0-P5, increased the skeletal muscle myoglobin mRNA 1.5- to 4.5-fold; a 2.5-fold increase was observed in ventricle muscle, but only when triiodothyronine treatment was extended to day P15. Conversely, hypothyroidism at P5 markedly decreased (60%) ventricular myoglobin mRNA. Moreover, transient hyperthyroidism in the neonatal period increased ventricle myoglobin mRNA (2-fold), and decreased heart rate (5%), fast muscle myoglobin mRNA (30%) and body weight (20%) in adulthood. Transient hypothyroidism in the neonatal period also permanently decreased fast muscle myoglobin mRNA (30%) and body weight (14%). These results indicated that changes in triiodothyronine supply in the neonatal period alter the myoglobin expression program in ventricle and skeletal muscle, leading to specific physiological repercussions and alterations in other parameters in adulthood.

Neonatal hyper- and hypothyroidism alter the myoglobin gene expression program in adulthood

PubMed Central

de Picoli Souza, K.; Nunes, M.T.

2014-01-01

Myoglobin acts as an oxygen store and a reactive oxygen species acceptor in muscles. We examined myoglobin mRNA in rat cardiac ventricle and skeletal muscles during the first 42 days of life and the impact of transient neonatal hypo- and hyperthyroidism on the myoglobin gene expression pattern. Cardiac ventricle and skeletal muscles of Wistar rats at 7-42 days of life were quickly removed, and myoglobin mRNA was determined by Northern blot analysis. Rats were treated with propylthiouracil (5-10 mg/100 g) and triiodothyronine (0.5-50 µg/100 g) for 5, 15, or 30 days after birth to induce hypo- and hyperthyroidism and euthanized either just after treatment or at 90 days. During postnatal (P) days 7-28, the ventricle myoglobin mRNA remained unchanged, but it gradually increased in skeletal muscle (12-fold). Triiodothyronine treatment, from days P0-P5, increased the skeletal muscle myoglobin mRNA 1.5- to 4.5-fold; a 2.5-fold increase was observed in ventricle muscle, but only when triiodothyronine treatment was extended to day P15. Conversely, hypothyroidism at P5 markedly decreased (60%) ventricular myoglobin mRNA. Moreover, transient hyperthyroidism in the neonatal period increased ventricle myoglobin mRNA (2-fold), and decreased heart rate (5%), fast muscle myoglobin mRNA (30%) and body weight (20%) in adulthood. Transient hypothyroidism in the neonatal period also permanently decreased fast muscle myoglobin mRNA (30%) and body weight (14%). These results indicated that changes in triiodothyronine supply in the neonatal period alter the myoglobin expression program in ventricle and skeletal muscle, leading to specific physiological repercussions and alterations in other parameters in adulthood. PMID:25098716

Patterns of saproxylic beetle succession in loblolly pine.

Treesearch

Michael Ulyshen; James Hanula

2010-01-01

Patterns of insect succession in dead wood remain unclear, particularly beyond the first several years of decay. In the present study, saproxylic beetles were sampled from loblolly pine (Pinus taeda L.) logs aged between 1 month and 9 years old using both emergence traps attached to logs in the field and rearing bags in the laboratory.

Zif268 mRNA Expression Patterns Reveal a Distinct Impact of Early Pattern Vision Deprivation on the Development of Primary Visual Cortical Areas in the Cat.

PubMed

Laskowska-Macios, Karolina; Zapasnik, Monika; Hu, Tjing-Tjing; Kossut, Malgorzata; Arckens, Lutgarde; Burnat, Kalina

2015-10-01

Pattern vision deprivation (BD) can induce permanent deficits in global motion perception. The impact of timing and duration of BD on the maturation of the central and peripheral visual field representations in cat primary visual areas 17 and 18 remains unknown. We compared early BD, from eye opening for 2, 4, or 6 months, with late onset BD, after 2 months of normal vision, using the expression pattern of the visually driven activity reporter gene zif268 as readout. Decreasing zif268 mRNA levels between months 2 and 4 characterized the normal maturation of the (supra)granular layers of the central and peripheral visual field representations in areas 17 and 18. In general, all BD conditions had higher than normal zif268 levels. In area 17, early BD induced a delayed decrease, beginning later in peripheral than in central area 17. In contrast, the decrease occurred between months 2 and 4 throughout area 18. Lack of pattern vision stimulation during the first 4 months of life therefore has a different impact on the development of areas 17 and 18. A high zif268 expression level at a time when normal vision is restored seems to predict the capacity of a visual area to compensate for BD. © The Author 2014. Published by Oxford University Press.

A new MIF4G domain-containing protein, CTIF, directs nuclear cap-binding protein CBP80/20-dependent translation

PubMed Central

Kim, Kyoung Mi; Cho, Hana; Choi, Kobong; Kim, Jaedong; Kim, Bong-Woo; Ko, Young-Gyu; Jang, Sung Key; Kim, Yoon Ki

2009-01-01

During or right after mRNA export via the nuclear pore complex (NPC) in mammalian cells, mRNAs undergo translation mediated by nuclear cap-binding proteins 80 and 20 (CBP80/20). After CBP80/20-dependent translation, CBP80/20 is replaced by cytoplasmic cap-binding protein eIF4E, which directs steady-state translation. Nonsense-mediated mRNA decay (NMD), one of the best-characterized mRNA surveillance mechanisms, has been shown to occur on CBP80/20-bound mRNAs. However, despite the tight link between CBP80/20-dependent translation and NMD, the underlying molecular mechanism and cellular factors that mediate CBP80/20-dependent translation remain obscure. Here, we identify a new MIF4G domain-containing protein, CTIF (CBP80/20-dependent translation initiation factor). CTIF interacts directly with CBP80 and is part of the CBP80/20-dependent translation initiation complex. Depletion of endogenous CTIF from an in vitro translation system selectively blocks the translation of CBP80-bound mRNAs, while addition of purified CTIF restores it. Accordingly, down-regulation of endogenous CTIF abrogates NMD. Confocal microscopy shows that CTIF is localized to the perinuclear region. Our observations demonstrate the existence of CBP80/20-dependent translation and support the idea that CBP80/20-dependent translation is mechanistically different from steady-state translation through identification of a specific cellular protein, CTIF. PMID:19648179

RNA granules: the good, the bad and the ugly

PubMed Central

Thomas, María Gabriela; Loschi, Mariela; Desbats, María Andrea; Boccaccio, Graciela Lidia

2010-01-01

Processing bodies (PBs) and Stress granules (SGs) are the founding members of a new class of RNA granules, known as mRNA silencing foci, as they harbor transcripts circumstantially excluded from the translationally active pool. PBs and SGs are able to release mRNAs thus allowing their translation. PBs are constitutive, but respond to stimuli that affect mRNA translation and decay, whereas SGs are specifically induced upon cellular stress, which triggers a global translational silencing by several pathways, including phosphorylation of the key translation initiation factor elF2alpha, and tRNA cleavage among others. PBs and SGs with different composition may coexist in a single cell. These macromolecular aggregates are highly conserved through evolution, from unicellular organisms to vertebrate neurons. Their dynamics is regulated by several signaling pathways, and depends on microfilaments and microtubules, and the cognate molecular motors myosin, dynein, and kinesin. SGs share features with aggresomes and related aggregates of unfolded proteins frequently present in neurodegenerative diseases, and may play a role in the pathology. Virus infections may induce or impair SG formation. Besides being important for mRNA regulation upon stress, SGs modulate the signaling balancing apoptosis and cell survival. Finally, the formation of nuclear stress bodies (nSBs), which share components with SGs, and the assembly of additional cytosolic aggregates containing RNA—the UV granules and the Ire1 foci—, all them induced by specific cell damage factors, contribute to cell survival. PMID:20813183

Derepression of microRNA-mediated protein translation inhibition by apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) and its family members.

PubMed

Huang, Jialing; Liang, Zhihui; Yang, Bin; Tian, Heng; Ma, Jin; Zhang, Hui

2007-11-16

The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G or A3G) and its fellow cytidine deaminase family members are potent restrictive factors for human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. A3G interacts with a vast spectrum of RNA-binding proteins and is located in processing bodies and stress granules. However, its cellular function remains to be further clarified. Using a luciferase reporter gene and green fluorescent protein reporter gene, we demonstrate that A3G and other APOBEC family members can counteract the inhibition of protein synthesis by various microRNAs (miRNAs) such as mir-10b, mir-16, mir-25, and let-7a. A3G could also enhance the expression level of miRNA-targeted mRNA. Further, A3G facilitated the association of microRNA-targeted mRNA with polysomes rather than with processing bodies. Intriguingly, experiments with a C288A/C291A A3G mutant indicated that this function of A3G is separable from its cytidine deaminase activity. Our findings suggest that the major cellular function of A3G, in addition to inhibiting the mobility of retrotransposons and replication of endogenous retroviruses, is most likely to prevent the decay of miRNA-targeted mRNA in processing bodies.

The decay of wood in landfills in contrasting climates in Australia

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

Ximenes, Fabiano, E-mail: fabiano.ximenes@dpi.nsw.gov.au; Björdal, Charlotte; Cowie, Annette

Highlights: • We examine decay in wood from landfills in contrasting environments in Australia. • Analysis is based on changes in chemical composition and microscopy. • Climate did not influence levels of decay observed. • Microscopy of retrieved samples revealed most of the decay was aerobic in nature. • Current default factors for wood decay in landfills overestimate methane emissions. - Abstract: Wood products in landfill are commonly assumed to decay within several decades, returning the carbon contained therein to the atmosphere, with about half the carbon released as methane. However, the rate and extent of decay is not wellmore » known, as very few studies have examined the decay of wood products in landfills. This study reports on the findings from landfill excavations conducted in the Australian cities of Sydney and Cairns located in temperate and tropical environments, respectively. The objective of this study was to determine whether burial of the wood in warmer, more tropical conditions in Cairns would result in greater levels of decay than occurs in the temperate environment of Sydney. Wood samples recovered after 16–44 years in landfill were examined through physical, chemical and microscopic analyses, and compared with control samples to determine the carbon loss. There was typically little or no decay in the wood samples analysed from the landfill in Sydney. Although there was significant decay in rainforest wood species excavated from Cairns, decay levels for wood types that were common to both Cairns and Sydney landfills were similar. The current Intergovernmental Panel on Climate Change (IPCC, 2006) default decay factor for organic materials in landfills is 50%. In contrast, the carbon loss determined for Pinus radiata recovered from Sydney and Cairns landfills was 7.9% and 4.4%, respectively, and 0% for Agathis sp. This suggests that climate did not influence decay, and that the more extensive levels of decay observed for some wood samples from Cairns indicates that those wood types were more susceptible to biodegradation. Microscopic analyses revealed that most decay patterns observed in samples analysed from Sydney were consistent with aerobic fungal decay. Only a minor portion of the microbial decay was due to erosion bacteria active in anaerobic/near anaerobic environments. The findings of this study strongly suggest that models that adopt current accepted default factors for the decay of wood in landfills greatly overestimate methane emissions.« less

Second stop and sbottom searches with a stealth stop

NASA Astrophysics Data System (ADS)

Cheng, Hsin-Chia; Li, Lingfeng; Qin, Qin

2016-11-01

The top squarks (stops) may be the most wanted particles after the Higgs boson discovery. The searches for the lightest stop have put strong constraints on its mass. However, there is still a search gap in the low mass region if the spectrum of the stop and the lightest neutralino is compressed. In that case, it may be easier to look for the second stop since naturalness requires both stops to be close to the weak scale. The current experimental searches for the second stop are based on the simplified model approach with the decay modes {overset{˜ }{t}}_2to {overset{˜ }{t}}_1Z and {overset{˜ }{t}}_2to {overset{˜ }{t}}_1h . However, in a realistic supersymmetric spectrum there is always a sbottom lighter than the second stop, hence the decay patterns are usually more complicated than the simplified model assumptions. In particular, there are often large branching ratios of the decays {overset{˜ }{t}}_2to {overset{˜ }{b}}_1W and {overset{˜ }{b}}_1to {overset{˜ }{t}}_1W as long as they are open. The decay chains can be even more complex if there are intermediate states of additional charginos and neutralinos in the decays. By studying several MSSM benchmark models at the 14 TeV LHC, we point out the importance of the multi- W final states in the second stop and the sbottom searches, such as the same-sign dilepton and multilepton signals, aside from the traditional search modes. The observed same-sign dilepton excesses at LHC Run 1 and Run 2 may be explained by some of our benchmark models. We also suggest that the vector boson tagging and a new kinematic variable may help to suppress the backgrounds and increase the signal significance for some search channels. Due to the complex decay patterns and lack of the dominant decay channels, the best reaches likely require a combination of various search channels at the LHC for the second stop and the lightest sbottom.

Comparative gene expression analysis of bovine nuclear-transferred embryos with different developmental potential by cDNA microarray and real-time PCR to determine genes that might reflect calf normality.

PubMed

Kato, Yoko; Li, Xiangping; Amarnath, Dasari; Ushizawa, Koichi; Hashizume, Kazuyoshi; Tokunaga, Tomoyuki; Taniguchi, Masanori; Tsunoda, Yukio

2007-01-01

Placental abnormalities are the main factor in the high incidence of somatic cell clone abnormalities. The expression of several trophoblast cell-specific molecules is enhanced during gestational days 7 to 14. To determine the possible genes whose expression patterns might reflect calf normality, we first compared the gene expression profiles on day 15 between in vitro-fertilized (IVF) embryos and two types of somatic cell nuclear-transferred embryos with either a high (FNT) or low (CNT) incidence of neonatal abnormalities using a cDNA microarray containing 16 of 21 placenta-specific genes developed from tissues collected across gestation. To identify significant genes from the screening of day 15 embryos, genes with a less than two-fold difference in expression between IVF and CNT embryos, and those with a greater than two-fold difference between IVF and FNT and between CNT and FNT were considered to contribute to clone abnormalities. These two comparisons revealed 18 down-regulated and 18 upregulated genes of the 1722 genes examined. We then examined the expression levels of 10 genes with known functions in eight-cell and blastocyst-stage embryos by real-time PCR. The mRNA expression pattern of interferon (IFN)-tau, a trophectoderm-related gene, differed between IVF, CNT, and FNT eight-cell embryos; few or none of the IVF or CNT eight-cell embryos expressed IFN-tau mRNA, but all eight-cell FNT embryos expressed IFN-tau. IFN-tau mRNA expression was significantly higher in IVF blastocysts, however, than in nuclear-transferred blastocysts. Average IFN-tau mRNA expression in FNT blastocysts was not different from that in CNT blastocysts, due to one CNT blastocyst with high expression. The precise relation between early expression of IFN-tau mRNA and inferior developmental potential in cloned embryos should be examined further.

Two novel LRR-only proteins in Chlamys farreri: Similar in structure, yet different in expression profile and pattern recognition.

PubMed

Wang, Mengqiang; Wang, Lingling; Xin, Lusheng; Wang, Xiudan; Wang, Lin; Xu, Jianchao; Jia, Zhihao; Yue, Feng; Wang, Hao; Song, Linsheng

2016-06-01

Leucine-rich repeat (LRR)-only proteins could mediate protein-ligand and protein-protein interactions and be involved in the immune response. In the present study, two novel LRR-only proteins, CfLRRop-2 and CfLRRop-3, were identified and characterized from scallop Chlamys farreri. They both contained nine LRR motifs with the consensus signature sequence LxxLxLxxNxL and formed typical horseshoe structure. The CfLRRop-2 and CfLRRop-3 mRNA transcripts were constitutively expressed in haemocytes, muscle, mantle, gill, haepatopancreas and gonad, with the highest expression level in haepatopancreas and gill, respectively. During the ontogenesis of scallop, the mRNA transcripts of CfLRRop-2 were kept at a high level in oocytes and embryos, while those of CfLRRop-3 were expressed at a rather low level from oocytes to blastula. Their mRNA transcripts were significantly increased after the stimulation of lipopolysaccharide (LPS), peptidoglycan (PGN), glucan (GLU) and polyinosinic-polycytidylic acid (poly I:C), and the mRNA expression of CfLRRop-2 rose more intensely than that of CfLRRop-3. After the suppression of CfTLR (previously identified Toll-like receptor in C. farreri) via RNA interference (RNAi), CfLRRop-3 mRNA transcripts increased more intensely and lastingly than those of CfLRRop-2. The rCfLRRop-3 protein could bind LPS, PGN, GLU and poly I:C, while rCfLRRop-2 exhibited no significant binding activity to them. Additionally, rCfLRRop-2 could significantly induce the release of TNF-α from the mixed primary cultured scallop haemocytes, but rCfLRRop-3 failed. These results collectively indicated that CfLRRop-2 might act as an immune effector or pro-inflammatory factor, while CfLRRop-3 would function as a pattern recognition receptor (PRR), suggesting the function of LRR-only protein family has differentiated in scallop. Copyright © 2016 Elsevier Ltd. All rights reserved.

Spatiotemporal and Long Lasting Modulation of 11 Key Nogo Signaling Genes in Response to Strong Neuroexcitation

PubMed Central

Karlsson, Tobias E.; Wellfelt, Katrin; Olson, Lars

2017-01-01

Inhibition of nerve growth and plasticity in the CNS is to a large part mediated by Nogo-like signaling, now encompassing a plethora of ligands, receptors, co-receptors and modulators. Here we describe the distribution and levels of mRNA encoding 11 key genes involved in Nogo-like signaling (Nogo-A, Oligodendrocyte-Myelin glycoprotein (OMgp), Nogo receptor 1 (NgR1), NgR2, NgR3, Lingo-1, TNF receptor orphan Y (Troy), Olfactomedin, Lateral olfactory tract usher substance (Lotus) and membrane-type matrix metalloproteinase-3 (MT3-MPP)), as well as BDNF and GAPDH. Expression was analyzed in nine different brain areas before, and at eight time points during the first 3 days after a strong neuroexcitatory stimulation, caused by one kainic acid injection. A temporo-spatial pattern of orderly transcriptional regulations emerges that strengthens the role of Nogo-signaling mechanisms for synaptic plasticity in synchrony with transcriptional increases of BDNF mRNA. For most Nogo-type signaling genes, the largest alterations of mRNA levels occur in the dentate gyrus, with marked alterations also in the CA1 region. Changes occurred somewhat later in several areas of the cerebral cortex. The detailed spatio-temporal pattern of mRNA presence and kainic acid-induced transcriptional response is gene-specific. We reveal that several different gene alterations combine to decrease (and later increase) Nogo-like signaling, as expected to allow structural plasticity responses. Other genes are altered in the opposite direction, suggesting that the system prepares in advance in order to rapidly restore balance. However, the fact that Lingo-1 shows a seemingly opposite, plasticity inhibiting response to kainic acid (strong increase of mRNA in the dentate gyrus), may instead suggest a plasticity-enhancing intracellular function of this presumed NgR1 co-receptor. PMID:28442990

Identification of tumor-restricted antigens NY-BR-1, SCP-1, and a new cancer/testis-like antigen NW-BR-3 by serological screening of a testicular library with breast cancer serum.

PubMed

Jäger, Dirk; Unkelbach, Marc; Frei, Claudia; Bert, Florian; Scanlan, Matthew J; Jäger, Elke; Old, Lloyd J; Chen, Yao-Tseng; Knuth, Alexander

2002-06-28

Serological analysis of recombinant cDNA expression libraries (SEREX) has led to the identification of several categories of new tumor antigens. We analyzed a testicular cDNA expression library with serum obtained from a breast cancer patient and isolated 13 genes designated NW-BR-1 through NW-BR-13. Of these, 3 showed tumor-restricted expression (NW-BR-1, -2 and -3), the others being expressed ubiquitously. NW-BR-3, representing 9 of 24 primary clones, showed tissue-restricted mRNA expression, being expressed in normal testis but not in 15 other normal tissues tested by Northern blotting. RT-PCR analysis showed strong NW-BR-3 expression in normal testis, weak expression in brain, kidney, trachea, uterus and normal prostate, and was negative in liver, heart, lung, colon, small intestine, bone marrow, breast, thymus, muscle, spleen, and stomach. NW-BR-3 mRNA expression was found in different tumor tissues and tumor cell lines by RT-PCR, thus showing a 'cancer/testis' (CT)-like mRNA expression pattern. NW-BR-3 shares 71% nucleotide and amino acid homology to a mouse gene cloned from mouse testicular tissue. Based on the mRNA expression pattern, NW-BR-3 represents a new candidate target gene for cancer immunotherapy. NW-BR-1 and NW-BR-2 also showed tumor-restricted mRNA expression. NW-BR-1 is a partial clone of the breast differentiation antigen NY-BR-1 previously identified by SEREX. NY-BR-1 is expressed in normal breast, testis and 80% of breast cancers. NW-BR-2 is identical to the CT antigen SCP-1, initially isolated by SEREX analysis of renal cancer. This study provides further evidence that SEREX is a powerful tool to identify new tumor antigens potentially relevant for immunotherapy approaches.

Nogo-receptor gene activity: cellular localization and developmental regulation of mRNA in mice and humans.

PubMed

Josephson, Anna; Trifunovski, Alexandra; Widmer, Hans Ruedi; Widenfalk, Johan; Olson, Lars; Spenger, Christian

2002-11-18

Nogo (reticulon-4) is a myelin-associated protein that is expressed in three different splice variants, Nogo-A, Nogo-B, and Nogo-C. Nogo-A inhibits neurite regeneration in the central nervous system. Messenger RNA encoding Nogo is expressed in oligodendrocytes and central and peripheral neurons, but not in astrocytes or Schwann cells. Nogo is a transmembraneous protein; the extracellular domain is termed Nogo-66, and a Nogo-66-receptor (Nogo-R) has been identified. We performed in situ hybridization in human and mouse nervous tissues to map the cellular distribution of Nogo-R gene activity patterns in fetal and adult human spinal cord and sensory ganglia, adult human brain, and the nervous systems of developing and adult mice. In the human fetus Nogo-R was transcribed in the ventral horn of the spinal cord and in dorsal root ganglia. In adult human tissues Nogo-R gene activity was found in neocortex, hippocampus, amygdala, and a subset of large and medium-sized neurons of the dorsal root ganglia. Nogo-R mRNA was not expressed in the adult human spinal cord at detectable levels. In the fetal mouse, Nogo-R was diffusely expressed in brain, brainstem, trigeminal ganglion, spinal cord, and dorsal root ganglia at all stages. In the adult mouse strong Nogo-R mRNA expression was found in neurons in neocortex, hippocampus, amygdala, habenula, thalamic nuclei, brainstem, the granular cell layer of cerebellum, and the mitral cell layer of the olfactory bulb. Neurons in the adult mouse striatum, the medial septal nucleus, and spinal cord did not express Nogo-R mRNA at detectable levels. In summary, Nogo-66-R mRNA expression in humans and mice was observed in neurons of the developing nervous system Expression was downregulated in the adult spinal cord of both species, and specific expression patterns were seen in the adult brain. Copyright 2002 Wiley-Liss, Inc.

Genes with mutation significance were highly associated with the clinical pattern of patients with breast cancer.

PubMed

Ding, Wan-Jun; Zeng, Tao; Wang, Li-Jun; Lei, Hong-Bo; Ge, Wei; Wang, Zhi

2017-11-17

In the United States, breast cancer is the second leading cause of cancer death in women. Over the past 20 years, breast cancer incidence and mortality rates increased rapidly in developing regions. We aimed to identify the gene mutation patterns that associated with the clinical patterns, including survival status, histo-pathological classes and so forth, of breast cancer. We retrieved 1098 cases of the clinical information, and level-3 legacy data of mRNA expression level, protein expression data and mutation files from GDC data portal. The genes with mutation significance were obtained. We studied the impacts of mutation types on the expression levels of mRNA and protein. Different statistics methods were used to calculate the correlation between the mutation types and the expression data or histo-clinical measures. There were 24 genes with mutation significance identified. The most mutated genes were selected to study the role of specific mutations played on the patients with breast cancer. One interesting finding was the missense mutations on TP53 were related with high expression levels of mRNA and protein. The missense mutations on TP53 were highly related with the morphology, race, ER status, PR status and HER2 Status, while the truncated mutations were only related with the morphology, ER status and PR status. The missense mutation on PIK3CA was highly associated with the morphology, race, ER status and PR status. The mutants with different mutants and the wild type of the most mutated genes had different impacts on the histo-clinical measures that might help personalized therapy.

DAILY PATTERNS OF CLOCK AND COGNITION-RELATED FACTORS ARE MODIFIED IN THE HIPPOCAMPUS OF VITAMIN A-DEFICIENT RATS

PubMed Central

Golini, Rebeca S.; Delgado, Silvia M.; Navigatore Fonzo, Lorena S.; Ponce, Ivana T.; Lacoste, María G.; Anzulovich, Ana C.

2012-01-01

The circadian expression of clock and clock-controlled cognition-related genes in the hippocampus would be essential to achieve an optimal daily cognitive performance. There is some evidence that retinoid nuclear receptors (RARs and RXRs) can regulate circadian gene expression in different tissues. In this study, Holtzman male rats from control and vitamin A-deficient groups were sacrificed throughout a 24-h period and hippocampus samples were isolated every 4 or 5 h. RARα and RXRβ expression level was quantified and daily expression patterns of clock BMAL1, PER1, RORα and REVERB genes, RORα and REVERB proteins, as well as temporal expression of cognition-related RC3 and BDNF genes were determined in the hippocampus of the two groups of rats. Our results show significant daily variations of BMAL1, PER1, RORα and REVERB genes, RORα and REVERB proteins and, consequently, daily oscillating expression of RC3 and BDNF genes in the rat hippocampus. Vitamin A deficiency reduced RXRβ mRNA level as well as the amplitude of PER1, REVERB gene and REVERB protein rhythms, and phase-shifted the daily peaks of BMAL1 and RORα mRNA, RORα protein and RC3 and BDNF mRNA levels. Thus, nutritional factors, such as vitamin A and its derivatives the retinoids, might modulate daily patterns of BDNF and RC3 expression in the hippocampus and they could be essential to maintain an optimal daily performance at molecular level in this learning-and-memory-related brain area. PMID:22434687

Repeated Ferret Odor Exposure Induces Different Temporal Patterns of Same-Stressor Habituation and Novel-Stressor Sensitization in Both Hypothalamic-Pituitary-Adrenal Axis Activity and Forebrain c-fos Expression in the Rat

PubMed Central

Weinberg, Marc S.; Bhatt, Aadra P.; Girotti, Milena; Masini, Cher V.; Day, Heidi E. W.; Campeau, Serge; Spencer, Robert L.

2009-01-01

Repeated exposure to a moderately intense stressor typically produces attenuation of the hypothalamic-pituitary-adrenal (HPA) axis response (habituation) on re-presentation of the same stressor; however, if a novel stressor is presented to the same animals, the HPA axis response may be augmented (sensitization). The extent to which this adaptation is also evident within neural activity patterns is unknown. This study tested whether repeated ferret odor (FO) exposure, a moderately intense psychological stressor for rats, leads to both same-stressor habituation and novel-stressor sensitization of the HPA axis response and neuronal activity as determined by immediate early gene induction (c-fos mRNA). Rats were presented with FO in their home cages for 30 min a day for up to 2 wk and subsequently challenged with FO or restraint. Rats displayed HPA axis activity habituation and widespread habituation of c-fos mRNA expression (in situ hybridization) throughout the brain in as few as three repeated presentations of FO. However, repeated FO exposure led to a more gradual development of sensitized HPA-axis and c-fos mRNA responses to restraint that were not fully evident until after 14 d of prior FO exposure. The sensitized response was evident in many of the same brain regions that displayed habituation, including primary sensory cortices and the prefrontal cortex. The shared spatial expression but distinct temporal development of habituation and sensitization neural response patterns suggests two independent processes with opposing influences across overlapping brain systems. PMID:18845631

Comparison of the age-related porcine endogenous retrovirus (PERV) expression using duplex RT-PCR

PubMed Central

Moon, Hyoung Joon; Kim, Hye Kwon; Park, Seong Jun; Lee, Chul Seung; Song, Dae Sub; Kang, Bo Kyu

2009-01-01

Porcine endogenous retroviruses (PERVs) are members of family Retroviridae, genus Gamma retrovirus, and transmitted by both horizontally and vertically like other endogenous retroviruses (ERVs). PERV was initially described in the 1970s having inserted its gene in the host genome of different pig breeds, and three classes, PERV-A, PERV-B, and PERV-C are known. The therapeutic use of living cells, tissues, and organs from animals called xenotransplantation might relieve the limited supply of allografts in the treatment of organ dysfunction. Because of ethical considerations, compatible organ sizes, and physiology, the pig has been regarded as an alternative source for xenotransplantation. Sensitive duplex reverse transcription-polymerase chain reaction protocols for simultaneously detecting PERV gag mRNA and porcine glyceraldehydes 3-phosphate dehydrogenase mRNA in one tube was established. To compare the age-related PERV expression patterns of the lung, liver, spleen, kidney, heart, and pancreas in commercial pigs, 20 pigs from four age groups (5 heads each in 10 days-, 40 days-, 70 days-, and 110 days-old, respectively) were used in this study. The expression patterns of PERV were statistically different among age groups in lung, liver, and kidney (ANOVA, p < 0.05). These data may support in the selection of appropriate donor pigs expressing low levels of PERV mRNA. PMID:19934597

Toll-Like Receptor and Accessory Molecule mRNA Expression in Humans and Mice as Well as in Murine Autoimmunity, Transient Inflammation, and Progressive Fibrosis

PubMed Central

Ramaiah, Santhosh Kumar Vankayala; Günthner, Roman; Lech, Maciej; Anders, Hans-Joachim

2013-01-01

The cell type-, organ-, and species-specific expression of the Toll-like receptors (TLRs) are well described, but little is known about the respective expression profiles of their accessory molecules. We therefore determined the mRNA expression levels of LBP, MD2, CD36, CD14, granulin, HMGB1, LL37, GRP94, UNC93b1, TRIL, PRAT4A, AP3B1, AEP and the respective TLRs in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. In addition, the expression profiles in transient tissue inflammation upon renal ischemia-reperfusion injury, in spleens and kidneys from mice with lupus-like systemic autoimmunity, and in progressive tissue fibrosis upon unilateral ureteral obstruction were studied. Several TLR co-factors were specifically regulated during the different phases of these disease entities, suggesting a functional involvement in the disease process. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to TLR-mediated innate immunity, which seems to be involved in the tissue injury phase, in the phase of tissue regeneration, and in progressive tissue remodelling. PMID:23803655

[Comparative study of expression of homeobox gene Msx-1, Msx-2 mRNA during the hard tissue formation of mouse tooth development].

PubMed

Wang, Y; Wang, J; Gao, Y

2001-07-01

To observe and compare the expression pattern of Msx-1, Msx-2 mRNA during the different stages of hard tissue formation in the first mandibular molar of mouse and investigate the relationship between the two genes. First mandibular molar germs from 1, 3, 7 and 14-days old mouse were separated and reverse transcription-polymerase chain reaction was performed on the total RNA of them using Msx-1, Msx-2 specific primers separately. Expression of both genes were detected during the different stages of hard tissue formation in the mouse first mandibular molars, but there was some interesting differences in the quantitiy between the two genes. Msx-1 transcripts appeared at the 1 day postnatally, and increase through 3 day, 7 day, then maximally expressed at 14 days postnatally; while Msx-2 mRNA was seen and expressed maximally at the 3 days postnatally, then there was a gradual reduction at 7 days, and 14 days postnatally. The homeobox gene Msx-1, Msx-2 may play a role in the events of the hard tissue formation. The complementary expression pattern of them during the specific stage of hard tissue formation indicates that there may be some functional redundancy between them during the biomineralization.

Extracellular Mitochondria and Mitochondrial Components Act as Damage-Associated Molecular Pattern Molecules in the Mouse Brain.

PubMed

Wilkins, Heather M; Koppel, Scott J; Weidling, Ian W; Roy, Nairita; Ryan, Lauren N; Stanford, John A; Swerdlow, Russell H

2016-12-01

Mitochondria and mitochondrial debris are found in the brain's extracellular space, and extracellular mitochondrial components can act as damage associated molecular pattern (DAMP) molecules. To characterize the effects of potential mitochondrial DAMP molecules on neuroinflammation, we injected either isolated mitochondria or mitochondrial DNA (mtDNA) into hippocampi of C57BL/6 mice and seven days later measured markers of inflammation. Brains injected with whole mitochondria showed increased Tnfα and decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation. Some of these effects were also observed in brains injected with mtDNA (decreased Trem2 mRNA, increased GFAP protein, and increased NFκB phosphorylation), and mtDNA injection also caused several unique changes including increased CSF1R protein and AKT phosphorylation. To further establish the potential relevance of this response to Alzheimer's disease (AD), a brain disorder characterized by neurodegeneration, mitochondrial dysfunction, and neuroinflammation we also measured App mRNA, APP protein, and Aβ 1-42 levels. We found mitochondria (but not mtDNA) injections increased these parameters. Our data show that in the mouse brain extracellular mitochondria and its components can induce neuroinflammation, extracellular mtDNA or mtDNA-associated proteins can contribute to this effect, and mitochondria derived-DAMP molecules can influence AD-associated biomarkers.

Expression of fibroblast growth factor receptors during development and regression of the bovine corpus luteum.

PubMed

Guerra, D M; Giometti, I C; Price, C A; Andrade, P B; Castilho, A C; Machado, M F; Ripamonte, P; Papa, P C; Buratini, J

2008-01-01

There is evidence that fibroblast growth factors (FGFs) are involved in the regulation of growth and regression of the corpus luteum (CL). However, the expression pattern of most FGF receptors (FGFRs) during CL lifespan is still unknown. The objective of the present study was to determine the pattern of expression of 'B' and 'C' splice variants of FGFRs in the bovine CL. Bovine CL were collected from an abattoir and classed as corpora hemorrhagica (Stage I), developing (Stage II), developed (Stage III) or regressed (Stage IV) CL. Expression of FGFR mRNA was measured by semiquantitative reverse transcription-polymerase chain reaction and FGFR protein was localised by immunohistochemistry. Expression of mRNA encoding the 'B' and 'C' spliced forms of FGFR1 and FGFR2 was readily detectable in the bovine CL and was accompanied by protein localisation. FGFR1C and FGFR2C mRNA expression did not vary throughout CL lifespan, whereas FGFR1B was upregulated in the developed (Stage III) CL. FGFR3B, FGFR3C and FGFR4 expression was inconsistent in the bovine CL. The present data indicate that FGFR1 and FGFR2 splice variants are the main receptors for FGF action in the bovine CL.

Decay pattern of the Pygmy Dipole Resonance in 140Ce

NASA Astrophysics Data System (ADS)

Isaak, J.; Löher, B.; Savran, D.; Aumann, T.; Beller, J.; Cooper, N.; Derya, V.; Duchêne, M.; Endres, J.; Fiori, E.; Kelley, J. H.; Knörzer, M.; Pietralla, N.; Ponomarev, V. Yu.; Romig, C.; Scheck, M.; Scheit, H.; Silva, J.; Tonchev, A. P.; Tornow, W.; Weller, H. R.; Werner, V.; Zilges, A.; Zweidinger, M.

2015-05-01

The decay behavior of low-lying dipole states in 140Ce was investigated exploiting the γ3-setup at the HIγS facility using quasi-monochromatic photon beams. Branching ratios of individual excited states as well as average branching ratios to low-lying states have been extracted using γ - γ coincidence measurements. The comparison of the average branching ratios to QPM calculations shows a remarkable agreement between experiment and theory in the energy range from 5.0 to 8.5 MeV.

Expression of aquaporin-3 and -8 mRNAs in the parr and smolt stages of sockeye salmon, Oncorhynchus nerka: effects of cortisol treatment and seawater acclimation.

PubMed

Choi, Young Jae; Shin, Hyun Suk; Kim, Na Na; Cho, Sung Hwoan; Yamamoto, Yuzo; Ueda, Hiroshi; Lee, Jehee; Choi, Cheol Young

2013-06-01

This study aimed to examine the role of 2 aquaporin (AQP) isoforms (AQP3, and -8) in sockeye salmon (Oncorhynchus nerka) in response to a hyperosmotic challenge from freshwater to seawater (SW) during the parr and smoltification (smolt) stages. AQP3 mRNA was primarily detected in the osmoregulatory organs, such as gills, while AQP8 mRNA was primarily found in the intestine. These results suggested that AQP isoforms play a role in osmoregulation in specific osmoregulatory organs. Similarly, AQP3 mRNA expression in the gills (mean values:1.06 ± 0.05 [parr] and 1.29 ± 0.07 [smolt]) was significantly higher than AQP8 mRNA levels (parr: 0.04 ± 0.003; smolt: 0.14 ± 0.004), and in the intestine, AQP8 mRNA expression (parr: 0.89 ± 0.007; smolt: 1.91 ± 0.03) was significantly higher than AQP3 mRNA levels (parr: 0.24 ± 0.006; smolt: 0.83 ± 0.005); these expression patterns were similar in vivo and in vitro. Additionally, AQP mRNA levels were lower in cortisol treated than in control groups. Therefore, these results suggest that AQPs play important roles in the water absorption mechanisms associated with multiple AQP isoforms, and that cortisol enhances the hypo-osmoregulatory capacity of fish in SW, and also controls the expression of AQPs in a hyperosmotic environment. Copyright © 2013 Elsevier Inc. All rights reserved.

The herpes simplex virus 1 virion host shutoff protein enhances translation of viral late mRNAs by preventing mRNA overload.

PubMed

Dauber, Bianca; Saffran, Holly A; Smiley, James R

2014-09-01

We recently demonstrated that the virion host shutoff (vhs) protein, an mRNA-specific endonuclease, is required for efficient herpes simplex virus 1 (HSV-1) replication and translation of viral true-late mRNAs, but not other viral and cellular mRNAs, in many cell types (B. Dauber, J. Pelletier, and J. R. Smiley, J. Virol. 85:5363-5373, 2011, http://dx.doi.org/10.1128/JVI.00115-11). Here, we evaluated whether the structure of true-late mRNAs or the timing of their transcription is responsible for the poor translation efficiency in the absence of vhs. To test whether the highly structured 5' untranslated region (5'UTR) of the true-late gC mRNA is the primary obstacle for translation initiation, we replaced it with the less structured 5'UTR of the γ-actin mRNA. However, this mutation did not restore translation in the context of a vhs-deficient virus. We then examined whether the timing of transcription affects translation efficiency at late times. To this end, we engineered a vhs-deficient virus mutant that transcribes the true-late gene US11 with immediate-early kinetics (IEUS11-ΔSma). Interestingly, IEUS11-ΔSma showed increased translational activity on the US11 transcript at late times postinfection, and US11 protein levels were restored to wild-type levels. These results suggest that mRNAs can maintain translational activity throughout the late stage of infection if they are present before translation factors and/or ribosomes become limiting. Taken together, these results provide evidence that in the absence of the mRNA-destabilizing function of vhs, accumulation of viral mRNAs overwhelms the capacity of the host translational machinery, leading to functional exclusion of the last mRNAs that are made during infection. The process of mRNA translation accounts for a significant portion of a cell's energy consumption. To ensure efficient use of cellular resources, transcription, translation, and mRNA decay are tightly linked and highly regulated. However, during virus infection, the overall amount of mRNA may increase drastically, possibly overloading the capacity of the translation apparatus. Our results suggest that the HSV-1 vhs protein, an mRNA-specific endoribonuclease, prevents mRNA overload during infection, thereby allowing translation of late viral mRNAs. The requirement for vhs varies between cell types. Further studies of the basis for this difference likely will offer insights into how cells regulate overall mRNA levels and access to the translational apparatus. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Decay characteristics of electroadhesive forces by periodic electrodes in dielectric layers

NASA Astrophysics Data System (ADS)

Lee, Junseok; Cha, Youngsu

2017-07-01

Electroadhesive force is the force generated by induced dipoles in the gradient of an electric field. Owing to its benefits of mechanical characteristics and versatility, it is widely used to hold and manipulate objects in robotic applications. So far, most studies in this field have been focused on the maximization of the magnitude of electroadhesive force. In this paper, we focus on the decay characteristics of electroadhesive force depending on the spatial distance from electrodes to employ the force to precisely separate a single layer from stacked dielectric layers. It turns out that all configurations with periodically repeating electrodes' arrangement, have the same decay characteristics which significantly depend on the geometrical period of the electrode patterns. Also, we find that the other parameters including the applied voltage and geometry of electrodes have little effect on the decay characteristics. The electric potential of an arbitrary electrode configuration is expanded in terms of the Fourier series, and we use it to analytically prove the high dependence of decay characteristics on the geometrical period. Numerical analysis is performed using the finite element method.

K selection in the decay of the (ν 5/2 [532 ] ⊗3/2 [411 ] ) 4- isomeric state in 102Zr

NASA Astrophysics Data System (ADS)

Browne, F.; Bruce, A. M.; Sumikama, T.; Nishizuka, I.; Nishimura, S.; Doornenbal, P.; Lorusso, G.; Söderström, P.-A.; Watanabe, H.; Daido, R.; Patel, Z.; Rice, S.; Sinclair, L.; Wu, J.; Xu, Z. Y.; Yagi, A.; Baba, H.; Chiga, N.; Carroll, R.; Didierjean, F.; Fang, Y.; Fukuda, N.; Gey, G.; Ideguchi, E.; Inabe, N.; Isobe, T.; Kameda, D.; Kojouharov, I.; Kurz, N.; Kubo, T.; Lalkovski, S.; Li, Z.; Lozeva, R.; Nishibata, N.; Odahara, A.; Podolyák, Zs.; Regan, P. H.; Roberts, O. J.; Sakurai, H.; Schaffner, H.; Simpson, G. S.; Suzuki, H.; Takeda, H.; Tanaka, M.; Taprogge, J.; Werner, V.; Wieland, O.

2017-08-01

The (ν 5/2 [532 ] ⊗3/2 [411 ] ) 4- state in 102Zr, populated in the β decay of 102Y, has been measured to be isomeric with a mean lifetime of 9.5(7) ns. It decays via four transitions, two of which are Δ K =2 (to the 3+ and 4+ members of the 2γ+ band) and one is Δ K =4 (to the 4+ member of the ground state 0+ band). The fourth (low-energy) transition is inferred to decay to an as-yet unassigned state. Hindrances of 106 were derived for the Δ K =2 transitions compared to Weisskopf estimates and the Δ K =4 transition hindered by a factor of 109. These values are consistent with the decay pattern of the analogous isomeric state in the neighboring N =62 nucleus 100Sr and with the broader systematics of such transitions. A comparison of the hindrances for the Δ K =4 transitions suggests that 102Zr is hardened against the γ degree of freedom compared to 100Sr.

Reduction in the mRNA expression of sVEGFR1 and sVEGFR2 is associated with the selection of dominant follicle in cows.

PubMed

Ortega Serrano, P V; Guzmán, A; Hernández-Coronado, C G; Castillo-Juárez, H; Rosales-Torres, A M

2016-12-01

The vascular endothelial growth factor (VEGF) is essential for follicular development by promoting follicular angiogenesis, as well as for the proliferation and survival of granulosa cells. The biological effects of VEGF are regulated by two membrane receptors, VEGFR1 and VEGFR2, and two soluble receptors, sVEGFR1 and sVEGFR2, which play an antagonistic role. Thus, the objective of this study was to identify the mRNA expression pattern of total VEGF, VEGFR1, VEGFR2, sVEGFR1 and sVEGFR2 in bovine preselected follicles (PRF) and post-selected follicles (POF). The mRNA expression of these five genes in both granulosa cells (GC) and theca cells (TC) was compared between follicles classified as PRF and POF based on their diameter and on their ratio of estradiol/progesterone (E2/P4). Results showed a lower expression of mRNA of sVEGFR1 and sVEGFR2 in POF than in PRF (p < .05). Regarding the mRNA expression of total VEGF, VEGFR1 and VEGFR2, there was no difference between POF and PRF follicles (p > .05). Our results showed that the mRNA expression of VEGFR2 and sVEGFR1 was more abundant than the expression of VEGFR1 and sVEGFR2, while GC was the main source of mRNA for total VEGF. On the other hand, TC was the follicular compartment where the receptors were most expressed. Our results suggest that non-dominant follicles maintain a greater concentration of the mRNA expression of both membrane and soluble VEGF receptors. On the other hand, follicular dominance is related to a reduction in the mRNA expression of sVEGFR1 and sVEGFR2, which may favour VEGF binding with VEGFR2 and, hence, improve the follicular health and development. © 2016 Blackwell Verlag GmbH.

Fungal and bacterial community succession differs for three wood types during decay in a forest soil.

PubMed

Prewitt, Lynn; Kang, Youngmin; Kakumanu, Madhavi L; Williams, Mark

2014-08-01

Wood decomposition by soil microorganisms is vital to carbon and nutrient cycles of forested ecosystems. Different wood types decompose at different rates; however, it is not known if there are differences in microbial community succession associated with the decay of different wood types. In this study, the microbial community associated with the decay of pine (decay-susceptible wood), western red cedar (decay resistant) and ACQ-treated pine (Ammoniacal Copper Quaternary, preservative-treated pine for decay resistance) in forest soil was characterized using DNA sequencing, phospholipid fatty acid (PLFA) analysis, and microbial activity over a 26-month period. Bray-Curtis ordination using an internal transcribed spacer (ITS) sequence and PLFA data indicated that fungal communities changed during succession and that wood type altered the pattern of succession. Nondecay fungi decreased over the 26 months of succession; however, by 18 months of decay, there was a major shift in the fungal communities. By this time, Trametes elegans dominated cedar and Phlebia radiata dominated pine and ACQ-treated pine. The description of PLFA associated with ACQ-treated pine resembled cedar more than pine; however, both PLFA and ITS descriptions indicated that fungal communities associated with ACQ-treated pine were less dynamic, perhaps a result of the inhibition by the ACQ preservative, compared with pine and cedar. Overall, fungal community composition and succession were associated with wood type. Further research into the differences in community composition will help to discern their functional importance to wood decay.

Detection of canine cytokine gene expression by reverse transcription-polymerase chain reaction.

PubMed

Pinelli, E; van der Kaaij, S Y; Slappendel, R; Fragio, C; Ruitenberg, E J; Bernadina, W; Rutten, V P

1999-08-02

Further characterization of the canine immune system will greatly benefit from the availability of tools to detect canine cytokines. Our interest concerns the study on the role of cytokines in canine visceral leishmaniasis. For this purpose, we have designed specific primers using previously published sequences for the detection of canine IL-2, IFN-gamma and IL10 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). For IL-4, we have cloned and sequenced this cytokine gene, and developed canine-specific primers. To control for sample-to-sample variation in the quantity of mRNA and variation in the RT and PCR reactions, the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a housekeeping gene, were determined in parallel. Primers to amplify G3PDH were designed from consensus sequences obtained from the Genbank database. The mRNA levels of the cytokines mentioned here were detected from ConA-stimulated peripheral mononuclear cells derived from Leishmania-infected dogs. A different pattern of cytokine production among infected animals was found.

Proteinases during Early Development of the Pacific Whiteleg Shrimp Penaeus vannamei.

PubMed

Hernandez-Cortes, Patricia; Rivera-Pérez, Crisalejandra; García-Carreño, Fernando; Martínez-Alarcón, Diana

2017-02-01

During shrimp larval development, changes occur in molecular components. Enzyme activity and mRNA expression of proteinases were assayed in Penaeus vannamei during larval development, which consists of 5 nauplius stages, 3 protozoeal stages, 3 mysis stages, and 12 postlarval stages. Trypsin activity reached a maximum at the beginning of postlarval stages 1 and 2, and significantly decreased in subsequent postlarval stages. Chymotrypsin activity increased at the third protozoeal stage, then significantly decreased in subsequent stages. Identification of proteinase by mass spectrometry and inhibitors allowed us to track their appearance in zymograms and to distinguish between isoenzymes. Chymotrypsin BI and BII had a distinguishing pattern of appearance during larval development, which could compensate for the reduction in trypsin activity. The mRNA content of isotrypsin 21, chymotrypsin 1, and zinc proteinase was differentially expressed in larvae. Zinc proteinase and chymotrypsin 1 mRNA were expressed at a basal content at the beginning of the protozoeal stages, increased by the end of the mysis stages and onward, while isotrypsin 21 mRNA had a peak at mysis stage 3. Transcript changes reflect transcriptional regulation of the proteinases tested. Proteinase mRNA in tissues, other than the digestive gland, suggests potentially different roles besides digestion during ontogeny.

Expression of klotho mRNA and protein in rat brain parenchyma from early postnatal development into adulthood

PubMed Central

Clinton, Sarah M.; Glover, Matthew E.; Maltare, Astha; Laszczyk, Ann M.; Mehi, Stephen J.; Simmons, Rebecca K.; King, Gwendalyn D.

2013-01-01

Without the age-regulating protein klotho, mouse lifespan is shortened and the rapid onset of age-related disorders occurs. Conversely, overexpression of klotho extends mouse lifespan. Klotho is most abundant in kidney and expressed in a limited number of other organs, including the brain, where klotho levels are highest in choroid plexus. Reports vary on where klotho is expressed within the brain parenchyma, and no data is available as to whether klotho levels change across postnatal development. We used in situ hybridization to map klotho mRNA expression in the developing and adult rat brain and report moderate, widespread expression across grey matter regions. mRNA expression levels in cortex, hippocampus, caudate putamen, and amygdala decreased during the second week of life and then gradually rose to adult levels by postnatal day 21. Immunohistochemistry revealed a protein expression pattern similar to the mRNA results, with klotho protein expressed widely throughout the brain. Klotho protein co-localized with both the neuronal marker NeuN, as well as, oligodendrocyte marker olig2. These results provide the first anatomical localization of klotho mRNA and protein in rat brain parenchyma and demonstrate that klotho levels vary during early postnatal development. PMID:23838326

Dispersal, environmental niches and oceanic-scale turnover in deep-sea bivalves

PubMed Central

McClain, Craig R.; Stegen, James C.; Hurlbert, Allen H.

2012-01-01

Patterns of beta-diversity or distance decay at oceanic scales are completely unknown for deep-sea communities. Even when appropriate data exist, methodological problems have made it difficult to discern the relative roles of environmental filtering and dispersal limitation for generating faunal turnover patterns. Here, we combine a spatially extensive dataset on deep-sea bivalves with a model incorporating ecological dynamics and shared evolutionary history to quantify the effects of environmental filtering and dispersal limitation. Both the model and empirical data are used to relate functional, taxonomic and phylogenetic similarity between communities to environmental and spatial distances separating them for 270 sites across the Atlantic Ocean. This study represents the first ocean-wide analysis examining distance decay as a function of a broad suite of explanatory variables. We find that both strong environmental filtering and dispersal limitation drive turnover in taxonomic, functional and phylogenetic composition in deep-sea bivalves, explaining 26 per cent, 34 per cent and 9 per cent of the variation, respectively. This contrasts with previous suggestions that dispersal is not limiting in broad-scale biogeographic and biodiversity patterning in marine systems. However, rates of decay in similarity with environmental distance were eightfold to 44-fold steeper than with spatial distance. Energy availability is the most influential environmental variable evaluated, accounting for 3.9 per cent, 9.4 per cent and 22.3 per cent of the variation in functional, phylogenetic and taxonomic similarity, respectively. Comparing empirical patterns with process-based theoretical predictions provided quantitative estimates of dispersal limitation and niche breadth, indicating that 95 per cent of deep-sea bivalve propagules will be able to persist in environments that deviate from their optimum by up to 2.1 g m−2 yr−1 and typically disperse 749 km from their natal site. PMID:22189399

Advanced cell-based modeling of the royal disease: characterization of the mutated F9 mRNA.

PubMed

Martorell, L; Luce, E; Vazquez, J L; Richaud-Patin, Y; Jimenez-Delgado, S; Corrales, I; Borras, N; Casacuberta-Serra, S; Weber, A; Parra, R; Altisent, C; Follenzi, A; Dubart-Kupperschmitt, A; Raya, A; Vidal, F; Barquinero, J

2017-11-01

Essentials The Royal disease (RD) is a form of hemophilia B predicted to be caused by a splicing mutation. We generated an iPSC-based model of the disease allowing mechanistic studies at the RNA level. F9 mRNA analysis in iPSC-derived hepatocyte-like cells showed the predicted abnormal splicing. Mutated F9 mRNA level was very low but we also found traces of wild type transcripts. Background The royal disease is a form of hemophilia B (HB) that affected many descendants of Queen Victoria in the 19th and 20th centuries. It was found to be caused by the mutation F9 c.278-3A>G. Objective To generate a physiological cell model of the disease and to study F9 expression at the RNA level. Methods Using fibroblasts from skin biopsies of a previously identified hemophilic patient bearing the F9 c.278-3A>G mutation and his mother, we generated induced pluripotent stem cells (iPSCs). Both the patient's and mother's iPSCs were differentiated into hepatocyte-like cells (HLCs) and their F9 mRNA was analyzed using next-generation sequencing (NGS). Results and Conclusion We demonstrated the previously predicted aberrant splicing of the F9 transcript as a result of an intronic nucleotide substitution leading to a frameshift and the generation of a premature termination codon (PTC). The F9 mRNA level in the patient's HLCs was significantly reduced compared with that of his mother, suggesting that mutated transcripts undergo nonsense-mediated decay (NMD), a cellular mechanism that degrades PTC-containing mRNAs. We also detected small proportions of correctly spliced transcripts in the patient's HLCs, which, combined with genetic variability in splicing and NMD machineries, could partially explain some clinical variability among affected members of the European royal families who had lifespans above the average. This work allowed the demonstration of the pathologic consequences of an intronic mutation in the F9 gene and represents the first bona fide cellular model of HB allowing the study of rare mutations at the RNA level. © 2017 International Society on Thrombosis and Haemostasis.

Differential expression of hsp70 stress proteins in human endothelial cells exposed to heat shock and hydrogen peroxide.

PubMed

Jornot, L; Mirault, M E; Junod, A F

1991-09-01

The potential role of oxidative stress conditions in the induction of heat shock proteins was studied in human umbilical vein endothelial cells. We compared the effects of temperature (43 to 45 degrees C), exposure to hydrogen peroxide (H2O2) and oxygen metabolites generated by the enzyme system hypoxanthine-xanthine oxidase (O2- plus H2O2), as well as exposure to 95% O2, on the expression of the major 70-kD heat shock proteins (hsp70). Northern blot analysis indicated that: (1) heat shock induced a rapid and marked increase in hsp70 mRNA levels that reached a maximum during recovery from a 30-min exposure to 45 degrees C; (2) treatment with a 5-mM H2O2 bolus or 50 mU/ml xanthine oxidase also increased hsp70 mRNA levels but to a lesser extent than heat shock (about 10 and 25 times less, respectively); (3) no change was detected after a 5-day exposure to 95% O2. Nuclear run on transcription data and kinetics of mRNA decay in the presence of actinomycin D indicated that the observed increase in hsp70 mRNA levels in both heat-shocked and H2O2-treated cells was mainly due to a transcriptional induction. The kinetics of hsp70 synthesis correlated with the accumulation of hsp70 mRNA. Two-dimensional gel electrophoresis and immunologic analysis of these heat shock proteins revealed a series of at least five distinct hsp70 isoforms induced in heat-shocked cells, whereas only a specific subset of these proteins, mainly one acidic isoform, was induced in very low amounts in response to H2O2 treatment. These results clearly indicate that the endothelial cell responses to oxidative stress and heat shock differ in both qualitative and quantitative terms in respect to hsp70 induction. They also suggest that the intensity of this response to oxidative stress conditions may vary depending on the nature of the oxidative challenge.

Peeling skin syndrome: genetic defects in late terminal differentiation of the epidermis.

PubMed

Bowden, Paul E

2011-03-01

In this issue, Israeli and colleagues confirm that homozygous mutations in corneodesmosin (CDSN) cause type B peeling skin syndrome (PSS), an autosomal recessive skin disorder. The deletion mutation described resulted in a frameshift, producing a downstream premature stop codon and early truncation of the protein. The recently described CDSN nonsense mutation in another PSS family also resulted in protein truncation and nonsense-mediated mRNA decay. Type B generalized PSS can now be clearly distinguished from acral PSS, caused by mutations in transglutaminase 5. This directly affects cornified envelope cross-linking rather than corneodesmosome adherence. These observations provide new insight into the molecular defects underlying two closely related forms of PSS.

Experimental taphonomy and the anatomy and diversity of the earliest fossil vertebrates (Chengjiang Biota, Cambrian, China)

NASA Astrophysics Data System (ADS)

Purnell, Mark; Gabbott, Sarah; Murdock, Duncan; Cong, Peiyun

2016-04-01

The oldest fossil vertebrates are from the Lower Cambrian Chengjiang biota of China, which contains four genera of fish-like, primitive vertebrates: Haikouichthys, Myllokunmingia, Zhongjianichthys and Zhongxiniscus. These fossils play key roles in calibrating molecular clocks and informing our view of the anatomy of animals close to the origin of vertebrates, potentially including transitional forms between vertebrates and their nearest relatives. Despite the evident importance of these fossils, the degree to which taphonomic processes have affected their anatomical completeness has not been investigated. For example, some or all might have been affected by stemward slippage - the pattern observed in experimental decay of non-biomineralised chordates in which preferential decay of synapomorphies and retention of plesiomorphic characters would cause fossil taxa to erroneously occupy more basal positions than they should. This hypothesis is based on experimental data derived from decay of non-biomineralised chordates under laboratory conditions. We have expanded this analysis to include a broader range of potentially significant environmental variables; we have also compared and combined the results of experiments from several taxa to identify general patterns of chordate decay. Examination of the Chengjiang vertebrates in the light of these results demonstrates that, contrary to some assertions, experimentally derived models of phylogenetic bias are applicable to fossils. Anatomical and phylogenetic interpretations of early vertebrates that do not take taphonomic biases into account risk overestimating diversity and the evolutionary significance of differences between fossil specimens.

Quantification of cytokine mRNA in peripheral blood mononuclear cells using branched DNA (bDNA) technology.

PubMed

Shen, L P; Sheridan, P; Cao, W W; Dailey, P J; Salazar-Gonzalez, J F; Breen, E C; Fahey, J L; Urdea, M S; Kolberg, J A

1998-06-01

Changes in the patterns of cytokine expression are thought to be of central importance in human infectious and inflammatory diseases. As such, there is a need for precise, reproducible assays for quantification of cytokine mRNA that are amenable to routine use in a clinical setting. In this report, we describe the design and performance of a branched DNA (bDNA) assay for the direct quantification of multiple cytokine mRNA levels in peripheral blood mononuclear cells (PBMCs). Oligonucleotide target probe sets were designed for several human cytokines, including TNFalpha, IL-2, IL-4, IL-6, IL-10, and IFNgamma. The bDNA assay yielded highly reproducible quantification of cytokine mRNAs, exhibited a broad linear dynamic range of over 3-log10, and showed a sensitivity sufficient to measure at least 3000 molecules. The potential clinical utility of the bDNA assay was explored by measuring cytokine mRNA levels in PBMCs from healthy and immunocompromised individuals. Cytokine expression levels in PBMCs from healthy blood donors were found to remain relatively stable over a one-month period of time. Elevated levels of IFNgamma mRNA were detected in PBMCs from HIV-1 seropositive individuals, but no differences in mean levels of TNFalpha or IL-6 mRNA were detected between seropositive and seronegative individuals. By providing a reproducible method for quantification of low abundance transcripts in clinical specimens, the bDNA assay may be useful for studies addressing the role of cytokine expression in disease.

Time-dependent effects of intermittent hydrostatic pressure on articular chondrocyte type II collagen and aggrecan mRNA expression.

PubMed

Smith, R L; Lin, J; Trindade, M C; Shida, J; Kajiyama, G; Vu, T; Hoffman, A R; van der Meulen, M C; Goodman, S B; Schurman, D J; Carter, D R

2000-01-01

The normal loading of joints during daily activities causes the articular cartilage to be exposed to high levels of intermittent hydrostatic pressure. This study quantified effects of intermittent hydrostatic pressure on expression of mRNA for important extracellular matrix constituents. Normal adult bovine articular chondrocytes were isolated and tested in primary culture, either as high-density monolayers or formed aggregates. Loaded cells were exposed to 10 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for periods of 2, 4, 8, 12, and 24 hrs. Other cells were intermittently loaded for a period of 4 hrs per day for 4 days. Semiquantitative reverse transcription polymerase chain reaction assays were used to assess mRNA signal levels for collagen types II and I and aggrecan. The results showed that type II collagen mRNA signal levels exhibited a biphasic pattern, with an initial increase of approximately five-fold at 4 and 8 hrs that subsequently decreased by 24 hrs. In contrast, aggrecan mRNA signal increased progressively up to three-fold throughout the loading period. Changing the loading profile to 4 hrs per day for 4 days increased the mRNA signal levels for type II collagen nine-fold and for aggrecan twenty-fold when compared to unloaded cultures. These data suggest that specific mechanical loading protocols may be required to optimally promote repair and regeneration of diseased joints.

Preformed mRNA in Cotyledons of Ungerminated Seeds of Cicer arietinum L. 1

PubMed Central

Matilla, Angel; Nicolás, Gregorio; Vicente, Oscar; Sierra, José Manuel

1980-01-01

Polyadenylated RNA was isolated from total RNA extracted from cotyledons of ungerminated or 18-hour-germinated chick-pea seeds by affinity chromatography on oligo(dT)-cellulose. Both poly(A)-containing RNA fractions exhibited a template activity when assayed in two cell-free translation systems, wheat germ extracts, and nuclease-treated reticulocyte lysates. Translation of preformed mRNA from cotyledons of dry seeds was completely abolished in the presence of several inhibitors of polypeptide chain initiation and also in the presence of the two “cap” analogues m7 GTP and m7 GMP. The patterns of polypeptides synthesized by translation of poly(A)-containing RNAs from cotyledons of ungerminated or 18-hour-germinated seeds, in the wheat germ system, analyzed by electrophoresis and autoradiography, were similar but not identical. It is concluded that cotyledons of dry Cicer arietinum L. seeds contain preformed mRNA. PMID:16661345

Cannabinoid receptor CB1 mRNA is highly expressed in the rat ciliary body: implications for the antiglaucoma properties of marihuana.

PubMed

Porcella, A; Casellas, P; Gessa, G L; Pani, L

1998-07-15

We used RT-PCR to measure relative differences in cannabinoid receptor (CB) mRNAs in the rat eye, comparing CB1 or CB2 transcripts to that of the normalizing reference gene beta2 microglobulin (beta2m). Significantly higher levels of CB1 mRNA levels were found in the ciliary body (0.84+/-0.05% of beta2m) than in the iris, (0.34+/-0.04% of beta2m), retina (0.07+/-0.005% of beta2m) and choroid (0.06+/-0.005% of beta2m). CB2 mRNA was undetectable. This expression pattern supports a specific role for the CB1 receptor in controlling intraocular pressure, helping to explain the antiglaucoma property of cannabinoids. Copyright 1998 Elsevier Science B.V. All rights reserved.

O6-Methylguanine-DNA Methyltransferase (MGMT) mRNA Expression Predicts Outcome in Malignant Glioma Independent of MGMT Promoter Methylation

PubMed Central

Kreth, Simone; Thon, Niklas; Eigenbrod, Sabina; Lutz, Juergen; Ledderose, Carola; Egensperger, Rupert; Tonn, Joerg C.; Kretzschmar, Hans A.; Hinske, Ludwig C.; Kreth, Friedrich W.

2011-01-01

Background We analyzed prospectively whether MGMT (O6-methylguanine-DNA methyltransferase) mRNA expression gains prognostic/predictive impact independent of MGMT promoter methylation in malignant glioma patients undergoing radiotherapy with concomitant and adjuvant temozolomide or temozolomide alone. As DNA-methyltransferases (DNMTs) are the enzymes responsible for setting up and maintaining DNA methylation patterns in eukaryotic cells, we analyzed further, whether MGMT promoter methylation is associated with upregulation of DNMT expression. Methodology/Principal Findings Adult patients with a histologically proven malignant astrocytoma (glioblastoma: N = 53, anaplastic astrocytoma: N = 10) were included. MGMT promoter methylation was determined by methylation-specific PCR (MSP) and sequencing analysis. Expression of MGMT and DNMTs mRNA were analysed by real-time qPCR. Prognostic factors were obtained from proportional hazards models. Correlation between MGMT mRNA expression and MGMT methylation status was validated using data from the Cancer Genome Atlas (TCGA) database (N = 229 glioblastomas). Low MGMT mRNA expression was strongly predictive for prolonged time to progression, treatment response, and length of survival in univariate and multivariate models (p<0.0001); the degree of MGMT mRNA expression was highly correlated with the MGMT promoter methylation status (p<0.0001); however, discordant findings were seen in 12 glioblastoma patients: Patients with methylated tumors with high MGMT mRNA expression (N = 6) did significantly worse than those with low transcriptional activity (p<0.01). Conversely, unmethylated tumors with low MGMT mRNA expression (N = 6) did better than their counterparts. A nearly identical frequency of concordant and discordant findings was obtained by analyzing the TCGA database (p<0.0001). Expression of DNMT1 and DNMT3b was strongly upregulated in tumor tissue, but not correlated with MGMT promoter methylation and MGMT mRNA expression. Conclusions/Significance MGMT mRNA expression plays a direct role for mediating tumor sensitivity to alkylating agents. Discordant findings indicate methylation-independent pathways of MGMT expression regulation. DNMT1 and DNMT3b are likely to be involved in CGI methylation. However, their exact role yet has to be defined. PMID:21365007

Deregulation of versican and elastin binding protein in solar elastosis.

PubMed

Knott, Anja; Reuschlein, Katja; Lucius, Ralph; Stäb, Franz; Wenck, Horst; Gallinat, Stefan

2009-04-01

Several changes in skin appearance including loss of elasticity and wrinkle formation are associated with alterations in the composition of the dermal extracellular matrix. They are induced by intrinsic aging or by environmental factors such as UV light referred to as photoaging. A general characteristic in the histology of photoaged skin is the accumulation of elastotic material suggesting impaired formation and/or massive breakdown of elastic fibres. In order to shed light on some of the underlying mechanisms we tracked two of the major players in elastic fibre formation in different skin conditions: EBP (elastin binding protein), a regulator of elastic fibre assembly and VER (versican), a component of functional elastic fibres as well as non-functional elastotic material. Using quantitative RT-PCR on skin biopsies we found that the expression levels of VER and EBP were unaltered during intrinsic skin aging. Upon acute UV stress however, VER and EBP showed different regulation patterns: VER mRNA increased after 6 h and was further up-regulated until 24 h. The EBP mRNA by contrast was reduced after 6 h but showed massive induction at 24 h after acute UV stress. In chronically sun-exposed skin, VER protein was accumulated similar to elastotic material in the extracellular space, whereas its mRNA level was consistently reduced compared to sun-protected skin. The EBP mRNA by contrast showed slightly increased expression levels in the sun-exposed area compared to its sun-protected counterpart. Based on these data we propose a model which may help to explain parts of the mechanisms leading to the formation of elastotic masses. We further hypothesize that the presence of elastotic material triggers some yet unknown feedback mechanism(s) resulting in altered expression patterns of VER and EBP in chronically sun-exposed skin.

Differential expression of the ufo/axl oncogene in human leukemia-lymphoma cell lines.

PubMed

Challier, C; Uphoff, C C; Janssen, J W; Drexler, H G

1996-05-01

The ufo protein (also termed axl) is a member of a new family of receptor tyrosine kinases and is encoded by a transforming gene that was initially isolated from primary human myeloid leukemia cells by DNA-mediated transformation of NIH/3T3 cells. The ligand, Gas6, a protein S-related molecule lacking any known function yet, has recently been identified. We report the expression pattern of ufo mRNA in a panel of 76 human continuous leukemia-lymphoma cell lines. The gene was not expressed in cell lines derived from lymphoid malignancies (n=28), but transcription was seen in 3/11 myeloid, 0/6 monocytic, 9/13 erythroid and 11/18 megakaryocytic cell lines. Several cell lines were treated with phorbol ester leading to significant upregulation of the ufo message in constitutively positive cells. An apparent ufo mRNA overexpression was not found in any of the positive leukemia cell lines, but was identified in the drug-resistant subclones of the cervix carcinoma cell line HeLa. Southern blot analysis of restriction enzyme-digested genomic DNA did not provide evidence for gene amplification, but the HeLa subclones showed banding patterns suggestive of gene rearrangement. Two main ufo mRNA bands of 3.2 and 5.0 kb were identified; no differences in the half-lives (t1/2 = 2.5 h) of these two mRNA species could be identified. In summary, ufo, representing a novel type of receptor tyrosine kinase, is expressed solely in myeloid and erythro-megakaryocytic leukemias but not in lymphoid malignancies. These and previous data suggest an involvement of the ufo receptor tyrosine kinase in normal and malignant myelopoiesis; however, its exact role, if any, and mode of operation in leukemogenesis remains to be determined.

Small and intermediate conductance Ca(2+)-activated K+ channels confer distinctive patterns of distribution in human tissues and differential cellular localisation in the colon and corpus cavernosum.

PubMed

Chen, Mao Xiang; Gorman, Shelby A; Benson, Bill; Singh, Kuljit; Hieble, J Paul; Michel, Martin C; Tate, Simon N; Trezise, Derek J

2004-06-01

The SK/IK family of small and intermediate conductance calcium-activated potassium channels contains four members, SK1, SK2, SK3 and IK1, and is important for the regulation of a variety of neuronal and non-neuronal functions. In this study we have analysed the distribution of these channels in human tissues and their cellular localisation in samples of colon and corpus cavernosum. SK1 mRNA was detected almost exclusively in neuronal tissues. SK2 mRNA distribution was restricted but more widespread than SK1, and was detected in adrenal gland, brain, prostate, bladder, liver and heart. SK3 mRNA was detected in almost every tissue examined. It was highly expressed in brain and in smooth muscle-rich tissues including the clitoris and the corpus cavernosum, and expression in the corpus cavernosum was upregulated up to 5-fold in patients undergoing sex-change operations. IK1 mRNA was present in surface-rich, secretory and inflammatory cell-rich tissues, highest in the trachea, prostate, placenta and salivary glands. In detailed immunohistochemical studies of the colon and the corpus cavernosum, SK1-like immunoreactivity was observed in the enteric neurons. SK3-like immunoreactivity was observed strongly in smooth muscle and vascular endothelium. IK1-like immunoreactivity was mainly observed in inflammatory cells and enteric neurons of the colon, but absent in corpus cavernosum. These distinctive patterns of distribution suggest that these channels are likely to have different biological functions and could be specifically targeted for a number of human diseases, such as irritable bowel syndrome, hypertension and erectile dysfunction.

Neurotensin gene expression increases during proestrus in the rostral medial preoptic nucleus: potential for direct communication with gonadotropin-releasing hormone neurons.

PubMed

Smith, M J; Wise, P M

2001-07-01

Neurotensin (NT)-containing neurons in the rostral portion of the medial preoptic nucleus (rMPN) of the brain may play a key role in regulating the pattern of secretion of GnRH, thereby influencing the reproductive cycle in females. The major goals of this study were to determine whether NT messenger RNA (mRNA) levels in the rMPN exhibit a unique pattern of expression in temporal association with the preovulatory LH surge and to assess whether NT neurons may communicate directly with GnRH neurons. We analyzed NT gene expression in rats using in situ hybridization over the day of proestrus and compared this with diestrous day 1. We also determined whether the high-affinity NT receptor (NT1) is expressed in GnRH neurons using dual-label in situ hybridization and whether this expression varies over the estrous cycle. We found that NT mRNA levels in the rMPN increase significantly on the day of proestrus, rising before the LH surge. No such change was detected on diestrous day 1, when the LH surge does not occur. Furthermore, we observed that a significant number of GnRH neurons coexpress NT1 mRNA and that the number of GnRH neurons expressing NT1 mRNA peaks on proestrus. Together with previous findings, our results suggest that increased expression of NT in the rMPN may directly stimulate GnRH neurons on proestrus, contributing to the LH surge. In addition, our results suggest that responsiveness of GnRH neurons to NT stimulation is enhanced on proestrus due to increased expression of NT receptors within GnRH neurons.

Distribution of glycinergic neuronal somata in the rat spinal cord.

PubMed

Hossaini, Mehdi; French, Pim J; Holstege, Jan C

2007-04-20

Glycine transporter 2 (GlyT2) mRNA is exclusively expressed in glycinergic neurons, and is presently considered a reliable marker for glycinergic neuronal somata. In this study, we have performed non-radioactive in situ hybridization to localize GlyT2 mRNA in fixed free-floating sections of cervical (C2 and C6), thoracic (T5), lumbar (L2 and L5) and sacral (S1) segments of the rat spinal cord. The results showed that in all segments the majority of the GlyT2 mRNA labeled (glycinergic) neuronal somata was present in the deep dorsal horn and the intermediate zone (laminae III-VIII), with around 50% (range 43.7-70.9%) in laminae VII&VIII. In contrast, the superficial dorsal horn, the motoneuronal cell groups and the area around the central canal contained only few glycinergic neuronal somata. The density (number of glycinergic neuronal somata per mm(2)) was also low in these areas, while the highest densities were found in laminae V to VIII. The lateral spinal nucleus and the lateral cervical nucleus also contained a limited number of glycinergic neurons. Our findings showed that the distribution pattern of the glycinergic neuronal somata is similar in all the examined segments. The few differences that were found in the relative laminar distribution between some of the segments, are most likely due to technical reasons. We therefore conclude that the observed distribution pattern of glycinergic neuronal somata is present throughout the spinal cord. Our findings further showed that the non-radioactive in situ hybridization technique for identifying GlyT2 mRNA in fixed free-floating sections is a highly efficient tool for identifying glycinergic neurons in the spinal cord.

Life cycles of persistent anomalies. I - Evolution of 500 mb height fields

NASA Technical Reports Server (NTRS)

Dole, Randall M.

1989-01-01

The life cycles of persistent anomalies of the extratropical Northern Hemisphere wintertime circulation are studied, focusing on the typical characteristics of the 500 mb height anomaly and flow patterns accompanying the development and breakdown of large-scale flow anomalies in the eastern North Atlantic and the northern Soviet Union. Following onset, anomaly centers develop and intensify in sequence downstream from the main center, forming a quasi-stationary wavetrain pattern. From development through decay, corresponding positive and negative patterns have similar evolutions.

Regnase-1 Maintains Iron Homeostasis via the Degradation of Transferrin Receptor 1 and Prolyl-Hydroxylase-Domain-Containing Protein 3 mRNAs.

PubMed

Yoshinaga, Masanori; Nakatsuka, Yoshinari; Vandenbon, Alexis; Ori, Daisuke; Uehata, Takuya; Tsujimura, Tohru; Suzuki, Yutaka; Mino, Takashi; Takeuchi, Osamu

2017-05-23

Iron metabolism is regulated by transcriptional and post-transcriptional mechanisms. The mRNA of the iron-controlling gene, transferrin receptor 1 (TfR1), has long been believed to be negatively regulated by a yet-unidentified endonuclease. Here, we show that the endonuclease Regnase-1 is critical for the degradation of mRNAs involved in iron metabolism in vivo. First, we demonstrate that Regnase-1 promotes TfR1 mRNA decay. Next, we show that Regnase-1 -/- mice suffer from severe iron deficiency anemia, although hepcidin expression is downregulated. The iron deficiency anemia is induced by a defect in duodenal iron uptake. We reveal that duodenal Regnase-1 controls the expression of PHD3, which impairs duodenal iron uptake via HIF2α suppression. Finally, we show that Regnase-1 is a HIF2α-inducible gene and thus provides a positive feedback loop for HIF2α activation via PHD3. Collectively, these results demonstrate that Regnase-1-mediated regulation of iron-related transcripts is essential for the maintenance of iron homeostasis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

The CCR4-NOT Complex Is Implicated in the Viability of Aneuploid Yeasts

PubMed Central

Tange, Yoshie; Kurabayashi, Atsushi; Goto, Bunshiro; Hoe, Kwang-Lae; Kim, Dong-Uk; Park, Han-Oh; Hayles, Jacqueline; Chikashige, Yuji; Tsutumi, Chihiro; Hiraoka, Yasushi; Yamao, Fumiaki; Nurse, Paul; Niwa, Osami

2012-01-01

To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability. PMID:22737087

UPF1 silenced cellular model systems for screening of read-through agents active on β039 thalassemia point mutation.

PubMed

Salvatori, Francesca; Pappadà, Mariangela; Breveglieri, Giulia; D'Aversa, Elisabetta; Finotti, Alessia; Lampronti, Ilaria; Gambari, Roberto; Borgatti, Monica

2018-05-15

Nonsense mutations promote premature translational termination, introducing stop codons within the coding region of mRNAs and causing inherited diseases, including thalassemia. For instance, in β 0 39 thalassemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, ribosomal read-through molecules, such as aminoglycoside antibiotics, have been tested on mRNAs carrying premature stop codons. These findings have introduced new hopes for the development of a pharmacological approach to the β 0 39 thalassemia therapy. While several strategies, designed to enhance translational read-through, have been reported to inhibit NMD efficiency concomitantly, experimental tools for systematic analysis of mammalian NMD inhibition by translational read-through are lacking. We developed a human cellular model of the β 0 39 thalassemia mutation with UPF-1 suppressed and showing a partial NMD suppression. This novel cellular model could be used for the screening of molecules exhibiting preferential read-through activity allowing a great rescue of the mutated transcripts.

Staying alive in adversity: transcriptome dynamics in the stress-resistant dauer larva.

PubMed

Holt, Suzan J

2006-10-01

In response to food depletion and overcrowding, the soil nematode Caenorhabditis elegans can arrest development and form an alternate third larval stage called the dauer. Though nonfeeding, the dauer larva is long lived and stress resistant. Metabolic and transcription rates are lowered but the transcriptome of the dauer is complex. In this study, distribution analysis of transcript profiles generated by Serial Analysis of Gene Expression (SAGE) in dauer larvae and in mixed developmental stages is presented. An inverse relationship was observed between frequency and abundance/copy number of SAGE tag types (transcripts) in both profiles. In the dauer profile, a relatively greater proportion of highly abundant transcripts was counterbalanced by a smaller fraction of low to moderately abundant transcripts. Comparisons of abundant tag counts between the two profiles revealed relative enrichment in the dauer profile of transcripts with predicted or known involvement in ribosome biogenesis and protein synthesis, membrane transport, and immune responses. Translation-coupled mRNA decay is proposed as part of an immune-like stress response in the dauer larva. An influence of genomic region on transcript level may reflect the coordination of transcription and mRNA turnover.

Extensive cross-regulation of post-transcriptional regulatory networks in Drosophila

DOE PAGES

Stoiber, Marcus H.; Olson, Sara; May, Gemma E.; ...

2015-08-20

In eukaryotic cells, RNAs exist as ribonucleoprotein particles (RNPs). Despite the importance of these complexes in many biological processes, including splicing, polyadenylation, stability, transportation, localization, and translation, their compositions are largely unknown. We affinity-purified 20 distinct RNA-binding proteins (RBPs) from cultured Drosophila melanogaster cells under native conditions and identified both the RNA and protein compositions of these RNP complexes. We identified “high occupancy target” (HOT) RNAs that interact with the majority of the RBPs we surveyed. HOT RNAs encode components of the nonsense-mediated decay and splicing machinery, as well as RNA-binding and translation initiation proteins. The RNP complexes contain proteinsmore » and mRNAs involved in RNA binding and post-transcriptional regulation. Genes with the capacity to produce hundreds of mRNA isoforms, ultracomplex genes, interact extensively with heterogeneous nuclear ribonuclear proteins (hnRNPs). Our data are consistent with a model in which subsets of RNPs include mRNA and protein products from the same gene, indicating the widespread existence of auto-regulatory RNPs. Lastly, from the simultaneous acquisition and integrative analysis of protein and RNA constituents of RNPs, we identify extensive cross-regulatory and hierarchical interactions in post-transcriptional control.« less

Extensive cross-regulation of post-transcriptional regulatory networks in Drosophila

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

Stoiber, Marcus H.; Olson, Sara; May, Gemma E.

In eukaryotic cells, RNAs exist as ribonucleoprotein particles (RNPs). Despite the importance of these complexes in many biological processes, including splicing, polyadenylation, stability, transportation, localization, and translation, their compositions are largely unknown. We affinity-purified 20 distinct RNA-binding proteins (RBPs) from cultured Drosophila melanogaster cells under native conditions and identified both the RNA and protein compositions of these RNP complexes. We identified “high occupancy target” (HOT) RNAs that interact with the majority of the RBPs we surveyed. HOT RNAs encode components of the nonsense-mediated decay and splicing machinery, as well as RNA-binding and translation initiation proteins. The RNP complexes contain proteinsmore » and mRNAs involved in RNA binding and post-transcriptional regulation. Genes with the capacity to produce hundreds of mRNA isoforms, ultracomplex genes, interact extensively with heterogeneous nuclear ribonuclear proteins (hnRNPs). Our data are consistent with a model in which subsets of RNPs include mRNA and protein products from the same gene, indicating the widespread existence of auto-regulatory RNPs. Lastly, from the simultaneous acquisition and integrative analysis of protein and RNA constituents of RNPs, we identify extensive cross-regulatory and hierarchical interactions in post-transcriptional control.« less

Growth cycle of Helicobacter pylori in gastric mucous layer.

PubMed

Nakazawa, Teruko

2002-12-01

Helicobacter pylori bacterium is characterized by its strong urease activity. Our studies on the role of H. pylori urease revealed; (i) it is essential for colonization, (ii) exogenous urea is required for acid resistance, (iii) the bacteria have the ability to move toward urea and sodium bicarbonate, (iv) urea hydrolysis accelerates chemotactic locomotion, and (v) decay of urease mRNA to accomplish the active center is pH-regulated; i.e., the mRNA is stabilized and destabilized under acidic and neutral conditions, respectively. Based on the above results, I propose the growth cycle of H. pylori in gastric mucous layer. H. pylori bacteria proliferate on the epithelial cell surface by utilizing nutrients derived from degraded cells. Proliferated bacteria leave the cell surface to pH-variable region where they encounter strong acid. Urease is activated with simultaneous opening of UreI channel so that urea is hydrolyzed to neutralize acid. Chemotaxis of H. pylori toward urea and sodium bicarbonate that are abundant on the cell surface is accelerated by urea hydrolysis so that the bacteria go back to the cell surface for the next round of proliferation. This growth cycle may allow the bacteria to infect persistently in the stomach.

Mediator binds to boundaries of chromosomal interaction domains and to proteins involved in DNA looping, RNA metabolism, chromatin remodeling, and actin assembly

PubMed Central

Chereji, Răzvan V.; Bharatula, Vasudha; Elfving, Nils; Blomberg, Jeanette; Larsson, Miriam; Morozov, Alexandre V.; Broach, James R.

2017-01-01

Abstract Mediator is a multi-unit molecular complex that plays a key role in transferring signals from transcriptional regulators to RNA polymerase II in eukaryotes. We have combined biochemical purification of the Saccharomyces cerevisiae Mediator from chromatin with chromatin immunoprecipitation in order to reveal Mediator occupancy on DNA genome-wide, and to identify proteins interacting specifically with Mediator on the chromatin template. Tandem mass spectrometry of proteins in immunoprecipitates of mediator complexes revealed specific interactions between Mediator and the RSC, Arp2/Arp3, CPF, CF 1A and Lsm complexes in chromatin. These factors are primarily involved in chromatin remodeling, actin assembly, mRNA 3′-end processing, gene looping and mRNA decay, but they have also been shown to enter the nucleus and participate in Pol II transcription. Moreover, we have found that Mediator, in addition to binding Pol II promoters, occupies chromosomal interacting domain (CID) boundaries and that Mediator in chromatin associates with proteins that have been shown to interact with CID boundaries, such as Sth1, Ssu72 and histone H4. This suggests that Mediator plays a significant role in higher-order genome organization. PMID:28575439

Hypomorphic NOTCH3 mutation in an Italian family with CADASIL features.

PubMed

Moccia, Marcello; Mosca, Lorena; Erro, Roberto; Cervasio, Mariarosaria; Allocca, Roberto; Vitale, Carmine; Leonardi, Antonio; Caranci, Ferdinando; Del Basso-De Caro, Maria Laura; Barone, Paolo; Penco, Silvana

2015-01-01

The cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is because of NOTCH3 mutations affecting the number of cysteine residues. In this view, the role of atypical NOTCH3 mutations is still debated. Therefore, we investigated a family carrying a NOTCH3 nonsense mutation, with dominantly inherited recurrent cerebrovascular disorders. Among 7 family members, 4 received a clinical diagnosis of CADASIL. A heterozygous truncating mutation in exon 3 (c.307C>T, p.Arg103X) was found in the 4 clinically affected subjects and in one 27-year old lady, only complaining of migraine with aura. Magnetic resonance imaging scans found typical signs of small-vessel disease in the 4 affected subjects, supporting the clinical diagnosis. Skin biopsies did not show the typical granular osmiophilic material, but only nonspecific signs of vascular damage, resembling those previously described in Notch3 knockout mice. Interestingly, messenger RNA (mRNA) analysis supports the hypothesis of an atypical NOTCH3 mutation, suggesting a nonsense-mediated mRNA decay. In conclusion, the present study broadens the spectrum of CADASIL mutations, and, therefore, opens new insights about Notch3 signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

Dynamic Maternal Gradients Control Timing and Shift-Rates for Drosophila Gap Gene Expression

PubMed Central

Verd, Berta; Crombach, Anton

2017-01-01

Pattern formation during development is a highly dynamic process. In spite of this, few experimental and modelling approaches take into account the explicit time-dependence of the rules governing regulatory systems. We address this problem by studying dynamic morphogen interpretation by the gap gene network in Drosophila melanogaster. Gap genes are involved in segment determination during early embryogenesis. They are activated by maternal morphogen gradients encoded by bicoid (bcd) and caudal (cad). These gradients decay at the same time-scale as the establishment of the antero-posterior gap gene pattern. We use a reverse-engineering approach, based on data-driven regulatory models called gene circuits, to isolate and characterise the explicitly time-dependent effects of changing morphogen concentrations on gap gene regulation. To achieve this, we simulate the system in the presence and absence of dynamic gradient decay. Comparison between these simulations reveals that maternal morphogen decay controls the timing and limits the rate of gap gene expression. In the anterior of the embyro, it affects peak expression and leads to the establishment of smooth spatial boundaries between gap domains. In the posterior of the embryo, it causes a progressive slow-down in the rate of gap domain shifts, which is necessary to correctly position domain boundaries and to stabilise the spatial gap gene expression pattern. We use a newly developed method for the analysis of transient dynamics in non-autonomous (time-variable) systems to understand the regulatory causes of these effects. By providing a rigorous mechanistic explanation for the role of maternal gradient decay in gap gene regulation, our study demonstrates that such analyses are feasible and reveal important aspects of dynamic gene regulation which would have been missed by a traditional steady-state approach. More generally, it highlights the importance of transient dynamics for understanding complex regulatory processes in development. PMID:28158178

Dynamic Maternal Gradients Control Timing and Shift-Rates for Drosophila Gap Gene Expression.

PubMed

Verd, Berta; Crombach, Anton; Jaeger, Johannes

2017-02-01

Pattern formation during development is a highly dynamic process. In spite of this, few experimental and modelling approaches take into account the explicit time-dependence of the rules governing regulatory systems. We address this problem by studying dynamic morphogen interpretation by the gap gene network in Drosophila melanogaster. Gap genes are involved in segment determination during early embryogenesis. They are activated by maternal morphogen gradients encoded by bicoid (bcd) and caudal (cad). These gradients decay at the same time-scale as the establishment of the antero-posterior gap gene pattern. We use a reverse-engineering approach, based on data-driven regulatory models called gene circuits, to isolate and characterise the explicitly time-dependent effects of changing morphogen concentrations on gap gene regulation. To achieve this, we simulate the system in the presence and absence of dynamic gradient decay. Comparison between these simulations reveals that maternal morphogen decay controls the timing and limits the rate of gap gene expression. In the anterior of the embyro, it affects peak expression and leads to the establishment of smooth spatial boundaries between gap domains. In the posterior of the embryo, it causes a progressive slow-down in the rate of gap domain shifts, which is necessary to correctly position domain boundaries and to stabilise the spatial gap gene expression pattern. We use a newly developed method for the analysis of transient dynamics in non-autonomous (time-variable) systems to understand the regulatory causes of these effects. By providing a rigorous mechanistic explanation for the role of maternal gradient decay in gap gene regulation, our study demonstrates that such analyses are feasible and reveal important aspects of dynamic gene regulation which would have been missed by a traditional steady-state approach. More generally, it highlights the importance of transient dynamics for understanding complex regulatory processes in development.

The impact of water loading on postglacial decay times in Hudson Bay

NASA Astrophysics Data System (ADS)

Han, Holly Kyeore; Gomez, Natalya

2018-05-01

Ongoing glacial isostatic adjustment (GIA) due to surface loading (ice and water) variations during the last glacial cycle has been contributing to sea-level changes globally throughout the Holocene, especially in regions like Canada that were heavily glaciated during the Last Glacial Maximum (LGM). The spatial and temporal distribution of GIA, as manifested in relative sea-level (RSL) change, are sensitive to the ice history and the rheological structure of the solid Earth, both of which are uncertain. It has been shown that RSL curves near the center of previously glaciated regions with no ongoing surface loading follow an exponential-like form, with the postglacial decay times associated with that form having a weak sensitivity to the details of the ice loading history. Postglacial decay time estimates thus provide a powerful datum for constraining the Earth's viscous structure and improving GIA predictions. We explore spatial patterns of postglacial decay time predictions in Hudson Bay by decomposing numerically modeled RSL changes into contributions from water and ice loading effects, and computing their relative impact on the decay times. We demonstrate that ice loading can contribute a strong geographic trend on the decay time estimates if the time window used to compute decay times includes periods that are temporally close to (i.e. contemporaneous with, or soon after) periods of active loading. This variability can be avoided by choosing a suitable starting point for the decay time window. However, more surprisingly, we show that across any adopted time window, water loading effects associated with inundation into, and postglacial flux out of, Hudson Bay and James Bay will impart significant geographic variability onto decay time estimates. We emphasize this issue by considering both maps of predicted decay times across the region and site-specific estimates, and we conclude that variability in observed decay times (whether based on existing or future data sets) may reflect this water loading signal.

Temporal and Spatial Post-Transcriptional Regulation of Zebrafish Tie1 mRNA by Long Noncoding RNA During Brain Vascular Assembly.

PubMed

Chowdhury, Tamjid A; Koceja, Chris; Eisa-Beygi, Shahram; Kleinstiver, Benjamin P; Kumar, Suresh N; Lin, Chien-Wei; Li, Keguo; Prabhudesai, Shubhangi; Joung, J Keith; Ramchandran, Ramani

2018-05-03

Tie1 (tyrosine kinase containing immunoglobulin and epidermal growth factor homology 1), an endothelial and hematopoietic cell-specific receptor tyrosine kinase, is an important regulator of angiogenesis and critical for maintaining vascular integrity. The post-transcriptional regulation of tie1 mRNA expression is not understood, but it might partly explain Tie1's differential expression pattern in endothelium. Following up on our previous work that identified natural antisense transcripts from the tie1 locus- tie1 antisense ( tie1AS ), which regulates tie1 mRNA levels in zebrafish-we attempted to identify the mechanism of this regulation. Through in vitro and in vivo ribonucleoprotein binding studies, we demonstrated that tie1AS long noncoding RNA interacts with an RNA binding protein-embryonic lethal and abnormal vision Drosophila-like 1 (Elavl1)-that regulates tie1 mRNA levels. When we disrupted the interaction between tie1AS and Elavl1 by using constitutively active antisense morpholino oligonucleotides or photoactivatable morpholino oligonucleotides, tie1 mRNA levels increased between 26 and 31 hours post-fertilization, particularly in the head. This increase correlated with dilation of primordial midbrain channels, smaller eyes, and reduced ventricular space. We also observed these phenotypes when we used CRISPR (clustered regularly interspaced short palindromic repeats)-mediated CRISPRi (CRISPR-mediated interference) to knock down tie1AS . Treatment of the morpholino oligonucleotide-injected embryos with a small molecule that decreased tie1 mRNA levels rescued all 3 abnormal phenotypes. We identified a novel mode of temporal and spatial post-transcriptional regulation of tie1 mRNA. It involves long noncoding RNA, tie1AS, and Elavl1 (an interactor of tie1AS ). © 2018 American Heart Association, Inc.

Molecular cloning of cDNAs for the nerve-cell specific phosphoprotein, synapsin I.

PubMed Central

Kilimann, M W; DeGennaro, L J

1985-01-01

To provide access to synapsin I-specific DNA sequences, we have constructed cDNA clones complementary to synapsin I mRNA isolated from rat brain. Synapsin I mRNA was specifically enriched by immunoadsorption of polysomes prepared from the brains of 10-14 day old rats. Employing this enriched mRNA, a cDNA library was constructed in pBR322 and screened by differential colony hybridization with single-stranded cDNA probes made from synapsin I mRNA and total polysomal poly(A)+ RNA. This screening procedure proved to be highly selective. Five independent recombinant plasmids which exhibited distinctly stronger hybridization with the synapsin I probe were characterized further by restriction mapping. All of the cDNA inserts gave restriction enzyme digestion patterns which could be aligned. In addition, some of the cDNA inserts were shown to contain poly(dA) sequences. Final identification of synapsin I cDNA clones relied on the ability of the cDNA inserts to hybridize specifically to synapsin I mRNA. Several plasmids were tested by positive hybridization selection. They specifically selected synapsin I mRNA which was identified by in vitro translation and immunoprecipitation of the translation products. The established cDNA clones were used for a blot-hybridization analysis of synapsin I mRNA. A fragment (1600 bases) from the longest cDNA clone hybridized with two discrete RNA species 5800 and 4500 bases long, in polyadenylated RNA from rat brain and PC12 cells. No hybridization was detected to RNA from rat liver, skeletal muscle or cardiac muscle. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. PMID:3933975

Dynamic proteomics emphasizes the importance of selective mRNA translation and protein turnover during Arabidopsis seed germination.

PubMed

Galland, Marc; Huguet, Romain; Arc, Erwann; Cueff, Gwendal; Job, Dominique; Rajjou, Loïc

2014-01-01

During seed germination, the transition from a quiescent metabolic state in a dry mature seed to a proliferative metabolic state in a vigorous seedling is crucial for plant propagation as well as for optimizing crop yield. This work provides a detailed description of the dynamics of protein synthesis during the time course of germination, demonstrating that mRNA translation is both sequential and selective during this process. The complete inhibition of the germination process in the presence of the translation inhibitor cycloheximide established that mRNA translation is critical for Arabidopsis seed germination. However, the dynamics of protein turnover and the selectivity of protein synthesis (mRNA translation) during Arabidopsis seed germination have not been addressed yet. Based on our detailed knowledge of the Arabidopsis seed proteome, we have deepened our understanding of seed mRNA translation during germination by combining two-dimensional gel-based proteomics with dynamic radiolabeled proteomics using a radiolabeled amino acid precursor, namely [(35)S]-methionine, in order to highlight de novo protein synthesis, stability, and turnover. Our data confirm that during early imbibition, the Arabidopsis translatome keeps reflecting an embryonic maturation program until a certain developmental checkpoint. Furthermore, by dividing the seed germination time lapse into discrete time windows, we highlight precise and specific patterns of protein synthesis. These data refine and deepen our knowledge of the three classical phases of seed germination based on seed water uptake during imbibition and reveal that selective mRNA translation is a key feature of seed germination. Beyond the quantitative control of translational activity, both the selectivity of mRNA translation and protein turnover appear as specific regulatory systems, critical for timing the molecular events leading to successful germination and seedling establishment.

Dynamic Proteomics Emphasizes the Importance of Selective mRNA Translation and Protein Turnover during Arabidopsis Seed Germination*

PubMed Central

Galland, Marc; Huguet, Romain; Arc, Erwann; Cueff, Gwendal; Job, Dominique; Rajjou, Loïc

2014-01-01

During seed germination, the transition from a quiescent metabolic state in a dry mature seed to a proliferative metabolic state in a vigorous seedling is crucial for plant propagation as well as for optimizing crop yield. This work provides a detailed description of the dynamics of protein synthesis during the time course of germination, demonstrating that mRNA translation is both sequential and selective during this process. The complete inhibition of the germination process in the presence of the translation inhibitor cycloheximide established that mRNA translation is critical for Arabidopsis seed germination. However, the dynamics of protein turnover and the selectivity of protein synthesis (mRNA translation) during Arabidopsis seed germination have not been addressed yet. Based on our detailed knowledge of the Arabidopsis seed proteome, we have deepened our understanding of seed mRNA translation during germination by combining two-dimensional gel-based proteomics with dynamic radiolabeled proteomics using a radiolabeled amino acid precursor, namely [35S]-methionine, in order to highlight de novo protein synthesis, stability, and turnover. Our data confirm that during early imbibition, the Arabidopsis translatome keeps reflecting an embryonic maturation program until a certain developmental checkpoint. Furthermore, by dividing the seed germination time lapse into discrete time windows, we highlight precise and specific patterns of protein synthesis. These data refine and deepen our knowledge of the three classical phases of seed germination based on seed water uptake during imbibition and reveal that selective mRNA translation is a key feature of seed germination. Beyond the quantitative control of translational activity, both the selectivity of mRNA translation and protein turnover appear as specific regulatory systems, critical for timing the molecular events leading to successful germination and seedling establishment. PMID:24198433

Expression patterns of the aquaporin gene family during renal development: influence of genetic variability.

PubMed

Parreira, Kleber S; Debaix, Huguette; Cnops, Yvette; Geffers, Lars; Devuyst, Olivier

2009-08-01

High-throughput analyses have shown that aquaporins (AQPs) belong to a cluster of genes that are differentially expressed during kidney organogenesis. However, the spatiotemporal expression patterns of the AQP gene family during tubular maturation and the potential influence of genetic variation on these patterns and on water handling remain unknown. We investigated the expression patterns of all AQP isoforms in fetal (E13.5 to E18.5), postnatal (P1 to P28), and adult (9 weeks) kidneys of inbred (C57BL/6J) and outbred (CD-1) mice. Using quantitative polymerase chain reaction (PCR), we evidenced two mRNA patterns during tubular maturation in C57 mice. The AQPs 1-7-11 showed an early (from E14.5) and progressive increase to adult levels, similar to the mRNA pattern observed for proximal tubule markers (Megalin, NaPi-IIa, OAT1) and reflecting the continuous increase in renal cortical structures during development. By contrast, AQPs 2-3-4 showed a later (E15.5) and more abrupt increase, with transient postnatal overexpression. Most AQP genes were expressed earlier and/or stronger in maturing CD-1 kidneys. Furthermore, adult CD-1 kidneys expressed more AQP2 in the collecting ducts, which was reflected by a significant delay in excreting a water load. The expression patterns of proximal vs. distal AQPs and the earlier expression in the CD-1 strain were confirmed by immunoblotting and immunostaining. These data (1) substantiate the clustering of important genes during tubular maturation and (2) demonstrate that genetic variability influences the regulation of the AQP gene family during tubular maturation and water handling by the mature kidney.

Chlorine decay and bacterial inactivation kinetics in drinking water in the tropics.

PubMed

Thøgersen, J; Dahi, E

1996-09-01

The decay of free chlorine (Cl2) and combined chlorine (mostly monochloramine: NH2Cl) and the inactivation of bacteria was examined in Dar es Salaam, Tanzania. Batch experiments, pilot-scale pipe experiments and full-scale pipe experiments were carried out to establish the kinetics for both decay and inactivation, and to compare the two disinfectants for use under tropical conditions. The decay of both disinfectants closely followed first order kinetics, with respect to the concentration of both disinfectant and disinfectant-consuming substances. Bacterial densities exhibited a kinetic pattern consisting of first order inactivation with respect to the density of the bacteria and the concentration of the disinfectant, and first order growth with respect to the bacterial density. The disinfection kinetic model takes the decaying concentration of the disinfectant into account. The decay rate constant for free chlorine was 114 lg(-1)h(-1), while the decay rate constant for combined chlorine was 1.84 lg(-1)h(-1) (1.6% of the decay rate for free chlorine). The average concentration of disinfectant consuming substances in the water phase was 2.6 mg Cl2/l for free chlorine and 5.6 mg NH2Cl/l for combined chlorine. The decay rate constant and the concentration of disinfectant consuming substances when water was pumped through pipes, depended on whether or not chlorination was continuous. Combined chlorine especially could clean the pipes of disinfectant consuming substances. The inactivation rate constant λ, was estimated at 3.06×10(4) lg(-1)h(-1). Based on the inactivation rate constant, and a growth rate constant determined in a previous study, the critical concentration of free chlorine was found to be 0.08 mg Cl2/l. The critical concentration is a value below which growth rates dominate over inactivation.

Epigenetic regulation of somatic angiotensin-converting enzyme by DNA methylation and histone acetylation.

PubMed

Rivière, Guillaume; Lienhard, Daniel; Andrieu, Thomas; Vieau, Didier; Frey, Brigitte M; Frey, Felix J

2011-04-01

Somatic angiotensin-converting enzyme (sACE) is crucial in cardiovascular homeostasis and displays a tissue-specific profile. Epigenetic patterns modulate genes expression and their alterations were implied in pathologies including hypertension. However, the influence of DNA methylation and chromatin condensation state on the expression of sACE is unknown. We examined whether such epigenetic mechanisms could participate in the control of sACE expression in vitro and in vivo. We identified two CpG islands in the human ace-1 gene 3 kb proximal promoter region. Their methylation abolished the luciferase activity of ace-1 promoter/reporter constructs transfected into human liver (HepG2), colon (HT29), microvascular endothelial (HMEC-1) and lung (SUT) cell lines (p < 0.001). Bisulphite sequencing revealed a cell-type specific basal methylation pattern of the ace-1 gene -1,466/+25 region. As assessed by RT-qPCR, inhibition of DNA methylation by 5-aza-2'-deoxycytidine and/or of histone deacetylation by trichostatin A highly stimulated sACE mRNA expression cell-type specifically (p < 0.001 vs. vehicle treated cells). In the rat, in vivo 5-aza-cytidine injections demethylated the ace-1 promoter and increased sACE mRNA expression in the lungs and liver (p = 0.05), but not in the kidney. In conclusion, the expression level of somatic ACE is modulated by CpG-methylation and histone deacetylases inhibition. The basal methylation pattern of the promoter of the ace-1 gene is cell-type specific and correlates to sACE transcription. DNMT inhibition is associated with altered methylation of the ace-1 promoter and a cell-type and tissue-specific increase of sACE mRNA levels. This study indicates a strong influence of epigenetic mechanisms on sACE expression.

Conserved regional patterns of GABA-related transcript expression in the neocortex of subjects with schizophrenia.

PubMed

Hashimoto, Takanori; Bazmi, H Holly; Mirnics, Karoly; Wu, Qiang; Sampson, Allan R; Lewis, David A

2008-04-01

Individuals with schizophrenia exhibit disturbances in a number of cognitive, affective, sensory, and motor functions that depend on the circuitry of different cortical areas. The cognitive deficits associated with dysfunction of the dorsolateral prefrontal cortex result, at least in part, from abnormalities in GABA neurotransmission, as reflected in a specific pattern of altered expression of GABA-related genes. Consequently, the authors sought to determine whether this pattern of altered gene expression is restricted to the dorsolateral prefrontal cortex or could also contribute to the dysfunction of other cortical areas in subjects with schizophrenia. Real-time quantitative polymerase chain reaction was used to assess the levels of eight GABA-related transcripts in four cortical areas (dorsolateral prefrontal cortex, anterior cingulate cortex, and primary motor and primary visual cortices) of subjects (N=12) with schizophrenia and matched normal comparison subjects. Expression levels of seven transcripts were lower in subjects with schizophrenia, with the magnitude of reduction for each transcript comparable across the four areas. The largest reductions were detected for mRNA encoding somatostatin and parvalbumin, followed by moderate decreases in mRNA expression for the 67-kilodalton isoform of glutamic acid decarboxylase, the GABA membrane transporter GAT-1, and the alpha 1 and delta subunits of GABA(A) receptors. In contrast, the expression of calretinin mRNA did not differ between the subject groups in any of the four areas. Because the areas examined represent the major functional domains (e.g., association, limbic, motor, and sensory) of the cerebral cortex, our findings suggest that a conserved set of molecular alterations affecting GABA neurotransmission contribute to the pathophysiology of different clinical features of schizophrenia.

CP violation in the decays B→KK¯ in the presence of DD¯↔KK¯ mixing

NASA Astrophysics Data System (ADS)

Barshay, S.; Sehgal, L. M.; van Leusen, J.

2004-08-01

We have recently shown that the large direct CP violation observed in the decay B0 →π+π-, and the enhanced branching ratio for B0 →π0π0, can be understood by invoking a small mixing of the ππ system with the dominant DDbar channel. We examine here the analogous effect of DDbar ↔ KKbar mixing on the rare decays B0 →K0Kbar0, B- →K-K0 and B0 →K+K-. We find (a) significant values for the asymmetry parameters C and S in B0 /Bbar0 →K0Kbar0, (b) a possible enhancement of the suppressed mode B0 →K+K-, (c) a correlation between the three decay channels following from a triangle relation between amplitudes AK0Kbar0 -AK+K- =AK-K0. The pattern of asymmetries and branching ratios is compared with that derived from the short-distance QCD penguin interaction.

Differential Regulation of Duplicate Light-Dependent Protochlorophyllide Oxidoreductases in the Diatom Phaeodactylum tricornutum

PubMed Central

Hunsperger, Heather M.; Cattolico, Rose Ann

2016-01-01

Background Diatoms (Bacilliariophyceae) encode two light-dependent protochlorophyllide oxidoreductases (POR1 and POR2) that catalyze the penultimate step of chlorophyll biosynthesis in the light. Algae live in dynamic environments whose changing light levels induce photoacclimative metabolic shifts, including altered cellular chlorophyll levels. We hypothesized that the two POR proteins may be differentially adaptive under varying light conditions. Using the diatom Phaeodactylum tricornutum as a test system, differences in POR protein abundance and por gene expression were examined when this organism was grown on an alternating light:dark cycles at different irradiances; exposed to continuous light; and challenged by a significant decrease in light availability. Results For cultures maintained on a 12h light: 12h dark photoperiod at 200μE m−2 s−1 (200L/D), both por genes were up-regulated during the light and down-regulated in the dark, though por1 transcript abundance rose and fell earlier than that of por2. Little concordance occurred between por1 mRNA and POR1 protein abundance. In contrast, por2 mRNA and POR2 protein abundances followed similar diurnal patterns. When 200L/D P. tricornutum cultures were transferred to continuous light (200L/L), the diurnal regulatory pattern of por1 mRNA abundance but not of por2 was disrupted, and POR1 but not POR2 protein abundance dropped steeply. Under 1200μE m−2 s−1 (1200L/D), both por1 mRNA and POR1 protein abundance displayed diurnal oscillations. A compromised diel por2 mRNA response under 1200L/D did not impact the oscillation in POR2 abundance. When cells grown at 1200L/D were then shifted to 50μE m−2 s−1 (50L/D), por1 and por2 mRNA levels decreased swiftly but briefly upon light reduction. Thereafter, POR1 but not POR2 protein levels rose significantly in response to this light stepdown. Conclusion Given the sensitivity of diatom por1/POR1 to real-time light cues and adherence of por2/POR2 regulation to the diurnal cycle, we suggest that POR1 supports photoacclimation, whereas POR2 is the workhorse for daily chlorophyll synthesis. PMID:27367227

Decay behaviors of the Pc hadronic molecules

NASA Astrophysics Data System (ADS)

Lin, Yong-Hui; Shen, Chao-Wei; Guo, Feng-Kun; Zou, Bing-Song

2017-06-01

The Pc(4380 ) and Pc(4450 ) states observed recently by the LHCb experiment were proposed to be either D ¯Σc* or D¯*Σc bound states. We analyze the decay behaviors of two such types of hadronic molecules within the effective Lagrangian framework. With branching ratios of ten possible decay channels calculated, it is found that the two types of hadronic molecules have distinguishable decay patterns. While the D ¯Σc* molecule decays dominantly to the D¯*Λc channel with a branching ratio by 2 orders of magnitude larger than to D ¯Λc, the D¯*Σc molecule decays to these two channels with a difference of less than a factor of 2. Our results show that the total decay width of Pc(4380 ) as the spin-parity-3/2- D ¯Σc* molecule is about a factor of 2 larger than the corresponding value for the D¯*Σc molecule. It suggests that the assignment of the D ¯Σc* molecule for Pc(4380 ) is more favorable than the D¯*Σc molecule. In addition, Pc(4450 ) seems to be a D¯*Σc molecule with JP=5/2+ in our scheme. Based on these partial decay widths of the Pc states, we estimate the cross sections for the reactions γ p →J /ψ p and π p →J /ψ p through the s-channel Pc states. The forthcoming γ p experiment at JLAB and the π p experiment at JPARC should be able to pin down the nature of these Pc states.

Rare Autosomal Recessive Cardiac Valvular Form of Ehlers-Danlos Syndrome Results from Mutations in the COL1A2 Gene That Activate the Nonsense-Mediated RNA Decay Pathway

PubMed Central

Schwarze, Ulrike; Hata, Ryu-Ichiro; McKusick, Victor A.; Shinkai, Hiroshi; Hoyme, H. Eugene; Pyeritz, Reed E.; Byers, Peter H.

2004-01-01

Splice site mutations in the COL1A2 gene of type I collagen can give rise to forms of Ehlers-Danlos syndrome (EDS) because of partial or complete skipping of exon 6, as well as to mild, moderate, or lethal forms of osteogenesis imperfecta as a consequence of skipping of other exons. We identified three unrelated individuals with a rare recessively inherited form of EDS (characterized by joint hypermobility, skin hyperextensibility, and cardiac valvular defects); in two of them, COL1A2 messenger RNA (mRNA) instability results from compound heterozygosity for splice site mutations in the COL1A2 gene, and, in the third, it results from homozygosity for a nonsense codon. The splice site mutations led to use of cryptic splice donor sites, creation of a downstream premature termination codon, and extremely unstable mRNA. In the wild-type allele, the two introns (IVS11 and IVS24) in which these mutations occurred were usually spliced slowly in relation to their respective immediate upstream introns. In the mutant alleles, the upstream intron was removed, so that exon skipping could not occur. In the context of the mutation in IVS24, computer-generated folding of a short stretch of mRNA surrounding the mutation site demonstrated realignment of the relationships between the donor and acceptor sites that could facilitate use of a cryptic donor site. These findings suggest that the order of intron removal is an important variable in prediction of mutation outcome at splice sites and that folding of the nascent mRNA could be one element that contributes to determination of order of splicing. The complete absence of proα2(I) chains has the surprising effect of producing cardiac valvular disease without bone involvement. PMID:15077201

Rare autosomal recessive cardiac valvular form of Ehlers-Danlos syndrome results from mutations in the COL1A2 gene that activate the nonsense-mediated RNA decay pathway.

PubMed

Schwarze, Ulrike; Hata, Ryu-Ichiro; McKusick, Victor A; Shinkai, Hiroshi; Hoyme, H Eugene; Pyeritz, Reed E; Byers, Peter H

2004-05-01

Splice site mutations in the COL1A2 gene of type I collagen can give rise to forms of Ehlers-Danlos syndrome (EDS) because of partial or complete skipping of exon 6, as well as to mild, moderate, or lethal forms of osteogenesis imperfecta as a consequence of skipping of other exons. We identified three unrelated individuals with a rare recessively inherited form of EDS (characterized by joint hypermobility, skin hyperextensibility, and cardiac valvular defects); in two of them, COL1A2 messenger RNA (mRNA) instability results from compound heterozygosity for splice site mutations in the COL1A2 gene, and, in the third, it results from homozygosity for a nonsense codon. The splice site mutations led to use of cryptic splice donor sites, creation of a downstream premature termination codon, and extremely unstable mRNA. In the wild-type allele, the two introns (IVS11 and IVS24) in which these mutations occurred were usually spliced slowly in relation to their respective immediate upstream introns. In the mutant alleles, the upstream intron was removed, so that exon skipping could not occur. In the context of the mutation in IVS24, computer-generated folding of a short stretch of mRNA surrounding the mutation site demonstrated realignment of the relationships between the donor and acceptor sites that could facilitate use of a cryptic donor site. These findings suggest that the order of intron removal is an important variable in prediction of mutation outcome at splice sites and that folding of the nascent mRNA could be one element that contributes to determination of order of splicing. The complete absence of pro alpha 2(I) chains has the surprising effect of producing cardiac valvular disease without bone involvement.

Position-dependent interactions of Y-box protein 2 (YBX2) with mRNA enable mRNA storage in round spermatids by repressing mRNA translation and blocking translation-dependent mRNA decay.

PubMed

Kleene, Kenneth C

2016-03-01

Many mRNAs encoding proteins needed for the construction of the specialized organelles of spermatozoa are stored as translationally repressed, free messenger ribonucleoproteins in round spermatids, to be actively translated in elongating and elongated spermatids. The factors that repress translation in round spermatids, however, have been elusive. Two lines of evidence implicate the highly abundant and well-known translational repressor, Y-box protein 2 (YBX2), as a critical factor: First, protamine 1 (Prm1) and sperm-mitochondria cysteine-rich protein (Smcp) mRNAs are prematurely recruited onto polysomes in Ybx2-knockout mouse round spermatids. Second, mutations in 3' untranslated region (3'UTR) cis-elements that abrogate YBX2 binding activate translation of Prm1 and Smcp mRNAs in round spermatids of transgenic mice. The abundance of YBX2 and its affinity for variable sequences, however, raise questions of how YBX2 targets specific mRNAs for repression. Mutations to the Prm1 and Smcp mRNAs in transgenic mice reveal that strong repression in round spermatids requires YBX2 binding sites located near the 3' ends of their 3'UTRs as locating the same sites in upstream positions produce negligible repression. This location-dependence implies that the assembly of repressive complexes is nucleated by adjacent cis-elements that enable cooperative interactions of YBX2 with co-factors. The available data suggest that, in vertebrates, YBX2 has the important role of coordinating the storage of translationally repressed mRNAs in round spermatids by inhibiting translational activity and the degradation of transcripts via translation-dependent deadenylation. These insights should facilitiate future experiments designed to unravel how YBX2 targets mRNAs for repression in round spermatids and how mutations in the YBX2 gene cause infertility in humans. Mol. Reprod. Dev. 83: 190-207, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

[Schizophrenia and cortical GABA neurotransmission].

PubMed

Hashimoto, Takanori; Matsubara, Takuro; Lewis, David A

2010-01-01

Individuals with schizophrenia show disturbances in a number of brain functions that regulate cognitive, affective, motor, and sensory processing. The cognitive deficits associated with dysfunction of the dorsolateral prefrontal cortex result, at least in part, from abnormalities in GABA neurotransmission, as reflected in a specific pattern of altered expression of GABA-related molecules. First, mRNA levels for the 67-kilodalton isoform of glutamic acid decarboxylase (GAD67), an enzyme principally responsible for GABA synthesis, and the GABA membrane transporter GAT1, which regulates the reuptake of synaptically released GABA, are decreased in a subset of GABA neurons. Second, affected GABA neurons include those that express the calcium-binding protein parvalbumin (PV), because PV mRNA levels are decreased in the prefrontal cortex of subjects with schizophrenia and GAD67 mRNA is undetectable in almost half of PV-containing neurons. These changes are accompanied by decreased GAT1 expression in the presynaptic terminals of PV-containing neurons and by increased postsynaptic GABA-A receptor alpha2 subunit expression at the axon initial segments of pyramidal neurons. These findings indicate decreased GABA synthesis/release by PV-containing GABA neurons and compensatory changes at synapses formed by these neurons. Third, another subset of GABA neurons that express the neuropeptide somatostatin (SST) also appear to be affected because their specific markers, SST and neuropeptide Y mRNAs, are decreased in a manner highly correlated with the decreases in GAD67 mRNA. Finally, mRNA levels for GABA-A receptor subunits for synaptic (alpha1 and gamma2) and extra-synaptic (delta) receptors are decreased, indicating alterations in both synaptic and extra-synaptic GABA neurotransmission. Together, this pattern of changes indicates that the altered GABA neurotransmission is specific to PV-containing and SST-containing GABA neuron subsets and involves both synaptic and extra-synaptic GABA-A receptors. Our recent analyses demonstrated that this pattern exists across diverse cortical areas including the prefrontal, anterior cingulate, primary motor, and primary visual cortices. GABA neurotransmission by PV-containing and SST-containing neurons is important for the generation of cortical oscillatory activities in the gamma (30-100 Hz) and theta (4-7 Hz) bands, respectively. These oscillatory activities have been proposed to play critical roles in regulating the efficiency of information transfer between neurons and neuronal networks in the cortex. Altered cortical GABA neurotransmission appears to contribute to disturbances in diverse functions through affecting the generation of cortical oscillations in schizophrenia.

Building the Vertebrate Spine

NASA Astrophysics Data System (ADS)

Pourquié, Olivier

2008-03-01

The vertebrate body can be subdivided along the antero-posterior (AP) axis into repeated structures called segments. This periodic pattern is established during embryogenesis by the somitogenesis process. Somites are generated in a rhythmic fashion from the paraxial mesoderm and subsequently differentiate to give rise to the vertebrae and skeletal muscles of the body. Somite formation involves an oscillator-the segmentation clock-whose periodic signal is converted into the periodic array of somite boundaries. This clock drives the dynamic expression of cyclic genes in the presomitic mesoderm and requires Notch and Wnt signaling. Microarray studies of the mouse presomitic mesoderm transcriptome reveal that the segmentation clock drives the periodic expression of a large network of cyclic genes involved in cell signaling. Mutually exclusive activation of the Notch/FGF and Wnt pathways during each cycle suggests that coordinated regulation of these three pathways underlies the clock oscillator. In humans, mutations in the genes associated to the function of this oscillator such as Dll3 or Lunatic Fringe result in abnormal segmentation of the vertebral column such as those seen in congenital scoliosis. Whereas the segmentation clock is thought to set the pace of vertebrate segmentation, the translation of this pulsation into the reiterated arrangement of segment boundaries along the AP axis involves dynamic gradients of FGF and Wnt signaling. The FGF signaling gradient is established based on an unusual mechanism involving mRNA decay which provides an efficient means to couple the spatio-temporal activation of segmentation to the posterior elongation of the embryo. Another striking aspect of somite production is the strict bilateral symmetry of the process. Retinoic acid was shown to control aspects of this coordination by buffering destabilizing effects from the embryonic left-right machinery. Defects in this embryonic program controlling vertebral symmetry might lead to scoliosis in humans. Finally, the subsequent regional differentiation of the precursors of the vertebrae is controlled by Hox genes, whose collinear expression controls both gastrulation of somite precursors and their subsequent patterning into region-specific types of structures. Therefore somite development provides an outstanding paradigm to study patterning and differentiation in vertebrate embryos.

Skin barrier disruption by sodium lauryl sulfate-exposure alters the expressions of involucrin, transglutaminase 1, profilaggrin, and kallikreins during the repair phase in human skin in vivo.

PubMed

Törmä, Hans; Lindberg, Magnus; Berne, Berit

2008-05-01

Detergents are skin irritants affecting keratinocytes. In this study, healthy volunteers were exposed to water (vehicle) and 1% sodium lauryl sulfate (SLS) under occlusive patch tests for 24 hours. The messenger RNA (mRNA) expression of keratinocyte differentiation markers and of enzymes involved in corneodesmosome degradation was examined in skin biopsies (n=8) during the repair phase (6 hours to 7 days postexposure) using real-time reverse-transcription PCR. It was found that the expression of involucrin was increased at 6 hours, but then rapidly normalized. The expression of transglutaminase 1 exhibited a twofold increase after 24 hours in the SLS-exposed skin. Profilaggrin was decreased after 6 hours. Later (4-7 days), the expression in SLS-exposed areas was >50% above than in control areas. An increased and altered immunofluorescence pattern of involucrin, transglutaminase 1, and filaggrin was also found (n=4). At 6 hours post-SLS exposure, the mRNA expression of kallikrein-7 (KLK-7) and kallikrein-5 (KLK-5) was decreased by 50 and 75%, respectively, as compared with control and water-exposed areas. Thereafter, the expression pattern of KLK-7 and KLK-5 was normalized. Changes in protein expression of KLK-5 were also found. In conclusion, SLS-induced skin barrier defects induce altered mRNA expression of keratinocyte differentiation markers and enzymes degrading corneodesmosomes.

The Yeast PUF Protein Puf5 Has Pop2-Independent Roles in Response to DNA Replication Stress

PubMed Central

Traven, Ana; Lo, Tricia L.; Lithgow, Trevor; Heierhorst, Jörg

2010-01-01

PUFs are RNA binding proteins that promote mRNA deadenylation and decay and inhibit translation. Yeast Puf5 is the prototype for studying PUF-dependent gene repression. Puf5 binds to the Pop2 subunit of the Ccr4-Pop2-NOT mRNA deadenylase, recruiting the deadenylase and associated translational repressors to mRNAs. Here we used yeast genetics to show that Puf5 has additional roles in vivo that do not require Pop2. Deletion of PUF5 caused increased sensitivity to DNA replication stress in cells lacking Pop2, as well as in cells mutated for two activities recruited to mRNAs by the Puf5-Pop2 interaction, the deadenylase Ccr4 and the translational repressor Dhh1. A functional Puf5 RNA binding domain was required, and Puf5 cytoplasmic localisation was sufficient for resistance to replication stress, indicating posttranscriptional gene expression control is involved. In contrast to DNA replication stress, in response to the cell wall integrity pathway activator caffeine, PUF5 and POP2 acted in the same genetic pathway, indicating that functions of Puf5 in the caffeine response are mediated by Pop2-dependent gene repression. Our results support a model in which Puf5 uses multiple, Pop2-dependent and Pop2-independent mechanisms to control mRNA expression. The Pop2-independent roles for Puf5 could involve spatial control of gene expression, a proposition supported by our data indicating that the active form of Puf5 is localised to cytoplasmic foci. PMID:20498834

Cell type-dependent gene regulation by Staufen2 in conjunction with Upf1

PubMed Central

2011-01-01

Background Staufen2 (Stau2), a double-stranded RNA-binding protein, is a component of neuronal RNA granules, which are dendritic mRNA transport machines. Although Stau2 is thought to be involved in the dendritic targeting of several mRNAs in neurons, the mechanism whereby Stau2 regulates these mRNAs is unknown. To elucidate the functions of Stau2, we screened for novel binding partners by affinity purification of GST-tagged Stau2 from 293F cells. Results Three RNA helicases, RNA helicase A, Upf1 and Mov10, were identified in Stau2-containing complexes. We focused our studies on Upf1, a key player in nonsense-mediated mRNA decay. Stau2 was found to bind directly to Upf1 in an RNA-independent manner in vitro. Tethering Stau2 to the 3'-untranslated region (UTR) of a reporter gene had little effect on its expression in HeLa cells. In contrast, when the same tethering assay was performed in 293F cells, we observed an increase in reporter protein levels. This upregulation of protein expression by Stau2 turned out to be dependent on Upf1. Moreover, we found that in 293F cells, Stau2 upregulates the reporter mRNA level in an Upf1-independent manner. Conclusions These results indicate that the recruitment of Stau2 alone or in combination with Upf1 differentially affects the fate of mRNAs. Moreover, the results suggest that Stau2-mediated fate determination could be executed in a cell type-specific manner. PMID:22087843

Two Ck1δ transcripts regulated by m6A methylation code for two antagonistic kinases in the control of the circadian clock

PubMed Central

Fustin, Jean-Michel; Kojima, Rika; Itoh, Kakeru; Chang, Hsin-Yi; Shiqi, Ye; Zhuang, Bowen; Oji, Asami; Gibo, Shingo; Narasimamurthy, Rajesh; Kurosawa, Gen; Doi, Masao; Manabe, Ichiro; Ishihama, Yasushi; Okamura, Hitoshi

2018-01-01

The N6-methylation of internal adenosines (m6A) in mRNA has been quantified and localized throughout the transcriptome. However, the physiological significance of m6A in most highly methylated mRNAs is unknown. It was demonstrated previously that the circadian clock, based on transcription-translation negative feedback loops, is sensitive to the general inhibition of m6A. Here, we show that the Casein Kinase 1 Delta mRNA (Ck1δ), coding for a critical kinase in the control of circadian rhythms, cellular growth, and survival, is negatively regulated by m6A. Inhibition of Ck1δ mRNA methylation leads to increased translation of two alternatively spliced CK1δ isoforms, CK1δ1 and CK1δ2, uncharacterized until now. The expression ratio between these isoforms is tissue-specific, CK1δ1 and CK1δ2 have different kinase activities, and they cooperate in the phosphorylation of the circadian clock protein PER2. While CK1δ1 accelerates the circadian clock by promoting the decay of PER2 proteins, CK1δ2 slows it down by stabilizing PER2 via increased phosphorylation at a key residue on PER2 protein. These observations challenge the previously established model of PER2 phosphorylation and, given the multiple functions and targets of CK1δ, the existence of two isoforms calls for a re-evaluation of past research when CK1δ1 and CK1δ2 were simply CK1δ. PMID:29784786

University of California San Francisco (UCSF-2): Integrative Genomic Approaches in Neuroblastoma (NBL) | Office of Cancer Genomics

Cancer.gov

The CTD2 Center at University of California San Francisco (UCSF-2) used an integrative genomics approach to reveal unidentified mRNA splicing patterns in neuroblastoma. Read the abstract Experimental Approaches Read the detailed Experimental Approaches

Expression of decay-accelerating factor (CD55), membrane cofactor protein (CD46) and CD59 in the human astroglioma cell line, D54-MG, and primary rat astrocytes.

PubMed

Yang, C; Jones, J L; Barnum, S R

1993-09-01

In this report, we have shown the expression of the complement regulatory proteins decay-accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and CD59 on human D54-MG astroglioma cells by several methods, including immunofluorescence, flow cytometry and Western blotting and Northern blot analysis. These studies demonstrate that all three proteins are structurally and antigenically similar to their counterparts expressed on HepG2 and SW480 cells (hepatocyte and epithelial cell lines, respectively). D54-MG cells express mRNA for all three proteins of the appropriate size(s). The phosphatidylinositol-specific enzyme, PIPLC, cleaved DAF from the surface of D54-MG cells, demonstrating that DAF is linked by a glycophospholipid anchor as has been shown for other cell types. Flow cytometry demonstrates that primary rat astrocytes also constitutively express all three regulatory proteins. These data are the first to demonstrate the expression of CD59 on astrocytes, and the presence of all three regulatory proteins on astrocytes suggests that regulation of complement activation in the central nervous system is important in neural host defense mechanisms.

Construction of a Lotus japonicus late nodulin expressed sequence tag library and identification of novel nodule-specific genes.

PubMed Central

Szczyglowski, K; Hamburger, D; Kapranov, P; de Bruijn, F J

1997-01-01

A range of novel expressed sequence tags (ESTs) associated with late developmental events during nodule organogenesis in the legume Lotus japonicus were identified using mRNA differential display; 110 differentially displayed polymerase chain reaction products were cloned and analyzed. Of 88 unique cDNAs obtained, 22 shared significant homology to DNA/protein sequences in the respective databases. This group comprises, among others, a nodule-specific homolog of protein phosphatase 2C, a peptide transporter protein, and a nodule-specific form of cytochrome P450. RNA gel-blot analysis of 16 differentially displayed ESTs confirmed their nodule-specific expression pattern. The kinetics of mRNA accumulation of the majority of the ESTs analyzed were found to resemble the expression pattern observed for the L. japonicus leghemoglobin gene. These results indicate that the newly isolated molecular markers correspond to genes induced during late developmental stages of L. japonicus nodule organogenesis and provide important, novel tools for the study of nodulation. PMID:9276951

Expression of c-Fes protein isoforms correlates with differentiation in myeloid leukemias.

PubMed

Carlson, Anne; Berkowitz, Jeanne McAdara; Browning, Damaris; Slamon, Dennis J; Gasson, Judith C; Yates, Karen E

2005-05-01

The cellular fes gene encodes a 93-kilodalton protein-tyrosine kinase (p93) that is expressed in both normal and neoplastic myeloid cells. Increased c-Fes expression is associated with differentiation in normal myeloid cells and cell lines. Our hypothesis was that primary leukemia cells would show a similar pattern of increased expression in more differentiated cells. Therefore, we compared c-Fes expression in cells with an undifferentiated, blast phenotype (acute myelogenous leukemia--AML) to cells with a differentiated phenotype (chronic myelogenous leukemia--CML). Instead of differences in p93 expression levels, we found complex patterns of c-Fes immunoreactive proteins that corresponded with differentiation in normal and leukemic myeloid cells. The "blast" pattern consisted of c-Fes immunoreactive proteins p93, p74, and p70; the "differentiated" pattern showed two additional c-Fes immunoreactive proteins, p67 and p62. Using mRNA from mouse and human cell lines, we found deletion of one or more exons in the c-fes mRNA. Those deletions predicted truncation of conserved domains (CDC15/FCH and SH2) involved in protein-protein interactions. No deletions were found, however, within the kinase domain. We infer that alternative splicing generates a family of c-Fes proteins. This may be a mechanism to direct the c-Fes kinase domain to different subcellular locations and/or substrates at specific stages of myeloid cell differentiation.

T cell source of type 1 cytokines determines illness patterns in respiratory syncytial virus-infected mice.

PubMed Central

Tang, Y. W.; Graham, B. S.

1997-01-01

Manipulation of the cytokine microenvironment at the time of vaccination can influence immune responses to remote challenge, providing a strategy to study the molecular pathogenesis of respiratory syncytial virus (RSV) vaccine-enhanced disease in the mouse model. Although treatment with antibody against IL-4 or recombinant IL-12 (rIL-12) at the time of formalin-inactivated RSV vaccination induced a similar shift in the pattern of cytokine mRNA expression upon live virus challenge, anti-IL-4 treated mice had increased CD8+ cytotoxic T lymphocyte activity and reduced illness compared with rIL-12-treated mice. To define effector mechanisms responsible for these patterns, CD4+ and/or CD8+ T lymphocytes were selectively depleted in vivo at the time of RSV challenge. In rIL-12-treated mice, CD4+ lymphocytes made the largest contribution to IFN-gamma mRNA, RSV clearance, and illness, while in anti-IL-4 treated mice, CD8+ lymphocytes were the major effector. The effector responsible for virus clearance also mediated illness, suggesting that efficiency of virus clearance determined disease expression. These results demonstrate that the phenotype of effector cells involved in the immune response to virus challenge may be a more important determinant of disease than patterns of cytokine expression classically assigned to Th1 and Th2 lymphocytes. PMID:9151790

Differential regulation of protease activated receptor-1 and tissue plasminogen activator expression by shear stress in vascular smooth muscle cells

NASA Technical Reports Server (NTRS)

Papadaki, M.; Ruef, J.; Nguyen, K. T.; Li, F.; Patterson, C.; Eskin, S. G.; McIntire, L. V.; Runge, M. S.

1998-01-01

Recent studies have demonstrated that vascular smooth muscle cells are responsive to changes in their local hemodynamic environment. The effects of shear stress on the expression of human protease activated receptor-1 (PAR-1) and tissue plasminogen activator (tPA) mRNA and protein were investigated in human aortic smooth muscle cells (HASMCs). Under conditions of low shear stress (5 dyn/cm2), PAR-1 mRNA expression was increased transiently at 2 hours compared with stationary control values, whereas at high shear stress (25 dyn/cm2), mRNA expression was decreased (to 29% of stationary control; P<0.05) at all examined time points (2 to 24 hours). mRNA half-life studies showed that this response was not due to increased mRNA instability. tPA mRNA expression was decreased (to 10% of stationary control; P<0.05) by low shear stress after 12 hours of exposure and was increased (to 250% of stationary control; P<0.05) after 24 hours at high shear stress. The same trends in PAR-1 mRNA levels were observed in rat smooth muscle cells, indicating that the effects of shear stress on human PAR-1 were not species-specific. Flow cytometry and ELISA techniques using rat smooth muscle cells and HASMCs, respectively, provided evidence that shear stress exerted similar effects on cell surface-associated PAR-1 and tPA protein released into the conditioned media. The decrease in PAR-1 mRNA and protein had functional consequences for HASMCs, such as inhibition of [Ca2+] mobilization in response to thrombin stimulation. These data indicate that human PAR-1 and tPA gene expression are regulated differentially by shear stress, in a pattern consistent with their putative roles in several arterial vascular pathologies.

Activation of the growth hormone/insulin-like growth factor axis by treatment with 17 alpha-methyltestosterone and seawater rearing in the tilapia, Oreochromis mossambicus.

PubMed

Riley, Larry G; Richman, Nurney H; Hirano, Tetsuya; Gordon Grau, E

2002-07-01

Effects of 17 alpha-methyltestosterone (MT) treatment and environmental salinity on the growth hormone (GH)/insulin-like growth factor (IGF) axis were examined in the euryhaline tilapia, Oreochromis mossambicus. Yolk-sac fry were collected from brood stock in fresh water (FW). After yolk-sac absorption, they were assigned randomly to 1 of 4 groups: FW, MT treatment in FW, SW, and MT treatment in seawater (SW). After 147 days, FW controls had the lowest levels of GH mRNA followed by FW fish treated with MT and SW control fish. Seawater fish fed with a diet containing MT, which grew the fastest, had significantly higher levels of GH mRNA than all the other groups. A significant correlation was observed between GH mRNA and the size of the individual fish. By contrast, plasma GH levels did not vary significantly among the groups. Pituitary GH mRNA levels, plasma IGF-I levels, and fish size varied in a correlated pattern, i.e., SW+MT>FW+MT=SW control>FW control. The tilapia pituitary produces two prolactins (PRLs), PRL(177) and PRL(188). Prolactin(177), but not PRL(188), exhibits growth-promoting actions in FW tilapia. Pituitary mRNA levels of both PRLs were significantly higher in fish reared in FW than those reared in SW. Treatment with MT significantly increased mRNA levels of both PRLs in FW, but had no effect on SW fish. No correlation was seen between plasma PRL levels and growth or between PRL mRNA levels and growth. These results indicate that SW rearing and MT treatment stimulate the GH/IGF-I axis, and suggest that pituitary GH mRNA at this stage of development is a better indicator of growth than plasma levels of GH and IGF-I.

Differential expression of chicken dimerization cofactor of hepatocyte nuclear factor-1 (DcoH) and its novel counterpart, DcoHalpha.

PubMed Central

Kim, H; You, S; Foster, L K; Farris, J; Choi, Y J; Foster, D N

2001-01-01

We have used differential display PCR to study altered gene expression in immortalized chicken embryo fibroblasts (CEFs) that have been established in our laboratory. This technique resulted in the cloning of a novel counterpart of the previously cloned chicken dimerization cofactor of hepatocyte nuclear factor (HNF)-1 (cDcoH), which was identified as cDcoHalpha. The steady-state mRNA levels of cDcoHalpha were up-regulated in all immortal CEFs tested compared with primary CEF cells. cDcoH and cDcoHalpha showed opposite patterns of mRNA expression due to differential regulation of transcription rates, but not mRNA half-lives, in primary and immortal CEFs. Expression of cDcoHalpha increased in the late G1 and early S phases of the cell cycle, while cDcoH mRNA increased in the late S and G2/M phases. In contrast with consistent expression of both genes in primary quiescent cells, cDcoH mRNA, but not cDcoHalpha mRNA, was dramatically decreased in primary senescent cells. The highest levels of cDcoHalpha mRNA were found in the kidney, liver, heart and ovarian follicles, while the major tissues expressing cDcoH were hypothalamus, kidney and liver. cDcoH and cDcoHalpha probes did not cross-hybridize to human hepatocyte mRNA. When transfected into human HepG2 cells, both cDcoH and cDcoHalpha showed similar functional activity as measured by increased expression of a reporter gene, as well as alpha-fetoprotein and albumin genes that both contain HNF-1 binding elements in their promoters. Our results suggest that the novel chicken DcoHalpha might function as a transcriptional cofactor for HNF-1 in specific cellular-environmental states. PMID:11237869

Wood-inhabiting fungi in southern Italy forest stands: morphogroups, vegetation types and decay classes.

PubMed

Granito, Vito Mario; Lunghini, Dario; Maggi, Oriana; Persiani, Anna Maria

2015-01-01

The authors conducted an ecological study of forests subjected to varying management. The aim of the study was to extend and integrate, within a multivariate context, knowledge of how saproxylic fungal communities behave along altitudinal/vegetational gradients in response to the varying features and quality of coarse woody debris (CWD). The intra-annual seasonal monitoring of saproxylic fungi, based on sporocarp inventories, was used to investigate saproxylic fungi in relation to vegetation types and management categories. We analyzed fungal species occurrence, recorded according to the presence/absence and frequency of sporocarps, on the basis of the harvest season, of coarse woody debris decay classes as well as other environmental and ecological variables. Two-way cluster analysis, DCA and Spearman's rank correlations, for indirect gradient analysis, were performed to identify any patterns of seasonality and decay. Most of the species were found on CWD in an intermediate decay stage. The first DCA axis revealed the vegetational/microclimate gradient as the main driver of fungal community composition, while the second axis corresponded to a strong gradient of CWD decay classes. © 2015 by The Mycological Society of America.

Closing in on the radiative weak chiral couplings

NASA Astrophysics Data System (ADS)

Cappiello, Luigi; Catà, Oscar; D'Ambrosio, Giancarlo

2018-03-01

We point out that, given the current experimental status of radiative kaon decays, a subclass of the O (p^4) counterterms of the weak chiral lagrangian can be determined in closed form. This involves in a decisive way the decay K^± → π ^± π ^0 l^+ l^-, currently being measured at CERN by the NA48/2 and NA62 collaborations. We show that consistency with other radiative kaon decay measurements leads to a rather clean prediction for the {O}(p^4) weak couplings entering this decay mode. This results in a characteristic pattern for the interference Dalitz plot, susceptible to be tested already with the limited statistics available at NA48/2. We also provide the first analysis of K_S→ π ^+π ^-γ ^*, which will be measured by LHCb and will help reduce (together with the related K_L decay) the experimental uncertainty on the radiative weak chiral couplings. A precise experimental determination of the {O}(p^4) weak couplings is important in order to assess the validity of the existing theoretical models in a conclusive way. We briefly comment on the current theoretical situation and discuss the merits of the different theoretical approaches.

Excitation of half-integer up-shifted decay channel and quasi-mode in plasma edge for high power electron Bernstein wave heating scenario

NASA Astrophysics Data System (ADS)

Ali Asgarian, M.; Abbasi, M.

2018-04-01

Electron Bernstein waves (EBW) consist of promising tools in driving localized off-axis current needed for sustained operation as well as effective selective heating scenarios in advanced over dense fusion plasmas like spherical tori and stellarators by applying high power radio frequency waves within the range of Megawatts. Here some serious non-linear effects like parametric decay modes are highly expect-able which have been extensively studied theoretically and experimentally. In general, the decay of an EBW depends on the ratio of the incident frequency and electron cyclotron frequency. At ratios less than two, parametric decay leads to a lower hybrid wave (or an ion Bernstein wave) and EBWs at a lower frequency. For ratios more than two, the daughter waves constitute either an electron cyclotron quasi-mode and another EBW or an ion wave and EBW. However, in contrast with these decay patterns, the excitation of an unusual up-shifted frequency decay channel for the ratio less than two is demonstrated in this study which is totally different as to its generation and persistence. It is shown that this mode varies from the conventional parametric decay channels which necessarily satisfy the matching conditions in frequency and wave-vector. Moreover, the excitation of some less-known local non-propagating quasi-modes (virtual modes) through weak-turbulence theory and their contributions to energy leakage from conversion process leading the reduction in conversion efficiency is assessed.

Implications of Secondary Aftershocks for Failure Processes

NASA Astrophysics Data System (ADS)

Gross, S. J.

2001-12-01

When a seismic sequence with more than one mainshock or an unusually large aftershock occurs, there is a compound aftershock sequence. The secondary aftershocks need not have exactly the same decay as the primary sequence, with the differences having implications for the failure process. When the stress step from the secondary mainshock is positive but not large enough to cause immediate failure of all the remaining primary aftershocks, failure processes which involve accelerating slip will produce secondary aftershocks that decay more rapidly than primary aftershocks. This is because the primary aftershocks are an accelerated version of the background seismicity, and secondary aftershocks are an accelerated version of the primary aftershocks. Real stress perturbations may be negative, and heterogeneities in mainshock stress fields mean that the real world situation is quite complicated. I will first describe and verify my picture of secondary aftershock decay with reference to a simple numerical model of slipping faults which obeys rate and state dependent friction and lacks stress heterogeneity. With such a model, it is possible to generate secondary aftershock sequences with perturbed decay patterns, quantify those patterns, and develop an analysis technique capable of correcting for the effect in real data. The secondary aftershocks are defined in terms of frequency linearized time s(T), which is equal to the number of primary aftershocks expected by a time T, $ s ≡ ∫ t=0T n(t) dt, where the start time t=0 is the time of the primary aftershock, and the primary aftershock decay function n(t) is extrapolated forward to the times of the secondary aftershocks. In the absence of secondary sequences the function s(T)$ re-scales the time so that approximately one event occurs per new time unit; the aftershock sequence is gone. If this rescaling is applied in the presence of a secondary sequence, the secondary sequence is shaped like a primary aftershock sequence, and can be fit by the same modeling techniques applied to simple sequences. The later part of the presentation will concern the decay of Hector Mine aftershocks as influenced by the Landers aftershocks. Although attempts to predict the abundance of Hector aftershocks based on stress overlap analysis are not very successful, the analysis does do a good job fitting the decay of secondary sequences.

Soil Biota and Litter Decay in High Arctic Ecosystems

NASA Astrophysics Data System (ADS)

González, G.; Rivera, F.; Makarova, O.; Gould, W. A.

2006-12-01

Frost heave action contributes to the formation of non-sorted circles in the High Arctic. Non-sorted circles tend to heave more than the surrounding tundra due to deeper thaw and the formation of ice lenses. Thus, the geomorphology, soils and vegetation on the centers of the patterned-ground feature (non-sorted circles) as compared to the surrounding soils (inter-circles) can be different. We established a decomposition experiment to look at in situ decay rates of the most dominant graminoid species on non-sorted circles and adjacent inter-circle soils along a climatic gradient in the Canadian High Arctic as a component of a larger study looking at the biocomplexity of small-featured patterned ground ecosystems. Additionally, we investigated variation in soil chemical properties and biota, including soil microarthropods and microbial composition and biomass, as they relate to climate, topographic position, and litter decay rates. Our three sites locations, from coldest to warmest, are Isachsen, Ellef Ringnes Island (ER), NU (bioclimatic subzone A); Mould Bay (MB), Prince Patrick Island, NT (bioclimatic subzone B), and Green Cabin (GC), Aulavik National Park, Thomsen River, Banks Island, NT (bioclimatic subzone C). Our sample design included the selection of 15 non-sorted circles and adjacent inter-circle areas within the zonal vegetation at each site (a total of 90 sites), and a second set of 3 non-sorted circles and adjacent inter-circle areas in dry, mesic and wet tundra at each of the sites. Soil invertebrates were sampled at each site using both pitfall traps, soil microbial biomass was determined using substrate induced respiration and bacterial populations were determined using the most probable number method. Decomposition rates were measured using litterbags and as the percent of mass remaining of Carex misandra, Luzula nivalis and Alopecuris alpinus in GC, MB and ER, respectively. Our findings indicate these graminoid species decayed significantly over time at a rate of 10-15 % mass loss / yr during the first year of decay. Decay rates are different in non-sorted circles vs. inter-circle soils along the climatic gradient. In MB, L. nivalis seems to decay faster in the inter-circle soils than in non-sorted circles (0.05

Succession pattern of carrion-feeding insects in Paramo, Colombia.

PubMed

Martinez, Efrain; Duque, Patricia; Wolff, Marta

2007-03-02

The minimum postmortem interval can be estimated based on knowledge of the pattern of insect succession on a corpse. To use this approach requires that we take into account the rates of insect development associated with particular climatological conditions of the region. This study is the first to look at insect succession on decomposing carcasses in the high altitude plains (Paramo) in Colombia, at 3035 m above sea level. Five stages of decomposition were designated with indicator species identified for each stage: Callíphora nigribasis at the fresh stage; Compsomyiops verena at the bloated stage; Compsomyiops boliviana during active decay; Stearibia nigriceps and Hydrotaea sp. during advanced decay and Leptocera sp. for dry remains. A succession table is presented for carrion-associated species of the region, which can be used for estimating time since death in similar areas. Compsomyiops boliviana is reported for the first time in Colombia.

Photoluminescence enhancement of monolayer tungsten disulfide in complicated plasmonic microstructures

NASA Astrophysics Data System (ADS)

Zhou, Yi; Hu, Xiaoyong; Gao, Wei; Song, Hanfa; Chu, Saisai; Yang, Hong; Gong, Qihuang

2018-06-01

Two-dimensional van der Waals materials are interesting for fundamental physics exploration and device applications because of their attractive physical properties. Here, we report a strategy to realize photoluminescence (PL) enhancement of two-dimensional transition-metal dichalcogenides (TMDCs) in the visible range using a plasmonic microstructure with patterned gold nanoantennas and a metal-insulator-semiconductor-insulator-metal structure. The PL intensity was enhanced by a factor of two under Y-polarization due to the increased radiative decay rate by the surface plasmon radiation channel in the gold nanoantennas and the decreased nonradiative decay rate by suppressing exciton quenching in the SiO2 isolation layer. The fluorescence lifetime of monolayer tungsten disulfide in this structure was shorter than that of a sample without patterned gold nanoantennas. Tailoring the light-matter interactions between two-dimensional TMDCs and plasmonic nanostructures may provide highly efficient optoelectronic devices such as TMDC-based light emitters.

Conformational changes in the P site and mRNA entry channel evoked by AUG recognition in yeast translation preinitiation complexes

PubMed Central

Zhang, Fan; Saini, Adesh K.; Shin, Byung-Sik; Nanda, Jagpreet; Hinnebusch, Alan G.

2015-01-01

The translation preinitiation complex (PIC) is thought to assume an open conformation when scanning the mRNA leader, with AUG recognition evoking a closed conformation and more stable P site interaction of Met-tRNAi; however, physical evidence is lacking that AUG recognition constrains interaction of mRNA with the 40S binding cleft. We compared patterns of hydroxyl radical cleavage of rRNA by Fe(II)-BABE tethered to unique sites in eIF1A in yeast PICs reconstituted with mRNA harboring an AUG or near-cognate (AUC) start codon. rRNA residues in the P site display reduced cleavage in AUG versus AUC PICs; and enhanced cleavage in the AUC complexes was diminished by mutations of scanning enhancer elements of eIF1A that increase near-cognate recognition in vivo. This suggests that accessibility of these rRNA residues is reduced by accommodation of Met-tRNAi in the P site (PIN state) and by their interactions with the anticodon stem of Met-tRNAi. Our cleavage data also provide evidence that AUG recognition evokes dissociation of eIF1 from its 40S binding site, ejection of the eIF1A-CTT from the P-site and rearrangement to a closed conformation of the entry channel with reduced mobility of mRNA. PMID:25670678

Uncovering Hidden Layers of Cell Cycle Regulation through Integrative Multi-omic Analysis

PubMed Central

Aviner, Ranen; Shenoy, Anjana; Elroy-Stein, Orna; Geiger, Tamar

2015-01-01

Studying the complex relationship between transcription, translation and protein degradation is essential to our understanding of biological processes in health and disease. The limited correlations observed between mRNA and protein abundance suggest pervasive regulation of post-transcriptional steps and support the importance of profiling mRNA levels in parallel to protein synthesis and degradation rates. In this work, we applied an integrative multi-omic approach to study gene expression along the mammalian cell cycle through side-by-side analysis of mRNA, translation and protein levels. Our analysis sheds new light on the significant contribution of both protein synthesis and degradation to the variance in protein expression. Furthermore, we find that translation regulation plays an important role at S-phase, while progression through mitosis is predominantly controlled by changes in either mRNA levels or protein stability. Specific molecular functions are found to be co-regulated and share similar patterns of mRNA, translation and protein expression along the cell cycle. Notably, these include genes and entire pathways not previously implicated in cell cycle progression, demonstrating the potential of this approach to identify novel regulatory mechanisms beyond those revealed by traditional expression profiling. Through this three-level analysis, we characterize different mechanisms of gene expression, discover new cycling gene products and highlight the importance and utility of combining datasets generated using different techniques that monitor distinct steps of gene expression. PMID:26439921

Distribution of androgen receptor mRNA expression in vocal, auditory, and neuroendocrine circuits in a teleost fish.

PubMed

Forlano, Paul M; Marchaterre, Margaret; Deitcher, David L; Bass, Andrew H

2010-02-15

Across all major vertebrate groups, androgen receptors (ARs) have been identified in neural circuits that shape reproductive-related behaviors, including vocalization. The vocal control network of teleost fishes presents an archetypal example of how a vertebrate nervous system produces social, context-dependent sounds. We cloned a partial cDNA of AR that was used to generate specific probes to localize AR expression throughout the central nervous system of the vocal plainfin midshipman fish (Porichthys notatus). In the forebrain, AR mRNA is abundant in proposed homologs of the mammalian striatum and amygdala, and in anterior and posterior parvocellular and magnocellular nuclei of the preoptic area, nucleus preglomerulosus, and posterior, ventral and anterior tuberal nuclei of the hypothalamus. Many of these nuclei are part of the known vocal and auditory circuitry in midshipman. The midbrain periaqueductal gray, an essential link between forebrain and hindbrain vocal circuitry, and the lateral line recipient nucleus medialis in the rostral hindbrain also express abundant AR mRNA. In the caudal hindbrain-spinal vocal circuit, high AR mRNA is found in the vocal prepacemaker nucleus and along the dorsal periphery of the vocal motor nucleus congruent with the known pattern of expression of aromatase-containing glial cells. Additionally, abundant AR mRNA expression is shown for the first time in the inner ear of a vertebrate. The distribution of AR mRNA strongly supports the role of androgens as modulators of behaviorally defined vocal, auditory, and neuroendocrine circuits in teleost fish and vertebrates in general. 2009 Wiley-Liss, Inc.

Effects of simulated microgravity on microRNA and mRNA expression profile of rat soleus

NASA Astrophysics Data System (ADS)

Xu, Hongjie; Wu, Feng; Cao, Hongqing; Kan, Guanghan; Zhang, Hongyu; Yeung, Ella W.; Shang, Peng; Dai, Zhongquan; Li, Yinghui

2015-02-01

Spaceflight induces muscle atrophy but mechanism is not well understood. Here, we quantified microRNAs (miRNAs) and mRNA shifts of rat soleus in response to microgravity. MiRNAs and mRNA microarray of soleus after tail suspension (TS) for 7 and 14 days were performed followed by target gene and function annotation analysis and qRT-PCR. Relative muscle mass lost by 37.0% in TS-7 but less than 10% in the following three weeks. TS altered 23 miRNAs and 1313 mRNAs with at least 2-fold. QRT-PCR confirmed some of these changes. MiR-214, miR-486-5p and miR-221 continuously decreased. MiR-674 and Let-7e decreased only in TS-7, while miR-320b and miR-187 decreased only in TS-14. But there was no alteration of miR-320 and miR-206 in both time point. For mRNA detection, actn3 (5.1-fold and 13.8-fold) and myh4 (38-fold and 51.6-fold) increased abundantly and a3galt2 decreased. Predicted targeted genes (whyz, ywhaz and SFRP2) of altered miRNAs decreased. GO terms and cellular pathway of these alteration showed enrichment in regulation of muscle metabolism. Integration analysis of the miRNA and mRNA expression profiles confirmed that eleven genes were differently regulated by four miRNAs. This is the first study that showed expression pattern and synergistical regulation of miRNA and mRNA in rat soleus of TS for up to 14 days.

Single-cell mRNA profiling reveals transcriptional heterogeneity among pancreatic circulating tumour cells.

PubMed

Lapin, Morten; Tjensvoll, Kjersti; Oltedal, Satu; Javle, Milind; Smaaland, Rune; Gilje, Bjørnar; Nordgård, Oddmund

2017-05-31

Single-cell mRNA profiling of circulating tumour cells may contribute to a better understanding of the biology of these cells and their role in the metastatic process. In addition, such analyses may reveal new knowledge about the mechanisms underlying chemotherapy resistance and tumour progression in patients with cancer. Single circulating tumour cells were isolated from patients with locally advanced or metastatic pancreatic cancer with immuno-magnetic depletion and immuno-fluorescence microscopy. mRNA expression was analysed with single-cell multiplex RT-qPCR. Hierarchical clustering and principal component analysis were performed to identify expression patterns. Circulating tumour cells were detected in 33 of 56 (59%) examined blood samples. Single-cell mRNA profiling of intact isolated circulating tumour cells revealed both epithelial-like and mesenchymal-like subpopulations, which were distinct from leucocytes. The profiled circulating tumour cells also expressed elevated levels of stem cell markers, and the extracellular matrix protein, SPARC. The expression of SPARC might correspond to an epithelial-mesenchymal transition in pancreatic circulating tumour cells. The analysis of single pancreatic circulating tumour cells identified distinct subpopulations and revealed elevated expression of transcripts relevant to the dissemination of circulating tumour cells to distant organ sites.

Longitudinal monitoring of bottlenose dolphins leukocyte cytokine mRNA responsiveness by qPCR

USDA-ARS?s Scientific Manuscript database

Both veterinarians caring for bottlenose dolphins (Tursiops truncatus) in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin health. Quantitative PCR (qPCR) can be used to assess cytokine expression patterns of peripheral blood m...

Longitudinal monitoring of bottlenose dolphin leukocyte cytokine mRNA responsiveness by qPCR

USDA-ARS?s Scientific Manuscript database

Both veterinarians caring for bottlenose dolphins (Tursiops truncatus) in managed populations and researchers monitoring wild populations use blood-based diagnostics to monitor bottlenose dolphin health. Quantitative PCR (qPCR) can be used to assess cytokine expression patterns of peripheral blood m...

Hedgehog signaling pathway is active in GBM with GLI1 mRNA expression showing a single continuous distribution rather than discrete high/low clusters.

PubMed

Chandra, Vikas; Das, Tapojyoti; Gulati, Puneet; Biswas, Nidhan K; Rote, Sarang; Chatterjee, Uttara; Ghosh, Samarendra N; Deb, Sumit; Saha, Suniti K; Chowdhury, Anup K; Ghosh, Subhashish; Rudin, Charles M; Mukherjee, Ankur; Basu, Analabha; Dhara, Surajit

2015-01-01

Hedgehog (Hh) signaling pathway is a valid therapeutic target in a wide range of malignancies. We focus here on glioblastoma multiforme (GBM), a lethal malignancy of the central nervous system (CNS). By analyzing RNA-sequencing based transcriptomics data on 149 clinical cases of TCGA-GBM database we show here a strong correlation (r = 0.7) between GLI1 and PTCH1 mRNA expression--as a hallmark of the canonical Hh-pathway activity in this malignancy. GLI1 mRNA expression varied in 3 orders of magnitude among the GBM patients of the same cohort showing a single continuous distribution-unlike the discrete high/low-GLI1 mRNA expressing clusters of medulloblastoma (MB). When compared with MB as a reference, the median GLI1 mRNA expression in GBM appeared 14.8 fold lower than that of the "high-Hh" cluster of MB but 5.6 fold higher than that of the "low-Hh" cluster of MB. Next, we demonstrated statistically significant up- and down-regulation of GLI1 mRNA expressions in GBM patient-derived low-passage neurospheres in vitro by sonic hedgehog ligand-enriched conditioned media (shh-CM) and by Hh-inhibitor drug vismodegib respectively. We also showed clinically achievable dose (50 μM) of vismodegib alone to be sufficient to induce apoptosis and cell cycle arrest in these low-passage GBM neurospheres in vitro. Vismodegib showed an effect on the neurospheres, both by down-regulating GLI1 mRNA expression and by inducing apoptosis/cell cycle arrest, irrespective of their relative endogenous levels of GLI1 mRNA expression. We conclude from our study that this single continuous distribution pattern of GLI1 mRNA expression technically puts almost all GBM patients in a single group rather than discrete high- or low-clusters in terms of Hh-pathway activity. That is suggestive of therapies with Hh-pathway inhibitor drugs in this malignancy without a need for further stratification of patients on the basis of relative levels of Hh-pathway activity among them.

Differential expression of cytokeratin mRNA and protein in normal prostate, prostatic intraepithelial neoplasia, and invasive carcinoma.

PubMed Central

Yang, Y.; Hao, J.; Liu, X.; Dalkin, B.; Nagle, R. B.

1997-01-01

The expression of cytokeratin (CK) mRNA for CK5, -8, -14, -16, and -19 was investigated in normal prostate, prostatic intraepithelial neoplasia (PIN) lesions, and invasive carcinoma using in situ hybridization. Protein localization was carried out in adjacent sections using immunohistochemistry and correlated with mRNA expression. Snap-frozen human prostate samples including 22 examples of normal glands, 20 cases of PIN lesions, and 12 cases of invasive carcinoma were examined. CK5 and -14 mRNA and protein were prominently expressed only in the basal cells of normal glands and PIN lesions. CK14 mRNA was absent in the luminal cells of the most of the PIN lesions but was seen at a low level in some PIN lesions. CK14 protein was not detected in any PIN lesion, suggesting that, if the cell that makes up the PIN lesions is derived from a basal cell, CK14 translation is depressed although a low level of CK14 mRNA may persist. CK8 mRNA and protein were constitutively expressed in all epithelia of normal and abnormal prostate tissues. CK19 mRNA and protein were persistently expressed in both basal and luminal cells of the tubular portion of normal glands as well as PIN lesions, but were expressed heterogeneously in both basal and luminal cells of normal alveoli. CK16 mRNA was expressed in a similar pattern as CK19, but CK16 protein was not detected either in normal or in abnormal prostate tissues. In conclusion, the expression of CK19 in PIN lesions is similar to its tubular expression and would support an origin of PIN lesions from this structure rather than the alveolar portion of the glands. The similar cytokeratin expression between PIN lesions and invasive carcinoma further supports the concept that PIN is a precursor lesion of invasive carcinoma. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:9033282

Indian summer monsoon rainfall variability in response to differences in the decay phase of El Niño

NASA Astrophysics Data System (ADS)

Chowdary, Jasti S.; Harsha, H. S.; Gnanaseelan, C.; Srinivas, G.; Parekh, Anant; Pillai, Prasanth; Naidu, C. V.

2017-04-01

In general the Indian summer monsoon (ISM) rainfall is near normal or excess during the El Niño decay phase. Nevertheless the impact of large variations in decaying El Niño on the ISM rainfall and circulation is not systematically examined. Based on the timing of El Niño decay with respect to boreal summer season, El Niño decay phases are classified into three types in this study using 142 years of sea surface temperature (SST) data, which are as follows: (1) early-decay (ED; decay during spring), (2) mid-summer decay (MD; decay by mid-summer) and (3) no-decay (ND; no decay in summer). It is observed that ISM rainfall is above normal/excess during ED years, normal during MD years and below normal/deficit in ND years, suggesting that the differences in El Niño decay phase display profound impact on the ISM rainfall. Tropical Indian Ocean (TIO) SST warming, induced by El Niño, decays rapidly before the second half of the monsoon season (August and September) in ED years, but persists up to the end of the season in MD years, whereas TIO warming maintained up to winter in ND case. Analysis reveals the existence of strong sub-seasonal ISM rainfall variations in the summer following El Niño years. During ED years, strong negative SST anomalies develop over the equatorial central-eastern Pacific by June and are apparent throughout the summer season accompanied by anomalous moisture divergence and high sea level pressure (SLP). The associated moisture convergence and low SLP over ISM region favour excess rainfall (mainly from July onwards). This circulation and rainfall anomalies are highly influenced by warm TIO SST and Pacific La Niña conditions in ED years. Convergence of southwesterlies from Arabian Sea and northeasterlies from Bay of Bengal leads to positive rainfall over most part of the Indian subcontinent from August onwards in MD years. ND years are characterized by negative rainfall anomaly spatial pattern and weaker circulation over India throughout the summer season, which are mainly due to persisting El Niño related warm SST anomalies over the Pacific. Atmospheric general circulation model simulation supports our hypothesis that El Niño decay variations modulate ISM rainfall and circulation.

[Regulation of the expression of coenzyme Q-synthesis complex during ageing].

PubMed

Campos-Silva, Carmen; Reyes-Torres, Iván; Rivera, Maximiliano; Meza-Torres, Catherine; Hernández-Camacho, Juan Diego; Rodríguez-Bies, Elisabet; Navas, Plácido; López-Lluch, Guillermo

Coenzyme Q is an essential component in the activity of the mitochondrial electron transport chain. Its synthesis involves, at least, a complex of ten different proteins. In this study, an attempt is made to determine the evolution of the expression of the genes involved in coenzyme Q synthesis during mouse ageing. The messenger RNA (mRNA) of different organs, such as brain, liver, kidney and skeletal muscle from young (8 months), mature (18 months), and old (24 months) mice was extracted by using Trizol and was then analysed by real time PCR (qPCR) using specific primers for all the known components of the coenzyme Q-synthesis complex (COQ genes). Liver showed the highest age-dependent changes in mRNA levels of the different components of Q-synthesis complex, affecting the extent of the variation as well as the significance of the change. In most of the cases, mRNA levels of the different components were higher in mature animals compared to young and old animals. When mRNAs of young and old animals were compared, only minor reductions of mRNA levels were found. Kidney showed a pattern similar to that found in liver as regards the changes in expression, although with lower increases in mature animals than those observed in the liver. Brain and skeletal muscle showed low variations, with muscle being the tissue with less changes, although a pattern similar to that found in liver and kidney was found, with slight increases in mature animals. The results of this study indicate that ageing is an important factor affecting COQ gene expression, but its effect depends on the organ, and that mature animals show higher levels of mRNA than young and old animals. Taken into consideration the importance of coenzyme Q in cell metabolism and ageing, a more detailed study is needed to understand the gene regulation of the coenzyme Q-synthesis mechanisms during ageing. Copyright © 2017 SEGG. Publicado por Elsevier España, S.L.U. All rights reserved.

Quantitative identification of proteins that influence miRNA biogenesis by RNA pull-down-SILAC mass spectrometry (RP-SMS).

PubMed

Choudhury, Nila Roy; Michlewski, Gracjan

2018-06-08

RNA-binding proteins mediate and control gene expression. As some examples, they regulate pre-mRNA synthesis and processing; mRNA localisation, translation and decay; and microRNA (miRNA) biogenesis and function. Here, we present a detailed protocol for RNA pull-down coupled to stable isotope labelling by amino acids in cell culture (SILAC) mass spectrometry (RP-SMS) that enables quantitative, fast and specific detection of RNA-binding proteins that regulate miRNA biogenesis. In general, this method allows for the identification of RNA-protein complexes formed using in vitro or chemically synthesized RNAs and protein extracts derived from cultured cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Impact of sediment particle size on biotransformation of 17β-estradiol and 17β-trenbolone.

PubMed

Zhang, Yun; Sangster, Jodi L; Gauza, Lukasz; Bartelt-Hunt, Shannon L

2016-12-01

Soil/sediment particle size has been reported to influence the sorption and bioavailability of steroid hormones in the environment. However, the impact of particle size on biotransformation has not been well elucidated. The present study investigated the dissipation of 17β-estradiol and 17β-trenbolone and the formation and degradation of the subsequent transformation products in different size fractions of a sandy and a silt loam sediment. The results showed that the decay of 17β-estradiol and 17β-trenbolone associated with fine particles followed a biphasic pattern with more rapid decay in the initial phase followed by a second phase with slower decay of the residues compared to their decay rates in the sand fraction. Estrone and trendione were detected as a primary biotransformation product for 17β-estradiol and 17β-trenbolone, respectively. The parent-to-product conversion ratios and the degradation rates of estrone and trendione varied among different size fractions, but no consistent correlation was observed between decay rates and sediment particle size. Estrone and trendione decayed in the whole sediments at rates not statistically different from those associated with the fine fractions. These results indicate that fine particles may play an important role in influencing the persistence of and the potential risk posed by steroid hormones in the aquatic systems. Copyright © 2016 Elsevier B.V. All rights reserved.

On the mechanism of non-radiative decay of blue fluorescent protein chromophore: New insight from the excited-state molecular dynamics simulations and potential energy calculations

NASA Astrophysics Data System (ADS)

Zhao, Li; Liu, Jian-Yong; Zhou, Pan-Wang

2017-11-01

A detailed theoretical investigation based on the ab initio on-the-fly surface hopping dynamics simulations and potential energy surfaces calculations has been performed to unveil the mechanism of the photoinduced non-adiabatic relaxation process of the isolated blue fluorescent protein (BFP) chromophore in gas phase. The data analysis presents that the dominant reaction coordinate of the BFP chromophore is driven by a rotation motion around the CC double bridging bond, which is in remarkable difference with a previous result which supports a Hula-Twist rotation pattern. Such behavior is consistent with the double bond rotation pattern of the GFP neutral chromophore. In addition, the dynamics simulations give an estimated decay time of 1.1 ps for the S1 state, which is agrees well with the experimental values measured in proteins. The present work offers a straightforward understanding for the decay mechanism of the BFP chromophore and suggestions of the photochemical properties of analogous protein chromophores. We hope the current work would be helpful for further exploration of the BFP photochemical and photophysical properties in various environments, and can provide guidance and prediction for rational design of the fluorescent proteins catering for different demands.

Patterns of mRNA and protein expression during minus-lens compensation and recovery in tree shrew sclera.

PubMed

Gao, Hong; Frost, Michael R; Siegwart, John T; Norton, Thomas T

2011-04-12

To increase our understanding of the mechanisms that remodel the sclera during the development of lens-induced myopia, when the sclera responds to putative "go" signals of retinal origin, and during recovery from lens-induced myopia, when the sclera responds to retinally-derived "stop" signals. Seven groups of tree shrews were used to examine mRNA levels during minus lens compensation and recovery. Starting 24 days after eye opening (days of visual experience [VE]) lens compensation animals wore a monocular -5D lens for 1, 4, or 11 days. Recovery animals wore the -5D lens for 11 days, which was then removed for 1 or 4 days. Normal animals were examined at 24 and 38 days of VE. All groups contained 8 animals. Scleral mRNA levels were examined in the treated and contralateral control eyes with quantitative real-time polymerase chain reaction (qPCR) for 27 genes divided into four categories: 1) signaling molecules, 2) matricellular proteins, 3) metalloproteinases (MPs) and tissue inhibitors of metalloproteinases (TIMPs), and 4) cell adhesion and other proteins. Four groups (n=5 per group) were used to examine protein levels. One group wore a -5D lens for 4 days. A second group recovered for 4 days after 11 days of -5D lens treatment. Two groups were used to examine age-matched normal protein levels at 28 and 39 days of VE. The levels of six scleral proteins that showed differential mRNA expression were examined with quantitative western blots. Nineteen of the genes showed differential (treated eye versus control eye) expression of mRNA levels in at least one group of animals. Which genes showed differential expression differed after 1 and 4 days of compensation and after 1 or 4 days of recovery. The mRNA level for one gene, a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), was upregulated in the treated eyes after 1 day of compensation. After 4 days, transforming growth factor beta receptor 3 (TGFBR3), transforming growth factor-beta-induced protein ig-h3 (TGFBI), and matrix metalloproteinase 14 (MMP14) mRNA levels were upregulated. Downregulated were mRNA levels for transforming growth factor beta-1 (TGFB1), transforming growth factor beta-2 (TGFB2), thrombospondin 1 (THBS1), tenascin (TNC), osteonectin (SPARC), osteopontin (SPP1), tissue inhibitor of metalloproteinases 3 (TIMP3), and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5). After 11 days of lens wear, there was no differential expression. During recovery, after 1 day, treated-eye mRNA downregulation was found for TGFB2, TGFBR1, TGFBR2, TGFBR3, SPARC, ADAMTS1, ADAMTS5, syndecan 4 (SDC4), and collagen type VI, alpha 1 (COL6A1). After 4 days, TGFB1, TGFB2, TGFB3, THBS2, and TIMP3 mRNA levels were upregulated in the recovering eye. Significant downregulation, relative to normal eyes, was found in both the control and treated eyes for most genes after 1 day of compensation; a similar decrease was found, compared to lens-compensated eyes, after one day of recovery. Protein levels for THBS1 showed positive correlation with the differential mRNA levels and TGFBR3 showed a negative correlation. No differential protein expression was found for TGFB2, TGFBI, MMP14, and TIMP3. The different patterns of differential mRNA expression during minus lens compensation (hyperopia) and recovery (myopia) show that scleral fibroblasts distinguish between "go" and "stop" conditions. There is evidence of binocular global downregulation of genes at the start of both lens wear and recovery. As additional information accumulates about changes in gene expression that occur during compensation and recovery the "signature" of differential changes may help us to understand in more detail how the sclera responds in "go" and "stop" conditions.

Transcriptional Activity, Chromosomal Distribution and Expression Effects of Transposable Elements in Coffea Genomes

PubMed Central

da Silva, Carlos R. M.; Andrade, Alan C.; Marraccini, Pierre; Teixeira, João B.; Carazzolle, Marcelo F.; Pereira, Gonçalo A. G.; Pereira, Luiz Filipe P.; Vanzela, André L. L.; Wang, Lu; Jordan, I. King; Carareto, Claudia M. A.

2013-01-01

Plant genomes are massively invaded by transposable elements (TEs), many of which are located near host genes and can thus impact gene expression. In flowering plants, TE expression can be activated (de-repressed) under certain stressful conditions, both biotic and abiotic, as well as by genome stress caused by hybridization. In this study, we examined the effects of these stress agents on TE expression in two diploid species of coffee, Coffea canephora and C. eugenioides, and their allotetraploid hybrid C. arabica. We also explored the relationship of TE repression mechanisms to host gene regulation via the effects of exonized TE sequences. Similar to what has been seen for other plants, overall TE expression levels are low in Coffea plant cultivars, consistent with the existence of effective TE repression mechanisms. TE expression patterns are highly dynamic across the species and conditions assayed here are unrelated to their classification at the level of TE class or family. In contrast to previous results, cell culture conditions per se do not lead to the de-repression of TE expression in C. arabica. Results obtained here indicate that differing plant drought stress levels relate strongly to TE repression mechanisms. TEs tend to be expressed at significantly higher levels in non-irrigated samples for the drought tolerant cultivars but in drought sensitive cultivars the opposite pattern was shown with irrigated samples showing significantly higher TE expression. Thus, TE genome repression mechanisms may be finely tuned to the ideal growth and/or regulatory conditions of the specific plant cultivars in which they are active. Analysis of TE expression levels in cell culture conditions underscored the importance of nonsense-mediated mRNA decay (NMD) pathways in the repression of Coffea TEs. These same NMD mechanisms can also regulate plant host gene expression via the repression of genes that bear exonized TE sequences. PMID:24244387

The decay of motor adaptation to novel movement dynamics reveals an asymmetry in the stability of motion state-dependent learning

PubMed Central

Hosseini, Eghbal A.; Nguyen, Katrina P.; Joiner, Wilsaan M.

2017-01-01

Motor adaptation paradigms provide a quantitative method to study short-term modification of motor commands. Despite the growing understanding of the role motion states (e.g., velocity) play in this form of motor learning, there is little information on the relative stability of memories based on these movement characteristics, especially in comparison to the initial adaptation. Here, we trained subjects to make reaching movements perturbed by force patterns dependent upon either limb position or velocity. Following training, subjects were exposed to a series of error-clamp trials to measure the temporal characteristics of the feedforward motor output during the decay of learning. The compensatory force patterns were largely based on the perturbation kinematic (e.g., velocity), but also showed a small contribution from the other motion kinematic (e.g., position). However, the velocity contribution in response to the position-based perturbation decayed at a slower rate than the position contribution to velocity-based training, suggesting a difference in stability. Next, we modified a previous model of motor adaptation to reflect this difference and simulated the behavior for different learning goals. We were interested in the stability of learning when the perturbations were based on different combinations of limb position or velocity that subsequently resulted in biased amounts of motion-based learning. We trained additional subjects on these combined motion-state perturbations and confirmed the predictions of the model. Specifically, we show that (1) there is a significant separation between the observed gain-space trajectories for the learning and decay of adaptation and (2) for combined motion-state perturbations, the gain associated to changes in limb position decayed at a faster rate than the velocity-dependent gain, even when the position-dependent gain at the end of training was significantly greater. Collectively, these results suggest that the state-dependent adaptation associated with movement velocity is relatively more stable than that based on position. PMID:28481891

Methamphetamine Users Have Increased Dental Disease: A Propensity Score Analysis.

PubMed

Shetty, V; Harrell, L; Clague, J; Murphy, D A; Dye, B A; Belin, T R

2016-07-01

Methamphetamine (MA) users are assumed to have a high burden of tooth decay. Less clear is how the distribution and severity of dental caries in MA users differ from the general population. Using a covariate-balancing propensity score strategy, we investigated the differential effects of MA use on dental caries by comparing the patterns of decayed, missing, and filled teeth in a community sample of 571 MA users with a subset of 2,755 demographically similar control individuals selected from a National Health and Nutrition Examination Survey (NHANES) cohort. Recruited over a 2-y period with a stratified sampling protocol, the MA users underwent comprehensive dental examinations by 3 trained and calibrated dentists using NHANES protocols. Propensity scores were estimated with logistic regression based on background characteristics, and a subset of closely matched subjects was stratified into quintiles for comparisons. MA users were twice as likely to have untreated caries (odds ratio [OR] = 2.08; 95% confidence interval [95% CI]: 1.55 to 2.78) and 4 times more likely to have caries experience (OR = 4.06; 95% CI: 2.24 to 7.34) than the control group of NHANES participants. Additionally, MA users were twice as likely to have 2 more decayed, missing, or filled teeth (OR = 2.08; 95% CI: 1.29 to 2.79) than the NHANES participants. The differential involvement of the teeth surfaces in MA users was quite distinctive, with carious surface involvement being highest for the maxillary central incisors, followed by maxillary posterior premolars and molars. Users injecting MA had significantly higher rates of tooth decay compared with noninjectors (P = 0.04). Although MA users experienced decayed and missing dental surfaces more frequently than NHANES participants, NHANES participants had more restored surfaces, especially on molars. The high rates and distinctive patterns of dental caries observed could be used 1) to alert dentists to covert MA use in their patients and 2) as the basis for comprehensive management strategies. © International & American Associations for Dental Research 2016.

Meat and Fish Freshness Inspection System Based on Odor Sensing

PubMed Central

Hasan, Najam ul; Ejaz, Naveed; Ejaz, Waleed; Kim, Hyung Seok

2012-01-01

We propose a method for building a simple electronic nose based on commercially available sensors used to sniff in the market and identify spoiled/contaminated meat stocked for sale in butcher shops. Using a metal oxide semiconductor-based electronic nose, we measured the smell signature from two of the most common meat foods (beef and fish) stored at room temperature. Food samples were divided into two groups: fresh beef with decayed fish and fresh fish with decayed beef. The prime objective was to identify the decayed item using the developed electronic nose. Additionally, we tested the electronic nose using three pattern classification algorithms (artificial neural network, support vector machine and k-nearest neighbor), and compared them based on accuracy, sensitivity, and specificity. The results demonstrate that the k-nearest neighbor algorithm has the highest accuracy. PMID:23202222

Transition in the decay rates of stationary distributions of Lévy motion in an energy landscape.

PubMed

Kaleta, Kamil; Lőrinczi, József

2016-02-01

The time evolution of random variables with Lévy statistics has the ability to develop jumps, displaying very different behaviors from continuously fluctuating cases. Such patterns appear in an ever broadening range of examples including random lasers, non-Gaussian kinetics, or foraging strategies. The penalizing or reinforcing effect of the environment, however, has been little explored so far. We report a new phenomenon which manifests as a qualitative transition in the spatial decay behavior of the stationary measure of a jump process under an external potential, occurring on a combined change in the characteristics of the process and the lowest eigenvalue resulting from the effect of the potential. This also provides insight into the fundamental question of what is the mechanism of the spatial decay of a ground state.

New signals for vector-like down-type quark in U(1) of E_6

NASA Astrophysics Data System (ADS)

Das, Kasinath; Li, Tianjun; Nandi, S.; Rai, Santosh Kumar

2018-01-01

We consider the pair production of vector-like down-type quarks in an E_6 motivated model, where each of the produced down-type vector-like quark decays into an ordinary Standard Model light quark and a singlet scalar. Both the vector-like quark and the singlet scalar appear naturally in the E_6 model with masses at the TeV scale with a favorable choice of symmetry breaking pattern. We focus on the non-standard decay of the vector-like quark and the new scalar which decays to two photons or two gluons. We analyze the signal for the vector-like quark production in the 2γ +≥ 2j channel and show how the scalar and vector-like quark masses can be determined at the Large Hadron Collider.

Anatomy of Bs → PV decays and effects of next-to-leading order contributions in the perturbative QCD factorization approach

NASA Astrophysics Data System (ADS)

Yan, Da-Cheng; Yang, Ping; Liu, Xin; Xiao, Zhen-Jun

2018-06-01

In this paper, we will make systematic calculations for the branching ratios and the CP-violating asymmetries of the twenty one Bbars0 → PV decays by employing the perturbative QCD (PQCD) factorization approach. Besides the full leading-order (LO) contributions, all currently known next-to-leading order (NLO) contributions are taken into account. We found numerically that: (a) the NLO contributions can provide ∼ 40% enhancement to the LO PQCD predictions for B (Bbars0 →K0K bar * 0) and B (Bbars0 →K±K*∓), or a ∼ 37% reduction to B (Bbars0 →π-K*+); and we confirmed that the inclusion of the known NLO contributions can improve significantly the agreement between the theory and those currently available experimental measurements; (b) the total effects on the PQCD predictions for the relevant Bs0 → P transition form factors after the inclusion of the NLO twist-2 and twist-3 contributions is generally small in magnitude: less than 10% enhancement respect to the leading order result; (c) for the "tree" dominated decay Bbars0 →K+ρ- and the "color-suppressed-tree" decay Bbars0 →π0K*0, the big difference between the PQCD predictions for their branching ratios are induced by different topological structure and by interference effects among the decay amplitude AT,C and AP: constructive for the first decay but destructive for the second one; and (d) for Bbars0 → V (η ,η‧) decays, the complex pattern of the PQCD predictions for their branching ratios can be understood by rather different topological structures and the interference effects between the decay amplitude A (Vηq) and A (Vηs) due to the η-η‧ mixing.

Enzymes Involved in Post-transcriptional RNA Metabolism in Gram-negative bacteria

PubMed Central

Mohanty, Bijoy K.

2018-01-01

Gene expression in Gram-negative bacteria is regulated at many levels, including transcription initiation, RNA processing, RNA/RNA interactions, mRNA decay, and translational controls involving enzymes that alter translational efficiency. In this chapter we discuss the various enzymes that control transcription, translation and RNA stability through RNA processing and degradation. RNA processing is essential to generate functional RNAs, while degradation helps control the steady-state level of each individual transcript. For example, all the pre-tRNAs are transcribed with extra nucleotides at both their 5′ and 3′ termini, which are subsequently processed to produce mature tRNAs that can be aminoacylated. Similarly, rRNAs that are transcribed as part of a 30S polycistronic transcript, are matured to individual 16S, 23S and 5S rRNAs. Decay of mRNAs plays a key role in gene regulation through controlling the steady-state level of each transcript, which is essential for maintaining appropriate protein levels. In addition, degradation of both translated and non-translated RNAs recycles nucleotides to facilitate new RNA synthesis. To carry out all these reactions Gram-negative bacteria employ a large number of endonucleases, exonucleases, RNA helicases, and poly(A) polymerase as well as proteins that regulate the catalytic activity of particular ribonucleases. Under certain stress conditions an additional group of specialized endonucleases facilitate the cell’s ability to adapt and survive. Many of the enzymes, such as RNase E, RNase III, polynucleotide phosphorylase, RNase R, and poly(A) polymerase I participate in multiple RNA processing and decay pathways. PMID:29676246

Phenotype-specific CpG island methylation events in a murine model of prostate cancer.

PubMed

Camoriano, Marta; Kinney, Shannon R Morey; Moser, Michael T; Foster, Barbara A; Mohler, James L; Trump, Donald L; Karpf, Adam R; Smiraglia, Dominic J

2008-06-01

Aberrant DNA methylation plays a significant role in nearly all human cancers and may contribute to disease progression to advanced phenotypes. Study of advanced prostate cancer phenotypes in the human disease is hampered by limited availability of tissues. We therefore took advantage of the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model to study whether three different phenotypes of TRAMP tumors (PRIM, late-stage primary tumors; AIP, androgen-independent primary tumors; and MET, metastases) displayed specific patterns of CpG island hypermethylation using Restriction Landmark Genomic Scanning. Each tumor phenotype displayed numerous hypermethylation events, with the most homogeneous methylation pattern in AIP and the most heterogeneous pattern in MET. Several loci displayed a phenotype-specific methylation pattern; the most striking pattern being loci methylated at high frequency in PRIM and AIP but rarely in MET. Examination of the mRNA expression of three genes, BC058385, Goosecoid, and Neurexin 2, which exhibited nonpromoter methylation, revealed increased expression associated with downstream methylation. Only methylated samples showed mRNA expression, in which tumor phenotype was a key factor determining the level of expression. The CpG island in the human orthologue of BC058385 was methylated in human AIP but not in primary androgen-stimulated prostate cancer or benign prostate. The clinical data show a proof-of-principle that the TRAMP model can be used to identify targets of aberrant CpG island methylation relevant to human disease. In conclusion, phenotype-specific hypermethylation events were associated with the overexpression of different genes and may provide new markers of prostate tumorigenesis.

Endometrial IL-1beta, IL-6 and TNF-alpha, mRNA expression in mares resistant or susceptible to post-breeding endometritis. Effects of estrous cycle, artificial insemination and immunomodulation.

PubMed

Fumuso, Elida; Giguère, Steeve; Wade, José; Rogan, Dragan; Videla-Dorna, Ignacio; Bowden, Raúl A

2003-11-15

Endometrial mRNA expression of the pro-inflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-alpha) was assessed in mares resistant (RM) or susceptible (SM) to persistent post-breeding endometritis (PPBE). Eight RM and eight SM, were selected based on reproductive records and functional tests out of a herd of 2,000 light cross-type mares. Three experiments were done to study transcription patterns in (i) basal conditions; (ii) after artificial insemination (AI); and (iii) after administration of an immunomodulator at time of artificial insemination. Endometrial biopsies were taken during consecutive cycles: (i) at estrus, when follicles reached 35 mm and at diestrus (7 +/- 1 days after ovulation); (ii) at 24 h post-AI, with dead semen (estrus) and in diestrus; (iii) at 24 h after treatment with a Mycobacterium phlei cell-wall extract (MCWE) preparation and AI (with dead semen), and at diestrus. mRNA expression was quantitated by real time PCR. Under basal conditions, SM had significantly higher mRNA expression of all cytokines in estrus and of IL-1beta and TNF-alpha in diestrus, compared to RM. After AI, there were no differences between RM and SM in estrus; however, mRNA expression for all three pro-inflammatory cytokines was higher than under basal conditions. In diestrus, RM showed significantly lower IL-1beta and TNF-alpha mRNA expression than SM. When MCWE was administered at time of AI, no differences between cytokine induction from RM and SM were found. Globally, mRNA expression for all three cytokines correlated well among themselves when expression was high. The present study showed that (i) in basal conditions RM had lower mRNA expression of pro-inflammatory cytokines than SM with no effect of estrous cycle; (ii) AI upregulated mRNA expression for all three cytokines in both RM and SM, with persistance in diestrus in the latter; (iii) treatment with MCWE at time of AI down-regulated mRNA expression of IL-1 with significant effects in SM which behaved like RM. Immunomodulation with MCWE could be of help in restoring homeostatic local inflammatory mechanisms, thus assisting in the prophylaxis of post-breeding endometritis in mares.

Physiological Challenges of Bone Repair

DTIC Science & Technology

2012-12-01

expression, in general, followed the same pattern in both groups, but significantly, lower levels of mRNA for Indian Hedgehog (ihh) and BMP-2 were detected in...the fracture calluses of the older rats. Indian Hedgehog is thought to be involved in chondrogenesis and bone repair, whereas BMP-2 stimulates bone

The histological characteristics, age-related thickness change of skin, and expression of the HSPs in the skin during hair cycle in yak (Bos grunniens)

PubMed Central

Yang, Xue; Cui, Yan; Yue, Jing; He, Honghong; Yu, Chuan; Liu, Penggang; Liu, Jun; Ren, Xiandong; Meng, Yun

2017-01-01

Objective This experiment was conducted to study the histological characteristics, age-related thickness changes, and expression of HSPs in the skin of yak. Methods A total of 20 yaks (10 males and 10 females) were used. Different regions of the normal skin of three different ages (newborn, half-year-old and adult) of yaks were harvested for histological study and thickness measurement. Biopsy samples were taken from the scapula regions of the skin from the same five approximately 1-year-old yaks during the hair cycle (telogen, anagen and catagen). RT-PCR, western blot and immunohistochemistry methods using the mRNA and protein levels were used to detect the expression of HSP27, HSP70 and HSP90. RT-PCR method was used to detect the mRNA expression of CGI-58 and KDF1. The IPP6.0 software was used to analyze the immunohistochemistry and measure the thickness of the skin. Results The general histological structure of hairy yak skin was similar to other domestic mammals. The unique features included prominent cutaneous vascular plexuses, underdeveloped sweat glands, a large number of nasolabial glands in the nasolabial plate, and hair follicle groups composed of one primary follicle and several secondary follicles. The skin, epidermis and dermis thickness did vary significantly between different body regions and different ages. The thickness of the skin, epidermis and dermis increased from newborn to adult in yaks. Yak skin thickness decreased from dorsally to ventrally on the trunk. The skin on the lateral surface was thicker than the skin on the medial surface on the limbs. HSP27, HSP70 and HSP90 showed different expression patterns during the hair cycle using RT-PCR, western blot and immunohistochemistry methods. The expression of HSP27 mRNA and protein in the anagen stage was the highest, followed by the catagen stage, and the expression in the telogen stage was the lowest. The expression of HSP70 mRNA and protein in the telogen stage was the highest, followed by the anagen stage, and the expression in the catagen stage was the lowest. The expression of HSP90 mRNA and protein in the anagen stage was the highest, followed by the telogen stage, and the expression in the catagen stage was the lowest. HSPs were mainly expressed in the outer root sheath of hair follicle during the hair cycle, also expressed in epidermis, sebaceous gland and sweat gland in the skin of Yak. The expression of CGI-58 mRNA in the anagen stage was the highest, followed by the catagen stage, and the expression in the telogen stage was the lowest. The expression of KDF1 mRNA in the telogen stage was the highest, followed by the catagen stage, and the expression in the anagen stage was the lowest. Meaning In this study, we examined and fully described the histology of normal skin in Yak and measured the skin thickness of different ages and different regions in Yak. These data may be useful to better understand and appreciate the adaptability features of yak skin. Our investigation reports the expression patterns of HSPs in yak skin for the first time. The different expression pattern of HSPs during the hair cycle suggests they may play different roles in yak hair follicle biology. PMID:28463974

The histological characteristics, age-related thickness change of skin, and expression of the HSPs in the skin during hair cycle in yak (Bos grunniens).

PubMed

Yang, Xue; Cui, Yan; Yue, Jing; He, Honghong; Yu, Chuan; Liu, Penggang; Liu, Jun; Ren, Xiandong; Meng, Yun

2017-01-01

This experiment was conducted to study the histological characteristics, age-related thickness changes, and expression of HSPs in the skin of yak. A total of 20 yaks (10 males and 10 females) were used. Different regions of the normal skin of three different ages (newborn, half-year-old and adult) of yaks were harvested for histological study and thickness measurement. Biopsy samples were taken from the scapula regions of the skin from the same five approximately 1-year-old yaks during the hair cycle (telogen, anagen and catagen). RT-PCR, western blot and immunohistochemistry methods using the mRNA and protein levels were used to detect the expression of HSP27, HSP70 and HSP90. RT-PCR method was used to detect the mRNA expression of CGI-58 and KDF1. The IPP6.0 software was used to analyze the immunohistochemistry and measure the thickness of the skin. The general histological structure of hairy yak skin was similar to other domestic mammals. The unique features included prominent cutaneous vascular plexuses, underdeveloped sweat glands, a large number of nasolabial glands in the nasolabial plate, and hair follicle groups composed of one primary follicle and several secondary follicles. The skin, epidermis and dermis thickness did vary significantly between different body regions and different ages. The thickness of the skin, epidermis and dermis increased from newborn to adult in yaks. Yak skin thickness decreased from dorsally to ventrally on the trunk. The skin on the lateral surface was thicker than the skin on the medial surface on the limbs. HSP27, HSP70 and HSP90 showed different expression patterns during the hair cycle using RT-PCR, western blot and immunohistochemistry methods. The expression of HSP27 mRNA and protein in the anagen stage was the highest, followed by the catagen stage, and the expression in the telogen stage was the lowest. The expression of HSP70 mRNA and protein in the telogen stage was the highest, followed by the anagen stage, and the expression in the catagen stage was the lowest. The expression of HSP90 mRNA and protein in the anagen stage was the highest, followed by the telogen stage, and the expression in the catagen stage was the lowest. HSPs were mainly expressed in the outer root sheath of hair follicle during the hair cycle, also expressed in epidermis, sebaceous gland and sweat gland in the skin of Yak. The expression of CGI-58 mRNA in the anagen stage was the highest, followed by the catagen stage, and the expression in the telogen stage was the lowest. The expression of KDF1 mRNA in the telogen stage was the highest, followed by the catagen stage, and the expression in the anagen stage was the lowest. In this study, we examined and fully described the histology of normal skin in Yak and measured the skin thickness of different ages and different regions in Yak. These data may be useful to better understand and appreciate the adaptability features of yak skin. Our investigation reports the expression patterns of HSPs in yak skin for the first time. The different expression pattern of HSPs during the hair cycle suggests they may play different roles in yak hair follicle biology.

Decay pattern of the Pygmy Dipole Resonance in 130Te

NASA Astrophysics Data System (ADS)

Isaak, J.; Beller, J.; Fiori, E.; Krtička, M.; Löher, B.; Pietralla, N.; Romig, C.; Rusev, G.; Savran, D.; Scheck, M.; Silva, J.; Sonnabend, K.; Tonchev, A.; Tornow, W.; Weller, H.; Zweidinger, M.

2014-03-01

The electric dipole strength distribution in 130Te has been investigated using the method of Nuclear Resonance Fluorescence. The experiments were performed at the Darmstadt High Intensity Photon Setup using bremsstrahlung as photon source and at the High Intensity overrightarrow γ -Ray Source, where quasi-monochromatic and polarized photon beams are provided. Average decay properties of 130Te below the neutron separation energy are determined. Comparing the experimental data to the predictions of the statistical model indicate, that nuclear structure effects play an important role even at sufficiently high excitation energies. Preliminary results will be presented.

Foxi2 Is an Animally Localized Maternal mRNA in Xenopus, and an Activator of the Zygotic Ectoderm Activator Foxi1e

PubMed Central

Cha, Sang-Wook; McAdams, Meredith; Kormish, Jay; Wylie, Christopher; Kofron, Matthew

2012-01-01

Foxi1e is a zygotic transcription factor that is essential for the expression of early ectodermal genes. It is expressed in a highly specific pattern, only in the deep cell layers of the animal hemisphere, and in a mosaic pattern in which expressing cells are interspersed with non-expressing cells. Previous work has shown that several signals in the blastula control this expression pattern, including nodals, the TGFβ family member Vg1, and Notch. However, these are all inhibitory, which raises the question of what activates Foxi1e. In this work, we show that a related Forkhead family protein, Foxi2, is a maternal activator of Foxi1e. Foxi2 mRNA is maternally encoded, and highly enriched in animal hemisphere cells of the blastula. ChIP assays show that it acts directly on upstream regulatory elements of Foxi1e. Its effect is specific, since animal cells depleted of Foxi2 are able to respond normally to mesoderm inducing signals from vegetal cells. Foxi2 thus acts as a link between the oocyte and the early pathway to ectoderm, in a similar fashion to the vegetally localized VegT acts to initiate endoderm and mesoderm formation. PMID:22848601

Influence of Genetic Variations in Selenoprotein Genes on the Pattern of Gene Expression after Supplementation with Brazil Nuts

PubMed Central

Rogero, Marcelo M.; Hesketh, John

2017-01-01

Selenium (Se) is an essential micronutrient for human health. Its beneficial effects are exerted by selenoproteins, which can be quantified in blood and used as molecular biomarkers of Se status. We hypothesize that the presence of genetic polymorphisms in selenoprotein genes may: (1) influence the gene expression of specific selenoproteins and (2) influence the pattern of global gene expression after Brazil nut supplementation. The study was conducted with 130 healthy volunteers in Sao Paulo, Brazil, who consumed one Brazil nut (300 μg/Se) a day for eight weeks. Gene expression of GPX1 and SELENOP and genotyping were measured by real-time PCR using TaqMan Assays. Global gene expression was assessed by microarray using Illumina HumanHT-12 v4 BeadChips. Brazil nut supplementation significantly increased GPX1 mRNA expression only in subjects with CC genotype at rs1050450 (p < 0.05). SELENOP mRNA expression was significantly higher in A-carriers at rs7579 either before or after supplementation (p < 0.05). Genotype for rs713041 in GPX4 affected the pattern of blood cell global gene expression. Genetic variations in selenoprotein genes modulated both GPX1 and SELENOP selenoprotein gene expression and global gene expression in response to Brazil nut supplementation. PMID:28696394

Post-transcriptional trafficking and regulation of neuronal gene expression.

PubMed

Goldie, Belinda J; Cairns, Murray J

2012-02-01

Intracellular messenger RNA (mRNA) traffic and translation must be highly regulated, both temporally and spatially, within eukaryotic cells to support the complex functional partitioning. This capacity is essential in neurons because it provides a mechanism for rapid input-restricted activity-dependent protein synthesis in individual dendritic spines. While this feature is thought to be important for synaptic plasticity, the structures and mechanisms that support this capability are largely unknown. Certainly specialized RNA binding proteins and binding elements in the 3' untranslated region (UTR) of translationally regulated mRNA are important, but the subtlety and complexity of this system suggests that an intermediate "specificity" component is also involved. Small non-coding microRNA (miRNA) are essential for CNS development and may fulfill this role by acting as the guide strand for mediating complex patterns of post-transcriptional regulation. In this review we examine post-synaptic gene regulation, mRNA trafficking and the emerging role of post-transcriptional gene silencing in synaptic plasticity.

Proteogenomic characterization of human colon and rectal cancer

PubMed Central

Zhang, Bing; Wang, Jing; Wang, Xiaojing; Zhu, Jing; Liu, Qi; Shi, Zhiao; Chambers, Matthew C.; Zimmerman, Lisa J.; Shaddox, Kent F.; Kim, Sangtae; Davies, Sherri R.; Wang, Sean; Wang, Pei; Kinsinger, Christopher R.; Rivers, Robert C.; Rodriguez, Henry; Townsend, R. Reid; Ellis, Matthew J.C.; Carr, Steven A.; Tabb, David L.; Coffey, Robert J.; Slebos, Robbert J.C.; Liebler, Daniel C.

2014-01-01

Summary We analyzed proteomes of colon and rectal tumors previously characterized by the Cancer Genome Atlas (TCGA) and performed integrated proteogenomic analyses. Somatic variants displayed reduced protein abundance compared to germline variants. mRNA transcript abundance did not reliably predict protein abundance differences between tumors. Proteomics identified five proteomic subtypes in the TCGA cohort, two of which overlapped with the TCGA “MSI/CIMP” transcriptomic subtype, but had distinct mutation, methylation, and protein expression patterns associated with different clinical outcomes. Although copy number alterations showed strong cis- and trans-effects on mRNA abundance, relatively few of these extend to the protein level. Thus, proteomics data enabled prioritization of candidate driver genes. The chromosome 20q amplicon was associated with the largest global changes at both mRNA and protein levels; proteomics data highlighted potential 20q candidates including HNF4A, TOMM34 and SRC. Integrated proteogenomic analysis provides functional context to interpret genomic abnormalities and affords a new paradigm for understanding cancer biology. PMID:25043054

Persisting PET-CT lesion activity and M. tuberculosis mRNA after pulmonary tuberculosis cure

PubMed Central

Malherbe, Stephanus T.; Shenai, Shubhada; Ronacher, Katharina; Loxton, Andre G.; Dolganov, Gregory; Kriel, Magdalena; Van, Tran; Chen, Ray Y.; Warwick, James; Via, Laura E.; Song, Taeksun; Lee, Myungsun; Schoolnik, Gary; Tromp, Gerard; Alland, David; Barry, Clifton E.; Winter, Jill; Walzl, Gerhard

2016-01-01

The absence of a gold standard to determine when antibiotics have induced sterilizing cure confounds the development of new approaches to treat pulmonary tuberculosis (PTB). We detected PET-CT imaging response patterns consistent with active disease along with the presence of Mycobacterium tuberculosis mRNA in sputum and bronchoalveolar lavage samples in a substantial proportion of adult, HIV-negative PTB patients after standard 6-month treatment plus one year follow-up, including patients with a durable cure and others who later developed recurrent disease. The presence of MTB mRNA in the context of non-resolving and intensifying lesions on PET-CT might indicate ongoing transcription, suggesting that even apparently curative PTB treatment may not eradicate all organisms in most patients. This suggests an important complementary role for the immune response in maintaining a disease-free state. Sterilizing drugs or host-directed therapies and better treatment response markers are likely needed for the successful development of improved and shortened PTB treatment strategies. PMID:27595324

The Crow Creek Site (39BF11) Massacre: A Preliminary Report,

DTIC Science & Technology

1981-02-01

components are typical of the Plains Village 13 pattern. This adaptation Is presumed to be a hybrid resulting from the diffusion of the Northwestern...Plains hunting tribes’ adaptation with that of the Eastern Woodland people (Lehmer 1971:65). The sub- sistence pattern is based on hunting and horticulture...sample, apparently most of the sample is partially decayed organic matter, very little or no charcoal present, few gastropod shells present in sample