Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

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Ahmed Kamel El-Sayed

2014-11-01

Full Text Available Reprogramming of somatic cells has great potential to provide therapeutic treatments for a number of diseases as well as provide insight into mechanisms underlying early embryonic development. Improvement of induced Pluripotent Stem Cells (iPSCs generation through mRNA-based methods is currently an area of intense research. This approach provides a number of advantages over previously used methods such as DNA integration and insertional mutagenesis. Using transfection of specifically synthesized mRNAs of various pluripotency factors, we generated iPSCs from mouse embryonic fibroblast (MEF cells. The genetic, epigenetic and functional properties of the iPSCs were evaluated at different times during the reprogramming process. We successfully introduced synthesized mRNAs, which localized correctly inside the cells and exhibited efficient and stable translation into proteins. Our work demonstrated a robust up-regulation and a gradual promoter de-methylation of the pluripotency markers, including non-transfected factors such as Nanog, SSEA-1 (stage-specific embryonic antigen 1 and Rex-1 (ZFP-42, zinc finger protein 42. Using embryonic stem cells (ESCs conditions to culture the iPS cells resulted in formation of ES-like colonies after approximately 12 days with only five daily repeated transfections. The colonies were positive for alkaline phosphatase and pluripotency-specific markers associated with ESCs. This study revealed the ability of pluripotency induction and generation of mouse mRNA induced pluripotent stem cells (mRNA iPSCs using transfection of specifically synthesized mRNAs of various pluripotency factors into mouse embryonic fibroblast (MEF cells. These generated iPSCs exhibited molecular and functional properties similar to ESCs, which indicate that this method is an efficient and viable alternative to ESCs and can be used for further biological, developmental and therapeutic investigations.

Translational coregulation of 5′TOP mRNAs by TIA-1 and TIAR

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Damgaard, Christian Kroun; Lykke-Andersen, Jens

2011-01-01

The response of cells to changes in their environment often requires coregulation of gene networks, but little is known about how this can occur at the post-transcriptional level. An important example of post-transcriptional coregulation is the selective translational regulation in response to growth conditions of mammalian mRNAs that encode protein biosynthesis factors and contain hallmark 5′-terminal oligopyrimidine tracts (5′TOP). However, the responsible trans-factors and the mechanism by which they coregulate 5′TOP mRNAs have remained elusive. Here we identify stress granule-associated TIA-1 and TIAR proteins as key factors in human 5′TOP mRNA regulation, which upon amino acid starvation assemble onto the 5′ end of 5′TOP mRNAs and arrest translation at the initiation step, as evidenced by TIA-1/TIAR-dependent 5′TOP mRNA translation repression, polysome release, and accumulation in stress granules. This requires starvation-mediated activation of the GCN2 (general control nonderepressible 2) kinase and inactivation of the mTOR (mammalian target of rapamycin) signaling pathway. Our findings provide a mechanistic explanation to the long-standing question of how the network of 5′TOP mRNAs are coregulated according to amino acid availability, thereby allowing redirection of limited resources to mount a nutrient deprivation response. This presents a fundamental example of how a group of mRNAs can be translationally coregulated in response to changes in the cellular environment. PMID:21979918

Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons.

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Pfeiffer-Guglielmi, Brigitte; Dombert, Benjamin; Jablonka, Sibylle; Hausherr, Vanessa; van Thriel, Christoph; Schöbel, Nicole; Jansen, Ralf-Peter

2014-06-04

Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission.

The RNA binding protein HuR differentially regulates unique subsets of mRNAs in estrogen receptor negative and estrogen receptor positive breast cancer

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Chen Jing

2010-04-01

Full Text Available Abstract Background The discordance between steady-state levels of mRNAs and protein has been attributed to posttranscriptional control mechanisms affecting mRNA stability and translation. Traditional methods of genome wide microarray analysis, profiling steady-state levels of mRNA, may miss important mRNA targets owing to significant posttranscriptional gene regulation by RNA binding proteins (RBPs. Methods The ribonomic approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip, provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich elements (ARE of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR has been described to control genes in several of the acquired capabilities of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established. Results We used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+ and MDA-MB-231 (estrogen receptor negative, ER- breast cancer cell lines. We identified unique subsets of HuR-associated mRNAs found individually or in both cell types. Two novel HuR targets, CD9 and CALM2 mRNAs, were identified and validated by quantitative RT-PCR and biotin pull-down analysis. Conclusion This is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer. We found distinct, differentially expressed subsets of cancer related genes in ER+ and ER- breast cancer cell lines, and noted that the differential regulation of two cancer-related genes by HuR was contingent upon the cellular

Sequestration of highly expressed mRNAs in cytoplasmic granules, P-bodies, and stress granules enhances cell viability.

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Anna Lavut

Full Text Available Transcriptome analyses indicate that a core 10%-15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and null mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical

Microarray-based identification and RT-PCR test screening for epithelial-specific mRNAs in peripheral blood of patients with colon cancer

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Coppola Domenico

2006-10-01

Full Text Available Abstract Background The efficacy of screening for colorectal cancer using a simple blood-based assay for the detection of tumor cells disseminated in the circulation at an early stage of the disease is gaining positive feedback from several lines of research. This method seems able to reduce colorectal cancer mortality and may replace colonoscopy as the most effective means of detecting colonic lesions. Methods In this work, we present a new microarray-based high-throughput screening method to identifying candidate marker mRNAs for the early detection of epithelial cells diluted in peripheral blood cells. This method includes 1. direct comparison of different samples of colonic mucosa and of blood cells to identify consistent epithelial-specific mRNAs from among 20,000 cDNA assayed by microarray slides; 2. identification of candidate marker mRNAs by data analysis, which allowed selection of only 10 putative differentially expressed genes; 3. Selection of some of the most suitable mRNAs (TMEM69, RANBP3 and PRSS22 that were assayed in blood samples from normal subjects and patients with colon cancer as possible markers for the presence of epithelial cells in the blood, using reverse transcription – polymerase chain reaction (RT-PCR. Results Our present results seem to provide an indication, for the first time obtained by genome-scale screening, that a suitable and consistent colon epithelium mRNA marker may be difficult to identify. Conclusion The design of new approaches to identify such markers is warranted.

The STAR protein QKI-7 recruits PAPD4 to regulate post-transcriptional polyadenylation of target mRNAs.

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Yamagishi, Ryota; Tsusaka, Takeshi; Mitsunaga, Hiroko; Maehata, Takaharu; Hoshino, Shin-ichi

2016-04-07

Emerging evidence has demonstrated that regulating the length of the poly(A) tail on an mRNA is an efficient means of controlling gene expression at the post-transcriptional level. In early development, transcription is silenced and gene expression is primarily regulated by cytoplasmic polyadenylation. In somatic cells, considerable progress has been made toward understanding the mechanisms of negative regulation by deadenylation. However, positive regulation through elongation of the poly(A) tail has not been widely studied due to the difficulty in distinguishing whether any observed increase in length is due to the synthesis of new mRNA, reduced deadenylation or cytoplasmic polyadenylation. Here, we overcame this barrier by developing a method for transcriptional pulse-chase analysis under conditions where deadenylases are suppressed. This strategy was used to show that a member of the Star family of RNA binding proteins, QKI, promotes polyadenylation when tethered to a reporter mRNA. Although multiple RNA binding proteins have been implicated in cytoplasmic polyadenylation during early development, previously only CPEB was known to function in this capacity in somatic cells. Importantly, we show that only the cytoplasmic isoform QKI-7 promotes poly(A) tail extension, and that it does so by recruiting the non-canonical poly(A) polymerase PAPD4 through its unique carboxyl-terminal region. We further show that QKI-7 specifically promotes polyadenylation and translation of three natural target mRNAs (hnRNPA1, p27(kip1)and β-catenin) in a manner that is dependent on the QKI response element. An anti-mitogenic signal that induces cell cycle arrest at G1 phase elicits polyadenylation and translation of p27(kip1)mRNA via QKI and PAPD4. Taken together, our findings provide significant new insight into a general mechanism for positive regulation of gene expression by post-transcriptional polyadenylation in somatic cells. © The Author(s) 2016. Published by Oxford

Regulation of mRNA translation during mitosis.

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Tanenbaum, Marvin E; Stern-Ginossar, Noam; Weissman, Jonathan S; Vale, Ronald D

2015-08-25

Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ~200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.

Nuclear-Encoded Mitochondrial mRNAs: A Powerful Force in Axonal Growth and Development.

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Gale, Jenna R; Aschrafi, Armaz; Gioio, Anthony E; Kaplan, Barry B

2018-04-01

Axons, their growth cones, and synaptic nerve terminals are neuronal subcompartments that have high energetic needs. As such, they are enriched in mitochondria, which supply the ATP necessary to meet these demands. To date, a heterogeneous population of nuclear-encoded mitochondrial mRNAs has been identified in distal axons and growth cones. Accumulating evidence suggests that the local translation of these mRNAs is required for mitochondrial maintenance and axonal viability. Here, we review evidence that suggests a critical role for axonal translation of nuclear-encoded mitochondrial mRNAs in axonal growth and development. Additionally, we explore the role that site-specific translation at the mitochondria itself may play in this process. Finally, we briefly review the clinical implications of dysregulation of local translation of mitochondrial-related mRNAs in neurodevelopmental disorders.

Stage-related expression of mRNAs during pollen development in lily and tobacco.

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Schrauwen, J A; de Groot, P F; van Herpen, M M; van der Lee, T; Reynen, W H; Weterings, K A; Wullems, G J

1990-09-01

Homogeneous populations of developing microspores and pollen from anthers of lily (Lilium longiflorum Thumb.) and tobacco (Nicotiana tabacum L.) show a continuous production of biomass, reaching a maximum in young pollen. The rate of RNA synthesis was 460 fg · h(-1) in young binucleate cells, 138 fg · h(-1) in late binucleate cells and 56 fg · h(-1) in microspores. The mRNA population in developing pollen can be separated into three groups. In the first group, certain types of mRNAs are present at a constant level during all stages of development. A second group is characteristic of young pollen and increases quantitatively until anthesis. A third group is seen transiently; to this belong mRNAs present only before mitosis or at a distinct cell stage after mitosis. Some of the translation products of this latter group of mRNAs showed similarities between lily and tobacco on two-dimensional gels in respect of molecular weight and isolectric point, indicating that those mRNAs and proteins play a role in the regulation of pollen development.

Identification of a chemical inhibitor for nuclear speckle formation: Implications for the function of nuclear speckles in regulation of alternative pre-mRNA splicing

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Kurogi, Yutaro; Matsuo, Yota; Mihara, Yuki; Yagi, Hiroaki; Shigaki-Miyamoto, Kaya; Toyota, Syukichi; Azuma, Yuko [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Chuo-ku, Kumamoto 860-8555 (Japan); Igarashi, Masayuki [Laboratory of Disease Biology, Institute of Microbial Chemistry, Shinagawa-ku, Tokyo 141-0021 (Japan); Tani, Tokio, E-mail: ttani@sci.kumamoto-u.ac.jp [Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University, Chuo-ku, Kumamoto 860-8555 (Japan)

2014-03-28

Highlights: • We identified tubercidin as a compound inducing aberrant formation of the speckles. • Tubercidin causes delocalization of poly (A){sup +}RNAs from nuclear speckles. • Tubercidin induces dispersion of splicing factors from nuclear speckles. • Tubercidin affects alternative pre-mRNA splicing. • Nuclear speckles play a role in regulation of alternative pre-mRNA splicing. - Abstract: Nuclear speckles are subnuclear structures enriched with RNA processing factors and poly (A){sup +} RNAs comprising mRNAs and poly (A){sup +} non-coding RNAs (ncRNAs). Nuclear speckles are thought to be involved in post-transcriptional regulation of gene expression, such as pre-mRNA splicing. By screening 3585 culture extracts of actinomycetes with in situ hybridization using an oligo dT probe, we identified tubercidin, an analogue of adenosine, as an inhibitor of speckle formation, which induces the delocalization of poly (A){sup +} RNA and dispersion of splicing factor SRSF1/SF2 from nuclear speckles in HeLa cells. Treatment with tubercidin also decreased steady-state MALAT1 long ncRNA, thought to be involved in the retention of SRSF1/SF2 in nuclear speckles. In addition, we found that tubercidin treatment promoted exon skipping in the alternative splicing of Clk1 pre-mRNA. These results suggest that nuclear speckles play a role in modulating the concentration of splicing factors in the nucleoplasm to regulate alternative pre-mRNA splicing.

Identification and analysis of pig chimeric mRNAs using RNA sequencing data

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2012-01-01

Background Gene fusion is ubiquitous over the course of evolution. It is expected to increase the diversity and complexity of transcriptomes and proteomes through chimeric sequence segments or altered regulation. However, chimeric mRNAs in pigs remain unclear. Here we identified some chimeric mRNAs in pigs and analyzed the expression of them across individuals and breeds using RNA-sequencing data. Results The present study identified 669 putative chimeric mRNAs in pigs, of which 251 chimeric candidates were detected in a set of RNA-sequencing data. The 618 candidates had clear trans-splicing sites, 537 of which obeyed the canonical GU-AG splice rule. Only two putative pig chimera variants whose fusion junction was overlapped with that of a known human chimeric mRNA were found. A set of unique chimeric events were considered middle variances in the expression across individuals and breeds, and revealed non-significant variance between sexes. Furthermore, the genomic region of the 5′ partner gene shares a similar DNA sequence with that of the 3′ partner gene for 458 putative chimeric mRNAs. The 81 of those shared DNA sequences significantly matched the known DNA-binding motifs in the JASPAR CORE database. Four DNA motifs shared in parental genomic regions had significant similarity with known human CTCF binding sites. Conclusions The present study provided detailed information on some pig chimeric mRNAs. We proposed a model that trans-acting factors, such as CTCF, induced the spatial organisation of parental genes to the same transcriptional factory so that parental genes were coordinatively transcribed to give birth to chimeric mRNAs. PMID:22925561

Identification and analysis of pig chimeric mRNAs using RNA sequencing data

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Ma Lei

2012-08-01

Full Text Available Abstract Background Gene fusion is ubiquitous over the course of evolution. It is expected to increase the diversity and complexity of transcriptomes and proteomes through chimeric sequence segments or altered regulation. However, chimeric mRNAs in pigs remain unclear. Here we identified some chimeric mRNAs in pigs and analyzed the expression of them across individuals and breeds using RNA-sequencing data. Results The present study identified 669 putative chimeric mRNAs in pigs, of which 251 chimeric candidates were detected in a set of RNA-sequencing data. The 618 candidates had clear trans-splicing sites, 537 of which obeyed the canonical GU-AG splice rule. Only two putative pig chimera variants whose fusion junction was overlapped with that of a known human chimeric mRNA were found. A set of unique chimeric events were considered middle variances in the expression across individuals and breeds, and revealed non-significant variance between sexes. Furthermore, the genomic region of the 5′ partner gene shares a similar DNA sequence with that of the 3′ partner gene for 458 putative chimeric mRNAs. The 81 of those shared DNA sequences significantly matched the known DNA-binding motifs in the JASPAR CORE database. Four DNA motifs shared in parental genomic regions had significant similarity with known human CTCF binding sites. Conclusions The present study provided detailed information on some pig chimeric mRNAs. We proposed a model that trans-acting factors, such as CTCF, induced the spatial organisation of parental genes to the same transcriptional factory so that parental genes were coordinatively transcribed to give birth to chimeric mRNAs.

Exposure to Pre- and Perinatal Risk Factors Partially Explains Mean Differences in Self-Regulation between Races.

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Barnes, J C; Boutwell, Brian B; Miller, J Mitchell; DeShay, Rashaan A; Beaver, Kevin M; White, Norman

2016-01-01

To examine whether differential exposure to pre- and perinatal risk factors explained differences in levels of self-regulation between children of different races (White, Black, Hispanic, Asian, and Other). Multiple regression models based on data from the Early Childhood Longitudinal Study, Birth Cohort (n ≈ 9,850) were used to analyze the impact of pre- and perinatal risk factors on the development of self-regulation at age 2 years. Racial differences in levels of self-regulation were observed. Racial differences were also observed for 9 of the 12 pre-/perinatal risk factors. Multiple regression analyses revealed that a portion of the racial differences in self-regulation was explained by differential exposure to several of the pre-/perinatal risk factors. Specifically, maternal age at childbirth, gestational timing, and the family's socioeconomic status were significantly related to the child's level of self-regulation. These factors accounted for a statistically significant portion of the racial differences observed in self-regulation. The findings indicate racial differences in self-regulation may be, at least partially, explained by racial differences in exposure to pre- and perinatal risk factors.

Aberrant Expression of miRNA and mRNAs in Lesioned Tissues of Graves' Disease

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Qiu Qin

2015-03-01

Full Text Available Background and Aims: Abnormal microRNA (miRNA expression is found in many diseases including autoimmune diseases. However, little is known about the role of miRNA regulation in Graves' disease (GD. Here, we simultaneously detected different expressions of miRNA and mRNAs in thyroid tissues via a high-throughput transcriptomics approach, known as microarray, in order to reveal the relationship between aberrant expression of miRNAs and mRNAs spectrum and GD. Methods: Totally 7 specimens of thyroid tissue from 4 GD patients and 3 controls were obtained by surgery for microarray analysis. Then, 30 thyroid specimens (18 GD and 12 controls were also collected for further validation by quantitative real-time PCR ( qRT-PCR . Results: Statistical analysis showed that the expressions of 5 specific miRNA were increased significantly while those of other 18 miRNA were decreased in thyroid tissue of GD patients (FC≥1.3 or≤0.77 and pConclusion: Our study highlights the possibility that miRNA-target gene network may be involved in the pathogenesis of GD and could provide new insights into understanding the pathophysiological mechanisms of GD.

Specific regulation of point-mutated K-ras-immortalized cell proliferation by a photodynamic antisense strategy.

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Higuchi, Maiko; Yamayoshi, Asako; Kato, Kiyoko; Kobori, Akio; Wake, Norio; Murakami, Akira

2010-02-01

It has been reported that point mutations in genes are responsible for various cancers, and the selective regulation of gene expression is an important factor in developing new types of anticancer drugs. To develop effective drugs for the regulation of point-mutated genes, we focused on photoreactive antisense oligonucleotides. Previously, we reported that photoreactive oligonucleotides containing 2'-O-psoralenylmethoxyethyl adenosine (2'-Ps-eom) showed drastic photoreactivity in a strictly sequence-specific manner. Here, we demonstrated the specific gene regulatory effects of 2'-Ps-eom on [(12)Val]K-ras mutant (GGT --> GTT). Photo-cross-linking between target mRNAs and 2'-Ps-eom was sequence-specific, and the effect was UVA irradiation-dependent. Furthermore, 2'-Ps-eom was able to inhibit K-ras-immortalized cell proliferation (K12V) but not Vco cells that have the wild-type K-ras gene. These results suggest that the 2'-Ps-eom will be a powerful nucleic acid drug to inhibit the expression of disease-causing point mutation genes, and has great therapeutic potential in the treatment of cancer.

Multiple Export Mechanisms for mRNAs

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Delaleau, Mildred; Borden, Katherine L. B.

2015-01-01

Nuclear mRNA export plays an important role in gene expression. We describe the mechanisms of mRNA export including the importance of mRNP assembly, docking with the nuclear basket of the nuclear pore complex (NPC), transit through the central channel of the NPC and cytoplasmic release. We describe multiple mechanisms of mRNA export including NXF1 and CRM1 mediated pathways. Selective groups of mRNAs can be preferentially transported in order to respond to cellular stimuli. RNAs can be selected based on the presence of specific cis-acting RNA elements and binding of specific adaptor proteins. The role that dysregulation of this process plays in human disease is also discussed. PMID:26343730

Exposure to Pre- and Perinatal Risk Factors Partially Explains Mean Differences in Self-Regulation between Races.

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J C Barnes

Full Text Available To examine whether differential exposure to pre- and perinatal risk factors explained differences in levels of self-regulation between children of different races (White, Black, Hispanic, Asian, and Other.Multiple regression models based on data from the Early Childhood Longitudinal Study, Birth Cohort (n ≈ 9,850 were used to analyze the impact of pre- and perinatal risk factors on the development of self-regulation at age 2 years.Racial differences in levels of self-regulation were observed. Racial differences were also observed for 9 of the 12 pre-/perinatal risk factors. Multiple regression analyses revealed that a portion of the racial differences in self-regulation was explained by differential exposure to several of the pre-/perinatal risk factors. Specifically, maternal age at childbirth, gestational timing, and the family's socioeconomic status were significantly related to the child's level of self-regulation. These factors accounted for a statistically significant portion of the racial differences observed in self-regulation.The findings indicate racial differences in self-regulation may be, at least partially, explained by racial differences in exposure to pre- and perinatal risk factors.

Accumulation of long-lived mRNAs associated with germination in embryos during seed development of rice

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Sano, Naoto; Ono, Hanako; Murata, Kazumasa; Yamada, Tetsuya; Hirasawa, Tadashi; Kanekatsu, Motoki

2015-01-01

Mature dry seeds contain translatable mRNAs called long-lived mRNAs. Early studies have shown that protein synthesis during the initial phase of seed germination occurs from long-lived mRNAs, without de novo transcription. However, the gene expression systems that generate long-lived mRNAs in seeds are not well understood. To examine the accumulation of long-lived mRNAs in developing rice embryos, germination tests using the transcriptional inhibitor actinomycin D (Act D) were performed with the Japonica rice cultivar Nipponbare. Although over 70% of embryos at 10 days after flowering (DAF) germinated in the absence of the inhibitor, germination was remarkably impaired in embryos treated with Act D. In contrast, more than 70% of embryos at 20, 25, 30 and 40 DAF germinated in the presence of Act D. The same results were obtained when another cultivar, Koshihikari, was used, indicating that the long-lived mRNAs required for germination predominantly accumulate in embryos between 10 and 20 DAF during seed development. RNA-Seq identified 529 long-lived mRNA candidates, encoding proteins such as ABA, calcium ion and phospholipid signalling-related proteins, and HSP DNA J, increased from 10 to 20 DAF and were highly abundant in 40 DAF embryos of Nipponbare and Koshihikari. We also revealed that these long-lived mRNA candidates are clearly up-regulated in 10 DAF germinating embryos after imbibition, suggesting that the accumulation of these mRNAs in embryos is indispensable for the induction of germination. The findings presented here may facilitate in overcoming irregular seed germination or producing more vigorous seedlings. PMID:25941326

Identification of an elaborate NK-specific system regulating HLA-C expression.

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Hongchuan Li

2018-01-01

Full Text Available The HLA-C gene appears to have evolved in higher primates to serve as a dominant source of ligands for the KIR2D family of inhibitory MHC class I receptors. The expression of NK cell-intrinsic MHC class I has been shown to regulate the murine Ly49 family of MHC class I receptors due to the interaction of these receptors with NK cell MHC in cis. However, cis interactions have not been demonstrated for the human KIR and HLA proteins. We report the discovery of an elaborate NK cell-specific system regulating HLA-C expression, indicating an important role for HLA-C in the development and function of NK cells. A large array of alternative transcripts with differences in intron/exon content are generated from an upstream NK-specific HLA-C promoter, and exon content varies between HLA-C alleles due to SNPs in splice donor/acceptor sites. Skipping of the first coding exon of HLA-C generates a subset of untranslatable mRNAs, and the proportion of untranslatable HLA-C mRNA decreases as NK cells mature, correlating with increased protein expression by mature NK cells. Polymorphism in a key Ets-binding site of the NK promoter has generated HLA-C alleles that lack significant promoter activity, resulting in reduced HLA-C expression and increased functional activity. The NK-intrinsic regulation of HLA-C thus represents a novel mechanism controlling the lytic activity of NK cells during development.

Granules Harboring Translationally Active mRNAs Provide a Platform for P-Body Formation following Stress

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Jennifer Lui

2014-11-01

Full Text Available 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.

Regional localization of suspensor mRNAs during early embryo development.

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Weterings, K; Apuya, N R; Bi, Y; Fischer, R L; Harada, J J; Goldberg, R B

2001-11-01

We investigated gene activity within the giant embryos of the scarlet runner bean (Phaseolus coccineus) to gain understanding of the processes by which the apical and basal cells become specified to follow different developmental pathways after division of the zygote. We identified two mRNAs, designated G564 and C541, that accumulate specifically within the suspensor of globular-stage embryos. G564 mRNA accumulates uniformly throughout the suspensor, whereas C541 mRNA accumulates to a higher level within the large basal cells of the suspensor that anchor the embryo to the surrounding seed tissue. Both G564 and C541 mRNAs begin to accumulate shortly after fertilization and are present within the two basal cells of embryos at the four-cell stage. In contrast, at the same stage, these mRNAs are not detectable within the two descendants of the apical cell. Nor are they detectable within cells of the embryo sac before fertilization, including the egg cell. We used a G564/beta-glucuronidase reporter gene to show that the G564 promoter is activated specifically within the basal region and suspensor of preglobular tobacco embryos. Analysis of the G564 promoter identified a sequence domain required for transcription within the suspensor that contains several copies of a conserved motif. These results show that derivatives of the apical and basal cells transcribe different genes as early as the four-cell stage of embryo development and suggest that the apical and basal cells are specified at the molecular level after division of the zygote.

Regional Localization of Suspensor mRNAs during Early Embryo Development

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Weterings, Koen; Apuya, Nestor R.; Bi, Yuping; Fischer, Robert L.; Harada, John J.; Goldberg, Robert B.

2001-01-01

We investigated gene activity within the giant embryos of the scarlet runner bean (Phaseolus coccineus) to gain understanding of the processes by which the apical and basal cells become specified to follow different developmental pathways after division of the zygote. We identified two mRNAs, designated G564 and C541, that accumulate specifically within the suspensor of globular-stage embryos. G564 mRNA accumulates uniformly throughout the suspensor, whereas C541 mRNA accumulates to a higher level within the large basal cells of the suspensor that anchor the embryo to the surrounding seed tissue. Both G564 and C541 mRNAs begin to accumulate shortly after fertilization and are present within the two basal cells of embryos at the four-cell stage. In contrast, at the same stage, these mRNAs are not detectable within the two descendants of the apical cell. Nor are they detectable within cells of the embryo sac before fertilization, including the egg cell. We used a G564/β-glucuronidase reporter gene to show that the G564 promoter is activated specifically within the basal region and suspensor of preglobular tobacco embryos. Analysis of the G564 promoter identified a sequence domain required for transcription within the suspensor that contains several copies of a conserved motif. These results show that derivatives of the apical and basal cells transcribe different genes as early as the four-cell stage of embryo development and suggest that the apical and basal cells are specified at the molecular level after division of the zygote. PMID:11701878

In situ localization of phenylpropanoid biosynthetic mRNAs and proteins in Parsley (Petroselinum crispum)

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Reinold, S.; Hahlbrock, K.

1997-01-01

Using in situ RNA/RNA hybridization, enzyme immunolocalization, and histochemical techniques, several phenylpropanoid biosynthetic activities and products were localized in tissue sections from various aerial parts of parsley (Petroselinum crispum) plants at different developmental stages. The enzymes and corresponding mRNAs analyzed included two representatives of general phenylpropanoid metabolism: phenylalanine ammonia-lyase (PAL) and 4-coumarate: CoA ligase (4CL), and one representative each from two distinct branch pathways: chalcone synthase (CHS; flavonoids) and S-adenosyl-L-methionine: bergaptol O-methyltransferase (BMT; furanocoumarins). In almost all cases, the relative timing of accumulation differed greatly for mRNA and protein and indicated short expression periods and short half-lives for all mRNAs as compared to the proteins. PAL and 4CL occurred almost ubiquitously in cell type-specific patterns, and their mRNAs and proteins were always coordinately expressed, whereas the cell type-specific localization of flavonoid and furanocoumarin biosynthetic activities was to a large extent mutually exclusive. However, the distribution patterns of CHS and BMT, when superimposed, closely matched those of PAL and 4CL in nearly all tissues analysed, suggesting that the flavonoid and furanocoumarin pathways together constituted a large majority of the total phenylpropanoid biosynthetic activity. Differential sites of synthesis and accumulation indicating intercellular translocation were observed both for flavonoids and for furanocoumarins in oil ducts and the surrounding tissue. The widespread occurrence of both classes of compounds, as well as selected, pathway-specific mRNAs and enzymes, in many cell types of all parsley organs including various flower parts suggests additional functions beyond the previously established roles of flavonoids in UV protection and furanocoumarins in pathogen defence. (author)

Expression of Each Cistron in the gal Operon Can Be Regulated by Transcription Termination and Generation of a galK-Specific mRNA, mK2

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Wang, Xun; Ji, Sang Chun; Yun, Sang Hoon; Jeon, Heung Jin; Kim, Si Wouk

2014-01-01

The gal operon of Escherichia coli has 4 cistrons, galE, galT, galK, and galM. In our previous report (H. J. Lee, H. J. Jeon, S. C. Ji, S. H. Yun, H. M. Lim, J. Mol. Biol. 378:318–327, 2008), we identified 6 different mRNA species, mE1, mE2, mT1, mK1, mK2, and mM1, in the gal operon and mapped these mRNAs. The mRNA map suggests a gradient of gene expression known as natural polarity. In this study, we investigated how the mRNAs are generated to understand the cause of natural polarity. Results indicated that mE1, mT1, mK1, and mM1, whose 3′ ends are located at the end of each cistron, are generated by transcription termination. Since each transcription termination is operating with a certain frequency and those 4 mRNAs have 5′ ends at the transcription initiation site(s), these transcription terminations are the basic cause of natural polarity. Transcription terminations at galE-galT and galT-galK junctions, making mE1 and mT1, are Rho dependent. However, the terminations to make mK1 and mM1 are partially Rho dependent. The 5′ ends of mK2 are generated by an endonucleolytic cleavage of a pre-mK2 by RNase P, and the 3′ ends are generated by Rho termination 260 nucleotides before the end of the operon. The 5′ portion of pre-mK2 is likely to become mE2. These results also suggested that galK expression could be regulated through mK2 production independent from natural polarity. PMID:24794565

LARP1 functions as a molecular switch for mTORC1-mediated translation of an essential class of mRNAs.

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Hong, Sungki; Freeberg, Mallory A; Han, Ting; Kamath, Avani; Yao, Yao; Fukuda, Tomoko; Suzuki, Tsukasa; Kim, John K; Inoki, Ken

2017-06-26

The RNA binding protein, LARP1, has been proposed to function downstream of mTORC1 to regulate the translation of 5'TOP mRNAs such as those encoding ribosome proteins (RP). However, the roles of LARP1 in the translation of 5'TOP mRNAs are controversial and its regulatory roles in mTORC1-mediated translation remain unclear. Here we show that LARP1 is a direct substrate of mTORC1 and Akt/S6K1. Deep sequencing of LARP1-bound mRNAs reveal that non-phosphorylated LARP1 interacts with both 5' and 3'UTRs of RP mRNAs and inhibits their translation. Importantly, phosphorylation of LARP1 by mTORC1 and Akt/S6K1 dissociates it from 5'UTRs and relieves its inhibitory activity on RP mRNA translation. Concomitantly, phosphorylated LARP1 scaffolds mTORC1 on the 3'UTRs of translationally-competent RP mRNAs to facilitate mTORC1-dependent induction of translation initiation. Thus, in response to cellular mTOR activity, LARP1 serves as a phosphorylation-sensitive molecular switch for turning off or on RP mRNA translation and subsequent ribosome biogenesis.

Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export.

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Larsen, Sean; Bui, Steven; Perez, Veronica; Mohammad, Adeba; Medina-Ramirez, Hilario; Newcomb, Laura L

2014-08-28

Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by influenza nucleoprotein and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins responsible for RNA synthesis in the nucleus of the host cell. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. Most cellular mRNA nuclear export is Nxf1-mediated, while select mRNAs utilize Crm1. Here we inhibited Nxf1 and Crm1 nuclear export prior to infection with influenza A/Udorn/307/1972(H3N2) virus and analyzed influenza intron-less mRNAs using cellular fractionation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We examined direct interaction between Nxf1 and influenza intron-less mRNAs using immuno purification of Nxf1 and RT-PCR of associated RNA. Inhibition of Nxf1 resulted in less influenza intron-less mRNA export into the cytoplasm for HA and NA influenza mRNAs in both human embryonic kidney cell line (293 T) and human lung adenocarcinoma epithelial cell line (A549). However, in 293 T cells no change was observed for mRNAs encoding the components of the viral ribonucleoproteins; NP, PA, PB1, and PB2, while in A549 cells, only PA, PB1, and PB2 mRNAs, encoding the RdRP, remained unaffected; NP mRNA was reduced in the cytoplasm. In A549 cells NP, NA, HA, mRNAs were found associated with Nxf1 but PA, PB1, and PB2 mRNAs were not. Crm1 inhibition also resulted in no significant difference in PA, PB1, and PB2 mRNA nuclear export. These results further confirm Nxf1-mediated nuclear export is functional during the influenza life cycle and hijacked for select influenza mRNA nuclear export. We reveal a cell type difference for Nxf1-mediated nuclear export of influenza NP mRNA, a reminder that cell type can influence molecular mechanisms. Importantly, we

Regulation of rice root development by a retrotransposon acting as a microRNA sponge.

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Cho, Jungnam; Paszkowski, Jerzy

2017-08-26

It is well documented that transposable elements (TEs) can regulate the expression of neighbouring genes. However, their ability to act in trans and influence ectopic loci has been reported rarely. We searched in rice transcriptomes for tissue-specific expression of TEs and found them to be regulated developmentally. They often shared sequence homology with co-expressed genes and contained potential microRNA-binding sites, which suggested possible contributions to gene regulation. In fact, we have identified a retrotransposon that is highly transcribed in roots and whose spliced transcript constitutes a target mimic for miR171. miR171 destabilizes mRNAs encoding the root-specific family of SCARECROW-Like transcription factors. We demonstrate that retrotransposon-derived transcripts act as decoys for miR171, triggering its degradation and thus results in the root-specific accumulation of SCARECROW-Like mRNAs. Such transposon-mediated post-transcriptional control of miR171 levels is conserved in diverse rice species.

TH and DCX mRNAs in peripheral blood and bone marrow predict outcome in metastatic neuroblastoma patients.

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Yáñez, Yania; Hervás, David; Grau, Elena; Oltra, Silvestre; Pérez, Gema; Palanca, Sarai; Bermúdez, Mar; Márquez, Catalina; Cañete, Adela; Castel, Victoria

2016-03-01

In metastatic neuroblastoma (NB) patients, accurate risk stratification and disease monitoring would reduce relapse probabilities. This study aims to evaluate the independent prognostic significance of detecting tyrosine hydroxylase (TH) and doublecortin (DCX) mRNAs by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in peripheral blood (PB) and bone marrow (BM) samples from metastatic NB patients. RT-qPCR was performed on PB and BM samples from metastatic NB patients at diagnosis, post-induction therapy and at the end of treatment for TH and DCX mRNAs detection. High levels of TH and DCX mRNAs when detected in PB and BM at diagnosis independently predicted worse outcome in a cohort of 162 metastatic NB. In the subgroup of high-risk metastatic NB, TH mRNA detected in PB remained as independent predictor of EFS and OS at diagnosis. After the induction therapy, high levels of TH mRNA in PB and DCX mRNA in BM independently predicted poor EFS and OS. Furthermore TH mRNA when detected in BM predicted worse EFS. TH mRNA in PB samples at the end of treatment is an independent predictor of worse outcome. TH and DCX mRNAs levels in PB and BM assessed by RT-qPCR should be considered in new pre-treatment risk stratification strategies to reliable estimate outcome differences in metastatic NB patients. In those high-risk metastatic NB, TH and DCX mRNA quantification could be used for the assessment of response to treatment and for early detection of progressive disease or relapses.

Microarray profiling and co-expression network analysis of circulating lncRNAs and mRNAs associated with major depressive disorder.

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Zhifen Liu

Full Text Available LncRNAs, which represent one of the most highly expressed classes of ncRNAs in the brain, are becoming increasingly interesting with regard to brain functions and disorders. However, changes in the expression of regulatory lncRNAs in Major Depressive Disorder (MDD have not yet been reported. Using microarrays, we profiled the expression of 34834 lncRNAs and 39224 mRNAs in peripheral blood sampled from MDD patients as well as demographically-matched controls. Among these, we found that 2007 lncRNAs and 1667 mRNAs were differentially expressed, 17 of which were documented as depression-related gene in previous studies. Gene Ontology (GO and pathway analyses indicated that the biological functions of differentially expressed mRNAs were related to fundamental metabolic processes and neurodevelopment diseases. To investigate the potential regulatory roles of the differentially expressed lncRNAs on the mRNAs, we also constructed co-expression networks composed of the lncRNAs and mRNAs, which shows significant correlated patterns of expression. In the MDD-derived network, there were a greater number of nodes and connections than that in the control-derived network. The lncRNAs located at chr10:874695-874794, chr10:75873456-75873642, and chr3:47048304-47048512 may be important factors regulating the expression of mRNAs as they have previously been reported associations with MDD. This study is the first to explore genome-wide lncRNA expression and co-expression with mRNA patterns in MDD using microarray technology. We identified circulating lncRNAs that are aberrantly expressed in MDD and the results suggest that lncRNAs may contribute to the molecular pathogenesis of MDD.

The Fragile X Protein binds mRNAs involved in cancer progression and modulates metastasis formation

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Lucá, Rossella; Averna, Michele; Zalfa, Francesca; Vecchi, Manuela; Bianchi, Fabrizio; Fata, Giorgio La; Del Nonno, Franca; Nardacci, Roberta; Bianchi, Marco; Nuciforo, Paolo; Munck, Sebastian; Parrella, Paola; Moura, Rute; Signori, Emanuela; Alston, Robert; Kuchnio, Anna; Farace, Maria Giulia; Fazio, Vito Michele; Piacentini, Mauro; De Strooper, Bart; Achsel, Tilmann; Neri, Giovanni; Neven, Patrick; Evans, D Gareth; Carmeliet, Peter; Mazzone, Massimiliano; Bagni, Claudia

2013-01-01

The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression. PMID:24092663

Somatomedin-C/insulin-like growth factor-I and Insulin-like growth factor-II mRNAs in rate fetal and adult tissues

International Nuclear Information System (INIS)

Lund, P.K.; Moats-Staats, B.M.; Hynes, M.A.; Simmons, J.G.; Jansen, M.; D'ercole, A.J.; Van Wyk, J.J.

1986-01-01

Somatomedin-C or insulin-like growth factor I (Sm-C/IGF-I) and insulin-like growth factor II (IGF-II) have been implicated in the regulation of fetal growth and development. In the present study 32 P-labeled complementary DNA probes encoding human and mouse Sm-C/IGF-I and human IGF-II were used in Northern blot hybridizations to analyze rat Sm-C/IGF-I and IGF-II mRNAs in poly(A + ) RNAs from intestine, liver, lung, and brain of adult rats and fetal rats between day 14 and 17 of gestation. In fetal rats, all four tissues contained a major mRNA of 1.7 kilobase (kb) that hybridized with the human Sm-C/IGF-I cDNA and mRNAs of 7.5, 4.7, 1.7, and 1.2 kb that hybridized with the mouse Sm-C/IGF-I cDNA. Adult rat intestine, liver, and lung also contained these mRNAs but Sm-C/IGF-I mRNAs were not detected in adult rat brain. These findings provide direct support for prior observations that multiple tissues in the fetus synthesize immunoreactive Sm-C/IGF-I and imply a role for Sm-C/IGF-I in fetal development as well as postnatally. Multiple IGF-II mRNAs of estimated sizes 4.7, 3.9, 2.2, 1.75, and 1.2 kb were observed in fetal rat intestine, liver, lung, and brain. The 4.7- and 3.9-kb mRNAs were the major hybridizing IGF-II mRNAs in all fetal tissues. Higher abundance of IGF-II mRNAs in rat fetal tissues compared with adult tissues supports prior hypotheses, based on serum IGF-II concentrations, that IGF-II is predominantly a fetal somatomedin. IGF-II mRNAs are present, however, in some poly(A + ) RNAs from adult rat tissues. The brain was the only tissue in the adult rat where the 4.7- and 3.9-kb IGF-II mRNAs were consistently detected. These findings suggest that a role for IGF-II in the adult rat, particularly in the central nervous system, cannot be excluded

Regulating Emotions during Difficult Multiattribute Decision Making: The Role of Pre-Decisional Coherence Shifting.

Science.gov (United States)

Carpenter, Stephanie M; Yates, J Frank; Preston, Stephanie D; Chen, Lydia

2016-01-01

Almost all real-life decisions entail attribute conflict; every serious choice alternative is better than its competitors on some attribute dimensions but worse on others. In pre-decisional "coherence shifting," the decision maker gradually softens that conflict psychologically to the point where one alternative is seen as dominant over its competitors, or nearly so. Specifically, weaknesses of the eventually chosen alternative come to be perceived as less severe and less important while its strengths seem more desirable and significant. The research described here demonstrates that difficult multiattribute decision problems are aversive and that pre-decisional coherence shifting aids individuals in regulating that emotional discomfort. Across three studies, attribute conflict was confirmed to be aversive (Study 1), and skin conductance responses and ratings of decision difficulty both decreased in participants who coherence shifted (Study 2). Coherence shifting was also diminished among decision makers who were depleted of regulatory resources, known to be required for common emotion regulation mechanisms. Further, coherence shifting was shown to be relatively common among people who reported strong suppression tendencies in everyday emotion regulation (Study 3). Overall, the data suggest that, at least in part, coherence shifting serves as a tool that helps decision makers manage the pre-decisional discomfort generated by attribute conflict. Theoretical and practical implications are discussed.

Translatome profiling in dormant and nondormant sunflower (Helianthus annuus) seeds highlights post-transcriptional regulation of germination.

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Layat, Elodie; Leymarie, Juliette; El-Maarouf-Bouteau, Hayat; Caius, José; Langlade, Nicolas; Bailly, Christophe

2014-12-01

Seed dormancy, which blocks germination in apparently favourable conditions, is a key regulatory control point of plant population establishment. As germination requires de novo translation, its regulation by dormancy is likely to be related to the association of individual transcripts to polysomes. Here, the polysome-associated mRNAs, that is, the translatome, were fractionated and characterized with microarrays in dormant and nondormant sunflower (Helianthus annuus) embryos during their imbibition at 10°C, a temperature preventing germination of dormant embryos. Profiling of mRNAs in polysomal complexes revealed that the translatome differs between germinating and nongerminating embryos. Association of transcripts with polysomes reached a maximum after 15 h of imbibition; at this time-point 194 polysome-associated transcripts were specifically found in nondormant embryos and 47 in dormant embryos only. The proteins corresponding to the polysomal mRNAs in nondormant embryos appeared to be very pertinent for germination and were involved mainly in transport, regulation of transcription or cell wall modifications. This work demonstrates that seed germination results from a timely regulated and selective recruitment of mRNAs to polysomes, thus opening novel fields of investigation for the understanding of this developmental process. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Muscle-specific splicing factors ASD-2 and SUP-12 cooperatively switch alternative pre-mRNA processing patterns of the ADF/cofilin gene in Caenorhabditis elegans.

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Genta Ohno

Full Text Available Pre-mRNAs are often processed in complex patterns in tissue-specific manners to produce a variety of protein isoforms from single genes. However, mechanisms orchestrating the processing of the entire transcript are not well understood. Muscle-specific alternative pre-mRNA processing of the unc-60 gene in Caenorhabditis elegans, encoding two tissue-specific isoforms of ADF/cofilin with distinct biochemical properties in regulating actin organization, provides an excellent in vivo model of complex and tissue-specific pre-mRNA processing; it consists of a single first exon and two separate series of downstream exons. Here we visualize the complex muscle-specific processing pattern of the unc-60 pre-mRNA with asymmetric fluorescence reporter minigenes. By disrupting juxtaposed CUAAC repeats and UGUGUG stretch in intron 1A, we demonstrate that these elements are required for retaining intron 1A, as well as for switching the processing patterns of the entire pre-mRNA from non-muscle-type to muscle-type. Mutations in genes encoding muscle-specific RNA-binding proteins ASD-2 and SUP-12 turned the colour of the unc-60 reporter worms. ASD-2 and SUP-12 proteins specifically and cooperatively bind to CUAAC repeats and UGUGUG stretch in intron 1A, respectively, to form a ternary complex in vitro. Immunohistochemical staining and RT-PCR analyses demonstrate that ASD-2 and SUP-12 are also required for switching the processing patterns of the endogenous unc-60 pre-mRNA from UNC-60A to UNC-60B in muscles. Furthermore, systematic analyses of partially spliced RNAs reveal the actual orders of intron removal for distinct mRNA isoforms. Taken together, our results demonstrate that muscle-specific splicing factors ASD-2 and SUP-12 cooperatively promote muscle-specific processing of the unc-60 gene, and provide insight into the mechanisms of complex pre-mRNA processing; combinatorial regulation of a single splice site by two tissue-specific splicing regulators

Isoforms of elongation factor eEF1A may be differently regulated at post-transcriptional level in breast cancer progression

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Vislovukh A. A.

2013-01-01

Full Text Available Eukaryotic translation elongation factor 1A exists as two 98 % homologous isoforms: eEF1A1 (A1 and eEF1A2 (A2 which are tissue and development specific. Despite high homology in an open reading frame (ORF region, mRNAs coding for eEF1A1 and eEF1A2 are different in their untranslated regions (UTR, suggesting a possibility of their dissimilar post-transcriptional regulation. Aim. To analyze the existence of cis-acting motifs in the UTRs of EEF1A1/A2 mRNAs, to confirm the possibility of post-transcriptional control of eEF1A1 and eEF1A2 expression. Methods. An ensemble of bioinformatic methods was applied to predict regulatory motifs in the UTRs of EEF1A1/A2 mRNAs. Dual-luciferase reporter assay was employed to detect post-transcriptional regulation of eEF1A1/A2 expression. Results. Numerous regulatory motifs in the UTR of EEF1A1/A2 mRNAs were found bioinformatically. The experimental evidence was obtained for the existence of negative regulation of EEF1A1 and positive regulation of EEF1A2 mRNA in the model of breast cancer development. Conclusions. EEF1A1 and EEF1A2 mRNAs contain distinct motifs in the UTRs and are differently regulated in cancer suggesting the possibility of their control by different cellular signals.

Sex-specific mouse liver gene expression: genome-wide analysis of developmental changes from pre-pubertal period to young adulthood

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Conforto Tara L

2012-04-01

Full Text Available Abstract Background Early liver development and the transcriptional transitions during hepatogenesis are well characterized. However, gene expression changes during the late postnatal/pre-pubertal to young adulthood period are less well understood, especially with regards to sex-specific gene expression. Methods Microarray analysis of male and female mouse liver was carried out at 3, 4, and 8 wk of age to elucidate developmental changes in gene expression from the late postnatal/pre-pubertal period to young adulthood. Results A large number of sex-biased and sex-independent genes showed significant changes during this developmental period. Notably, sex-independent genes involved in cell cycle, chromosome condensation, and DNA replication were down regulated from 3 wk to 8 wk, while genes associated with metal ion binding, ion transport and kinase activity were up regulated. A majority of genes showing sex differential expression in adult liver did not display sex differences prior to puberty, at which time extensive changes in sex-specific gene expression were seen, primarily in males. Thus, in male liver, 76% of male-specific genes were up regulated and 47% of female-specific genes were down regulated from 3 to 8 wk of age, whereas in female liver 67% of sex-specific genes showed no significant change in expression. In both sexes, genes up regulated from 3 to 8 wk were significantly enriched (p p Ihh; female-specific Cdx4, Cux2, Tox, and Trim24 and may contribute to the developmental changes that lead to global acquisition of liver sex-specificity by 8 wk of age. Conclusions Overall, the observed changes in gene expression during postnatal liver development reflect the deceleration of liver growth and the induction of specialized liver functions, with widespread changes in sex-specific gene expression primarily occurring in male liver.

The fragile X protein binds mRNAs involved in cancer progression and modulates metastasis formation.

Science.gov (United States)

Lucá, Rossella; Averna, Michele; Zalfa, Francesca; Vecchi, Manuela; Bianchi, Fabrizio; La Fata, Giorgio; Del Nonno, Franca; Nardacci, Roberta; Bianchi, Marco; Nuciforo, Paolo; Munck, Sebastian; Parrella, Paola; Moura, Rute; Signori, Emanuela; Alston, Robert; Kuchnio, Anna; Farace, Maria Giulia; Fazio, Vito Michele; Piacentini, Mauro; De Strooper, Bart; Achsel, Tilmann; Neri, Giovanni; Neven, Patrick; Evans, D Gareth; Carmeliet, Peter; Mazzone, Massimiliano; Bagni, Claudia

2013-10-01

The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression. © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

Endogenous short RNAs generated by Dicer 2 and RNA-dependent RNA polymerase 1 regulate mRNAs in the basal fungus Mucor circinelloides

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Nicolas, Francisco Esteban; Moxon, Simon; de Haro, Juan P.; Calo, Silvia; Grigoriev, Igor V.; Torres-Martínez, Santiago; Moulton, Vincent; Ruiz-Vázquez, Rosa M.; Dalmay, Tamas

2010-01-01

Endogenous short RNAs (esRNAs) play diverse roles in eukaryotes and usually are produced from double-stranded RNA (dsRNA) by Dicer. esRNAs are grouped into different classes based on biogenesis and function but not all classes are present in all three eukaryotic kingdoms. The esRNA register of fungi is poorly described compared to other eukaryotes and it is not clear what esRNA classes are present in this kingdom and whether they regulate the expression of protein coding genes. However, evidence that some dicer mutant fungi display altered phenotypes suggests that esRNAs play an important role in fungi. Here, we show that the basal fungus Mucor circinelloides produces new classes of esRNAs that map to exons and regulate the expression of many protein coding genes. The largest class of these exonic-siRNAs (ex-siRNAs) are generated by RNA-dependent RNA Polymerase 1 (RdRP1) and dicer-like 2 (DCL2) and target the mRNAs of protein coding genes from which they were produced. Our results expand the range of esRNAs in eukaryotes and reveal a new role for esRNAs in fungi. PMID:20427422

Endogenous short RNAs generated by Dicer 2 and RNA-dependent RNA polymerase 1 regulate mRNAs in the basal fungus Mucor circinelloides

Energy Technology Data Exchange (ETDEWEB)

Grigoriev, Igor; Nicolas, Francisco; Moxon, Simon; Haro, Juan de; Calo, Silvia; Torres-Martinez, Santiago; Moulton, Vincent; Ruiz-Vazquez, Rosa; Dalmay, Tamas

2011-09-01

Endogenous short RNAs (esRNAs) play diverse roles in eukaryotes and usually are produced from double-stranded RNA (dsRNA) by Dicer. esRNAs are grouped into different classes based on biogenesis and function but not all classes are present in all three eukaryotic kingdoms. The esRNA register of fungi is poorly described compared to other eukaryotes and it is not clear what esRNA classes are present in this kingdom and whether they regulate the expression of protein coding genes. However, evidence that some dicer mutant fungi display altered phenotypes suggests that esRNAs play an important role in fungi. Here, we show that the basal fungus Mucor circinelloides produces new classes of esRNAs that map to exons and regulate the expression of many protein coding genes. The largest class of these exonic-siRNAs (ex-siRNAs) are generated by RNA-dependent RNA Polymerase 1 (RdRP1) and dicer-like 2 (DCL2) and target the mRNAs of protein coding genes from which they were produced. Our results expand the range of esRNAs in eukaryotes and reveal a new role for esRNAs in fungi

Regulating Emotions during Difficult Multiattribute Decision Making: The Role of Pre-Decisional Coherence Shifting.

Directory of Open Access Journals (Sweden)

Stephanie M Carpenter

Full Text Available Almost all real-life decisions entail attribute conflict; every serious choice alternative is better than its competitors on some attribute dimensions but worse on others. In pre-decisional "coherence shifting," the decision maker gradually softens that conflict psychologically to the point where one alternative is seen as dominant over its competitors, or nearly so. Specifically, weaknesses of the eventually chosen alternative come to be perceived as less severe and less important while its strengths seem more desirable and significant. The research described here demonstrates that difficult multiattribute decision problems are aversive and that pre-decisional coherence shifting aids individuals in regulating that emotional discomfort. Across three studies, attribute conflict was confirmed to be aversive (Study 1, and skin conductance responses and ratings of decision difficulty both decreased in participants who coherence shifted (Study 2. Coherence shifting was also diminished among decision makers who were depleted of regulatory resources, known to be required for common emotion regulation mechanisms. Further, coherence shifting was shown to be relatively common among people who reported strong suppression tendencies in everyday emotion regulation (Study 3. Overall, the data suggest that, at least in part, coherence shifting serves as a tool that helps decision makers manage the pre-decisional discomfort generated by attribute conflict. Theoretical and practical implications are discussed.

Ovarian steroid regulation of monoamine oxidase-A and -B mRNAs in the macaque dorsal raphe and hypothalamic nuclei.

Science.gov (United States)

Gundlah, Chrisana; Lu, Nick Z; Bethea, Cynthia L

2002-03-01

The serotonin neural system plays a pivotal role in mood, affective regulation and integrative cognition, as well as numerous autonomic functions. We have shown that ovarian steroids alter the expression of several genes in the dorsal raphe of macaques, which may increase serotonin synthesis and decrease serotonin autoinhibition. Another control point in aminergic neurotransmission involves degradation by MAO. This enzyme occurs in two isoforms, A and B, which have different substrate preferences. We questioned the effect of ovarian steroid hormones on MAO-A and MAO-B mRNA expression in the dorsal raphe nucleus and hypothalamus using in situ hybridization in non-human primates. Rhesus monkeys ( Macaca mulatta; n=5/group) were spayed and either placebo treated (controls), estrogen (E) treated (28 days), progesterone (P) treated (14 days placebo+14 days P), or E+P treated (14 days E+14 days E+P). Perfusion-fixed sections (25 microm) were hybridized with a 233 bp MAO-A, or a 373 bp MAO-B, radiolabeled-antisense monkey specific probes. Autoradiographic films were analyzed by densitometry, which was performed with NIH Image Software. MAO-A and -B mRNAs were detected in the dorsal raphe nucleus (DRN) and in the hypothalamic suprachiasmatic nucleus (SCN), preoptic area (POA), paraventricular nucleus (PVN), supraoptic nucleus (SON), lateral hypothalamus (LH) and ventromedial nucleus (VMN). MAO-A mRNA optical density was significantly decreased by E, P, and E+P in the DRN and in the hypothalamic PVN, LH and VMN. Ovarian hormones had no effect on MAO-B mRNA expression in the DRN. However, there was a significant decrease in MAO-B optical density in the hypothalamic POA, LH and VMN with E, P or E+P treatment. Pixel area generally reflected optical density. Ovarian steroids decreased MAO-A, but not B, in the raphe nucleus. However, both MAO-A and B were decreased in discrete hypothalamic nuclei by hormone replacement. These data suggest that the transcriptional regulation of

Splicing of goose parvovirus pre-mRNA influences cytoplasmic translation of the processed mRNA

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Li, Long; Pintel, David J., E-mail: pinteld@missouri.edu

2012-04-25

Translation of goose parvovirus (GPV) 72 kDa Rep 1 is initiated from unspliced P9-generated mRNAs in ORF1 from the first in-frame AUG (537 AUG); however, this AUG is bypassed in spliced P9-generated RNA: translation of the 52 kDa Rep 2 protein from spliced RNA is initiated in ORF2 at the next AUG downstream (650 AUG). Usage of the 537 AUG was restored in spliced RNA when the GPV intron was replaced with a chimeric SV40 intron, or following specific mutations of the GPV intron which did not appear in the final spliced mRNA. Additionally, 650 AUG usage was gained in unspliced RNA when the GPV intron splice sites were debilitated. Splicing-dependent regulation of translation initiation was mediated in cis by GPV RNA surrounding the target AUGs. Thus, nuclear RNA processing of GPV P9-generated pre-mRNAs has a complex, but significant, effect on alternative translation initiation of the GPV Rep proteins.

Splicing of goose parvovirus pre-mRNA influences cytoplasmic translation of the processed mRNA

International Nuclear Information System (INIS)

Li, Long; Pintel, David J.

2012-01-01

Translation of goose parvovirus (GPV) 72 kDa Rep 1 is initiated from unspliced P9-generated mRNAs in ORF1 from the first in-frame AUG (537 AUG); however, this AUG is bypassed in spliced P9-generated RNA: translation of the 52 kDa Rep 2 protein from spliced RNA is initiated in ORF2 at the next AUG downstream (650 AUG). Usage of the 537 AUG was restored in spliced RNA when the GPV intron was replaced with a chimeric SV40 intron, or following specific mutations of the GPV intron which did not appear in the final spliced mRNA. Additionally, 650 AUG usage was gained in unspliced RNA when the GPV intron splice sites were debilitated. Splicing-dependent regulation of translation initiation was mediated in cis by GPV RNA surrounding the target AUGs. Thus, nuclear RNA processing of GPV P9-generated pre-mRNAs has a complex, but significant, effect on alternative translation initiation of the GPV Rep proteins.

Capture of microRNA-bound mRNAs identifies the tumor suppressor miR-34a as a regulator of growth factor signaling.

Directory of Open Access Journals (Sweden)

Ashish Lal

2011-11-01

Full Text Available A simple biochemical method to isolate mRNAs pulled down with a transfected, biotinylated microRNA was used to identify direct target genes of miR-34a, a tumor suppressor gene. The method reidentified most of the known miR-34a regulated genes expressed in K562 and HCT116 cancer cell lines. Transcripts for 982 genes were enriched in the pull-down with miR-34a in both cell lines. Despite this large number, validation experiments suggested that ~90% of the genes identified in both cell lines can be directly regulated by miR-34a. Thus miR-34a is capable of regulating hundreds of genes. The transcripts pulled down with miR-34a were highly enriched for their roles in growth factor signaling and cell cycle progression. These genes form a dense network of interacting gene products that regulate multiple signal transduction pathways that orchestrate the proliferative response to external growth stimuli. Multiple candidate miR-34a-regulated genes participate in RAS-RAF-MAPK signaling. Ectopic miR-34a expression reduced basal ERK and AKT phosphorylation and enhanced sensitivity to serum growth factor withdrawal, while cells genetically deficient in miR-34a were less sensitive. Fourteen new direct targets of miR-34a were experimentally validated, including genes that participate in growth factor signaling (ARAF and PIK3R2 as well as genes that regulate cell cycle progression at various phases of the cell cycle (cyclins D3 and G2, MCM2 and MCM5, PLK1 and SMAD4. Thus miR-34a tempers the proliferative and pro-survival effect of growth factor stimulation by interfering with growth factor signal transduction and downstream pathways required for cell division.

Capture of microRNA-bound mRNAs identifies the tumor suppressor miR-34a as a regulator of growth factor signaling.

Science.gov (United States)

Lal, Ashish; Thomas, Marshall P; Altschuler, Gabriel; Navarro, Francisco; O'Day, Elizabeth; Li, Xiao Ling; Concepcion, Carla; Han, Yoon-Chi; Thiery, Jerome; Rajani, Danielle K; Deutsch, Aaron; Hofmann, Oliver; Ventura, Andrea; Hide, Winston; Lieberman, Judy

2011-11-01

A simple biochemical method to isolate mRNAs pulled down with a transfected, biotinylated microRNA was used to identify direct target genes of miR-34a, a tumor suppressor gene. The method reidentified most of the known miR-34a regulated genes expressed in K562 and HCT116 cancer cell lines. Transcripts for 982 genes were enriched in the pull-down with miR-34a in both cell lines. Despite this large number, validation experiments suggested that ~90% of the genes identified in both cell lines can be directly regulated by miR-34a. Thus miR-34a is capable of regulating hundreds of genes. The transcripts pulled down with miR-34a were highly enriched for their roles in growth factor signaling and cell cycle progression. These genes form a dense network of interacting gene products that regulate multiple signal transduction pathways that orchestrate the proliferative response to external growth stimuli. Multiple candidate miR-34a-regulated genes participate in RAS-RAF-MAPK signaling. Ectopic miR-34a expression reduced basal ERK and AKT phosphorylation and enhanced sensitivity to serum growth factor withdrawal, while cells genetically deficient in miR-34a were less sensitive. Fourteen new direct targets of miR-34a were experimentally validated, including genes that participate in growth factor signaling (ARAF and PIK3R2) as well as genes that regulate cell cycle progression at various phases of the cell cycle (cyclins D3 and G2, MCM2 and MCM5, PLK1 and SMAD4). Thus miR-34a tempers the proliferative and pro-survival effect of growth factor stimulation by interfering with growth factor signal transduction and downstream pathways required for cell division.

Capture of MicroRNA–Bound mRNAs Identifies the Tumor Suppressor miR-34a as a Regulator of Growth Factor Signaling

Science.gov (United States)

O'Day, Elizabeth; Li, Xiao Ling; Concepcion, Carla; Han, Yoon-Chi; Thiery, Jerome; Rajani, Danielle K.; Deutsch, Aaron; Hofmann, Oliver; Ventura, Andrea; Hide, Winston; Lieberman, Judy

2011-01-01

A simple biochemical method to isolate mRNAs pulled down with a transfected, biotinylated microRNA was used to identify direct target genes of miR-34a, a tumor suppressor gene. The method reidentified most of the known miR-34a regulated genes expressed in K562 and HCT116 cancer cell lines. Transcripts for 982 genes were enriched in the pull-down with miR-34a in both cell lines. Despite this large number, validation experiments suggested that ∼90% of the genes identified in both cell lines can be directly regulated by miR-34a. Thus miR-34a is capable of regulating hundreds of genes. The transcripts pulled down with miR-34a were highly enriched for their roles in growth factor signaling and cell cycle progression. These genes form a dense network of interacting gene products that regulate multiple signal transduction pathways that orchestrate the proliferative response to external growth stimuli. Multiple candidate miR-34a–regulated genes participate in RAS-RAF-MAPK signaling. Ectopic miR-34a expression reduced basal ERK and AKT phosphorylation and enhanced sensitivity to serum growth factor withdrawal, while cells genetically deficient in miR-34a were less sensitive. Fourteen new direct targets of miR-34a were experimentally validated, including genes that participate in growth factor signaling (ARAF and PIK3R2) as well as genes that regulate cell cycle progression at various phases of the cell cycle (cyclins D3 and G2, MCM2 and MCM5, PLK1 and SMAD4). Thus miR-34a tempers the proliferative and pro-survival effect of growth factor stimulation by interfering with growth factor signal transduction and downstream pathways required for cell division. PMID:22102825

Multiple-targeted graphene-based nanocarrier for intracellular imaging of mRNAs

International Nuclear Information System (INIS)

Wang, Ying; Li, Zhaohui; Liu, Misha; Xu, Jinjin; Hu, Dehong; Lin, Yuehe; Li, Jinghong

2017-01-01

Simultaneous detection and imaging of multiple intracellular messenger RNA (mRNAs) hold great significant for early cancer diagnostics and preventive medicine development. Herein, we propose a multiple-targeted graphene oxide (GO) nanocarrier that can simultaneously detect and image different type mRNAs in living cells. First of all, in vitro detection of multiple targets have been realized successfully based on the multiple-targeted GO nanocarrier with linear relationship ranging from 3 nM to 200 nM, as well as sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) mRNA and 2.45 nM for β-actin mRNA. Additionally, this nanosensing platform composed of fluorescent labelled single strand DNA probes and GO nanocarrier can identify Mn-SOD mRNA and endogenous mRNA of β-actin in living cancer cells, showing rapid response, high specificity, nuclease stability, and good biocompatibility during the cell imaging. Thirdly, changes of the expression levels of mRNA in living cells before or after the drug treatment can be monitored successfully. By using multiple ssDNA as probes and GO nanocarrier as the cellular delivery cargo, the proposed simultaneous multiple-targeted sensing platform will be of great potential as a powerful tool for intracellular trafficking process from basic research to clinical diagnosis. - Graphical abstract: Schematic illustration of simultaneously multiple mRNAs monitoring inside single living breast cancer cell based on GO nanocarrier. In particular, the fluorescent signals could be monitored when Mn-SOD probe (red) and β-actin probe (green) hybridizes with their mRNA targets inside the living cells. Random probe (orange) was regarded as control probe for the sensing strategy. - Highlights: • A multiple-targeted GO nanocarrier was used for mRNAs imaging and expression changes after drug treatment can be monitored successfully. • Sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) m

Genomic analysis suggests that mRNA destabilization by the microprocessor is specialized for the auto-regulation of Dgcr8.

Directory of Open Access Journals (Sweden)

Archana Shenoy

2009-09-01

Full Text Available The Microprocessor, containing the RNA binding protein Dgcr8 and RNase III enzyme Drosha, is responsible for processing primary microRNAs to precursor microRNAs. The Microprocessor regulates its own levels by cleaving hairpins in the 5'UTR and coding region of the Dgcr8 mRNA, thereby destabilizing the mature transcript.To determine whether the Microprocessor has a broader role in directly regulating other coding mRNA levels, we integrated results from expression profiling and ultra high-throughput deep sequencing of small RNAs. Expression analysis of mRNAs in wild-type, Dgcr8 knockout, and Dicer knockout mouse embryonic stem (ES cells uncovered mRNAs that were specifically upregulated in the Dgcr8 null background. A number of these transcripts had evolutionarily conserved predicted hairpin targets for the Microprocessor. However, analysis of deep sequencing data of 18 to 200nt small RNAs in mouse ES, HeLa, and HepG2 indicates that exonic sequence reads that map in a pattern consistent with Microprocessor activity are unique to Dgcr8.We conclude that the Microprocessor's role in directly destabilizing coding mRNAs is likely specifically targeted to Dgcr8 itself, suggesting a specialized cellular mechanism for gene auto-regulation.

Epigenetic microRNA Regulation

DEFF Research Database (Denmark)

Wiklund, Erik Digman

2011-01-01

MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) that negatively regulate gene expression post-transcriptionally by binding to complementary sequences in the 3’UTR of target mRNAs in the cytoplasm. However, recent evidence suggests that certain miRNAs are enriched in the nucleus, and their t......MicroRNAs (miRNAs) are small non-coding RNAs (ncRNAs) that negatively regulate gene expression post-transcriptionally by binding to complementary sequences in the 3’UTR of target mRNAs in the cytoplasm. However, recent evidence suggests that certain miRNAs are enriched in the nucleus...

The Analysis of the Pre-Emption Right under the Contract of Sale in the Regulation of New Civil Code

Directory of Open Access Journals (Sweden)

Mirela Costache

2011-05-01

Full Text Available In this paper we will keep under review the specificity of the reported pre-emption right to the sale contract, according to the article 1730-1740 of the New Civil Code. With the entry into force of the new future regulation, the pre-emption right will acquire a separate status, being currently known that the legal status of the right under the review is diverse; there are many legal provisions which provide this right in various areas, being excedentary to the sale contract, such as culture, privatization, franchising, intellectual property. According to the analysis of the future legal deposition, it shows that pre-emption right may have as a source both the law and the contract, in this case it is referred to the legal and conventional right of pre-emption. We note also that, in light of the new regulations, the mechanism for exercising the right of pre-emption is similar to the one applicable to the right of preference. Objectives: The purpose of this paper is to focus on the usefulness of this new legislative measure designed to establish a proper legal support specific to the holder of this right in the conclusion of a contract in relation to third parties. Approach: This topic emphasizes the use of the following methods: observation, comparison and interpretation of laws.

Identification of microRNAs and mRNAs associated with multidrug resistance of human laryngeal cancer Hep-2 cells

Energy Technology Data Exchange (ETDEWEB)

Yin, Wanzhong; Wang, Ping; Wang, Xin [Department of Otorhinolaryngology, Head and Neck Surgery, The First Clinical Hospital, Norman Bethune College of Medicine, Jilin University, Changchun (China); Song, Wenzhi [Department of Stomatology, China-Japan Friendship Hospital, Jilin University, Changchun (China); Cui, Xiangyan; Yu, Hong; Zhu, Wei [Department of Otorhinolaryngology, Head and Neck Surgery, The First Clinical Hospital, Norman Bethune College of Medicine, Jilin University, Changchun (China)

2013-06-12

Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96'µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (∼45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33±1.76 vs 28.75±1.12'h, P<0.05), higher percentage of cells in G0/G1 phase (80.98±0.52 vs 69.14±0.89, P<0.05), increased efflux of rhodamine 123 (95.97±0.56 vs 12.40±0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer.

Identification of microRNAs and mRNAs associated with multidrug resistance of human laryngeal cancer Hep-2 cells

International Nuclear Information System (INIS)

Yin, Wanzhong; Wang, Ping; Wang, Xin; Song, Wenzhi; Cui, Xiangyan; Yu, Hong; Zhu, Wei

2013-01-01

Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96'µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (∼45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33±1.76 vs 28.75±1.12'h, P<0.05), higher percentage of cells in G0/G1 phase (80.98±0.52 vs 69.14±0.89, P<0.05), increased efflux of rhodamine 123 (95.97±0.56 vs 12.40±0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer

Characterization of mTOR-Responsive Truncated mRNAs in Cell Proliferation

Science.gov (United States)

2017-07-01

These findings identify a previously uncharacterized role for mTOR in modulating 3’- UTR length of mRNAs by alternative polyadenylation ( APA ). Another...outcome of APA in the mTOR-activated transcriptome is an early termination of mRNA transcription to produce truncated mRNAs with polyadenylation in...for exhaustive analysis of Alternative cleavage and polyadenylation ( APA ) events (Figure 1). In IntMAP, first the position of multiple

A Requirement for Mena, an Actin Regulator, in Local mRNA Translation in Developing Neurons.

Science.gov (United States)

Vidaki, Marina; Drees, Frauke; Saxena, Tanvi; Lanslots, Erwin; Taliaferro, Matthew J; Tatarakis, Antonios; Burge, Christopher B; Wang, Eric T; Gertler, Frank B

2017-08-02

During neuronal development, local mRNA translation is required for axon guidance and synaptogenesis, and dysregulation of this process contributes to multiple neurodevelopmental and cognitive disorders. However, regulation of local protein synthesis in developing axons remains poorly understood. Here, we uncover a novel role for the actin-regulatory protein Mena in the formation of a ribonucleoprotein complex that involves the RNA-binding proteins HnrnpK and PCBP1 and regulates local translation of specific mRNAs in developing axons. We find that translation of dyrk1a, a Down syndrome- and autism spectrum disorders-related gene, is dependent on Mena, both in steady-state conditions and upon BDNF stimulation. We identify hundreds of additional mRNAs that associate with the Mena complex, suggesting that it plays broader role(s) in post-transcriptional gene regulation. Our work establishes a dual role for Mena in neurons, providing a potential link between regulation of actin dynamics and local translation. Copyright © 2017 Elsevier Inc. All rights reserved.

Redox regulation of photosynthetic gene expression.

Science.gov (United States)

Queval, Guillaume; Foyer, Christine H

2012-12-19

Redox chemistry and redox regulation are central to the operation of photosynthesis and respiration. However, the roles of different oxidants and antioxidants in the regulation of photosynthetic or respiratory gene expression remain poorly understood. Leaf transcriptome profiles of a range of Arabidopsis thaliana genotypes that are deficient in either hydrogen peroxide processing enzymes or in low molecular weight antioxidant were therefore compared to determine how different antioxidant systems that process hydrogen peroxide influence transcripts encoding proteins targeted to the chloroplasts or mitochondria. Less than 10 per cent overlap was observed in the transcriptome patterns of leaves that are deficient in either photorespiratory (catalase (cat)2) or chloroplastic (thylakoid ascorbate peroxidase (tapx)) hydrogen peroxide processing. Transcripts encoding photosystem II (PSII) repair cycle components were lower in glutathione-deficient leaves, as were the thylakoid NAD(P)H (nicotinamide adenine dinucleotide (phosphate)) dehydrogenases (NDH) mRNAs. Some thylakoid NDH mRNAs were also less abundant in tAPX-deficient and ascorbate-deficient leaves. Transcripts encoding the external and internal respiratory NDHs were increased by low glutathione and low ascorbate. Regulation of transcripts encoding specific components of the photosynthetic and respiratory electron transport chains by hydrogen peroxide, ascorbate and glutathione may serve to balance non-cyclic and cyclic electron flow pathways in relation to oxidant production and reductant availability.

5'-Terminal AUGs in Escherichia coli mRNAs with Shine-Dalgarno Sequences: Identification and Analysis of Their Roles in Non-Canonical Translation Initiation.

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Heather J Beck

Full Text Available Analysis of the Escherichia coli transcriptome identified a unique subset of messenger RNAs (mRNAs that contain a conventional untranslated leader and Shine-Dalgarno (SD sequence upstream of the gene's start codon while also containing an AUG triplet at the mRNA's 5'- terminus (5'-uAUG. Fusion of the coding sequence specified by the 5'-terminal putative AUG start codon to a lacZ reporter gene, as well as primer extension inhibition assays, reveal that the majority of the 5'-terminal upstream open reading frames (5'-uORFs tested support some level of lacZ translation, indicating that these mRNAs can function both as leaderless and canonical SD-leadered mRNAs. Although some of the uORFs were expressed at low levels, others were expressed at levels close to that of the respective downstream genes and as high as the naturally leaderless cI mRNA of bacteriophage λ. These 5'-terminal uORFs potentially encode peptides of varying lengths, but their functions, if any, are unknown. In an effort to determine whether expression from the 5'-terminal uORFs impact expression of the immediately downstream cistron, we examined expression from the downstream coding sequence after mutations were introduced that inhibit efficient 5'-uORF translation. These mutations were found to affect expression from the downstream cistrons to varying degrees, suggesting that some 5'-uORFs may play roles in downstream regulation. Since the 5'-uAUGs found on these conventionally leadered mRNAs can function to bind ribosomes and initiate translation, this indicates that canonical mRNAs containing 5'-uAUGs should be examined for their potential to function also as leaderless mRNAs.

Anchoring a Defined Sequence to the 55' Ends of mRNAs : The Bolt to Clone Rare Full Length mRNAs and Generate cDNA Libraries porn a Few Cells.

Science.gov (United States)

Baptiste, J; Milne Edwards, D; Delort, J; Mallet, J

1993-01-01

Among numerous applications, the polymerase chain reaction (PCR) (1,2) provides a convenient means to clone 5' ends of rare mRNAs and to generate cDNA libraries from tissue available in amounts too low to be processed by conventional methods. Basically, the amplification of cDNAs by the PCR requires the availability of the sequences of two stretches of the molecule to be amplified. A sequence can easily be imposed at the 5' end of the first-strand cDNAs (corresponding to the 3' end of the mRNAs) by priming the reverse transcription with a specific primer (for cloning the 5' end of rare messenger) or with an oligonucleotide tailored with a poly (dT) stretch (for cDNA library construction), taking advantage of the poly (A) sequence that is located at the 3' end of mRNAs. Several strategies have been devised to tag the 3' end of the ss-cDNAs (corresponding to the 55' end of the mRNAs). We (3) and others have described strategies based on the addition of a homopolymeric dG (4,5) or dA (6,7) tail using terminal deoxyribonucleotide transferase (TdT) ("anchor-PCR" [4]). However, this strategy has important limitations. The TdT reaction is difficult to control and has a low efficiency (unpublished observations). But most importantly, the return primers containing a homopolymeric (dC or dT) tail generate nonspecific amplifications, a phenomenon that prevents the isolation of low abundance mRNA species and/or interferes with the relative abundance of primary clones in the library. To circumvent these drawbacks, we have used two approaches. First, we devised a strategy based on a cRNA enrichment procedure, which has been useful to eliminate nonspecific-PCR products and to allow detection and cloning of cDNAs of low abundance (3). More recently, to avoid the nonspecific amplification resulting from the annealing of the homopolymeric tail oligonucleotide, we have developed a novel anchoring strategy that is based on the ligation of an oligonucleotide to the 35' end of ss

Organ-Specific and Age-Dependent Expression of Insulin-like Growth Factor-I (IGF-I) mRNA Variants: IGF-IA and IB mRNAs in the Mouse

OpenAIRE

Ohtsuki, Takashi; Otsuki, Mariko; Murakami, Yousuke; Maekawa, Tetsuya; Yamamoto, Takashi; Akasaka, Koji; Takeuchi, Sakae; Takahashi, Sumio

2005-01-01

Insulin-like growth factor-I (IGF-I) gene generates several IGF-I mRNA variants by alternative splicing. Two promoters are present in mouse IGF-I gene. Each promoter encodes two IGF-I mRNA variants (IGF-IA and IGF-IB mRNAs). Variants differ by the presence (IGF-IB) or absence (IGF-IA) of a 52-bp insert in the E domain-coding region. Functional differences among IGF-I mRNAs, and regulatory mechanisms for alternative splicing of IGF-I mRNA are not yet known. We analyzed the expression of mouse ...

Multiple-Targeted Graphene-based Nanocarrier for Intracellular Imaging of mRNAs

Energy Technology Data Exchange (ETDEWEB)

Wang, Ying; Li, Zhaohui; Liu, Misha; Hu, Dehong; Lin, Yuehe; Li, Jinghong

2017-08-29

Simultaneous detection and imaging of multiple intracellular messenger RNA (mRNAs) hold great significant for early cancer diagnostics and preventive medicine development. Herein, we propose a multiple-targeted graphene oxide (GO) nanocarrier that can simultaneously detect and image different type mRNAs in living cells. First of all, in vitro detection of multiple targets have been realized successfully based on the multiple-targeted GO nanocarrier with linear relationship ranging from 3 nM to 200 nM, as well as sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) mRNA and 2.45 nM for β-actin mRNA. Additionally, this nanosensing platform composed of fluorescent labeled single strand DNA probes and GO nanocarrier can identify Mn-SOD mRNA and endogenous mRNA of β-actin in living cancer cells, showing rapid response, high specificity, nuclease stability, and good biocompatibility during the cell imaging. Thirdly, changes of the expression levels of mRNA in living cells before or after the drug treatment can be monitored successfully. By using multiple ssDNA as probes and GO nanocarrier as the cellular delivery cargo, the proposed simultaneous multiple-targeted sensing platform will be of great potential as a powerful tool for intracellular trafficking process from basic research to clinical diagnosis.

Negative regulation of EGFR/MAPK pathway by Pumilio in Drosophila melanogaster.

Directory of Open Access Journals (Sweden)

Sung Yun Kim

Full Text Available In Drosophila melanogaster, specification of wing vein cells and sensory organ precursor (SOP cells, which later give rise to a bristle, requires EGFR signaling. Here, we show that Pumilio (Pum, an RNA-binding translational repressor, negatively regulates EGFR signaling in wing vein and bristle development. We observed that loss of Pum function yielded extra wing veins and additional bristles. Conversely, overexpression of Pum eliminated wing veins and bristles. Heterozygotes for Pum produced no phenotype on their own, but greatly enhanced phenotypes caused by the enhancement of EGFR signaling. Conversely, over-expression of Pum suppressed the effects of ectopic EGFR signaling. Components of the EGFR signaling pathway are encoded by mRNAs that have Nanos Response Element (NRE-like sequences in their 3'UTRs; NREs are known to bind Pum to confer regulation in other mRNAs. We show that these NRE-like sequences bind Pum and confer repression on a luciferase reporter in heterologous cells. Taken together, our evidence suggests that Pum functions as a negative regulator of EGFR signaling by directly targeting components of the pathway in Drosophila.

Potato spindle tuber viroid infection triggers degradation of chloride channel protein CLC-b-like and Ribosomal protein S3a-like mRNAs in tomato plants.

Science.gov (United States)

Adkar-Purushothama, Charith Raj; Iyer, Pavithran Sridharan; Perreault, Jean-Pierre

2017-08-21

It is well established that viroid derived small RNA (vd-sRNA) induces RNA silencing of endogenous mRNA. However, it remains not clear how exactly viroid infections can lead to severe symptom induction given the fact that fewer vd-sRNAs binding the specific target mRNAs were recovered from the infected plants. To answer this question, the two least expressed (+) and (-) strand vd-sRNAs of potato spindle tuber viroid (PSTVd) binding to both the 3' UTR and the coding region of tomato mRNAs were analyzed by infecting tomato plants with two variants of PSTVd. As products of these putative target mRNAs are involved in plant phenotype, the effect of this viroid on these genes were analyzed by infecting tomato plants with two variants of PSTVd. The direct interaction between the vd-sRNAs and putative mRNAs was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Parallel analysis of RNA ends of viroid infected plants revealed the widespread cleavage of the target mRNAs in locations other than the vd-sRNA binding site during the viroid infection implying the viroid-infection induced vd-sRNA independent degradation of endogenous mRNAs during viroid infection.

SAD-B kinase regulates pre-synaptic vesicular dynamics at hippocampal Schaffer collateral synapses and affects contextual fear memory.

Science.gov (United States)

Watabe, Ayako M; Nagase, Masashi; Hagiwara, Akari; Hida, Yamato; Tsuji, Megumi; Ochiai, Toshitaka; Kato, Fusao; Ohtsuka, Toshihisa

2016-01-01

Synapses of amphids defective (SAD)-A/B kinases control various steps in neuronal development and differentiation, such as axon specifications and maturation in central and peripheral nervous systems. At mature pre-synaptic terminals, SAD-B is associated with synaptic vesicles and the active zone cytomatrix; however, how SAD-B regulates neurotransmission and synaptic plasticity in vivo remains unclear. Thus, we used SAD-B knockout (KO) mice to study the function of this pre-synaptic kinase in the brain. We found that the paired-pulse ratio was significantly enhanced at Shaffer collateral synapses in the hippocampal CA1 region in SAD-B KO mice compared with wild-type littermates. We also found that the frequency of the miniature excitatory post-synaptic current was decreased in SAD-B KO mice. Moreover, synaptic depression following prolonged low-frequency synaptic stimulation was significantly enhanced in SAD-B KO mice. These results suggest that SAD-B kinase regulates vesicular release probability at pre-synaptic terminals and is involved in vesicular trafficking and/or regulation of the readily releasable pool size. Finally, we found that hippocampus-dependent contextual fear learning was significantly impaired in SAD-B KO mice. These observations suggest that SAD-B kinase plays pivotal roles in controlling vesicular release properties and regulating hippocampal function in the mature brain. Synapses of amphids defective (SAD)-A/B kinases control various steps in neuronal development and differentiation, but their roles in mature brains were only partially known. Here, we demonstrated, at mature pre-synaptic terminals, that SAD-B regulates vesicular release probability and synaptic plasticity. Moreover, hippocampus-dependent contextual fear learning was significantly impaired in SAD-B KO mice, suggesting that SAD-B kinase plays pivotal roles in controlling vesicular release properties and regulating hippocampal function in the mature brain. © 2015 International

Do pre-drinkers consume more alcohol than non-pre-drinkers on an event-specific night out?

DEFF Research Database (Denmark)

Østergaard, Jeanette; Skov, Peter Rohde

2014-01-01

Introduction and Aims Young people drinking heavily before going out to bars and clubs is associated with alcohol-related harm and therefore of great public concern. This study examines whether pre-drinkers consume more alcohol than non-pre-drinkers on an event-specific night out in England...... demographics, socioeconomic status, frequency of intoxication and alcohol unit intake before and during the young people's night out. Results A mixed linear model performed on the panel mobile survey shows that pre-drinkers in England and Denmark consume 9.185 (P ..., respectively, more than the non-pre-drinkers. However, in both countries pre-drinkers consume 3.430 (P alcohol on-premises than the non-pre-drinkers. Discussion and Conclusion Pre-drinking is a widespread phenomenon in England and Denmark, with more than half of young...

Culture medium, gas atmosphere and MAPK inhibition affect regulation of RNA-binding protein targets during mouse preimplantation development.

Science.gov (United States)

Calder, Michele D; Watson, Patricia H; Watson, Andrew J

2011-11-01

During oogenesis, mammalian oocytes accumulate maternal mRNAs that support the embryo until embryonic genome activation. RNA-binding proteins (RBP) may regulate the stability and turnover of maternal and embryonic mRNAs. We hypothesised that varying embryo culture conditions, such as culture medium, oxygen tension and MAPK inhibition, affects regulation of RBPs and their targets during preimplantation development. STAU1, ELAVL1, KHSRP and ZFP36 proteins and mRNAs were detected throughout mouse preimplantation development, whereas Elavl2 mRNA decreased after the two-cell stage. Potential target mRNAs of RBP regulation, Gclc, Slc2a1 and Slc7a1 were detected during mouse preimplantation development. Gclc mRNA was significantly elevated in embryos cultured in Whitten's medium compared with embryos cultured in KSOMaa, and Gclc mRNA was elevated under high-oxygen conditions. Inhibition of the p38 MAPK pathway reduced Slc7a1 mRNA expression while inhibition of ERK increased Slc2a1 mRNA expression. The half-lives of the potential RBP mRNA targets are not regulated in parallel; Slc2a1 mRNA displayed the longest half-life. Our results indicate that mRNAs and proteins encoding five RBPs are present during preimplantation development and more importantly, demonstrate that expression of RBP target mRNAs are regulated by culture medium, gas atmosphere and MAPK pathways.

Properties and regulation of biosynthesis of cottonseed storage proteins. Comprehensive progress report, December 1, 1976 to September 1, 1979

Energy Technology Data Exchange (ETDEWEB)

Dure, III, L S

1979-01-01

The regulation of gene expression in cotton seed embryogenesis was studied by attempting to define what gene products are likely to be highly regulated during this developmental progression. The flow of nitrogen into the free amino acids pools of the developing cotyledons, and into the principal nitrogen nutritional reserve of the seed, the storage proteins was measured. This was continued by following the flow of nitrogen from the storage proteins to the principal exported amino acid asparagine that occurs during the first several days of germination. In this fashion the rise and fall of certain enzymes of amino acid intermediary metabolism could be postulated, and in some cases, verified. The subsets of abundant mRNAs whose appearance and disappearance coincided with developmental events in cotyledon embryogenesis/germination with the short range goal of identifying proteins/enzyme activities were delineated as well as their mRNAs that represent specific developmental stages and the long range goal of using these representatives as probes for studying the mechanisms controlling the rise and fall of these mRNAs and their protein products.

HNRNPLL stabilizes mRNAs for DNA replication proteins and promotes cell cycle progression in colorectal cancer cells.

Science.gov (United States)

Sakuma, Keiichiro; Sasaki, Eiichi; Kimura, Kenya; Komori, Koji; Shimizu, Yasuhiro; Yatabe, Yasushi; Aoki, Masahiro

2018-06-05

HNRNPLL (heterogeneous nuclear ribonucleoprotein L-like), an RNA-binding protein that regulates alternative splicing of pre-mRNAs, has been shown to regulate differentiation of lymphocytes, as well as metastasis of colorectal cancer cells. Here we show that HNRNPLL promotes cell cycle progression and hence proliferation of colorectal cancer cells. Functional annotation analysis of those genes whose expression levels were changed by three-fold or more in RNA sequencing analysis between SW480 cells overexpressing HNRNPLL and those knocked down for HNRNPLL revealed enrichment of DNA replication-related genes by HNRNPLL overexpression. Among 13 genes detected in the DNA replication pathway, PCNA, RFC3, and FEN1 showed reproducible upregulation by HNRNPLL overexpression both at mRNA and protein levels in SW480 and HT29 cells. Importantly, knockdown of any of these genes alone suppressed the proliferation promoting effect induced by HNRNPLL overexpression. RNA-immunoprecipitation assay presented a binding of FLAG-tagged HNRNPLL to mRNA of these genes, and HNRNPLL overexpression significantly suppressed the downregulation of these genes during 12 hours of actinomycin D treatment, suggesting a role of HNRNPLL in mRNA stability. Finally, analysis of a public RNA sequencing dataset of clinical samples suggested a link between overexpression of HNRNPLL and that of PCNA, RFC3, and FEN1. This link was further supported by immunohistochemistry of colorectal cancer clinical samples, whereas expression of CDKN1A, which is known to inhibit the cooperative function of PCNA, RFC3, and FEN1, was negatively associated with HNRNPLL expression. These results indicate that HNRNPLL stabilizes mRNAs encoding regulators of DNA replication and promotes colorectal cancer cell proliferation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

PuF, an antimetastatic and developmental signaling protein, interacts with the Alzheimer’s amyloid-β precursor protein via a tissue-specific proximal regulatory element (PRE

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Lahiri Debomoy K

2013-01-01

Full Text Available Abstract Background Alzheimer’s disease (AD is intimately tied to amyloid-β (Aβ peptide. Extraneuronal brain plaques consisting primarily of Aβ aggregates are a hallmark of AD. Intraneuronal Aβ subunits are strongly implicated in disease progression. Protein sequence mutations of the Aβ precursor protein (APP account for a small proportion of AD cases, suggesting that regulation of the associated gene (APP may play a more important role in AD etiology. The APP promoter possesses a novel 30 nucleotide sequence, or “proximal regulatory element” (PRE, at −76/−47, from the +1 transcription start site that confers cell type specificity. This PRE contains sequences that make it vulnerable to epigenetic modification and may present a viable target for drug studies. We examined PRE-nuclear protein interaction by gel electrophoretic mobility shift assay (EMSA and PRE mutant EMSA. This was followed by functional studies of PRE mutant/reporter gene fusion clones. Results EMSA probed with the PRE showed DNA-protein interaction in multiple nuclear extracts and in human brain tissue nuclear extract in a tissue-type specific manner. We identified transcription factors that are likely to bind the PRE, using competition gel shift and gel supershift: Activator protein 2 (AP2, nm23 nucleoside diphosphate kinase/metastatic inhibitory protein (PuF, and specificity protein 1 (SP1. These sites crossed a known single nucleotide polymorphism (SNP. EMSA with PRE mutants and promoter/reporter clone transfection analysis further implicated PuF in cells and extracts. Functional assays of mutant/reporter clone transfections were evaluated by ELISA of reporter protein levels. EMSA and ELISA results correlated by meta-analysis. Conclusions We propose that PuF may regulate the APP gene promoter and that AD risk may be increased by interference with PuF regulation at the PRE. PuF is targeted by calcium/calmodulin-dependent protein kinase II inhibitor 1, which also

Differential trypanosome surface coat regulation by a CCCH protein that co-associates with procyclin mRNA cis-elements.

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Pegine Walrad

2009-02-01

Full Text Available The genome of Trypanosoma brucei is unusual in being regulated almost entirely at the post-transcriptional level. In terms of regulation, the best-studied genes are procyclins, which encode a family of major surface GPI-anchored glycoproteins (EP1, EP2, EP3, GPEET that show differential expression in the parasite's tsetse-fly vector. Although procyclin mRNA cis-regulatory sequences have provided the paradigm for post-transcriptional control in kinetoplastid parasites, trans-acting regulators of procyclin mRNAs are unidentified, despite intensive effort over 15 years. Here we identify the developmental regulator, TbZFP3, a CCCH-class predicted RNA binding protein, as an isoform-specific regulator of Procyclin surface coat expression in trypanosomes. We demonstrate (i that endogenous TbZFP3 shows sequence-specific co-precipitation of EP1 and GPEET, but not EP2 and EP3, procyclin mRNA isoforms, (ii that ectopic overexpression of TbZFP3 does not perturb the mRNA abundance of procyclin transcripts, but rather that (iii their protein expression is regulated in an isoform-specific manner, as evidenced by mass spectrometric analysis of the Procyclin expression signature in the transgenic cell lines. The TbZFP3 mRNA-protein complex (TbZFP3mRNP is identified as a trans-regulator of differential surface protein expression in trypanosomes. Moreover, its sequence-specific interactions with procyclin mRNAs are compatible with long-established predictions for Procyclin regulation. Combined with the known association of TbZFP3 with the translational apparatus, this study provides a long-sought missing link between surface protein cis-regulatory signals and the gene expression machinery in trypanosomes.

Expression of cytochrome P450 regulators in cynomolgus macaque.

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Uno, Yasuhiro; Yamazaki, Hiroshi

2017-09-11

1. Cytochrome P450 (P450) regulators including nuclear receptors and transcription factors have not been fully investigated in cynomolgus macaques, an important species used in drug metabolism studies. In this study, we analyzed 17 P450 regulators by sequence and phylogenetic analysis, and tissue expression. 2. Gene and genome structures of 17 P450 regulators were similar to the human orthologs, and the deduced amino acid sequences showed high sequence identities (92-95%) and more closely clustered in a phylogenetic tree, with the human orthologs. 3. Many of the P450 regulator mRNAs were preferentially expressed in the liver, kidney, and/or jejunum. Among the P450 regulator mRNAs, PXR was most abundant in the liver and jejunum, and HNF4α in the kidney. In the liver, the expression of most P450 regulator mRNAs did not show significant differential expression (>2.5-fold) between cynomolgus macaques bred in Cambodia, China, and Indonesia, or rhesus macaques. 4. By correlation analysis, most of the P450 regulators were significantly (p < 0.05) correlated to other P450 regulators, and many of them were also significantly (p < 0.05) correlated with P450s. 5. These results suggest that 17 P450 regulators of cynomolgus macaques had similar molecular characteristics to the human orthologs.

Circadian control of mRNA polyadenylation dynamics regulates rhythmic protein expression

OpenAIRE

Kojima, Shihoko; Sher-Chen, Elaine L.; Green, Carla B.

2012-01-01

Green and colleagues perform a global analysis of circadian-controlled poly(A) tails and identify hundreds of mRNAs that display dynamic rhythmic polyadenylation states. They identify three distinct classes of mRNAs with rhythmic poly(A) tails. Interestingly, class III mRNAs are controlled not by transcription, but by rhythmic cytoplasmic polyadenylation, and are regulated by the components of the cytoplasmic polyadenylation machinery, CPEB2 in particular, which are themselves rhythmically ex...

Roles of viral and cellular proteins in the expression of alternatively spliced HTLV-1 pX mRNAs

International Nuclear Information System (INIS)

Princler, Gerald L.; Julias, John G.; Hughes, Stephen H.; Derse, David

2003-01-01

The human T cell leukemia virus type 1 (HTLV-1) genome contains a cluster of at least five open reading frames (ORFs) near the 3' terminus within the pX region. The pX ORFs are encoded by mono- or bicistronic mRNAs that are generated by alternative splicing. The various pX mRNAs result from skipping of the internal exon (2-exon versus 3-exon isofoms) or from the utilization of alternative splice acceptor sites in the terminal exon. The Rex and Tax proteins, encoded by ORFs X-III and X-IV, have been studied intensively and are encoded by the most abundant of the alternative 3-exon mRNAs. The protein products of the other pX ORFs have not been detected in HTLV-1-infected cell lines and the levels of the corresponding mRNAs have not been accurately established. We have used real-time RT-PCR with splice-site specific primers to accurately measure the levels of individual pX mRNA species in chronically infected T cell lines. We have asked whether virus regulatory proteins or ectopic expression of cellular factors influence pX mRNA splicing in cells that were transfected with HTLV-1 provirus clones. In chronically infected cell lines, the pX-tax/rex mRNA was present at 500- to 2500-fold higher levels than the pX-tax-orfII mRNA and at approximately 1000-fold higher levels than pX-rex-orfI mRNA. Chronically infected cell lines that contain numerous defective proviruses expressed 2-exon forms of pX mRNAs at significantly higher levels compared to cell lines that contain a single full-length provirus. Cells transfected with provirus expression plasmids expressed similar relative amounts of 3-exon pX mRNAs but lower levels of 2-exon mRNA forms compared to cells containing a single, full-length provirus. The pX mRNA expression patterns were nearly identical in cells transfected with wild-type, Tax-minus, or Rex-minus proviruses. Cotransfection of cells with HTLV-1 provirus in combination with SF2/ASF expression plasmid resulted in a relative increase in pX-tax/rex m

Genes involved in the astrocyte-neuron lactate shuttle (ANLS) are specifically regulated in cortical astrocytes following sleep deprivation in mice.

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Petit, Jean-Marie; Gyger, Joël; Burlet-Godinot, Sophie; Fiumelli, Hubert; Martin, Jean-Luc; Magistretti, Pierre J

2013-10-01

There is growing evidence indicating that in order to meet the neuronal energy demands, astrocytes provide lactate as an energy substrate for neurons through a mechanism called "astrocyte-neuron lactate shuttle" (ANLS). Since neuronal activity changes dramatically during vigilance states, we hypothesized that the ANLS may be regulated during the sleep-wake cycle. To test this hypothesis we investigated the expression of genes associated with the ANLS specifically in astrocytes following sleep deprivation. Astrocytes were purified by fluorescence-activated cell sorting from transgenic mice expressing the green fluorescent protein (GFP) under the control of the human astrocytic GFAP-promoter. 6-hour instrumental sleep deprivation (TSD). Animal sleep research laboratory. Young (P23-P27) FVB/N-Tg (GFAP-GFP) 14Mes/J (Tg) mice of both sexes and 7-8 week male Tg and FVB/Nj mice. Basal sleep recordings and sleep deprivation achieved using a modified cage where animals were gently forced to move. Since Tg and FVB/Nj mice displayed a similar sleep-wake pattern, we performed a TSD in young Tg mice. Total RNA was extracted from the GFP-positive and GFP-negative cells sorted from cerebral cortex. Quantitative RT-PCR analysis showed that levels of Glut1, α-2-Na/K pump, Glt1, and Ldha mRNAs were significantly increased following TSD in GFP-positive cells. In GFP-negative cells, a tendency to increase, although not significant, was observed for Ldha, Mct2, and α-3-Na/K pump mRNAs. This study shows that TSD induces the expression of genes associated with ANLS specifically in astrocytes, underlying the important role of astrocytes in the maintenance of the neuro-metabolic coupling across the sleep-wake cycle.

Evaluation of bias associated with high-multiplex, target-specific pre-amplification

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Steven T. Okino

2016-01-01

Full Text Available We developed a novel PCR-based pre-amplification (PreAmp technology that can increase the abundance of over 350 target genes one million-fold. To assess potential bias introduced by PreAmp we utilized ERCC RNA reference standards, a model system that quantifies measurement error in RNA analysis. We assessed three types of bias: amplification bias, dynamic range bias and fold-change bias. We show that our PreAmp workflow introduces only minimal amplification and fold-change bias under stringent conditions. We do detect dynamic range bias if a target gene is highly abundant and PreAmp occurred for 16 or more PCR cycles; however, this type of bias is easily correctable. To assess PreAmp bias in a gene expression profiling experiment, we analyzed a panel of genes that are regulated during differentiation using the NTera2 stem cell model system. We find that results generated using PreAmp are similar to results obtained using standard qPCR (without the pre-amplification step. Importantly, PreAmp maintains patterns of gene expression changes across samples; the same biological insights would be derived from a PreAmp experiment as with a standard gene expression profiling experiment. We conclude that our PreAmp technology can facilitate analysis of extremely limited samples in gene expression quantification experiments.

Development of an in situ hybridization assay for the detection of ostreid herpesvirus type 1 mRNAs in the Pacific oyster, Crassostrea gigas.

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Corbeil, Serge; Faury, Nicole; Segarra, Amélie; Renault, Tristan

2015-01-01

An in situ hybridization protocol for detecting mRNAs of ostreid herpesvirus type 1 (OsHV-1) which infects Pacific oysters, Crassostrea gigas, was developed. Three RNA probes were synthesized by cloning three partial OsHV-1 genes into plasmids using three specific primer pairs, and performing a transcription in the presence of digoxigenin dUTP. The RNA probes were able to detect the virus mRNAs in paraffin sections of experimentally infected oysters 26 h post-injection. The in situ hybridization showed that the OsHV-1 mRNAs were mainly present in connective tissues in gills, mantle, adductor muscle, digestive gland and gonads. DNA detection by in situ hybridization using a DNA probe and viral DNA quantitation by real-time PCR were also performed and results were compared with those obtained using RNA probes. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterization and distribution of GHRH, PACAP, TRH, SST and IGF1 mRNAs in the green iguana.

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Ávila-Mendoza, José; Pérez-Rueda, Ernesto; Urban-Sosa, Valeria; Carranza, Martha; Martínez-Moreno, Carlos G; Luna, Maricela; Arámburo, Carlos

2018-01-01

The somatotropic axis (SA) regulates numerous aspects of vertebrate physiology such as development, growth, and metabolism and has influence on several tissues including neural, immune, reproductive and gastric tract. Growth hormone (GH) is a key component of SA, it is synthesized and released mainly by pituitary somatotrophs, although now it is known that virtually all tissues can express GH, which, in addition to its well-described endocrine roles, also has autocrine/paracrine/intracrine actions. In the pituitary, GH expression is regulated by several hypothalamic neuropeptides including GHRH, PACAP, TRH and SST. GH, in turn, regulates IGF1 synthesis in several target tissues, adding complexity to the system since GH effects can be exerted either directly or mediated by IGF1. In reptiles, little is known about the SA components and their functional interactions. The aim of this work was to characterize the mRNAs of the principal SA components in the green iguana and to develop the tools that allow the study of the structural and functional evolution of this system in reptiles. By employing RT-PCR and RACE, the cDNAs encoding for GHRH, PACAP, TRH, SST and IGF1 were amplified and sequenced. Results showed that these cDNAs coded for the corresponding protein precursors of 154, 170, 243, 113, and 131 amino acids, respectively. Of these, GHRH, PACAP, SST and IGF1 precursors exhibited a high structural conservation with respect to its counterparts in other vertebrates. On the other hand, iguana's TRH precursor showed 7 functional copies of mature TRH (pyr-QHP-NH 2 ), as compared to 4 and 6 copies of TRH in avian and mammalian proTRH sequences, respectively. It was found that in addition to its primary production site (brain for GHRH, PACAP, TRH and SST, and liver for IGF1), they were also expressed in other peripheral tissues, i.e. testes and ovaries expressed all the studied mRNAs, whereas TRH and IGF1 mRNAs were observed ubiquitously in all tissues considered. These

Intrinsic noise of microRNA-regulated genes and the ceRNA hypothesis.

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Javad Noorbakhsh

Full Text Available MicroRNAs are small noncoding RNAs that regulate genes post-transciptionally by binding and degrading target eukaryotic mRNAs. We use a quantitative model to study gene regulation by inhibitory microRNAs and compare it to gene regulation by prokaryotic small non-coding RNAs (sRNAs. Our model uses a combination of analytic techniques as well as computational simulations to calculate the mean-expression and noise profiles of genes regulated by both microRNAs and sRNAs. We find that despite very different molecular machinery and modes of action (catalytic vs stoichiometric, the mean expression levels and noise profiles of microRNA-regulated genes are almost identical to genes regulated by prokaryotic sRNAs. This behavior is extremely robust and persists across a wide range of biologically relevant parameters. We extend our model to study crosstalk between multiple mRNAs that are regulated by a single microRNA and show that noise is a sensitive measure of microRNA-mediated interaction between mRNAs. We conclude by discussing possible experimental strategies for uncovering the microRNA-mRNA interactions and testing the competing endogenous RNA (ceRNA hypothesis.

Features of CRISPR-Cas Regulation Key to Highly Efficient and Temporally-Specific crRNA Production

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Andjela Rodic

2017-11-01

Full Text Available Bacterial immune systems, such as CRISPR-Cas or restriction-modification (R-M systems, affect bacterial pathogenicity and antibiotic resistance by modulating horizontal gene flow. A model system for CRISPR-Cas regulation, the Type I-E system from Escherichia coli, is silent under standard laboratory conditions and experimentally observing the dynamics of CRISPR-Cas activation is challenging. Two characteristic features of CRISPR-Cas regulation in E. coli are cooperative transcription repression of cas gene and CRISPR array promoters, and fast non-specific degradation of full length CRISPR transcripts (pre-crRNA. In this work, we use computational modeling to understand how these features affect the system expression dynamics. Signaling which leads to CRISPR-Cas activation is currently unknown, so to bypass this step, we here propose a conceptual setup for cas expression activation, where cas genes are put under transcription control typical for a restriction-modification (R-M system and then introduced into a cell. Known transcription regulation of an R-M system is used as a proxy for currently unknown CRISPR-Cas transcription control, as both systems are characterized by high cooperativity, which is likely related to similar dynamical constraints of their function. We find that the two characteristic CRISPR-Cas control features are responsible for its temporally-specific dynamical response, so that the system makes a steep (switch-like transition from OFF to ON state with a time-delay controlled by pre-crRNA degradation rate. We furthermore find that cooperative transcription regulation qualitatively leads to a cross-over to a regime where, at higher pre-crRNA processing rates, crRNA generation approaches the limit of an infinitely abrupt system induction. We propose that these dynamical properties are associated with rapid expression of CRISPR-Cas components and efficient protection of bacterial cells against foreign DNA. In terms of synthetic

Expression of Rac1 alternative 3' UTRs is a cell specific mechanism with a function in dendrite outgrowth in cortical neurons.

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Braz, Sandra Oliveira; Cruz, Andrea; Lobo, Andrea; Bravo, Joana; Moreira-Ribeiro, Joana; Pereira-Castro, Isabel; Freitas, Jaime; Relvas, Joao B; Summavielle, Teresa; Moreira, Alexandra

2017-06-01

The differential expression of mRNAs containing tandem alternative 3' UTRs, achieved by mechanisms of alternative polyadenylation and post-transcriptional regulation, has been correlated with a variety of cellular states. In differentiated cells and brain tissues there is a general use of distal polyadenylation signals, originating mRNAs with longer 3' UTRs, in contrast with proliferating cells and other tissues such as testis, where most mRNAs contain shorter 3' UTRs. Although cell type and state are relevant in many biological processes, how these mechanisms occur in specific brain cell types is still poorly understood. Rac1 is a member of the Rho family of small GTPases with essential roles in multiple cellular processes, including cell differentiation and axonal growth. Here we used different brain cell types and tissues, including oligodendrocytes, microglia, astrocytes, cortical and hippocampal neurons, and optical nerve, to show that classical Rho GTPases express mRNAs with alternative 3' UTRs differently, by gene- and cell- specific mechanisms. In particular, we show that Rac1 originate mRNA isoforms with longer 3' UTRs specifically during neurite growth of cortical, but not hippocampal neurons. Furthermore, we demonstrate that the longest Rac1 3' UTR is necessary for driving the mRNA to the neurites, and also for neurite outgrowth in cortical neurons. Our results indicate that the expression of Rac1 longer 3' UTR is a gene and cell-type specific mechanism in the brain, with a new physiological function in cortical neuron differentiation. Copyright © 2017 Elsevier B.V. All rights reserved.

Opposite responses of rabbit and human globin mRNAs to translational inhibition by cap analogues

International Nuclear Information System (INIS)

Shakin, S.H.; Liebhaber, S.A.

1987-01-01

The translational efficiency of an mRNA may be determined at the step of translational initiation by the efficiency of its interaction with the cap binding protein complex. To further investigate the role of these interactions in translational control, the authors compare in vitro the relative sensitivities of rabbit and human α- and β-globin mRNAs to translational inhibition by cap analogues. They find that rabbit β-globin mRNA is more resistant to translational inhibition by cap analogues than rabbit α-globin mRNA, while in contrast, human β-globin mRNA is more sensitive to cap analogue inhibition than human α- and β-globin mRNAs is unexpected as direct in vivo and in vitro comparisons of polysome profiles reveal parallel translational handling of the α- and β-globin mRNAs from these two species. This discordance between the relative translational sensitivities of these mRNAs to cap analogues and their relative ribosome loading activities suggests that cap-dependent events may not be rate limiting in steady-state globin translation

Single-molecule imaging of {beta}-actin mRNAs in the cytoplasm of a living cell

Energy Technology Data Exchange (ETDEWEB)

Yamagishi, Mai; Ishihama, Yo [Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033 (Japan); Shirasaki, Yoshitaka [Laboratory of Genome Technology, Department of Human Genome Research, Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818 (Japan); Kurama, Hideki [Major in Integrative Bioscience and Biomedical Engineering, Graduate School of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 169-8555 (Japan); Funatsu, Takashi, E-mail: funatsu@mail.ecc.u-tokyo.ac.jp [Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033 (Japan); Center for NanoBio Integration, The University of Tokyo, Bunkyo-ku, Tokyo 113-8656 (Japan)

2009-04-15

{beta}-Actin mRNA labeled with an MS2-EGFP fusion protein was expressed in chicken embryo fibroblasts and its localization and movement were analyzed by single-molecule imaging. Most {beta}-Actin mRNAs localized to the leading edge, while some others were observed in the perinuclear region. Singe-molecule tracking of individual mRNAs revealed that the majority of mRNAs were in unrestricted Brownian motion at the leading edge and in restricted Brownian motion in the perinuclear region. The macroscopic diffusion coefficient of mRNA (D{sub MACRO}) at the leading edge was 0.3 {mu}m{sup 2}/s. On the other hand, D{sub MACRO} in the perinuclear region was 0.02 {mu}m{sup 2}/s. The destruction of microfilaments with cytochalasin D, which is known to delocalize {beta}-actin mRNAs, led to an increase in D{sub MACRO} to 0.2 {mu}m{sup 2}/s in the perinuclear region. These results suggest that the microstructure, composed of microfilaments, serves as a barrier for the movement of {beta}-actin mRNA.

The Crc global regulator inhibits the Pseudomonas putida pWW0 toluene/xylene assimilation pathway by repressing the translation of regulatory and structural genes.

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Moreno, Renata; Fonseca, Pilar; Rojo, Fernando

2010-08-06

In Pseudomonas putida, the expression of the pWW0 plasmid genes for the toluene/xylene assimilation pathway (the TOL pathway) is subject to complex regulation in response to environmental and physiological signals. This includes strong inhibition via catabolite repression, elicited by the carbon sources that the cells prefer to hydrocarbons. The Crc protein, a global regulator that controls carbon flow in pseudomonads, has an important role in this inhibition. Crc is a translational repressor that regulates the TOL genes, but how it does this has remained unknown. This study reports that Crc binds to sites located at the translation initiation regions of the mRNAs coding for XylR and XylS, two specific transcription activators of the TOL genes. Unexpectedly, eight additional Crc binding sites were found overlapping the translation initiation sites of genes coding for several enzymes of the pathway, all encoded within two polycistronic mRNAs. Evidence is provided supporting the idea that these sites are functional. This implies that Crc can differentially modulate the expression of particular genes within polycistronic mRNAs. It is proposed that Crc controls TOL genes in two ways. First, Crc inhibits the translation of the XylR and XylS regulators, thereby reducing the transcription of all TOL pathway genes. Second, Crc inhibits the translation of specific structural genes of the pathway, acting mainly on proteins involved in the first steps of toluene assimilation. This ensures a rapid inhibitory response that reduces the expression of the toluene/xylene degradation proteins when preferred carbon sources become available.

Resveratrol, by modulating RNA processing factor levels, can influence the alternative splicing of pre-mRNAs.

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Markus, M Andrea; Marques, Francine Z; Morris, Brian J

2011-01-01

Alternative pre-mRNA splicing defects can contribute to, or result from, various diseases, including cancer. Aberrant mRNAs, splicing factors and other RNA processing factors have therefore become targets for new therapeutic interventions. Here we report that the natural polyphenol resveratrol can modulate alternative splicing in a target-specific manner. We transfected minigenes of several alternatively spliceable primary mRNAs into HEK293 cells in the presence or absence of 1, 5, 20 and 50 µM resveratrol and measured exon levels by semi-quantitative PCR after separation by agarose gel electrophoresis. We found that 20 µg/ml and 50 µg/ml of resveratrol affected exon inclusion of SRp20 and SMN2 pre-mRNAs, but not CD44v5 or tau pre-mRNAs. By Western blotting and immunofluorescence we showed that this effect may be due to the ability of resveratrol to change the protein level but not the localization of several RNA processing factors. The processing factors that increased significantly were ASF/SF2, hnRNPA1 and HuR, but resveratrol did not change the levels of RBM4, PTBP1 and U2AF35. By means of siRNA-mediated knockdown we depleted cells of SIRT1, regarded as a major target of resveratrol, and showed that the effect on splicing was not dependent on SIRT1. Our results suggest that resveratrol might be an attractive small molecule to treat diseases in which aberrant splicing has been implicated, and justify more extensive research on the effects of resveratrol on the splicing machinery.

Regulation of Flavonoid Biosynthetic Genes in Germinating Arabidopsis Seedlings.

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Kubasek, WL; Shirley, BW; McKillop, A; Goodman, HM; Briggs, W; Ausubel, FM

1992-01-01

Many higher plants, including Arabidopsis, transiently display purple anthocyanin pigments just after seed germination. We observed that steady state levels of mRNAs encoded by four flavonoid biosynthetic genes, PAL1 (encoding phenylalanine ammonia-lyase 1), CHS (encoding chalcone synthase), CHI (encoding chalcone isomerase), and DFR (encoding dihydroflavonol reductase), were temporally regulated, peaking in 3-day-old seedlings grown in continuous white light. Except for the case of PAL1 mRNA, mRNA levels for these flavonoid genes were very low in seedlings grown in darkness. Light induction studies using seedlings grown in darkness showed that PAL1 mRNA began to accumulate before CHS and CHI mRNAs, which, in turn, began to accumulate before DFR mRNA. This order of induction is the same as the order of the biosynthetic steps in flavonoid biosynthesis. Our results suggest that the flavonoid biosynthetic pathway is coordinately regulated by a developmental timing mechanism during germination. Blue light and UVB light induction experiments using red light- and dark-grown seedlings showed that the flavonoid biosynthetic genes are induced most effectively by UVB light and that blue light induction is mediated by a specific blue light receptor. PMID:12297632

Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein.

Science.gov (United States)

Chen, Linan; Dumelie, Jason G; Li, Xiao; Cheng, Matthew Hk; Yang, Zhiyong; Laver, John D; Siddiqui, Najeeb U; Westwood, J Timothy; Morris, Quaid; Lipshitz, Howard D; Smibert, Craig A

2014-01-07

Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown. To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug's target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes. Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism.

Intracellular distribution of histone mRNAs in human fibroblasts studied by in situ hybridization

International Nuclear Information System (INIS)

Lawrence, J.B.; Singer, R.H.; Villnave, C.A.; Stein, J.L.; Stein, G.S.

1988-01-01

We have used in situ hybridization to study the intracellular distribution of mRNAs for cell cycle-dependent core and H1 histone proteins in human WI-38 fibroblasts. Because histones are abundant nuclear proteins and histone mRNA expression is tightly coupled to DNA synthesis, it was of interest to determine whether histone mRNAs are localized near the nucleus. Cells were hybridized with tritiated DNA probes specific for either histone H1, histone H4, actin, or poly(A)+ mRNA and were processed for autoradiography. In exponentially growing cultures, the fraction of histone mRNA-positive cells correlated well with the fraction of cells in S phase and was eliminated by hydroxyurea inhibition of DNA synthesis. Within individual cells the label for histone mRNA was widely distributed throughout the cytoplasm and did not appear to be more heavily concentrated near the nucleus. However, histone mRNA appeared to exhibit patchy, nonhomogeneous localization, and a quantitative evaluation confirmed that grain distributions were not as uniform as they were after hybridizations to poly(A)+ mRNA. Actin mRNA in WI-38 cells was also widely distributed throughout the cytoplasm but differed from histone mRNA in that label for actin mRNA was frequently most dense at the outermost region of narrow cell extensions. The localization of actin mRNA was less pronounced but qualitatively very similar to that previously described for chicken embryonic myoblasts and fibroblasts. We conclude that localization of histones in WI-38 cells is not facilitated by localization of histone protein synthesis near the nucleus and that there are subtle but discrete and potentially functional differences in the distributions of histone, actin, and poly(A)+ mRNAs

Improved patient specific seizure detection during pre-surgical evaluation.

LENUS (Irish Health Repository)

Chua, Eric C-P

2011-04-01

There is considerable interest in improved off-line automated seizure detection methods that will decrease the workload of EEG monitoring units. Subject-specific approaches have been demonstrated to perform better than subject-independent ones. However, for pre-surgical diagnostics, the traditional method of obtaining a priori data to train subject-specific classifiers is not practical. We present an alternative method that works by adapting the threshold of a subject-independent to a specific subject based on feedback from the user.

An innovative pre-targeting strategy for tumor cell specific imaging and therapy.

Science.gov (United States)

Qin, Si-Yong; Peng, Meng-Yun; Rong, Lei; Jia, Hui-Zhen; Chen, Si; Cheng, Si-Xue; Feng, Jun; Zhang, Xian-Zheng

2015-09-21

A programmed pre-targeting system for tumor cell imaging and targeting therapy was established based on the "biotin-avidin" interaction. In this programmed functional system, transferrin-biotin can be actively captured by tumor cells with the overexpression of transferrin receptors, thus achieving the pre-targeting modality. Depending upon avidin-biotin recognition, the attachment of multivalent FITC-avidin to biotinylated tumor cells not only offered the rapid fluorescence labelling, but also endowed the pre-targeted cells with targeting sites for the specifically designed biotinylated peptide nano-drug. Owing to the successful pre-targeting, tumorous HepG2 and HeLa cells were effectively distinguished from the normal 3T3 cells via fluorescence imaging. In addition, the self-assembled peptide nano-drug resulted in enhanced cell apoptosis in the observed HepG2 cells. The tumor cell specific pre-targeting strategy is applicable for a variety of different imaging and therapeutic agents for tumor treatments.

Antagonism of microRNA-122 in mice by systemically administered LNA-antimiR leads to up-regulation of a large set of predicted target mRNAs in the liver

DEFF Research Database (Denmark)

Elmen, Joachim; Lindow, Morten; Silahtaroglu, Asli

2008-01-01

’end of miR-122 leads to specific, dose-dependent silencing of miR-122 and shows no hepatotoxicity in mice. Antagonism of miR-122 is due to formation of stable heteroduplexes between the LNA-antimiR and miR-122 as detected by northern analysis. Fluorescence in situ hybridization demonstrated uptake...... of the LNA-antimiR in mouse liver cells, which was accompanied by markedly reduced hybridization signals for mature miR-122 in treated mice. Functional antagonism of miR-122 was inferred from a low cholesterol phenotype and derepression within 24 h of 199 liver mRNAs showing significant enrichment for mi...

The translation initiation factor eIF4E regulates the sex-specific expression of the master switch gene Sxl in Drosophila melanogaster.

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Patricia L Graham

2011-07-01

Full Text Available In female fruit flies, Sex-lethal (Sxl turns off the X chromosome dosage compensation system by a mechanism involving a combination of alternative splicing and translational repression of the male specific lethal-2 (msl-2 mRNA. A genetic screen identified the translation initiation factor eif4e as a gene that acts together with Sxl to repress expression of the Msl-2 protein. However, eif4e is not required for Sxl mediated repression of msl-2 mRNA translation. Instead, eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs. Like other factors required for Sxl regulation of splicing, eif4e shows maternal-effect female-lethal interactions with Sxl. This female lethality can be enhanced by mutations in other co-factors that promote female-specific splicing and is caused by a failure to properly activate the Sxl-positive autoregulatory feedback loop in early embryos. In this feedback loop Sxl proteins promote their own synthesis by directing the female-specific alternative splicing of Sxl-Pm pre-mRNAs. Analysis of pre-mRNA splicing when eif4e activity is compromised demonstrates that Sxl-dependent female-specific splicing of both Sxl-Pm and msl-2 pre-mRNAs requires eif4e activity. Consistent with a direct involvement in Sxl-dependent alternative splicing, eIF4E is associated with unspliced Sxl-Pm pre-mRNAs and is found in complexes that contain early acting splicing factors--the U1/U2 snRNP protein Sans-fils (Snf, the U1 snRNP protein U1-70k, U2AF38, U2AF50, and the Wilms' Tumor 1 Associated Protein Fl(2d--that have been directly implicated in Sxl splicing regulation.

Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis

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Keisuke Nakajima

2015-01-01

Full Text Available Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3′UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3′UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf. In contrast, TALEN mRNAs without this 3′UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

Expression Pattern of Myogenic Regulatory Transcription Factor mRNAs in the Embryo and Adult Labeo rohita (Hamilton, 1822

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Archya Sengupta

2014-01-01

Full Text Available Understanding the regulation of skeletal muscle development is important to meet the increasing demand of Indian major carp Labeo rohita. Myogenic regulatory factors (MRFs along with myocyte specific enhancer factor 2 (MEF2 play the pivotal role in the determination and differentiation of skeletal muscle. The majority of skeletal muscle genes require both MRFs and MEF2 family members to activate their transcription. In this study, the expression pattern of MyoD, myf-5, myogenin, and MEF2A was observed from 6 h after fertilization to 12 months of age using semiquantitative RT-PCR as well as real-time PCR method. MyoD and myf-5 mRNAs were expressed at high level at the early embryonic stages. Myogenin and MEF2A were expressed after MyoD and myf-5 and remained active up to adult stage. Expression of MyoD was lower than that of Myf-5 after the 5th month. Partial sequencing of MyoD, myf-5, and MEF2A was done to draw phylogeny. In phylogenetic study, Labeo MyoD, MEF2A and myf-5 were found to be closely related to those of common carp. The present investigation suggests that the four transcription factors play pivotal role in the regulation of muscle growth of Labeo rohita in an overlapping and interconnected way.

Regulation of protein synthesis during sea urchin early development

International Nuclear Information System (INIS)

Kelso, L.C.

1989-01-01

Fertilization of the sea urchin egg results in a 20-40 fold increase in the rate of protein synthesis. The masked message hypothesis proposes that mRNAs are masked or unavailable for translation in the egg. We devised an in vivo assay to test this hypothesis. Our results show that masked mRNAs limit protein synthesis in the unfertilized egg. In addition, we show that protein synthesis is also regulated at the level of translational machinery. Following fertilization is a period of rapid cell divisions. This period, known as the rapid cleavage stage, is characterized by the transient synthesis of a novel set of proteins. The synthesis of these proteins is programmed by maternal mRNAs stored in the unfertilized egg. To study the behavior of these mRNAs, we prepared a cDNA library from polysomal poly (A+) RNA from 2-hour embryos. [ 32 P] labeled probes, prepared from the cDNA library, were used to monitor the levels of individual mRNAs in polysomes at fertilization and during early development

Active pre-filters for dc/dc Boost regulators

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Carlos Andrés Ramos-Paja

2014-05-01

Full Text Available This paper proposes an active pre-filter to mitigate the current harmonics generated by classical dc/dc Boost regulators, which generate current ripples proportional to the duty cycle. Therefore, high output voltage conditions, i.e., high voltage conversion ratios, produce high current harmonics that must be filtered to avoid damage or source losses. Traditionally, these current components are filtered using electrolytic capacitors, which introduce reliability problems because of their high failure rate. The solution introduced in this paper instead uses a dc/dc converter based on the parallel connection of the Boost canonical cells to filter the current ripples generated by the Boost regulator, improving the system reliability. This solution provides the additional benefits of improving the overall efficiency and the voltage conversion ratio. Finally, the solution is validated with simulations and experimental results.

Predicted RNA Binding Proteins Pes4 and Mip6 Regulate mRNA Levels, Translation, and Localization during Sporulation in Budding Yeast.

Science.gov (United States)

Jin, Liang; Zhang, Kai; Sternglanz, Rolf; Neiman, Aaron M

2017-05-01

In response to starvation, diploid cells of Saccharomyces cerevisiae undergo meiosis and form haploid spores, a process collectively referred to as sporulation. The differentiation into spores requires extensive changes in gene expression. The transcriptional activator Ndt80 is a central regulator of this process, which controls many genes essential for sporulation. Ndt80 induces ∼300 genes coordinately during meiotic prophase, but different mRNAs within the NDT80 regulon are translated at different times during sporulation. The protein kinase Ime2 and RNA binding protein Rim4 are general regulators of meiotic translational delay, but how differential timing of individual transcripts is achieved was not known. This report describes the characterization of two related NDT80 -induced genes, PES4 and MIP6 , encoding predicted RNA binding proteins. These genes are necessary to regulate the steady-state expression, translational timing, and localization of a set of mRNAs that are transcribed by NDT80 but not translated until the end of meiosis II. Mutations in the predicted RNA binding domains within PES4 alter the stability of target mRNAs. PES4 and MIP6 affect only a small portion of the NDT80 regulon, indicating that they act as modulators of the general Ime2/Rim4 pathway for specific transcripts. Copyright © 2017 American Society for Microbiology.

Roles and regulation of Epstein-Barr virus microRNAs

NARCIS (Netherlands)

Hooykaas, M.J.G.

2016-01-01

MicroRNAs are posttranscriptional gene regulators that play important roles in many cellular processes. These short non-coding RNA molecules regulate gene expression by binding to complementary target mRNAs, thereby inducing RNA destabilization and inhibition of translation. Several DNA viruses

Resveratrol, by modulating RNA processing factor levels, can influence the alternative splicing of pre-mRNAs.

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M Andrea Markus

Full Text Available Alternative pre-mRNA splicing defects can contribute to, or result from, various diseases, including cancer. Aberrant mRNAs, splicing factors and other RNA processing factors have therefore become targets for new therapeutic interventions. Here we report that the natural polyphenol resveratrol can modulate alternative splicing in a target-specific manner. We transfected minigenes of several alternatively spliceable primary mRNAs into HEK293 cells in the presence or absence of 1, 5, 20 and 50 µM resveratrol and measured exon levels by semi-quantitative PCR after separation by agarose gel electrophoresis. We found that 20 µg/ml and 50 µg/ml of resveratrol affected exon inclusion of SRp20 and SMN2 pre-mRNAs, but not CD44v5 or tau pre-mRNAs. By Western blotting and immunofluorescence we showed that this effect may be due to the ability of resveratrol to change the protein level but not the localization of several RNA processing factors. The processing factors that increased significantly were ASF/SF2, hnRNPA1 and HuR, but resveratrol did not change the levels of RBM4, PTBP1 and U2AF35. By means of siRNA-mediated knockdown we depleted cells of SIRT1, regarded as a major target of resveratrol, and showed that the effect on splicing was not dependent on SIRT1. Our results suggest that resveratrol might be an attractive small molecule to treat diseases in which aberrant splicing has been implicated, and justify more extensive research on the effects of resveratrol on the splicing machinery.

Endogenous Methanol Regulates Mammalian Gene Activity

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Komarova, Tatiana V.; Petrunia, Igor V.; Shindyapina, Anastasia V.; Silachev, Denis N.; Sheshukova, Ekaterina V.; Kiryanov, Gleb I.; Dorokhov, Yuri L.

2014-01-01

We recently showed that methanol emitted by wounded plants might function as a signaling molecule for plant-to-plant and plant-to-animal communications. In mammals, methanol is considered a poison because the enzyme alcohol dehydrogenase (ADH) converts methanol into toxic formaldehyde. However, the detection of methanol in the blood and exhaled air of healthy volunteers suggests that methanol may be a chemical with specific functions rather than a metabolic waste product. Using a genome-wide analysis of the mouse brain, we demonstrated that an increase in blood methanol concentration led to a change in the accumulation of mRNAs from genes primarily involved in detoxification processes and regulation of the alcohol/aldehyde dehydrogenases gene cluster. To test the role of ADH in the maintenance of low methanol concentration in the plasma, we used the specific ADH inhibitor 4-methylpyrazole (4-MP) and showed that intraperitoneal administration of 4-MP resulted in a significant increase in the plasma methanol, ethanol and formaldehyde concentrations. Removal of the intestine significantly decreased the rate of methanol addition to the plasma and suggested that the gut flora may be involved in the endogenous production of methanol. ADH in the liver was identified as the main enzyme for metabolizing methanol because an increase in the methanol and ethanol contents in the liver homogenate was observed after 4-MP administration into the portal vein. Liver mRNA quantification showed changes in the accumulation of mRNAs from genes involved in cell signalling and detoxification processes. We hypothesized that endogenous methanol acts as a regulator of homeostasis by controlling the mRNA synthesis. PMID:24587296

Endogenous methanol regulates mammalian gene activity.

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Tatiana V Komarova

Full Text Available We recently showed that methanol emitted by wounded plants might function as a signaling molecule for plant-to-plant and plant-to-animal communications. In mammals, methanol is considered a poison because the enzyme alcohol dehydrogenase (ADH converts methanol into toxic formaldehyde. However, the detection of methanol in the blood and exhaled air of healthy volunteers suggests that methanol may be a chemical with specific functions rather than a metabolic waste product. Using a genome-wide analysis of the mouse brain, we demonstrated that an increase in blood methanol concentration led to a change in the accumulation of mRNAs from genes primarily involved in detoxification processes and regulation of the alcohol/aldehyde dehydrogenases gene cluster. To test the role of ADH in the maintenance of low methanol concentration in the plasma, we used the specific ADH inhibitor 4-methylpyrazole (4-MP and showed that intraperitoneal administration of 4-MP resulted in a significant increase in the plasma methanol, ethanol and formaldehyde concentrations. Removal of the intestine significantly decreased the rate of methanol addition to the plasma and suggested that the gut flora may be involved in the endogenous production of methanol. ADH in the liver was identified as the main enzyme for metabolizing methanol because an increase in the methanol and ethanol contents in the liver homogenate was observed after 4-MP administration into the portal vein. Liver mRNA quantification showed changes in the accumulation of mRNAs from genes involved in cell signalling and detoxification processes. We hypothesized that endogenous methanol acts as a regulator of homeostasis by controlling the mRNA synthesis.

Profiling of Long Non-coding RNAs and mRNAs by RNA-Sequencing in the Hippocampi of Adult Mice Following Propofol Sedation.

Science.gov (United States)

Fan, Jun; Zhou, Quan; Li, Yan; Song, Xiuling; Hu, Jijie; Qin, Zaisheng; Tang, Jing; Tao, Tao

2018-01-01

Propofol is a frequently used intravenous anesthetic agent. The impairment caused by propofol on the neural system, especially the hippocampus, has been widely reported. However, the molecular mechanism underlying the effects of propofol on learning and memory functions in the hippocampus is still unclear. In the present study we performed lncRNA and mRNA analysis in the hippocampi of adult mice, after propofol sedation, through RNA-Sequencing (RNA-Seq). A total of 146 differentially expressed lncRNAs and 1103 mRNAs were identified. Bioinformatics analysis, including gene ontology (GO) analysis, pathway analysis and network analysis, were done for the identified dysregulated genes. Pathway analysis indicated that the FoxO signaling pathway played an important role in the effects of propofol on the hippocampus. Finally, four lncRNAs and three proteins were selected from the FoxO-related network for further validation. The up-regulation of lncE230001N04Rik and the down-regulation of lncRP23-430H21.1 and lncB230206L02Rik showed the same fold change tendencies but changes in Gm26532 were not statistically significant in the RNA-Seq results, following propofol sedation. The FoxO pathway-related proteins, PI3K and AKT, are up-regulated in propofol-exposed group. FoxO3a is down-regulated at both mRNA and protein levels. Our study reveals that propofol sedation can influence the expression of lncRNAs and mRNAs in the hippocampus, and bioinformatics analysis have identified key biological processes and pathways associated with propofol sedation. Cumulatively, our results provide a framework for further study on the role of lncRNAs in propofol-induced or -related neurotoxicity, particularly with regards to hippocampus-related dysfunction.

Profiling of Long Non-coding RNAs and mRNAs by RNA-Sequencing in the Hippocampi of Adult Mice Following Propofol Sedation

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Jun Fan

2018-03-01

Full Text Available Propofol is a frequently used intravenous anesthetic agent. The impairment caused by propofol on the neural system, especially the hippocampus, has been widely reported. However, the molecular mechanism underlying the effects of propofol on learning and memory functions in the hippocampus is still unclear. In the present study we performed lncRNA and mRNA analysis in the hippocampi of adult mice, after propofol sedation, through RNA-Sequencing (RNA-Seq. A total of 146 differentially expressed lncRNAs and 1103 mRNAs were identified. Bioinformatics analysis, including gene ontology (GO analysis, pathway analysis and network analysis, were done for the identified dysregulated genes. Pathway analysis indicated that the FoxO signaling pathway played an important role in the effects of propofol on the hippocampus. Finally, four lncRNAs and three proteins were selected from the FoxO-related network for further validation. The up-regulation of lncE230001N04Rik and the down-regulation of lncRP23-430H21.1 and lncB230206L02Rik showed the same fold change tendencies but changes in Gm26532 were not statistically significant in the RNA-Seq results, following propofol sedation. The FoxO pathway-related proteins, PI3K and AKT, are up-regulated in propofol-exposed group. FoxO3a is down-regulated at both mRNA and protein levels. Our study reveals that propofol sedation can influence the expression of lncRNAs and mRNAs in the hippocampus, and bioinformatics analysis have identified key biological processes and pathways associated with propofol sedation. Cumulatively, our results provide a framework for further study on the role of lncRNAs in propofol-induced or -related neurotoxicity, particularly with regards to hippocampus-related dysfunction.

Testosterone stimulates progesterone production and STAR, P450 cholesterol side-chain cleavage and LH receptor mRNAs expression in hen (Gallus domesticus) granulosa cells.

Science.gov (United States)

Rangel, P L; Rodríguez, A; Rojas, S; Sharp, P J; Gutierrez, C G

2009-12-01

The chicken ovary is organized into a hierarchy of yellow yolky follicles that ovulate on successive days. Active or passive immunization of laying hens against testosterone blocks ovulation without affecting follicle development. Testosterone may play a role in pre-ovulatory follicle maturation by stimulating granulosa progesterone production. We assessed whether this stimulus is dose-related and depends on the maturity of the donor follicle, and if it does so by stimulating granulosa cell STAR, P450 cholesterol side-chain cleavage (P450scc), and LH receptor (LHCGR) mRNAs expression. Progesterone production by granulosa cells from F1, F3, and F4 follicles, cultured for 3 h without testosterone was greater in cells collected 11-14 h than 1-4 h after ovulation. These differences in progesterone production were less pronounced after granulosa cells had been cultured for 24 h. Culture of granulosa cells for 3 or 24 h with testosterone (1-100 ng/ml) stimulated progesterone production in cells collected from F4, F3, or F1 follicles 1-4, or 11-14 h after ovulation. Testosterone (0-4000 ng/ml) alone or in combination with LH (0-100 ng/ml) increased progesterone production by F1 granulosa cells, collected 1-4 and 11-14 h after ovulation and cultured for 3 h. Finally, testosterone (10 or 100 ng/ml) increased STAR, P450scc, and LHCGR mRNAs, when added to 3 h cultures of F1 granulosa cells. In conclusion, testosterone stimulates granulosa cell progesterone production in hen pre-ovulatory hierarchical follicles irrespective of maturational state, acting alone or additively with LH. We propose that testosterone promotes granulosa cell maturation to facilitate the pre-ovulatory release of LH.

Unusually effective microRNA targeting within repeat-rich coding regions of mammalian mRNAs

Science.gov (United States)

Schnall-Levin, Michael; Rissland, Olivia S.; Johnston, Wendy K.; Perrimon, Norbert; Bartel, David P.; Berger, Bonnie

2011-01-01

MicroRNAs (miRNAs) regulate numerous biological processes by base-pairing with target messenger RNAs (mRNAs), primarily through sites in 3′ untranslated regions (UTRs), to direct the repression of these targets. Although miRNAs have sometimes been observed to target genes through sites in open reading frames (ORFs), large-scale studies have shown such targeting to be generally less effective than 3′ UTR targeting. Here, we show that several miRNAs each target significant groups of genes through multiple sites within their coding regions. This ORF targeting, which mediates both predictable and effective repression, arises from highly repeated sequences containing miRNA target sites. We show that such sequence repeats largely arise through evolutionary duplications and occur particularly frequently within families of paralogous C2H2 zinc-finger genes, suggesting the potential for their coordinated regulation. Examples of ORFs targeted by miR-181 include both the well-known tumor suppressor RB1 and RBAK, encoding a C2H2 zinc-finger protein and transcriptional binding partner of RB1. Our results indicate a function for repeat-rich coding sequences in mediating post-transcriptional regulation and reveal circumstances in which miRNA-mediated repression through ORF sites can be reliably predicted. PMID:21685129

The Binding Sites of miR-619-5p in the mRNAs of Human and Orthologous Genes.

Science.gov (United States)

Atambayeva, Shara; Niyazova, Raigul; Ivashchenko, Anatoliy; Pyrkova, Anna; Pinsky, Ilya; Akimniyazova, Aigul; Labeit, Siegfried

2017-06-01

Normally, one miRNA interacts with the mRNA of one gene. However, there are miRNAs that can bind to many mRNAs, and one mRNA can be the target of many miRNAs. This significantly complicates the study of the properties of miRNAs and their diagnostic and medical applications. The search of 2,750 human microRNAs (miRNAs) binding sites in 12,175 mRNAs of human genes using the MirTarget program has been completed. For the binding sites of the miR-619-5p the hybridization free energy of the bonds was equal to 100% of the maximum potential free energy. The mRNAs of 201 human genes have complete complementary binding sites of miR-619-5p in the 3'UTR (214 sites), CDS (3 sites), and 5'UTR (4 sites). The mRNAs of CATAD1, ICA1L, GK5, POLH, and PRR11 genes have six miR-619-5p binding sites, and the mRNAs of OPA3 and CYP20A1 genes have eight and ten binding sites, respectively. All of these miR-619-5p binding sites are located in the 3'UTRs. The miR-619-5p binding site in the 5'UTR of mRNA of human USP29 gene is found in the mRNAs of orthologous genes of primates. Binding sites of miR-619-5p in the coding regions of mRNAs of C8H8orf44, C8orf44, and ISY1 genes encode the WLMPVIP oligopeptide, which is present in the orthologous proteins. Binding sites of miR-619-5p in the mRNAs of transcription factor genes ZNF429 and ZNF429 encode the AHACNP oligopeptide in another reading frame. Binding sites of miR-619-5p in the 3'UTRs of all human target genes are also present in the 3'UTRs of orthologous genes of mammals. The completely complementary binding sites for miR-619-5p are conservative in the orthologous mammalian genes. The majority of miR-619-5p binding sites are located in the 3'UTRs but some genes have miRNA binding sites in the 5'UTRs of mRNAs. Several genes have binding sites for miRNAs in the CDSs that are read in different open reading frames. Identical nucleotide sequences of binding sites encode different amino acids in different proteins. The binding sites of miR-619-5p

The significance of translation regulation in the stress response

Science.gov (United States)

2013-01-01

Background The stress response in bacteria involves the multistage control of gene expression but is not entirely understood. To identify the translational response of bacteria in stress conditions and assess its contribution to the regulation of gene expression, the translational states of all mRNAs were compared under optimal growth condition and during nutrient (isoleucine) starvation. Results A genome-scale study of the translational response to nutritional limitation was performed in the model bacterium Lactococcus lactis. Two measures were used to assess the translational status of each individual mRNA: the fraction engaged in translation (ribosome occupancy) and ribosome density (number of ribosomes per 100 nucleotides). Under isoleucine starvation, half of the mRNAs considered were translationally down-regulated mainly due to decreased ribosome density. This pattern concerned genes involved in growth-related functions such as translation, transcription, and the metabolism of fatty acids, phospholipids and bases, contributing to the slowdown of growth. Only 4% of the mRNAs were translationally up-regulated, mostly related to prophagic expression in response to stress. The remaining genes exhibited antagonistic regulations of the two markers of translation. Ribosome occupancy increased significantly for all the genes involved in the biosynthesis of isoleucine, although their ribosome density had decreased. The results revealed complex translational regulation of this pathway, essential to cope with isoleucine starvation. To elucidate the regulation of global gene expression more generally, translational regulation was compared to transcriptional regulation under isoleucine starvation and to other post-transcriptional regulations related to mRNA degradation and mRNA dilution by growth. Translational regulation appeared to accentuate the effects of transcriptional changes for down-regulated growth-related functions under isoleucine starvation although m

Gibberellin-induced changes in the populations of translatable mRNAs and accumulated polypeptides in dwarfs of maize and pea

International Nuclear Information System (INIS)

Chory, J.; Voytas, D.F.; Olszewski, N.E.; Ausubel, F.M.

1987-01-01

Two-dimensional gel electrophoresis was used to characterize the molecular mechanism of gibberellin-induced stem elongation in maize and pea. Dwarf mutants of maize and pea lack endogenous gibberellin (GA 1 ) but become phenotypically normal with exogenous applications of this hormone. Sections from either etiolated maize or green pea seedlings were incubated in the presence of [ 35 S] methionine for 3 hours with or without gibberellin. Labeled proteins from soluble and particulate fractions were analyzed by two-dimensional gel electrophoresis and specific changes in the patterns of protein synthesis were observed upon treatment with gibberellin. Polyadenylated mRNAs from etiolated or green maize shoots and green pea epicotyls treated or not with gibberellin (a 0.5 to 16 hour time course) were assayed by translation in a rabbit reticulocyte extract and separation of products by two-dimensional gel electrophoresis. Both increases and decreases in the levels of specific polypeptides were seen for pea and corn, and these changes were observed within 30 minutes of treatment with gibberellin. Together, these data indicate that gibberellin induces changes in the expression of a subset of gene products within elongating dwarfs. This may be due to changes in transcription rate, mRNA stability, or increased efficiency of translation of certain mRNAs

Pre-service science teachers' teaching self-efficacy in relation to personality traits and academic self-regulation.

Science.gov (United States)

Senler, Burcu; Sungur-Vural, Semra

2013-01-01

The aim of this study is to examine the relationship among pre-service science teachers' personality traits, academic self-regulation and teaching self-efficacy by proposing and testing a conceptual model. For the specified purpose, 1794 pre-service science teachers participated in the study. The Teachers' Sense of Efficacy Scale, the NEO Five-Factor Inventory, and the Motivated Strategies for Learning Questionnaire were administered to assess pre-service science teachers' teaching self-efficacy, personality, and academic self-regulation respectively. Results showed that agreeableness, neuroticism, performance approach goals, and use of metacognitive strategies are positively linked to different dimensions of teaching self-efficacy, namely self-efficacy for student engagement, instructional strategies, and classroom management. In general, while agreeableness and neuroticism were found to be positively associated with different facets of self-regulation and teaching self-efficacy, openness was found to be negatively linked to these adaptive outcomes.

Regulation of ARE-mRNA Stability by Cellular Signaling

DEFF Research Database (Denmark)

Damgaard, Christian Kroun; Lykke-Andersen, Jens

2013-01-01

but as a response to different cellular cues they can become either stabilized, allowing expression of a given gene, or further destabilized to silence their expression. These tightly regulated mRNAs include many that encode growth factors, proto-oncogenes, cytokines, and cell cycle regulators. Failure to properly...

RNA-binding proteins involved in post-transcriptional regulation in bacteria

Directory of Open Access Journals (Sweden)

Elke eVan Assche

2015-03-01

Full Text Available Post-transcriptional regulation is a very important mechanism to control gene expression in changing environments. In the past decade, a lot of interest has been directed towards the role of small RNAs in bacterial post-transcriptional regulation. However, small RNAs are not the only molecules controlling gene expression at this level, RNA-binding proteins play an important role as well. CsrA and Hfq are the two best studied bacterial proteins of this type, but recently, additional proteins involved in post-transcriptional control have been identified. This review focuses on the general working mechanisms of post-transcriptionally active RNA-binding proteins, which include (i adaptation of the susceptibility of mRNAs and sRNAs to RNases, (ii modulating the accessibility of the ribosome binding site of mRNAs, (iii recruiting and assisting in the interaction of mRNAs with other molecules and (iv regulating transcription terminator / antiterminator formation, and gives an overview of both the well-studied and the newly identified proteins that are involved in post-transcriptional regulatory processes. Additionally, the post-transcriptional mechanisms by which the expression or the activity of these proteins is regulated, are described. For many of the newly identified proteins, however, mechanistic questions remain. Most likely, more post-transcriptionally active proteins will be identified in the future.

Network analysis of microRNAs and their regulation in human ovarian cancer

KAUST Repository

Schmeier, Sebastian; Schaefer, Ulf; Essack, Magbubah; Bajic, Vladimir B.

2011-01-01

Background: MicroRNAs (miRNAs) are small non-coding RNA molecules that repress the translation of messenger RNAs (mRNAs) or degrade mRNAs. These functions of miRNAs allow them to control key cellular processes such as development, differentiation and apoptosis, and they have also been implicated in several cancers such as leukaemia, lung, pancreatic and ovarian cancer (OC). Unfortunately, the specific machinery of miRNA regulation, involving transcription factors (TFs) and transcription co-factors (TcoFs), is not well understood. In the present study we focus on computationally deciphering the underlying network of miRNAs, their targets, and their control mechanisms that have an influence on OC development.Results: We analysed experimentally verified data from multiple sources that describe miRNA influence on diseases, miRNA targeting of mRNAs, and on protein-protein interactions, and combined this data with ab initio transcription factor binding site predictions within miRNA promoter regions. From these analyses, we derived a network that describes the influence of miRNAs and their regulation in human OC. We developed a methodology to analyse the network in order to find the nodes that have the largest potential of influencing the network's behaviour (network hubs). We further show the potentially most influential miRNAs, TFs and TcoFs, showing subnetworks illustrating the involved mechanisms as well as regulatory miRNA network motifs in OC. We find an enrichment of miRNA targeted OC genes in the highly relevant pathways cell cycle regulation and apoptosis.Conclusions: We combined several sources of interaction and association data to analyse and place miRNAs within regulatory pathways that influence human OC. These results represent the first comprehensive miRNA regulatory network analysis for human OC. This suggests that miRNAs and their regulation may play a major role in OC and that further directed research in this area is of utmost importance to enhance our

Network analysis of microRNAs and their regulation in human ovarian cancer

KAUST Repository

Schmeier, Sebastian

2011-11-03

Background: MicroRNAs (miRNAs) are small non-coding RNA molecules that repress the translation of messenger RNAs (mRNAs) or degrade mRNAs. These functions of miRNAs allow them to control key cellular processes such as development, differentiation and apoptosis, and they have also been implicated in several cancers such as leukaemia, lung, pancreatic and ovarian cancer (OC). Unfortunately, the specific machinery of miRNA regulation, involving transcription factors (TFs) and transcription co-factors (TcoFs), is not well understood. In the present study we focus on computationally deciphering the underlying network of miRNAs, their targets, and their control mechanisms that have an influence on OC development.Results: We analysed experimentally verified data from multiple sources that describe miRNA influence on diseases, miRNA targeting of mRNAs, and on protein-protein interactions, and combined this data with ab initio transcription factor binding site predictions within miRNA promoter regions. From these analyses, we derived a network that describes the influence of miRNAs and their regulation in human OC. We developed a methodology to analyse the network in order to find the nodes that have the largest potential of influencing the network\\'s behaviour (network hubs). We further show the potentially most influential miRNAs, TFs and TcoFs, showing subnetworks illustrating the involved mechanisms as well as regulatory miRNA network motifs in OC. We find an enrichment of miRNA targeted OC genes in the highly relevant pathways cell cycle regulation and apoptosis.Conclusions: We combined several sources of interaction and association data to analyse and place miRNAs within regulatory pathways that influence human OC. These results represent the first comprehensive miRNA regulatory network analysis for human OC. This suggests that miRNAs and their regulation may play a major role in OC and that further directed research in this area is of utmost importance to enhance

SUMOylation Is an Inhibitory Constraint that Regulates the Prion-like Aggregation and Activity of CPEB3

Directory of Open Access Journals (Sweden)

Bettina Drisaldi

2015-06-01

Full Text Available Protein synthesis is crucial for the maintenance of long-term-memory-related synaptic plasticity. The prion-like cytoplasmic polyadenylation element-binding protein 3 (CPEB3 regulates the translation of several mRNAs important for long-term synaptic plasticity in the hippocampus. Here, we provide evidence that the prion-like aggregation and activity of CPEB3 is controlled by SUMOylation. In the basal state, CPEB3 is a repressor and is soluble. Under these circumstances, CPEB3 is SUMOylated in hippocampal neurons both in vitro and in vivo. Following neuronal stimulation, CPEB3 is converted into an active form that promotes the translation of target mRNAs, and this is associated with a decrease of SUMOylation and an increase of aggregation. A chimeric CPEB3 protein fused to SUMO cannot form aggregates and cannot activate the translation of target mRNAs. These findings suggest a model whereby SUMO regulates translation of mRNAs and structural synaptic plasticity by modulating the aggregation of the prion-like protein CPEB3.

TiGER: a database for tissue-specific gene expression and regulation.

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Liu, Xiong; Yu, Xueping; Zack, Donald J; Zhu, Heng; Qian, Jiang

2008-06-09

Understanding how genes are expressed and regulated in different tissues is a fundamental and challenging question. However, most of currently available biological databases do not focus on tissue-specific gene regulation. The recent development of computational methods for tissue-specific combinational gene regulation, based on transcription factor binding sites, enables us to perform a large-scale analysis of tissue-specific gene regulation in human tissues. The results are stored in a web database called TiGER (Tissue-specific Gene Expression and Regulation). The database contains three types of data including tissue-specific gene expression profiles, combinatorial gene regulations, and cis-regulatory module (CRM) detections. At present the database contains expression profiles for 19,526 UniGene genes, combinatorial regulations for 7,341 transcription factor pairs and 6,232 putative CRMs for 2,130 RefSeq genes. We have developed and made publicly available a database, TiGER, which summarizes and provides large scale data sets for tissue-specific gene expression and regulation in a variety of human tissues. This resource is available at 1.

TiGER: A database for tissue-specific gene expression and regulation

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Zack Donald J

2008-06-01

Full Text Available Abstract Background Understanding how genes are expressed and regulated in different tissues is a fundamental and challenging question. However, most of currently available biological databases do not focus on tissue-specific gene regulation. Results The recent development of computational methods for tissue-specific combinational gene regulation, based on transcription factor binding sites, enables us to perform a large-scale analysis of tissue-specific gene regulation in human tissues. The results are stored in a web database called TiGER (Tissue-specific Gene Expression and Regulation. The database contains three types of data including tissue-specific gene expression profiles, combinatorial gene regulations, and cis-regulatory module (CRM detections. At present the database contains expression profiles for 19,526 UniGene genes, combinatorial regulations for 7,341 transcription factor pairs and 6,232 putative CRMs for 2,130 RefSeq genes. Conclusion We have developed and made publicly available a database, TiGER, which summarizes and provides large scale data sets for tissue-specific gene expression and regulation in a variety of human tissues. This resource is available at 1.

The Arabidopsis TOR Kinase Specifically Regulates the Expression of Nuclear Genes Coding for Plastidic Ribosomal Proteins and the Phosphorylation of the Cytosolic Ribosomal Protein S6.

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Dobrenel, Thomas; Mancera-Martínez, Eder; Forzani, Céline; Azzopardi, Marianne; Davanture, Marlène; Moreau, Manon; Schepetilnikov, Mikhail; Chicher, Johana; Langella, Olivier; Zivy, Michel; Robaglia, Christophe; Ryabova, Lyubov A; Hanson, Johannes; Meyer, Christian

2016-01-01

Protein translation is an energy consuming process that has to be fine-tuned at both the cell and organism levels to match the availability of resources. The target of rapamycin kinase (TOR) is a key regulator of a large range of biological processes in response to environmental cues. In this study, we have investigated the effects of TOR inactivation on the expression and regulation of Arabidopsis ribosomal proteins at different levels of analysis, namely from transcriptomic to phosphoproteomic. TOR inactivation resulted in a coordinated down-regulation of the transcription and translation of nuclear-encoded mRNAs coding for plastidic ribosomal proteins, which could explain the chlorotic phenotype of the TOR silenced plants. We have identified in the 5' untranslated regions (UTRs) of this set of genes a conserved sequence related to the 5' terminal oligopyrimidine motif, which is known to confer translational regulation by the TOR kinase in other eukaryotes. Furthermore, the phosphoproteomic analysis of the ribosomal fraction following TOR inactivation revealed a lower phosphorylation of the conserved Ser240 residue in the C-terminal region of the 40S ribosomal protein S6 (RPS6). These results were confirmed by Western blot analysis using an antibody that specifically recognizes phosphorylated Ser240 in RPS6. Finally, this antibody was used to follow TOR activity in plants. Our results thus uncover a multi-level regulation of plant ribosomal genes and proteins by the TOR kinase.

Learning Pre-Played Solos: Self-Regulated Learning Strategies in Jazz/Improvised Music

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Nielsen, Siw G.

2015-01-01

This article reports on the self-regulated learning strategies of two advanced students in jazz/improvised music education when learning pre-played solos over well-known jazz tunes. The students were enrolled in a well-established performance degree programme in a music conservatoire, and videotaped their own individual practice sessions. In…

Expression analysis of miRNA and target mRNAs in esophageal cancer

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Meng, X.R. [Oncology Department, The First Affiliated Hospital of Zhengzhou University, Zhengzhou (China); Lu, P. [Gastrointestinal Surgery Department, People' s Hospital of Zhengzhou, Zhengzhou (China); Mei, J.Z.; Liu, G.J. [Medical Oncology Department, People' s Hospital of Zhengzhou, Zhengzhou (China); Fan, Q.X. [Oncology Department, The First Affiliated Hospital of Zhengzhou University, Zhengzhou (China)

2014-08-01

We aimed to investigate miRNAs and related mRNAs through a network-based approach in order to learn the crucial role that they play in the biological processes of esophageal cancer. Esophageal squamous-cell carcinoma (ESCC) and adenocarcinoma (EAC)-related miRNA and gene expression data were downloaded from the Gene Expression Omnibus database, and differentially expressed miRNAs and genes were selected. Target genes of differentially expressed miRNAs were predicted and their regulatory networks were constructed. Differentially expressed miRNA analysis selected four miRNAs associated with EAC and ESCC, among which hsa-miR-21 and hsa-miR-202 were shared by both diseases. hsa-miR-202 was reported for the first time to be associated with esophageal cancer in the present study. Differentially expressed miRNA target genes were mainly involved in cancer-related and signal-transduction pathways. Functional categories of these target genes were related to transcriptional regulation. The results may indicate potential target miRNAs and genes for future investigations of esophageal cancer.

Exposure of Lactating Dairy Cows to Acute Pre-Ovulatory Heat Stress Affects Granulosa Cell-Specific Gene Expression Profiles in Dominant Follicles

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Vanselow, Jens; Vernunft, Andreas; Koczan, Dirk; Spitschak, Marion; Kuhla, Björn

2016-01-01

High environmental temperatures induce detrimental effects on various reproductive processes in cattle. According to the predicted global warming the number of days with unfavorable ambient temperatures will further increase. The objective of this study was to investigate effects of acute heat stress during the late pre-ovulatory phase on morphological, physiological and molecular parameters of dominant follicles in cycling cows during lactation. Eight German Holstein cows in established lactation were exposed to heat stress (28°C) or thermoneutral conditions (15°C) with pair-feeding for four days. After hormonal heat induction growth of the respective dominant follicles was monitored by ultrasonography for two days, then an ovulatory GnRH dose was given and follicular steroid hormones and granulosa cell-specific gene expression profiles were determined 23 hrs thereafter. The data showed that the pre-ovulatory growth of dominant follicles and the estradiol, but not the progesterone concentrations tended to be slightly affected. mRNA microarray and hierarchical cluster analysis revealed distinct expression profiles in granulosa cells derived from heat stressed compared to pair-fed animals. Among the 255 affected genes heatstress-, stress- or apoptosis associated genes were not present. But instead, we found up-regulation of genes essentially involved in G-protein coupled signaling pathways, extracellular matrix composition, and several members of the solute carrier family as well as up-regulation of FST encoding follistatin. In summary, the data of the present study show that acute pre-ovulatory heat stress can specifically alter gene expression profiles in granulosa cells, however without inducing stress related genes and pathways and suggestively can impair follicular growth due to affecting the activin-inhibin-follistatin system. PMID:27532452

Genome-wide RIP-Chip analysis of translational repressor-bound mRNAs in the Plasmodium gametocyte

KAUST Repository

Guerreiro, Ana

2014-11-03

Background Following fertilization, the early proteomes of metazoans are defined by the translation of stored but repressed transcripts; further embryonic development relies on de novo transcription of the zygotic genome. During sexual development of Plasmodium berghei, a rodent model for human malaria species including P. falciparum, the stability of repressed mRNAs requires the translational repressors DOZI and CITH. When these repressors are absent, Plasmodium zygote development and transmission to the mosquito vector is halted, as hundreds of transcripts become destabilized. However, which mRNAs are direct targets of these RNA binding proteins, and thus subject to translational repression, is unknown. Results We identify the maternal mRNA contribution to post-fertilization development of P. berghei using RNA immunoprecipitation and microarray analysis. We find that 731 mRNAs, approximately 50% of the transcriptome, are associated with DOZI and CITH, allowing zygote development to proceed in the absence of RNA polymerase II transcription. Using GFP-tagging, we validate the repression phenotype of selected genes and identify mRNAs relying on the 5′ untranslated region for translational control. Gene deletion reveals a novel protein located in the ookinete crystalloid with an essential function for sporozoite development. Conclusions Our study details for the first time the P. berghei maternal repressome. This mRNA population provides the developing ookinete with coding potential for key molecules required for life-cycle progression, and that are likely to be critical for the transmission of the malaria parasite from the rodent and the human host to the mosquito vector.

Fragile x mental retardation protein regulates proliferation and differentiation of adult neural stem/progenitor cells.

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Yuping Luo

2010-04-01

Full Text Available Fragile X syndrome (FXS, the most common form of inherited mental retardation, is caused by the loss of functional fragile X mental retardation protein (FMRP. FMRP is an RNA-binding protein that can regulate the translation of specific mRNAs. Adult neurogenesis, a process considered important for neuroplasticity and memory, is regulated at multiple molecular levels. In this study, we investigated whether Fmrp deficiency affects adult neurogenesis. We show that in a mouse model of fragile X syndrome, adult neurogenesis is indeed altered. The loss of Fmrp increases the proliferation and alters the fate specification of adult neural progenitor/stem cells (aNPCs. We demonstrate that Fmrp regulates the protein expression of several components critical for aNPC function, including CDK4 and GSK3beta. Dysregulation of GSK3beta led to reduced Wnt signaling pathway activity, which altered the expression of neurogenin1 and the fate specification of aNPCs. These data unveil a novel regulatory role for Fmrp and translational regulation in adult neurogenesis.

Profiling Pre-Service Teachers' Awareness and Regulation of Their Own Thinking: Evidence from an Asian Country

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Lee, Chwee Beng; Teo, Timothy; Chai, Ching Sing

2010-01-01

This study aims to examine pre-service teachers' knowledge and regulation of cognition. The authors administered Schraw and Dennison's Metacognitive Awareness Inventory to 254 pre-service teachers in Singapore. The results showed no significant difference by educational level on all subscales except for evaluation, which is a subscale of…

Sequence features of E. coli mRNAs affect their degradation.

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Gal Lenz

Full Text Available Degradation of mRNA in bacteria is a regulatory mechanism, providing an efficient way to fine-tune protein abundance in response to environmental changes. While the mechanisms responsible for initiation and subsequent propagation of mRNA degradation are well studied, the mRNA features that affect its stability are yet to be elucidated. We calculated three properties for each mRNA in the E. coli transcriptome: G+C content, tRNA adaptation index (tAI and folding energy. Each of these properties were then correlated with the experimental transcript half life measured for each transcript and detected significant correlations. A sliding window analysis identified the regions that displayed the maximal signal. The correlation between transcript half life and both G+C content and folding energy was strongest at the 5' termini of the mRNAs. Partial correlations showed that each of the parameters contributes separately to mRNA half life. Notably, mRNAs of recently-acquired genes in the E. coli genome, which have a distinct nucleotide composition, tend to be highly stable. This high stability may aid the evolutionary fixation of horizontally acquired genes.

Epigenetic regulation of lncRNA connects ubiquitin-proteasome system with infection-inflammation in preterm births and preterm premature rupture of membranes.

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Luo, Xiucui; Pan, Jing; Wang, Leilei; Wang, Peirong; Zhang, Meijiao; Liu, Meilin; Dong, Ziqing; Meng, Qian; Tao, Xuguang; Zhao, Xinliang; Zhong, Julia; Ju, Weina; Gu, Yang; Jenkins, Edmund C; Brown, W Ted; Shi, Qingxi; Zhong, Nanbert

2015-02-15

Preterm premature rupture of membranes (PPROM) is responsible for one third of all preterm births (PTBs). We have recently demonstrated that long noncoding RNAs (lncRNAs) are differentially expressed in human placentas derived from PPROM, PTB, premature rupture of the membranes (PROM), and full-term birth (FTB), and determined the major biological pathways involved in PPROM. Here, we further investigated the relationship of lncRNAs, which are differentially expressed in spontaneous PTB (sPTB) and PPROM placentas and are found to overlap a coding locus, with the differential expression of transcribed mRNAs at the same locus. Ten lncRNAs (five up-regulated and five down-regulated) and the lncRNA-associated 10 mRNAs (six up- and four down-regulated), which were identified by microarray in comparing PPROM vs. sPTB, were then validated by real-time quantitative PCR. A total of 62 (38 up- and 24 down-regulated) and 1,923 (790 up- and 1,133 down-regulated) lncRNAs were identified from placentas of premature labor (sPTB + PPROM), as compared to those from full-term labor (FTB + PROM) and from premature rupture of membranes (PPROM + PROM), as compared to those from non-rupture of membranes (sPTB + FTB), respectively. We found that a correlation existed between differentially expressed lncRNAs and their associated mRNAs, which could be grouped into four categories based on the gene strand (sense or antisense) of lncRNA and its paired transcript. These findings suggest that lncRNA regulates mRNA transcription through differential mechanisms. Differential expression of the transcripts PPP2R5C, STAM, TACC2, EML4, PAM, PDE4B, STAM, PPP2R5C, PDE4B, and EGFR indicated a co-expression among these mRNAs, which are involved in the ubiquitine-proteasome system (UPS), in addition to signaling transduction and beta adrenergic signaling, suggesting that imbalanced regulation of UPS may present an additional mechanism underlying the premature rupture of membrane in PPROM

Noise-induced multistability in the regulation of cancer by genes and pseudogenes

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Petrosyan, K. G., E-mail: pkaren@phys.sinica.edu.tw [Institute of Physics, Academia Sinica, Nankang, Taipei 11529, Taiwan (China); Hu, Chin-Kun, E-mail: huck@phys.sinica.edu.tw [Institute of Physics, Academia Sinica, Nankang, Taipei 11529, Taiwan (China); National Center for Theoretical Sciences, National Tsing Hua University, Hsinchu 30013, Taiwan (China); Business School, University of Shanghai for Science and Technology, Shanghai 200093 (China)

2016-07-28

We extend a previously introduced model of stochastic gene regulation of cancer to a nonlinear case having both gene and pseudogene messenger RNAs (mRNAs) self-regulated. The model consists of stochastic Boolean genetic elements and possesses noise-induced multistability (multimodality). We obtain analytical expressions for probabilities for the case of constant but finite number of microRNA molecules which act as a noise source for the competing gene and pseudogene mRNAs. The probability distribution functions display both the global bistability regime as well as even-odd number oscillations for a certain range of model parameters. Statistical characteristics of the mRNA’s level fluctuations are evaluated. The obtained results of the extended model advance our understanding of the process of stochastic gene and pseudogene expressions that is crucial in regulation of cancer.

Transcript specificity in yeast pre-mRNA splicing revealed by mutations in core spliceosomal components.

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Jeffrey A Pleiss

2007-04-01

Full Text Available Appropriate expression of most eukaryotic genes requires the removal of introns from their pre-messenger RNAs (pre-mRNAs, a process catalyzed by the spliceosome. In higher eukaryotes a large family of auxiliary factors known as SR proteins can improve the splicing efficiency of transcripts containing suboptimal splice sites by interacting with distinct sequences present in those pre-mRNAs. The yeast Saccharomyces cerevisiae lacks functional equivalents of most of these factors; thus, it has been unclear whether the spliceosome could effectively distinguish among transcripts. To address this question, we have used a microarray-based approach to examine the effects of mutations in 18 highly conserved core components of the spliceosomal machinery. The kinetic profiles reveal clear differences in the splicing defects of particular pre-mRNA substrates. Most notably, the behaviors of ribosomal protein gene transcripts are generally distinct from other intron-containing transcripts in response to several spliceosomal mutations. However, dramatically different behaviors can be seen for some pairs of transcripts encoding ribosomal protein gene paralogs, suggesting that the spliceosome can readily distinguish between otherwise highly similar pre-mRNAs. The ability of the spliceosome to distinguish among its different substrates may therefore offer an important opportunity for yeast to regulate gene expression in a transcript-dependent fashion. Given the high level of conservation of core spliceosomal components across eukaryotes, we expect that these results will significantly impact our understanding of how regulated splicing is controlled in higher eukaryotes as well.

TBX3 regulates splicing in vivo: a novel molecular mechanism for Ulnar-mammary syndrome.

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Pavan Kumar P

2014-03-01

Full Text Available TBX3 is a member of the T-box family of transcription factors with critical roles in development, oncogenesis, cell fate, and tissue homeostasis. TBX3 mutations in humans cause complex congenital malformations and Ulnar-mammary syndrome. Previous investigations into TBX3 function focused on its activity as a transcriptional repressor. We used an unbiased proteomic approach to identify TBX3 interacting proteins in vivo and discovered that TBX3 interacts with multiple mRNA splicing factors and RNA metabolic proteins. We discovered that TBX3 regulates alternative splicing in vivo and can promote or inhibit splicing depending on context and transcript. TBX3 associates with alternatively spliced mRNAs and binds RNA directly. TBX3 binds RNAs containing TBX binding motifs, and these motifs are required for regulation of splicing. Our study reveals that TBX3 mutations seen in humans with UMS disrupt its splicing regulatory function. The pleiotropic effects of TBX3 mutations in humans and mice likely result from disrupting at least two molecular functions of this protein: transcriptional regulation and pre-mRNA splicing.

TBX3 regulates splicing in vivo: a novel molecular mechanism for Ulnar-mammary syndrome.

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Kumar P, Pavan; Franklin, Sarah; Emechebe, Uchenna; Hu, Hao; Moore, Barry; Lehman, Chris; Yandell, Mark; Moon, Anne M

2014-03-01

TBX3 is a member of the T-box family of transcription factors with critical roles in development, oncogenesis, cell fate, and tissue homeostasis. TBX3 mutations in humans cause complex congenital malformations and Ulnar-mammary syndrome. Previous investigations into TBX3 function focused on its activity as a transcriptional repressor. We used an unbiased proteomic approach to identify TBX3 interacting proteins in vivo and discovered that TBX3 interacts with multiple mRNA splicing factors and RNA metabolic proteins. We discovered that TBX3 regulates alternative splicing in vivo and can promote or inhibit splicing depending on context and transcript. TBX3 associates with alternatively spliced mRNAs and binds RNA directly. TBX3 binds RNAs containing TBX binding motifs, and these motifs are required for regulation of splicing. Our study reveals that TBX3 mutations seen in humans with UMS disrupt its splicing regulatory function. The pleiotropic effects of TBX3 mutations in humans and mice likely result from disrupting at least two molecular functions of this protein: transcriptional regulation and pre-mRNA splicing.

77 FR 3636 - Federal Acquisition Regulation; Brand-Name Specifications; Correction

Science.gov (United States)

2012-01-25

... 26] RIN 9000-AK55 Federal Acquisition Regulation; Brand-Name Specifications; Correction AGENCY... Regulation (FAR) to implement the Office of Management and Budget memoranda on brand-name specifications, FAR Case 2005-037, Brand-Name Specifications, which published in the Federal Register at 77 FR 189 on...

Localization of nuclear retained mRNAs in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Thomsen, Rune; Libri, Domenico; Boulay, Jocelyne

2003-01-01

site of transcription, and known S. cerevisiae nuclear structures such as the nucleolus and the nucleolar body. Our results show that retained SSA4 RNA localizes to an area in close proximity to the SSA4 locus. On deletion of Rrp6p and release from the genomic locus, heat shock mRNAs produced...

Comparison of a preQ1 riboswitch aptamer in metabolite-bound and free states with implications for gene regulation.

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Jenkins, Jermaine L; Krucinska, Jolanta; McCarty, Reid M; Bandarian, Vahe; Wedekind, Joseph E

2011-07-15

Riboswitches are RNA regulatory elements that govern gene expression by recognition of small molecule ligands via a high affinity aptamer domain. Molecular recognition can lead to active or attenuated gene expression states by controlling accessibility to mRNA signals necessary for transcription or translation. Key areas of inquiry focus on how an aptamer attains specificity for its effector, the extent to which the aptamer folds prior to encountering its ligand, and how ligand binding alters expression signal accessibility. Here we present crystal structures of the preQ(1) riboswitch from Thermoanaerobacter tengcongensis in the preQ(1)-bound and free states. Although the mode of preQ(1) recognition is similar to that observed for preQ(0), surface plasmon resonance revealed an apparent K(D) of 2.1 ± 0.3 nm for preQ(1) but a value of 35.1 ± 6.1 nm for preQ(0). This difference can be accounted for by interactions between the preQ(1) methylamine and base G5 of the aptamer. To explore conformational states in the absence of metabolite, the free-state aptamer structure was determined. A14 from the ceiling of the ligand pocket shifts into the preQ(1)-binding site, resulting in "closed" access to the metabolite while simultaneously increasing exposure of the ribosome-binding site. Solution scattering data suggest that the free-state aptamer is compact, but the "closed" free-state crystal structure is inadequate to describe the solution scattering data. These observations are distinct from transcriptional preQ(1) riboswitches of the same class that exhibit strictly ligand-dependent folding. Implications for gene regulation are discussed.

TCR Signal Strength Regulates Akt Substrate Specificity To Induce Alternate Murine Th and T Regulatory Cell Differentiation Programs.

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Hawse, William F; Boggess, William C; Morel, Penelope A

2017-07-15

The Akt/mTOR pathway is a key driver of murine CD4 + T cell differentiation, and induction of regulatory T (Treg) cells results from low TCR signal strength and low Akt/mTOR signaling. However, strong TCR signals induce high Akt activity that promotes Th cell induction. Yet, it is unclear how Akt controls alternate T cell fate decisions. We find that the strength of the TCR signal results in differential Akt enzymatic activity. Surprisingly, the Akt substrate networks associated with T cell fate decisions are qualitatively different. Proteomic profiling of Akt signaling networks during Treg versus Th induction demonstrates that Akt differentially regulates RNA processing and splicing factors to drive T cell differentiation. Interestingly, heterogeneous nuclear ribonucleoprotein (hnRNP) L or hnRNP A1 are Akt substrates during Treg induction and have known roles in regulating the stability and splicing of key mRNAs that code for proteins in the canonical TCR signaling pathway, including CD3ζ and CD45. Functionally, inhibition of Akt enzymatic activity results in the dysregulation of splicing during T cell differentiation, and knockdown of hnRNP L or hnRNP A1 results in the lower induction of Treg cells. Together, this work suggests that a switch in substrate specificity coupled to the phosphorylation status of Akt may lead to alternative cell fates and demonstrates that proteins involved with alternative splicing are important factors in T cell fate decisions. Copyright © 2017 by The American Association of Immunologists, Inc.

Transcriptome-wide analysis of the Trypanosoma cruzi proliferative cycle identifies the periodically expressed mRNAs and their multiple levels of control.

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Santiago Chávez

Full Text Available Trypanosoma cruzi is the protozoan parasite causing American trypanosomiasis or Chagas disease, a neglected parasitosis with important human health impact in Latin America. The efficacy of current therapy is limited, and its toxicity is high. Since parasite proliferation is a fundamental target for rational drug design, we sought to progress into its understanding by applying a genome-wide approach. Treating a TcI linage strain with hydroxyurea, we isolated epimastigotes in late G1, S and G2/M cell cycle stages at 70% purity. The sequencing of each phase identified 305 stage-specific transcripts (1.5-fold change, p≤0.01, coding for conserved cell cycle regulated proteins and numerous proteins whose cell cycle dependence has not been recognized before. Comparisons with the parasite T. brucei and the human host reveal important differences. The meta-analysis of T. cruzi transcriptomic and ribonomic data indicates that cell cycle regulated mRNAs are subject to sub-cellular compartmentalization. Compositional and structural biases of these genes- including CAI, GC content, UTR length, and polycistron position- may contribute to their regulation. To discover nucleotide motifs responsible for the co-regulation of cell cycle regulated genes, we looked for overrepresented motifs at their UTRs and found a variant of the cell cycle sequence motif at the 3' UTR of most of the S and G2 stage genes. We additionally identified hairpin structures at the 5' UTRs of a high proportion of the transcripts, suggesting that periodic gene expression might also rely on translation initiation in T. cruzi. In summary, we report a comprehensive list of T. cruzi cell cycle regulated genes, including many previously unstudied proteins, we show evidence favoring a multi-step control of their expression, and we identify mRNA motifs that may mediate their regulation. Our results provide novel information of the T. cruzi proliferative proteins and the integrated levels of

Genome-wide analysis of uncapped mRNAs under heat stress in Arabidopsis

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Emilio Gutierrez-Beltran

2015-09-01

Full Text Available Recently, we have showed that Tudor Staphylococcal Nuclease (TSN or Tudor-SN proteins (TSN1 and TSN2 are localized in cytoplasmic messenger ribonucleoprotein (mRNP complexes called stress granules (SG and processing bodies (PB under heat stress in Arabidopsis. One of the primary functions of these mRNP complexes is mRNA decay, which generates uncapped mRNAs by the action of endonucleases and decapping enzymes (Thomas et al., 2011 [1]. In order to figure out whether TSN proteins could be implicated in mRNA decay, we isolated uncapped and total mRNAs of Wild type (WT; Col and Ler and TSN double knock-out (tsn1tsn2 seedlings grown under heat stress (39 °C for 40 min and control (23 °C conditions. Here, we provide the experimental procedure to reproduce the results (NCBI GEO accession number GSE63522 published by Gutierrez-Beltran et al. (2015 in The Plant Cell [2].

A Tool that Can be Effective in the Self-Regulated Learning of Pre-Service Teachers: The Mind Map

Science.gov (United States)

Tanriseven, Isil

2014-01-01

The aim of this study is to analyse the effect of task planning with mind maps on the self-regulation strategies and motivational beliefs of pre-service teachers. A quasi-experimental design, with a pre-test and post-test control group, was applied in the research. The research group comprised of 60 pre-service teachers taking "Teaching…

Specificity of emotion regulation deficits in social anxiety: an internet study.

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Rusch, Silke; Westermann, Stefan; Lincoln, Tania M

2012-09-01

There is evidence for an association between social anxiety and emotion regulation difficulties. This study investigates that emotion regulation difficulties are specific to two domains of social anxiety. An explorative study was conducted to examine the associations between emotion regulation facets and social anxiety in the normal population. N= 149 healthy volunteers participated in an internet-based survey. Emotion regulation deficits were measured by the Difficulties in Emotion Regulation Scale which consists of six subscales. Social anxiety was measured by the Social Phobia Scale and the Social Interaction Anxiety Scale. Hierarchical regression analyses showed that anxiety of interactive social situations is associated with non-acceptance of negative emotions, impulse control difficulties, and lack of functional emotion regulation strategies over and above the impact of age and general psychopathology. In contrast, anxiety of being observed by others was not specifically associated with emotion regulation strategies. The results support the hypothesis that specific emotion regulation deficits are relevant to specific aspects of social anxiety. Implications for further research and therapy are discussed. © 2011 The British Psychological Society.

An "up, no change, or down" system: Time-dependent expression of mRNAs in contused skeletal muscle of rats used for wound age estimation.

Science.gov (United States)

Sun, Jun-Hong; Zhu, Xi-Yan; Dong, Ta-Na; Zhang, Xiao-Hong; Liu, Qi-Qing; Li, San-Qiang; Du, Qiu-Xiang

2017-03-01

The combined use of multiple markers is considered a promising strategy in estimating the age of wounds. We sought to develop an "up, no change, or down" system and to explore how to combine and use various parameters. In total, 78 Sprague Dawley rats were divided randomly into a control group and contusion groups of 4-, 8-, 12-, 16-, 20-, 24-, 28-, 32-, 36-, 40-, 44-, and 48-h post-injury (n=6 per group). A contusion was produced in the right limb of the rats under diethyl ether anesthesia by a drop-ball technique; the animals were sacrificed at certain time points thereafter, using a lethal dose of pentobarbital. Levels of PUM2, TAB2, GJC1, and CHRNA1 mRNAs were detected in contused muscle using real-time PCR. An up, no change, or down system was developed with the relative quantities of the four mRNAs recorded as black, dark gray, or light gray boxes, representing up-, no change, or down-regulation of the gene of interest during wound repair. The four transcripts were combined and used as a marker cluster for color model analysis of each contusion group. Levels of PUM2, TAB2, and GJC1 mRNAs decreased, whereas that of CHRNA1 increased in wound repair (Psystem was adequate to distinguish most time groups with the color model. Thus, the proposed up, no change, or down system provide the means to determine the minimal periods of early wounds. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Life cycle and production of Baetis rhodani in a regulated river in Western Norway: comparison of pre- and post-regulation conditions

International Nuclear Information System (INIS)

Raddum, G.G.; Fjellheim, A.

1993-01-01

The benthic invertebrate fauna of the lowland part of the Aurland River was investigated in 1966-7 over a full year cycle. The watershed was built out for hydropower production during 1979-83. In this study the life cycle and production of Baetis rhodani from pre-regulated conditions (1966-7) was compared with two types of post-regulation streams(1988-9). Before regulation the water flow was high during May-June and in autumn, combined with heavy rainfall. The lowest flow was in winter and early spring. The pre-regulated temperature was low in winter, increasing from April to a maximum in August (10-14 o C). After regulation one part of the river received reduced flow and increased summer temperatures (upper part) and one received hypolimnion release and reduced summer temperatures (lower part). In the upper part the density of B.rhodani increased between 10 and 20 times. The reason for this seems to be increased temperature, reduced accidental drift of larvae and increased amount of stored organic material on the bottom. In the lower part the density increase was two to five times and the production two times higher after regulation. The lower increase in production than density was due to a much higher proportion of small larvae. B.rhodani had mainly an univoltine life cycle before regulation, but a small part of the population was bivoltine. After regulation the species was univoltine in the lower part. In the upper part the life cycle was about one month faster, but no clear indication of bivoltinism was seen. The absence of the second generation is due to low temperatures and probably a lack of signals connected with temperature. (Author)

Dissecting specific and global transcriptional regulation of bacterial gene expression

NARCIS (Netherlands)

Gerosa, Luca; Kochanowski, Karl; Heinemann, Matthias; Sauer, Uwe

Gene expression is regulated by specific transcriptional circuits but also by the global expression machinery as a function of growth. Simultaneous specific and global regulation thus constitutes an additional-but often neglected-layer of complexity in gene expression. Here, we develop an

Using auditory pre-information to solve the cocktail-party problem: electrophysiological evidence for age-specific differences.

Science.gov (United States)

Getzmann, Stephan; Lewald, Jörg; Falkenstein, Michael

2014-01-01

Speech understanding in complex and dynamic listening environments requires (a) auditory scene analysis, namely auditory object formation and segregation, and (b) allocation of the attentional focus to the talker of interest. There is evidence that pre-information is actively used to facilitate these two aspects of the so-called "cocktail-party" problem. Here, a simulated multi-talker scenario was combined with electroencephalography to study scene analysis and allocation of attention in young and middle-aged adults. Sequences of short words (combinations of brief company names and stock-price values) from four talkers at different locations were simultaneously presented, and the detection of target names and the discrimination between critical target values were assessed. Immediately prior to speech sequences, auditory pre-information was provided via cues that either prepared auditory scene analysis or attentional focusing, or non-specific pre-information was given. While performance was generally better in younger than older participants, both age groups benefited from auditory pre-information. The analysis of the cue-related event-related potentials revealed age-specific differences in the use of pre-cues: Younger adults showed a pronounced N2 component, suggesting early inhibition of concurrent speech stimuli; older adults exhibited a stronger late P3 component, suggesting increased resource allocation to process the pre-information. In sum, the results argue for an age-specific utilization of auditory pre-information to improve listening in complex dynamic auditory environments.

Using auditory pre-information to solve the cocktail-party problem: electrophysiological evidence for age-specific differences

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Stephan eGetzmann

2014-12-01

Full Text Available Speech understanding in complex and dynamic listening environments requires (a auditory scene analysis, namely auditory object formation and segregation, and (b allocation of the attentional focus to the talker of interest. There is evidence that pre-information is actively used to facilitate these two aspects of the so-called cocktail-party problem. Here, a simulated multi-talker scenario was combined with electroencephalography to study scene analysis and allocation of attention in young and middle-aged adults. Sequences of short words (combinations of brief company names and stock-price values from four talkers at different locations were simultaneously presented, and the detection of target names and the discrimination between critical target values were assessed. Immediately prior to speech sequences, auditory pre-information was provided via cues that either prepared auditory scene analysis or attentional focusing, or non-specific pre-information was given. While performance was generally better in younger than older participants, both age groups benefited from auditory pre-information. The analysis of the cue-related event-related potentials revealed age-specific differences in the use of pre-cues: Younger adults showed a pronounced N2 component, suggesting early inhibition of concurrent speech stimuli; older adults exhibited a stronger late P3 component, suggesting increased resource allocation to process the pre-information. In sum, the results argue for an age-specific utilization of auditory pre-information to improve listening in complex dynamic auditory environments.

Development of the NUMO pre-selection, site-specific safety case

International Nuclear Information System (INIS)

Fujiyama, Tetsuo; Suzuki, Satoru; Deguchi, Akira; Umeki, Hiroyuki

2016-01-01

Key conclusions: ◆ “The NUMO pre-selection, site-specific safety case” provides the basic structure for subsequent safety cases that will be applied to any selected site, emphasising practical approaches and methodology which will be applicable for the conditions/constraints during an actual siting process. ◆ The preliminary results of the design and safety assessment would underpin the feasibility and safety of geological disposal in Japan.

FRUITING GENES OF SCHIZOPHYLLUM-COMMUNE ARE TRANSCRIPTIONALLY REGULATED

NARCIS (Netherlands)

SCHUREN, FHJ; VANDERLENDE, TR; WESSELS, JGH

Fruiting genes in Schizophyllum commune are controlled by the mating-type genes and other regulatory genes. To examine whether differential accumulation of mRNAs for these fruiting genes is caused by transcriptional regulation, run-on transcription assaYs were performed with nuclei isolated from

Time spent studying on a pre-registration nursing programme module: an exploratory study and implications for regulation.

Science.gov (United States)

Snelling, Paul C; Lipscomb, Martin; Lockyer, Lesley; Yates, Sue; Young, Pat

2010-11-01

European Union (EU) regulations require that university programmes are of specified duration. Additional EU regulations apply specifically to university based nurse education, enacted in the UK by the Nursing and Midwifery Council (NMC). However, little is known about how much time student nurses spend on their studies. In this exploratory study, students undertaking a single module in the pre-registration diploma programme at an English university were asked to keep a log of learning activity for the duration of the module. Twenty-six students completed the log. These students achieved higher grades and attended more lectures than the average for the module. The mean study time was 128.4 h against a regulatory assumption that the module should take 200 h. More than half of the 26 students undertook paid work during the module run, though this work was not associated with poorer performance. Problems in regulation for course duration are discussed and it is suggested that undertaking a 4600 h course in 3 years is problematic. More research is required so that patterns of study can be better understood and student centred programmes meeting regulatory requirements developed. Copyright © 2010 Elsevier Ltd. All rights reserved.

Cis-regulatory RNA elements that regulate specialized ribosome activity.

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Xue, Shifeng; Barna, Maria

2015-01-01

Recent evidence has shown that the ribosome itself can play a highly regulatory role in the specialized translation of specific subpools of mRNAs, in particular at the level of ribosomal proteins (RP). However, the mechanism(s) by which this selection takes place has remained poorly understood. In our recent study, we discovered a combination of unique RNA elements in the 5'UTRs of mRNAs that allows for such control by the ribosome. These mRNAs contain a Translation Inhibitory Element (TIE) that inhibits general cap-dependent translation, and an Internal Ribosome Entry Site (IRES) that relies on a specific RP for activation. The unique combination of an inhibitor of general translation and an activator of specialized translation is key to ribosome-mediated control of gene expression. Here we discuss how these RNA regulatory elements provide a new level of control to protein expression and their implications for gene expression, organismal development and evolution.

Deadenylase depletion protects inherited mRNAs in primordial germ cells.

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Swartz, S Zachary; Reich, Adrian M; Oulhen, Nathalie; Raz, Tal; Milos, Patrice M; Campanale, Joseph P; Hamdoun, Amro; Wessel, Gary M

2014-08-01

A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a 'time capsule' model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo. © 2014. Published by The Company of Biologists Ltd.

Stress and Withdrawal from Chronic Ethanol Induce Selective Changes in Neuroimmune mRNAs in Differing Brain Sites

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Darin J. Knapp

2016-07-01

Full Text Available Stress is a strong risk factor in alcoholic relapse and may exert effects that mimic aspects of chronic alcohol exposure on neurobiological systems. With the neuroimmune system becoming a prominent focus in the study of the neurobiological consequences of stress, as well as chronic alcohol exposure proving to be a valuable focus in this regard, the present study sought to compare the effects of stress and chronic ethanol exposure on induction of components of the neuroimmune system. Rats were exposed to either 1 h exposure to a mild stressor (restraint or exposure to withdrawal from 15 days of chronic alcohol exposure (i.e., withdrawal from chronic ethanol, WCE and assessed for neuroimmune mRNAs in brain. Restraint stress alone elevated chemokine (C–C motif ligand 2 (CCL2, interleukin-1-beta (IL-1β, tumor necrosis factor alpha (TNFα and toll-like receptor 4 (TLR4 mRNAs in the cerebral cortex within 4 h with a return to a control level by 24 h. These increases were not accompanied by an increase in corresponding proteins. Withdrawal from WCE also elevated cytokines, but did so to varying degrees across different cytokines and brain regions. In the cortex, stress and WCE induced CCL2, TNFα, IL-1β, and TLR4 mRNAs. In the hypothalamus, only WCE induced cytokines (CCL2 and IL-1β while in the hippocampus, WCE strongly induced CCL2 while stress and WCE induced IL-1β. In the amygdala, only WCE induced CCL2. Finally—based on the previously demonstrated role of corticotropin-releasing factor 1 (CRF1 receptor inhibition in blocking WCE-induced cytokine mRNAs—the CRF1 receptor antagonist CP154,526 was administered to a subgroup of stressed rats and found to be inactive against induction of CCL2, TNFα, or IL-1β mRNAs. These differential results suggest that stress and WCE manifest broad neuroimmune effects in brain depending on the cytokine and brain region, and that CRF inhibition may not be a relevant mechanism in non-alcohol exposed animals

A quantitative comparison of cell-type-specific microarray gene expression profiling methods in the mouse brain.

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Benjamin W Okaty

Full Text Available Expression profiling of restricted neural populations using microarrays can facilitate neuronal classification and provide insight into the molecular bases of cellular phenotypes. Due to the formidable heterogeneity of intermixed cell types that make up the brain, isolating cell types prior to microarray processing poses steep technical challenges that have been met in various ways. These methodological differences have the potential to distort cell-type-specific gene expression profiles insofar as they may insufficiently filter out contaminating mRNAs or induce aberrant cellular responses not normally present in vivo. Thus we have compared the repeatability, susceptibility to contamination from off-target cell-types, and evidence for stress-responsive gene expression of five different purification methods--Laser Capture Microdissection (LCM, Translating Ribosome Affinity Purification (TRAP, Immunopanning (PAN, Fluorescence Activated Cell Sorting (FACS, and manual sorting of fluorescently labeled cells (Manual. We found that all methods obtained comparably high levels of repeatability, however, data from LCM and TRAP showed significantly higher levels of contamination than the other methods. While PAN samples showed higher activation of apoptosis-related, stress-related and immediate early genes, samples from FACS and Manual studies, which also require dissociated cells, did not. Given that TRAP targets actively translated mRNAs, whereas other methods target all transcribed mRNAs, observed differences may also reflect translational regulation.

Death of a dogma: eukaryotic mRNAs can code for more than one protein.

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Mouilleron, Hélène; Delcourt, Vivian; Roucou, Xavier

2016-01-08

mRNAs carry the genetic information that is translated by ribosomes. The traditional view of a mature eukaryotic mRNA is a molecule with three main regions, the 5' UTR, the protein coding open reading frame (ORF) or coding sequence (CDS), and the 3' UTR. This concept assumes that ribosomes translate one ORF only, generally the longest one, and produce one protein. As a result, in the early days of genomics and bioinformatics, one CDS was associated with each protein-coding gene. This fundamental concept of a single CDS is being challenged by increasing experimental evidence indicating that annotated proteins are not the only proteins translated from mRNAs. In particular, mass spectrometry (MS)-based proteomics and ribosome profiling have detected productive translation of alternative open reading frames. In several cases, the alternative and annotated proteins interact. Thus, the expression of two or more proteins translated from the same mRNA may offer a mechanism to ensure the co-expression of proteins which have functional interactions. Translational mechanisms already described in eukaryotic cells indicate that the cellular machinery is able to translate different CDSs from a single viral or cellular mRNA. In addition to summarizing data showing that the protein coding potential of eukaryotic mRNAs has been underestimated, this review aims to challenge the single translated CDS dogma. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genome-wide discovery and differential regulation of conserved and novel microRNAs in chickpea via deep sequencing.

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Jain, Mukesh; Chevala, V V S Narayana; Garg, Rohini

2014-11-01

MicroRNAs (miRNAs) are essential components of complex gene regulatory networks that orchestrate plant development. Although several genomic resources have been developed for the legume crop chickpea, miRNAs have not been discovered until now. For genome-wide discovery of miRNAs in chickpea (Cicer arietinum), we sequenced the small RNA content from seven major tissues/organs employing Illumina technology. About 154 million reads were generated, which represented more than 20 million distinct small RNA sequences. We identified a total of 440 conserved miRNAs in chickpea based on sequence similarity with known miRNAs in other plants. In addition, 178 novel miRNAs were identified using a miRDeep pipeline with plant-specific scoring. Some of the conserved and novel miRNAs with significant sequence similarity were grouped into families. The chickpea miRNAs targeted a wide range of mRNAs involved in diverse cellular processes, including transcriptional regulation (transcription factors), protein modification and turnover, signal transduction, and metabolism. Our analysis revealed several miRNAs with differential spatial expression. Many of the chickpea miRNAs were expressed in a tissue-specific manner. The conserved and differential expression of members of the same miRNA family in different tissues was also observed. Some of the same family members were predicted to target different chickpea mRNAs, which suggested the specificity and complexity of miRNA-mediated developmental regulation. This study, for the first time, reveals a comprehensive set of conserved and novel miRNAs along with their expression patterns and putative targets in chickpea, and provides a framework for understanding regulation of developmental processes in legumes. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Sink limitation induces the expression of multiple soybean vegetative lipoxygenase mRNAs while the endogenous jasmonic acid level remains low.

Science.gov (United States)

Bunker, T W; Koetje, D S; Stephenson, L C; Creelman, R A; Mullet, J E; Grimes, H D

1995-08-01

The response of individual members of the lipoxygenase multigene family in soybeans to sink deprivation was analyzed. RNase protection assays indicated that a novel vegetative lipoxygenase gene, vlxC, and three other vegetative lipoxygenase mRNAs accumulated in mature leaves in response to a variety of sink limitations. These data suggest that several members of the lipoxygenase multigene family are involved in assimilate partitioning. The possible involvement of jasmonic acid as a signaling molecule regulating assimilate partitioning into the vegetative storage proteins and lipoxygenases was directly assessed by determining the endogenous level of jasmonic acid in leaves from plants with their pods removed. There was no rise in the level of endogenous jasmonic acid coincident with the strong increase in both vlxC and vegetative storage protein VspB transcripts in response to sink limitation. Thus, expression of the vegetative lipoxygenases and vegetative storage proteins is not regulated by jasmonic acid in sink-limited leaves.

Regnase-1 Maintains Iron Homeostasis via the Degradation of Transferrin Receptor 1 and Prolyl-Hydroxylase-Domain-Containing Protein 3 mRNAs

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Masanori Yoshinaga

2017-05-01

Full Text Available 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.

Developmental expression of the G protein-coupled receptor 54 and three GnRH mRNAs in the teleost fish cobia.

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Mohamed, J Shaik; Benninghoff, Abby D; Holt, G Joan; Khan, Izhar A

2007-02-01

The cDNAs of the G protein-coupled receptor 54 (GPR54) and three prepro-gonadotropin-releasing hormones, GnRH-I (seabream GnRH), GnRH-II (chicken GnRH-II), and GnRH-III (salmon GnRH) were isolated and cloned from the brain of the teleost fish cobia, Rachycentron canadum. The cobia GPR54 cDNA was 95 and 51-56% identical to those of tilapia and mammalian models respectively. The GnRH cDNA sequences of cobia showed strong identities to those of tilapia, Atlantic croaker, red drum, and the seabass and seabream species. The real-time quantitative RT-PCR methods allowed detection of all three GnRH mRNAs on the first day after hatching (DAH). The GnRH-I mRNA levels, which were the lowest among the three GnRHs, increased gradually with two distinct peaks in larvae at 3 and 4 DAH. On the other hand, GnRH-II and GnRH-III mRNAs were significantly higher in larvae at 2 and 6 DAH compared with those on the preceding days. In addition, significant peaks of all the three GnRH mRNAs were observed in the brains of 26-day-old fish. The finding of higher GnRH-I and GnRH-II mRNAs in males than females at 153 DAH may be related to early puberty observed during the first year in laboratory-reared male cobia. Moreover, this study demonstrates for the first time the expression of GPR54 mRNA during larval development in a vertebrate species. The concomitant expression patterns of GPR54 and GnRH mRNAs during different stages of larval and juvenile developments, and during early puberty in male cobia suggest a potential relationship between GPR54 and multiple GnRHs during these stages of development consistent with the role of GPR54 in controlling GnRH release in mammals. The increase in GPR54 and GnRH mRNAs observed during early puberty in cobia is consistent with a similar change reported in pubertal rats. This finding together with the localization of GPR54 mRNAs on GnRH neurons in fish and mammals suggests that the GPR54-GnRH interactions may be conserved in different vertebrate groups.

Combinatorial regulation of tissue specification by GATA and FOG factors

Science.gov (United States)

Chlon, Timothy M.; Crispino, John D.

2012-01-01

The development of complex organisms requires the formation of diverse cell types from common stem and progenitor cells. GATA family transcriptional regulators and their dedicated co-factors, termed Friend of GATA (FOG) proteins, control cell fate and differentiation in multiple tissue types from Drosophila to man. FOGs can both facilitate and antagonize GATA factor transcriptional regulation depending on the factor, cell, and even the specific gene target. In this review, we highlight recent studies that have elucidated mechanisms by which FOGs regulate GATA factor function and discuss how these factors use these diverse modes of gene regulation to control cell lineage specification throughout metazoans. PMID:23048181

77 FR 189 - Federal Acquisition Regulation; Brand-Name Specifications

Science.gov (United States)

2012-01-03

... 2006-0020, Sequence 26] RIN 9000-AK55 Federal Acquisition Regulation; Brand-Name Specifications... Management and Budget memoranda on brand-name specifications. DATES: Effective Date: February 2, 2012. FOR... brand- name specifications. Eight respondents submitted 32 comments in response to the interim rule. The...

Expression of the somatostatin receptor family mRNAs in lung cancer

International Nuclear Information System (INIS)

Wang Jing; Wang Liangang; Deng Jinglan; Wu Shengxi

2000-01-01

To investigate the characteristics of expression and distribution of 5 subtypes of somatostatin receptors (SSTR1-5) in lung cancer, in situ hybridization was used to examine the expression patterns of SSTR mRNAs in 21 cases of different pathologic types of lung cancer tissues with [α- 35 S]dATP labelled oligonucleotides of the 5 SSTR subtypes as probes. Additionally, Leica Q-500 image analysis processing system was employed for the semi-quantitatively analysis of the hybridization signals. Patterns of SSTR1-5 expression in lung cancer tissues were found as follows. SSTR2 was prominent in small cell lung cancer (SCLC), whereas in non-small cell lung cancer (NSCLC) including the adenous cancer (Ad) and the squamous cancer (Sq), the expression of SSTR1 mRNA was stronger than that of the other 4 types. the expression density of SSTR1-5 in the NSCLC was higher that the SCLC (p < 0.01). The expression patterns and densities of the SSTR subtypes showed heterogeneity in different pathologic types of lung cancer. The expressions of the SSTR mRNAs in both SCLC and NSCLC indicated the positive prospects for somatostatin analog (SSA)-oriented agents in the treatment of both types of the lung cancer

Affect and person specificity in mood regulation

OpenAIRE

Corby, Emma Kate

2007-01-01

489 university students in three countries completed questionnaires in a study investigating affect and person specificity in the use of mood regulation strategies. The major aims of the study were to (1) describe the relationship between specific affective states and the strategies utilised, (2) explore the role that individual differences variables played in the tendency to use particular strategies, and (3) measure the impact that the use of different strategies had upon subjective well-b...

Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs.

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Sarit Edelheit

2013-06-01

Full Text Available The presence of 5-methylcytidine (m(5C in tRNA and rRNA molecules of a wide variety of organisms was first observed more than 40 years ago. However, detection of this modification was limited to specific, abundant, RNA species, due to the usage of low-throughput methods. To obtain a high resolution, systematic, and comprehensive transcriptome-wide overview of m(5C across the three domains of life, we used bisulfite treatment on total RNA from both gram positive (B. subtilis and gram negative (E. coli bacteria, an archaeon (S. solfataricus and a eukaryote (S. cerevisiae, followed by massively parallel sequencing. We were able to recover most previously documented m(5C sites on rRNA in the four organisms, and identified several novel sites in yeast and archaeal rRNAs. Our analyses also allowed quantification of methylated m(5C positions in 64 tRNAs in yeast and archaea, revealing stoichiometric differences between the methylation patterns of these organisms. Molecules of tRNAs in which m(5C was absent were also discovered. Intriguingly, we detected m(5C sites within archaeal mRNAs, and identified a consensus motif of AUCGANGU that directs methylation in S. solfataricus. Our results, which were validated using m(5C-specific RNA immunoprecipitation, provide the first evidence for mRNA modifications in archaea, suggesting that this mode of post-transcriptional regulation extends beyond the eukaryotic domain.

CSR-1 and P granules suppress sperm-specific transcription in the C. elegans germline.

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Campbell, Anne C; Updike, Dustin L

2015-05-15

Germ granules (P granules) in C. elegans are required for fertility and function to maintain germ cell identity and pluripotency. Sterility in the absence of P granules is often accompanied by the misexpression of soma-specific proteins and the initiation of somatic differentiation in germ cells. To investigate whether this is caused by the accumulation of somatic transcripts, we performed mRNA-seq on dissected germlines with and without P granules. Strikingly, we found that somatic transcripts do not increase in the young adult germline when P granules are impaired. Instead, we found that impairing P granules causes sperm-specific mRNAs to become highly overexpressed. This includes the accumulation of major sperm protein (MSP) transcripts in germ cells, a phenotype that is suppressed by feminization of the germline. A core component of P granules, the endo-siRNA-binding Argonaute protein CSR-1, has recently been ascribed with the ability to license transcripts for germline expression. However, impairing CSR-1 has very little effect on the accumulation of its mRNA targets. Instead, we found that CSR-1 functions with P granules to prevent MSP and sperm-specific mRNAs from being transcribed in the hermaphrodite germline. These findings suggest that P granules protect germline integrity through two different mechanisms, by (1) preventing the inappropriate expression of somatic proteins at the level of translational regulation, and by (2) functioning with CSR-1 to limit the domain of sperm-specific expression at the level of transcription. © 2015. Published by The Company of Biologists Ltd.

Thyroid hormone coordinately regulates Na sup + -K sup + -ATPase. alpha. - and. beta. -subunit mRNA levels in kidney

Energy Technology Data Exchange (ETDEWEB)

McDonough, A.A.; Brown, T.A.; Horowitz, B.; Chiu, R.; Schlotterbeck, J.; Bowen, J.; Schmitt, C.A. (Univ. of Southern California School of Medicine, Los Angeles (USA))

1988-02-01

Synthesis of the sodium pump, Na{sup +}-K{sup +}-ATPase, is regulated by thyroid hormone in responsive tissues. The purpose of this study was to determine if triiodothyronine (T{sub 3}) regulates the concentration of the mRNAs coding for the two enzyme subunits, {alpha} and {beta}, and the time course of the response. A single dose of T{sub 3} was administered to hypothyroid rats that were killed at various times after injection. In the kidney cortexes of the T{sub 3}-injected animals, as well as hypothyroid and euthyroid rats, {alpha}- and {beta}-mRNA concentrations were measured by dot blot using cDNAs corresponding to the two mRNAs; {alpha}-subunit abundance was measured by Western blot using antibodies to the enzyme, and Na{sup +}-K{sup +}-ATPase activity was measured enzymatically. {alpha}- and {beta}-mRNAs increased coordinately to 1.6-fold over hypothyroid levels by 12 h after T{sub 3}. The authors conclude that T{sub 3} regulates Na{sup +}-K{sup +}-ATPase synthesis and activity by coordinately increasing the mRNAs of both the {alpha}- and {beta}-subunits of the enzyme.

Rasputin functions as a positive regulator of orb in Drosophila oogenesis.

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Alexandre Costa

Full Text Available The determination of cell fate and the establishment of polarity axes during Drosophila oogenesis depend upon pathways that localize mRNAs within the egg chamber and control their on-site translation. One factor that plays a central role in regulating on-site translation of mRNAs is Orb. Orb is a founding member of the conserved CPEB family of RNA-binding proteins. These proteins bind to target sequences in 3' UTRs and regulate mRNA translation by modulating poly(A tail length. In addition to controlling the translation of axis-determining mRNAs like grk, fs(1K10, and osk, Orb protein autoregulates its own synthesis by binding to orb mRNA and activating its translation. We have previously shown that Rasputin (Rin, the Drosophila homologue of Ras-GAP SH3 Binding Protein (G3BP, associates with Orb in a messenger ribonucleoprotein (mRNP complex. Rin is an evolutionarily conserved RNA-binding protein believed to function as a link between Ras signaling and RNA metabolism. Here we show that Orb and Rin form a complex in the female germline. Characterization of a new rin allele shows that rin is essential for oogenesis. Co-localization studies suggest that Orb and Rin form a complex in the oocyte at different stages of oogenesis. This is supported by genetic and biochemical analyses showing that rin functions as a positive regulator in the orb autoregulatory pathway by increasing Orb protein expression. Tandem Mass Spectrometry analysis shows that several canonical stress granule proteins are associated with the Orb-Rin complex suggesting that a conserved mRNP complex regulates localized translation during oogenesis in Drosophila.

Rasputin functions as a positive regulator of orb in Drosophila oogenesis.

Science.gov (United States)

Costa, Alexandre; Pazman, Cecilia; Sinsimer, Kristina S; Wong, Li Chin; McLeod, Ian; Yates, John; Haynes, Susan; Schedl, Paul

2013-01-01

The determination of cell fate and the establishment of polarity axes during Drosophila oogenesis depend upon pathways that localize mRNAs within the egg chamber and control their on-site translation. One factor that plays a central role in regulating on-site translation of mRNAs is Orb. Orb is a founding member of the conserved CPEB family of RNA-binding proteins. These proteins bind to target sequences in 3' UTRs and regulate mRNA translation by modulating poly(A) tail length. In addition to controlling the translation of axis-determining mRNAs like grk, fs(1)K10, and osk, Orb protein autoregulates its own synthesis by binding to orb mRNA and activating its translation. We have previously shown that Rasputin (Rin), the Drosophila homologue of Ras-GAP SH3 Binding Protein (G3BP), associates with Orb in a messenger ribonucleoprotein (mRNP) complex. Rin is an evolutionarily conserved RNA-binding protein believed to function as a link between Ras signaling and RNA metabolism. Here we show that Orb and Rin form a complex in the female germline. Characterization of a new rin allele shows that rin is essential for oogenesis. Co-localization studies suggest that Orb and Rin form a complex in the oocyte at different stages of oogenesis. This is supported by genetic and biochemical analyses showing that rin functions as a positive regulator in the orb autoregulatory pathway by increasing Orb protein expression. Tandem Mass Spectrometry analysis shows that several canonical stress granule proteins are associated with the Orb-Rin complex suggesting that a conserved mRNP complex regulates localized translation during oogenesis in Drosophila.

MAPK Signal Transduction Pathway Regulation: A Novel Mechanism of Rat HSC-T6 Cell Apoptosis Induced by FUZHENGHUAYU Tablet

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Qi Wang

2013-01-01

Full Text Available FUZHENGHUAYU Tablets have been widely used in the treatment of liver fibrosis in China. Here, we investigate the apoptotic effect of FUZHENGHUAYU Tablet in rat liver stellate cell line HSC-T6. HSC-T6 cells were incubated with control serum or drug serum from rats fed with 0.9% NaCl or FUZHENGHUAYU Tablet, respectively. Cells exposed to drug serum showed higher proportions of early and late apoptotic cells than controls. The mRNA levels of collagens I and III, TGF-β1 and α-SMA were reduced by drug serum compared to control serum. Differentially expressed mRNAs and miRNAs were analyzed by microarray and sequencing, respectively. We identified 334 differentially expressed mRNAs and also 60 GOs and two pathways related to the mRNAs. Seventy-five differentially expressed miRNAs were down-regulated by drug serum and 1963 target genes were predicted. 134 GOs up-regulated in drug serum group were linked to miRNA targets, and drug serum also regulated 43 miRNA signal transduction pathways. Protein levels were evaluated by Western blot. Drug serum down-regulated (phospho-SAPK/JNK/(SAPK/JNK and up-regulated phospho-p38/p38 ratios. The study showed that FUZHENGHUAYU Tablet induced apoptosis in rat HSC-T6 cells possibly in part by activating p38 and inhibiting SAPK/JNK.

Theoretical studies on sRNA-mediated regulation in bacteria

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Chang, Xiao-Xue; Xu, Liu-Fang; Shi, Hua-Lin

2015-12-01

Small RNA(sRNA)-mediated post-transcriptional regulation differs from protein-mediated regulation. Through base-pairing, sRNA can regulate the target mRNA in a catalytic or stoichiometric manner. Some theoretical models were built for comparison of the protein-mediated and sRNA-mediated modes in the steady-state behaviors and noise properties. Many experiments demonstrated that a single sRNA can regulate several mRNAs, which causes crosstalk between the targets. Here, we focus on some models in which two target mRNAs are silenced by the same sRNA to discuss their crosstalk features. Additionally, the sequence-function relationship of sRNA and its role in the kinetic process of base-pairing have been highlighted in model building. Project supported by the National Basic Research Program of China (Grant No. 2013CB834100), the National Natural Science Foundation of China (Grant Nos. 11121403 and 11274320), the Open Project Program of State Key Laboratory of Theoretical Physics, Institute of Theoretical Physics, Chinese Academy of Sciences, China (Grant No. Y4KF171CJ1), the National Natural Science Foundation for Young Scholar of China (Grant No. 11304115), and the China Postdoctoral Science Foundation (Grant No. 2013M541282).

The fission yeast RNA binding protein Mmi1 regulates meiotic genes by controlling intron specific splicing and polyadenylation coupled RNA turnover.

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Huei-Mei Chen

Full Text Available The polyA tails of mRNAs are monitored by the exosome as a quality control mechanism. We find that fission yeast, Schizosaccharomyces pombe, adopts this RNA quality control mechanism to regulate a group of 30 or more meiotic genes at the level of both splicing and RNA turnover. In vegetative cells the RNA binding protein Mmi1 binds to the primary transcripts of these genes. We find the novel motif U(U/C/GAAAC highly over-represented in targets of Mmi1. Mmi1 can specifically regulate the splicing of particular introns in a transcript: it inhibits the splicing of introns that are in the vicinity of putative Mmi1 binding sites, while allowing the splicing of other introns that are far from such sites. In addition, binding of Mmi1, particularly near the 3' end, alters 3' processing to promote extremely long polyA tails of up to a kilobase. The hyperadenylated transcripts are then targeted for degradation by the nuclear exonuclease Rrp6. The nuclear polyA binding protein Pab2 assists this hyperadenylation-mediated RNA decay. Rrp6 also targets other hyperadenylated transcripts, which become hyperadenylated in an unknown, but Mmi1-independent way. Thus, hyperadenylation may be a general signal for RNA degradation. In addition, binding of Mmi1 can affect the efficiency of 3' cleavage. Inactivation of Mmi1 in meiosis allows meiotic expression, through splicing and RNA stabilization, of at least 29 target genes, which are apparently constitutively transcribed.

Tribolium castaneum Transformer-2 regulates sex determination and development in both males and females.

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Shukla, Jayendra Nath; Palli, Subba Reddy

2013-12-01

Tribolium castaneum Transformer (TcTra) is essential for female sex determination and maintenance through the regulation of sex-specific splicing of doublesex (dsx) pre-mRNA. In females, TcTra also regulates the sex-specific splicing of its own pre-mRNA to ensure continuous production of functional Tra protein. Transformer protein is absent in males and hence dsx pre-mRNA is spliced in a default mode. The mechanisms by which males inhibit the production of functional Tra protein are not known. Here, we report on functional characterization of transformer-2 (tra-2) gene (an ortholog of Drosophila transformer-2) in T. castaneum. RNA interference-mediated knockdown in the expression of gene coding for tra-2 in female pupae or adults resulted in the production of male-specific isoform of dsx and both female and male isoforms of tra suggesting that Tra-2 is essential for the female-specific splicing of tra and dsx pre-mRNAs. Interestingly, knockdown of tra-2 in males did not affect the splicing of dsx but resulted in the production of both female and male isoforms of tra suggesting that Tra-2 suppresses female-specific splicing of tra pre-mRNA in males. This dual regulation of sex-specific splicing of tra pre-mRNA ensures a tight regulation of sex determination and maintenance. These data suggest a critical role for Tra-2 in suppression of female sex determination cascade in males. In addition, RNAi studies showed that Tra-2 is also required for successful embryonic and larval development in both sexes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Regulation of Translation Initiation under Biotic and Abiotic Stresses

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Ana B. Castro-Sanz

2013-02-01

Full Text Available Plants have developed versatile strategies to deal with the great variety of challenging conditions they are exposed to. Among them, the regulation of translation is a common target to finely modulate gene expression both under biotic and abiotic stress situations. Upon environmental challenges, translation is regulated to reduce the consumption of energy and to selectively synthesize proteins involved in the proper establishment of the tolerance response. In the case of viral infections, the situation is more complex, as viruses have evolved unconventional mechanisms to regulate translation in order to ensure the production of the viral encoded proteins using the plant machinery. Although the final purpose is different, in some cases, both plants and viruses share common mechanisms to modulate translation. In others, the mechanisms leading to the control of translation are viral- or stress-specific. In this paper, we review the different mechanisms involved in the regulation of translation initiation under virus infection and under environmental stress in plants. In addition, we describe the main features within the viral RNAs and the cellular mRNAs that promote their selective translation in plants undergoing biotic and abiotic stress situations.

Genome-wide RIP-Chip analysis of translational repressor-bound mRNAs in the Plasmodium gametocyte

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Guerreiro, Ana; Deligianni, Elena; Santos, Jorge M; Silva, Patricia AGC; Louis, Christos; Pain, Arnab; Janse, Chris J; Franke-Fayard, Blandine; Carret, Celine K; Siden-Kiamos, Inga; Mair, Gunnar R

2014-01-01

of RNA polymerase II transcription. Using GFP-tagging, we validate the repression phenotype of selected genes and identify mRNAs relying on the 5′ untranslated region for translational control. Gene deletion reveals a novel protein located in the ookinete

Cup regulates oskar mRNA stability during oogenesis.

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Broyer, Risa M; Monfort, Elena; Wilhelm, James E

2017-01-01

The proper regulation of the localization, translation, and stability of maternally deposited transcripts is essential for embryonic development in many organisms. These different forms of regulation are mediated by the various protein subunits of the ribonucleoprotein (RNP) complexes that assemble on maternal mRNAs. However, while many of the subunits that regulate the localization and translation of maternal transcripts have been identified, relatively little is known about how maternal mRNAs are stockpiled and stored in a stable form to support early development. One of the best characterized regulators of maternal transcripts is Cup - a broadly conserved component of the maternal RNP complex that in Drosophila acts as a translational repressor of the localized message oskar. In this study, we have found that loss of cup disrupts the localization of both the oskar mRNA and its associated proteins to the posterior pole of the developing oocyte. This defect is not due to a failure to specify the oocyte or to disruption of RNP transport. Rather, the localization defects are due to a drop in oskar mRNA levels in cup mutant egg chambers. Thus, in addition to its role in regulating oskar mRNA translation, Cup also plays a critical role in controlling the stability of the oskar transcript. This suggests that Cup is ideally positioned to coordinate the translational control function of the maternal RNP complex with its role in storing maternal transcripts in a stable form. Published by Elsevier Inc.

Increases of heat shock proteins and their mRNAs at high hydrostatic pressure in a deep-sea piezophilic bacterium, Shewanella violacea.

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Sato, Hiroshi; Nakasone, Kaoru; Yoshida, Takao; Kato, Chiaki; Maruyama, Tadashi

2015-07-01

When non-extremophiles encounter extreme environmental conditions, which are natural for the extremophiles, stress reactions, e.g., expression of heat shock proteins (HSPs), are thought to be induced for survival. To understand how the extremophiles live in such extreme environments, we studied the effects of high hydrostatic pressure on cellular contents of HSPs and their mRNAs during growth in a piezophilic bacterium, Shewanella violacea. HSPs increased at high hydrostatic pressures even when optimal for growth. The mRNAs and proteins of these HSPs significantly increased at higher hydrostatic pressure in S. violacea. In the non-piezophilic Escherichia coli, however, their mRNAs decreased, while their proteins did not change. Several transcriptional start sites (TSSs) for HSP genes were determined by the primer extension method and some of them showed hydrostatic pressure-dependent increase of the mRNAs. A major refolding target of one of the HSPs, chaperonin, at high hydrostatic pressure was shown to be RplB, a subunit of the 50S ribosome. These results suggested that in S. violacea, HSPs play essential roles, e.g., maintaining protein complex machinery including ribosomes, in the growth and viability at high hydrostatic pressure, and that, in their expression, the transcription is under the control of σ(32).

Primary Auditory Cortex Regulates Threat Memory Specificity

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Wigestrand, Mattis B.; Schiff, Hillary C.; Fyhn, Marianne; LeDoux, Joseph E.; Sears, Robert M.

2017-01-01

Distinguishing threatening from nonthreatening stimuli is essential for survival and stimulus generalization is a hallmark of anxiety disorders. While auditory threat learning produces long-lasting plasticity in primary auditory cortex (Au1), it is not clear whether such Au1 plasticity regulates memory specificity or generalization. We used…

The specificity of long noncoding RNA expression.

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Gloss, Brian S; Dinger, Marcel E

2016-01-01

Over the last decade, long noncoding RNAs (lncRNAs) have emerged as a fundamental molecular class whose members play pivotal roles in the regulation of the genome. The observation of pervasive transcription of mammalian genomes in the early 2000s sparked a revolution in the understanding of information flow in eukaryotic cells and the incredible flexibility and dynamic nature of the transcriptome. As a molecular class, distinct loci yielding lncRNAs are set to outnumber those yielding mRNAs. However, like many important discoveries, the road leading to uncovering this diverse class of molecules that act through a remarkable repertoire of mechanisms, was not a straight one. The same characteristic that most distinguishes lncRNAs from mRNAs, i.e. their developmental-stage, tissue-, and cell-specific expression, was one of the major impediments to their discovery and recognition as potentially functional regulatory molecules. With growing numbers of lncRNAs being assigned to biological functions, the specificity of lncRNA expression is now increasingly recognized as a characteristic that imbues lncRNAs with great potential as biomarkers and for the development of highly targeted therapeutics. Here we review the history of lncRNA research and how technological advances and insight into biological complexity have gone hand-in-hand in shaping this revolution. We anticipate that as increasing numbers of these molecules, often described as the dark matter of the genome, are characterized and the structure-function relationship of lncRNAs becomes better understood, it may ultimately be feasible to decipher what these non-(protein)-coding genes encode. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. Copyright © 2015 Elsevier B.V. All rights reserved.

Regnase-1 Maintains Iron Homeostasis via the Degradation of Transferrin Receptor 1 and Prolyl-Hydroxylase-Domain-Containing Protein 3 mRNAs.

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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.

Circulating mRNAs and miRNAs as candidate markers for the diagnosis and prognosis of prostate cancer

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Souza, Marilesia Ferreira de; Kuasne, Hellen; Barros-Filho, Mateus de Camargo

2017-01-01

Circulating nucleic acids are found in free form in body fluids and may serve as minimally invasive tools for cancer diagnosis and prognosis. Only a few studies have investigated the potential application of circulating mRNAs and microRNAs (miRNAs) in prostate cancer (PCa). The Cancer Genome Atlas......RNA expression revealed eleven genes and eight miRNAs which were validated by RT-qPCR in plasma samples from 102 untreated PCa patients and 50 cancer-free individuals. Two genes, OR51E2 and SIM2, and two miRNAs, miR-200c and miR-200b, showed significant association with PCa. Expression levels...... of these transcripts distinguished PCa patients from controls (67% sensitivity and 75% specificity). PCa patients and controls with prostate-specific antigen (PSA) ≤ 4.0 ng/mL were discriminated based on OR51E2 and SIM2 expression levels. The miR-200c expression showed association with Gleason score and miR-200b...

The NS1 Protein from Influenza Virus Stimulates Translation Initiation by Enhancing Ribosome Recruitment to mRNAs.

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Panthu, Baptiste; Terrier, Olivier; Carron, Coralie; Traversier, Aurélien; Corbin, Antoine; Balvay, Laurent; Lina, Bruno; Rosa-Calatrava, Manuel; Ohlmann, Théophile

2017-10-27

The non-structural protein NS1 of influenza A viruses exerts pleiotropic functions during infection. Among these functions, NS1 was shown to be involved in the control of both viral and cellular translation; however, the mechanism by which this occurs remains to be determined. Thus, we have revisited the role of NS1 in translation by using a combination of influenza infection, mRNA reporter transfection, and in vitro functional and biochemical assays. Our data show that the NS1 protein is able to enhance the translation of virtually all tested mRNAs with the exception of constructs bearing the Dicistroviruses Internal ribosome entry segment (IRESes) (DCV and CrPV), suggesting a role at the level of translation initiation. The domain of NS1 required for translation stimulation was mapped to the RNA binding amino-terminal motif of the protein with residues R38 and K41 being critical for activity. Although we show that NS1 can bind directly to mRNAs, it does not correlate with its ability to stimulate translation. This activity rather relies on the property of NS1 to associate with ribosomes and to recruit them to target mRNAs. Copyright © 2017 Elsevier Ltd. All rights reserved.

MicroRNA-212 post-transcriptionally regulates oocyte-specific basic-helix-loop-helix transcription factor, factor in the germline alpha (FIGLA, during bovine early embryogenesis.

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Swamy K Tripurani

Full Text Available Factor in the germline alpha (FIGLA is an oocyte-specific basic helix-loop-helix transcription factor essential for primordial follicle formation and expression of many genes required for folliculogenesis, fertilization and early embryonic survival. Here we report the characterization of bovine FIGLA gene and its regulation during early embryogenesis. Bovine FIGLA mRNA expression is restricted to gonads and is detected in fetal ovaries harvested as early as 90 days of gestation. FIGLA mRNA and protein are abundant in germinal vesicle and metaphase II stage oocytes, as well as in embryos from pronuclear to eight-cell stage but barely detectable at morula and blastocyst stages, suggesting that FIGLA might be a maternal effect gene. Recent studies in zebrafish and mice have highlighted the importance of non-coding small RNAs (microRNAs as key regulatory molecules targeting maternal mRNAs for degradation during embryonic development. We hypothesized that FIGLA, as a maternal transcript, is regulated by microRNAs during early embryogenesis. Computational predictions identified a potential microRNA recognition element (MRE for miR-212 in the 3' UTR of the bovine FIGLA mRNA. Bovine miR-212 is expressed in oocytes and tends to increase in four-cell and eight-cell stage embryos followed by a decline at morula and blastocyst stages. Transient transfection and reporter assays revealed that miR-212 represses the expression of FIGLA in a MRE dependent manner. In addition, ectopic expression of miR-212 mimic in bovine early embryos dramatically reduced the expression of FIGLA protein. Collectively, our results demonstrate that FIGLA is temporally regulated during bovine early embryogenesis and miR-212 is an important negative regulator of FIGLA during the maternal to zygotic transition in bovine embryos.

SRSF1 Prevents DNA Damage and Promotes Tumorigenesis through Regulation of DBF4B Pre-mRNA Splicing

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Linlin Chen

2017-12-01

Full Text Available Dysregulated alternative splicing events have been implicated in many types of cancer, but the underlying molecular mechanisms remain unclear. Here, we observe that the splicing factor SRSF1 regulates DBF4B exon6 splicing by specifically binding and promoting its inclusion. Knockdown of the exon6-containing isoform (DBF4B-FL significantly inhibits the tumorigenic potential of colon cancer cells in vitro and in mice, and SRSF1 inactivation phenocopies DBF4B-FL depletion. DBF4B-FL and SRSF1 are required for cancer cell proliferation and for the maintenance of genomic stability. Overexpression of DBF4B-FL can protect against DNA damage induced by SRSF1 knockdown and rescues growth defects in SRSF1-depleted cells. Increased DBF4B exon6 inclusion parallels SRSF1 upregulation in clinical colorectal cancer samples. Taken together, our findings identify SRSF1 as a key regulator of DBF4B pre-mRNA splicing dysregulation in colon cancer, with possible clinical implications as candidate prognostic factors in cancer patients.

miRNA and mRNA Expression Profiles Reveal Insight into Chitosan-Mediated Regulation of Plant Growth.

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Zhang, Xiaoqian; Li, Kecheng; Xing, Ronge; Liu, Song; Chen, Xiaolin; Yang, Haoyue; Li, Pengcheng

2018-04-18

Chitosan has been numerously studied as a plant growth regulator and stress tolerance inducer. To investigate the roles of chitosan as bioregulator on plant and unravel its possible metabolic responses mechanisms, we simultaneously investigated mRNAs and microRNAs (miRNAs) expression profiles of wheat seedlings in response to chitosan heptamer. We found 400 chitosan-responsive differentially expressed genes, including 268 up-regulated and 132 down-regulated mRNAs, many of which were related to photosynthesis, primary carbon and nitrogen metabolism, defense responses, and transcription factors. Moreover, miRNAs also participate in chitosan-mediated regulation on plant growth. We identified 87 known and 21 novel miRNAs, among which 56 miRNAs were induced or repressed by chitosan heptamer, such as miRNA156, miRNA159a, miRNA164, miRNA171a, miRNA319, and miRNA1127. The integrative analysis of miRNA and mRNA expression profiles in this case provides fundamental information for further investigation of regulation mechanisms of chitosan on plant growth and will facilitate its application in agriculture.

Selective nuclear export of specific classes of mRNA from mammalian nuclei is promoted by GANP

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Wickramasinghe, Vihandha O.; Andrews, Robert; Ellis, Peter; Langford, Cordelia; Gurdon, John B.; Stewart, Murray; Venkitaraman, Ashok R.; Laskey, Ronald A.

2014-01-01

The nuclear phase of the gene expression pathway culminates in the export of mature messenger RNAs (mRNAs) to the cytoplasm through nuclear pore complexes. GANP (germinal- centre associated nuclear protein) promotes the transfer of mRNAs bound to the transport factor NXF1 to nuclear pore complexes. Here, we demonstrate that GANP, subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex, promotes selective nuclear export of a specific subset of mRNAs whose transport depends on NXF1. Genome-wide gene expression profiling showed that half of the transcripts whose nuclear export was impaired following NXF1 depletion also showed reduced export when GANP was depleted. GANP-dependent transcripts were highly expressed, yet short-lived, and were highly enriched in those encoding central components of the gene expression machinery such as RNA synthesis and processing factors. After injection into Xenopus oocyte nuclei, representative GANP-dependent transcripts showed faster nuclear export kinetics than representative transcripts that were not influenced by GANP depletion. We propose that GANP promotes the nuclear export of specific classes of mRNAs that may facilitate rapid changes in gene expression. PMID:24510098

RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells.

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Pandolfini, Luca; Luzi, Ettore; Bressan, Dario; Ucciferri, Nadia; Bertacchi, Michele; Brandi, Rossella; Rocchiccioli, Silvia; D'Onofrio, Mara; Cremisi, Federico

2016-05-06

Embryonic stem cells are intrinsically unstable and differentiate spontaneously if they are not shielded from external stimuli. Although the nature of such instability is still controversial, growing evidence suggests that protein translation control may play a crucial role. We performed an integrated analysis of RNA and proteins at the transition between naïve embryonic stem cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators are specifically released from translational inhibition mediated by RNA-induced silencing complex (RISC). This suggests that, prior to differentiation, the propensity of embryonic stem cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators is reinstated following acute inactivation of RISC and it correlates with loss of stemness markers and activation of early cell differentiation markers in treated embryonic stem cells. We propose that RISC-mediated inhibition of specific sets of chromatin regulators is a primary mechanism for preserving embryonic stem cell pluripotency while inhibiting the onset of embryonic developmental programs.

MicroRNAs, Regulatory Networks, and Comorbidities

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Russo, Francesco; Belling, Kirstine; Jensen, Anders Boeck

2017-01-01

MicroRNAs (miRNAs) are small noncoding RNAs involved in the posttranscriptional regulation of messenger RNAs (mRNAs). Each miRNA targets a specific set of mRNAs. Upon binding the miRNA inhibits mRNA translation or facilitate mRNA degradation. miRNAs are frequently deregulated in several pathologies...

Pre-Ischemic Treadmill Training for Prevention of Ischemic Brain Injury via Regulation of Glutamate and Its Transporter GLT-1

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Jingchun Guo

2012-07-01

Full Text Available Pre-ischemic treadmill training exerts cerebral protection in the prevention of cerebral ischemia by alleviating neurotoxicity induced by excessive glutamate release following ischemic stroke. However, the underlying mechanism of this process remains unclear. Cerebral ischemia-reperfusion injury was observed in a rat model after 2 weeks of pre-ischemic treadmill training. Cerebrospinal fluid was collected using the microdialysis sampling method, and the concentration of glutamate was determined every 40 min from the beginning of ischemia to 4 h after reperfusion with high-performance liquid chromatography (HPLC-fluorescence detection. At 3, 12, 24, and 48 h after ischemia, the expression of the glutamate transporter-1 (GLT-1 protein in brain tissues was determined by Western blot respectively. The effect of pre-ischemic treadmill training on glutamate concentration and GLT-1 expression after cerebral ischemia in rats along with changes in neurobehavioral score and cerebral infarct volume after 24 h ischemia yields critical information necessary to understand the protection mechanism exhibited by pre-ischemic treadmill training. The results demonstrated that pre-ischemic treadmill training up-regulates GLT-1 expression, decreases extracellular glutamate concentration, reduces cerebral infarct volume, and improves neurobehavioral score. Pre-ischemic treadmill training is likely to induce neuroprotection after cerebral ischemia by regulating GLT-1 expression, which results in re-uptake of excessive glutamate.

Antisense sequences and antagomiR 155 in therapy for B lymphomas over expressing miR-155: preclinical models and identification of target mRNAs

International Nuclear Information System (INIS)

Marziali, G.; Peschle, C.

2009-01-01

Micro RNAs (miRNAs) are a conserved class of small noncoding RNAs (22-25 nucleotides), which modulate gene expression at post-transcriptional level by base pairing to the 3'UTR of the target mRNAs, thus causing messenger degradation or inhibiting its translation. Experimental evidence indicate that several miRNAs are deregulated in human tumors. MIRN155 has been shown to be highly expressed in a variety of human B cell lymphomas, especially diffuse large B cells, Hodgkin, and a subset of Burkitt lymphomas. Its expression is physiologically increased in activated B and T cells and it plays a key role in regulating the homeostasis and function of the immune system

Cell Volume Regulation and Signaling in 3T3-L1 Pre-adipocytes and Adipocytes

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Eduardsen, Kathrine; Larsen, Susanne; Novak, Ivana

2011-01-01

Caveolae have been implicated in sensing of cell volume perturbations, yet evidence is still limited and findings contradictory. Here, we investigated the possible role of caveolae in cell volume regulation and volume sensitive signaling in an adipocyte system with high (3T3-L1 adipocytes......); intermediate (3T3-L1 pre-adipocytes); and low (cholesterol-depleted 3T3-L1 pre-adipocytes) caveolae levels. Using large-angle light scattering, we show that compared to pre-adipocytes, differentiated adipocytes exhibit several-fold increased rates of volume restoration following osmotic cell swelling (RVD......) and osmotic cell shrinkage (RVI), accompanied by increased swelling-activated taurine efflux. However, caveolin-1 distribution was not detectably altered after osmotic swelling or shrinkage, and caveolae integrity, as studied by cholesterol depletion or expression of dominant negative Cav-1, was not required...

Nascent-Seq reveals novel features of mouse circadian transcriptional regulation

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Menet, Jerome S; Rodriguez, Joseph; Abruzzi, Katharine C; Rosbash, Michael

2012-01-01

A substantial fraction of the metazoan transcriptome undergoes circadian oscillations in many cells and tissues. Based on the transcription feedback loops important for circadian timekeeping, it is commonly assumed that this mRNA cycling reflects widespread transcriptional regulation. To address this issue, we directly measured the circadian dynamics of mouse liver transcription using Nascent-Seq (genome-wide sequencing of nascent RNA). Although many genes are rhythmically transcribed, many rhythmic mRNAs manifest poor transcriptional rhythms, indicating a prominent contribution of post-transcriptional regulation to circadian mRNA expression. This analysis of rhythmic transcription also showed that the rhythmic DNA binding profile of the transcription factors CLOCK and BMAL1 does not determine the transcriptional phase of most target genes. This likely reflects gene-specific collaborations of CLK:BMAL1 with other transcription factors. These insights from Nascent-Seq indicate that it should have broad applicability to many other gene expression regulatory issues. DOI: http://dx.doi.org/10.7554/eLife.00011.001 PMID:23150795

TTP specifically regulates the internalization of the transferrin receptor

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Tosoni, Daniela; Puri, Claudia; Confalonieri, Stefano

2005-01-01

Different plasma membrane receptors are internalized through saturable/noncompetitive pathways, suggesting cargo-specific regulation. Here, we report that TTP (SH3BP4), a SH3-containing protein, specifically regulates the internalization of the transferrin receptor (TfR). TTP interacts...... with endocytic proteins, including clathrin, dynamin, and the TfR, and localizes selectively to TfR-containing coated-pits (CCP) and -vesicles (CCV). Overexpression of TTP specifically inhibits TfR internalization, and causes the formation of morphologically aberrant CCP, which are probably fission impaired....... This effect is mediated by the SH3 of TTP, which can bind to dynamin, and it is rescued by overexpression of dynamin. Functional ablation of TTP causes a reduction in TfR internalization, and reduced cargo loading and size of TfR-CCV. Tyrosine phosphorylation of either TTP or dynamin prevents...

Activity-dependent expression of ELAV/Hu RBPs and neuronal mRNAs in seizure and cocaine brain.

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Tiruchinapalli, Dhanrajan M; Caron, Marc G; Keene, Jack D

2008-12-01

Growing evidence indicates that both seizure (glutamate) and cocaine (dopamine) treatment modulate synaptic plasticity within the mesolimbic region of the CNS. Activation of glutamatergic neurons depends on the localized translation of neuronal mRNA products involved in modulating synaptic plasticity. In this study, we demonstrate the dendritic localization of HuR and HuD RNA-binding proteins (RBPs) and their association with neuronal mRNAs following these two paradigms of seizure and cocaine treatment. Both the ubiquitously expressed HuR and neuronal HuD RBPs were detected in different regions as well as within dendrites of the brain and in dissociated neurons. Quantitative analysis revealed an increase in HuR, HuD and p-glycogen synthase kinase 3beta (GSK3beta) protein levels as well as neuronal mRNAs encoding Homer, CaMKIIalpha, vascular early response gene, GAP-43, neuritin, and neuroligin protein products following either seizure or cocaine treatment. Inhibition of the Akt/GSK3beta signaling pathway by acute or chronic LiCl treatment revealed changes in HuR, HuD, pGSK3beta, p-Akt, and beta-catenin protein levels. In addition, a genetically engineered hyperdopaminergic mouse model (dopamine transporter knockout) revealed decreased expression of HuR protein levels, but no significant change was observed in HuD or fragile-X mental retardation protein RBPs. Finally, our data suggest that HuR and HuD RBPs potentially interact directly with neuronal mRNAs important for differentiation and synaptic plasticity.

Ribonuclease inhibitor 1 regulates erythropoiesis by controlling GATA1 translation.

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Chennupati, Vijaykumar; Veiga, Diogo Ft; Maslowski, Kendle M; Andina, Nicola; Tardivel, Aubry; Yu, Eric Chi-Wang; Stilinovic, Martina; Simillion, Cedric; Duchosal, Michel A; Quadroni, Manfredo; Roberts, Irene; Sankaran, Vijay G; MacDonald, H Robson; Fasel, Nicolas; Angelillo-Scherrer, Anne; Schneider, Pascal; Hoang, Trang; Allam, Ramanjaneyulu

2018-04-02

Ribosomal proteins (RP) regulate specific gene expression by selectively translating subsets of mRNAs. Indeed, in Diamond-Blackfan anemia and 5q- syndrome, mutations in RP genes lead to a specific defect in erythroid gene translation and cause anemia. Little is known about the molecular mechanisms of selective mRNA translation and involvement of ribosomal-associated factors in this process. Ribonuclease inhibitor 1 (RNH1) is a ubiquitously expressed protein that binds to and inhibits pancreatic-type ribonucleases. Here, we report that RNH1 binds to ribosomes and regulates erythropoiesis by controlling translation of the erythroid transcription factor GATA1. Rnh1-deficient mice die between embryonic days E8.5 and E10 due to impaired production of mature erythroid cells from progenitor cells. In Rnh1-deficient embryos, mRNA levels of Gata1 are normal, but GATA1 protein levels are decreased. At the molecular level, we found that RNH1 binds to the 40S subunit of ribosomes and facilitates polysome formation on Gata1 mRNA to confer transcript-specific translation. Further, RNH1 knockdown in human CD34+ progenitor cells decreased erythroid differentiation without affecting myelopoiesis. Our results reveal an unsuspected role for RNH1 in the control of GATA1 mRNA translation and erythropoiesis.

Daughter-specific transcription factors regulate cell size control in budding yeast.

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Di Talia, Stefano; Wang, Hongyin; Skotheim, Jan M; Rosebrock, Adam P; Futcher, Bruce; Cross, Frederick R

2009-10-01

In budding yeast, asymmetric cell division yields a larger mother and a smaller daughter cell, which transcribe different genes due to the daughter-specific transcription factors Ace2 and Ash1. Cell size control at the Start checkpoint has long been considered to be a main regulator of the length of the G1 phase of the cell cycle, resulting in longer G1 in the smaller daughter cells. Our recent data confirmed this concept using quantitative time-lapse microscopy. However, it has been proposed that daughter-specific, Ace2-dependent repression of expression of the G1 cyclin CLN3 had a dominant role in delaying daughters in G1. We wanted to reconcile these two divergent perspectives on the origin of long daughter G1 times. We quantified size control using single-cell time-lapse imaging of fluorescently labeled budding yeast, in the presence or absence of the daughter-specific transcriptional regulators Ace2 and Ash1. Ace2 and Ash1 are not required for efficient size control, but they shift the domain of efficient size control to larger cell size, thus increasing cell size requirement for Start in daughters. Microarray and chromatin immunoprecipitation experiments show that Ace2 and Ash1 are direct transcriptional regulators of the G1 cyclin gene CLN3. Quantification of cell size control in cells expressing titrated levels of Cln3 from ectopic promoters, and from cells with mutated Ace2 and Ash1 sites in the CLN3 promoter, showed that regulation of CLN3 expression by Ace2 and Ash1 can account for the differential regulation of Start in response to cell size in mothers and daughters. We show how daughter-specific transcriptional programs can interact with intrinsic cell size control to differentially regulate Start in mother and daughter cells. This work demonstrates mechanistically how asymmetric localization of cell fate determinants results in cell-type-specific regulation of the cell cycle.

Daughter-Specific Transcription Factors Regulate Cell Size Control in Budding Yeast

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Di Talia, Stefano; Wang, Hongyin; Skotheim, Jan M.; Rosebrock, Adam P.; Futcher, Bruce; Cross, Frederick R.

2009-01-01

In budding yeast, asymmetric cell division yields a larger mother and a smaller daughter cell, which transcribe different genes due to the daughter-specific transcription factors Ace2 and Ash1. Cell size control at the Start checkpoint has long been considered to be a main regulator of the length of the G1 phase of the cell cycle, resulting in longer G1 in the smaller daughter cells. Our recent data confirmed this concept using quantitative time-lapse microscopy. However, it has been proposed that daughter-specific, Ace2-dependent repression of expression of the G1 cyclin CLN3 had a dominant role in delaying daughters in G1. We wanted to reconcile these two divergent perspectives on the origin of long daughter G1 times. We quantified size control using single-cell time-lapse imaging of fluorescently labeled budding yeast, in the presence or absence of the daughter-specific transcriptional regulators Ace2 and Ash1. Ace2 and Ash1 are not required for efficient size control, but they shift the domain of efficient size control to larger cell size, thus increasing cell size requirement for Start in daughters. Microarray and chromatin immunoprecipitation experiments show that Ace2 and Ash1 are direct transcriptional regulators of the G1 cyclin gene CLN3. Quantification of cell size control in cells expressing titrated levels of Cln3 from ectopic promoters, and from cells with mutated Ace2 and Ash1 sites in the CLN3 promoter, showed that regulation of CLN3 expression by Ace2 and Ash1 can account for the differential regulation of Start in response to cell size in mothers and daughters. We show how daughter-specific transcriptional programs can interact with intrinsic cell size control to differentially regulate Start in mother and daughter cells. This work demonstrates mechanistically how asymmetric localization of cell fate determinants results in cell-type-specific regulation of the cell cycle. PMID:19841732

Daughter-specific transcription factors regulate cell size control in budding yeast.

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Stefano Di Talia

2009-10-01

Full Text Available In budding yeast, asymmetric cell division yields a larger mother and a smaller daughter cell, which transcribe different genes due to the daughter-specific transcription factors Ace2 and Ash1. Cell size control at the Start checkpoint has long been considered to be a main regulator of the length of the G1 phase of the cell cycle, resulting in longer G1 in the smaller daughter cells. Our recent data confirmed this concept using quantitative time-lapse microscopy. However, it has been proposed that daughter-specific, Ace2-dependent repression of expression of the G1 cyclin CLN3 had a dominant role in delaying daughters in G1. We wanted to reconcile these two divergent perspectives on the origin of long daughter G1 times. We quantified size control using single-cell time-lapse imaging of fluorescently labeled budding yeast, in the presence or absence of the daughter-specific transcriptional regulators Ace2 and Ash1. Ace2 and Ash1 are not required for efficient size control, but they shift the domain of efficient size control to larger cell size, thus increasing cell size requirement for Start in daughters. Microarray and chromatin immunoprecipitation experiments show that Ace2 and Ash1 are direct transcriptional regulators of the G1 cyclin gene CLN3. Quantification of cell size control in cells expressing titrated levels of Cln3 from ectopic promoters, and from cells with mutated Ace2 and Ash1 sites in the CLN3 promoter, showed that regulation of CLN3 expression by Ace2 and Ash1 can account for the differential regulation of Start in response to cell size in mothers and daughters. We show how daughter-specific transcriptional programs can interact with intrinsic cell size control to differentially regulate Start in mother and daughter cells. This work demonstrates mechanistically how asymmetric localization of cell fate determinants results in cell-type-specific regulation of the cell cycle.

Regulation of host-pathogen interactions via the post-transcriptional Csr/Rsm system.

Science.gov (United States)

Kusmierek, Maria; Dersch, Petra

2018-02-01

A successful colonization of specific hosts requires a rapid and efficient adaptation of the virulence-relevant gene expression program by bacterial pathogens. An important element in this endeavor is the Csr/Rsm system. This multi-component, post-transcriptional control system forms a central hub within complex regulatory networks and coordinately adjusts virulence properties with metabolic and physiological attributes of the pathogen. A key function is elicited by the RNA-binding protein CsrA/RsmA. CsrA/RsmA interacts with numerous target mRNAs, many of which encode crucial virulence factors, and alters their translation, stability or elongation of transcription. Recent studies highlighted that important colonization factors, toxins, and bacterial secretion systems are under CsrA/RsmA control. CsrA/RsmA deficiency impairs host colonization and attenuates virulence, making this post-transcriptional regulator a suitable drug target. The CsrA/RsmA protein can be inactivated through sequestration by non-coding RNAs, or via binding to specific highly abundant mRNAs and interacting proteins. The wide range of interaction partners and RNA targets, as well as the overarching, interlinked genetic control circuits illustrate the complexity of this regulatory system in the different pathogens. Future work addressing spatio-temporal changes of Csr/Rsm-mediated control during the course of an infection will help us to understand how bacteria reprogram their expression profile to cope with continuous changes experienced in colonized niches. Copyright © 2017 Elsevier Ltd. All rights reserved.

Isolation and characterization of human glycophorin A cDNAs using a synthetic oligonucleotide approach: nucleotide sequence, mRNA structure and regulation by 12-O-tetradecanoylphorbol 13-acetate (TPA)

International Nuclear Information System (INIS)

Siebert, P.D.; Fukuda, M.

1986-01-01

The authors have previously shown that treatment of human erythroleukemic K562 cells with the tumor-promoting phorbol ester, TPA, results in a diminished expression of glycophorin A at the level of protein biosynthesis and in vitro mRNA translation activity. To further examine the structure, relationships and expression of human glycophorins they have successfully isolated and sequenced several glycophorin A specific cDNA clones derived from K562 cells, by making extensive use of mixed and exact synthetic oligonucleotides as primers and radioactively labeled probes. The nucleotide sequence obtained from the largest glycophorin A cDNA suggests the presence of a hydrophobic leader-like peptide of at least 19 amino acids. Northern gel analysis using both whole cDNA-plasmid and synthetic oligonucleotide probes revealed the existence of multiple mRNAs, three of which they believe to be glycophorin A-specific, whereas a fourth and smaller mRNA appears to be glycophorin B-specific. Furthermore, the abundance of all four glycophorin mRNAs were found to be extensively reduced following treatment of K562 cells with TPA suggesting coordinate regulation, possibly at the level of gene transcription

Regulation of cytokines by small RNAs during skin inflammation

Directory of Open Access Journals (Sweden)

Mikkelsen Jacob G

2010-07-01

Full Text Available Abstract Intercellular signaling by cytokines is a vital feature of the innate immune system. In skin, an inflammatory response is mediated by cytokines and an entwined network of cellular communication between T-cells and epidermal keratinocytes. Dysregulated cytokine production, orchestrated by activated T-cells homing to the skin, is believed to be the main cause of psoriasis, a common inflammatory skin disorder. Cytokines are heavily regulated at the transcriptional level, but emerging evidence suggests that regulatory mechanisms that operate after transcription play a key role in balancing the production of cytokines. Herein, we review the nature of cytokine signaling in psoriasis with particular emphasis on regulation by mRNA destabilizing elements and the potential targeting of cytokine-encoding mRNAs by miRNAs. The proposed linkage between mRNA decay mediated by AU-rich elements and miRNA association is described and discussed as a possible general feature of cytokine regulation in skin. Moreover, we describe the latest attempts to therapeutically target cytokines at the RNA level in psoriasis by exploiting the cellular RNA interference machinery. The applicability of cytokine-encoding mRNAs as future clinical drug targets is evaluated, and advances and obstacles related to topical administration of RNA-based drugs targeting the cytokine circuit in psoriasis are described.

WNT2B2 mRNA, up-regulated in primary gastric cancer, is a positive regulator of the WNT- beta-catenin-TCF signaling pathway.

Science.gov (United States)

Katoh, M; Kirikoshi, H; Terasaki, H; Shiokawa, K

2001-12-21

Genetic alterations of WNT signaling molecules lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway. We have previously cloned and characterized WNT2B/WNT13 gene on human chromosome 1p13, which is homologous to proto-oncogene WNT2 on human chromosome 7q31. WNT2B1 and WNT2B2 mRNAs, generated from the WNT2B gene due to alternative splicing of the alternative promoter type, encode almost identical polypeptides with divergence in the N-terminal region. WNT2B2 mRNA rather than WNT2B1 mRNA is preferentially expressed in NT2 cells with the potential of neuronal differentiation. Here, we describe our investigations of expression of WNT2B mRNAs in various types of human primary cancer. Matched tumor/normal expression array analysis revealed that WNT2B mRNAs were significantly up-regulated in 2 of 8 cases of primary gastric cancer. WNT2B2 mRNA rather than WNT2B1 mRNA was found to be preferentially up-regulated in a case of primary gastric cancer (signet ring cell carcinoma). Function of WNT2B1 mRNA and that of WNT2B2 mRNA were investigated by using Xenopus axis duplication assay. Injection of synthetic WNT2B1 mRNA into the ventral marginal zone of fertilized Xenopus eggs at the 4-cell stage did not induce axis duplication. In contrast, ventral injection of synthetic WNT2B2 mRNA induced axis duplication in 90% of embryos (complete axis duplication, 24%). These results strongly suggest that WNT2B2 up-regulation in some cases of gastric cancer might lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway.

Site-Specific Pre-Swelling-Directed Morphing Structures of Patterned Hydrogels.

Science.gov (United States)

Wang, Zhi Jian; Hong, Wei; Wu, Zi Liang; Zheng, Qiang

2017-12-11

Morphing materials have promising applications in various fields, yet how to program the self-shaping process for specific configurations remains a challenge. Herein we show a versatile approach to control the buckling of individual domains and thus the outcome configurations of planar-patterned hydrogels. By photolithography, high-swelling disc gels were positioned in a non-swelling gel sheet; the swelling mismatch resulted in out-of-plain buckling of the disc gels. To locally control the buckling direction, masks with holes were used to guide site-specific swelling of the high-swelling gel under the holes, which built a transient through-thickness gradient and thus directed the buckling during the subsequent unmasked swelling process. Therefore, various configurations of an identical patterned hydrogel can be programmed by the pre-swelling step with different masks to encode the buckling directions of separate domains. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Human thyroid peroxidase: complete cDNA and protein sequence, chromosome mapping, and identification of two alternately spliced mRNAs

International Nuclear Information System (INIS)

Kimura, S.; Kotani, T.; McBride, O.W.; Umeki, K.; Hirai, K.; Nakayama, T.; Ohtaki, S.

1987-01-01

Two forms of human thyroid peroxidase cDNAs were isolated from a λgt11 cDNA library, prepared from Graves disease thyroid tissue mRNA, by use of oligonucleotides. The longest complete cDNA, designated phTPO-1, has 3048 nucleotides and an open reading frame consisting of 933 amino acids, which would encode a protein with a molecular weight of 103,026. Five potential asparagine-linked glycosylation sites are found in the deduced amino acid sequence. The second peroxidase cDNA, designated phTPO-2, is almost identical to phTPO-1 beginning 605 base pairs downstream except that it contains 1-base-pair difference and lacks 171 base pairs in the middle of the sequence. This results in a loss of 57 amino acids corresponding to a molecular weight of 6282. Interestingly, this 171-nucleotide sequence has GT and AG at its 5' and 3' boundaries, respectively, that are in good agreement with donor and acceptor splice site consensus sequences. Using specific oligonucleotide probes for the mRNAs derived from the cDNA sequences hTOP-1 and hTOP-2, the authors show that both are expressed in all thyroid tissues examined and the relative level of two mRNAs is different in each sample. The results suggest that two thyroid peroxidase proteins might be generated through alternate splicing of the same gene. By using somatic cell hybrid lines, the thyroid peroxidase gene was mapped to the short arm of human chromosome 2

Water pollution - control of pollution regulations: water pollution from abandoned mines; pre-notification of mine abandonment consultation paper and draft regulations

Energy Technology Data Exchange (ETDEWEB)

NONE

1997-02-01

The paper contains proposals to specify the content of the pre-abandonment notices which UK mine operators will be required to give in the future. The proposals are designed to enhance the Environment Agency`s ability to tackle water pollution from abandoned mines. They set out the precise contents of the notification, which mine operators will have to send to the Agency at least six months in advance of any intended abandonment. The regulations will cover coal and non-coal mines. Estimated compliance costs to business have been drawn up following consultation with the Department of Trade and Industry, the Health and Safety Executive, the Coal Authority and the Environment Agency. The regulations are due in late spring 1998.

Nephron segment specific microRNA biomarkers of pre-clinical drug-induced renal toxicity: Opportunities and challenges

Energy Technology Data Exchange (ETDEWEB)

Nassirpour, Rounak, E-mail: Rounak.nassirpour@pfizer.com [Drug Safety, Pfizer Worldwide Research and Development, 1 Burtt Rd, Andover, MA 01810 (United States); Ramaiah, Shashi K. [Drug Safety, Pfizer Worldwide Research and Development, 610 Main Street, Cambridge, MA 02139 (United States); Whiteley, Laurence O. [Drug Safety, Pfizer Worldwide Research and Development, 1 Burtt Rd, Andover, MA 01810 (United States)

2016-12-01

Drug-induced nephrotoxicity is a common drug development complication for pharmaceutical companies. Sensitive, specific, translatable and non-invasive biomarkers of renal toxicity are urgently needed to diagnose nephron segment specific injury. The currently available gold standard biomarkers for nephrotoxicity are not kidney-specific, lack sensitivity for early detection, and are not suitable for renal damage localization (glomerular vs tubulointerstitial injury). MicroRNAs (miRNAs) are increasingly gaining momentum as promising biomarkers of various organ toxicities, including drug induced renal injury. This is mostly due to their stability in easily accessible biofluids, ease of developing nucleic acids detection compared to protein detection assays, as well as their interspecies translatability. Increasing concordance of miRNA findings by standardizing methodology most suitable for their detection and quantitation, as well as characterization of their expression pattern in a cell type specific manner, will accelerate progress toward validation of these miRNAs as biomarkers in pre-clinical, and clinical settings. This review aims to highlight the current pre-clinical findings surrounding miRNAs as biomarkers in two important segments of the nephron, the glomerulus and tubules. - Highlights: • miRNAs are promising biomarkers of drug-induced kidney injury. • Summarized pre-clinical miRNA biomarkers of drug-induced nephrotoxicity. • Described the strengths and challenges associated with miRNAs as biomarkers.

Identification of the miRNA-mRNA regulatory network of small cell osteosarcoma based on RNA-seq.

Science.gov (United States)

Xie, Lin; Liao, Yedan; Shen, Lida; Hu, Fengdi; Yu, Sunlin; Zhou, Yonghong; Zhang, Ya; Yang, Yihao; Li, Dongqi; Ren, Minyan; Yuan, Zhongqin; Yang, Zuozhang

2017-06-27

Small cell osteosarcoma (SCO) is a rare subtype of osteosarcoma characterized by highly aggressive progression and a poor prognosis. The miRNA and mRNA expression profiles of peripheral blood mononuclear cells (PBMCs) were obtained in 3 patients with SCO and 10 healthy individuals using high-throughput RNA-sequencing. We identified 37 dysregulated miRNAs and 1636 dysregulated mRNAs in patients with SCO compared to the healthy controls. Specifically, the 37 dysregulated miRNAs consisted of 27 up-regulated miRNAs and 10 down-regulated miRNAs; the 1636 dysregulated mRNAs consisted of 555 up-regulated mRNAs and 1081 down-regulated mRNAs. The target-genes of miRNAs were predicted, and 1334 negative correlations between miRNAs and mRNAs were used to construct an miRNA-mRNA regulatory network. Dysregulated genes were significantly enriched in pathways related to cancer, mTOR signaling and cell cycle signaling. Specifically, hsa-miR-26b-5p, hsa-miR-221-3p and hsa-miR-125b-2-3p were significantly dysregulated miRNAs and exhibited a high degree of connectivity with target genes. Overall, the expression of dysregulated genes in tumor tissues and peripheral blood samples of patients with SCO measured by quantitative real-time polymerase chain reaction corroborated with our bioinformatics analyses based on the expression profiles of PBMCs from patients with SCO. Thus, hsa-miR-26b-5p, hsa-miR-221-3p and hsa-miR-125b-2-3p may be involved in SCO tumorigenesis.

Tissue-specific expression and regulation by 1,25(OH)2D3 of chick protein kinase inhibitor (PKI) mRNA.

Science.gov (United States)

Marchetto, G S; Henry, H L

1997-02-01

The heat-stable protein kinase inhibitor (PKI) protein is a specific and potent competitive inhibitor of the catalytic subunit of cAMP-dependent protein kinase (PKA). Previously, it has been shown that vitamin D status affects chick kidney PKI activity: a 5- to 10-fold increase in PKI activity was observed in kidneys of chronically vitamin D-deficient chicks and treatment with 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) in cultured kidney cells resulted in a 95% decrease in PKI activity. The authors have recently cloned the cDNA for chick kidney PKI and have used the coding sequence to study the regulation of PKI mRNA. Northern analysis showed the expression of two PKI messages, which are 2.7 and 3.3 kb in size. These mRNAs are expressed in brain, muscle, testis, and kidney, but not in pancreas, liver, or intestine. PKI mRNA steady-state levels are downregulated by 47% in kidneys from vitamin D-replete chicks as compared to vitamin D-deficient chicks. PKI mRNA levels in brain, muscle, and testis are not affected by vitamin D status. Treatment of primary chick kidney cultures treated with 10(-7) M 1,25(OH)2D3 for 24h resulted in a 20-30% decrease in PKI mRNA. 1,25(OH)2D3 treatment does not affect the stability of PKI mRNA as determined by treatment of cell cultures with actinomycin D. This study shows that 1,25(OH)2D3 directly and tissue-specifically downregulates PKI mRNA in the chick kidney.

Differential Binding of Mitochondrial Transcripts by MRB8170 and MRB4160 Regulates Distinct Editing Fates of Mitochondrial mRNA in Trypanosomes

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Sameer Dixit

2017-01-01

Full Text Available A dozen mRNAs are edited by multiple insertions and/or deletions of uridine residues in the mitochondrion of Trypanosoma brucei. Several protein complexes have been implicated in performing this type of RNA editing, including the mitochondrial RNA-binding complex 1 (MRB1. Two paralogous novel RNA-binding proteins, MRB8170 and MRB4160, are loosely associated with the core MRB1 complex. Their roles in RNA editing and effects on target mRNAs are so far not well understood. In this study, individual-nucleotide-resolution UV-cross-linking and affinity purification (iCLAP revealed a preferential binding of both proteins to mitochondrial mRNAs, which was positively correlated with their extent of editing. Integrating additional in vivo and in vitro data, we propose that binding of MRB8170 and/or MRB4160 onto pre-mRNA marks it for the initiation of editing and that initial binding of both proteins may facilitate the recruitment of other components of the RNA editing/processing machinery to ensure efficient editing. Surprisingly, MRB8170 also binds never-edited mRNAs, suggesting that at least this paralog has an additional role outside RNA editing to shape the mitochondrial transcriptome.

Analyzing the interactions of mRNAs, miRNAs, lncRNAs and circRNAs to predict competing endogenous RNA networks in glioblastoma.

Science.gov (United States)

Yuan, Yang; Jiaoming, Li; Xiang, Wang; Yanhui, Liu; Shu, Jiang; Maling, Gou; Qing, Mao

2018-05-01

Cross-talk between competitive endogenous RNAs (ceRNAs) may play a critical role in revealing potential mechanisms of tumor development and physiology. Glioblastoma is the most common type of malignant primary brain tumor, and the mechanisms of tumor genesis and development in glioblastoma are unclear. Here, to investigate the role of non-coding RNAs and the ceRNA network in glioblastoma, we performed paired-end RNA sequencing and microarray analyses to obtain the expression profiles of mRNAs, lncRNAs, circRNAs and miRNAs. We identified that the expression of 501 lncRNAs, 1999 mRNAs, 2038 circRNAs and 143 miRNAs were often altered between glioblastoma and matched normal brain tissue. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed on these differentially expressed mRNAs and miRNA-mediated target genes of lncRNAs and circRNAs. Furthermore, we used a multi-step computational framework and several bioinformatics methods to construct a ceRNA network combining mRNAs, miRNAs, lncRNAs and circRNA, based on co-expression analysis between the differentially expressed RNAs. We identified that plenty of lncRNAs, CircRNAs and their downstream target genes in the ceRNA network are related to glutamatergic synapse, suggesting that glutamate metabolism is involved in glioma biological functions. Our results will accelerate the understanding of tumorigenesis, cancer progression and even therapeutic targeting in glioblastoma.

Shine-dalgarno sequences play an essential role in the translation of plastid mRNAs in tobacco

DEFF Research Database (Denmark)

Scharff, Lars; Ehrnthaler, Miriam; Janowski, Marcin

2017-01-01

SD]). Although many chloroplast mRNAs harbor putative SDs in their 5' untranslated regions and the aSD displays strong conservation, the functional relevance of SD-aSD interactions in plastid translation is unclear. Here, by generating transplastomic tobacco (Nicotiana tabacum) mutants with point mutations...

Macoilin, a conserved nervous system-specific ER membrane protein that regulates neuronal excitability.

Directory of Open Access Journals (Sweden)

Fausto Arellano-Carbajal

2011-03-01

Full Text Available Genome sequence comparisons have highlighted many novel gene families that are conserved across animal phyla but whose biological function is unknown. Here, we functionally characterize a member of one such family, the macoilins. Macoilins are characterized by several highly conserved predicted transmembrane domains towards the N-terminus and by coiled-coil regions C-terminally. They are found throughout Eumetazoa but not in other organisms. Mutants for the single Caenorhabditis elegans macoilin, maco-1, exhibit a constellation of behavioral phenotypes, including defects in aggregation, O₂ responses, and swimming. MACO-1 protein is expressed broadly and specifically in the nervous system and localizes to the rough endoplasmic reticulum; it is excluded from dendrites and axons. Apart from subtle synapse defects, nervous system development appears wild-type in maco-1 mutants. However, maco-1 animals are resistant to the cholinesterase inhibitor aldicarb and sensitive to levamisole, suggesting pre-synaptic defects. Using in vivo imaging, we show that macoilin is required to evoke Ca²(+ transients, at least in some neurons: in maco-1 mutants the O₂-sensing neuron PQR is unable to generate a Ca²(+ response to a rise in O₂. By genetically disrupting neurotransmission, we show that pre-synaptic input is not necessary for PQR to respond to O₂, indicating that the response is mediated by cell-intrinsic sensory transduction and amplification. Disrupting the sodium leak channels NCA-1/NCA-2, or the N-,P/Q,R-type voltage-gated Ca²(+ channels, also fails to disrupt Ca²(+ responses in the PQR cell body to O₂ stimuli. By contrast, mutations in egl-19, which encodes the only Caenorhabditis elegans L-type voltage-gated Ca²(+ channel α1 subunit, recapitulate the Ca²(+ response defect we see in maco-1 mutants, although we do not see defects in localization of EGL-19. Together, our data suggest that macoilin acts in the ER to regulate assembly or

Crystal Structure of the HEAT Domain from the Pre-mRNA Processing Factor Symplekin

Energy Technology Data Exchange (ETDEWEB)

Kennedy, Sarah A.; Frazier, Monica L.; Steiniger, Mindy; Mast, Ann M.; Marzluff, William F.; Redinbo, Matthew R.; (UNC)

2010-09-30

The majority of eukaryotic pre-mRNAs are processed by 3'-end cleavage and polyadenylation, although in metazoa the replication-dependent histone mRNAs are processed by 3'-end cleavage but not polyadenylation. The macromolecular complex responsible for processing both canonical and histone pre-mRNAs contains the {approx} 1160-residue protein Symplekin. Secondary-structural prediction algorithms identified putative HEAT domains in the 300 N-terminal residues of all Symplekins of known sequence. The structure and dynamics of this domain were investigated to begin elucidating the role Symplekin plays in mRNA maturation. The crystal structure of the Drosophila melanogaster Symplekin HEAT domain was determined to 2.4 {angstrom} resolution with single-wavelength anomalous dispersion phasing methods. The structure exhibits five canonical HEAT repeats along with an extended 31-amino-acid loop (loop 8) between the fourth and fifth repeat that is conserved within closely related Symplekin sequences. Molecular dynamics simulations of this domain show that the presence of loop 8 dampens correlated and anticorrelated motion in the HEAT domain, therefore providing a neutral surface for potential protein-protein interactions. HEAT domains are often employed for such macromolecular contacts. The Symplekin HEAT region not only structurally aligns with several established scaffolding proteins, but also has been reported to contact proteins essential for regulating 3'-end processing. Together, these data support the conclusion that the Symplekin HEAT domain serves as a scaffold for protein-protein interactions essential to the mRNA maturation process.

Pancreas lineage allocation and specification are regulated by sphingosine-1-phosphate signalling

Science.gov (United States)

Serafimidis, Ioannis; Rodriguez-Aznar, Eva; Lesche, Mathias; Yoshioka, Kazuaki; Takuwa, Yoh; Dahl, Andreas; Pan, Duojia; Gavalas, Anthony

2017-01-01

During development, progenitor expansion, lineage allocation, and implementation of differentiation programs need to be tightly coordinated so that different cell types are generated in the correct numbers for appropriate tissue size and function. Pancreatic dysfunction results in some of the most debilitating and fatal diseases, including pancreatic cancer and diabetes. Several transcription factors regulating pancreas lineage specification have been identified, and Notch signalling has been implicated in lineage allocation, but it remains unclear how these processes are coordinated. Using a combination of genetic approaches, organotypic cultures of embryonic pancreata, and genomics, we found that sphingosine-1-phosphate (S1p), signalling through the G protein coupled receptor (GPCR) S1pr2, plays a key role in pancreas development linking lineage allocation and specification. S1pr2 signalling promotes progenitor survival as well as acinar and endocrine specification. S1pr2-mediated stabilisation of the yes-associated protein (YAP) is essential for endocrine specification, thus linking a regulator of progenitor growth with specification. YAP stabilisation and endocrine cell specification rely on Gαi subunits, revealing an unexpected specificity of selected GPCR intracellular signalling components. Finally, we found that S1pr2 signalling posttranscriptionally attenuates Notch signalling levels, thus regulating lineage allocation. Both S1pr2-mediated YAP stabilisation and Notch attenuation are necessary for the specification of the endocrine lineage. These findings identify S1p signalling as a novel key pathway coordinating cell survival, lineage allocation, and specification and linking these processes by regulating YAP levels and Notch signalling. Understanding lineage allocation and specification in the pancreas will shed light in the origins of pancreatic diseases and may suggest novel therapeutic approaches. PMID:28248965

AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes.

Science.gov (United States)

Young, Nathan P; Kamireddy, Anwesh; Van Nostrand, Jeanine L; Eichner, Lillian J; Shokhirev, Maxim Nikolaievich; Dayn, Yelena; Shaw, Reuben J

2016-03-01

Faithful execution of developmental programs relies on the acquisition of unique cell identities from pluripotent progenitors, a process governed by combinatorial inputs from numerous signaling cascades that ultimately dictate lineage-specific transcriptional outputs. Despite growing evidence that metabolism is integrated with many molecular networks, how pathways that control energy homeostasis may affect cell fate decisions is largely unknown. Here, we show that AMP-activated protein kinase (AMPK), a central metabolic regulator, plays critical roles in lineage specification. Although AMPK-deficient embryonic stem cells (ESCs) were normal in the pluripotent state, these cells displayed profound defects upon differentiation, failing to generate chimeric embryos and preferentially adopting an ectodermal fate at the expense of the endoderm during embryoid body (EB) formation. AMPK(-/-) EBs exhibited reduced levels of Tfeb, a master transcriptional regulator of lysosomes, leading to diminished endolysosomal function. Remarkably, genetic loss of Tfeb also yielded endodermal defects, while AMPK-null ESCs overexpressing this transcription factor normalized their differential potential, revealing an intimate connection between Tfeb/lysosomes and germ layer specification. The compromised endolysosomal system resulting from AMPK or Tfeb inactivation blunted Wnt signaling, while up-regulating this pathway restored expression of endodermal markers. Collectively, these results uncover the AMPK pathway as a novel regulator of cell fate determination during differentiation. © 2016 Young et al.; Published by Cold Spring Harbor Laboratory Press.

Voltage pre-regulator without insulation with high efficiency; Pre-regulador de tension sin aislamiento con alta eficiencia

Energy Technology Data Exchange (ETDEWEB)

Lopez Ramos, Octaviano

2003-04-15

Nowadays there are problems caused by the inadequate and inefficient use of the electrical energy in the supplying systems, these problems are becoming more complicated with the passage of time, due to the proliferation of electronic equipment that does not incorporate some solutions to such problems. This has originated a tendency towards the development of systems that process with higher efficiency ({eta}) the electrical energy. In addition of making good use of the energy, they are designed so that they can correct the power factor (PF) and therefore reduce the contamination of harmonics in the network; which causes faults or destruction in other electronic equipment. Additionally it is possible to design them to obtain a good dynamic response, which assures that they can efficiently counteract load changes. The non-lineal characteristic that is present in all of the power supplies, which base its entrance on a rectification and filtration stage and, causes a low power factor (PF) and a high total harmonic distortion (THD). The factor is to contaminate the electric network, which can cause faults or breakdowns of other equipment. Since approximately 15 years the concern for solving this problem of the inadequate use of the energy has been taking importance, developing converters that correct the PF (pre-regulator) and that traditionally are connected in cascade with the typical stage of conversion of direct current to direct current (DC-DC) used to regulate the voltage; this solution has a low efficiency ({eta}) and high cost, by the excess of components connected in cascade. Nevertheless the dynamic response (DR) is adequate since it counts with separate stages for the correction of the PF and the regulation. From a revision of the state-of-the-art, in this thesis a new topology of pre-regulator for power supplies with a high efficiency is presented ({eta}), due to a novel strategy of managing the energy; also it corrects the PF. This represents an advance

Structural Studies of RNA Helicases Involved in Eukaryotic Pre-mRNA Splicing, Ribosome Biogenesis, and Translation Initiation

DEFF Research Database (Denmark)

He, Yangzi

and ligates the neighbouring exons to generate mature mRNAs. Prp43 is an RNA helicase of the DEAH/RHA family. In yeast, once mRNAs are released, Prp43 catalyzes the disassembly of spliceosomes. The 18S, 5.8S and 25S rRNAs are transcribed as a single polycistronic transcript—the 35S pre......-rRNA. It is nucleolytically cleaved and chemically modified to generate mature rRNAs, which assemble with ribosomal proteins to form the ribosome. Prp43 is required for the processing of the 18S rRNA. Using X-ray crystallography, I determined a high resolution structure of Prp43 bound to ADP, the first structure of a DEAH....../RHA helicase. It defined the conserved structural features of all DEAH/RHA helicases, and unveiled a novel nucleotide binding site. Additionally a preliminary low resolution structure of a ternary complex comprising Prp43, a non-hydrolyzable ATP analogue, and a single-stranded RNA, was obtained. The ribosome...

50 CFR 32.6 - What are the procedures for publication of refuge-specific sport fishing regulations?

Science.gov (United States)

2010-10-01

... refuge-specific sport fishing regulations? 32.6 Section 32.6 Wildlife and Fisheries UNITED STATES FISH... sport fishing regulations? (a) Refuge-specific fishing regulations are issued only at the time of or after the opening of a wildlife refuge area to sport fishing. (b) Refuge-specific fishing regulations...

Identifying functional cancer-specific miRNA-mRNA interactions in testicular germ cell tumor.

Science.gov (United States)

Sedaghat, Nafiseh; Fathy, Mahmood; Modarressi, Mohammad Hossein; Shojaie, Ali

2016-09-07

Testicular cancer is the most common cancer in men aged between 15 and 35 and more than 90% of testicular neoplasms are originated at germ cells. Recent research has shown the impact of microRNAs (miRNAs) in different types of cancer, including testicular germ cell tumor (TGCT). MicroRNAs are small non-coding RNAs which affect the development and progression of cancer cells by binding to mRNAs and regulating their expressions. The identification of functional miRNA-mRNA interactions in cancers, i.e. those that alter the expression of genes in cancer cells, can help delineate post-regulatory mechanisms and may lead to new treatments to control the progression of cancer. A number of sequence-based methods have been developed to predict miRNA-mRNA interactions based on the complementarity of sequences. While necessary, sequence complementarity is, however, not sufficient for presence of functional interactions. Alternative methods have thus been developed to refine the sequence-based interactions using concurrent expression profiles of miRNAs and mRNAs. This study aims to find functional cancer-specific miRNA-mRNA interactions in TGCT. To this end, the sequence-based predicted interactions are first refined using an ensemble learning method, based on two well-known methods of learning miRNA-mRNA interactions, namely, TaLasso and GenMiR++. Additional functional analyses were then used to identify a subset of interactions to be most likely functional and specific to TGCT. The final list of 13 miRNA-mRNA interactions can be potential targets for identifying TGCT-specific interactions and future laboratory experiments to develop new therapies. Copyright © 2016 Elsevier Ltd. All rights reserved.

Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to fungal elicitor and infection.

Science.gov (United States)

Showalter, A M; Bell, J N; Cramer, C L; Bailey, J A; Varner, J E; Lamb, C J

1985-10-01

Hydroxyproline-rich glycoproteins (HRGPs) are important structural components of plant cell walls and also accumulate in response to infection as an apparent defense mechanism. Accumulation of HRGP mRNA in biologically stressed bean (Phaseolus vulgaris L.) cells was monitored by blot hybridization with (32)P-labeled tomato genomic HRGP sequences. Elicitor treatment of suspension-cultured cells caused a marked increase in hybridizable HRGP mRNA. The response was less rapid but more prolonged than that observed for mRNAs encoding enzymes of phytoalexin biosynthesis. HRGP mRNA also accumulated during race:cultivar-specific interactions between bean hypocotyls and the partially biotrophic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. In an incompatible interaction (host resistant) there was an early increase in HRGP mRNA correlated with expression of hypersensitive resistance; whereas, in a compatible interaction (host susceptible), marked accumulation of HRGP mRNA occurred as a delayed response at the onset of lesion formation. In both interactions, mRNA accumulation was observed in uninfected cells distant from the site of fungal inoculation, indicating intercellular transmission of an elicitation signal.

TCR Down-Regulation Controls Virus-Specific CD8+ T Cell Responses

DEFF Research Database (Denmark)

Bonefeld, Charlotte Menné; Haks, Mariëlle; Nielsen, Bodil

2008-01-01

The CD3gamma di-leucine-based motif plays a central role in TCR down-regulation. However, little is understood about the role of the CD3gamma di-leucine-based motif in physiological T cell responses. In this study, we show that the expansion in numbers of virus-specific CD8(+) T cells is impaired...... in mice with a mutated CD3gamma di-leucine-based motif. The CD3gamma mutation did not impair early TCR signaling, nor did it compromise recruitment or proliferation of virus-specific T cells, but it increased the apoptosis rate of the activated T cells by increasing down-regulation of the antiapoptotic...... molecule Bcl-2. This resulted in a 2-fold reduction in the clonal expansion of virus-specific CD8(+) T cells during the acute phase of vesicular stomatitis virus and lymphocytic choriomeningitis virus infections. These results identify an important role of CD3gamma-mediated TCR down-regulation in virus...

Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae

OpenAIRE

Gadir, Noga; Haim-Vilmovsky, Liora; Kraut-Cohen, Judith; Gerst, Jeffrey E.

2011-01-01

Targeted mRNA localization is a likely determinant of localized protein synthesis. To investigate whether mRNAs encoding mitochondrial proteins (mMPs) localize to mitochondria and, thus, might confer localized protein synthesis and import, we visualized endogenously expressed mMPs in vivo for the first time. We determined the localization of 24 yeast mMPs encoding proteins of the mitochondrial matrix, outer and inner membrane, and intermembrane space and found that many mMPs colocalize with m...

No significant regulation of bicoid mRNA by Pumilio or Nanos in the early Drosophila embryo.

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Wharton, Tammy H; Nomie, Krystle J; Wharton, Robin P

2018-01-01

Drosophila Pumilio (Pum) is a founding member of the conserved Puf domain class of RNA-binding translational regulators. Pum binds with high specificity, contacting eight nucleotides, one with each of the repeats in its RNA-binding domain. In general, Pum is thought to block translation in collaboration with Nanos (Nos), which exhibits no binding specificity in isolation but is recruited jointly to regulatory sequences containing a Pum binding site in the 3'-UTRs of target mRNAs. Unlike Pum, which is ubiquitous in the early embryo, Nos is tightly restricted to the posterior, ensuring that repression of its best-characterized target, maternal hunchback (hb) mRNA, takes place exclusively in the posterior. An exceptional case of Nos-independent regulation by Pum has been described-repression of maternal bicoid (bcd) mRNA at the anterior pole of the early embryo, dependent on both Pum and conserved Pum binding sites in the 3'-UTR of the mRNA. We have re-investigated regulation of bcd in the early embryo; our experiments reveal no evidence of a role for Pum or its conserved binding sites in regulation of the perdurance of bcd mRNA or protein. Instead, we find that Pum and Nos control the accumulation of bcd mRNA in testes.

Epigenetic regulation of normal human mammary cell type-specific miRNAs

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Vrba, Lukas [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center; Inst. of Plant Molecular Biology, Ceske Budejovice (Czech Republic). Biology Centre ASCR; Garbe, James C. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Center; Stampfer, Martha R. [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Center; Futscher, Bernard W. [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center and Dept. of Pharmacology & Toxicology

2011-08-26

Epigenetic mechanisms are important regulators of cell type–specific genes, including miRNAs. In order to identify cell type-specific miRNAs regulated by epigenetic mechanisms, we undertook a global analysis of miRNA expression and epigenetic states in three isogenic pairs of human mammary epithelial cells (HMEC) and human mammary fibroblasts (HMF), which represent two differentiated cell types typically present within a given organ, each with a distinct phenotype and a distinct epigenotype. While miRNA expression and epigenetic states showed strong interindividual concordance within a given cell type, almost 10% of the expressed miRNA showed a cell type–specific pattern of expression that was linked to the epigenetic state of their promoter. The tissue-specific miRNA genes were epigenetically repressed in nonexpressing cells by DNA methylation (38%) and H3K27me3 (58%), with only a small set of miRNAs (21%) showing a dual epigenetic repression where both DNA methylation and H3K27me3 were present at their promoters, such as MIR10A and MIR10B. Individual miRNA clusters of closely related miRNA gene families can each display cell type–specific repression by the same or complementary epigenetic mechanisms, such as the MIR200 family, and MIR205, where fibroblasts repress MIR200C/141 by DNA methylation, MIR200A/200B/429 by H3K27me3, and MIR205 by both DNA methylation and H3K27me3. Since deregulation of many of the epigenetically regulated miRNAs that we identified have been linked to disease processes such as cancer, it is predicted that compromise of the epigenetic control mechanisms is important for this process. Overall, these results highlight the importance of epigenetic regulation in the control of normal cell type–specific miRNA expression.

Specific Increase of Protein Levels by Enhancing Translation Using Antisense Oligonucleotides Targeting Upstream Open Frames.

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Liang, Xue-Hai; Shen, Wen; Crooke, Stanley T

2017-01-01

A number of diseases are caused by low levels of key proteins; therefore, increasing the amount of specific proteins in human bodies is of therapeutic interest. Protein expression is downregulated by some structural or sequence elements present in the 5' UTR of mRNAs, such as upstream open reading frames (uORF). Translation initiation from uORF(s) reduces translation from the downstream primary ORF encoding the main protein product in the same mRNA, leading to a less efficient protein expression. Therefore, it is possible to use antisense oligonucleotides (ASOs) to specifically inhibit translation of the uORF by base-pairing with the uAUG region of the mRNA, redirecting translation machinery to initiate from the primary AUG site. Here we review the recent findings that translation of specific mRNAs can be enhanced using ASOs targeting uORF regions. Appropriately designed and optimized ASOs are highly specific, and they act in a sequence- and position-dependent manner, with very minor off-target effects. Protein levels can be increased using this approach in different types of human and mouse cells, and, importantly, also in mice. Since uORFs are present in around half of human mRNAs, the uORF-targeting ASOs may thus have valuable potential as research tools and as therapeutics to increase the levels of proteins for a variety of genes.

Mixed messages: Re-initiation factors regulate translation of animal mRNAs

OpenAIRE

Obermayer, Benedikt; Rajewsky, Nikolaus

2014-01-01

When ribosomes encounter upstream open reading frames (uORFs) during scanning of the 5' untranslated region (5' UTR), translation of the downstream ORF requires re-initiation. In a recent paper in Nature, Schleich et al. describe metazoan factors which specifically promote re-initiation.

TFPI alpha and beta regulate mRNAs and microRNAs involved in cancer biology and in the immune system in breast cancer cells.

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Benedicte Stavik

Full Text Available Emerging evidence indicate a new role of TFPI in cancer biology. We recently reported that both isoforms of TFPI induced apoptosis and inhibited proliferation of cancer cells. The signaling pathway(s mediating the effects of TFPI is, however, presently still unclear. Our goal was to further investigate the cellular processes affected by TFPI and to get insight into the molecular mechanisms involved in the effects of TFPI, using a global gene expression study approach. TFPIα or TFPIβ cDNA were transfected into SK-BR-3 breast cancer cells for stable overexpression. Global mRNA and microRNA (miRNA expressions were measured and functional annotation of the differentially expressed genes and miRNAs according to gene ontology terms was conducted. Selected results were validated using qRT-PCR and Western blot. A total of 242 and 801 mRNA transcripts and 120 and 46 miRNAs were differentially expressed in cells overexpressing TFPIα or TFPIβ, respectively. Overexpression of either isoform significantly affected the expression of genes involved in cell development (apoptosis, cell movement, migration, invasion, colony formation, growth, and adhesion and immune response. Network analyses revealed biological interactions between these genes and implied that several of the genes may be involved in both processes. The expression profiles also correlated significantly with clinical phenotype and outcome. Functional cluster analyses indicated altered activity of the epidermal growth factor receptor, small GTPases, and the NF-κB and JAK/STAT cascades when TFPI was overexpressed, and increased activity of the transcription factors NF-κB and Elk-1 and phospho-Akt levels was observed. Integrated mRNA-miRNA analyses showed that 19% and 32% of the differentially expressed genes in cells overexpressing TFPIα or TFPIβ, respectively, may have been regulated by miRNAs. Overexpression of TFPI in breast cancer cells affected the expression of mRNAs and mi

TFPI alpha and beta regulate mRNAs and microRNAs involved in cancer biology and in the immune system in breast cancer cells.

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Stavik, Benedicte; Skretting, Grethe; Olstad, Ole Kristoffer; Sletten, Marit; Dehli Vigeland, Magnus; Sandset, Per Morten; Iversen, Nina

2012-01-01

Emerging evidence indicate a new role of TFPI in cancer biology. We recently reported that both isoforms of TFPI induced apoptosis and inhibited proliferation of cancer cells. The signaling pathway(s) mediating the effects of TFPI is, however, presently still unclear. Our goal was to further investigate the cellular processes affected by TFPI and to get insight into the molecular mechanisms involved in the effects of TFPI, using a global gene expression study approach. TFPIα or TFPIβ cDNA were transfected into SK-BR-3 breast cancer cells for stable overexpression. Global mRNA and microRNA (miRNA) expressions were measured and functional annotation of the differentially expressed genes and miRNAs according to gene ontology terms was conducted. Selected results were validated using qRT-PCR and Western blot. A total of 242 and 801 mRNA transcripts and 120 and 46 miRNAs were differentially expressed in cells overexpressing TFPIα or TFPIβ, respectively. Overexpression of either isoform significantly affected the expression of genes involved in cell development (apoptosis, cell movement, migration, invasion, colony formation, growth, and adhesion) and immune response. Network analyses revealed biological interactions between these genes and implied that several of the genes may be involved in both processes. The expression profiles also correlated significantly with clinical phenotype and outcome. Functional cluster analyses indicated altered activity of the epidermal growth factor receptor, small GTPases, and the NF-κB and JAK/STAT cascades when TFPI was overexpressed, and increased activity of the transcription factors NF-κB and Elk-1 and phospho-Akt levels was observed. Integrated mRNA-miRNA analyses showed that 19% and 32% of the differentially expressed genes in cells overexpressing TFPIα or TFPIβ, respectively, may have been regulated by miRNAs. Overexpression of TFPI in breast cancer cells affected the expression of mRNAs and miRNAs involved in processes

Investigating Relationships among Pre-Service Science Teachers' Conceptual Knowledge of Electric Current, Motivational Beliefs and Self-Regulation

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Inaltun, Hüseyin; Ates, Salih

2015-01-01

The purpose of this study is to examine relationships among pre-service science teachers' conceptual knowledge of electric current, motivational beliefs, and self-regulation. One hundred and twenty-seven students (female = 107, male = 20) enrolled in the science education program of a public university in Ankara participated the study. A concept…

Eukaryotic initiation factor 3 (eIF3) and 5’ mRNA leader sequences as agents of translational regulation in Arabidopsis. Final report

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von Arnim, Albrecht G. [Univ. of Tennessee, Knoxville, TN (United States)

2015-02-04

Protein synthesis, or translation, consumes a sizable fraction of the cell’s energy budget, estimated at 5% and up to 50% in differentiated and growing cells, respectively. Plants also invest significant energy and biomass to construct and maintain the translation apparatus. Translation is regulated by a variety of external stimuli. Compared to transcriptional control, attributes of translational control include reduced sensitivity to stochastic fluctuation, a finer gauge of control, and more rapid responsiveness to environmental stimuli. Yet, our murky understanding of translational control allows few generalizations. Consequently, translational regulation is underutilized in the context of transgene regulation, although synthetic biologists are now beginning to appropriate RNA-level gene regulation into their regulatory circuits. We also know little about how translational control contributes to the diversity of plant form and function. This project explored how an emerging regulatory mRNA sequence element, upstream open reading frames (uORFs), is integrated with the general translation initiation machinery to permit translational regulation on specific mRNAs.

Optimized Mitochondrial Targeting of Proteins Encoded by Modified mRNAs Rescues Cells Harboring Mutations in mtATP6

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Randall Marcelo Chin

2018-03-01

Full Text Available Summary: Mitochondrial disease may be caused by mutations in the protein-coding genes of the mitochondrial genome. A promising strategy for treating such diseases is allotopic expression—the translation of wild-type copies of these proteins in the cytosol, with subsequent translocation into the mitochondria, resulting in rescue of mitochondrial function. In this paper, we develop an automated, quantitative, and unbiased screening platform to evaluate protein localization and mitochondrial morphology. This platform was used to compare 31 mitochondrial targeting sequences and 15 3′ UTRs in their ability to localize up to 9 allotopically expressed proteins to the mitochondria and their subsequent impact on mitochondrial morphology. Taking these two factors together, we synthesized chemically modified mRNAs that encode for an optimized allotopic expression construct for mtATP6. These mRNAs were able to functionally rescue a cell line harboring the 8993T > G point mutation in the mtATP6 gene. : Allotopic expression of proteins normally encoded by mtDNA is a promising therapy for mitochondrial disease. Chin et al. use an unbiased and high-content imaging-based screening platform to optimize allotopic expression. Modified mRNAs encoding for the optimized allotopic expression constructs rescued the respiration and growth of mtATP6-deficient cells. Keywords: mitochondria, mitochondrial disease, mRNA, modified mRNA, ATP6, allotopic expression, rare disease, gene therapy, screening, high content imaging

Identity-specific motivation: How distinct identities direct self-regulation across distinct situations.

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Browman, Alexander S; Destin, Mesmin; Molden, Daniel C

2017-12-01

Research on self-regulation has traditionally emphasized that people's thoughts and actions are guided by either (a) domain-general motivations that emerge from a cumulative history of life experiences, or (b) situation-specific motivations that emerge in immediate response to the incentives present in a particular context. However, more recent studies have illustrated the importance of understanding the interplay between such domain-general and situation-specific motivations across the types of contexts people regularly encounter. The present research, therefore, expands existing perspectives on self-regulation by investigating how people's identities -the internalized roles, relationships, and social group memberships that define who they are-systemically guide when and how different domain-general motivations are activated within specific types of situations. Using the motivational framework described by regulatory focus theory (Higgins, 1997), Studies 1 and 2 demonstrate that people indeed have distinct, identity-specific motivations that uniquely influence their current self-regulation when such identities are active. Studies 3-5 then begin to explore how identity-specific motivations are situated within people's larger self-concept. Studies 3a and 3b demonstrate that the less compatible people's specific identities, the more distinct are the motivations connected to those identities. Studies 4-5 then provide some initial, suggestive evidence that identity-specific motivations are not a separate, superordinate feature of people's identities that then alter how they pursue any subordinate, identity-relevant traits, but instead that such motivations emerge from the cumulative motivational significance of the subordinate traits to which the identities themselves become attached. Implications for understanding the role of the self-concept in self-regulation are discussed. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Regulation of collagen biosynthesis in cultured bovine aortic smooth muscle cells

International Nuclear Information System (INIS)

Stepp, M.A.

1986-01-01

Aortic smooth muscles cells have been implicated in the etiology of lesions which occur in atherosclerosis and hypertension. Both diseases involve proliferation of smooth muscle cells and accumulation of excessive amounts of extracellular matrix proteins, including collagen type I and type III produced by the smooth muscle cells. To better understand the sites of regulation of collagen biosynthesis and to correlate these with the growth rate of the cells, cultured bovine aortic smooth muscle cells were studied as a function of the number of days (3 to 14) in second passage. Cells grew rapidly up to day 6 when confluence was reached. The total incorporation of [ 3 H]-proline into proteins was highest at day 3 and decreased to a constant level after the cultures reached confluence. In contrast, collagen protein production was lowest before confluence and continued to increase over the entire time course of the experiments. cDNA clones for the α1 and α2 chains of type I and the α1 chain of type III collagen were used to quantitate the steady state level of collagen mRNAs. RNA was tested in a cell-free translation system. Changes in the translational activity of collagen mRNAs parallelled the observed increases in collagen protein production. Thus, at later time points, collagen mRNAs are more active in directing synthesis of preprocollagens, even though less collagen mRNA is present. The conclusion is that the site of regulation of the expression of collagen genes is a function of the growth rate of cultured smooth muscle cells

Proteomic analysis of polyribosomes identifies splicing factors as potential regulators of translation during mitosis.

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Aviner, Ranen; Hofmann, Sarah; Elman, Tamar; Shenoy, Anjana; Geiger, Tamar; Elkon, Ran; Ehrlich, Marcelo; Elroy-Stein, Orna

2017-06-02

Precise regulation of mRNA translation is critical for proper cell division, but little is known about the factors that mediate it. To identify mRNA-binding proteins that regulate translation during mitosis, we analyzed the composition of polysomes from interphase and mitotic cells using unbiased quantitative mass-spectrometry (LC-MS/MS). We found that mitotic polysomes are enriched with a subset of proteins involved in RNA processing, including alternative splicing and RNA export. To demonstrate that these may indeed be regulators of translation, we focused on heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a test case and confirmed that it is recruited to elongating ribosomes during mitosis. Then, using a combination of pulsed SILAC, metabolic labeling and ribosome profiling, we showed that knockdown of hnRNP C affects both global and transcript-specific translation rates and found that hnRNP C is specifically important for translation of mRNAs that encode ribosomal proteins and translation factors. Taken together, our results demonstrate how proteomic analysis of polysomes can provide insight into translation regulation under various cellular conditions of interest and suggest that hnRNP C facilitates production of translation machinery components during mitosis to provide daughter cells with the ability to efficiently synthesize proteins as they enter G1 phase. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Pre-attentive, context-specific representation of fear memory in the auditory cortex of rat.

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Akihiro Funamizu

Full Text Available Neural representation in the auditory cortex is rapidly modulated by both top-down attention and bottom-up stimulus properties, in order to improve perception in a given context. Learning-induced, pre-attentive, map plasticity has been also studied in the anesthetized cortex; however, little attention has been paid to rapid, context-dependent modulation. We hypothesize that context-specific learning leads to pre-attentively modulated, multiplex representation in the auditory cortex. Here, we investigate map plasticity in the auditory cortices of anesthetized rats conditioned in a context-dependent manner, such that a conditioned stimulus (CS of a 20-kHz tone and an unconditioned stimulus (US of a mild electrical shock were associated only under a noisy auditory context, but not in silence. After the conditioning, although no distinct plasticity was found in the tonotopic map, tone-evoked responses were more noise-resistive than pre-conditioning. Yet, the conditioned group showed a reduced spread of activation to each tone with noise, but not with silence, associated with a sharpening of frequency tuning. The encoding accuracy index of neurons showed that conditioning deteriorated the accuracy of tone-frequency representations in noisy condition at off-CS regions, but not at CS regions, suggesting that arbitrary tones around the frequency of the CS were more likely perceived as the CS in a specific context, where CS was associated with US. These results together demonstrate that learning-induced plasticity in the auditory cortex occurs in a context-dependent manner.

Pre-attentive, context-specific representation of fear memory in the auditory cortex of rat.

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Funamizu, Akihiro; Kanzaki, Ryohei; Takahashi, Hirokazu

2013-01-01

Neural representation in the auditory cortex is rapidly modulated by both top-down attention and bottom-up stimulus properties, in order to improve perception in a given context. Learning-induced, pre-attentive, map plasticity has been also studied in the anesthetized cortex; however, little attention has been paid to rapid, context-dependent modulation. We hypothesize that context-specific learning leads to pre-attentively modulated, multiplex representation in the auditory cortex. Here, we investigate map plasticity in the auditory cortices of anesthetized rats conditioned in a context-dependent manner, such that a conditioned stimulus (CS) of a 20-kHz tone and an unconditioned stimulus (US) of a mild electrical shock were associated only under a noisy auditory context, but not in silence. After the conditioning, although no distinct plasticity was found in the tonotopic map, tone-evoked responses were more noise-resistive than pre-conditioning. Yet, the conditioned group showed a reduced spread of activation to each tone with noise, but not with silence, associated with a sharpening of frequency tuning. The encoding accuracy index of neurons showed that conditioning deteriorated the accuracy of tone-frequency representations in noisy condition at off-CS regions, but not at CS regions, suggesting that arbitrary tones around the frequency of the CS were more likely perceived as the CS in a specific context, where CS was associated with US. These results together demonstrate that learning-induced plasticity in the auditory cortex occurs in a context-dependent manner.

Two cis-acting elements responsible for posttranscriptional trans-regulation of gene expression of human T-cell leukemia virus type I

International Nuclear Information System (INIS)

Seiki, Motoharu; Inoue, Junichiro; Hidaka, Makoto; Yoshida, Mitsuaki

1988-01-01

The pX sequence of human T-cell leukemia virus type I codes for two nuclear proteins, p40 tax and p27 rex and a cytoplasmic protein, p21 X-III . p40 tax activates transcription from the long terminal repeat (LTR), whereas p27 rex modulates posttranscriptional processing to accumulate gag and env mRNAs that retain intron sequences. In this paper, the authors identify two cis-acting sequence elements needed for regulation by p27 rex : a 5' splice signal and a specific sequence in the 3' LTR. These two sequence elements are sufficient for regulation by p27 rex ; expression of a cellular gene (metallothionein I) became sensitive to rex regulation when the LTR was inserted at the 3' end of this gene. The requirement for these two elements suggests and unusual regulatory mechanism of RNA processing in the nucleus

Tissue-specifically regulated site-specific excision of selectable marker genes in bivalent insecticidal, genetically-modified rice.

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Hu, Zhan; Ding, Xuezhi; Hu, Shengbiao; Sun, Yunjun; Xia, Liqiu

2013-12-01

Marker-free, genetically-modified rice was created by the tissue-specifically regulated Cre/loxP system, in which the Cre recombinase gene and hygromycin phosphotransferase gene (hpt) were flanked by two directly oriented loxP sites. Cre expression was activated by the tissue-specific promoter OsMADS45 in flower or napin in seed, resulting in simultaneous excision of the recombinase and marker genes. Segregation of T1 progeny was performed to select recombined plants. The excision was confirmed by PCR, Southern blot and sequence analyses indicating that efficiency varied from 10 to 53 % for OsMADS45 and from 12 to 36 % for napin. The expression of cry1Ac and vip3A was detected by RT-PCR analysis in marker-free transgenic rice. These results suggested that our tissue-specifically regulated Cre/loxP system could auto-excise marker genes from transgenic rice and alleviate public concerns about the security of GM crops.

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

Science.gov (United States)

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

1999-08-01

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

Rapid dopaminergic modulation of the fish hypothalamic transcriptome and proteome.

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Jason T Popesku

2010-08-01

Full Text Available Dopamine (DA is a major neurotransmitter playing an important role in the regulation of vertebrate reproduction. We developed a novel method for the comparison of transcriptomic and proteomic data obtained from in vivo experiments designed to study the neuroendocrine actions of DA.Female goldfish were injected (i.p. with DA agonists (D1-specific; SKF 38393, or D2-specific; LY 171555 and sacrificed after 5 h. Serum LH levels were reduced by 57% and 75% by SKF 38393 and LY 171555, respectively, indicating that the treatments produced physiologically relevant responses in vivo. Bioinformatic strategies and a ray-finned fish database were established for microarray and iTRAQ proteomic analysis of the hypothalamus, revealing a total of 3088 mRNAs and 42 proteins as being differentially regulated by the treatments. Twenty one proteins and mRNAs corresponding to these proteins appeared on both lists. Many of the mRNAs and proteins affected by the treatments were grouped into the Gene Ontology categorizations of protein complex, signal transduction, response to stimulus, and regulation of cellular processes. There was a 57% and 14% directional agreement between the differentially-regulated mRNAs and proteins for SKF 38393 and LY 171555, respectively.The results demonstrate the applicability of advanced high-throughput genomic and proteomic analyses in an amendable well-studied teleost model species whose genome has yet to be sequenced. We demonstrate that DA rapidly regulates multiple hypothalamic pathways and processes that are also known to be involved in pathologies of the central nervous system.

The STAR protein QKI-7 recruits PAPD4 to regulate post-transcriptional polyadenylation of target mRNAs

OpenAIRE

Yamagishi, Ryota; Tsusaka, Takeshi; Mitsunaga, Hiroko; Maehata, Takaharu; Hoshino, Shin-ichi

2016-01-01

Emerging evidence has demonstrated that regulating the length of the poly(A) tail on an mRNA is an efficient means of controlling gene expression at the post-transcriptional level. In early development, transcription is silenced and gene expression is primarily regulated by cytoplasmic polyadenylation. In somatic cells, considerable progress has been made toward understanding the mechanisms of negative regulation by deadenylation. However, positive regulation through elongation of the poly(A)...

Ghrelin: an emerging player in the regulation of reproduction in non-mammalian vertebrates.

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Unniappan, Suraj

2010-07-01

The endocrine regulation of vertebrate reproduction is achieved by the coordinated actions of multiple endocrine factors mainly produced from the brain, pituitary, and gonads. In addition to these, several other tissues including the fat and gut produce factors that have reproductive effects. Ghrelin is one such gut/brain hormone with species-specific effects in the regulation of mammalian reproduction. Recent studies have shown that ghrelin and ghrelin receptor mRNAs, and protein are expressed in the ovary and testis of mammals, indicating a direct effect for ghrelin in the control of reproduction. Ghrelin regulates mammalian reproduction by modulating hormone secretion from the brain and pituitary, and by acting directly on the gonads to influence reproductive tissue development and steroid hormone release. Based on the studies reported so far, ghrelin seems to have a predominantly inhibitory role on mammalian reproduction. The presence of ghrelin and ghrelin receptor has been found in the brain, pituitary and gonads of several non-mammalian vertebrates. In contrast to mammals, ghrelin seems to have a stimulatory role in the regulation of non-mammalian reproduction. The main objective of this review is to do a perspective analysis of the comparative aspects of ghrelin regulation of reproduction. (c) 2009 Elsevier Inc. All rights reserved.

A novel mammal-specific three partite enhancer element regulates node and notochord-specific Noto expression.

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Leonie Alten

Full Text Available The vertebrate organizer and notochord have conserved, essential functions for embryonic development and patterning. The restricted expression of developmental regulators in these tissues is directed by specific cis-regulatory modules (CRMs whose sequence conservation varies considerably. Some CRMs have been conserved throughout vertebrates and likely represent ancestral regulatory networks, while others have diverged beyond recognition but still function over a wide evolutionary range. Here we identify and characterize a mammalian-specific CRM required for node and notochord specific (NNC expression of NOTO, a transcription factor essential for node morphogenesis, nodal cilia movement and establishment of laterality in mouse. A 523 bp enhancer region (NOCE upstream the Noto promoter was necessary and sufficient for NNC expression from the endogenous Noto locus. Three subregions in NOCE together mediated full activity in vivo. Binding sites for known transcription factors in NOCE were functional in vitro but dispensable for NOCE activity in vivo. A FOXA2 site in combination with a novel motif was necessary for NOCE activity in vivo. Strikingly, syntenic regions in non-mammalian vertebrates showed no recognizable sequence similarities. In contrast to its activity in mouse NOCE did not drive NNC expression in transgenic fish. NOCE represents a novel, mammal-specific CRM required for the highly restricted Noto expression in the node and nascent notochord and thus regulates normal node development and function.

A novel mammal-specific three partite enhancer element regulates node and notochord-specific Noto expression.

Science.gov (United States)

Alten, Leonie; Schuster-Gossler, Karin; Eichenlaub, Michael P; Wittbrodt, Beate; Wittbrodt, Joachim; Gossler, Achim

2012-01-01

The vertebrate organizer and notochord have conserved, essential functions for embryonic development and patterning. The restricted expression of developmental regulators in these tissues is directed by specific cis-regulatory modules (CRMs) whose sequence conservation varies considerably. Some CRMs have been conserved throughout vertebrates and likely represent ancestral regulatory networks, while others have diverged beyond recognition but still function over a wide evolutionary range. Here we identify and characterize a mammalian-specific CRM required for node and notochord specific (NNC) expression of NOTO, a transcription factor essential for node morphogenesis, nodal cilia movement and establishment of laterality in mouse. A 523 bp enhancer region (NOCE) upstream the Noto promoter was necessary and sufficient for NNC expression from the endogenous Noto locus. Three subregions in NOCE together mediated full activity in vivo. Binding sites for known transcription factors in NOCE were functional in vitro but dispensable for NOCE activity in vivo. A FOXA2 site in combination with a novel motif was necessary for NOCE activity in vivo. Strikingly, syntenic regions in non-mammalian vertebrates showed no recognizable sequence similarities. In contrast to its activity in mouse NOCE did not drive NNC expression in transgenic fish. NOCE represents a novel, mammal-specific CRM required for the highly restricted Noto expression in the node and nascent notochord and thus regulates normal node development and function.

Fetal sex-specific differences in gestational age at delivery in pre-eclampsia

DEFF Research Database (Denmark)

Schalekamp-Timmermans, Sarah; Arends, Lidia R.; Alsaker, Elin

2017-01-01

Background: Pre-eclampsia (PE) is a major pregnancy disorder complicating up to 8% of pregnancies. Increasing evidence indicates a sex-specific interplay between the mother,placenta and fetus. This may lead to different adaptive mechanisms during pregnancy. Methods: We performed an individual...... fetus as compared with pregnancies with a male fetus (OR 1.36, 95% CI 1.17-1.59). Conclusions: Sexual dimorphic differences in the occurrence of PE exist, with preterm PE being more prevalent among pregnancies with a female fetus as compared with pregnancies with a male fetus and with no differences...

CELF family RNA-binding protein UNC-75 regulates two sets of mutually exclusive exons of the unc-32 gene in neuron-specific manners in Caenorhabditis elegans.

Directory of Open Access Journals (Sweden)

Hidehito Kuroyanagi

Full Text Available An enormous number of alternative pre-mRNA splicing patterns in multicellular organisms are coordinately defined by a limited number of regulatory proteins and cis elements. Mutually exclusive alternative splicing should be strictly regulated and is a challenging model for elucidating regulation mechanisms. Here we provide models of the regulation of two sets of mutually exclusive exons, 4a-4c and 7a-7b, of the Caenorhabditis elegans uncoordinated (unc-32 gene, encoding the a subunit of V0 complex of vacuolar-type H(+-ATPases. We visualize selection patterns of exon 4 and exon 7 in vivo by utilizing a trio and a pair of symmetric fluorescence splicing reporter minigenes, respectively, to demonstrate that they are regulated in tissue-specific manners. Genetic analyses reveal that RBFOX family RNA-binding proteins ASD-1 and FOX-1 and a UGCAUG stretch in intron 7b are involved in the neuron-specific selection of exon 7a. Through further forward genetic screening, we identify UNC-75, a neuron-specific CELF family RNA-binding protein of unknown function, as an essential regulator for the exon 7a selection. Electrophoretic mobility shift assays specify a short fragment in intron 7a as the recognition site for UNC-75 and demonstrate that UNC-75 specifically binds via its three RNA recognition motifs to the element including a UUGUUGUGUUGU stretch. The UUGUUGUGUUGU stretch in the reporter minigenes is actually required for the selection of exon 7a in the nervous system. We compare the amounts of partially spliced RNAs in the wild-type and unc-75 mutant backgrounds and raise a model for the mutually exclusive selection of unc-32 exon 7 by the RBFOX family and UNC-75. The neuron-specific selection of unc-32 exon 4b is also regulated by UNC-75 and the unc-75 mutation suppresses the Unc phenotype of the exon-4b-specific allele of unc-32 mutants. Taken together, UNC-75 is the neuron-specific splicing factor and regulates both sets of the mutually exclusive

Induction of CYP1A1, CYP1A2, and CYP1B1 mRNAs by nitropolycyclic aromatic hydrocarbons in various human tissue-derived cells: chemical-, cytochrome P450 isoform-, and cell-specific differences

Energy Technology Data Exchange (ETDEWEB)

Iwanari, M.; Nakajima, M.; Yokoi, T. [Div. of Drug Metabolism, Kanazawa Univ., Kanazawa (Japan); Kizu, R.; Hayakawa, K. [Lab. of Hygienic Chemistry, Kanazawa Univ., Kanazawa (Japan)

2002-06-01

Nitropolycyclic aromatic hydrocarbons (NPAHs) are found in diesel exhaust and ambient air. NPAHs as well as polycyclic aromatic hydrocarbons (PAHs) are known to have mutagenicity, carcinogenicity, and endocrine-disruptive effects. In the present study, the inducibility of the human cytochrome P450-1 (CYP1) family by NPAHs was compared with those produced by their parent PAHs and some reductive metabolites, amino-PAHs. Furthermore, to investigate the differences in the inducibility of the CYP1 family in human tissues, various human tissue-derived cell lines, namely HepG2 (hepatocellular carcinoma), ACHN (renal carcinoma), A549 (lung carcinoma), MCF-7 (breast carcinoma), LS-180 (colon carcinoma), HT-1197 (bladder carcinoma), HeLa (cervix of uterus adenocarcinoma), OMC-3 (ovarian carcinoma), and NEC14 (testis embryonal carcinoma), were treated with NPAHs, PAHs, or amino-PAHs. The mRNA levels of CYP1A1, CYP1A2, and CYP1B1 were determined with reverse transcription-polymerase chain reaction (RT-PCR). The cell lines were classified into two groups: CYP1 inducible cell lines, comprising HepG2, MCF-7, LS-180, and OMC-3 cells, and CYP1 non-inducible cell lines, comprising ACHN, A549, HT-1197, HeLa, and NEC14 cells. In inducible cell lines, the induction profile of chemical specificity was similar for CYP1A1, CYP1A2, and CYP1B1, although the extent of induction differed among the cell lines and for the CYP isoforms. Pyrene, 1-nitropyrene, 1-aminopyrene, 1,3-, 1,6-, and 1,8-dinitropyrenes slightly induced CYP1 mRNAs, but 1,3-dinitropyrene produced a 6-fold induction of CYP1A1 mRNA in MCF-7 cells. 2-Nitrofluoranthene and 3-nitrofluoranthene exhibited stronger inducibility than fluoranthene in the inducible cell lines. 6-Nitrochrysene induced CYP1 mRNAs to the same extent or more potently than chrysene. The induction potencies of 6-nitrobenzo[a]pyrene and 7-nitrobenz[a]anthracene were weaker than those of their parents benzo[a]pyrene and benz[a]anthracene, respectively. This

Regulation and registration as drivers of continuous professional competence for Irish pre-hospital practitioners: a discussion paper.

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Knox, S; Dunne, S S; Hughes, M; Cheeseman, S; Dunne, C P

2016-05-01

The regulatory body responsible for the registration of Irish pre-hospital practitioners, the Pre-Hospital Emergency Care Council (PHECC), identified the need to implement a continuing professional competence (CPC) framework. The first cycle of CPC (focused on emergency medical technicians) commenced in November 2013 creating for the first time a formal relationship between continuing competence and registration to practice. To review current literature and to describe benefits and challenges relevant to CPC, regulation, registration and their respective contributions to professionalism of pre-hospital practitioners: advanced paramedics, paramedics and emergency medical technicians. Online search of cumulative index to nursing and allied health literature (CINAHL Plus with Full Text), Allied and Complementary Medicine (AMED) and 'Pubmed' databases using: 'Continuous Professional Development'; 'Continuous Professional Development'; 'emergency medical technician'; 'paramedic'; 'registration'; 'regulation'; and "profession' for relevant articles published since 2004. Additional policy documents, discussion papers, and guidance documents were identified from bibliographies of papers found. Reports, governmental policies for other healthcare professions, and professional developments internationally for allied professions (e.g., nursing, physiotherapy and medicine) link maintenance of competence with requirements for registration to practice. We suggest that evolving professionalisation of Irish paramedics should be affirmed through behaviours and competencies that incorporate adherence to professional codes of conduct, reflective practice, and commitment to continuing professional development. While the need for ambulance practitioner CPD was identified in Ireland almost a decade ago, PHECC now has the opportunity to introduce a model of CPD for paramedics linking competence and professionalism to annual registration.

A hairpin within YAP mRNA 3′UTR functions in regulation at post-transcription level

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Gao, Yuen; Wang, Yuan; Feng, Jinyan; Feng, Guoxing; Zheng, Minying; Yang, Zhe; Xiao, Zelin; Lu, Zhanping [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin 300071 (China)

2015-04-03

The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. Recently, it has been reported that mRNAs display regulatory roles that rely on their ability to compete for microRNA binding, independent of their protein-coding function. However, the regulatory mechanism of mRNAs remains poorly understood. Here, we report that a hairpin within YAP mRNA 3′untranslated region (3′UTR) functions in regulation at post-transcription level through generating endogenous siRNAs (esiRNAs). Bioinformatics analysis for secondary structure showed that YAP mRNA displayed a hairpin structure (termed standard hairpin, S-hairpin) within its 3′UTR. Surprisingly, we observed that the overexpression of S-hairpin derived from YAP 3′UTR (YAP-sh) increased the luciferase reporter activities of transcriptional factor NF-κB and AP-1 in 293T cells. Moreover, we identified that a fragment from YAP-sh, an esiRNA, was able to target mRNA 3′UTR of NF2 (a member of Hippo-signaling pathway) and YAP mRNA 3′UTR itself in hepatoma cells. Thus, we conclude that the YAP-sh within YAP mRNA 3′UTR may serve as a novel regulatory element, which functions in regulation at post-transcription level. Our finding provides new insights into the mechanism of mRNAs in regulatory function. - Highlights: • An S-hairpin within YAP mRNA 3′UTR possesses regulatory function. • YAP-sh acts as a regulatory element for YAP at post-transcription level. • YAP-sh-3p20, an esiRNA derived from YAP-sh, targets mRNAs of YAP and NF2. • YAP-sh-3p20 depresses the proliferation of HepG2 cells in vitro.

A hairpin within YAP mRNA 3′UTR functions in regulation at post-transcription level

International Nuclear Information System (INIS)

Gao, Yuen; Wang, Yuan; Feng, Jinyan; Feng, Guoxing; Zheng, Minying; Yang, Zhe; Xiao, Zelin; Lu, Zhanping; Ye, Lihong; Zhang, Xiaodong

2015-01-01

The central dogma of gene expression is that DNA is transcribed into messenger RNAs, which in turn serve as the template for protein synthesis. Recently, it has been reported that mRNAs display regulatory roles that rely on their ability to compete for microRNA binding, independent of their protein-coding function. However, the regulatory mechanism of mRNAs remains poorly understood. Here, we report that a hairpin within YAP mRNA 3′untranslated region (3′UTR) functions in regulation at post-transcription level through generating endogenous siRNAs (esiRNAs). Bioinformatics analysis for secondary structure showed that YAP mRNA displayed a hairpin structure (termed standard hairpin, S-hairpin) within its 3′UTR. Surprisingly, we observed that the overexpression of S-hairpin derived from YAP 3′UTR (YAP-sh) increased the luciferase reporter activities of transcriptional factor NF-κB and AP-1 in 293T cells. Moreover, we identified that a fragment from YAP-sh, an esiRNA, was able to target mRNA 3′UTR of NF2 (a member of Hippo-signaling pathway) and YAP mRNA 3′UTR itself in hepatoma cells. Thus, we conclude that the YAP-sh within YAP mRNA 3′UTR may serve as a novel regulatory element, which functions in regulation at post-transcription level. Our finding provides new insights into the mechanism of mRNAs in regulatory function. - Highlights: • An S-hairpin within YAP mRNA 3′UTR possesses regulatory function. • YAP-sh acts as a regulatory element for YAP at post-transcription level. • YAP-sh-3p20, an esiRNA derived from YAP-sh, targets mRNAs of YAP and NF2. • YAP-sh-3p20 depresses the proliferation of HepG2 cells in vitro

Post-Transcriptional Regulation by the Csr Global Regulatory System in Escherichia coli

OpenAIRE

Suzuki, Kazushi; 鈴木, 一史

2007-01-01

In many species of bacteria, the Csr (carbon storage regulator) global regulatory system coordinates the expression of various genes. In Escherichia coli, the central component of this system, CsrA, is a RNA-binding protein. The CsrA is a homodimer and binds to leader segments of target mRNAs, affecting their translation and stability. CsrA activity is regulated by two small non-coding RNAs, CsrB and CsrC. These RNAs contain multiple CsrA-binding sequences and act by sequestering CsrA. In thi...

Effects of pathogen reduction systems on platelet microRNAs, mRNAs, activation, and function.

Science.gov (United States)

Osman, Abdimajid; Hitzler, Walter E; Meyer, Claudius U; Landry, Patricia; Corduan, Aurélie; Laffont, Benoit; Boilard, Eric; Hellstern, Peter; Vamvakas, Eleftherios C; Provost, Patrick

2015-01-01

Pathogen reduction (PR) systems for platelets, based on chemically induced cross-linking and inactivation of nucleic acids, potentially prevent transfusion transmission of infectious agents, but can increase clinically significant bleeding in some clinical studies. Here, we documented the effects of PR systems on microRNA and mRNA levels of platelets stored in the blood bank, and assessed their impact on platelet activation and function. Unlike platelets subjected to gamma irradiation or stored in additive solution, platelets treated with Intercept (amotosalen+ ultraviolet-A [UVA] light) exhibited significantly reduced levels of 6 of the 11 microRNAs, and 2 of the 3 anti-apoptotic mRNAs (Bcl-xl and Clusterin) that we monitored, compared with platelets stored in plasma. Mirasol (riboflavin+ UVB light) treatment of platelets did not produce these effects. PR neither affected platelet microRNA synthesis or function nor induced cross-linking of microRNA-sized endogenous platelet RNA species. However, the reduction in the platelet microRNA levels induced by Intercept correlated with the platelet activation (p < 0.05) and an impaired platelet aggregation response to ADP (p < 0.05). These results suggest that Intercept treatment may induce platelet activation, resulting in the release of microRNAs and mRNAs from platelets. The clinical implications of this reduction in platelet nucleic acids secondary to Intercept remain to be established.

Minoxidil specifically decreases the expression of lysine hydroxylase in cultured human skin fibroblasts.

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Hautala, T; Heikkinen, J; Kivirikko, K I; Myllylä, R

1992-01-01

The levels of lysine hydroxylase protein and the levels of the mRNAs for lysine hydroxylase and the alpha- and beta-subunits of proline 4-hydroxylase were measured in cultured human skin fibroblasts treated with 1 mM-minoxidil. The data demonstrate that minoxidil decreases the amount of lysine hydroxylase protein, this being due to a decrease in the level of lysine hydroxylase mRNA. The effect of minoxidil appears to be highly specific, as no changes were observed in the amounts of mRNAs for the alpha- and beta-subunits of proline 4-hydroxylase. Images Fig. 1. Fig. 2. Fig. 3. PMID:1314568

Pretreatment antigen-specific immunity and regulation - association with subsequent immune response to anti-tumor DNA vaccination.

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Johnson, Laura E; Olson, Brian M; McNeel, Douglas G

2017-07-18

Immunotherapies have demonstrated clinical benefit for many types of cancers, however many patients do not respond, and treatment-related adverse effects can be severe. Hence many efforts are underway to identify treatment predictive biomarkers. We have reported the results of two phase I trials using a DNA vaccine encoding prostatic acid phosphatase (PAP) in patients with biochemically recurrent prostate cancer. In both trials, persistent PAP-specific Th1 immunity developed in some patients, and this was associated with favorable changes in serum PSA kinetics. In the current study, we sought to determine if measures of antigen-specific or antigen non-specific immunity were present prior to treatment, and associated with subsequent immune response, to identify possible predictive immune biomarkers. Patients who developed persistent PAP-specific, IFNγ-secreting immune responses were defined as immune "responders." The frequency of peripheral T cell and B cell lymphocytes, natural killer cells, monocytes, dendritic cells, myeloid derived suppressor cells, and regulatory T cells were assessed by flow cytometry and clinical laboratory values. PAP-specific immune responses were evaluated by cytokine secretion in vitro, and by antigen-specific suppression of delayed-type hypersensitivity to a recall antigen in an in vivo SCID mouse model. The frequency of peripheral blood cell types did not differ between the immune responder and non-responder groups. Non-responder patients tended to have higher PAP-specific IL-10 production pre-vaccination (p = 0.09). Responder patients had greater preexisting PAP-specific bystander regulatory responses that suppressed DTH to a recall antigen (p = 0.016). While our study population was small (n = 38), these results suggest that different measures of antigen-specific tolerance or regulation might help predict immunological outcome from DNA vaccination. These will be prospectively evaluated in an ongoing randomized, phase II trial.

76 FR 10947 - Registration and Regulation of Security-Based Swap Execution Facilities

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2011-02-28

... SB swaps is opaque, with little, if any, pre-trade transparency (the ability of market participants... development of the SB swap market may alter some of the specific calculus for future regulation of SB SEFs..., there may be no pre-trade or post-trade transparency available to the marketplace because only the two...

Targeted Regression of Hepatocellular Carcinoma by Cancer-Specific RNA Replacement through MicroRNA Regulation.

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Kim, Juhyun; Won, Ranhui; Ban, Guyee; Ju, Mi Ha; Cho, Kyung Sook; Young Han, Sang; Jeong, Jin-Sook; Lee, Seong-Wook

2015-07-20

Hepatocellular carcinoma (HCC) has a high fatality rate and limited therapeutic options with side effects and low efficacy. Here, we proposed a new anti-HCC approach based on cancer-specific post-transcriptional targeting. To this end, trans-splicing ribozymes from Tetrahymena group I intron were developed, which can specifically induce therapeutic gene activity through HCC-specific replacement of telomerase reverse transcriptase (TERT) RNA. To circumvent side effects due to TERT expression in regenerating liver tissue, liver-specific microRNA-regulated ribozymes were constructed by incorporating complementary binding sites for the hepatocyte-selective microRNA-122a (miR-122a), which is down-regulated in HCC. The ribozyme activity in vivo was assessed in mouse models orthotopically implanted with HCC. Systemic administration of adenovirus encoding the developed ribozymes caused efficient anti-cancer effect and the least hepatotoxicity with regulation of ribozyme expression by miR-122a in both xenografted and syngeneic orthotopic murine model of multifocal HCC. Of note, the ribozyme induced local and systemic antitumor immunity, thereby completely suppressing secondary tumor challenge in the syngeneic mouse. The cancer specific trans-splicing ribozyme system, which mediates tissue-specific microRNA-regulated RNA replacement, provides a clinically relevant, safe, and efficient strategy for HCC treatment.

Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance.

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Morimoto, Shimpei; Yahara, Koji

2018-03-01

Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes ( ADC17 and KIN1 ) that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning) that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after accounting for potential

Interplay of noncoding RNAs, mRNAs, and proteins during the growth of eukaryotic cells

International Nuclear Information System (INIS)

Zhdanov, V. P.

2010-01-01

Numerous biological functions of noncoding RNAs (ncRNAs) in eukaryotic cells are based primarily on their ability to pair with target mRNAs and then either to prevent translation or to result in rapid degradation of the mRNA-ncRNA complex. Using a general model describing this scenario, we show that ncRNAs may help to maintain constant mRNA and protein concentrations during the growth of cells. The possibility of observation of this effect on the global scale is briefly discussed.

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication.

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Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

2017-01-09

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The significance of translation regulation in the stress response

OpenAIRE

Picard, Flora; Loubière, Pascal; Girbal, Laurence; Bousquet, Muriel

2013-01-01

Background: The stress response in bacteria involves the multistage control of gene expression but is not entirely understood. To identify the translational response of bacteria in stress conditions and assess its contribution to the regulation of gene expression, the translational states of all mRNAs were compared under optimal growth condition and during nutrient (isoleucine) starvation. Results: A genome-scale study of the translational response to nutritional limitation was performed in t...

Nicotine induces cell proliferation in association with cyclin D1 up-regulation and inhibits cell differentiation in association with p53 regulation in a murine pre-osteoblastic cell line

International Nuclear Information System (INIS)

Sato, Tsuyoshi; Abe, Takahiro; Nakamoto, Norimichi; Tomaru, Yasuhisa; Koshikiya, Noboru; Nojima, Junya; Kokabu, Shoichiro; Sakata, Yasuaki; Kobayashi, Akio; Yoda, Tetsuya

2008-01-01

Recent studies have suggested that nicotine critically affects bone metabolism. Many studies have examined the effects of nicotine on proliferation and differentiation, but the underlying molecular mechanisms remain unclear. We examined cell cycle regulators involved in the proliferation and differentiation of MC3T3-E1 cells. Nicotine induced cell proliferation in association with p53 down-regulation and cyclin D1 up-regulation. In differentiated cells, nicotine reduced alkaline phosphatase activity and mineralized nodule formation in dose-dependent manners. Furthermore, p53 expression was sustained in nicotine-treated cells during differentiation. These findings indicate that nicotine promotes the cell cycle and inhibits differentiation in association with p53 regulation in pre-osteoblastic cells

The Role of Pre-Service Teachers' Perceived Instrumentality, Goal Commitment, and Motivation in Their Self-Regulation Strategies for Learning in Teacher Education Courses

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Lee, Jumi; Turner, Jeannine

2017-01-01

This study investigated the influence of pre-service teachers' (n = 142) perceived endogenous/exogenous instrumentality, goal commitment, and intrinsic/extrinsic motivation on their use of self-regulation strategies (effort regulation, management of time and study environment) for their teacher-education courses. Data were drawn from a customised…

Human trabecular meshwork cells express BMP antagonist mRNAs and proteins.

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Tovar-Vidales, Tara; Fitzgerald, Ashley M; Clark, Abbot F

2016-06-01

Glaucoma patients have elevated aqueous humor and trabecular meshwork (TM) levels of transforming growth factor-beta2 (TGF-β2). TGF-β2 has been associated with increased extracellular matrix (ECM) deposition (i.e. fibronectin), which is attributed to the increased resistance of aqueous humor outflow through the TM. We have previously demonstrated that bone morphogenetic protein (BMP) 4 selectively counteracts the profibrotic effect of TGF-β2 with respect to ECM synthesis in the TM, and this action is reversed by the BMP antagonist gremlin. Thus, the BMP and TGF-β signaling pathways antagonize each other's antifibrotic and profibrotic roles. The purpose of this study was to determine whether cultured human TM cells: (a) express other BMP antagonists including noggin, chordin, BMPER, BAMBI, Smurf1 and 2, and (b) whether expression of these proteins is regulated by exogenous TGF-β2 treatment. Primary human trabecular meshwork (TM) cells were grown to confluency and treated with TGF-β2 (5 ng/ml) for 24 or 48 h in serum-free medium. Untreated cell served as controls. qPCR and Western immunoblots (WB) determined that human TM cells expressed mRNAs and proteins for the BMP antagonist proteins: noggin, chordin, BMPER, BAMBI, and Smurf1/2. Exogenous TGF-β2 decreased chordin, BMPER, BAMBI, and Smurf1 mRNA and protein expression. In contrast, TGF-β2 increased secreted noggin and Smurf2 mRNA and protein levels. BMP antagonist members are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of BMP antagonist members may lead to functional changes in the human TM. Copyright © 2016 Elsevier Ltd. All rights reserved.

MicroRNA-dependent regulation of transcription in non-small cell lung cancer.

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Sonia Molina-Pinelo

Full Text Available Squamous cell lung cancer (SCC and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC, and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA and mRNA profiling (Whole Genome 44 K array G112A, Agilent was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a and miR-708 and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1 were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies.

Do People With Psychosis Have Specific Difficulties Regulating Emotions?

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Lincoln, Tania M; Hartmann, Maike; Köther, Ulf; Moritz, Steffen

2015-01-01

Difficulties in emotion regulation (ER) are present in psychotic disorders, but their precise nature is not yet fully understood and it is unclear which difficulties are unique to psychosis compared with other disorders. This study investigated whether ER difficulties in psychosis are more prominent for the ability to modify emotions or for the ability to tolerate and accept them. Furthermore, it investigated whether ER difficulties occur for sadness, anxiety, anger and shame likewise. ER skills were assessed in participants with psychotic disorders (n = 37), participants with depression (n = 30) and healthy controls (n = 28) using the Emotion Regulation Skill Questionnaire that asks participants to rate the intensity of different emotions over the past week and the skills employed to handle each of them. Compared with healthy controls, participants with psychosis showed reduced skills related to awareness, understanding and acceptance of potentially distressing emotions, but not in the ability to modify them. These differences remained significant after controlling for depression. Participants with psychosis showed reduced ER skills in regard to all of the assessed emotions compared with the healthy controls, despite the fact that they only reported sadness as being significantly more intense. The participants with depression showed a similar pattern of ER skills to the psychosis sample, although with a tendency towards even more pronounced difficulties. It is concluded that psychosis is characterized by difficulties in using specific ER skills related to awareness, understanding and acceptance to regulate anger, shame, anxiety and sadness. These difficulties are not unique to psychosis but nevertheless present a promising treatment target. The participants with psychosis found it more difficult to be aware of their emotions, to understand them and to accept them than the healthy control group. However, they reported equal skills when it came to

Localization of hyaluronan with a hyaluronan-specific hyaluronic acid binding protein in the placenta in pre-eclampsia.

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Matejevic, D; Neudeck, H; Graf, R; Müller, T; Dietl, J

2001-01-01

Hyaluronan (HA), a high molecular weight polysaccharide, is a major component of connective tissue and is thus present in the extracellular matrix of most tissues. Increased serum concentrations have been reported in association with pre-eclampsia and liver malfunction, amongst other disorders. We have performed histochemical investigations with a HA-specific hyaluronic acid binding protein in placentas from uncomplicated pregnancies and from patients with pre-eclampsia. Staining for HA was found in the stroma and blood vessel walls of stem villi in all the placentas investigated. The syncytiotrophoblast and cytotrophoblast cells usually remained unstained. In addition, reactivity for HA was found within and on the surface of intervillous and perivillous fibrinoid deposits. Since fibrinoid deposits are increased in pre-eclampsia, our findings suggest that the increased HA serum concentrations in cases of pre-eclampsia could result from the stroma of the infarcted villi and from the fibrinoid deposits. HA may reach the maternal blood through fibrinoid gaps. Copyright 2001 S. Karger AG, Basel

Coordinated Regulations of mRNA Synthesis and Decay during Cold Acclimation in Arabidopsis Cells.

KAUST Repository

Arae, Toshihiro

2017-04-18

Plants possess a cold acclimation system to acquire freezing tolerance through pre-exposure to non-freezing low temperatures. The transcriptional cascade of C-repeat binding factors (CBFs)/dehydration response element-binding factors (DREBs) is considered a major transcriptional regulatory pathway during cold acclimation. However, little is known regarding the functional significance of mRNA stability regulation in the response of gene expression to cold stress. The actual level of individual mRNAs is determined by a balance between mRNA synthesis and degradation. Therefore, it is important to assess the regulatory steps to increase our understanding of gene regulation. Here, we analyzed temporal changes in mRNA amounts and half-lives in response to cold stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were estimated from the above two parameters using a mathematical approach. Our results showed that several cold-responsive genes, including Cold-regulated 15a, were relatively destabilized, whereas the mRNA amounts were increased during cold treatment by accelerating the transcription rate to overcome the destabilization. Considering the kinetics of mRNA synthesis and degradation, this apparently contradictory result supports that mRNA destabilization is advantageous for the swift increase in CBF-responsive genes in response to cold stress.

Novel mRNA-specific effects of ribosome drop-off on translation rate and polysome profile.

Directory of Open Access Journals (Sweden)

Pierre Bonnin

2017-05-01

Full Text Available The well established phenomenon of ribosome drop-off plays crucial roles in translational accuracy and nutrient starvation responses during protein translation. When cells are under stress conditions, such as amino acid starvation or aminoacyl-tRNA depletion due to a high level of recombinant protein expression, ribosome drop-off can substantially affect the efficiency of protein expression. Here we introduce a mathematical model that describes the effects of ribosome drop-off on the ribosome density along the mRNA and on the concomitant protein synthesis rate. Our results show that ribosome premature termination may lead to non-intuitive ribosome density profiles, such as a ribosome density which increases from the 5' to the 3' end. Importantly, the model predicts that the effects of ribosome drop-off on the translation rate are mRNA-specific, and we quantify their resilience to drop-off, showing that the mRNAs which present ribosome queues are much less affected by ribosome drop-off than those which do not. Moreover, among those mRNAs that do not present ribosome queues, resilience to drop-off correlates positively with the elongation rate, so that sequences using fast codons are expected to be less affected by ribosome drop-off. This result is consistent with a genome-wide analysis of S. cerevisiae, which reveals that under favourable growth conditions mRNAs coding for proteins involved in the translation machinery, known to be highly codon biased and using preferentially fast codons, are highly resilient to ribosome drop-off. Moreover, in physiological conditions, the translation rate of mRNAs coding for regulatory, stress-related proteins, is less resilient to ribosome drop-off. This model therefore allows analysis of variations in the translational efficiency of individual mRNAs by accounting for the full range of known ribosome behaviours, as well as explaining mRNA-specific variations in ribosome density emerging from ribosome profiling

CKI isoforms α and ε regulate Star–PAP target messages by controlling Star–PAP poly(A) polymerase activity and phosphoinositide stimulation

Science.gov (United States)

Laishram, Rakesh S.; Barlow, Christy A.; Anderson, Richard A.

2011-01-01

Star–PAP is a non-canonical, nuclear poly(A) polymerase (PAP) that is regulated by the lipid signaling molecule phosphatidylinositol 4,5 bisphosphate (PI4,5P2), and is required for the expression of a select set of mRNAs. It was previously reported that a PI4,5P2 sensitive CKI isoform, CKIα associates with and phosphorylates Star–PAP in its catalytic domain. Here, we show that the oxidative stress-induced by tBHQ treatment stimulates the CKI mediated phosphorylation of Star–PAP, which is critical for both its polyadenylation activity and stimulation by PI4,5P2. CKI activity was required for the expression and efficient 3′-end processing of its target mRNAs in vivo as well as the polyadenylation activity of Star–PAP in vitro. Specific CKI activity inhibitors (IC261 and CKI7) block in vivo Star–PAP activity, but the knockdown of CKIα did not equivalently inhibit the expression of Star–PAP targets. We show that in addition to CKIα, Star–PAP associates with another CKI isoform, CKIε in the Star–PAP complex that phosphorylates Star–PAP and complements the loss of CKIα. Knockdown of both CKI isoforms (α and ε) resulted in the loss of expression and the 3′-end processing of Star–PAP targets similar to the CKI activity inhibitors. Our results demonstrate that CKI isoforms α and ε modulate Star–PAP activity and regulates Star–PAP target messages. PMID:21729869

Identification of mRNAs that move over long distances using an RNA-Seq analysis of Arabidopsis/Nicotiana benthamiana heterografts.

Science.gov (United States)

Notaguchi, Michitaka; Higashiyama, Tetsuya; Suzuki, Takamasa

2015-02-01

Phloem is a conductive tissue that allocates nutrients from mature source leaves to sinks such as young developing tissues. Phloem also delivers proteins and RNA species, such as small RNAs and mRNAs. Intensive studies on plant systemic signaling revealed the essential roles of proteins and RNA species. However, many of their functions are still largely unknown, with the roles of transported mRNAs being particularly poorly understood. A major difficulty is the absence of an accurate and comprehensive list of mobile transcripts. In this study, we used a hetero-graft system with Nicotiana benthamiana as the recipient scion and Arabidopsis as the donor stock, to identify transcripts that moved long distances across the graft union. We identified 138 Arabidopsis transcripts as mobile mRNAs, which we collectively termed the mRNA mobilome. Reverse transcription-PCR, quantitative real-time PCR and droplet digital PCR analyses confirmed the mobility. The transcripts included potential signaling factors and, unexpectedly, more general factors. In our investigations, we found no preferred transcript length, no previously known sequence motifs in promoter or transcript sequences and no similarities between the level of the transcripts and that in the source leaves. Grafting experiments regarding the function of ERECTA, an identified transcript, showed that no function of the transcript mobilized. To our knowledge, this is the first report identifying transcripts that move over long distances using a hetero-graft system between different plant taxa. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

No evidence that mRNAs have lower folding free energies than random sequences with the same dinucleotide distribution

DEFF Research Database (Denmark)

Workman, Christopher; Krogh, Anders Stærmose

1999-01-01

This work investigates whether mRNA has a lower estimated folding free energy than random sequences. The free energy estimates are calculated by the mfold program for prediction of RNA secondary structures. For a set of 46 mRNAs it is shown that the predicted free energy is not significantly diff...

The Human Splicing Factor ASF/SF2 can Specifically Recognize Pre-mRNA 5' Splice Sites

Science.gov (United States)

Zuo, Ping; Manley, James L.

1994-04-01

ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.

Specificity of the E. coli LysR-type transcriptional regulators.

Directory of Open Access Journals (Sweden)

Gwendowlyn S Knapp

2010-12-01

Full Text Available Families of paralogous oligomeric proteins are common in biology. How the specificity of assembly evolves is a fundamental question of biology. The LysR-Type Transcriptional Regulators (LTTR form perhaps the largest family of transcriptional regulators in bacteria. Because genomes often encode many LTTR family members, it is assumed that many distinct homooligomers are formed simultaneously in the same cell without interfering with each other's activities, suggesting specificity in the interactions. However, this assumption has not been systematically tested.A negative-dominant assay with λcI repressor fusions was used to evaluate the assembly of the LTTRs in E. coli K-12. Thioredoxin (Trx-LTTR fusions were used to challenge the homooligomeric interactions of λcI-LTTR fusions. Eight cI-LTTR fusions were challenged with twenty-eight Trx fusions. LTTRs could be divided into three classes based on their interactions with other LTTRs.Multimerization of LTTRs in E. coli K-12 is mostly specific. However, under the conditions of the assay, many LTTRs interact with more than one noncognate partner. The physiological significance and physical basis for these interactions are not known.

cis-Acting and trans-acting modulation of equine infectious anemia virus alternative RNA splicing

International Nuclear Information System (INIS)

Liao, Huey-Jane; Baker, Carl C.; Princler, Gerald L.; Derse, David

2004-01-01

Equine infectious anemia virus (EIAV), a lentivirus distantly related to HIV-1, encodes regulatory proteins, EIAV Tat (ETat) and Rev (ERev), from a four-exon mRNA. Exon 3 of the tat/rev mRNA contains a 30-nucleotide purine-rich element (PRE) which binds both ERev and SF2/ASF, a member of the SR family of RNA splicing factors. To better understand the role of this element in the regulation of EIAV pre-mRNA splicing, we quantified the effects of mutation or deletion of the PRE on exon 3 splicing in vitro and on alternative splicing in vivo. We also determined the branch point elements upstream of exons 3 and 4. In vitro splicing of exon 3 to exon 4 was not affected by mutation of the PRE, and addition of purified SR proteins enhanced splicing independently of the PRE. In vitro splicing of exon 2 to exon 3 was dependent on the PRE; under conditions of excess SR proteins, either the PRE or the 5' splice site of exon 3 was sufficient to activate splicing. We applied isoform-specific primers in real-time RT-PCR reactions to quantitatively analyze alternative splicing in cells transfected with rev-minus EIAV provirus constructs. In the context of provirus with wild-type exon 3, greater than 80% of the viral mRNAs were multiply spliced, and of these, less than 1% excluded exon 3. Deletion of the PRE resulted in a decrease in the relative amount of multiply spliced mRNA to about 40% of the total and approximately 39% of the viral mRNA excluded exon 3. Ectopic expression of ERev caused a decrease in the relative amount of multiply spliced mRNA to approximately 50% of the total and increased mRNAs that excluded exon 3 to about 4%. Over-expression of SF2/ASF in cells transfected with wild-type provirus constructs inhibited splicing but did not significantly alter exon 3 skipping

Does Preschool Self-Regulation Predict Later Behavior Problems in General or Specific Problem Behaviors?

Science.gov (United States)

Lonigan, Christopher J; Spiegel, Jamie A; Goodrich, J Marc; Morris, Brittany M; Osborne, Colleen M; Lerner, Matthew D; Phillips, Beth M

2017-11-01

Findings from prior research have consistently indicated significant associations between self-regulation and externalizing behaviors. Significant associations have also been reported between children's language skills and both externalizing behaviors and self-regulation. Few studies to date, however, have examined these relations longitudinally, simultaneously, or with respect to unique clusters of externalizing problems. The current study examined the influence of preschool self-regulation on general and specific externalizing behavior problems in early elementary school and whether these relations were independent of associations between language, self-regulation, and externalizing behaviors in a sample of 815 children (44% female). Additionally, given a general pattern of sex differences in the presentations of externalizing behavior problems, self-regulation, and language skills, sex differences for these associations were examined. Results indicated unique relations of preschool self-regulation and language with both general externalizing behavior problems and specific problems of inattention. In general, self-regulation was a stronger longitudinal correlate of externalizing behavior for boys than it was for girls, and language was a stronger longitudinal predictor of hyperactive/impulsive behavior for girls than it was for boys.

Pre- and postnatal stress and asthma in children: Temporal- and sex-specific associations

Science.gov (United States)

Lee, Alison; Chiu, Yueh-Hsiu Mathilda; Rosa, Maria José; Jara, Calvin; Wright, Robert O.; Coull, Brent A.; Wright, Rosalind J.

2016-01-01

BACKGROUND Temporal- and sex-specific effects of perinatal stress have not been examined for childhood asthma. OBJECTIVES We examined associations between pre- and/or postnatal stress and children's asthma (n=765) and effect modification by sex in a prospective cohort study. METHODS Maternal negative life events (NLEs) were ascertained prenatally and postpartum. NLEs scores were categorized as 0, 1-2, 3-4, or ≥5 to assess exposure-response relationships. We examined effects of pre- and postnatal stress on children's asthma by age 6 years modeling each as independent predictors; mutually adjusting for prenatal and postnatal stress; and finally considering interactions between pre- and postnatal stress. Effect modification by sex was examined in stratified analyses and by fitting interaction terms. RESULTS When considering stress in each period independently, among boys a dose-response relationship was evident for each level increase on the ordinal scale prenatally (OR=1.38, 95% CI 1.06, 1.79; p-for-trend=0.03) and postnatally (OR=1.53, 95% CI 1.16, 2.01; p-for-trend=0.001); among girls only the postnatal trend was significant (OR=1.60, 95% CI 1.14, 2.22; p-for-trend=0.005). Higher stress in both the pre- and postnatal periods was associated with increased odds of being diagnosed with asthma in girls [OR=1.37, 95% CI 0.98, 1.91 (pinteraction=0.07)] but not boys [OR=1.08, 95% CI 0.82, 1.42 (pinteraction=0.61)]. CONCLUSIONS While boys were more vulnerable to stress during the prenatal period, girls were more impacted by postnatal stress and cumulative stress across both periods in relation to asthma. Understanding sex and temporal differences in response to early life stress may provide unique insight into asthma etiology and natural history. PMID:26953156

Region-specific RNA m6A methylation represents a new layer of control in the gene regulatory network in the mouse brain.

Science.gov (United States)

Chang, Mengqi; Lv, Hongyi; Zhang, Weilong; Ma, Chunhui; He, Xue; Zhao, Shunli; Zhang, Zhi-Wei; Zeng, Yi-Xin; Song, Shuhui; Niu, Yamei; Tong, Wei-Min

2017-09-01

N 6 -methyladenosine (m 6 A) is the most abundant epitranscriptomic mark found on mRNA and has important roles in various physiological processes. Despite the relatively high m 6 A levels in the brain, its potential functions in the brain remain largely unexplored. We performed a transcriptome-wide methylation analysis using the mouse brain to depict its region-specific methylation profile. RNA methylation levels in mouse cerebellum are generally higher than those in the cerebral cortex. Heterogeneity of RNA methylation exists across different brain regions and different types of neural cells including the mRNAs to be methylated, their methylation levels and methylation site selection. Common and region-specific methylation have different preferences for methylation site selection and thereby different impacts on their biological functions. In addition, high methylation levels of fragile X mental retardation protein (FMRP) target mRNAs suggest that m 6 A methylation is likely to be used for selective recognition of target mRNAs by FMRP in the synapse. Overall, we provide a region-specific map of RNA m 6 A methylation and characterize the distinct features of specific and common methylation in mouse cerebellum and cerebral cortex. Our results imply that RNA m 6 A methylation is a newly identified element in the region-specific gene regulatory network in the mouse brain. © 2017 The Authors.

Specific microRNAs Regulate Heat Stress Responses in Caenorhabditis elegans

DEFF Research Database (Denmark)

Nehammer, Camilla; Podolska, Agnieszka; Mackowiak, Sebastian D

2015-01-01

have identified additional functions for already known players (mir-71 and mir-239) as well as identifying mir-80 and the mir-229 mir-64-66 cluster as important regulators of the heat stress response in C. elegans. These findings uncover an additional layer of complexity to the regulation of stress...... to heat stress in Caenorhabditis elegans and show that a discrete subset of miRNAs is thermoregulated. Using in-depth phenotypic analyses of miRNA deletion mutant strains we reveal multiple developmental and post-developmental survival and behavioral functions for specific miRNAs during heat stress. We...

A 3'-coterminal nested set of independently transcribed mRNAs is generated during Berne virus replication

International Nuclear Information System (INIS)

Snijder, E.J.; Horzinek, M.C.; Spaan, W.J.

1990-01-01

By using poly(A)-selected RNA from Berne virus (BEV)-infected embryonic mule skin cells as a template, cDNA was prepared and cloned in plasmid pUC9. Recombinants covering a contiguous sequence of about 10 kilobases were identified. Northern (RNA) blot hybridizations with various restriction fragments from these clones showed that the five BEV mRNAs formed a 3'-coterminal nested set. Sequence analysis revealed the presence of four complete open reading frames of 4743, 699, 426, and 480 nucleotides, with initiation codons coinciding with the 5' ends of BEV RNAs 2 through 5, respectively. By using primer extension analysis and oligonucleotide hybridizations, RNA 5 was found to be contiguous on the consensus sequence. The transcription of BEV mRNAs was studied by means of UV mapping. BEV RNAs 1, 2, and 3 were shown to be transcribed independently, which is also likely--although not rigorously proven--for RNAs 4 and 5. Upstream of the AUG codon of each open reading frame a conserved sequence pattern was observed which is postulated to function as a core promoter sequence in subgenomic RNA transcription. In the area surrounding the core promoter region of the two most abundant subgenomic BEV RNAs, a number of homologous sequence motifs were identified

Tissue-specific 5' heterogeneity of PPARα transcripts and their differential regulation by leptin.

Directory of Open Access Journals (Sweden)

Emma S Garratt

Full Text Available The genes encoding nuclear receptors comprise multiple 5'untranslated exons, which give rise to several transcripts encoding the same protein, allowing tissue-specific regulation of expression. Both human and mouse peroxisome proliferator activated receptor (PPAR α genes have multiple promoters, although their function is unknown. Here we have characterised the rat PPARα promoter region and have identified three alternative PPARα transcripts, which have different transcription start sites owing to the utilisation of distinct first exons. Moreover these alternative PPARα transcripts were differentially expressed between adipose tissue and liver. We show that while the major adipose (P1 and liver (P2 transcripts were both induced by dexamethasone, they were differentially regulated by the PPARα agonist, clofibric acid, and leptin. Leptin had no effect on the adipose-specific P1 transcript, but induced liver-specific P2 promoter activity via a STAT3/Sp1 mechanism. Moreover in Wistar rats, leptin treatment between postnatal day 3-13 led to an increase in P2 but not P1 transcription in adipose tissue which was sustained into adulthood. This suggests that the expression of the alternative PPARα transcripts are in part programmed by early life exposure to leptin leading to persistent change in adipose tissue fatty acid metabolism through specific activation of a quiescent PPARα promoter. Such complexity in the regulation of PPARα may allow the expression of PPARα to be finely regulated in response to environmental factors.

76 FR 59304 - 2011-2012 Refuge-Specific Hunting and Sport Fishing Regulations; Correction

Science.gov (United States)

2011-09-26

..., upland game hunting, big game hunting, and sport fishing for the 2011-2012 season. Inadvertently, this...-0038; 93270-1265-0000-4A] RIN 1018-AX54 2011-2012 Refuge-Specific Hunting and Sport Fishing Regulations... our regulations concerning hunting and sport fishing programs at national wildlife refuges...

Can a genetically-modified organism-containing diet influence embryo development? A preliminary study on pre-implantation mouse embryos.

Science.gov (United States)

Cisterna, B; Flach, F; Vecchio, L; Barabino, S M L; Battistelli, S; Martin, T E; Malatesta, M; Biggiogera, M

2008-01-01

In eukaryotic cells, pre-mRNAs undergo several transformation steps to generate mature mRNAs. Recent studies have demonstrated that a diet containing a genetically modified (GM) soybean can induce modifications of nuclear constituents involved in RNA processing in some tissues of young, adult and old mice. On this basis, we have investigated the ultrastructural and immunocytochemical features of pre-implantation embryos from mice fed either GM or non- GM soybean in order to verify whether the parental diet can affect the morpho-functional development of the embryonic ribonucleoprotein structural constituents involved in pre-mRNA pathways. Morphological observations revealed that the general aspect of embryo nuclear components is similar in the two experimental groups. However, immunocytochemical and in situ hybridization results suggest a temporary decrease of pre-mRNA transcription and splicing in 2-cell embryos and a resumption in 4-8-cell embryos from mice fed GM soybean; moreover, pre-mRNA maturation seems to be less efficient in both 2-cell and 4-8-cell embryos from GM-fed mice than in controls. Although our results are still preliminary and limited to the pre-implantation phases, the results of this study encourage deepening on the effects of food components and/or contaminants on embryo development.

The Drosophila Translational Control Element (TCE is required for high-level transcription of many genes that are specifically expressed in testes.

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Rebeccah J Katzenberger

Full Text Available To investigate the importance of core promoter elements for tissue-specific transcription of RNA polymerase II genes, we examined testis-specific transcription in Drosophila melanogaster. Bioinformatic analyses of core promoter sequences from 190 genes that are specifically expressed in testes identified a 10 bp A/T-rich motif that is identical to the translational control element (TCE. The TCE functions in the 5' untranslated region of Mst(3CGP mRNAs to repress translation, and it also functions in a heterologous gene to regulate transcription. We found that among genes with focused initiation patterns, the TCE is significantly enriched in core promoters of genes that are specifically expressed in testes but not in core promoters of genes that are specifically expressed in other tissues. The TCE is variably located in core promoters and is conserved in melanogaster subgroup species, but conservation dramatically drops in more distant species. In transgenic flies, short (300-400 bp genomic regions containing a TCE directed testis-specific transcription of a reporter gene. Mutation of the TCE significantly reduced but did not abolish reporter gene transcription indicating that the TCE is important but not essential for transcription activation. Finally, mutation of testis-specific TFIID (tTFIID subunits significantly reduced the transcription of a subset of endogenous TCE-containing but not TCE-lacking genes, suggesting that tTFIID activity is limited to TCE-containing genes but that tTFIID is not an obligatory regulator of TCE-containing genes. Thus, the TCE is a core promoter element in a subset of genes that are specifically expressed in testes. Furthermore, the TCE regulates transcription in the context of short genomic regions, from variable locations in the core promoter, and both dependently and independently of tTFIID. These findings set the stage for determining the mechanism by which the TCE regulates testis-specific transcription and

The Drosophila Translational Control Element (TCE) is required for high-level transcription of many genes that are specifically expressed in testes.

Science.gov (United States)

Katzenberger, Rebeccah J; Rach, Elizabeth A; Anderson, Ashley K; Ohler, Uwe; Wassarman, David A

2012-01-01

To investigate the importance of core promoter elements for tissue-specific transcription of RNA polymerase II genes, we examined testis-specific transcription in Drosophila melanogaster. Bioinformatic analyses of core promoter sequences from 190 genes that are specifically expressed in testes identified a 10 bp A/T-rich motif that is identical to the translational control element (TCE). The TCE functions in the 5' untranslated region of Mst(3)CGP mRNAs to repress translation, and it also functions in a heterologous gene to regulate transcription. We found that among genes with focused initiation patterns, the TCE is significantly enriched in core promoters of genes that are specifically expressed in testes but not in core promoters of genes that are specifically expressed in other tissues. The TCE is variably located in core promoters and is conserved in melanogaster subgroup species, but conservation dramatically drops in more distant species. In transgenic flies, short (300-400 bp) genomic regions containing a TCE directed testis-specific transcription of a reporter gene. Mutation of the TCE significantly reduced but did not abolish reporter gene transcription indicating that the TCE is important but not essential for transcription activation. Finally, mutation of testis-specific TFIID (tTFIID) subunits significantly reduced the transcription of a subset of endogenous TCE-containing but not TCE-lacking genes, suggesting that tTFIID activity is limited to TCE-containing genes but that tTFIID is not an obligatory regulator of TCE-containing genes. Thus, the TCE is a core promoter element in a subset of genes that are specifically expressed in testes. Furthermore, the TCE regulates transcription in the context of short genomic regions, from variable locations in the core promoter, and both dependently and independently of tTFIID. These findings set the stage for determining the mechanism by which the TCE regulates testis-specific transcription and understanding the

Translation initiation on mRNAs bound by nuclear cap-binding protein complex CBP80/20 requires interaction between CBP80/20-dependent translation initiation factor and eukaryotic translation initiation factor 3g.

Science.gov (United States)

Choe, Junho; Oh, Nara; Park, Sungjin; Lee, Ye Kyung; Song, Ok-Kyu; Locker, Nicolas; Chi, Sung-Gil; Kim, Yoon Ki

2012-05-25

In the cytoplasm of mammalian cells, either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF) 4E can direct the initiation of translation. Although the recruitment of ribosomes to mRNAs during eIF4E-dependent translation (ET) is well characterized, the molecular mechanism for CBP80/20-dependent translation (CT) remains obscure. Here, we show that CBP80/20-dependent translation initiation factor (CTIF), which has been shown to be preferentially involved in CT but not ET, specifically interacts with eIF3g, a component of the eIF3 complex involved in ribosome recruitment. By interacting with eIF3g, CTIF serves as an adaptor protein to bridge the CBP80/20 and the eIF3 complex, leading to efficient ribosome recruitment during CT. Accordingly, down-regulation of CTIF using a small interfering RNA causes a redistribution of CBP80 from polysome fractions to subpolysome fractions, without significant consequence to eIF4E distribution. In addition, down-regulation of eIF3g inhibits the efficiency of nonsense-mediated mRNA decay, which is tightly coupled to CT but not to ET. Moreover, the artificial tethering of CTIF to an intercistronic region of dicistronic mRNA results in translation of the downstream cistron in an eIF3-dependent manner. These findings support the idea that CT mechanistically differs from ET.

A novel CDX2 isoform regulates alternative splicing.

Directory of Open Access Journals (Sweden)

Matthew E Witek

Full Text Available Gene expression is a dynamic and coordinated process coupling transcription with pre-mRNA processing. This regulation enables tissue-specific transcription factors to induce expression of specific transcripts that are subsequently amplified by alternative splicing allowing for increased proteome complexity and functional diversity. The intestine-specific transcription factor CDX2 regulates development and maintenance of the intestinal epithelium by inducing expression of genes characteristic of the mature enterocyte phenotype. Here, sequence analysis of CDX2 mRNA from colonic mucosa-derived tissues revealed an alternatively spliced transcript (CDX2/AS that encodes a protein with a truncated homeodomain and a novel carboxy-terminal domain enriched in serine and arginine residues (RS domain. CDX2 and CDX2/AS exhibited distinct nuclear expression patterns with minimal areas of co-localization. CDX2/AS did not activate the CDX2-dependent promoter of guanylyl cyclase C nor inhibit transcriptional activity of CDX2. Unlike CDX2, CDX2/AS co-localized with the putative splicing factors ASF/SF2 and SC35. CDX2/AS altered splicing patterns of CD44v5 and Tra2-β1 minigenes in Lovo colon cancer cells independent of CDX2 expression. These data demonstrate unique dual functions of the CDX2 gene enabling it to regulate gene expression through both transcription (CDX2 and pre-mRNA processing (CDX2/AS.

Increased expression of alpha- and beta-globin mRNAs at the pituitary following exposure to estrogen during the critical period of neonatal sex differentiation in the rat

DEFF Research Database (Denmark)

Leffers, H; Navarro, V M; Nielsen, John E

2006-01-01

Deterioration of reproductive health in human and wildlife species during the past decades has drawn considerable attention to the potential adverse effects of exposure to xenosteroids during sensitive periods of sex development. The hypothalamic-pituitary (HP) unit is a key element in the neuroe......Deterioration of reproductive health in human and wildlife species during the past decades has drawn considerable attention to the potential adverse effects of exposure to xenosteroids during sensitive periods of sex development. The hypothalamic-pituitary (HP) unit is a key element......, we screened for differentially expressed genes at the pituitary and hypothalamus of rats after neonatal exposure to estradiol benzoate. Our analyses identified persistent up-regulation of alpha- and beta-globin mRNAs at the pituitary following neonatal estrogenization. This finding was confirmed...... by combination of RT-PCR analyses and in situ hybridization. Induction of alpha- and beta-globin mRNA expression at the pituitary by neonatal exposure to estrogen was demonstrated as dose-dependent and it was persistently detected up to puberty. In contrast, durable up-regulation of alpha- and beta-globin genes...

The development of self-regulated learning during the pre-clinical stage of medical school: a comparison between a lecture-based and a problem-based curriculum.

Science.gov (United States)

Lucieer, Susanna M; van der Geest, Jos N; Elói-Santos, Silvana M; de Faria, Rosa M Delbone; Jonker, Laura; Visscher, Chris; Rikers, Remy M J P; Themmen, Axel P N

2016-03-01

Society expects physicians to always improve their competencies and to be up to date with developments in their field. Therefore, an important aim of medical schools is to educate future medical doctors to become self-regulated, lifelong learners. However, it is unclear if medical students become better self-regulated learners during the pre-clinical stage of medical school, and whether students develop self-regulated learning skills differently, dependent on the educational approach of their medical school. In a cross-sectional design, we investigated the development of 384 medical students' self-regulated learning skills with the use of the Self-Regulation of Learning Self-Report Scale. Next, we compared this development in students who enrolled in two distinct medical curricula: a problem-based curriculum and a lectured-based curriculum. Analysis showed that more skills decreased than increased during the pre-clinical stage of medical school, and that the difference between the curricula was mainly caused by a decrease in the skill evaluation in the lecture-based curriculum. These findings seem to suggest that, irrespective of the curriculum, self-regulated learning skills do not develop during medical school.

MicroRNA signature of the human developing pancreas

Directory of Open Access Journals (Sweden)

Correa-Medina Mayrin

2010-09-01

Full Text Available Abstract Background MicroRNAs are non-coding RNAs that regulate gene expression including differentiation and development by either inhibiting translation or inducing target degradation. The aim of this study is to determine the microRNA expression signature during human pancreatic development and to identify potential microRNA gene targets calculating correlations between the signature microRNAs and their corresponding mRNA targets, predicted by bioinformatics, in genome-wide RNA microarray study. Results The microRNA signature of human fetal pancreatic samples 10-22 weeks of gestational age (wga, was obtained by PCR-based high throughput screening with Taqman Low Density Arrays. This method led to identification of 212 microRNAs. The microRNAs were classified in 3 groups: Group number I contains 4 microRNAs with the increasing profile; II, 35 microRNAs with decreasing profile and III with 173 microRNAs, which remain unchanged. We calculated Pearson correlations between the expression profile of microRNAs and target mRNAs, predicted by TargetScan 5.1 and miRBase altgorithms, using genome-wide mRNA expression data. Group I correlated with the decreasing expression of 142 target mRNAs and Group II with the increasing expression of 876 target mRNAs. Most microRNAs correlate with multiple targets, just as mRNAs are targeted by multiple microRNAs. Among the identified targets are the genes and transcription factors known to play an essential role in pancreatic development. Conclusions We have determined specific groups of microRNAs in human fetal pancreas that change the degree of their expression throughout the development. A negative correlative analysis suggests an intertwined network of microRNAs and mRNAs collaborating with each other. This study provides information leading to potential two-way level of combinatorial control regulating gene expression through microRNAs targeting multiple mRNAs and, conversely, target mRNAs regulated in

Direct Binding between Pre-S1 and TRP-like Domains in TRPP Channels Mediates Gating and Functional Regulation by PIP2

OpenAIRE

Wang Zheng; Ruiqi Cai; Laura Hofmann; Vasyl Nesin; Qiaolin Hu; Wentong Long; Mohammad Fatehi; Xiong Liu; Shaimaa Hussein; Tim Kong; Jingru Li; Peter E. Light; Jingfeng Tang; Veit Flockerzi; Leonidas Tsiokas

2018-01-01

Transient receptor potential (TRP) channels are regulated by diverse stimuli comprising thermal, chemical, and mechanical modalities. They are also commonly regulated by phosphatidylinositol-4,5-bisphosphate (PIP2), with underlying mechanisms largely unknown. We here revealed an intramolecular interaction of the TRPP3 N and C termini (N-C) that is functionally essential. The interaction was mediated by aromatic Trp81 in pre-S1 domain and cationic Lys568 in TRP-like domain. Structure-function ...

How can we characterize nano-specific soft regulation? Lessons from occupational health and safety governance

NARCIS (Netherlands)

Reichow, Aline; Dorbeck-Jung, Barbel R.; Konrad, Kornelia; Coenen, Christopher; Dijkstra, Anne; Milburn, Colin; van Lente, Harro

2013-01-01

Soft regulation is a widely used instrument in the governance of emerging technologies, especially in the governance of nanotechnologies. So far, evaluations on the effects of nano-specific soft regulation cannot build on a coherent and consistent typology. Characterization of soft regulation is

The NO signaling pathway differentially regulates KCC3a and KCC3b mRNA expression.

Science.gov (United States)

Di Fulvio, Mauricio; Lauf, Peter K; Adragna, Norma C

2003-11-01

Nitric oxide (NO) donors and protein kinase G (PKG) acutely up-regulate K-Cl cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in vascular smooth muscle cells (VSMCs). Here, we report the presence, relative abundance, and regulation by sodium nitroprusside (SNP) of the novel KCC3a and KCC3b mRNAs, in primary cultures of rat VSMCs. KCC3a and KCC3b mRNAs were expressed in an approximate 3:1 ratio, as determined by semiquantitative RT-PCR analysis. SNP as well as YC-1 and 8-Br-cGMP, a NO-independent stimulator of soluble guanylyl cyclase (sGC) and PKG, respectively, increased KCC3a and KCC3b mRNA expression by 2.5-fold and 8.1-fold in a time-dependent manner, following a differential kinetics. Stimulation of the NO/sGC/PKG signaling pathway with either SNP, YC-1, or 8-Br-cGMP decreased the KCC3a/KCC3b ratio from 3.0+/-0.4 to 0.9+/-0.1. This is the first report on a differential regulation by the NO/sGC/PKG signaling pathway of a cotransporter and of KCC3a and KCC3b mRNA expression.

Growth Arrest-Specific 6 (Gas6) and TAM Receptors in Mouse Platelets.

Science.gov (United States)

Uras, Fikriye; Küçük, Burhanettin; Bingöl Özakpınar, Özlem; Demir, Ahmet Muzaffer

2015-03-05

Growth arrest-specific 6 (Gas6) is a newly discovered vitamin K-dependent protein, which is a ligand for TAM receptors [Tyro3 (Sky), Axl, and Mer] from the tyrosine kinase family. Gas6 knockout mice were resistant to venous and arterial thrombosis. There are contradictory reports on the presence of Gas6 and its receptors in mouse platelets. The objective of this study was to investigate whether Gas6 and its receptors were present in mouse platelets or not. Specific pathogen-free BALB/c male and female mice of 8-10 weeks old and 25-30 g in weight were anesthetized under light ether anesthesia and blood samples were taken from their hearts. RNAs were isolated from isolated platelets, and then mRNAs encoding Gas6 and TAM receptors were detected by reverse transcription-polymerase chain reaction (RT-PCR). Protein concentrations of Gas6 and TAM receptors in platelets were measured by ELISA, but not those of Mer, because of the absence of any commercial ELISA kit for mouse specimens. RT-PCR results indicated the presence of mRNAs encoding Gas6 and Mer in mouse platelets. However, although RT-PCR reactions were performed at various temperatures and cycles, we could not detect the presence of mRNAs encoding Axl and Tyro3 (Sky). Receptor protein levels of Axl and Tyro3 were below the detection limits of the ELISA method. We found the presence of mRNAs encoding Gas6 and the receptor Mer in mouse platelets, but not Axl and Tyro3. Gas6, Axl, and Tyro3 protein levels were below the detection limits of the ELISA. The presence of mRNA is not obvious evidence of protein expression in platelets that have no nucleus or DNA. Further studies are required to clarify the presence of Gas6/TAM receptors in platelets using real-time PCR and more sensitive immunological methods, and future studies on mechanisms will indicate whether the Gas6/TAM pathway is a strategy for treatment of disorders.

Differential regulation of the transcriptional activity of the glucocorticoid receptor through site-specific phosphorylation

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Raj Kumar

2008-08-01

Full Text Available Raj Kumar1, William J Calhoun21Division of Gastroenterology; 2Division of Allergy, Pulmonary, Immunology, Critical Care, and Sleep (APICS, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX, USAAbstract: Post-translational modifications such as phosphorylation are known to play an important role in the gene regulation by the transcription factors including the nuclear hormone receptor superfamily of which the glucocorticoid receptor (GR is a member. Protein phosphorylation often switches cellular activity from one state to another. Like many other transcription factors, the GR is a phosphoprotein, and phosphorylation plays an important role in the regulation of GR activity. Cell signaling pathways that regulate phosphorylation of the GR and its associated proteins are important determinants of GR function under various physiological conditions. While the role of many phosphorylation sites in the GR is still not fully understood, the role of others is clearer. Several aspects of transcription factor function, including DNA binding affinity, interaction of transactivation domains with the transcription initiation complex, and shuttling between the cytoplasmic compartments, have all been linked to site-specific phosphorylation. All major phosphorylation sites in the human GR are located in the N-terminal domain including the major transactivation domain, AF1. Available literature clearly indicates that many of these potential phosphorylation sites are substrates for multiple kinases, suggesting the potential for a very complex regulatory network. Phosphorylated GR interacts favorably with critical coregulatory proteins and subsequently enhances transcriptional activity. In addition, the activities and specificities of coregulators may be subject to similar regulation by phosphorylation. Regulation of the GR activity due to phosphorylation appears to be site-specific and dependent upon specific cell signaling cascade

Toward epigenetic and gene regulation models of specific language impairment: looking for links among growth, genes, and impairments

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Rice Mabel L

2012-11-01

Full Text Available Abstract Children with specific language impairment (SLI are thought to have an inherited form of language impairment that spares other developmental domains. SLI shows strong heritability and recent linkage and association studies have replicated results for candidate genes. Regulatory regions of the genes may be involved. Behavioral growth models of language development of children with SLI reveal that the onset of language is delayed, and the growth trajectories of children with SLI parallel those of younger children without SLI. The rate of language acquisition decelerates in the pre-adolescent period, resulting in immature language levels for the children with SLI that persist into adolescence and beyond. Recent genetic and epigenetic discoveries and models relevant to language impairment are reviewed. T cell regulation of onset, acceleration, and deceleration signaling are described as potential conceptual parallels to the growth timing elements of language acquisition and impairment. A growth signaling disruption (GSD hypothesis is proposed for SLI, which posits that faulty timing mechanisms at the cellular level, intrinsic to neurocortical functioning essential for language onset and growth regulation, are at the core of the growth outcomes of SLI. The GSD highlights the need to document and account for growth patterns over childhood and suggests needed directions for future investigation.

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

Science.gov (United States)

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

2013-11-01

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

A viral microRNA functions as an ortholog of cellular miR-155

Science.gov (United States)

Gottwein, Eva; Mukherjee, Neelanjan; Sachse, Christoph; Frenzel, Corina; Majoros, William H.; Chi, Jen-Tsan A.; Braich, Ravi; Manoharan, Muthiah; Soutschek, Jürgen; Ohler, Uwe; Cullen, Bryan R.

2008-01-01

All metazoan eukaryotes express microRNAs (miRNAs), ∼22 nt regulatory RNAs that can repress the expression of mRNAs bearing complementary sequences1. Several DNA viruses also express miRNAs in infected cells, suggesting a role in viral replication and pathogenesis2. While specific viral miRNAs have been shown to autoregulate viral mRNAs3,4 or downregulate cellular mRNAs5,6, the function of the majority of viral miRNAs remains unknown. Here, we report that the miR-K12−11 miRNA encoded by Kaposi's Sarcoma Associated Herpesvirus (KSHV) shows significant homology to cellular miR-155, including the entire miRNA “seed” region7. Using a range of assays, we demonstrate that expression of physiological levels of miR-K12−11 or miR-155 results in the downregulation of an extensive set of common mRNA targets, including genes with known roles in cell growth regulation. Our findings indicate that viral miR-K12−11 functions as an ortholog of cellular miR-155 and has likely evolved to exploit a pre-existing gene regulatory pathway in B-cells. Moreover, the known etiological role of miR-155 in B-cell transformation8-10 suggests that miR-K12−11 may contribute to the induction of KSHV-positive B-cell tumors in infected patients. PMID:18075594

Beliefs about family-school relationships. Changes in pre-service teachers after receiving specific training

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Cristina Vázquez

2014-12-01

Full Text Available Teachers’ beliefs about family-school relationship vary in a continuum according to the role that parents and teachers have, and the power that they hold. Pre-service teachers also have beliefs about this relationship and their own competence to develop it. Two groups of pre-service teachers (second year students participated in this study. One group received specific training on family-school relationship and its improvement (116 students attending a degree in Early Childhood Education, who constituted the experimental group, EG. The other group was not trained (92 students attending a degree in Primary Education, who made up the control group, CG. The Beliefs about family-school Questionnaire (CCR was developed and applied before and after the EG was trained. Results show that students in the EG increased their beliefs about family-school collaboration in the post-test and decreased their beliefs about parental subordination to teachers’ authority and parents’ carelessness. Students in the CG kept their beliefs unchanged, which were significantly more prone to support teachers’ authority and parental subordination and parents’ carelessness compared to the EG.. Perceived competence for family-school relationship did not change significantly in either group. However, significant correlations between beliefs and perceived competence were found, pointing out the importance of working pre-service teachers’ beliefs about family-school collaboration.

BDNF deficiency and young-adult methamphetamine induce sex-specific effects on prepulse inhibition regulation

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Elizabeth E Manning

2013-06-01

Full Text Available Brain-derived neurotrophic factor (BDNF has been implicated in the pathophysiology of schizophrenia, yet its role in the development of specific symptoms is unclear. Methamphetamine (METH users have an increased risk of psychosis and schizophrenia, and METH-treated animals have been used extensively as a model to study the positive symptoms of schizophrenia. We investigated whether METH treatment in BDNF heterozygous mutant mice (HET has cumulative effects on sensorimotor gating, including the disruptive effects of psychotropic drugs. BDNF HETs and WT littermates were treated during young-adulthood with METH and, following a two-week break, prepulse inhibition (PPI was examined. At baseline, BDNF HETs showed reduced PPI compared to WT mice irrespective of METH pre-treatment. An acute challenge with amphetamine (AMPH disrupted PPI but male BDNF HETs were more sensitive to this effect, irrespective of METH pre-treatment. In contrast, female mice treated with METH were less sensitive to the disruptive effects of AMPH, and there were no effects of BDNF genotype. Similar changes were not observed in the response to an acute apomorphine or MK-801 challenge. These results show that genetically-induced reduction of BDNF caused changes in a behavioural endophenotype relevant to the positive symptoms of schizophrenia. However, major sex differences were observed in the effects of a psychotropic drug challenge on this behaviour. These findings suggest sex differences in the effects of BDNF depletion and METH treatment on the monoamine signaling pathways that regulate PPI. Given that these same pathways are thought to contribute to the expression of positive symptoms in schizophrenia, this work suggests that there may be significant sex differences in the pathophysiology underlying these symptoms. Elucidating these sex differences may be important for our understanding of the neurobiology of schizophrenia and developing better treatments strategies for the

Co-ordinate expression of activin A and its type I receptor mRNAs during phorbol ester-induced differentiation of human K562 erythroleukemia cells.

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Hildén, K; Tuuri, T; Erämaa, M; Ritvos, O

1999-07-20

Activins were originally isolated based on their ability to stimulate follicle-stimulating hormone secretion but later they have been shown to regulate a number of different cellular functions such as nerve cell survival, mesoderm induction during early embryogenesis as well as hematopoiesis. We studied the regulation of activin A, a homodimer of betaA-subunits, mRNA and protein in K562 erythroleukemia cells, which are known to be induced toward the erythroid lineage in response to activin or TGF-beta or toward the megakaryocytic lineage by the phorbol ester protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). Here we show by Northern blot analysis as well as by Western and ligand blotting that TPA strongly promotes activin betaA-subunit mRNA and activin A protein expression in K562 cells in time- and concentration dependent manner. In contrast, neither activin A nor TGF-beta induced betaA-subunit mRNA expression during erythroid differentiation in K562 cells. Interestingly, whereas activin type II receptors are not regulated during K562 cell differentiation (Hilden et al. (1994) Blood 83, 2163-2170), we now show that the activin type I and IB receptor mRNAs are clearly induced by TPA but not by activin or TGF-beta. We also show that the inducing effect of TPA on expression of activin betaA-subunit mRNA is potentiated by the protein kinase A activator 8-bromo-cAMP. We conclude that activin A and its type I receptors appear to be co-ordinately up-regulated during megakaryocytic differentiation of K562 cells.

Specific micro RNA-regulated TetR-KRAB transcriptional control of transgene expression in viral vector-transduced cells.

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Virginie Pichard

Full Text Available Precise control of transgene expression in a tissue-specific and temporally regulated manner is desirable for many basic and applied investigations gene therapy applications. This is important to regulate dose of transgene products and minimize unwanted effects. Previously described methods have employed tissue specific promoters, miRNA-based transgene silencing or tetR-KRAB-mediated suppression of transgene promoters. To improve on versatility of transgene expression control, we have developed expression systems that use combinations of a tetR-KRAB artificial transgene-repressor, endogenous miRNA silencing machinery and tissue specific promoters. Precise control of transgene expression was demonstrated in liver-, macrophage- and muscle-derived cells. Efficiency was also demonstrated in vivo in murine muscle. This multicomponent and modular regulatory system provides a robust and easily adaptable method for achieving regulated transgene expression in different tissue types. The improved precision of regulation will be useful for many gene therapy applications requiring specific spatiotemporal transgene regulation.

Mining the 30UTR of Autism-implicated Genes for SNPs Perturbing MicroRNA Regulation

Institute of Scientific and Technical Information of China (English)

Varadharajan Vaishnavi; Mayakannan Manikandan; Arasambattu Kannan Munirajan

2014-01-01

Autism spectrum disorder (ASD) refers to a group of childhood neurodevelopmental dis-orders with polygenic etiology. The expression of many genes implicated in ASD is tightly regulated by various factors including microRNAs (miRNAs), a class of noncoding RNAs 22 nucleotides in length that function to suppress translation by pairing with‘miRNA recognition elements’ (MREs) present in the 30untranslated region (30UTR) of target mRNAs. This emphasizes the role played by miRNAs in regulating neurogenesis, brain development and differentiation and hence any perturba-tions in this regulatory mechanism might affect these processes as well. Recently, single nucleotide polymorphisms (SNPs) present within 30UTRs of mRNAs have been shown to modulate existing MREs or even create new MREs. Therefore, we hypothesized that SNPs perturbing miRNA-medi-ated gene regulation might lead to aberrant expression of autism-implicated genes, thus resulting in disease predisposition or pathogenesis in at least a subpopulation of ASD individuals. We developed a systematic computational pipeline that integrates data from well-established databases. By following a stringent selection criterion, we identified 9 MRE-modulating SNPs and another 12 MRE-creating SNPs in the 30UTR of autism-implicated genes. These high-confidence candidate SNPs may play roles in ASD and hence would be valuable for further functional validation.

A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking.

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Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

2015-05-18

An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes.

Structural and functional conservation of CLEC-2 with the species-specific regulation of transcript expression in evolution.

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Wang, Lan; Ren, Shifang; Zhu, Haiyan; Zhang, Dongmei; Hao, Yuqing; Ruan, Yuanyuan; Zhou, Lei; Lee, Chiayu; Qiu, Lin; Yun, Xiaojing; Xie, Jianhui

2012-08-01

CLEC-2 was first identified by sequence similarity to C-type lectin-like molecules with immune functions and has been reported as a receptor for the platelet-aggregating snake venom toxin rhodocytin and the endogenous sialoglycoprotein podoplanin. Recent researches indicate that CLEC-2-deficient mice were lethal at the embryonic stage associated with disorganized and blood-filled lymphatic vessels and severe edema. In view of a necessary role of CLEC-2 in the individual development, it is of interest to investigate its phylogenetic homology and highly conserved functional regions. In this work, we reported that CLEC-2 from different species holds with an extraordinary conservation by sequence alignment and phylogenetic tree analysis. The functional structures including N-linked oligosaccharide sites and ligand-binding domain implement a structural and functional conservation in a variety of species. The glycosylation sites (N120 and N134) are necessary for the surface expression CLEC-2. CLEC-2 from different species possesses the binding activity of mouse podoplanin. Nevertheless, the expression of CLEC-2 is regulated with a species-specific manner. The alternative splicing of pre-mRNA, a regulatory mechanism of gene expression, and the binding sites on promoter for several key transcription factors vary between different species. Therefore, CLEC-2 shares high sequence homology and functional identity. However the transcript expression might be tightly regulated by different mechanisms in evolution.

Linking high-resolution metabolic flux phenotypes and transcriptional regulation in yeast modulated by the global regulator Gcn4p

DEFF Research Database (Denmark)

Moxley, Joel F.; Jewett, Michael Christopher; Antoniewicz, Maciek R.

2009-01-01

. However, the potential of systems biology approaches is limited by difficulties in integrating metabolic measurements across the functional levels of the cell despite their being most closely linked to cellular phenotype. To address this limitation, we developed a model-based approach to correlate m......RNA and metabolic flux data that combines information from both interaction network models and flux determination models. We started by quantifying 5,764 mRNAs, 54 metabolites, and 83 experimental C-13-based reaction fluxes in continuous cultures of yeast under stress in the absence or presence of global regulator...... of metabolic flux (i.e., use of different reaction pathways) by transcriptional regulation and metabolite interaction density (i.e., level of pairwise metabolite-protein interactions) as a key biosynthetic control determinant. Furthermore, this model predicted flux rewiring in studies of follow...

Effects of Larval Density on Gene Regulation in Caenorhabditis elegans During Routine L1 Synchronization.

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Chan, Io Long; Rando, Oliver J; Conine, Colin C

2018-05-04

Bleaching gravid C. elegans followed by a short period of starvation of the L1 larvae is a routine method performed by worm researchers for generating synchronous populations for experiments. During the process of investigating dietary effects on gene regulation in L1 stage worms by single-worm RNA-Seq, we found that the density of resuspended L1 larvae affects expression of many mRNAs. Specifically, a number of genes related to metabolism and signaling are highly expressed in worms arrested at low density, but are repressed at higher arrest densities. We generated a GFP reporter strain based on one of the most density-dependent genes in our dataset - lips-15 - and confirmed that this reporter was expressed specifically in worms arrested at relatively low density. Finally, we show that conditioned media from high density L1 cultures was able to downregulate lips-15 even in L1 animals arrested at low density, and experiments using daf-22 mutant animals demonstrated that this effect is not mediated by the ascaroside family of signaling pheromones. Together, our data implicate a soluble signaling molecule in density sensing by L1 stage C. elegans , and provide guidance for design of experiments focused on early developmental gene regulation. Copyright © 2018 Chan et al.

Protein tyrosine phosphatase SAP-1 protects against colitis through regulation of CEACAM20 in the intestinal epithelium.

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Murata, Yoji; Kotani, Takenori; Supriatna, Yana; Kitamura, Yasuaki; Imada, Shinya; Kawahara, Kohichi; Nishio, Miki; Daniwijaya, Edwin Widyanto; Sadakata, Hisanobu; Kusakari, Shinya; Mori, Munemasa; Kanazawa, Yoshitake; Saito, Yasuyuki; Okawa, Katsuya; Takeda-Morishita, Mariko; Okazawa, Hideki; Ohnishi, Hiroshi; Azuma, Takeshi; Suzuki, Akira; Matozaki, Takashi

2015-08-04

Intestinal epithelial cells contribute to regulation of intestinal immunity in mammals, but the detailed molecular mechanisms of such regulation have remained largely unknown. Stomach-cancer-associated protein tyrosine phosphatase 1 (SAP-1, also known as PTPRH) is a receptor-type protein tyrosine phosphatase that is localized specifically at microvilli of the brush border in gastrointestinal epithelial cells. Here we show that SAP-1 ablation in interleukin (IL)-10-deficient mice, a model of inflammatory bowel disease, resulted in a marked increase in the severity of colitis in association with up-regulation of mRNAs for various cytokines and chemokines in the colon. Tyrosine phosphorylation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvillus-specific transmembrane protein of the Ig superfamily, was greatly increased in the intestinal epithelium of the SAP-1-deficient animals, suggesting that this protein is a substrate for SAP-1. Tyrosine phosphorylation of CEACAM20 by the protein tyrosine kinase c-Src and the consequent association of CEACAM20 with spleen tyrosine kinase (Syk) promoted the production of IL-8 in cultured cells through the activation of nuclear factor-κB (NF-κB). In addition, SAP-1 and CEACAM20 were found to form a complex through interaction of their ectodomains. SAP-1 and CEACAM20 thus constitute a regulatory system through which the intestinal epithelium contributes to intestinal immunity.

Specification of Drosophila corpora cardiaca neuroendocrine cells from mesoderm is regulated by Notch signaling.

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Sangbin Park

2011-08-01

Full Text Available Drosophila neuroendocrine cells comprising the corpora cardiaca (CC are essential for systemic glucose regulation and represent functional orthologues of vertebrate pancreatic α-cells. Although Drosophila CC cells have been regarded as developmental orthologues of pituitary gland, the genetic regulation of CC development is poorly understood. From a genetic screen, we identified multiple novel regulators of CC development, including Notch signaling factors. Our studies demonstrate that the disruption of Notch signaling can lead to the expansion of CC cells. Live imaging demonstrates localized emergence of extra precursor cells as the basis of CC expansion in Notch mutants. Contrary to a recent report, we unexpectedly found that CC cells originate from head mesoderm. We show that Tinman expression in head mesoderm is regulated by Notch signaling and that the combination of Daughterless and Tinman is sufficient for ectopic CC specification in mesoderm. Understanding the cellular, genetic, signaling, and transcriptional basis of CC cell specification and expansion should accelerate discovery of molecular mechanisms regulating ontogeny of organs that control metabolism.

Differential stability of host mRNAs in Friend erythroleukemia cells infected with herpes simplex virus type 1

International Nuclear Information System (INIS)

Mayman, B.A.; Nishioka, Y.

1985-01-01

The consequences of herpes simplex virus type 1 infection on cellular macromolecules were investigated in Friend erythroleukemia cells. The patterns of protein synthesis, examined by polyacrylamide gel electrophoresis, demonstrated that by 4 h postinfection the synthesis of many host proteins, with the exception of histones, was inhibited. Examination of the steady-state level of histone H3 mRNA by molecular hybridization of total RNA to a cloned mouse histone H3 complementary DNA probe demonstrated that the ratio of histone H3 mRNA to total RNA remained unchanged for the first 4 h postinfection. In contrast, the steady-state levels of globin and actin mRNAs decreased progressively at early intervals postinfection. Studies on RNA synthesis in isolated nuclei demonstrated that the transcription of the histone H3 gene was inhibited to approximately the same extent as that of actin gene. It was concluded that the stabilization of preexisting histone H3 mRNA was responsible for the persistence of H3 mRNA and histone protein synthesis in herpes simplex virus type 1-infected Friend erythroleukemia cells. The possible mechanisms influencing the differential stability of host mRNAs during the course of productive infection with herpes simplex virus type 1 are discussed

Cup Blocks the Precocious Activation of the Orb Autoregulatory Loop

Science.gov (United States)

Wong, Li Chin; Schedl, Paul

2011-01-01

Translational regulation of localized mRNAs is essential for patterning and axes determination in many organisms. In the Drosophila ovary, the germline-specific Orb protein mediates the translational activation of a variety of mRNAs localized in the oocyte. One of the Orb target mRNAs is orb itself, and this autoregulatory activity ensures that Orb proteins specifically accumulate in the developing oocyte. Orb is an RNA-binding protein and is a member of the cytoplasmic polyadenylation element binding (CPEB) protein family. We report here that Cup forms a complex in vivo with Orb. We also show that cup negatively regulates orb and is required to block the precocious activation of the orb positive autoregulatory loop. In cup mutant ovaries, high levels of Orb accumulate in the nurse cells, leading to what appears to be a failure in oocyte specification as a number of oocyte markers inappropriately accumulate in nurse cells. In addition, while orb mRNA is mislocalized and destabilized, a longer poly(A) tail is maintained than in wild type ovaries. Analysis of Orb phosphoisoforms reveals that loss of cup leads to the accumulation of hyperphosphorylated Orb, suggesting that an important function of cup in orb-dependent mRNA localization pathways is to impede Orb activation. PMID:22164257

G3BP1, G3BP2 and CAPRIN1 are required for translation of interferon stimulated mRNAs and are targeted by a dengue virus non-coding RNA.

Science.gov (United States)

Bidet, Katell; Dadlani, Dhivya; Garcia-Blanco, Mariano A

2014-07-01

Viral RNA-host protein interactions are critical for replication of flaviviruses, a genus of positive-strand RNA viruses comprising major vector-borne human pathogens including dengue viruses (DENV). We examined three conserved host RNA-binding proteins (RBPs) G3BP1, G3BP2 and CAPRIN1 in dengue virus (DENV-2) infection and found them to be novel regulators of the interferon (IFN) response against DENV-2. The three RBPs were required for the accumulation of the protein products of several interferon stimulated genes (ISGs), and for efficient translation of PKR and IFITM2 mRNAs. This identifies G3BP1, G3BP2 and CAPRIN1 as novel regulators of the antiviral state. Their antiviral activity was antagonized by the abundant DENV-2 non-coding subgenomic flaviviral RNA (sfRNA), which bound to G3BP1, G3BP2 and CAPRIN1, inhibited their activity and lead to profound inhibition of ISG mRNA translation. This work describes a new and unexpected level of regulation for interferon stimulated gene expression and presents the first mechanism of action for an sfRNA as a molecular sponge of anti-viral effectors in human cells.

Can a genetically-modified organism-containing diet influence embryo development? A preliminary study on pre-implantation mouse embryos

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B Cisterna

2009-08-01

Full Text Available In eukaryotic cells, pre-mRNAs undergo several transformation steps to generate mature mRNAs. Recent studies have demonstrated that a diet containing a genetically modified (GM soybean can induce modifications of nuclear constituents involved in RNA processing in some tissues of young, adult and old mice. On this basis, we have investigated the ultrastructural and immunocytochemical features of pre-implantation embryos from mice fed either GM or non- GM soybean in order to verify whether the parental diet can affect the morpho-functional development of the embryonic ribonucleoprotein structural constituents involved in premRNA pathways. Morphological observations revealed that the general aspect of embryo nuclear components is similar in the two experimental groups. However, immunocytochemical and in situ hybridization results suggest a temporary decrease of pre-mRNA transcription and splicing in 2-cell embryos and a resumption in 4-8-cell embryos from mice fed GM soybean; moreover, pre-mRNA maturation seems to be less efficient in both 2-cell and 4-8-cell embryos from GM-fed mice than in controls. Although our results are still preliminary and limited to the pre-implantation phases, the results of this study encourage deepening on the effects of food components and/or contaminants on embryo development.

Literature-based condition-specific miRNA-mRNA target prediction.

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Minsik Oh

Full Text Available miRNAs are small non-coding RNAs that regulate gene expression by binding to the 3'-UTR of genes. Many recent studies have reported that miRNAs play important biological roles by regulating specific mRNAs or genes. Many sequence-based target prediction algorithms have been developed to predict miRNA targets. However, these methods are not designed for condition-specific target predictions and produce many false positives; thus, expression-based target prediction algorithms have been developed for condition-specific target predictions. A typical strategy to utilize expression data is to leverage the negative control roles of miRNAs on genes. To control false positives, a stringent cutoff value is typically set, but in this case, these methods tend to reject many true target relationships, i.e., false negatives. To overcome these limitations, additional information should be utilized. The literature is probably the best resource that we can utilize. Recent literature mining systems compile millions of articles with experiments designed for specific biological questions, and the systems provide a function to search for specific information. To utilize the literature information, we used a literature mining system, BEST, that automatically extracts information from the literature in PubMed and that allows the user to perform searches of the literature with any English words. By integrating omics data analysis methods and BEST, we developed Context-MMIA, a miRNA-mRNA target prediction method that combines expression data analysis results and the literature information extracted based on the user-specified context. In the pathway enrichment analysis using genes included in the top 200 miRNA-targets, Context-MMIA outperformed the four existing target prediction methods that we tested. In another test on whether prediction methods can re-produce experimentally validated target relationships, Context-MMIA outperformed the four existing target prediction

Cytoplasmic protein binding to highly conserved sequences in the 3' untranslated region of mouse protamine 2 mRNA, a translationally regulated transcript of male germ cells

International Nuclear Information System (INIS)

Kwon, Y.K.; Hecht, N.B.

1991-01-01

The expression of the protamines, the predominant nuclear proteins of mammalian spermatozoa, is regulated translationally during male germ-cell development. The 3' untranslated region (UTR) of protamine 1 mRNA has been reported to control its time of translation. To understand the mechanisms controlling translation of the protamine mRNAs, we have sought to identify cis elements of the 3' UTR of protamine 2 mRNA that are recognized by cytoplasmic factors. From gel retardation assays, two sequence elements are shown to form specific RNA-protein complexes. Protein binding sites of the two complexes were determined by RNase T1 mapping, by blocking the putative binding sites with antisense oligonucleotides, and by competition assays. The sequences of these elements, located between nucleotides + 537 and + 572 in protamine 2 mRNA, are highly conserved among postmeiotic translationally regulated nuclear proteins of the mammalian testis. Two closely linked protein binding sites were detected. UV-crosslinking studies revealed that a protein of about 18 kDa binds to one of the conserved sequences. These data demonstrate specific protein binding to a highly conserved 3' UTR of translationally regulated testicular mRNA

The FasX Small Regulatory RNA Negatively Regulates the Expression of Two Fibronectin-Binding Proteins in Group A Streptococcus.

Science.gov (United States)

Danger, Jessica L; Makthal, Nishanth; Kumaraswami, Muthiah; Sumby, Paul

2015-12-01

The group A Streptococcus (GAS; Streptococcus pyogenes) causes more than 700 million human infections each year. The success of this pathogen can be traced in part to the extensive arsenal of virulence factors that are available for expression in temporally and spatially specific manners. To modify the expression of these virulence factors, GAS use both protein- and RNA-based regulators, with the best-characterized RNA-based regulator being the small regulatory RNA (sRNA) FasX. FasX is a 205-nucleotide sRNA that contributes to GAS virulence by enhancing the expression of the thrombolytic secreted virulence factor streptokinase and by repressing the expression of the collagen-binding cell surface pili. Here, we have expanded the FasX regulon, showing that this sRNA also negatively regulates the expression of the adhesion- and internalization-promoting, fibronectin-binding proteins PrtF1 and PrtF2. FasX posttranscriptionally regulates the expression of PrtF1/2 through a mechanism that involves base pairing to the prtF1 and prtF2 mRNAs within their 5' untranslated regions, overlapping the mRNA ribosome-binding sites. Thus, duplex formation between FasX and the prtF1 and prtF2 mRNAs blocks ribosome access, leading to an inhibition of mRNA translation. Given that FasX positively regulates the expression of the spreading factor streptokinase and negatively regulates the expression of the collagen-binding pili and of the fibronectin-binding PrtF1/2, our data are consistent with FasX functioning as a molecular switch that governs the transition of GAS between the colonization and dissemination stages of infection. More than half a million deaths each year are a consequence of infections caused by GAS. Insights into how this pathogen regulates the production of proteins during infection may facilitate the development of novel therapeutic or preventative regimens aimed at inhibiting this activity. Here, we have expanded insight into the regulatory activity of the GAS small

Cigarette smoking during pregnancy regulates the expression of specific nicotinic acetylcholine receptor (nAChR) subunits in the human placenta

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Machaalani, R., E-mail: rita.machaalani@sydney.edu.au [Department of Medicine, The University of Sydney, NSW 2006 (Australia); Bosch Institute, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia); Ghazavi, E. [Bosch Institute, The University of Sydney, NSW 2006 (Australia); School of Medical Sciences (Pharmacology), The University of Sydney, NSW 2006 (Australia); Hinton, T. [School of Medical Sciences (Pharmacology), The University of Sydney, NSW 2006 (Australia); Waters, K.A. [Department of Medicine, The University of Sydney, NSW 2006 (Australia); The Children' s Hospital at Westmead, NSW 2145 (Australia); Hennessy, A. [School of Medicine, University of Western Sydney, NSW 2751 (Australia); Heart Research Institute, 7 Eliza St Newtown, NSW 2042 (Australia)

2014-05-01

Smoking during pregnancy is associated with low birth weight, premature delivery, and neonatal morbidity and mortality. Nicotine, a major pathogenic compound of cigarette smoke, binds to the nicotinic acetylcholine receptors (nAChRs). A total of 16 nAChR subunits have been identified in mammals (9 α, 4 β, and 1 δ, γ and ε subunits). The effect of cigarette smoking on the expression of these subunits in the placenta has not yet been determined, thus constituting the aim of this study. Using RT-qPCR and western blotting, this study investigated all 16 mammalian nAChR subunits in the normal healthy human placenta, and compared mRNA and protein expressions in the placentas from smokers (n = 8) to controls (n = 8). Our data show that all 16 subunit mRNAs are expressed in the normal, non-diseased human placenta and that the expression of α2, α3, α4, α9, β2 and β4 subunits is greater than the other subunits. For mRNA, cigarette smoke exposure was associated with increased expression of the α9 subunit, and decreased expression of the δ subunit. At the protein level, expression of both α9 and δ was increased. Thus, cigarette smoking in pregnancy is sufficient to regulate nAChR subunits in the placenta, specifically α9 and δ subunits, and could contribute to the adverse effects of vasoconstriction and decreased re-epithelialisation (α9), and increased calcification and apoptosis (δ), seen in the placentas of smoking women. - Highlights: • All 16 mammalian nAChR subunits are expressed in the human placenta. • Cigarette smoking increases α9 mRNA and protein in the placenta. • Cigarette smoking decreases δ mRNA but increases δ protein in the placenta.

Cigarette smoking during pregnancy regulates the expression of specific nicotinic acetylcholine receptor (nAChR) subunits in the human placenta

International Nuclear Information System (INIS)

Machaalani, R.; Ghazavi, E.; Hinton, T.; Waters, K.A.; Hennessy, A.

2014-01-01

Smoking during pregnancy is associated with low birth weight, premature delivery, and neonatal morbidity and mortality. Nicotine, a major pathogenic compound of cigarette smoke, binds to the nicotinic acetylcholine receptors (nAChRs). A total of 16 nAChR subunits have been identified in mammals (9 α, 4 β, and 1 δ, γ and ε subunits). The effect of cigarette smoking on the expression of these subunits in the placenta has not yet been determined, thus constituting the aim of this study. Using RT-qPCR and western blotting, this study investigated all 16 mammalian nAChR subunits in the normal healthy human placenta, and compared mRNA and protein expressions in the placentas from smokers (n = 8) to controls (n = 8). Our data show that all 16 subunit mRNAs are expressed in the normal, non-diseased human placenta and that the expression of α2, α3, α4, α9, β2 and β4 subunits is greater than the other subunits. For mRNA, cigarette smoke exposure was associated with increased expression of the α9 subunit, and decreased expression of the δ subunit. At the protein level, expression of both α9 and δ was increased. Thus, cigarette smoking in pregnancy is sufficient to regulate nAChR subunits in the placenta, specifically α9 and δ subunits, and could contribute to the adverse effects of vasoconstriction and decreased re-epithelialisation (α9), and increased calcification and apoptosis (δ), seen in the placentas of smoking women. - Highlights: • All 16 mammalian nAChR subunits are expressed in the human placenta. • Cigarette smoking increases α9 mRNA and protein in the placenta. • Cigarette smoking decreases δ mRNA but increases δ protein in the placenta

Tissue-Specific 5′ Heterogeneity of PPARα Transcripts and Their Differential Regulation by Leptin

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Garratt, Emma S.; Vickers, Mark H.; Gluckman, Peter D.; Hanson, Mark A.

2013-01-01

The genes encoding nuclear receptors comprise multiple 5′untranslated exons, which give rise to several transcripts encoding the same protein, allowing tissue-specific regulation of expression. Both human and mouse peroxisome proliferator activated receptor (PPAR) α genes have multiple promoters, although their function is unknown. Here we have characterised the rat PPARα promoter region and have identified three alternative PPARα transcripts, which have different transcription start sites owing to the utilisation of distinct first exons. Moreover these alternative PPARα transcripts were differentially expressed between adipose tissue and liver. We show that while the major adipose (P1) and liver (P2) transcripts were both induced by dexamethasone, they were differentially regulated by the PPARα agonist, clofibric acid, and leptin. Leptin had no effect on the adipose-specific P1 transcript, but induced liver-specific P2 promoter activity via a STAT3/Sp1 mechanism. Moreover in Wistar rats, leptin treatment between postnatal day 3–13 led to an increase in P2 but not P1 transcription in adipose tissue which was sustained into adulthood. This suggests that the expression of the alternative PPARα transcripts are in part programmed by early life exposure to leptin leading to persistent change in adipose tissue fatty acid metabolism through specific activation of a quiescent PPARα promoter. Such complexity in the regulation of PPARα may allow the expression of PPARα to be finely regulated in response to environmental factors. PMID:23825665

Neuroblastoma mRNAs predict outcome in children with stage 4 neuroblastoma: a European HR-NBL1/SIOPEN study.

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Viprey, Virginie F; Gregory, Walter M; Corrias, Maria V; Tchirkov, Andrei; Swerts, Katrien; Vicha, Ales; Dallorso, Sandro; Brock, Penelope; Luksch, Roberto; Valteau-Couanet, Dominique; Papadakis, Vassilios; Laureys, Genevieve; Pearson, Andrew D; Ladenstein, Ruth; Burchill, Susan A

2014-04-01

To evaluate the hypothesis that detection of neuroblastoma mRNAs by reverse transcriptase quantitative polymerase chain reaction (RTqPCR) in peripheral blood (PB) and bone marrow aspirates (BM) from children with stage 4 neuroblastoma are clinically useful biomarkers of risk. RTqPCR for paired-like homeobox 2b (PHOX2B), tyrosine hydroxylase (TH), and doublecortin (DCX) mRNA in PB and BM of children enrolled onto the High-Risk Neuroblastoma Trial-1 of the European Society of Pediatric Oncology Neuroblastoma Group (HR-NBL1/SIOPEN) was performed at diagnosis and after induction therapy. High levels of TH, PHOX2B, or DCX mRNA in PB or BM at diagnosis strongly predicted for worse event-free survival (EFS) and overall survival (OS) in a cohort of 290 children. After induction therapy, high levels of these mRNAs predicted worse EFS and OS in BM but not in PB. Combinations of mRNAs in BM did not add to the predictive power of any single mRNA. However, in the original (n = 182) and validation (n = 137) PB cohorts, high TH (log10TH > 0.8) or high PHOX2B (log10PHOX2B > 0.28) identify 19% of children as ultrahigh risk, with 5-year EFS and OS rates of 0%; OS rate was 25% (95% CI, 16% to 36%) and EFS rate was 38% (95% CI, 28% to 49%) in the remaining children. The magnitude of reduction in mRNA level between diagnosis and postinduction therapy in BM or PB was not of additional predictive value. High levels of TH and PHOX2B mRNA in PB at diagnosis objectively identify children with ultrahigh-risk disease who may benefit from novel treatment approaches. The level of TH, PHOX2B, and DCX mRNA in BM and/or PB at diagnosis might contribute to an algorithm to improve stratification of children for treatment.

Tissue-specific regulation of BMP signaling by Drosophila N-glycanase 1.

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Galeone, Antonio; Han, Seung Yeop; Huang, Chengcheng; Hosomi, Akira; Suzuki, Tadashi; Jafar-Nejad, Hamed

2017-08-04

Mutations in the human N- glycanase 1 ( NGLY1 ) cause a rare, multisystem congenital disorder with global developmental delay. However, the mechanisms by which NGLY1 and its homologs regulate embryonic development are not known. Here we show that Drosophila Pngl encodes an N -glycanase and exhibits a high degree of functional conservation with human NGLY1. Loss of Pngl results in developmental midgut defects reminiscent of midgut-specific loss of BMP signaling. Pngl mutant larvae also exhibit a severe midgut clearance defect, which cannot be fully explained by impaired BMP signaling. Genetic experiments indicate that Pngl is primarily required in the mesoderm during Drosophila development. Loss of Pngl results in a severe decrease in the level of Dpp homodimers and abolishes BMP autoregulation in the visceral mesoderm mediated by Dpp and Tkv homodimers. Thus, our studies uncover a novel mechanism for the tissue-specific regulation of an evolutionarily conserved signaling pathway by an N -glycanase enzyme.

Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance

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Shimpei Morimoto

2018-03-01

Full Text Available Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes (ADC17 and KIN1 that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after

Architecture and dynamics of overlapped RNA regulatory networks.

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Lapointe, Christopher P; Preston, Melanie A; Wilinski, Daniel; Saunders, Harriet A J; Campbell, Zachary T; Wickens, Marvin

2017-11-01

A single protein can bind and regulate many mRNAs. Multiple proteins with similar specificities often bind and control overlapping sets of mRNAs. Yet little is known about the architecture or dynamics of overlapped networks. We focused on three proteins with similar structures and related RNA-binding specificities-Puf3p, Puf4p, and Puf5p of S. cerevisiae Using RNA Tagging, we identified a "super-network" comprised of four subnetworks: Puf3p, Puf4p, and Puf5p subnetworks, and one controlled by both Puf4p and Puf5p. The architecture of individual subnetworks, and thus the super-network, is determined by competition among particular PUF proteins to bind mRNAs, their affinities for binding elements, and the abundances of the proteins. The super-network responds dramatically: The remaining network can either expand or contract. These strikingly opposite outcomes are determined by an interplay between the relative abundance of the RNAs and proteins, and their affinities for one another. The diverse interplay between overlapping RNA-protein networks provides versatile opportunities for regulation and evolution. © 2017 Lapointe et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Pre-cultivation with Selected Prebiotics Enhances the Survival and the Stress Response of Lactobacillus rhamnosus Strains in Simulated Gastrointestinal Transit

Directory of Open Access Journals (Sweden)

Mariantonietta Succi

2017-06-01

Full Text Available In our study, we dwelled upon combinations of lactobacilli/prebiotics, considering four different strains belonging to the Lactobacillus rhamnosus species, including Lactobacillus rhamnosus GG (LGG, and different prebiotics often found in commercial synbiotic products, such as inulin, lactulose and polyols mannitol and sorbitol. In the first step of the research, the survival, the growth kinetic parameters and the protein expression of Lb. rhamnosus strains cultivated in presence of the different prebiotics as a unique carbon source were evaluated. In the second step, the influence of pre-cultivation in medium added of metabolizable prebiotics on the strains survival to simulated gastrointestinal (GI transit, assayed without prebiotics addition, was estimated. Our results showed that the presence in the medium of certain low fermented prebiotics, specific for each strain, represents a stress factor that significantly affects the growth of Lb. rhamnosus strains, inducing the up-regulation of several proteins. In detail, all added prebiotics used as unique carbon source caused a growth retard compared with glucose, as testified by increased values of the lag phase and decreased values of the μmax. Mannitol evidenced intermediate μmax values between those registered with glucose and those detected with the other assayed prebiotics. Moreover, the cultivation with prebiotics induced the over expression of 7 protein bands. Interestingly, we found a correlation between the up-regulation of two specific stress proteins, called P4 (ATP-binding subunit Clpx and P7 (GrpE, and the death kinetic parameters (resistance and cells viability registered during the simulated GI transit of strains pre-cultivated with specific, low fermented prebiotics. Specifically, the highest resistance and gastric-vitality scores were highlighted for the strain AT195 when pre-cultivated in presence of sorbitol. Conversely, the lowest values were found in the case of DSM20021

Pre-cultivation with Selected Prebiotics Enhances the Survival and the Stress Response of Lactobacillus rhamnosus Strains in Simulated Gastrointestinal Transit.

Science.gov (United States)

Succi, Mariantonietta; Tremonte, Patrizio; Pannella, Gianfranco; Tipaldi, Luca; Cozzolino, Autilia; Romaniello, Rossana; Sorrentino, Elena; Coppola, Raffaele

2017-01-01

In our study, we dwelled upon combinations of lactobacilli/prebiotics, considering four different strains belonging to the Lactobacillus rhamnosus species, including Lactobacillus rhamnosus GG (LGG), and different prebiotics often found in commercial synbiotic products, such as inulin, lactulose and polyols mannitol and sorbitol. In the first step of the research, the survival, the growth kinetic parameters and the protein expression of Lb. rhamnosus strains cultivated in presence of the different prebiotics as a unique carbon source were evaluated. In the second step, the influence of pre-cultivation in medium added of metabolizable prebiotics on the strains survival to simulated gastrointestinal (GI) transit, assayed without prebiotics addition, was estimated. Our results showed that the presence in the medium of certain low fermented prebiotics, specific for each strain, represents a stress factor that significantly affects the growth of Lb. rhamnosus strains, inducing the up-regulation of several proteins. In detail, all added prebiotics used as unique carbon source caused a growth retard compared with glucose, as testified by increased values of the lag phase and decreased values of the μmax. Mannitol evidenced intermediate μmax values between those registered with glucose and those detected with the other assayed prebiotics. Moreover, the cultivation with prebiotics induced the over expression of 7 protein bands. Interestingly, we found a correlation between the up-regulation of two specific stress proteins, called P4 (ATP-binding subunit Clpx) and P7 (GrpE), and the death kinetic parameters (resistance and cells viability) registered during the simulated GI transit of strains pre-cultivated with specific, low fermented prebiotics. Specifically, the highest resistance and gastric-vitality scores were highlighted for the strain AT195 when pre-cultivated in presence of sorbitol. Conversely, the lowest values were found in the case of DSM20021 pre

Nuclear Export of Pre-Ribosomal Subunits Requires Dbp5, but Not as an RNA-Helicase as for mRNA Export.

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Neumann, Bettina; Wu, Haijia; Hackmann, Alexandra; Krebber, Heike

2016-01-01

The DEAD-box RNA-helicase Dbp5/Rat8 is known for its function in nuclear mRNA export, where it displaces the export receptor Mex67 from the mRNA at the cytoplasmic side of the nuclear pore complex (NPC). Here we show that Dbp5 is also required for the nuclear export of both pre-ribosomal subunits. Yeast temperature-sensitive dbp5 mutants accumulate both ribosomal particles in their nuclei. Furthermore, Dbp5 genetically and physically interacts with known ribosomal transport factors such as Nmd3. Similar to mRNA export we show that also for ribosomal transport Dbp5 is required at the cytoplasmic side of the NPC. However, unlike its role in mRNA export, Dbp5 does not seem to undergo its ATPase cycle for this function, as ATPase-deficient dbp5 mutants that selectively inhibit mRNA export do not affect ribosomal transport. Furthermore, mutants of GLE1, the ATPase stimulating factor of Dbp5, show no major ribosomal export defects. Consequently, while Dbp5 uses its ATPase cycle to displace the export receptor Mex67 from the translocated mRNAs, Mex67 remains bound to ribosomal subunits upon transit to the cytoplasm, where it is detectable on translating ribosomes. Therefore, we propose a model, in which Dbp5 supports ribosomal transport by capturing ribosomal subunits upon their cytoplasmic appearance at the NPC, possibly by binding export factors such as Mex67. Thus, our findings reveal that although different ribonucleoparticles, mRNAs and pre-ribosomal subunits, use shared export factors, they utilize different transport mechanisms.

Overexpression of LncRNA AC067945.2 Down-Regulates Collagen Expression in Skin Fibroblasts and Possibly Correlates with the VEGF and Wnt Signalling Pathways.

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Chen, Ling; Li, Jingyun; Li, Qian; Li, Xue; Gao, Yanli; Hua, Xiangdong; Zhou, Bei; Li, Jun

2018-01-01

Long non-coding RNAs (lncRNAs) are thought to play crucial roles in human diseases. However, the function of lncRNAs in hypertrophic scar formation remains poorly understood. Utilizing qRT-PCR, we explored the expression changes of AC067945.2. Overexpression of AC067945.2 in normal skin fibroblasts was performed by transient plasmid transfection. Western blot was used to check the proteins' expression changes. Cell Counting Kit-8 (CCK-8) assay and Annexin V/7-AAD staining were used to examine cell proliferation and apoptosis, respectively. mRNA-seq was applied to dissect the differentially expressed mRNAs in AC067945.2 overexpressed cells. We also performed ELISA to detect the VEGF secretion. AC067945.2 was down-regulated in hypertrophic scar tissues. Overexpression of AC067945.2 did not affect cell proliferation, but it mildly promoted early apoptosis in normal skin fibroblasts. Furthermore, AC067945.2 overexpression inhibited the expression of COL1A1, COL1A2, COL3A1 and α-SMA proteins. Transforming growth factor-β1 (TGF-β1) could inhibit the expression of AC067945.2. Based on mRNA-seq data, compared with mRNAs in the control group, 138 mRNAs were differentially expressed, including 14 up-regulated and 124 down-regulated transcripts, in the AC067945.2 overexpression group. Gene ontology and pathway analyses revealed that AC067945.2 overexpression was correlated with developmental processes, binding, extracellular region, and the vascular endothelial cell growth factor (VEGF) and Wnt signalling pathways. ELISA confirmed that AC067945.2 overexpression could repress VEGF secretion. Taken together, our data uncovered the functions of a novel lncRNA AC067945.2, which might help us understand the mechanisms regulated by AC067945.2 in the pathogenesis of hypertrophic scar formation. © 2018 The Author(s). Published by S. Karger AG, Basel.

Towards an understanding of miRNA regulation

DEFF Research Database (Denmark)

Jensen, Trine Ilsø

miRNAs are well-known regulators of gene expression. They function post-transcriptionally by binding to complementary sites within the 3´UTR of target mRNAs, which mediates translational repression and destabilization. However, miRNA expression itself is also subjected to regulation. Here, we...... report a new method to investigate and potentially characterize the pri-miRNA transcript. Overexpression of a transdominant Drosha mutant, which is unable to cleave its substrate, enables stabilization of the pri-miRNA transcript. Drosha mutant immunoprecipitation from the nuclear compartment...... is performed followed by high-throughput sequencing (nuclear Drosha Mt2 RIPseq). This method allows for the detection of global pri-miRNA signature and also provides a method to potentially identify new Drosha substrates. Furthermore, data on the identification of a novel endogenous circular RNA sponge (ciRS-7...

Angiotensin II Regulates microRNA-132/-212 in Hypertensive Rats and Humans

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Eskildsen, Tilde V; Jeppesen, Pia L; Schneider, Mikael

2013-01-01

MicroRNAs (miRNAs), a group of small non-coding RNAs that fine tune translation of multiple target mRNAs, are emerging as key regulators in cardiovascular development and disease. MiRNAs are involved in cardiac hypertrophy, heart failure and remodeling following cardiac infarction; however, mi...... no effect. Taken together, these data suggest that miR-132 and miR-212 are involved in AngII induced hypertension, providing a new perspective in hypertensive disease mechanisms....

Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio.

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Weidmann, Chase A; Qiu, Chen; Arvola, René M; Lou, Tzu-Fang; Killingsworth, Jordan; Campbell, Zachary T; Tanaka Hall, Traci M; Goldstrohm, Aaron C

2016-08-02

Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation by Drosophila Pumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulated in vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.

Myb-binding protein 1a (Mybbp1a) regulates levels and processing of pre-ribosomal RNA.

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Hochstatter, Julia; Hölzel, Michael; Rohrmoser, Michaela; Schermelleh, Lothar; Leonhardt, Heinrich; Keough, Rebecca; Gonda, Thomas J; Imhof, Axel; Eick, Dirk; Längst, Gernot; Németh, Attila

2012-07-13

Ribosomal RNA gene transcription, co-transcriptional processing, and ribosome biogenesis are highly coordinated processes that are tightly regulated during cell growth. In this study we discovered that Mybbp1a is associated with both the RNA polymerase I complex and the ribosome biogenesis machinery. Using a reporter assay that uncouples transcription and RNA processing, we show that Mybbp1a represses rRNA gene transcription. In addition, overexpression of the protein reduces RNA polymerase I loading on endogenous rRNA genes as revealed by chromatin immunoprecipitation experiments. Accordingly, depletion of Mybbp1a results in an accumulation of the rRNA precursor in vivo but surprisingly also causes growth arrest of the cells. This effect can be explained by the observation that the modulation of Mybbp1a protein levels results in defects in pre-rRNA processing within the cell. Therefore, the protein may play a dual role in the rRNA metabolism, potentially linking and coordinating ribosomal DNA transcription and pre-rRNA processing to allow for the efficient synthesis of ribosomes.

BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism.

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Rodríguez, Andrea E; López-Crisosto, Camila; Peña-Oyarzún, Daniel; Salas, Daniela; Parra, Valentina; Quiroga, Clara; Morawe, Tobias; Chiong, Mario; Behl, Christian; Lavandero, Sergio

2016-01-01

Autophagy is mainly regulated by post-translational and lipid modifications of ATG proteins. In some scenarios, the induction of autophagy is accompanied by increased levels of certain ATG mRNAs such as MAP1LC3B/LC3B, ATG5 or ATG12. However, little is known about the regulation of ATG protein synthesis at the translational level. The cochaperone of the HSP70 system BAG3 (BCL2-associated athanogene 3) has been associated to LC3B lipidation through an unknown mechanism. In the present work, we studied how BAG3 controls autophagy in HeLa and HEK293 cells. Our results showed that BAG3 regulates the basal amount of total cellular LC3B protein by controlling its mRNA translation. This effect was apparently specific to LC3B because other ATG protein levels were not affected. BAG3 knockdown did not affect LC3B lipidation induced by nutrient deprivation or proteasome inhibition. We concluded that BAG3 maintains the basal amount of LC3B protein by controlling the translation of its mRNA in HeLa and HEK293 cells.

GDE2 regulates subtype-specific motor neuron generation through inhibition of Notch signaling.

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Sabharwal, Priyanka; Lee, Changhee; Park, Sungjin; Rao, Meenakshi; Sockanathan, Shanthini

2011-09-22

The specification of spinal interneuron and motor neuron identities initiates within progenitor cells, while motor neuron subtype diversification is regulated by hierarchical transcriptional programs implemented postmitotically. Here we find that mice lacking GDE2, a six-transmembrane protein that triggers motor neuron generation, exhibit selective losses of distinct motor neuron subtypes, specifically in defined subsets of limb-innervating motor pools that correlate with the loss of force-generating alpha motor neurons. Mechanistically, GDE2 is expressed by postmitotic motor neurons but utilizes extracellular glycerophosphodiester phosphodiesterase activity to induce motor neuron generation by inhibiting Notch signaling in neighboring motor neuron progenitors. Thus, neuronal GDE2 controls motor neuron subtype diversity through a non-cell-autonomous feedback mechanism that directly regulates progenitor cell differentiation, implying that subtype specification initiates within motor neuron progenitor populations prior to their differentiation into postmitotic motor neurons. Copyright © 2011 Elsevier Inc. All rights reserved.

Possible role of TIEG1 as a feedback regulator of myostatin and TGF-{beta} in myoblasts

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Miyake, Masato; Hayashi, Shinichiro; Iwasaki, Shunsuke; Chao, Guozheng; Takahashi, Hideyuki; Watanabe, Kouichi; Ohwada, Shyuichi; Aso, Hisashi [Laboratory of Functional Morphology, Department of Animal Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-Ku, Sendai 981-8555 (Japan); Yamaguchi, Takahiro, E-mail: ty1010@bios.tohoku.ac.jp [Laboratory of Functional Morphology, Department of Animal Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-Ku, Sendai 981-8555 (Japan)

2010-03-19

Myostatin and TGF-{beta} negatively regulate skeletal muscle development and growth. Both factors signal through the Smad2/3 pathway. However, the regulatory mechanism of myostatin and TGF-{beta} signaling remains unclear. TGF-{beta} inducible early gene (TIEG) 1 is highly expressed in skeletal muscle and has been implicated in the modulation of TGF-{beta} signaling. These findings prompted us to investigate the effect of TIEG1 on myostatin and TGF-{beta} signaling using C2C12 myoblasts. Myostatin and TGF-{beta} induced the expression of TIEG1 and Smad7 mRNAs, but not TIEG2 mRNA, in proliferating C2C12 cells. When differentiating C2C12 myoblasts were stimulated by myostatin, TIEG1 mRNA was up-regulated at a late stage of differentiation. In contrast, TGF-{beta} enhanced TIEG1 expression at an early stage. Overexpression of TIEG1 prevented the transcriptional activation of Smad by myostatin and TGF-{beta} in both proliferating or differentiating C2C12 cells, but the expression of Smad2 and Smad7 mRNAs was not affected. Forced expression of TIEG1 inhibited myogenic differentiation but did not cause more inhibition than the empty vector in the presence of myostatin or TGF-{beta}. These results demonstrate that TIEG1 is one possible feedback regulator of myostatin and TGF-{beta} that prevents excess action in myoblasts.

Possible role of TIEG1 as a feedback regulator of myostatin and TGF-β in myoblasts

International Nuclear Information System (INIS)

Miyake, Masato; Hayashi, Shinichiro; Iwasaki, Shunsuke; Chao, Guozheng; Takahashi, Hideyuki; Watanabe, Kouichi; Ohwada, Shyuichi; Aso, Hisashi; Yamaguchi, Takahiro

2010-01-01

Myostatin and TGF-β negatively regulate skeletal muscle development and growth. Both factors signal through the Smad2/3 pathway. However, the regulatory mechanism of myostatin and TGF-β signaling remains unclear. TGF-β inducible early gene (TIEG) 1 is highly expressed in skeletal muscle and has been implicated in the modulation of TGF-β signaling. These findings prompted us to investigate the effect of TIEG1 on myostatin and TGF-β signaling using C2C12 myoblasts. Myostatin and TGF-β induced the expression of TIEG1 and Smad7 mRNAs, but not TIEG2 mRNA, in proliferating C2C12 cells. When differentiating C2C12 myoblasts were stimulated by myostatin, TIEG1 mRNA was up-regulated at a late stage of differentiation. In contrast, TGF-β enhanced TIEG1 expression at an early stage. Overexpression of TIEG1 prevented the transcriptional activation of Smad by myostatin and TGF-β in both proliferating or differentiating C2C12 cells, but the expression of Smad2 and Smad7 mRNAs was not affected. Forced expression of TIEG1 inhibited myogenic differentiation but did not cause more inhibition than the empty vector in the presence of myostatin or TGF-β. These results demonstrate that TIEG1 is one possible feedback regulator of myostatin and TGF-β that prevents excess action in myoblasts.

Nuclear pre-mRNA processing in plants

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Reddy, A.S.N. [Colorado State Univ., Fort Collins, CO (United States). Dept. of Biology and Program in Molecular Plant Biology; Golovkin, M. (eds.) [Thomas Jefferson Univ., Philadelphia, PA (United States). Dept. of Microbiology

2008-07-01

This volume of CTMI, entitled Nuclear premRNA Processing in Plants, with 16 chapters from leading scientists in this area, summarizes recent advances in nuclear pre-mRNA processing and its role in plant growth and development. It provides researchers in the field, as well as those in related areas, with an up-to-date and comprehensive, yet concise, overview of the current status and future potential of this research in understanding plant biology. The first four chapters focus on spliceosome composition, genome-wide alternative splicing, and splice site requirements for U1 and U12 introns using computational and empirical approaches. Analysis of sequenced plant genomes has revealed that 80% of all protein-coding nuclear genes contain one or more introns. The lack of an in vitro plant splicing system has made it difficult to identify general and plant-specific components of splicing machinery in plants. The next three chapters focus on serine/arginine-rich (SR) proteins, a family of highly conserved proteins, which are known to play key roles in constitutive and regulated splicing of pre-mRNA and other aspects of RNA metabolism in metazoans. These proteins engage both in RNA binding and protein.protein interactions and function as splicing regulators at multiple stages of spliceosome assembly. This family of proteins has expanded considerably in plants with several plant-specific SR proteins. Several serendipitous discoveries made using forward genetics are indicating that RNA metabolism (alternative splicing, alternative polyadenylation, mRNA transport) plays an important role in many aspects of plant growth and development and in plant responses to biotic and abiotic stresses. The next seven chapters focus on these aspects of RNA metabolism. The plant hormone abscisic acid (ABA) regulates a number of physiological processes during plant growth and development. The next chapter or A.B. Rose discusses the ways introns affect gene expression both positively and

Nuclear pre-mRNA processing in plants

International Nuclear Information System (INIS)

Reddy, A.S.N.; Golovkin, M.

2008-01-01

This volume of CTMI, entitled Nuclear premRNA Processing in Plants, with 16 chapters from leading scientists in this area, summarizes recent advances in nuclear pre-mRNA processing and its role in plant growth and development. It provides researchers in the field, as well as those in related areas, with an up-to-date and comprehensive, yet concise, overview of the current status and future potential of this research in understanding plant biology. The first four chapters focus on spliceosome composition, genome-wide alternative splicing, and splice site requirements for U1 and U12 introns using computational and empirical approaches. Analysis of sequenced plant genomes has revealed that 80% of all protein-coding nuclear genes contain one or more introns. The lack of an in vitro plant splicing system has made it difficult to identify general and plant-specific components of splicing machinery in plants. The next three chapters focus on serine/arginine-rich (SR) proteins, a family of highly conserved proteins, which are known to play key roles in constitutive and regulated splicing of pre-mRNA and other aspects of RNA metabolism in metazoans. These proteins engage both in RNA binding and protein.protein interactions and function as splicing regulators at multiple stages of spliceosome assembly. This family of proteins has expanded considerably in plants with several plant-specific SR proteins. Several serendipitous discoveries made using forward genetics are indicating that RNA metabolism (alternative splicing, alternative polyadenylation, mRNA transport) plays an important role in many aspects of plant growth and development and in plant responses to biotic and abiotic stresses. The next seven chapters focus on these aspects of RNA metabolism. The plant hormone abscisic acid (ABA) regulates a number of physiological processes during plant growth and development. The next chapter or A.B. Rose discusses the ways introns affect gene expression both positively and

Chl1 DNA helicase regulates Scc2 deposition specifically during DNA-replication in Saccharomyces cerevisiae.

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Soumya Rudra

Full Text Available The conserved family of cohesin proteins that mediate sister chromatid cohesion requires Scc2, Scc4 for chromatin-association and Eco1/Ctf7 for conversion to a tethering competent state. A popular model, based on the notion that cohesins form huge ring-like structures, is that Scc2, Scc4 function is essential only during G1 such that sister chromatid cohesion results simply from DNA replisome passage through pre-loaded cohesin rings. In such a scenario, cohesin deposition during G1 is temporally uncoupled from Eco1-dependent establishment reactions that occur during S-phase. Chl1 DNA helicase (homolog of human ChlR1/DDX11 and BACH1/BRIP1/FANCJ helicases implicated in Fanconi anemia, breast and ovarian cancer and Warsaw Breakage Syndrome plays a critical role in sister chromatid cohesion, however, the mechanism through which Chl1 promotes cohesion remains poorly understood. Here, we report that Chl1 promotes Scc2 loading unto DNA such that both Scc2 and cohesin enrichment to chromatin are defective in chl1 mutant cells. The results further show that both Chl1 expression and chromatin-recruitment are tightly regulated through the cell cycle, peaking during S-phase. Importantly, kinetic ChIP studies reveals that Chl1 is required for Scc2 chromatin-association specifically during S-phase, but not during G1. Despite normal chromatin enrichment of both Scc2 and cohesin during G1, chl1 mutant cells exhibit severe chromosome segregation and cohesion defects--revealing that G1-loaded cohesins is insufficient to promote cohesion. Based on these findings, we propose a new model wherein S-phase cohesin loading occurs during DNA replication and in concert with both cohesion establishment and chromatin assembly reactions--challenging the notion that DNA replication fork navigates through or around pre-loaded cohesin rings.

Pre-screening Discussions and Prostate-Specific Antigen Testing for Prostate Cancer Screening.

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Li, Jun; Zhao, Guixiang; Hall, Ingrid J

2015-08-01

For many men, the net benefit of prostate cancer screening with prostate-specific antigen (PSA) tests may be small. Many major medical organizations have issued recommendations for prostate cancer screening, stressing the need for shared decision making before ordering a test. The purpose of this study is to better understand associations between discussions about benefits and harms of PSA testing and uptake of the test among men aged ≥40 years. Associations between pre-screening discussions and PSA testing were examined using self-reported data from the 2012 Behavioral Risk Factor Surveillance System. Unadjusted prevalence of PSA testing was estimated and AORs were calculated using logistic regression in 2014. The multivariate analysis showed that men who had ever discussed advantages of PSA testing only or discussed both advantages and disadvantages were more likely, respectively, to report having had a test within the past year than men who had no discussions (ptesting with their healthcare providers were more likely (AOR=2.75, 95% CI=2.00, 3.79) to report getting tested than men who had no discussions. Discussions of the benefits or harms of PSA testing are positively associated with increased uptake of the test. Given the conflicting recommendations for prostate cancer screening and increasing importance of shared decision making, this study points to the need for understanding how pre-screening discussions are being conducted in clinical practice and the role played by patients' values and preferences in decisions about PSA testing. Published by Elsevier Inc.

TCR down-regulation controls virus-specific CD8+ T cell responses

DEFF Research Database (Denmark)

Bonefeld, Charlotte Menné; Haks, Mariëlle; Nielsen, Bodil

2008-01-01

in mice with a mutated CD3gamma di-leucine-based motif. The CD3gamma mutation did not impair early TCR signaling, nor did it compromise recruitment or proliferation of virus-specific T cells, but it increased the apoptosis rate of the activated T cells by increasing down-regulation of the antiapoptotic...

Emergence of differentially regulated pathways associated with the development of regional specificity in chicken skin.

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Chang, Kai-Wei; Huang, Nancy A; Liu, I-Hsuan; Wang, Yi-Hui; Wu, Ping; Tseng, Yen-Tzu; Hughes, Michael W; Jiang, Ting Xin; Tsai, Mong-Hsun; Chen, Chien-Yu; Oyang, Yen-Jen; Lin, En-Chung; Chuong, Cheng-Ming; Lin, Shau-Ping

2015-01-23

Regional specificity allows different skin regions to exhibit different characteristics, enabling complementary functions to make effective use of the integumentary surface. Chickens exhibit a high degree of regional specificity in the skin and can serve as a good model for when and how these regional differences begin to emerge. We used developing feather and scale regions in embryonic chickens as a model to gauge the differences in their molecular pathways. We employed cosine similarity analysis to identify the differentially regulated and co-regulated genes. We applied low cell techniques for expression validation and chromatin immunoprecipitation (ChIP)-based enhancer identification to overcome limited cell availabilities from embryonic chicken skin. We identified a specific set of genes demonstrating a high correlation as being differentially expressed during feather and scale development and maturation. Some members of the WNT, TGF-beta/BMP, and Notch family known to be involved in feathering skin differentiation were found to be differentially regulated. Interestingly, we also found genes along calcium channel pathways that are differentially regulated. From the analysis of differentially regulated pathways, we used calcium signaling pathways as an example for further verification. Some voltage-gated calcium channel subunits, particularly CACNA1D, are expressed spatio-temporally in the skin epithelium. These calcium signaling pathway members may be involved in developmental decisions, morphogenesis, or epithelial maturation. We further characterized enhancers associated with histone modifications, including H3K4me1, H3K27ac, and H3K27me3, near calcium channel-related genes and identified signature intensive hotspots that may be correlated with certain voltage-gated calcium channel genes. We demonstrated the applicability of cosine similarity analysis for identifying novel regulatory pathways that are differentially regulated during development. Our study

49 CFR 663.23 - Description of pre-award audit.

Science.gov (United States)

2010-10-01

... 49 Transportation 7 2010-10-01 2010-10-01 false Description of pre-award audit. 663.23 Section 663.23 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL TRANSIT ADMINISTRATION, DEPARTMENT OF TRANSPORTATION PRE-AWARD AND POST-DELIVERY AUDITS OF ROLLING STOCK PURCHASES Pre-Award Audits § 663.23 Description of pre-award...

Regulation of bacterial photosynthesis genes by the small noncoding RNA PcrZ.

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Mank, Nils N; Berghoff, Bork A; Hermanns, Yannick N; Klug, Gabriele

2012-10-02

The small RNA PcrZ (photosynthesis control RNA Z) of the facultative phototrophic bacterium Rhodobacter sphaeroides is induced upon a drop of oxygen tension with similar kinetics to those of genes for components of photosynthetic complexes. High expression of PcrZ depends on PrrA, the response regulator of the PrrB/PrrA two-component system with a central role in redox regulation in R. sphaeroides. In addition the FnrL protein, an activator of some photosynthesis genes at low oxygen tension, is involved in redox-dependent expression of this small (s)RNA. Overexpression of full-length PcrZ in R. sphaeroides affects expression of a small subset of genes, most of them with a function in photosynthesis. Some mRNAs from the photosynthetic gene cluster were predicted to be putative PcrZ targets and results from an in vivo reporter system support these predictions. Our data reveal a negative effect of PcrZ on expression of its target mRNAs. Thus, PcrZ counteracts the redox-dependent induction of photosynthesis genes, which is mediated by protein regulators. Because PrrA directly activates photosynthesis genes and at the same time PcrZ, which negatively affects photosynthesis gene expression, this is one of the rare cases of an incoherent feed-forward loop including an sRNA. Our data identified PcrZ as a trans acting sRNA with a direct regulatory function in formation of photosynthetic complexes and provide a model for the control of photosynthesis gene expression by a regulatory network consisting of proteins and a small noncoding RNA.

The chromatin remodeler SPLAYED regulates specific stress signaling pathways.

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Justin W Walley

2008-12-01

Full Text Available Organisms are continuously exposed to a myriad of environmental stresses. Central to an organism's survival is the ability to mount a robust transcriptional response to the imposed stress. An emerging mechanism of transcriptional control involves dynamic changes in chromatin structure. Alterations in chromatin structure are brought about by a number of different mechanisms, including chromatin modifications, which covalently modify histone proteins; incorporation of histone variants; and chromatin remodeling, which utilizes ATP hydrolysis to alter histone-DNA contacts. While considerable insight into the mechanisms of chromatin remodeling has been gained, the biological role of chromatin remodeling complexes beyond their function as regulators of cellular differentiation and development has remained poorly understood. Here, we provide genetic, biochemical, and biological evidence for the critical role of chromatin remodeling in mediating plant defense against specific biotic stresses. We found that the Arabidopsis SWI/SNF class chromatin remodeling ATPase SPLAYED (SYD is required for the expression of selected genes downstream of the jasmonate (JA and ethylene (ET signaling pathways. SYD is also directly recruited to the promoters of several of these genes. Furthermore, we show that SYD is required for resistance against the necrotrophic pathogen Botrytis cinerea but not the biotrophic pathogen Pseudomonas syringae. These findings demonstrate not only that chromatin remodeling is required for selective pathogen resistance, but also that chromatin remodelers such as SYD can regulate specific pathways within biotic stress signaling networks.

Regulation and regulatory role of WNT signaling in potentiating FSH action during bovine dominant follicle selection.

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P S P Gupta

Full Text Available Follicular development occurs in wave like patterns in monotocous species such as cattle and humans and is regulated by a complex interaction of gonadotropins with local intrafollicular regulatory molecules. To further elucidate potential mechanisms controlling dominant follicle selection, granulosa cell RNA harvested from F1 (largest and F2 (second largest follicles isolated at predeviation (PD and onset of diameter deviation (OD stages of the first follicular wave was subjected to preliminary RNA transcriptome analysis. Expression of numerous WNT system components was observed. Hence experiments were performed to test the hypothesis that WNT signaling modulates FSH action on granulosa cells during follicular waves. Abundance of mRNA for WNT pathway members was evaluated in granulosa cells harvested from follicles at emergence (EM, PD, OD and early dominance (ED stages of the first follicular wave. In F1 follicles, abundance of CTNNB1 and DVL1 mRNAs was higher and AXIN2 mRNA was lower at ED versus EM stages and DVL1 and FZD6 mRNAs were higher and AXIN2 mRNA was lower in F1 versus F2 follicle at the ED stage. Bovine granulosa cells were treated in vitro with increasing doses of the WNT inhibitor IWR-1+/- maximal stimulatory dose of FSH. IWR-1 treatment blocked the FSH-induced increase in granulosa cell numbers and reduced the FSH-induced increase in estradiol. Granulosa cells were also cultured in the presence or absence of FSH +/- IWR-1 and hormonal regulation of mRNA for WNT pathway members and known FSH targets determined. FSH treatment increased CYP19A1, CCND2, CTNNB1, AXIN2 and FZD6 mRNAs and the stimulatory effect on CYP19A1 mRNA was reduced by IWR-1. In contrast, FSH reduced CARTPT mRNA and IWR-1 partially reversed the inhibitory effect of FSH. Results support temporal and hormonal regulation and a potential role for WNT signaling in potentiating FSH action during dominant follicle selection.

Integrated Analysis of Long Noncoding RNA and mRNA Expression Profile in Advanced Laryngeal Squamous Cell Carcinoma.

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Ling Feng

Full Text Available Long non-coding RNA (lncRNA plays an important role in tumorigenesis. However, the expression pattern and function of lncRNAs in laryngeal squamous cell carcinoma (LSCC are still unclear. To investigate the aberrantly expressed lncRNAs and mRNAs in advanced LSCC, we screened lncRNA and mRNA expression profiles in 9 pairs of primary Stage IVA LSCC tissues and adjacent non-neoplastic tissues by lncRNA and mRNA integrated microarrays. Gene Ontology and pathway analysis were performed to find out the significant function and pathway of the differentially expressed mRNAs, gene-gene functional interaction network and ceRNA network were constructed to select core mRNAs, and lncRNA-mRNA expression correlation network was built to identify the interactions between lncRNA and mRNA. qRT-PCR was performed to further validate the expressions of selected lncRNAs and mRNAs in advanced LSCC. We found 1459 differentially expressed lncRNAs and 2381 differentially expressed mRNAs, including 846 up-regulated lncRNAs and 613 down-regulated lncRNAs, 1542 up-regulated mRNAs and 839 down-regulated mRNAs. The mRNAs ITGB1, HIF1A, and DDIT4 were selected as core mRNAs, which are mainly involved in biological processes, such as matrix organization, cell cycle, adhesion, and metabolic pathway. LncRNA-mRNA expression correlation network showed LncRNA NR_027340, MIR31HG were positively correlated with ITGB1, HIF1A respectively. LncRNA SOX2-OT was negatively correlated with DDIT4. qRT-PCR further validated the expression of these lncRNAs and mRNAs. The work provides convincing evidence that the identified lncRNAs and mRNAs are potential biomarkers in advanced LSCC for further future studies.

Human muscle fibre type-specific regulation of AMPK and downstream targets by exercise

DEFF Research Database (Denmark)

Kristensen, Dorte Enggaard; Albers, Peter Hjorth; Prats, Clara

2015-01-01

are expressed in a fibre type-dependent manner and that fibre type-specific activation of AMPK and downstream targets is dependent on exercise intensity. Pools of type I and II fibres were prepared from biopsies of m. vastus lateralis from healthy men before and after two exercise trials; A) continuous cycling......AMP-activated protein kinase (AMPK) is a regulator of energy homeostasis during exercise. Studies suggest muscle fibre type-specific AMPK expression. However, fibre type-specific regulation of AMPK and downstream targets during exercise has not been proven. We hypothesized that AMPK subunits...... (CON) 30 min at 69 ± 1% VO2peak or B) interval cycling (INT) 30 min with 6 × 1.5 min high-intense bouts peaking at 95 ± 2% VO2peak . In type I vs. II fibres a higher β1 AMPK (+215%) and lower γ3 AMPK expression (-71%) was found. α1 , α2 , β2 and γ1 AMPK expression was similar between fibre types...

Identification of microRNAs regulating the developmental pathways of bone marrow derived mast cells.

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Yang Xiang

Full Text Available MicroRNAs (miRNAs play important roles in leukocyte differentiation, although those utilised for specific programs and key functions remain incompletely characterised. As a global approach to gain insights into the potential regulatory role of miRNA in mast cell differentiation we characterised expression in BM cultures from the initiation of differentiation. In cultures enriched in differentiating mast cells we characterised miRNA expression and identified miRNA targeting the mRNA of putative factors involved in differentiation pathways and cellular identity. Detailed pathway analysis identified a unique miRNA network that is intimately linked to the mast cell differentiation program.We identified 86 unique miRNAs with expression patterns that were up- or down- regulated at 5-fold or more during bone marrow derived mast cells (BMMC development. By employing TargetScan and MeSH databases, we identified 524 transcripts involved in 30 canonical pathways as potentially regulated by these specific 86 miRNAs. Furthermore, by applying miRanda and IPA analyses, we predict that 7 specific miRNAs of this group are directly associated with the expression of c-Kit and FcεRIα and likewise, that 18 miRNAs promote expression of Mitf, GATA1 and c/EBPα three core transcription factors that direct mast cell differentiation. Furthermore, we have identified 11 miRNAs that may regulate the expression of STATs-3, -5a/b, GATA2 and GATA3 during differentiation, along with 13 miRNAs that target transcripts encoding Ndst2, mMCP4 and mMCP6 and thus may regulate biosynthesis of mast cell secretory mediators.This investigation characterises changes in miRNA expression in whole BM cultures during the differentiation of mast cells and predicts functional links between miRNAs and their target mRNAs for the regulation of development. This information provides an important resource for further investigations of the contributions of miRNAs to mast cell differentiation and

A FYVE zinc finger domain protein specifically links mRNA transport to endosome trafficking

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Pohlmann, Thomas; Baumann, Sebastian; Haag, Carl; Albrecht, Mario; Feldbrügge, Michael

2015-01-01

An emerging theme in cellular logistics is the close connection between mRNA and membrane trafficking. A prominent example is the microtubule-dependent transport of mRNAs and associated ribosomes on endosomes. This coordinated process is crucial for correct septin filamentation and efficient growth of polarised cells, such as fungal hyphae. Despite detailed knowledge on the key RNA-binding protein and the molecular motors involved, it is unclear how mRNAs are connected to membranes during transport. Here, we identify a novel factor containing a FYVE zinc finger domain for interaction with endosomal lipids and a new PAM2-like domain required for interaction with the MLLE domain of the key RNA-binding protein. Consistently, loss of this FYVE domain protein leads to specific defects in mRNA, ribosome, and septin transport without affecting general functions of endosomes or their movement. Hence, this is the first endosomal component specific for mRNP trafficking uncovering a new mechanism to couple mRNPs to endosomes. DOI: http://dx.doi.org/10.7554/eLife.06041.001 PMID:25985087

An approach to analyse the specific impact of rapamycin on mRNA-ribosome association

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Jaquier-Gubler Pascale

2008-08-01

Full Text Available Abstract Background Recent work, using both cell culture model systems and tumour derived cell lines, suggests that the differential recruitment into polysomes of mRNA populations may be sufficient to initiate and maintain tumour formation. Consequently, a major effort is underway to use high density microarray profiles to establish molecular fingerprints for cells exposed to defined drug regimes. The aim of these pharmacogenomic approaches is to provide new information on how drugs can impact on the translational read-out within a defined cellular background. Methods We describe an approach that permits the analysis of de-novo mRNA-ribosome association in-vivo during short drug exposures. It combines hypertonic shock, polysome fractionation and high-throughput analysis to provide a molecular phenotype of translationally responsive transcripts. Compared to previous translational profiling studies, the procedure offers increased specificity due to the elimination of the drugs secondary effects (e.g. on the transcriptional read-out. For this pilot "proof-of-principle" assay we selected the drug rapamycin because of its extensively studied impact on translation initiation. Results High throughput analysis on both the light and heavy polysomal fractions has identified mRNAs whose re-recruitment onto free ribosomes responded to short exposure to the drug rapamycin. The results of the microarray have been confirmed using real-time RT-PCR. The selective down-regulation of TOP transcripts is also consistent with previous translational profiling studies using this drug. Conclusion The technical advance outlined in this manuscript offers the possibility of new insights into mRNA features that impact on translation initiation and provides a molecular fingerprint for transcript-ribosome association in any cell type and in the presence of a range of drugs of interest. Such molecular phenotypes defined pre-clinically may ultimately impact on the evaluation of

[Regulation of heat shock gene expression in response to stress].

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Garbuz, D G

2017-01-01

Heat shock (HS) genes, or stress genes, code for a number of proteins that collectively form the most ancient and universal stress defense system. The system determines the cell capability of adaptation to various adverse factors and performs a variety of auxiliary functions in normal physiological conditions. Common stress factors, such as higher temperatures, hypoxia, heavy metals, and others, suppress transcription and translation for the majority of genes, while HS genes are upregulated. Transcription of HS genes is controlled by transcription factors of the HS factor (HSF) family. Certain HSFs are activated on exposure to higher temperatures or other adverse factors to ensure stress-induced HS gene expression, while other HSFs are specifically activated at particular developmental stages. The regulation of the main mammalian stress-inducible factor HSF1 and Drosophila melanogaster HSF includes many components, such as a variety of early warning signals indicative of abnormal cell activity (e.g., increases in intracellular ceramide, cytosolic calcium ions, or partly denatured proteins); protein kinases, which phosphorylate HSFs at various Ser residues; acetyltransferases; and regulatory proteins, such as SUMO and HSBP1. Transcription factors other than HSFs are also involved in activating HS gene transcription; the set includes D. melanogaster GAF, mammalian Sp1 and NF-Y, and other factors. Transcription of several stress genes coding for molecular chaperones of the glucose-regulated protein (GRP) family is predominantly regulated by another stress-detecting system, which is known as the unfolded protein response (UPR) system and is activated in response to massive protein misfolding in the endoplasmic reticulum and mitochondrial matrix. A translational fine tuning of HS protein expression occurs via changing the phosphorylation status of several proteins involved in translation initiation. In addition, specific signal sequences in the 5'-UTRs of some HS

Identification of the Elusive Mammalian Enzyme Phosphatidylcholine-Specific Phospholipase C

Science.gov (United States)

2015-09-01

Summary of Results. Task 1. To identify mammalian PC- PLC . Based on results published by other groups, we proposed to identify candidate PC- PLC mRNAs by...establishing the role of the elusive mammalian protein, phosphatidycholine- specific phospholipase C (PC- PLC ) in the inflammatory processes involved in...progression of rheumatoid arthritis (RA). Thus, the main scopes of this proposal are: 1. to identify the PC- PLC gene and protein; and 2. to test PC- PLC

G3BP1, G3BP2 and CAPRIN1 are required for translation of interferon stimulated mRNAs and are targeted by a dengue virus non-coding RNA.

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Katell Bidet

2014-07-01

Full Text Available Viral RNA-host protein interactions are critical for replication of flaviviruses, a genus of positive-strand RNA viruses comprising major vector-borne human pathogens including dengue viruses (DENV. We examined three conserved host RNA-binding proteins (RBPs G3BP1, G3BP2 and CAPRIN1 in dengue virus (DENV-2 infection and found them to be novel regulators of the interferon (IFN response against DENV-2. The three RBPs were required for the accumulation of the protein products of several interferon stimulated genes (ISGs, and for efficient translation of PKR and IFITM2 mRNAs. This identifies G3BP1, G3BP2 and CAPRIN1 as novel regulators of the antiviral state. Their antiviral activity was antagonized by the abundant DENV-2 non-coding subgenomic flaviviral RNA (sfRNA, which bound to G3BP1, G3BP2 and CAPRIN1, inhibited their activity and lead to profound inhibition of ISG mRNA translation. This work describes a new and unexpected level of regulation for interferon stimulated gene expression and presents the first mechanism of action for an sfRNA as a molecular sponge of anti-viral effectors in human cells.

Extensive gene-specific translational reprogramming in a model of B cell differentiation and Abl-dependent transformation.

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Jamie G Bates

Full Text Available To what extent might the regulation of translation contribute to differentiation programs, or to the molecular pathogenesis of cancer? Pre-B cells transformed with the viral oncogene v-Abl are suspended in an immortalized, cycling state that mimics leukemias with a BCR-ABL1 translocation, such as Chronic Myelogenous Leukemia (CML and Acute Lymphoblastic Leukemia (ALL. Inhibition of the oncogenic Abl kinase with imatinib reverses transformation, allowing progression to the next stage of B cell development. We employed a genome-wide polysome profiling assay called Gradient Encoding to investigate the extent and potential contribution of translational regulation to transformation and differentiation in v-Abl-transformed pre-B cells. Over half of the significantly translationally regulated genes did not change significantly at the level of mRNA abundance, revealing biology that might have been missed by measuring changes in transcript abundance alone. We found extensive, gene-specific changes in translation affecting genes with known roles in B cell signaling and differentiation, cancerous transformation, and cytoskeletal reorganization potentially affecting adhesion. These results highlight a major role for gene-specific translational regulation in remodeling the gene expression program in differentiation and malignant transformation.

A 3'-untranslated region (3'UTR) induces organ adhesion by regulating miR-199a* functions.

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Lee, Daniel Y; Shatseva, Tatiana; Jeyapalan, Zina; Du, William W; Deng, Zhaoqun; Yang, Burton B

2009-01-01

Mature microRNAs (miRNAs) are single-stranded RNAs of 18-24 nucleotides that repress post-transcriptional gene expression. However, it is unknown whether the functions of mature miRNAs can be regulated. Here we report that expression of versican 3'UTR induces organ adhesion in transgenic mice by modulating miR-199a* activities. The study was initiated by the hypothesis that the non-coding 3'UTR plays a role in the regulation of miRNA function. Transgenic mice expressing a construct harboring the 3'UTR of versican exhibits the adhesion of organs. Computational analysis indicated that a large number of microRNAs could bind to this fragment potentially including miR-199a*. Expression of versican and fibronectin, two targets of miR-199a*, are up-regulated in transgenic mice, suggesting that the 3'UTR binds and modulates miR-199a* activities, freeing mRNAs of versican and fibronectin from being repressed by miR-199a*. Confirmation of the binding was performed by PCR using mature miR-199a* as a primer and the targeting was performed by luciferase assays. Enhanced adhesion by expression of the 3'UTR was confirmed by in vitro assays. Our results demonstrated that upon arrival in cytoplasm, miRNA activities can be modulated locally by the 3'UTR. Our assay may be developed as sophisticated approaches for studying the mutual regulation of miRNAs and mRNAs in vitro and in vivo. We anticipate that expression of the 3'UTR may be an approach in the development of gene therapy.

mRNA localization mechanisms in Trypanosoma cruzi.

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Lysangela R Alves

Full Text Available Asymmetric mRNA localization is a sophisticated tool for regulating and optimizing protein synthesis and maintaining cell polarity. Molecular mechanisms involved in the regulated localization of transcripts are widespread in higher eukaryotes and fungi, but not in protozoa. Trypanosomes are ancient eukaryotes that branched off early in eukaryote evolution. We hypothesized that these organisms would have basic mechanisms of mRNA localization. FISH assays with probes against transcripts coding for proteins with restricted distributions showed a discrete localization of the mRNAs in the cytoplasm. Moreover, cruzipain mRNA was found inside reservosomes suggesting new unexpected functions for this vacuolar organelle. Individual mRNAs were also mobilized to RNA granules in response to nutritional stress. The cytoplasmic distribution of these transcripts changed with cell differentiation, suggesting that localization mechanisms might be involved in the regulation of stage-specific protein expression. Transfection assays with reporter genes showed that, as in higher eukaryotes, 3'UTRs were responsible for guiding mRNAs to their final location. Our results strongly suggest that Trypanosoma cruzi have a core, basic mechanism of mRNA localization. This kind of controlled mRNA transport is ancient, dating back to early eukaryote evolution.

Translational Capacity of a Cell Is Determined during Transcription Elongation via the Ccr4-Not Complex

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Ishaan Gupta

2016-05-01

Full Text Available The current understanding of gene expression considers transcription and translation to be independent processes. Challenging this notion, we found that translation efficiency is determined during transcription elongation through the imprinting of mRNAs with Not1, the central scaffold of the Ccr4-Not complex. We determined that another subunit of the complex, Not5, defines Not1 binding to specific mRNAs, particularly those produced from ribosomal protein genes. This imprinting mechanism specifically regulates ribosomal protein gene expression, which in turn determines the translational capacity of cells. We validate our model by SILAC and polysome profiling experiments. As a proof of concept, we demonstrate that enhanced translation compensates for transcriptional elongation stress. Taken together, our data indicate that in addition to defining mRNA stability, components of the Ccr4-Not imprinting complex regulate RNA translatability, thus ensuring global gene expression homeostasis.

Patient-specific three-dimensional printing for pre-surgical planning in hepatocellular carcinoma treatment.

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Perica, Elizabeth; Sun, Zhonghua

2017-12-01

Recently, three-dimensional (3D) printing has shown great interest in medicine, and 3D printed models may be rendered as part of the pre-surgical planning process in order to better understand the complexities of an individual's anatomy. The aim of this study is to investigate the feasibility of utilising 3D printed liver models as clinical tools in pre-operative planning for resectable hepatocellular carcinoma (HCC) lesions. High-resolution contrast-enhanced computed tomography (CT) images were acquired and utilized to generate a patient-specific 3D printed liver model. Hepatic structures were segmented and edited to produce a printable model delineating intrahepatic anatomy and a resectable HCC lesion. Quantitative assessment of 3D model accuracy compared measurements of critical anatomical landmarks acquired from the original CT images, standard tessellation language (STL) files, and the 3D printed liver model. Comparative analysis of surveys completed by two radiologists investigated the clinical value of 3D printed liver models in radiology. The application of utilizing 3D printed liver models as tools in surgical planning for resectable HCC lesions was evaluated through kappa analysis of questionnaires completed by two abdominal surgeons. A scaled down multi-material 3D liver model delineating patient-specific hepatic anatomy and pathology was produced, requiring a total production time of 25.25 hours and costing a total of AUD $1,250. A discrepancy was found in the total mean of measurements at each stage of production, with a total mean of 18.28±9.31 mm for measurements acquired from the original CT data, 15.63±8.06 mm for the STL files, and 14.47±7.71 mm for the 3D printed liver model. The 3D liver model did not enhance the radiologists' perception of patient-specific anatomy or pathology. Kappa analysis of the surgeon's responses to survey questions yielded a percentage agreement of 80%, and a κ value of 0.38 (P=0.24) indicating fair agreement. Study

Analysis of transcription factor mRNAs in identified oxytocin and vasopressin magnocellular neurons isolated by laser capture microdissection.

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Madison Humerick

Full Text Available The oxytocin (Oxt and vasopressin (Avp magnocellular neurons (MCNs in the hypothalamus are the only neuronal phenotypes that are present in the supraoptic nucleus (SON, and are characterized by their robust and selective expression of either the Oxt or Avp genes. In this paper, we take advantage of the differential expression of these neuropeptide genes to identify and isolate these two individual phenotypes from the rat SON by laser capture microdissection (LCM, and to analyze the differential expression of several of their transcription factor mRNAs by qRT-PCR. We identify these neuronal phenotypes by stereotaxically injecting recombinant Adeno-Associated Viral (rAAV vectors which contain cell-type specific Oxt or Avp promoters that drive expression of EGFP selectively in either the Oxt or Avp MCNs into the SON. The fluorescent MCNs are then dissected by LCM using a novel Cap Road Map protocol described in this paper, and the purified MCNs are extracted for their RNAs. qRT-PCR of these RNAs show that some transcription factors (RORA and c-jun are differentially expressed in the Oxt and Avp MCNs.

Holding on and letting go: the perspectives of pre-seniors and seniors on driving self-regulation in later life.

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Rudman, Deborah Laliberte; Friedland, Judith; Chipman, Mary; Sciortino, Paola

2006-01-01

Although decisions related to driving are vital to well-being in later life, little is known about how aging drivers who do not experience a medical condition that requires driving cessation regulate their driving. This exploratory, qualitative study used focus groups with 79 such community-dwelling individuals to examine driving self-regulation from the perspective of pre-senior (aged 55-64) drivers, senior (aged 65 years or over) drivers, and senior ex-drivers. Themes resulting from inductive analysis addressed the importance of driving, mechanisms of self-monitoring and self-regulation, people who influenced decision making, and opinions regarding licensing regulations. A preliminary model of the process of self-regulation that highlights intrapersonal, interpersonal, and environmental influences on why, how, and when aging drivers adapt or cease driving is presented. The model identifies areas for future research to enhance understanding of this process, including the effectiveness of self-regulation. Findings suggest that increased public awareness of issues related to driving and aging could assist aging drivers, their families, and their family physicians in optimizing driving safety for this population. Since a near accident or accident was seen as the only factor that would lead many informants to stop driving and few informants planned for driving cessation, there is a need for interventions that help aging drivers make the transition to ex-driver in a timely and personally acceptable way.

Dietary methanol regulates human gene activity.

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Anastasia V Shindyapina

Full Text Available Methanol (MeOH is considered to be a poison in humans because of the alcohol dehydrogenase (ADH-mediated conversion of MeOH to formaldehyde (FA, which is toxic. Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and a modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC from volunteers after pectin intake showed various responses for 30 significantly differentially regulated mRNAs, most of which were somehow involved in the pathogenesis of Alzheimer's disease (AD. There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes were not significantly expressed. A qRT-PCR analysis of volunteer WBCs after pectin and red wine intake confirmed the complicated relationship between the plasma MeOH content and the mRNA accumulation of both genes that were previously identified, namely, GAPDH and SNX27, and genes revealed in this study, including MME, SORL1, DDIT4, HBA and HBB. We hypothesized that human plasma MeOH has an impact on the WBC mRNA levels of genes involved in cell signaling.

24 CFR 203.370 - Pre-foreclosure sales.

Science.gov (United States)

2010-04-01

... 24 Housing and Urban Development 2 2010-04-01 2010-04-01 false Pre-foreclosure sales. 203.370...-foreclosure sales. (a) General. HUD will pay FHA insurance benefits to mortgagees in cases where, in accordance with all regulations and procedures applicable to pre-foreclosure sales, the mortgaged property is...

Physiological roles of Regulated Ire1 Dependent Decay

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Dina S. Coelho

2014-04-01

Full Text Available Ire1 is an important transducer of the Unfolded Protein Response (UPR that is activated by the accumulation of misfolded proteins in the Endoplamic Reticulum (ER stress. Activated Ire1 mediates the splicing of an intron from the mRNA of Xbp1, causing a frame-shift during translation and introducing a new carboxyl domain in the Xbp1 protein, which only then becomes a fully functional transcription factor. Studies using cell culture systems demonstrated that Ire1 also promotes the degradation of mRNAs encoding mostly ER-targeted proteins, to reduce the load of incoming ER client proteins during ER stress. This process was called RIDD (regulated Ire1-dependent decay, but its physiological significance remained poorly characterized beyond cell culture systems. Here we review several recent studies that have highlighted the physiological roles of RIDD in specific biological paradigms, such as photoreceptor differentiation in Drosophila or mammalian liver and endocrine pancreas function. These studies demonstrate the importance of RIDD in tissues undergoing intense secretory function and highlight the physiologic role of RIDD during UPR activation in cells and organisms.

Challenges of animal models in SCI research: Effects of pre-injury task-specific training in adult rats before lesion.

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May, Zacnicte; Fouad, Karim; Shum-Siu, Alice; Magnuson, David S K

2015-09-15

A rarely explored subject in animal research is the effect of pre-injury variables on behavioral outcome post-SCI. Low reporting of such variables may underlie some discrepancies in findings between laboratories. Particularly, intensive task-specific training before a SCI might be important, considering that sports injuries are one of the leading causes of SCI. Thus, individuals with SCI often underwent rigorous training before their injuries. In the present study, we asked whether training before SCI on a grasping task or a swimming task would influence motor recovery in rats. Swim pre-training impaired recovery of swimming 2 and 4 weeks post-injury. This result fits with the idea of motor learning interference, which posits that learning something new may disrupt learning of a new task; in this case, learning strategies to compensate for functional loss after SCI. In contrast to swimming, grasp pre-training did not influence grasping ability after SCI at any time point. However, grasp pre-trained rats attempted to grasp more times than untrained rats in the first 4 weeks post-injury. Also, lesion volume of grasp pre-trained rats was greater than that of untrained rats, a finding which may be related to stress or activity. The increased participation in rehabilitative training of the pre-trained rats in the early weeks post-injury may have potentiated spontaneous plasticity in the spinal cord and counteracted the deleterious effect of interference and bigger lesions. Thus, our findings suggest that pre-training plays a significant role in recovery after CNS damage and needs to be carefully controlled for. Copyright © 2015 Elsevier B.V. All rights reserved.

The hypoxic proteome is influenced by gene-specific changes in mRNA translation

International Nuclear Information System (INIS)

Koritzinsky, Marianne; Seigneuric, Renaud; Magagnin, Michael G.; Beucken, Twan van den; Lambin, Philippe; Wouters, Bradly G.

2005-01-01

Background and purpose: Hypoxia causes a rapid reduction in mRNA translation efficiency. This inhibition does not affect all mRNA species to the same extent and can therefore contribute significantly to hypoxia-induced differential protein expression. Our aim in this study was to characterize changes in gene expression during acute hypoxia and evaluate the contribution of regulation via mRNA translation on these changes. For each gene, the contribution of changes in mRNA abundance versus mRNA translation was determined. Materials and methods: DU145 prostate carcinoma cells were exposed to 4 h of hypoxia ( 2 ). Efficiently translated mRNAs were isolated by sedimentation through a sucrose gradient. Affymetrix microarray technology was used to evaluate both the transcriptional and translational contribution to gene expression. Results were validated by quantitative PCR. Results: One hundred and twenty genes were more than 4-fold upregulated by hypoxia in the efficiently translated fraction of mRNA, in comparison to only 76 genes at the level of transcription. Of the 50 genes demonstrating the largest changes in translation, 11 were found to be more than 2-fold over represented in the translated fraction in comparison to their overall transcriptional level. The gene with the highest translational contribution to its induction was CITED-2, which is a negative regulator of HIF-1 transcriptional activity. Conclusions: Gene-specific regulation of mRNA translation contributes significantly to differential gene expression during hypoxia

Small kinetochore associated protein (SKAP promotes UV-induced cell apoptosis through negatively regulating pre-mRNA processing factor 19 (Prp19.

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Shan Lu

Full Text Available Apoptosis is a regulated cellular suicide program that is critical for the development and maintenance of healthy tissues. Previous studies have shown that small kinetochore associated protein (SKAP cooperates with kinetochore and mitotic spindle proteins to regulate mitosis. However, the role of SKAP in apoptosis has not been investigated. We have identified a new interaction involving SKAP, and we propose a mechanism through which SKAP regulates cell apoptosis. Our experiments demonstrate that both overexpression and knockdown of SKAP sensitize cells to UV-induced apoptosis. Further study has revealed that SKAP interacts with Pre-mRNA processing Factor 19 (Prp19. We find that UV-induced apoptosis can be inhibited by ectopic expression of Prp19, whereas silencing Prp19 has the opposite effect. Additionally, SKAP negatively regulates the protein levels of Prp19, whereas Prp19 does not alter SKAP expression. Finally, rescue experiments demonstrate that the pro-apoptotic role of SKAP is executed through Prp19. Taken together, these findings suggest that SKAP promotes UV-induced cell apoptosis by negatively regulating the anti-apoptotic protein Prp19.

Tissue-specific down-regulation of RIPK 2 in Mycobacterium leprae-infected nu/nu mice

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Gue-Tae Chae

1992-01-01

Full Text Available RIPK 2 is adapter molecule in the signal pathway involved in Toll-like receptors. However, there has been no reported association between receptor-interacting serine/threonine kinase 2 (RIPK 2 expression and the infectious diseases involving mycobacterial infection. This study found that its expression was down-regulated in the footpads and skin but was up-regulated in the liver of Mycobacterium leprae-infected nu/nu mice compared with those of the M. leprae non-infected nu/nu mice. It was observed that the interlukin-12p40 and interferon-γ genes involved in the susceptibility of M. leprae were down-regulated in the skin but were up-regulated in the liver. Overall, this suggests that regulation of RIPK 2 expression is tissue-specifically associated with M. leprae infection.

An intronic microRNA silences genes that are functionally antagonistic to its host gene.

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Barik, Sailen

2008-09-01

MicroRNAs (miRNAs) are short noncoding RNAs that down-regulate gene expression by silencing specific target mRNAs. While many miRNAs are transcribed from their own genes, nearly half map within introns of 'host' genes, the significance of which remains unclear. We report that transcriptional activation of apoptosis-associated tyrosine kinase (AATK), essential for neuronal differentiation, also generates miR-338 from an AATK gene intron that silences a family of mRNAs whose protein products are negative regulators of neuronal differentiation. We conclude that an intronic miRNA, transcribed together with the host gene mRNA, may serve the interest of its host gene by silencing a cohort of genes that are functionally antagonistic to the host gene itself.

Gene expression in the developing cerebellum during perinatal hypo- and hyperthyroidism.

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Figueiredo, B C; Almazan, G; Ma, Y; Tetzlaff, W; Miller, F D; Cuello, A C

1993-03-01

The intensity of p75NGFR receptor-like immunoreactivity and the mRNAs encoding p75NGFR, T alpha 1 alpha-tubulin, GAP-43 and the myelin proteins MBP and PLP were measured in the developing cerebellum to study the effects of perinatal thyroid hormone imbalance in rats. Results compared to age-matched controls provide in vivo evidence for differential gene regulation by thyroid hormone in the developing cerebellum. We found that p75NGFR immunoreactivity was strikingly elevated in hypothyroid rats, whereas p75NGFR mRNA content remained only twice as high as that of control levels on postnatal day 15 (P15). When p75NGFR immunoreactivity was still elevated in hypothyroid rats, Purkinje cells exhibited proximal axonal varicosities, axonal twisting and differences in axonal caliber. The mRNAs encoding proteins involved with neurite growth-promoting elements, T alpha 1 alpha-tubulin and GAP-43, were also increased in hypothyroidism, possibly reflecting a neuronal response to a deficiency in, or damage to, cerebellar neurons, or a general delay in their down regulation. Similar increases were not observed for the myelin specific genes. MBP and PLP mRNAs were first detected on P2 of hyperthyroid rats, and they increased with age. Hypo- or hyperthyroidism did not affect the initial onset of MBP and PLP expression, however, hyperthyroidism increased levels of PLP and MBP mRNAs between P2 and P10. By contrast, the most consistent decrease in MBP and PLP mRNAs in rats with thyroid hormone deficiency was observed only on P10. At later times (P15 and P30), the two mRNA levels were similar to controls in all groups. These results are consistent with a role for thyroid hormone in the earlier stages of cerebellar myelination. Hypothryoidism led to specific increases in T alpha 1 alpha-tubulin and GAP-43 mRNAs, and in the immunoreactivity and mRNA levels of p75NGFR receptor--all changes that may play a role in the observed abnormal neuronal outgrowth.

MicroRNA-124 inhibits the progression of adjuvant-induced arthritis in rats.

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Nakamachi, Yuji; Ohnuma, Kenichiro; Uto, Kenichi; Noguchi, Yoriko; Saegusa, Jun; Kawano, Seiji

2016-03-01

MicroRNAs (miRNAs) are small endogenous, non-coding RNAs that act as post-transcriptional regulators. We analysed the in vivo effect of miRNA-124 (miR-124, the rat analogue of human miR-124a) on adjuvant-induced arthritis (AIA) in rats. AIA was induced in Lewis rats by injecting incomplete Freund's adjuvant with heat-killed Mycobacterium tuberculosis. Precursor (pre)-miR-124 was injected into the right hind ankle on day 9. Morphological changes in the ankle joint were assessed by micro-CT and histopathology. Cytokine expression was examined by western blotting and real-time RT-PCR. The effect of miR-124 on predicted target messenger RNAs (mRNAs) was examined by luciferase reporter assays. The effect of pre-miR-124 or pre-miR-124a on the differentiation of human osteoclasts was examined by tartrate-resistant acid phosphatase staining. We found that miR-124 suppressed AIA in rats, as demonstrated by decreased synoviocyte proliferation, leucocyte infiltration and cartilage or bone destruction. Osteoclast counts and expression level of receptor activator of the nuclear factor κB ligand (RANKL), integrin β1 (ITGB1) and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) were reduced in AIA rats treated with pre-miR-124. Luciferase analysis showed that miR-124 directly targeted the 3'UTR of the rat NFATc1, ITGB1, specificity protein 1 and CCAAT/enhancer-binding protein α mRNAs. Pre-miR-124 also suppressed NFATc1 expression in RAW264.7 cells. Both miR-124 and miR-124a directly targeted the 3'-UTR of human NFATc1 mRNA, and both pre-miR-124 and pre-miR-124a suppressed the differentiation of human osteoclasts. We found that miR-124 ameliorated AIA by suppressing critical prerequisites for arthritis development, such as RANKL and NFATc1. Thus, miR-124a is a candidate for therapeutic use for human rheumatoid arthritis. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Amplified in Breast Cancer Regulates Transcription and Translation in Breast Cancer Cells.

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Ochnik, Aleksandra M; Peterson, Mark S; Avdulov, Svetlana V; Oh, Annabell S; Bitterman, Peter B; Yee, Douglas

2016-02-01

Control of mRNA translation is fundamentally altered in cancer. Insulin-like growth factor-I (IGF-I) signaling regulates key translation mediators to modulate protein synthesis (e.g. eIF4E, 4E-BP1, mTOR, and S6K1). Importantly the Amplified in Breast Cancer (AIB1) oncogene regulates transcription and is also a downstream mediator of IGF-I signaling. To determine if AIB1 also affects mRNA translation, we conducted gain and loss of AIB1 function experiments in estrogen receptor alpha (ERα)(+) (MCF-7L) and ERα(-) (MDA-MB-231, MDA-MB-435 and LCC6) breast cancer cells. AIB1 positively regulated IGF-I-induced mRNA translation in both ERα(+) and ERα(-) cells. Formation of the eIF4E-4E-BP1 translational complex was altered in the AIB1 ERα(+) and ERα(-) knockdown cells, leading to a reduction in the eIF4E/4E-BP1 and eIF4G/4E-BP1 ratios. In basal and IGF-I stimulated MCF-7 and LCC6 cells, knockdown of AIB1 decreased the integrity of the cap-binding complex, reduced global IGF-I stimulated polyribosomal mRNA recruitment with a concomitant decrease in ten of the thirteen genes tested in polysome-bound mRNAs mapping to proliferation, cell cycle, survival, transcription, translation and ribosome biogenesis ontologies. Specifically, knockdown of AIB1 decreased ribosome-bound mRNA and steady-state protein levels of the transcription factors ERα and E2F1 in addition to reduced ribosome-bound mRNA of the ribosome biogenesis factor BYSL in a cell-line specific manner to regulate mRNA translation. The oncogenic transcription factor AIB1 has a novel role in the regulation of polyribosome recruitment and formation of the translational complex. Combinatorial therapies targeting IGF signaling and mRNA translation in AIB1 expressing breast cancers may have clinical benefit and warrants further investigation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

JPP: A Java Pre-Processor

OpenAIRE

Kiniry, Joseph R.; Cheong, Elaine

1998-01-01

The Java Pre-Processor, or JPP for short, is a parsing pre-processor for the Java programming language. Unlike its namesake (the C/C++ Pre-Processor, cpp), JPP provides functionality above and beyond simple textual substitution. JPP's capabilities include code beautification, code standard conformance checking, class and interface specification and testing, and documentation generation.

Select Skeletal Muscle mRNAs Related to Exercise Adaptation Are Minimally Affected by Different Pre-exercise Meals that Differ in Macronutrient Profile

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Pim Knuiman

2018-01-01

Full Text Available Background: Substantial research has been done on the impact of carbohydrate and fat availability on endurance exercise adaptation, though its role in the acute adaptive response to resistance exercise has yet to be fully characterized.Purpose: We aimed to assess the effects of a pre-resistance exercise isocaloric mixed meal containing different amounts of carbohydrates and fat, on post-resistance exercise gene expression associated with muscle adaptation.Methods: Thirteen young (age 21.2 ± 1.6 year, recreationally trained (VO2max 51.3 ± 4.8 ml/kg/min men undertook an aerobic exercise session of 90-min continuous cycling (70% VO2max in the morning with pre- and post-exercise protein ingestion (10 and 15 g casein in a 500 ml beverage pre- and post-exercise, respectively. Subjects then rested for 2 h and were provided with a meal consisting of either 3207 kJ; 52 g protein; 51 g fat; and 23 g carbohydrate (FAT or 3124 kJ; 53 g protein; 9 g fat; and 109 g carbohydrate (CHO. Two hours after the meal, subjects completed 5 × 8 repetitions (80% 1-RM for both bilateral leg press and leg extension directly followed by 25 g of whey protein (500 ml beverage. Muscle biopsies were obtained from the vastus lateralis at baseline (morning and 1 and 3 h post-resistance exercise (afternoon to determine intramuscular mRNA response.Results: Muscle glycogen levels were significantly decreased post-resistance exercise, without any differences between conditions. Plasma free fatty acids increased significantly after the mixed meal in the FAT condition, while glucose and insulin were higher in the CHO condition. However, PDK4 mRNA quantity was significantly higher in the FAT condition at 3 h post-resistance exercise compared to CHO. HBEGF, INSIG1, MAFbx, MURF1, SIRT1, and myostatin responded solely as a result of exercise without any differences between the CHO and FAT group. FOXO3A, IGF-1, PGC-1α, and VCP expression levels remained unchanged over the course of the

PHF6 regulates phenotypic plasticity through chromatin organization within lineage-specific genes.

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Soto-Feliciano, Yadira M; Bartlebaugh, Jordan M E; Liu, Yunpeng; Sánchez-Rivera, Francisco J; Bhutkar, Arjun; Weintraub, Abraham S; Buenrostro, Jason D; Cheng, Christine S; Regev, Aviv; Jacks, Tyler E; Young, Richard A; Hemann, Michael T

2017-05-15

Developmental and lineage plasticity have been observed in numerous malignancies and have been correlated with tumor progression and drug resistance. However, little is known about the molecular mechanisms that enable such plasticity to occur. Here, we describe the function of the plant homeodomain finger protein 6 (PHF6) in leukemia and define its role in regulating chromatin accessibility to lineage-specific transcription factors. We show that loss of Phf6 in B-cell leukemia results in systematic changes in gene expression via alteration of the chromatin landscape at the transcriptional start sites of B-cell- and T-cell-specific factors. Additionally, Phf6 KO cells show significant down-regulation of genes involved in the development and function of normal B cells, show up-regulation of genes involved in T-cell signaling, and give rise to mixed-lineage lymphoma in vivo. Engagement of divergent transcriptional programs results in phenotypic plasticity that leads to altered disease presentation in vivo, tolerance of aberrant oncogenic signaling, and differential sensitivity to frontline and targeted therapies. These findings suggest that active maintenance of a precise chromatin landscape is essential for sustaining proper leukemia cell identity and that loss of a single factor (PHF6) can cause focal changes in chromatin accessibility and nucleosome positioning that render cells susceptible to lineage transition. © 2017 Soto-Feliciano et al.; Published by Cold Spring Harbor Laboratory Press.

Regulating expressin of cell and tissue-specific genes by modifying transcription

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Beachy, Roger N. [Donald Danforth Plant Science Center, St. Louis, MO (United States); Dai, Shunhong [Donald Danforth Plant Science Center, St. Louis, MO (United States)

2009-12-15

Transcriptional regulation is the primary step to control gene expression, therefore function. Such regulation is achieved primarily via a combination of the activities of the promoter cis regulatory DNA elements and trans regulatory proteins that function through binding to these DNA elements. Our research supported by this program has led to the identification of rice bZIP transcription factors RF2a, RF2b and RLP1 that play key roles in regulating the activity of a vascular tissue specific promoter isolated from Rice Tungro Bacilliform Virus (RTBV) through their interactions with the Box II essential cis element located in the promoter. RF2a, RF2b and RLP1 possess multiple regulatory domains. Functional characterization reveals that those domains can activate or repress the activity of the RTBV promoter. Studies of transcriptional regulation of the RTBV promoter by this group of bZIP proteins not only provide insights about gene expression in the vascular tissue, but also insights about general mechanisms of transcription activation and repression. The knowledge gained from this research will also enable us to develop a well-described set of tools that can be used to control expression of multiple genes in transgenic plants and to improve biofuel feedstock.

An Elongin-Cullin-SOCS Box Complex Regulates Stress-Induced Serotonergic Neuromodulation

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Xicotencatl Gracida

2017-12-01

Full Text Available Neuromodulatory cells transduce environmental information into long-lasting behavioral responses. However, the mechanisms governing how neuronal cells influence behavioral plasticity are difficult to characterize. Here, we adapted the translating ribosome affinity purification (TRAP approach in C. elegans to profile ribosome-associated mRNAs from three major tissues and the neuromodulatory dopaminergic and serotonergic cells. We identified elc-2, an Elongin C ortholog, specifically expressed in stress-sensing amphid neuron dual ciliated sensory ending (ADF serotonergic sensory neurons, and we found that it plays a role in mediating a long-lasting change in serotonin-dependent feeding behavior induced by heat stress. We demonstrate that ELC-2 and the von Hippel-Lindau protein VHL-1, components of an Elongin-Cullin-SOCS box (ECS E3 ubiquitin ligase, modulate this behavior after experiencing stress. Also, heat stress induces a transient redistribution of ELC-2, becoming more nuclearly enriched. Together, our results demonstrate dynamic regulation of an E3 ligase and a role for an ECS complex in neuromodulation and control of lasting behavioral states.

Human hnRNP Q re-localizes to cytoplasmic granules upon PMA, thapsigargin, arsenite and heat-shock treatments

International Nuclear Information System (INIS)

Quaresma, Alexandre J.C.; Bressan, G.C.; Gava, L.M.; Lanza, D.C.F.; Ramos, C.H.I; Kobarg, Joerg

2009-01-01

Eukaryotic gene expression is regulated on different levels ranging from pre-mRNA processing to translation. One of the most characterized families of RNA-binding proteins is the group of hnRNPs: heterogenous nuclear ribonucleoproteins. Members of this protein family play important roles in gene expression control and mRNAs metabolism. In the cytoplasm, several hnRNPs proteins are involved in RNA-related processes and they can be frequently found in two specialized structures, known as GW-bodies (GWbs), previously known as processing bodies: PBs, and stress granules, which may be formed in response to specific stimuli. GWbs have been early reported to be involved in the mRNA decay process, acting as a site of mRNA degradation. In a similar way, stress granules (SGs) have been described as cytoplasmic aggregates, which contain accumulated mRNAs in cells under stress conditions and present reduced or inhibited translation. Here, we characterized the hnRNP Q localization after different stress conditions. hnRNP Q is a predominantly nuclear protein that exhibits a modular organization and several RNA-related functions. Our data suggest that the nuclear localization of hnRNP Q might be modified after different treatments, such as: PMA, thapsigargin, arsenite and heat shock. Under different stress conditions, hnRNP Q can fully co-localize with the endoplasmatic reticulum specific chaperone, BiP. However, under stress, this protein only co-localizes partially with the proteins: GW182 - GWbs marker protein and TIA-1 stress granule component

The translational repressor Crc controls the Pseudomonas putida benzoate and alkane catabolic pathways using a multi-tier regulation strategy.

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Hernández-Arranz, Sofía; Moreno, Renata; Rojo, Fernando

2013-01-01

Metabolically versatile bacteria usually perceive aromatic compounds and hydrocarbons as non-preferred carbon sources, and their assimilation is inhibited if more preferable substrates are available. This is achieved via catabolite repression. In Pseudomonas putida, the expression of the genes allowing the assimilation of benzoate and n-alkanes is strongly inhibited by catabolite repression, a process controlled by the translational repressor Crc. Crc binds to and inhibits the translation of benR and alkS mRNAs, which encode the transcriptional activators that induce the expression of the benzoate and alkane degradation genes respectively. However, sequences similar to those recognized by Crc in benR and alkS mRNAs exist as well in the translation initiation regions of the mRNA of several structural genes of the benzoate and alkane pathways, which suggests that Crc may also regulate their translation. The present results show that some of these sites are functional, and that Crc inhibits the induction of both pathways by limiting not only the translation of their transcriptional activators, but also that of genes coding for the first enzyme in each pathway. Crc may also inhibit the translation of a gene involved in benzoate uptake. This multi-tier approach probably ensures the rapid regulation of pathway genes, minimizing the assimilation of non-preferred substrates when better options are available. A survey of possible Crc sites in the mRNAs of genes associated with other catabolic pathways suggested that targeting substrate uptake, pathway induction and/or pathway enzymes may be a common strategy to control the assimilation of non-preferred compounds. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

Arabidopsis phosphoinositide-specific phospholipase C 4 negatively regulates seedling salt tolerance.

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Xia, Keke; Wang, Bo; Zhang, Jiewei; Li, Yuan; Yang, Hailian; Ren, Dongtao

2017-08-01

Previous physiological and pharmacological studies have suggested that the activity of phosphoinositide-specific phospholipase C (PI-PLC) plays an important role in regulating plant salt stress responses by altering the intracellular Ca 2+ concentration. However, the individual members of plant PLCs involved in this process need to be identified. Here, the function of AtPLC4 in the salt stress response of Arabidopsis seedlings was analysed. plc4 mutant seedlings showed hyposensitivity to salt stress compared with Col-0 wild-type seedlings, and the salt hyposensitive phenotype could be complemented by the expression of native promoter-controlled AtPLC4. Transgenic seedlings with AtPLC4 overexpression (AtPLC4 OE) exhibited a salt-hypersensitive phenotype, while transgenic seedlings with its inactive mutant expression (AtPLC4m OE) did not exhibit this phenotype. Using aequorin as a Ca 2+ indicator in plc4 mutant and AtPLC4 OE seedlings, AtPLC4 was shown to positively regulate the salt-induced Ca 2+ increase. The salt-hypersensitive phenotype of AtPLC4 OE seedlings was partially rescued by EGTA. An analysis of salt-responsive genes revealed that the transcription of RD29B, MYB15 and ZAT10 was inversely regulated in plc4 mutant and AtPLC4 OE seedlings. Our findings suggest that AtPLC4 negatively regulates the salt tolerance of Arabidopsis seedlings, and Ca 2+ may be involved in regulating this process. © 2017 John Wiley & Sons Ltd.

Regulation and specificity of antifungal metapleural gland secretion in leaf-cutting ants

DEFF Research Database (Denmark)

Yek, Sze Huei; Nash, David Richard; Jensen, Annette Bruun

2012-01-01

significantly larger for ants challenged with virulent and mild pathogens/weeds than for controls and Escovopsis-challenged ants. We conclude that the MG defence system of leaf-cutting ants has characteristics reminiscent of an additional cuticular immune system, with specific and non-specific components......Ants have paired metapleural glands (MGs) to produce secretions for prophylactic hygiene. These exocrine glands are particularly well developed in leaf-cutting ants, but whether the ants can actively regulate MG secretion is unknown. In a set of controlled experiments using conidia of five fungi...

Specific DNA-binding proteins and DNA sequences involved in steroid hormone regulation of gene expression

International Nuclear Information System (INIS)

Spelsberg, T.; Hora, J.; Horton, M.; Goldberger, A.; Littlefield, B.; Seelke, R.; Toyoda, H.

1987-01-01

Steroid hormones circulate in the blood and are taken by target cells via complexes with intracellular binding proteins termed receptors, that are hormone and tissue specific. Each receptor binds it specific steroid with very high affinity, having an equilibrium dissociation constant (K/sub d/) in the range of 10 -9 to 10 -10 M. Once bound by their specific steroid hormones, the steroid receptors undergo a conformational change which allows them to bind with high affinity to sites on chromatin, termed nuclear acceptor sites. There are estimated 5,000 to 10,000 of these sites expressed with an equal number not expressed (''masked'') in intact chromatin. The result of the binding to nuclear acceptor sites is an alteration of gene transcription or, in some cases, gene expression as measured by the changing levels of specific RNAs and proteins in that target tissue. Each steroid regulates specific effects on the RNA and protein profiles. The chronology of the above mechanism of action after injection of radiolabelled steroid as is follows: Steroid-receptor complex formation (1 minute), nuclear acceptor sites (2 minutes), effects on RNA synthesis (10 to 30 minutes), and finally the changing protein profiles via changes in protein synthesis and protein turnover (1 to 6 hours). Thus steroid receptors represent one of the first identified intracellular gene regulation proteins. The receptor molecules themselves are regulated by the presence or absence of the steroid molecule

Selecting "saviour siblings": reconsidering the regulation in Australia of pre-implantation genetic diagnosis in conjunction with tissue typing.

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Taylor-Sands, Michelle

2007-05-01

In recent years, pre-implantation genetic diagnosis (PGD) has been developed to enable the selection of a tissue type matched "saviour sibling" for a sick child. This article examines the current regulatory framework governing PGD in Australia. The availability of PGD in Australia to create a saviour sibling depends on the regulation of ART services by each State and Territory. The limitations on the use of PGD vary throughout Australia, according to the level of regulation of ART in each jurisdiction. This article considers the limitations on the use of PGD for tissue typing in Australia and argues that some of these should be removed for a more consistent national approach. In particular, the focus in ART legislation on the "paramount interests" of the child to be born is inappropriate for the application of tissue typing, which necessarily involves the interests of other family members.

Identification of Wnt Pathway Target Genes Regulating the Division and Differentiation of Larval Seam Cells and Vulval Precursor Cells in Caenorhabditis elegans.

Science.gov (United States)

Gorrepati, Lakshmi; Krause, Michael W; Chen, Weiping; Brodigan, Thomas M; Correa-Mendez, Margarita; Eisenmann, David M

2015-06-05

The evolutionarily conserved Wnt/β-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector β-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/β-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type-specific "mRNA tagging" to enrich for VPC and seam cell-specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type-specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells. Copyright © 2015 Gorrepati et al.

Fat-specific protein 27 regulates storage of triacylglycerol

DEFF Research Database (Denmark)

Keller, P.; Petrie, J.T.; Rose, P. De

2008-01-01

FSP27 (fat-specific protein 27) is a member of the cell death-inducing DNA fragmentation factor-alpha-like effector (CIDE) family. Although Cidea and Cideb were initially characterized as activators of apoptosis, recent studies have demonstrated important metabolic roles for these proteins...... in several cell types without induction of adipocyte genes. Increased triacylglycerol is likely due to decreased beta-oxidation of nonesterified fatty acids. Altered flux of fatty acids into triacylglycerol may be a direct effect of FSP27 function, which is localized to lipid droplets in 293T cells and 3T3-L...... decreases with total fat mass but is not associated with measures of insulin resistance (e.g. homeostasis model assessment). Together, these data indicate that FSP27 binds to lipid droplets and regulates their enlargement Udgivelsesdato: 2008/5/23...

Improvement of the design and generation of highly specific plant knockdown lines using primary synthetic microRNAs (pri-smiRNAs

Directory of Open Access Journals (Sweden)

Alves Leonardo

2010-03-01

Full Text Available Abstract Background microRNAs (miRNAs are endogenous small non-coding RNAs that post-transcriptionally regulate gene expression. In plants, they typically show high complementarity to a single sequence motif within their target mRNAs and act by catalyzing specific mRNA cleavage and degradation. miRNAs are processed from much longer primary transcripts via precursor miRNAs containing fold-back structures. Leaving these secondary structures intact, miRNAs can be re-designed experimentally to target mRNAs of choice. Results We designed primary synthetic miRNAs (pri-smiRNAs on the basis of the primary transcript of the Arabidopsis MIR159A gene by replacing the original miR159a and the corresponding miR159a* with novel sequences, keeping the overall secondary structure as predicted by the program RNAfold. We used the program RNAhybrid to optimize smiRNA design and to screen the complete Arabidopsis transcriptome for potential off-targets. To improve the molecular cloning of the pri-smiRNA we inserted restriction sites in the original MIR159A primary transcript to easily accommodate the smiRNA/smiRNA* DNA fragment. As a proof-of-concept, we targeted the single gene encoding chalcone synthase (CHS in Arabidopsis. We demonstrate smiRNA(CHS expression and CHS mRNA cleavage in different transgenic lines. Phenotypic changes in these lines were observed for seed color and flavonol derivatives, and quantified with respect to anthocyanin content. We also tested the effect of mismatches and excess G:U base pairs on knockdown efficiency. Conclusions RNAhybrid-assisted design of smiRNAs and generation of pri-smiRNAs using a novel vector containing restriction sites greatly improves specificity and speed of the generation of stable knockdown lines for functional analyses in plants.

Overview of OVATE FAMILY PROTEINS, a novel class of plant-specific growth regulators

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Shucai eWang

2016-03-01

Full Text Available OVATE FAMILY PROTEINS (OFPs are a class of proteins with a conserved OVATE domain. OVATE protein was first identified in tomato as a key regulator of fruit shape. OFPs are plant-specific proteins that are widely distributed in the plant kingdom including mosses and lycophytes. Transcriptional activity analysis of Arabidopsis OFPs (AtOFPs in protoplasts suggests that they act as transcription repressors. Functional characterization of OFPs from different plant species including Arabidopsis, rice, tomato, pepper and banana suggests that OFPs regulate multiple aspects of plant growth and development, which is likely achieved by interacting with different types of transcription factors including the KNOX and BELL classes, and/or directly regulating the expression of target genes such as Gibberellin 20 oxidase (GA20ox. Here, we examine how OVATE was originally identified, summarize recent progress in elucidation of the roles of OFPs in regulating plant growth and development, and describe possible mechanisms underpinning this regulation. Finally, we review potential new research directions that could shed additional light on the functional biology of OFPs in plants.

LIN28 expression in malignant germ cell tumors down-regulates let-7 and increases oncogene levels

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Murray, Matthew J.; Saini, Harpreet K.; Siegler, Charlotte A.; Hanning, Jennifer E.; Barker, Emily M.; van Dongen, Stijn; Ward, Dawn M.; Raby, Katie L.; Groves, Ian J.; Scarpini, Cinzia G.; Pett, Mark R.; Thornton, Claire M.; Enright, Anton J.; Nicholson, James C.; Coleman, Nicholas

2013-01-01

Despite their clinico-pathologic heterogeneity, malignant germ-cell-tumors (GCTs) share molecular abnormalities that are likely to be functionally important. In this study, we investigated the potential significance of down-regulation of the let-7 family of tumor-suppressor microRNAs in malignant-GCTs. Microarray results from pediatric and adult samples (n=45) showed that LIN28, the negative-regulator of let-7 biogenesis, was abundant in malignant-GCTs, regardless of patient age, tumor site or histologic subtype. Indeed, a strong negative-correlation existed between LIN28 and let-7 levels in specimens with matched datasets. Low let-7 levels were biologically significant, since the sequence complementary to the 2-7nt common let-7 seed ‘GAGGUA’ was enriched in the 3′untranslated regions of mRNAs up-regulated in pediatric and adult malignant-GCTs, compared with normal gonads (a mixture of germ cells and somatic cells). We identified 27 mRNA targets of let-7 that were up-regulated in malignant-GCT cells, confirming significant negative-correlations with let-7 levels. Among 16 mRNAs examined in a largely independent set of specimens by qRT-PCR, we defined negative-associations with let-7e levels for six oncogenes, including MYCN, AURKB, CCNF, RRM2, MKI67 and C12orf5 (when including normal control tissues). Importantly, LIN28 depletion in malignant-GCT cells restored let-7 levels and repressed all of these oncogenic let-7 mRNA targets, with LIN28 levels correlating with cell proliferation and MYCN levels. Conversely, ectopic expression of let-7e was sufficient to reduce proliferation and down-regulate MYCN, AURKB and LIN28, the latter via a double-negative feedback loop. We concluded that the LIN28/let-7 pathway has a critical pathobiological role in malignant-GCTs and therefore offers a promising target for therapeutic intervention. PMID:23774216

Tissue-specific expression of insulin-like growth factor II mRNAs with distinct 5' untranslated regions

International Nuclear Information System (INIS)

Irminger, J.C.; Rosen, K.M.; Humble, R.E.; Villa-Komaroff, L.

1987-01-01

The authors have used RNA from human hypothalamus as template for the production of cDNAs encoding insulin-like growth factor II (IGF-II). The prohormone coding sequence of brain IGF-II RNA is identical to that found in liver; however, the 5' untranslated sequence of the brain cDNA has no homology to the 5' untranslated sequence of the previously reported liver cDNAs. By using hybridization to specific probes as well as a method based on the properties of RNase H, they found that the human IGF-II gene has at least three exons that encode alternative 5' untranslated regions and that are expressed in a tissue-specific manner. A probe specific to the brain cDNA 5' untranslated region hybridizes to a 6.0-kilobase transcript present in placenta, hypothalamus, adrenal gland, kidney, Wilms tumor, and a pheochromocytoma. The 5' untranslated sequence of the brain cDNA does not hybridize to a 5.3-kilobase transcript found in liver or to a 5.0-kb transcript found in pheochromocytoma. By using RNase H to specifically fragment the IGF-II transcripts into 3' and 5' fragments, they found that the RNAs vary in size due to differences in the 5' end but not the 3' end

50 CFR 32.3 - What are the procedures for publication of refuge-specific hunting regulations?

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2010-10-01

... and publication of the opening of a wildlife refuge area to migratory game bird, upland game or big game hunting. (b) Refuge-specific hunting regulations may contain the following items: (1) Wildlife... FISHING General Provisions § 32.3 What are the procedures for publication of refuge-specific hunting...

Drosophila Nanos acts as a molecular clamp that modulates the RNA-binding and repression activities of Pumilio

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Weidmann, Chase A.; Qiu, Chen; Arvola, René M.; Lou, Tzu-Fang; Killingsworth, Jordan; Campbell, Zachary T.; Tanaka Hall, Traci M.; Goldstrohm, Aaron C.

2016-08-02

Collaboration among the multitude of RNA-binding proteins (RBPs) is ubiquitous, yet our understanding of these key regulatory complexes has been limited to single RBPs. We investigated combinatorial translational regulation byDrosophilaPumilio (Pum) and Nanos (Nos), which control development, fertility, and neuronal functions. Our results show how the specificity of one RBP (Pum) is modulated by cooperative RNA recognition with a second RBP (Nos) to synergistically repress mRNAs. Crystal structures of Nos-Pum-RNA complexes reveal that Nos embraces Pum and RNA, contributes sequence-specific contacts, and increases Pum RNA-binding affinity. Nos shifts the recognition sequence and promotes repression complex formation on mRNAs that are not stably bound by Pum alone, explaining the preponderance of sub-optimal Pum sites regulatedin vivo. Our results illuminate the molecular mechanism of a regulatory switch controlling crucial gene expression programs, and provide a framework for understanding how the partnering of RBPs evokes changes in binding specificity that underlie regulatory network dynamics.

Occupational health hazards of trichloroethylene among workers in relation to altered mRNA expression of cell cycle regulating genes (p53, p21, bax and bcl-2 and PPARA

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Meenu Varshney

2015-01-01

Full Text Available Trichloroethylene (TCE is widely used as a metal degreaser in industrial processes. The present study reports on the effects of TCE exposure on workers employed in the lock industries. To ensure exposure of the workers to TCE, its toxic metabolites, trichloroacetic acid (TCA, dichloroacetic acid (DCA and trichloroethanol (TCEOH were detected in the plasma of the subjects through solid phase microextraction-gas chromatography-electron capture detection. TCA, DCA and TCEOH were detected in the range of 0.004–2.494 μg/mL, 0.01–3.612 μg/mL and 0.002–0.617 μg/mL, respectively. Quantitative reverse transcription polymerase chain reaction analysis revealed up-regulated expression of p53 (2.4-fold; p < 0.05, p21 (2-fold; p < 0.01, bax (2.9-fold; p < 0.01 mRNAs and down-regulated expression of bcl-2 (67%; p < 0.05 mRNAs, indicating DNA damaging potential of these metabolites. No effects were observed on the levels of p16 and c-myc mRNAs. Further, as TCA and DCA, the ligand of peroxisome proliferator activated receptor alpha (PPARA, are involved in the process of hepatocarcinogenesis in rodents, we examined expression of PPARA mRNA and let-7c miRNA in the workers. No statistically significant differences in expression of PPARA mRNA and let-7c miRNA in patients were observed as compared to values in controls. Dehydroepiandosterone sulfate (DHEAS is a reported endogenous ligand of PPARA so its competitive role was also studied. We observed decreased levels of DHEAS hormone in the subjects. Hence, its involvement in mediation of the observed changes in the levels of various mRNAs analyzed in this study appears unlikely.

A 3'-untranslated region (3'UTR induces organ adhesion by regulating miR-199a* functions.

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Daniel Y Lee

Full Text Available Mature microRNAs (miRNAs are single-stranded RNAs of 18-24 nucleotides that repress post-transcriptional gene expression. However, it is unknown whether the functions of mature miRNAs can be regulated. Here we report that expression of versican 3'UTR induces organ adhesion in transgenic mice by modulating miR-199a* activities. The study was initiated by the hypothesis that the non-coding 3'UTR plays a role in the regulation of miRNA function. Transgenic mice expressing a construct harboring the 3'UTR of versican exhibits the adhesion of organs. Computational analysis indicated that a large number of microRNAs could bind to this fragment potentially including miR-199a*. Expression of versican and fibronectin, two targets of miR-199a*, are up-regulated in transgenic mice, suggesting that the 3'UTR binds and modulates miR-199a* activities, freeing mRNAs of versican and fibronectin from being repressed by miR-199a*. Confirmation of the binding was performed by PCR using mature miR-199a* as a primer and the targeting was performed by luciferase assays. Enhanced adhesion by expression of the 3'UTR was confirmed by in vitro assays. Our results demonstrated that upon arrival in cytoplasm, miRNA activities can be modulated locally by the 3'UTR. Our assay may be developed as sophisticated approaches for studying the mutual regulation of miRNAs and mRNAs in vitro and in vivo. We anticipate that expression of the 3'UTR may be an approach in the development of gene therapy.

Systemic responses to inhaled ozone in mice: cachexia and down-regulation of liver xenobiotic metabolizing genes

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Last, Jerold A [Pulmonary and Critical Care Medicine, School of Medicine, Toxic Substances Program, 1131 Surge I, University of California, Davis, CA 95616-8723 (United States); Gohil, Kishorchandra [Pulmonary and Critical Care Medicine, School of Medicine, Toxic Substances Program, 1131 Surge I, University of California, Davis, CA 95616-8723 (United States); Mathrani, Vivek C [Pulmonary and Critical Care Medicine, School of Medicine, Toxic Substances Program, 1131 Surge I, University of California, Davis, CA 95616-8723 (United States); Kenyon, Nicholas J [Pulmonary and Critical Care Medicine, School of Medicine, Toxic Substances Program, 1131 Surge I, University of California, Davis, CA 95616-8723 (United States)

2005-10-15

Rats or mice acutely exposed to high concentrations of ozone show an immediate and significant weight loss, even when allowed free access to food and water. The mechanisms underlying this systemic response to ozone have not been previously elucidated. We have applied the technique of global gene expression analysis to the livers of C57BL mice acutely exposed to ozone. Mice lost up to 14% of their original body weight, with a 42% decrease in total food consumption. We previously had found significant up-regulation of genes encoding proliferative enzymes, proteins related to acute phase reactions and cytoskeletal functions, and other biomarkers of a cachexia-like inflammatory state in lungs of mice exposed to ozone. These results are consistent with a general up-regulation of different gene families responsive to NF-{kappa}B in the lungs of the exposed mice. In the present study, we observed significant down-regulation of different families of mRNAs in the livers of the exposed mice, including genes related to lipid and fatty acid metabolism, and to carbohydrate metabolism in this tissue, consistent with a systemic cachexic response. Several interferon-dependent genes were down-regulated in the liver, suggesting a possible role for interferon as a signaling molecule between lung and liver. In addition, transcription of several mRNAs encoding enzymes of xenobiotic metabolism in the livers of mice exposed to ozone was decreased, suggesting cytokine-mediated suppression of cytochrome P450 expression. This finding may explain a previously controversial report from other investigators more than 20 years ago of prolongation of pentobarbital sleeping time in mice exposed to ozone.

Systemic responses to inhaled ozone in mice: cachexia and down-regulation of liver xenobiotic metabolizing genes

International Nuclear Information System (INIS)

Last, Jerold A.; Gohil, Kishorchandra; Mathrani, Vivek C.; Kenyon, Nicholas J.

2005-01-01

Rats or mice acutely exposed to high concentrations of ozone show an immediate and significant weight loss, even when allowed free access to food and water. The mechanisms underlying this systemic response to ozone have not been previously elucidated. We have applied the technique of global gene expression analysis to the livers of C57BL mice acutely exposed to ozone. Mice lost up to 14% of their original body weight, with a 42% decrease in total food consumption. We previously had found significant up-regulation of genes encoding proliferative enzymes, proteins related to acute phase reactions and cytoskeletal functions, and other biomarkers of a cachexia-like inflammatory state in lungs of mice exposed to ozone. These results are consistent with a general up-regulation of different gene families responsive to NF-κB in the lungs of the exposed mice. In the present study, we observed significant down-regulation of different families of mRNAs in the livers of the exposed mice, including genes related to lipid and fatty acid metabolism, and to carbohydrate metabolism in this tissue, consistent with a systemic cachexic response. Several interferon-dependent genes were down-regulated in the liver, suggesting a possible role for interferon as a signaling molecule between lung and liver. In addition, transcription of several mRNAs encoding enzymes of xenobiotic metabolism in the livers of mice exposed to ozone was decreased, suggesting cytokine-mediated suppression of cytochrome P450 expression. This finding may explain a previously controversial report from other investigators more than 20 years ago of prolongation of pentobarbital sleeping time in mice exposed to ozone

High-Throughput Screening to Identify Regulators of Meiosis-Specific Gene Expression in Saccharomyces cerevisiae.

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Kassir, Yona

2017-01-01

Meiosis and gamete formation are processes that are essential for sexual reproduction in all eukaryotic organisms. Multiple intracellular and extracellular signals feed into pathways that converge on transcription factors that induce the expression of meiosis-specific genes. Once triggered the meiosis-specific gene expression program proceeds in a cascade that drives progress through the events of meiosis and gamete formation. Meiosis-specific gene expression is tightly controlled by a balance of positive and negative regulatory factors that respond to a plethora of signaling pathways. The budding yeast Saccharomyces cerevisiae has proven to be an outstanding model for the dissection of gametogenesis owing to the sophisticated genetic manipulations that can be performed with the cells. It is possible to use a variety selection and screening methods to identify genes and their functions. High-throughput screening technology has been developed to allow an array of all viable yeast gene deletion mutants to be screened for phenotypes and for regulators of gene expression. This chapter describes a protocol that has been used to screen a library of homozygous diploid yeast deletion strains to identify regulators of the meiosis-specific IME1 gene.

Epigenetic regulation of RELN and GAD1 in the frontal cortex (FC) of autism spectrum disorder (ASD) subjects.

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Zhubi, Adrian; Chen, Ying; Guidotti, Alessandro; Grayson, Dennis R

2017-11-01

Both Reelin (RELN) and glutamate decarboxylase 67 (GAD1) have been implicated in the pathophysiology of Autism Spectrum Disorders (ASD). We have previously shown that both mRNAs are reduced in the cerebella (CB) of ASD subjects through a mechanism that involves increases in the amounts of MECP2 binding to the corresponding promoters. In the current study, we examined the expression of RELN, GAD1, GAD2, and several other mRNAs implicated in this disorder in the frontal cortices (FC) of ASD and CON subjects. We also focused on the role that epigenetic processes play in the regulation of these genes in ASD brain. Our goal is to better understand the molecular basis for the down-regulation of genes expressed in GABAergic neurons in ASD brains. We measured mRNA levels corresponding to selected GABAergic genes using qRT-PCR in RNA isolated from both ASD and CON groups. We determined the extent of binding of MECP2 and DNMT1 repressor proteins by chromatin immunoprecipitation (ChIP) assays. The amount of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) present in the promoters of the target genes was quantified by methyl DNA immunoprecipitation (MeDIP) and hydroxyl MeDIP (hMeDIP). We detected significant reductions in the mRNAs associated with RELN and GAD1 and significant increases in mRNAs encoding the Ten-eleven Translocation (TET) enzymes 1, 2, and 3. We also detected increased MECP2 and DNMT1 binding to the corresponding promoter regions of GAD1, RELN, and GAD2. Interestingly, there were decreased amounts of 5mC at both promoters and little change in 5hmC content in these same DNA fragments. Our data demonstrate that RELN, GAD1, and several other genes selectively expressed in GABAergic neurons, are down-regulated in post-mortem ASD FC. In addition, we observed increased DNMT1 and MECP2 binding at the corresponding promoters of these genes. The finding of increased MECP2 binding to the RELN, GAD1 and GAD2 promoters, with reduced amounts of 5mC and unchanged

Structure analysis of the global metabolic regulator Crc from Pseudomonas aeruginosa.

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Wei, Yong; Zhang, Heng; Gao, Zeng-Qiang; Xu, Jian-Hua; Liu, Quan-Sheng; Dong, Yu-Hui

2013-01-01

The global metabolic regulator catabolite repression control (Crc) has recently been found to modulate the susceptibility to antibiotics and virulence in the opportunistic pathogen Pseudomonas aeruginosa and been suggested as a nonlethal target for novel antimicrobials. In P. aeruginosa, Crc couples with the CA motifs from the small RNA CrcZ to form a post-transcriptional regulator system and is removed from the 5'-end of the target mRNAs. In this study, we first reported the crystal structure of Crc from P. aeruginosa refined to 2.20 Å. The structure showed that it consists of two halves with similar overall topology and there are 11 β strands surrounded by 13 helices, forming a four-layered α/β-sandwich. The circular dichroism spectroscopy revealed that it is thermostable in solution and shares similar characteristics to that in crystal. Comprehensive structural analysis and comparison with the homologies of Crc showed high similarity with several known nucleases and consequently may be classified into a member exodeoxyribonuclease III. However, it shows distinct substrate specificity (RNA as the preferred substrate) compared to these DNA endonucleases. Structural comparisons also revealed potential RNA recognition and binding region mainly consisting of five flexible loops. Our structure study provided the basis for the future application of Crc as a target to develop new antibiotics. Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.

Plasticity during Sleep Is Linked to Specific Regulation of Cortical Circuit Activity

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Niels Niethard

2017-09-01

Full Text Available Sleep is thought to be involved in the regulation of synaptic plasticity in two ways: by enhancing local plastic processes underlying the consolidation of specific memories and by supporting global synaptic homeostasis. Here, we briefly summarize recent structural and functional studies examining sleep-associated changes in synaptic morphology and neural excitability. These studies point to a global down-scaling of synaptic strength across sleep while a subset of synapses increases in strength. Similarly, neuronal excitability on average decreases across sleep, whereas subsets of neurons increase firing rates across sleep. Whether synapse formation and excitability is down or upregulated across sleep appears to partly depend on the cell’s activity level during wakefulness. Processes of memory-specific upregulation of synapse formation and excitability are observed during slow wave sleep (SWS, whereas global downregulation resulting in elimination of synapses and decreased neural firing is linked to rapid eye movement sleep (REM sleep. Studies of the excitation/inhibition balance in cortical circuits suggest that both processes are connected to a specific inhibitory regulation of cortical principal neurons, characterized by an enhanced perisomatic inhibition via parvalbumin positive (PV+ cells, together with a release from dendritic inhibition by somatostatin positive (SOM+ cells. Such shift towards increased perisomatic inhibition of principal cells appears to be a general motif which underlies the plastic synaptic changes observed during sleep, regardless of whether towards up or downregulation.

The transcription factor Lc-Maf participates in Col27a1 regulation during chondrocyte maturation

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Mayo, Jaime L.; Holden, Devin N. [Department of Microbiology and Molecular Biology, Brigham Young University, 591 WIDB, Provo, UT 84602 (United States); Barrow, Jeffery R. [Department of Physiology and Developmental Biology, Brigham Young University, Provo, UT 84602 (United States); Bridgewater, Laura C., E-mail: laura_bridgewater@byu.edu [Department of Microbiology and Molecular Biology, Brigham Young University, 591 WIDB, Provo, UT 84602 (United States)

2009-08-01

The transcription factor Lc-Maf, which is a splice variant of c-Maf, is expressed in cartilage undergoing endochondral ossification and participates in the regulation of type II collagen through a cartilage-specific Col2a1 enhancer element. Type XXVII and type XI collagens are also expressed in cartilage during endochondral ossification, and so enhancer/reporter assays were used to determine whether Lc-Maf could regulate cartilage-specific enhancers from the Col27a1 and Col11a2 genes. The Col27a1 enhancer was upregulated over 4-fold by Lc-Maf, while the Col11a2 enhancer was downregulated slightly. To confirm the results of these reporter assays, rat chondrosarcoma (RCS) cells were transiently transfected with an Lc-Maf expression plasmid, and quantitative RT-PCR was performed to measure the expression of endogenous Col27a1 and Col11a2 genes. Endogenous Col27a1 was upregulated 6-fold by Lc-Maf overexpression, while endogenous Col11a2 was unchanged. Finally, in situ hybridization and immunohistochemistry were performed in the radius and ulna of embryonic day 17 mouse forelimbs undergoing endochondral ossification. Results demonstrated that Lc-Maf and Col27a1 mRNAs are coexpressed in proliferating and prehypertrophic regions, as would be predicted if Lc-Maf regulates Col27a1 expression. Type XXVII collagen protein was also most abundant in prehypertrophic and proliferating chondrocytes. Others have shown that mice that are null for Lc-Maf and c-Maf have expanded hypertrophic regions with reduced ossification and delayed vascularization. Separate studies have indicated that Col27a1 may serve as a scaffold for ossification and vascularization. The work presented here suggests that Lc-Maf may affect the process of endochondral ossification by participating in the regulation of Col27a1 expression.

Transforming growth factor-β1 regulates fibronectin isoform expression and splicing factor SRp40 expression during ATDC5 chondrogenic maturation

International Nuclear Information System (INIS)

Han Fei; Gilbert, James R.; Harrison, Gerald; Adams, Christopher S.; Freeman, Theresa; Tao Zhuliang; Zaka, Raihana; Liang Hongyan; Williams, Charlene; Tuan, Rocky S.; Norton, Pamela A.; Hickok, Noreen J.

2007-01-01

Fibronectin (FN) isoform expression is altered during chondrocyte commitment and maturation, with cartilage favoring expression of FN isoforms that includes the type II repeat extra domain B (EDB) but excludes extra domain A (EDA). We and others have hypothesized that the regulated splicing of FN mRNAs is necessary for the progression of chondrogenesis. To test this, we treated the pre-chondrogenic cell line ATDC5 with transforming growth factor-β1, which has been shown to modulate expression of the EDA and EDB exons, as well as the late markers of chondrocyte maturation; it also slightly accelerates the early acquisition of a sulfated proteoglycan matrix without affecting cell proliferation. When chondrocytes are treated with TGF-β1, the EDA exon is preferentially excluded at all times whereas the EDB exon is relatively depleted at early times. This regulated alternative splicing of FN correlates with the regulation of alternative splicing of SRp40, a splicing factor facilitating inclusion of the EDA exon. To determine if overexpression of the SRp40 isoforms altered FN and FN EDA organization, cDNAs encoding these isoforms were overexpressed in ATDC5 cells. Overexpression of the long-form of SRp40 yielded an FN organization similar to TGF-β1 treatment; whereas overexpression of the short form of SRp40 (which facilitates EDA inclusion) increased formation of long-thick FN fibrils. Therefore, we conclude that the effects of TGF-β1 on FN splicing during chondrogenesis may be largely dependent on its effect on SRp40 isoform expression

Direct Binding between Pre-S1 and TRP-like Domains in TRPP Channels Mediates Gating and Functional Regulation by PIP2

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Wang Zheng

2018-02-01

Full Text Available Transient receptor potential (TRP channels are regulated by diverse stimuli comprising thermal, chemical, and mechanical modalities. They are also commonly regulated by phosphatidylinositol-4,5-bisphosphate (PIP2, with underlying mechanisms largely unknown. We here revealed an intramolecular interaction of the TRPP3 N and C termini (N-C that is functionally essential. The interaction was mediated by aromatic Trp81 in pre-S1 domain and cationic Lys568 in TRP-like domain. Structure-function analyses revealed similar N-C interaction in TRPP2 as well as TRPM8/-V1/-C4 via highly conserved tryptophan and lysine/arginine residues. PIP2 bound to cationic residues in TRPP3, including K568, thereby disrupting the N-C interaction and negatively regulating TRPP3. PIP2 had similar negative effects on TRPP2. Interestingly, we found that PIP2 facilitates the N-C interaction in TRPM8/-V1, resulting in channel potentiation. The intramolecular N-C interaction might represent a shared mechanism underlying the gating and PIP2 regulation of TRP channels.

Development of transgenic rats producing human β-amyloid precursor protein as a model for Alzheimer's disease: Transgene and endogenous APP genes are regulated tissue-specifically

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Chan Anthony WS

2008-02-01

Full Text Available Abstract Background Alzheimer's disease (AD is a devastating neurodegenerative disorder that affects a large and growing number of elderly individuals. In addition to idiopathic disease, AD is also associated with autosomal dominant inheritance, which causes a familial form of AD (FAD. Some instances of FAD have been linked to mutations in the β-amyloid protein precursor (APP. Although there are numerous mouse AD models available, few rat AD models, which have several advantages over mice, have been generated. Results Fischer 344 rats expressing human APP driven by the ubiquitin-C promoter were generated via lentiviral vector infection of Fischer 344 zygotes. We generated two separate APP-transgenic rat lines, APP21 and APP31. Serum levels of human amyloid-beta (Aβ40 were 298 pg/ml for hemizygous and 486 pg/ml for homozygous APP21 animals. Serum Aβ42 levels in APP21 homozygous rats were 135 pg/ml. Immunohistochemistry in brain showed that the human APP transgene was expressed in neurons, but not in glial cells. These findings were consistent with independent examination of enhanced green fluorescent protein (eGFP in the brains of eGFP-transgenic rats. APP21 and APP31 rats expressed 7.5- and 3-times more APP mRNA, respectively, than did wild-type rats. Northern blots showed that the human APP transgene, driven by the ubiquitin-C promoter, is expressed significantly more in brain, kidney and lung compared to heart and liver. A similar expression pattern was also seen for the endogenous rat APP. The unexpected similarity in the tissue-specific expression patterns of endogenous rat APP and transgenic human APP mRNAs suggests regulatory elements within the cDNA sequence of APP. Conclusion This manuscript describes the generation of APP-transgenic inbred Fischer 344 rats. These are the first human AD model rat lines generated by lentiviral infection. The APP21 rat line expresses high levels of human APP and could be a useful model for AD. Tissue-specific

Complex mutual regulation of facilitates chromatin transcription (FACT) subunits on both mRNA and protein levels in human cells.

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Safina, Alfiya; Garcia, Henry; Commane, Mairead; Guryanova, Olga; Degan, Seamus; Kolesnikova, Kateryna; Gurova, Katerina V

2013-08-01

Facilitates chromatin transcription (FACT) is a chromatin remodeling complex with two subunits: SSRP1 and SPT16. Mechanisms controlling FACT levels are of interest, since the complex is not expressed in most differentiated cells, but is frequently upregulated in cancer, particularly in poorly differentiated, aggressive tumors. Moreover, inhibition of FACT expression or function in tumor cells interferes with their survival. Here we demonstrate that SSRP1 and SPT16 protein levels decline upon induction of cellular differentiation or senescence in vitro and that similar declines in protein levels for both SSRP1 and SPT16 occur upon RNAi-mediated knockdown of either SSRP1 or SPT16. The interdependence of SSRP1 and SPT16 protein levels was found to be due to their association with SSRP1 and SPT16 mRNAs, which stabilizes the proteins. In particular, presence of SSRP1 mRNA is critical for SPT16 protein stability. In addition, binding of SSRP1 and SPT16 mRNAs to the FACT complex increases the stability and efficiency of translation of the mRNAs. These data support a model in which the FACT complex is stable when SSRP1 mRNA is present, but quickly degrades when SSRP1 mRNA levels drop. In the absence of FACT complex, SSRP1 and SPT16 mRNAs are unstable and inefficiently translated, making reactivation of FACT function unlikely in normal cells. Thus, we have described a complex and unusual mode of regulation controlling cellular FACT levels that results in amplified and stringent control of FACT activity. The FACT dependence of tumor cells suggests that mechanisms controlling FACT levels could be targeted for anticancer therapy.

Patient-specific surgical simulator for the pre-operative planning of single-incision laparoscopic surgery with bimanual robots.

Science.gov (United States)

Turini, Giuseppe; Moglia, Andrea; Ferrari, Vincenzo; Ferrari, Mauro; Mosca, Franco

2012-01-01

The trend of surgical robotics is to follow the evolution of laparoscopy, which is now moving towards single-incision laparoscopic surgery. The main drawback of this approach is the limited maneuverability of the surgical tools. Promising solutions to improve the surgeon's dexterity are based on bimanual robots. However, since both robot arms are completely inserted into the patient's body, issues related to possible unwanted collisions with structures adjacent to the target organ may arise. This paper presents a simulator based on patient-specific data for the positioning and workspace evaluation of bimanual surgical robots in the pre-operative planning of single-incision laparoscopic surgery. The simulator, designed for the pre-operative planning of robotic laparoscopic interventions, was tested by five expert surgeons who evaluated its main functionalities and provided an overall rating for the system. The proposed system demonstrated good performance and usability, and was designed to integrate both present and future bimanual surgical robots.

WIPP - Pre-Licensing and Operations: Developer and Regulator Perspectives

International Nuclear Information System (INIS)

Peake, Tom; Patterson, R.

2014-01-01

The Waste Isolation Pilot Plant (WIPP) is a disposal system for defense-related transuranic (TRU) radioactive waste. Developed by the Department of Energy (DOE), WIPP is located in Southeastern New Mexico: radioactive waste is disposed of 2,150 feet underground in an ancient layer of salt with a total capacity of 6.2 million cubic feet of waste. Congress authorized the development and construction of WIPP in 1980 for the express purpose of providing a research and development facility to demonstrate the safe disposal of radioactive wastes resulting from the defense activities and programs of the United States. This paper makes a historical review of the site development, site operations (waste disposal operations started in 1999), communications between US EPA and DOE, the chronology of pre-licensing and pre-operations, the operational phase and the regulatory challenges, and the lessons learned after 12 years of operations

What's the FOX Got to Do with the KITten? Regulating the Lineage-Specific Transcriptional Landscape in GIST.

Science.gov (United States)

Lee, Donna M; Duensing, Anette

2018-02-01

Transcriptional regulation of the KIT receptor tyrosine kinase, a master regulator in gastrointestinal stromal tumors (GIST) and their precursors, the interstitial cells of Cajal (ICC), is part of a positive feedback loop involving the transcription factor ETV1. A new study now shows that the forkhead box (FOX) family transcription factor FOXF1 not only is an upstream regulator of ETV1 and hence ICC/GIST lineage-specific gene transcription, but also functions as lineage-specific pioneer factor with an active role in chromatin rearrangement to facilitate ETV1 binding and transcriptional activity. Cancer Discov; 8(2); 146-9. ©2018 AACR See related article by Ran et al., p. 234 . ©2018 American Association for Cancer Research.

Replicative Stress Induces Intragenic Transcription of the ASE1 Gene that Negatively Regulates Ase1 Activity

OpenAIRE

McKnight, Kelly; Liu, Hong; Wang, Yanchang

2014-01-01

Intragenic transcripts initiate within the coding region of a gene, thereby producing shorter mRNAs and proteins. Although intragenic transcripts are widely expressed [1], their role in the functional regulation of genes remains largely unknown. In budding yeast, DNA replication stress activates the S-phase checkpoint that stabilizes replication forks and arrests cells in S-phase with a short spindle [2-4]. When yeast cells were treated with hydroxyurea (HU) to block DNA synthesis and induce ...

An extracellular-matrix-specific GEF-GAP interaction regulates Rho GTPase crosstalk for 3D collagen migration.

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Kutys, Matthew L; Yamada, Kenneth M

2014-09-01

Rho-family GTPases govern distinct types of cell migration on different extracellular matrix proteins in tissue culture or three-dimensional (3D) matrices. We searched for mechanisms selectively regulating 3D cell migration in different matrix environments and discovered a form of Cdc42-RhoA crosstalk governing cell migration through a specific pair of GTPase activator and inhibitor molecules. We first identified βPix, a guanine nucleotide exchange factor (GEF), as a specific regulator of migration in 3D collagen using an affinity-precipitation-based GEF screen. Knockdown of βPix specifically blocks cell migration in fibrillar collagen microenvironments, leading to hyperactive cellular protrusion accompanied by increased collagen matrix contraction. Live FRET imaging and RNAi knockdown linked this βPix knockdown phenotype to loss of polarized Cdc42 but not Rac1 activity, accompanied by enhanced, de-localized RhoA activity. Mechanistically, collagen phospho-regulates βPix, leading to its association with srGAP1, a GTPase-activating protein (GAP), needed to suppress RhoA activity. Our results reveal a matrix-specific pathway controlling migration involving a GEF-GAP interaction of βPix with srGAP1 that is critical for maintaining suppressive crosstalk between Cdc42 and RhoA during 3D collagen migration.

Selective increases of AMPA, NMDA and kainate receptor subunit mRNAs in the hippocampus and orbitofrontal cortex but not in prefrontal cortex of human alcoholics

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Zhe eJin

2014-01-01

Full Text Available Glutamate is the main excitatory transmitter in the human brain. Drugs that affect the glutamatergic signaling will alter neuronal excitability. Ethanol inhibits glutamate receptors. We examined the expression level of glutamate receptor subunit mRNAs in human post-mortem samples from alcoholics and compared the results to brain samples from control subjects. RNA from hippocampal dentate gyrus (HP-DG, orbitofrontal cortex (OFC, and dorso-lateral prefrontal cortex (DL-PFC samples from 21 controls and 19 individuals with chronic alcohol dependence were included in the study. Total RNA was assayed using quantitative RT-PCR. Out of the 16 glutamate receptor subunits, mRNAs encoding two AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-ylpropanoic acid receptor subunits GluA2 and GluA3; three kainate receptor subunits GluK2, GluK3 and GluK5 and five NMDA (N-methyl-D-aspartate receptor subunits GluN1, GluN2A, GluN2C, GluN2D and GluN3A were significantly increased in the HP-DG region in alcoholics. In the OFC, mRNA encoding the NMDA receptor subunit GluN3A was increased, whereas in the DL-PFC, no differences in mRNA levels were observed. Our laboratory has previously shown that the expression of genes encoding inhibitory GABA-A receptors is altered in the HP-DG and OFC of alcoholics (Jin et al., 2011. Whether the changes in one neurotransmitter system drives changes in the other or if they change independently is currently not known. The results demonstrate that excessive long-term alcohol consumption is associated with altered expression of genes encoding glutamate receptors in a brain region-specific manner. It is an intriguing possibility that genetic predisposition to alcoholism may contribute to these gene expression changes.

Internet-Specific Epistemic Beliefs and Self-Regulated Learning in Online Academic Information Searching

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Chiu, Yen-Lin; Liang, Jyh-Chong; Tsai, Chin-Chung

2013-01-01

Epistemic beliefs have been considered as important components of the self-regulatory model; however, their relationships with self-regulated learning processes in the Internet context need further research. The main purpose of this study was to examine the relationships between Internet-specific epistemic belief dimensions and self-regulated…

Patient-specific instrumentation for total knee arthroplasty does not match the pre-operative plan as assessed by intra-operative computer-assisted navigation.

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Scholes, Corey; Sahni, Varun; Lustig, Sebastien; Parker, David A; Coolican, Myles R J

2014-03-01

The introduction of patient-specific instruments (PSI) for guiding bone cuts could increase the incidence of malalignment in primary total knee arthroplasty. The purpose of this study was to assess the agreement between one type of patient-specific instrumentation (Zimmer PSI) and the pre-operative plan with respect to bone cuts and component alignment during TKR using imageless computer navigation. A consecutive series of 30 femoral and tibial guides were assessed in-theatre by the same surgeon using computer navigation. Following surgical exposure, the PSI cutting guides were placed on the joint surface and alignment assessed using the navigation tracker. The difference between in-theatre data and the pre-operative plan was recorded and analysed. The error between in-theatre measurements and pre-operative plan for the femoral and tibial components exceeded 3° for 3 and 17% of the sample, respectively, while the error for total coronal alignment exceeded 3° for 27% of the sample. The present results indicate that alignment with Zimmer PSI cutting blocks, assessed by imageless navigation, does not match the pre-operative plan in a proportion of cases. To prevent unnecessary increases in the incidence of malalignment in primary TKR, it is recommended that these devices should not be used without objective verification of alignment, either in real-time or with post-operative imaging. Further work is required to identify the source of discrepancies and validate these devices prior to routine use. II.

Goldfish neurokinin B: Cloning, tissue distribution, and potential role in regulating reproduction.

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Qi, Xin; Zhou, Wenyi; Li, Shuisheng; Liu, Yun; Ye, Gang; Liu, Xiaochun; Peng, Chun; Zhang, Yong; Lin, Haoran

2015-09-15

Neurokinin B (NKB) is a member of the tackykinin (TAC) family known to play a critical role in the neuroendocrine regulation of reproduction in mammals. However, its biological functions in teleosts are less clear. The aim of this study was to determine the role of NKB in fish reproduction using goldfish as a model. Two transcripts, TAC3a and TAC3b, which encode several NKBs, including NKBa-13, NKBa-10, NKBb-13, and NKBb-11, were cloned. Phylogenetic analysis revealed that NKBa-10 and NKBb-11 are closely related to mammalian NKB, while NKB-13s are more conserved in teleosts. Quantitative real-time PCR analyses in various tissues showed that TAC3a and TAC3b mRNAs were mainly expressed in the brain. In situ hybridization further detected TAC3a and TAC3b mRNAs in several regions of the brain known to be involved in the regulation of reproduction and metabolism, as well as in the neurohypophysis of the pituitary. To investigate the potential role of NKBs in reproduction, goldfish were injected intraperitoneally with synthetic NKBa-13, -10, NKBb-13, or -11 peptides and the mRNA levels of hypothalamic gonadotropin-releasing hormone (GnRH) and pituitary gonadotropin subunits were measured. NKBa-13, -10, or NKBb-13, but not -11, significantly increased hypothalamic salmon GnRH and pituitary FSHβ and LHβ mRNA levels in both female and male goldfish. Finally, ovariectomy increased, while estradiol replacement reduced, TAC3a mRNA levels without affecting TAC3b expression in the hypothalamus. These data suggest that NKBa-13, -10, and NKBb-13 play a role in mediating the estrogen negative feedback regulation of gonadotropins. Copyright © 2014 Elsevier Inc. All rights reserved.

Microarray-based approach identifies microRNAs and their target functional patterns in polycystic kidney disease

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Boehn Susanne NE

2008-12-01

Full Text Available Abstract Background MicroRNAs (miRNAs play key roles in mammalian gene expression and several cellular processes, including differentiation, development, apoptosis and cancer pathomechanisms. Recently the biological importance of primary cilia has been recognized in a number of human genetic diseases. Numerous disorders are related to cilia dysfunction, including polycystic kidney disease (PKD. Although involvement of certain genes and transcriptional networks in PKD development has been shown, not much is known how they are regulated molecularly. Results Given the emerging role of miRNAs in gene expression, we explored the possibilities of miRNA-based regulations in PKD. Here, we analyzed the simultaneous expression changes of miRNAs and mRNAs by microarrays. 935 genes, classified into 24 functional categories, were differentially regulated between PKD and control animals. In parallel, 30 miRNAs were differentially regulated in PKD rats: our results suggest that several miRNAs might be involved in regulating genetic switches in PKD. Furthermore, we describe some newly detected miRNAs, miR-31 and miR-217, in the kidney which have not been reported previously. We determine functionally related gene sets, or pathways to reveal the functional correlation between differentially expressed mRNAs and miRNAs. Conclusion We find that the functional patterns of predicted miRNA targets and differentially expressed mRNAs are similar. Our results suggest an important role of miRNAs in specific pathways underlying PKD.

RRM domain of Arabidopsis splicing factor SF1 is important for pre-mRNA splicing of a specific set of genes

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Lee, Keh Chien

2017-04-11

The RNA recognition motif of Arabidopsis splicing factor SF1 affects the alternative splicing of FLOWERING LOCUS M pre-mRNA and a heat shock transcription factor HsfA2 pre-mRNA. Splicing factor 1 (SF1) plays a crucial role in 3\\' splice site recognition by binding directly to the intron branch point. Although plant SF1 proteins possess an RNA recognition motif (RRM) domain that is absent in its fungal and metazoan counterparts, the role of the RRM domain in SF1 function has not been characterized. Here, we show that the RRM domain differentially affects the full function of the Arabidopsis thaliana AtSF1 protein under different experimental conditions. For example, the deletion of RRM domain influences AtSF1-mediated control of flowering time, but not the abscisic acid sensitivity response during seed germination. The alternative splicing of FLOWERING LOCUS M (FLM) pre-mRNA is involved in flowering time control. We found that the RRM domain of AtSF1 protein alters the production of alternatively spliced FLM-β transcripts. We also found that the RRM domain affects the alternative splicing of a heat shock transcription factor HsfA2 pre-mRNA, thereby mediating the heat stress response. Taken together, our results suggest the importance of RRM domain for AtSF1-mediated alternative splicing of a subset of genes involved in the regulation of flowering and adaptation to heat stress.

TargetMiner: microRNA target prediction with systematic identification of tissue-specific negative examples.

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Bandyopadhyay, Sanghamitra; Mitra, Ramkrishna

2009-10-15

Prediction of microRNA (miRNA) target mRNAs using machine learning approaches is an important area of research. However, most of the methods suffer from either high false positive or false negative rates. One reason for this is the marked deficiency of negative examples or miRNA non-target pairs. Systematic identification of non-target mRNAs is still not addressed properly, and therefore, current machine learning approaches are compelled to rely on artificially generated negative examples for training. In this article, we have identified approximately 300 tissue-specific negative examples using a novel approach that involves expression profiling of both miRNAs and mRNAs, miRNA-mRNA structural interactions and seed-site conservation. The newly generated negative examples are validated with pSILAC dataset, which elucidate the fact that the identified non-targets are indeed non-targets.These high-throughput tissue-specific negative examples and a set of experimentally verified positive examples are then used to build a system called TargetMiner, a support vector machine (SVM)-based classifier. In addition to assessing the prediction accuracy on cross-validation experiments, TargetMiner has been validated with a completely independent experimental test dataset. Our method outperforms 10 existing target prediction algorithms and provides a good balance between sensitivity and specificity that is not reflected in the existing methods. We achieve a significantly higher sensitivity and specificity of 69% and 67.8% based on a pool of 90 feature set and 76.5% and 66.1% using a set of 30 selected feature set on the completely independent test dataset. In order to establish the effectiveness of the systematically generated negative examples, the SVM is trained using a different set of negative data generated using the method in Yousef et al. A significantly higher false positive rate (70.6%) is observed when tested on the independent set, while all other factors are kept the

A conserved RNA structural element within the hepatitis B virus post-transcriptional regulatory element enhance nuclear export of intronless transcripts and repress the splicing mechanism.

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Visootsat, Akasit; Payungporn, Sunchai; T-Thienprasert, Nattanan P

2015-12-01

Hepatitis B virus (HBV) infection is a primary cause of hepatocellular carcinoma and liver cirrhosis worldwide. To develop novel antiviral drugs, a better understanding of HBV gene expression regulation is vital. One important aspect is to understand how HBV hijacks the cellular machinery to export unspliced RNA from the nucleus. The HBV post-transcriptional regulatory element (HBV PRE) has been proposed to be the HBV RNA nuclear export element. However, the function remains controversial, and the core element is unclear. This study, therefore, aimed to identify functional regulatory elements within the HBV PRE and investigate their functions. Using bioinformatics programs based on sequence conservation and conserved RNA secondary structures, three regulatory elements were predicted, namely PRE 1151-1410, PRE 1520-1620 and PRE 1650-1684. PRE 1151-1410 significantly increased intronless and unspliced luciferase activity in both HepG2 and COS-7 cells. Likewise, PRE 1151-1410 significantly elevated intronless and unspliced HBV surface transcripts in liver cancer cells. Moreover, motif analysis predicted that PRE 1151-1410 contains several regulatory motifs. This study reported the roles of PRE 1151-1410 in intronless transcript nuclear export and the splicing mechanism. Additionally, these results provide knowledge in the field of HBV RNA regulation. Moreover, PRE 1151-1410 may be used to enhance the expression of other mRNAs in intronless reporter plasmids.

Regulation of platelet activating factor receptor coupled phosphoinositide-specific phospholipase C activity

International Nuclear Information System (INIS)

Morrison, W.J.

1988-01-01

The major objectives of this study were two-fold. The first was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of polyphosphoinositide-specific phospholipase C (PLC) in rabbit platelets. The second was to determine regulatory features of this receptor-coupled mechanism. [ 3 H]PAF binding demonstrated two binding sites, a high affinity site with a inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 μM. PAF receptor coupled activation of phosphoinositide-specific PLC was studied in platelets which were made refractory, by short term pretreatments, to either PAF or thrombin. Saponin-permeabilized rabbit platelets continue to regulate the mechanism(s) coupling PAF receptors to PLC stimulation. However, TRPγS and GDPβS, which affect guanine nucleotide regulatory protein functions, were unable to modulate the PLC activity to any appreciable extent as compared to PAF. The possible involvement of protein kinase C (PKC) activation in regulating PAF-stimulated PLC activity was studied in rabbit platelets pretreated with staurosporine followed by pretreatments with PAF or phorbol 12-myristate 13-acetate (PMA)

Co-expression networks reveal the tissue-specific regulation of transcription and splicing.

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Saha, Ashis; Kim, Yungil; Gewirtz, Ariel D H; Jo, Brian; Gao, Chuan; McDowell, Ian C; Engelhardt, Barbara E; Battle, Alexis

2017-11-01

Gene co-expression networks capture biologically important patterns in gene expression data, enabling functional analyses of genes, discovery of biomarkers, and interpretation of genetic variants. Most network analyses to date have been limited to assessing correlation between total gene expression levels in a single tissue or small sets of tissues. Here, we built networks that additionally capture the regulation of relative isoform abundance and splicing, along with tissue-specific connections unique to each of a diverse set of tissues. We used the Genotype-Tissue Expression (GTEx) project v6 RNA sequencing data across 50 tissues and 449 individuals. First, we developed a framework called Transcriptome-Wide Networks (TWNs) for combining total expression and relative isoform levels into a single sparse network, capturing the interplay between the regulation of splicing and transcription. We built TWNs for 16 tissues and found that hubs in these networks were strongly enriched for splicing and RNA binding genes, demonstrating their utility in unraveling regulation of splicing in the human transcriptome. Next, we used a Bayesian biclustering model that identifies network edges unique to a single tissue to reconstruct Tissue-Specific Networks (TSNs) for 26 distinct tissues and 10 groups of related tissues. Finally, we found genetic variants associated with pairs of adjacent nodes in our networks, supporting the estimated network structures and identifying 20 genetic variants with distant regulatory impact on transcription and splicing. Our networks provide an improved understanding of the complex relationships of the human transcriptome across tissues. © 2017 Saha et al.; Published by Cold Spring Harbor Laboratory Press.

Tissue-specific expression and regulatory networks of pig microRNAome.

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Paolo Martini

Full Text Available BACKGROUND: Despite the economic and medical importance of the pig, knowledge about its genome organization, gene expression regulation, and molecular mechanisms involved in physiological processes is far from that achieved for mouse and rat, the two most used model organisms in biomedical research. MicroRNAs (miRNAs are a wide class of molecules that exert a recognized role in gene expression modulation, but only 280 miRNAs in pig have been characterized to date. RESULTS: We applied a novel computational approach to predict species-specific and conserved miRNAs in the pig genome, which were then subjected to experimental validation. We experimentally identified candidate miRNAs sequences grouped in high-confidence (424 and medium-confidence (353 miRNAs according to RNA-seq results. A group of miRNAs was also validated by PCR experiments. We established the subtle variability in expression of isomiRs and miRNA-miRNA star couples supporting a biological function for these molecules. Finally, miRNA and mRNA expression profiles produced from the same sample of 20 different tissue of the animal were combined, using a correlation threshold to filter miRNA-target predictions, to identify tissue-specific regulatory networks. CONCLUSIONS: Our data represent a significant progress in the current understanding of miRNAome in pig. The identification of miRNAs, their target mRNAs, and the construction of regulatory circuits will provide new insights into the complex biological networks in several tissues of this important animal model.

32 CFR 1800.14 - Fee estimates (pre-request option).

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2010-07-01

... 32 National Defense 6 2010-07-01 2010-07-01 false Fee estimates (pre-request option). 1800.14 Section 1800.14 National Defense Other Regulations Relating to National Defense NATIONAL... Requests § 1800.14 Fee estimates (pre-request option). In order to avoid unanticipated or potentially large...

A 3′-Untranslated Region (3′UTR) Induces Organ Adhesion by Regulating miR-199a* Functions

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Lee, Daniel Y.; Shatseva, Tatiana; Jeyapalan, Zina; Du, William W.; Deng, Zhaoqun; Yang, Burton B.

2009-01-01

Mature microRNAs (miRNAs) are single-stranded RNAs of 18–24 nucleotides that repress post-transcriptional gene expression. However, it is unknown whether the functions of mature miRNAs can be regulated. Here we report that expression of versican 3′UTR induces organ adhesion in transgenic mice by modulating miR-199a* activities. The study was initiated by the hypothesis that the non-coding 3′UTR plays a role in the regulation of miRNA function. Transgenic mice expressing a construct harboring the 3′UTR of versican exhibits the adhesion of organs. Computational analysis indicated that a large number of microRNAs could bind to this fragment potentially including miR-199a*. Expression of versican and fibronectin, two targets of miR-199a*, are up-regulated in transgenic mice, suggesting that the 3′UTR binds and modulates miR-199a* activities, freeing mRNAs of versican and fibronectin from being repressed by miR-199a*. Confirmation of the binding was performed by PCR using mature miR-199a* as a primer and the targeting was performed by luciferase assays. Enhanced adhesion by expression of the 3′UTR was confirmed by in vitro assays. Our results demonstrated that upon arrival in cytoplasm, miRNA activities can be modulated locally by the 3′UTR. Our assay may be developed as sophisticated approaches for studying the mutual regulation of miRNAs and mRNAs in vitro and in vivo. We anticipate that expression of the 3′UTR may be an approach in the development of gene therapy. PMID:19223980

Zonation of heme synthesis enzymes in mouse liver and their regulation by β-catenin and Ha-ras.

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Braeuning, Albert; Schwarz, Michael

2010-11-01

Cytochrome P450 (CYP) hemoproteins play an important role in hepatic biotransformation. Recently, β-catenin and Ha-ras signaling have been identified as players controlling transcription of various CYP genes in mouse liver. The aim of the present study was to analyze the role of β-catenin and Ha-ras in the regulation of heme synthesis. Heme synthesis-related gene expression was analyzed in normal liver, in transgenic mice expressing activated β-catenin or Ha-ras, and in hepatomas. Regulation of the aminolevulinate dehydratase promoter was studied in vitro. Elevated expression of mRNAs and proteins involved in heme biosynthesis was linked to β-catenin activation in perivenous hepatocytes, in transgenic hepatocytes, and in hepatocellular tumors. Stimulation of the aminolevulinate dehydratase promoter by β-catenin was independent of the β-catenin/T-cell-specific transcription factor dimer. By contrast, activation of Ha-ras repressed heme synthesis-related gene expression. The present data suggest that β-catenin enhances the expression of both CYPs and heme synthesis-related genes, thus coordinating the availability of CYP apoprotein and its prosthetic group heme. The reciprocal regulation of heme synthesis by β-catenin and Ha-ras-dependent signaling supports our previous hypothesis that antagonistic action of these pathways plays a major role in the control of zonal gene expression in healthy mouse liver and aberrant expression patterns in hepatocellular tumors.

Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

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Li, Ying [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Department of Clinical Laboratory, Second Affiliated Hospital of Dalian Medical University, Dalian 116023 (China); Huang, Xiaohua [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Department of Clinical Biochemistry, College of Laboratory Medicine, Dalian Medical University, Dalian 116044 (China); An, Yue [Department of Clinical Laboratory, Second Affiliated Hospital of Dalian Medical University, Dalian 116023 (China); Ren, Feng [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Yang, Zara Zhuyun; Zhu, Hongmei; Zhou, Lei [The Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650228 (China); Department of Anatomy and Developmental Biology, Monash University, Clayton 3800 (Australia); He, Xiaowen; Schachner, Melitta [Keck Center for Collaborative Neuroscience and Department of Cell Biology and Neuroscience, Rutgers University, New Brunswick, NJ (United States); Xiao, Zhicheng, E-mail: zhicheng.xiao@monash.edu [The Key Laboratory of Stem Cell and Regenerative Medicine, Institute of Molecular and Clinical Medicine, Kunming Medical University, Kunming 650228 (China); Department of Anatomy and Developmental Biology, Monash University, Clayton 3800 (Australia); Ma, Keli, E-mail: makeli666@aliyun.com [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Li, Yali, E-mail: yalilipaper@gmail.com [Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044 (China); Department of Anatomy, National University of Singapore, Singapore 119078 (Singapore)

2013-10-25

Highlights: •Down-regulating FUT9 and ST3Gal4 expression blocks L1-induced neuronal differentiation of ESCs. •Up-regulating FUT9 and ST3Gal4 expression in L1-ESCs depends on the activation of PLCγ. •L1 promotes ESCs to differentiate into neuron through regulating cell surface glycosylation. -- Abstract: Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression.

Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

International Nuclear Information System (INIS)

Li, Ying; Huang, Xiaohua; An, Yue; Ren, Feng; Yang, Zara Zhuyun; Zhu, Hongmei; Zhou, Lei; He, Xiaowen; Schachner, Melitta; Xiao, Zhicheng; Ma, Keli; Li, Yali

2013-01-01

Highlights: •Down-regulating FUT9 and ST3Gal4 expression blocks L1-induced neuronal differentiation of ESCs. •Up-regulating FUT9 and ST3Gal4 expression in L1-ESCs depends on the activation of PLCγ. •L1 promotes ESCs to differentiate into neuron through regulating cell surface glycosylation. -- Abstract: Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression

Alba-domain proteins of Trypanosoma brucei are cytoplasmic RNA-binding proteins that interact with the translation machinery.

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Jan Mani

Full Text Available Trypanosoma brucei and related pathogens transcribe most genes as polycistronic arrays that are subsequently processed into monocistronic mRNAs. Expression is frequently regulated post-transcriptionally by cis-acting elements in the untranslated regions (UTRs. GPEET and EP procyclins are the major surface proteins of procyclic (insect midgut forms of T. brucei. Three regulatory elements common to the 3' UTRs of both mRNAs regulate mRNA turnover and translation. The glycerol-responsive element (GRE is unique to the GPEET 3' UTR and regulates its expression independently from EP. A synthetic RNA encompassing the GRE showed robust sequence-specific interactions with cytoplasmic proteins in electromobility shift assays. This, combined with column chromatography, led to the identification of 3 Alba-domain proteins. RNAi against Alba3 caused a growth phenotype and reduced the levels of Alba1 and Alba2 proteins, indicative of interactions between family members. Tandem-affinity purification and co-immunoprecipitation verified these interactions and also identified Alba4 in sub-stoichiometric amounts. Alba proteins are cytoplasmic and are recruited to starvation granules together with poly(A RNA. Concomitant depletion of all four Alba proteins by RNAi specifically reduced translation of a reporter transcript flanked by the GPEET 3' UTR. Pulldown of tagged Alba proteins confirmed interactions with poly(A binding proteins, ribosomal protein P0 and, in the case of Alba3, the cap-binding protein eIF4E4. In addition, Alba2 and Alba3 partially cosediment with polyribosomes in sucrose gradients. Alba-domain proteins seem to have exhibited great functional plasticity in the course of evolution. First identified as DNA-binding proteins in Archaea, then in association with nuclear RNase MRP/P in yeast and mammalian cells, they were recently described as components of a translationally silent complex containing stage-regulated mRNAs in Plasmodium. Our results are

MicroRNAs - A New Generation Molecular Targets for Treating Cellular Diseases

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Paulmurugan, Ramasamy

2013-01-01

MicroRNAs (miRNAs) are a unique class of non-coding, small RNAs, similar to mRNAs, transcribed by cells, but for entirely different reasons. While mRNAs are transcribed to code for proteins, miRNAs are produced to regulate the production of proteins from mRNAs. miRNAs are central components that tightly and temporally regulating gene expression in cells. Dysregulation of miRNAs expressions in cellular pathogenesis, including cancer, has been reported, and it clearly supports the importance of...

Regulation of cuticle-degrading subtilisin proteases from the entomopathogenic fungi, Lecanicillium spp: implications for host specificity.

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Bye, Natasha J; Charnley, A Keith

2008-01-01

The ability to produce cuticle-degrading proteases to facilitate host penetration does not distinguish per se entomopathogenic fungi from saprophytes. However, adapted pathogens may produce host-protein specific enzymes in response to cues. This possibility prompted an investigation of the regulation of isoforms of the subtilisin Pr1-like proteases from five aphid-pathogenic isolates of Lecanicillium spp. Significant differences were found in substrate specificity and regulation of Pr1-like proteases between isoforms of the same isolate and between different isolates. For example, the pI 8.6 isoform from KV71 was considerably more active against aphid than locust cuticle and was induced specifically by N-acetylglucosamine (NAG). Isoform pI 9.1 from the same isolate was only produced on insect cuticle while most other isoforms were more prominent on chitin containing substrates but not induced by NAG. The ability to regulate isoforms independently may allow production at critical points in host penetration. Appearance of proteases (not subtilisins) with pI 4.2 and 4.4 only on aphid cuticle was a possible link with host specificity of KV71. The absence of C or N metabolite repression in subtilisins from KV42 is unusual for pathogen proteases and may help to account for differences in virulence strategy between aphid-pathogenic isolates of Lecanicillium longisporum (unpublished data).

miRNAs in Normal and Malignant Hematopoiesis