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Showing papers on "Gene expression published in 2008"


Journal ArticleDOI
TL;DR: It is found that all five members of the microRNA-200 family were markedly downregulated in cells that had undergone EMT in response to transforming growth factor (TGF)-β or to ectopic expression of the protein tyrosine phosphatase Pez, suggesting that downregulation of themicroRNAs may be an important step in tumour progression.
Abstract: Epithelial to mesenchymal transition (EMT) facilitates tissue remodelling during embryonic development and is viewed as an essential early step in tumour metastasis. We found that all five members of the microRNA-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) and miR-205 were markedly downregulated in cells that had undergone EMT in response to transforming growth factor (TGF)-β or to ectopic expression of the protein tyrosine phosphatase Pez. Enforced expression of the miR-200 family alone was sufficient to prevent TGF-β-induced EMT. Together, these microRNAs cooperatively regulate expression of the E-cadherin transcriptional repressors ZEB1 (also known as δEF1) and SIP1 (also known as ZEB2), factors previously implicated in EMT and tumour metastasis. Inhibition of the microRNAs was sufficient to induce EMT in a process requiring upregulation of ZEB1 and/or SIP1. Conversely, ectopic expression of these microRNAs in mesenchymal cells initiated mesenchymal to epithelial transition (MET). Consistent with their role in regulating EMT, expression of these microRNAs was found to be lost in invasive breast cancer cell lines with mesenchymal phenotype. Expression of the miR-200 family was also lost in regions of metaplastic breast cancer specimens lacking E-cadherin. These data suggest that downregulation of the microRNAs may be an important step in tumour progression. MicroRNAs are small, non-coding RNAs that modulate gene expression post-transcriptionally. In metazoa, they act predominantly to inhibit translation of their specific targets, but they also typically cause a modest reduction in the level of their target mRNAs 1,2 . Hundreds of microRNAs have been identified in vertebrates, with varying patterns of expression that range from ubiquitous to highly tissue- or developmental-stage-restricted. In some cases, an individual microRNA can act as a developmental switch by regulating a key target mRNA 3 . Speculating that switching between cell phenotypes that occurs during EMT may be specified to some extent by microRNAs, we searched for microRNAs whose expression changed during EMT. To this end, we used an in vitro model of EMT, which was generated by stable transfection of Madin Darby canine kidney (MDCK) epithelial cells with the protein tyrosine phosphatase Pez (PTP-Pez). Overexpression of PTP-Pez caused MDCK cells to undergo EMT, as indicated by loss of E-cadherin expression, gain in expression of the mesenchymal markers fibronectin, ZEB1 and SIP1, loss of cohesion, induction of cell motility and a change in cell morphology

3,640 citations


Journal ArticleDOI
04 Sep 2008-Nature
TL;DR: The impact of micro RNAs on the proteome indicated that for most interactions microRNAs act as rheostats to make fine-scale adjustments to protein output.
Abstract: MicroRNAs are endogenous ∼23-nucleotide RNAs that can pair to sites in the messenger RNAs of protein-coding genes to downregulate the expression from these messages. MicroRNAs are known to influence the evolution and stability of many mRNAs, but their global impact on protein output had not been examined. Here we use quantitative mass spectrometry to measure the response of thousands of proteins after introducing microRNAs into cultured cells and after deleting mir-223 in mouse neutrophils. The identities of the responsive proteins indicate that targeting is primarily through seed-matched sites located within favourable predicted contexts in 3′ untranslated regions. Hundreds of genes were directly repressed, albeit each to a modest degree, by individual microRNAs. Although some targets were repressed without detectable changes in mRNA levels, those translationally repressed by more than a third also displayed detectable mRNA destabilization, and, for the more highly repressed targets, mRNA destabilization usually comprised the major component of repression. The impact of microRNAs on the proteome indicated that for most interactions microRNAs act as rheostats to make fine-scale adjustments to protein output. MicroRNAs can regulate gene expression by either inhibiting translation of a messenger RNA, or inducing its degradation. While previous studies have measured regulation at the mRNA level, it was unknown how much regulation occurred at the protein level. Now two groups led by David Bartel and Nikolaus Rajewsky have used variants of the technique known as SILAC (stable isotope labelling with amino acids in cell culture) to measure proteome-wide changes in protein level as a function of expression of endogenous and exogenous microRNAs. They find that while microRNAs can directly repress the translation of hundreds of genes, additional indirect effects result in changes in expression of thousands of genes. Many of the changes observed are less than twofold in magnitude, however, indicating either directly or indirectly, microRNAs can act as rheostats to fine-tune protein synthesis to match the needs of the cell at any given time. In one of two studies, a technique known as SILAC is used to measure, on a large scale, changes in protein level as a function of expression of endogenous and exogenous miRNAs. It is found that although miRNAs directly repress the translation of hundreds of genes, additional indirect effects result in changes in expression of thousands of genes.

3,562 citations


Journal ArticleDOI
04 Sep 2008-Nature
TL;DR: It is shown that a single miRNA can repress the production of hundreds of proteins, but that this repression is typically relatively mild, and the data suggest that a mi RNA can, by direct or indirect effects, tune protein synthesis from thousands of genes.
Abstract: Animal microRNAs (miRNAs) regulate gene expression by inhibiting translation and/or by inducing degradation of target messenger RNAs. It is unknown how much translational control is exerted by miRNAs on a genome-wide scale. We used a new proteomic approach to measure changes in synthesis of several thousand proteins in response to miRNA transfection or endogenous miRNA knockdown. In parallel, we quantified mRNA levels using microarrays. Here we show that a single miRNA can repress the production of hundreds of proteins, but that this repression is typically relatively mild. A number of known features of the miRNA-binding site such as the seed sequence also govern repression of human protein synthesis, and we report additional target sequence characteristics. We demonstrate that, in addition to downregulating mRNA levels, miRNAs also directly repress translation of hundreds of genes. Finally, our data suggest that a miRNA can, by direct or indirect effects, tune protein synthesis from thousands of genes.

3,412 citations


Journal ArticleDOI
TL;DR: This protocol describes ISH of digoxigenin-labeled antisense RNA probes to whole-mount zebrafish embryos and uses conditions that favor specific hybridization to complementary mRNA sequences in the tissue(s) expressing the corresponding gene.
Abstract: The in situ hybridization (ISH) technique allows the sites of expression of particular genes to be detected. This protocol describes ISH of digoxigenin-labeled antisense RNA probes to whole-mount zebrafish embryos. In our method, PCR-amplified sequence of a gene of interest is used as a template for the synthesis of an antisense RNA probe, which is labeled with digoxigenin-linked nucleotides. Embryos are fixed and permeabilized before being soaked in the digoxigenin-labeled probe. We use conditions that favor specific hybridization to complementary mRNA sequences in the tissue(s) expressing the corresponding gene. After washing away excess probe, hybrids are detected by immunohistochemistry using an alkaline phosphatase-conjugated antibody against digoxigenin and a chromogenic substrate. The whole procedure takes only 3 days and, because ISH conditions are the same for each probe tested, allows high throughput analysis of zebrafish gene expression during embryogenesis.

2,323 citations


Journal ArticleDOI
TL;DR: Loss of expression of the miR-200 family members may play a critical role in the repression of E-cadherin by ZEB1 and ZEB2 during EMT, thereby enhancing migration and invasion during cancer progression.

1,577 citations


Journal ArticleDOI
20 Jun 2008-Science
TL;DR: It is found that states of increased proliferation are associated with widespread reductions in the 3′UTR-based regulatory capacity of mRNAs, which is a characteristic of gene expression during immune cell activation and correlates with proliferation across diverse cell types and tissues.
Abstract: Messenger RNA (mRNA) stability, localization, and translation are largely determined by sequences in the 3' untranslated region (3'UTR). We found a conserved increase in expression of mRNAs terminating at upstream polyadenylation sites after activation of primary murine CD4+ T lymphocytes. This program, resulting in shorter 3'UTRs, is a characteristic of gene expression during immune cell activation and correlates with proliferation across diverse cell types and tissues. Forced expression of full-length 3'UTRs conferred reduced protein expression. In some cases the reduction in protein expression could be reversed by deletion of predicted microRNA target sites in the variably included region. Our data indicate that gene expression is coordinately regulated, such that states of increased proliferation are associated with widespread reductions in the 3'UTR-based regulatory capacity of mRNAs.

1,284 citations


Journal ArticleDOI
TL;DR: This study examines transgene expression and biodistribution of adeno-associated virus (AAV) pseudotyped 1-9 after tail vein (TV) injection in male mice and finds AAV9 had the best viral genome distribution and highest protein levels.

1,209 citations


Journal ArticleDOI
TL;DR: Findings reveal a new mode by which miRNAs may regulate gene expression, and identify a miRNA that targets promoter sequences and induces gene expression.
Abstract: Recent studies have shown that microRNA (miRNA) regulates gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report new evidence in which miRNA may also function to induce gene expression. By scanning gene promoters in silico for sequences complementary to known miRNAs, we identified a putative miR-373 target site in the promoter of E-cadherin. Transfection of miR-373 and its precursor hairpin RNA (pre-miR-373) into PC-3 cells readily induced E-cadherin expression. Knockdown experiments confirmed that induction of E-cadherin by pre-miR-373 required the miRNA maturation protein Dicer. Further analysis revealed that cold-shock domain-containing protein C2 (CSDC2), which possesses a putative miR-373 target site within its promoter, was also readily induced in response to miR-373 and pre-miR-373. Furthermore, enrichment of RNA polymerase II was detected at both E-cadherin and CSDC2 promoters after miR-373 transfection. Mismatch mutations to miR-373 indicated that gene induction was specific to the miR-373 sequence. Transfection of promoter-specific dsRNAs revealed that the concurrent induction of E-cadherin and CSDC2 by miR-373 required the miRNA target sites in both promoters. In conclusion, we have identified a miRNA that targets promoter sequences and induces gene expression. These findings reveal a new mode by which miRNAs may regulate gene expression.

1,194 citations


Journal ArticleDOI
21 May 2008-PLOS ONE
TL;DR: The studies indicate that replicative senescence of MSC preparations is a continuous process starting from the first passage onwards, which includes far reaching alterations in phenotype, differentiation potential, global gene expression patterns, and miRNA profiles that need to be considered for therapeutic application of M SC preparations.
Abstract: Mesenchymal stem cells (MSC) comprise a promising tool for cellular therapy. These cells are usually culture expanded prior to their application. However, a precise molecular definition of MSC and the sequel of long-term in vitro culture are yet unknown. In this study, we have addressed the impact of replicative senescence on human MSC preparations. Within 43 to 77 days of cultivation (7 to 12 passages), MSC demonstrated morphological abnormalities, enlargement, attenuated expression of specific surface markers, and ultimately proliferation arrest. Adipogenic differentiation potential decreased whereas the propensity for osteogenic differentiation increased. mRNA expression profiling revealed a consistent pattern of alterations in the global gene expression signature of MSC at different passages. These changes are not restricted to later passages, but are continuously acquired with increasing passages. Genes involved in cell cycle, DNA replication and DNA repair are significantly down-regulated in late passages. Genes from chromosome 4q21 were over-represented among differentially regulated transcripts. Differential expression of 10 genes has been verified in independent donor samples as well as in MSC that were isolated under different culture conditions. Furthermore, miRNA expression profiling revealed an up-regulation of hsa-mir-371, hsa-mir-369-5P, hsa-mir-29c, hsa-mir-499 and hsa-let-7f upon in vitro propagation. Our studies indicate that replicative senescence of MSC preparations is a continuous process starting from the first passage onwards. This process includes far reaching alterations in phenotype, differentiation potential, global gene expression patterns, and miRNA profiles that need to be considered for therapeutic application of MSC preparations.

1,016 citations


Journal ArticleDOI
11 Jan 2008-Cell
TL;DR: Current models for miRNA-mediated gene silencing are discussed, and a hypothesis to reconcile differences is formed that could help clarify the mechanisms behind the silencing of genes by miRNAs.

1,006 citations


Journal ArticleDOI
24 Jan 2008-Nature
TL;DR: This work characterize a nuclear protein interaction cascade mediating transduction of GA signals to the activity regulation of a light-responsive transcription factor in the presence of GA, and releases PIF3 from the negative effect of DELLA proteins.
Abstract: Light and gibberellins (GAs) mediate many essential and partially overlapping plant developmental processes. DELLA proteins are GA-signalling repressors that block GA-induced development. GA induces degradation of DELLA proteins via the ubiquitin/proteasome pathway, but light promotes accumulation of DELLA proteins by reducing GA levels. It was proposed that DELLA proteins restrain plant growth largely through their effect on gene expression. However, the precise mechanism of their function in coordinating GA signalling and gene expression remains unknown. Here we characterize a nuclear protein interaction cascade mediating transduction of GA signals to the activity regulation of a light-responsive transcription factor. In the absence of GA, nuclear-localized DELLA proteins accumulate to higher levels, interact with phytochrome-interacting factor 3 (PIF3, a bHLH-type transcription factor) and prevent PIF3 from binding to its target gene promoters and regulating gene expression, and therefore abrogate PIF3-mediated light control of hypocotyl elongation. In the presence of GA, GID1 proteins (GA receptors) elevate their direct interaction with DELLA proteins in the nucleus, trigger DELLA protein's ubiquitination and proteasome-mediated degradation, and thus release PIF3 from the negative effect of DELLA proteins.

Journal ArticleDOI
28 Mar 2008-Science
TL;DR: Common principles of transcription factor– and microRNA-mediated gene regulatory events are reviewed and conceptual differences in how these factors control gene expression are discussed.
Abstract: The properties of a cell are determined by the genetic information encoded in its genome. Understanding how such information is differentially and dynamically retrieved to define distinct cell types and cellular states is a major challenge facing molecular biology. Gene regulatory factors that control the expression of genomic information come in a variety of flavors, with transcription factors and microRNAs representing the most numerous gene regulatory factors in multicellular genomes. Here, I review common principles of transcription factor- and microRNA-mediated gene regulatory events and discuss conceptual differences in how these factors control gene expression.

Journal ArticleDOI
TL;DR: Findings point to these miRNAs as critical components of an SRF-dependent myogenic transcriptional circuit in orchestrating cardiac development, gene expression, and function.
Abstract: MicroRNAs (miRNAs) modulate gene expression by inhibiting mRNA translation and promoting mRNA degradation, but little is known of their potential roles in organ formation or function. miR-133a-1 and miR-133a-2 are identical, muscle-specific miRNAs that are regulated during muscle development by the SRF transcription factor. We show that mice lacking either miR-133a-1 or miR-133a-2 are normal, whereas deletion of both miRNAs causes lethal ventricular-septal defects in approximately half of double-mutant embryos or neonates; miR-133a double-mutant mice that survive to adulthood succumb to dilated cardiomyopathy and heart failure. The absence of miR-133a expression results in ectopic expression of smooth muscle genes in the heart and aberrant cardiomyocyte proliferation. These abnormalities can be attributed, at least in part, to elevated expression of SRF and cyclin D2, which are targets for repression by miR-133a. These findings reveal essential and redundant roles for miR-133a-1 and miR-133a-2 in orchestrating cardiac development, gene expression, and function and point to these miRNAs as critical components of an SRF-dependent myogenic transcriptional circuit.

Journal ArticleDOI
TL;DR: Results suggest epigenetic modification of the bdnf gene as a mechanism for isoform-specific gene readout during memory consolidation in the hippocampus suggests selective changes in gene expression are required for long-term memory formation.
Abstract: Long-term memory formation requires selective changes in gene expression. Here, we determined the contribution of chromatin remodeling to learning-induced changes in brain-derived neurotrophic factor (bdnf) gene expression in the adult hippocampus. Contextual fear learning induced differential regulation of exon-specific bdnf mRNAs (I, IV, VI, IX) that was associated with changes in bdnf DNA methylation and altered local chromatin structure. Infusions of zebularine (a DNA methyltransferase inhibitor) significantly altered bdnf DNA methylation and triggered changes in exon-specific bdnf mRNA levels, indicating that altered DNA methylation is sufficient to drive differential bdnf transcript regulation in the hippocampus. In addition, NMDA receptor blockade prevented memory-associated alterations in bdnf DNA methylation, resulting in a block of altered bdnf gene expression in hippocampus and a deficit in memory formation. These results suggest epigenetic modification of the bdnf gene as a mechanism for isoform-specific gene readout during memory consolidation.

Journal ArticleDOI
TL;DR: Global analysis of expressed genes in a naturally occurring microbial community revealed not only indigenous gene- and taxon-specific expression patterns but also gene categories undetected in previous DNA-based metagenomic surveys.
Abstract: Metagenomics is expanding our knowledge of the gene content, functional significance, and genetic variability in natural microbial communities. Still, there exists limited information concerning the regulation and dynamics of genes in the environment. We report here global analysis of expressed genes in a naturally occurring microbial community. We first adapted RNA amplification technologies to produce large amounts of cDNA from small quantities of total microbial community RNA. The fidelity of the RNA amplification procedure was validated with Prochlorococcus cultures and then applied to a microbial assemblage collected in the oligotrophic Pacific Ocean. Microbial community cDNAs were analyzed by pyrosequencing and compared with microbial community genomic DNA sequences determined from the same sample. Pyrosequencing-based estimates of microbial community gene expression compared favorably to independent assessments of individual gene expression using quantitative PCR. Genes associated with key metabolic pathways in open ocean microbial species—including genes involved in photosynthesis, carbon fixation, and nitrogen acquisition—and a number of genes encoding hypothetical proteins were highly represented in the cDNA pool. Genes present in the variable regions of Prochlorococcus genomes were among the most highly expressed, suggesting these encode proteins central to cellular processes in specific genotypes. Although many transcripts detected were highly similar to genes previously detected in ocean metagenomic surveys, a significant fraction (≈50%) were unique. Thus, microbial community transcriptomic analyses revealed not only indigenous gene- and taxon-specific expression patterns but also gene categories undetected in previous DNA-based metagenomic surveys.

Journal ArticleDOI
TL;DR: Combining single-transcript measurements with computational modeling indicates that low expression variation is achieved by transcribing genes using single transcription-initiation events that are clearly separated in time, rather than by transcriptional bursts.
Abstract: Understanding the kinetics of gene expression involves accurate quantitation of gene expression. This is now undertaken by quantifying nascent-RNA levels and relating this indication of transcriptional activity to mRNA abundance in single yeast cells. Combining these measurements with computational modeling indicates that the tested yeast housekeeping genes are probably expressed through single initiation events, whereas a SAGA-transcribed gene shows behavior consistent with transcriptional bursting. Proper execution of transcriptional programs is a key requirement of gene expression regulation, demanding accurate control of timing and amplitude. How precisely the transcription machinery fulfills this task is not known. Using an in situ hybridization approach that detects single mRNA molecules, we measured mRNA abundance and transcriptional activity within single Saccharomyces cerevisiae cells. We found that expression levels for particular genes are higher than initially reported and can vary substantially among cells. However, variability for most constitutively expressed genes is unexpectedly small. Combining single-transcript measurements with computational modeling indicates that low expression variation is achieved by transcribing genes using single transcription-initiation events that are clearly separated in time, rather than by transcriptional bursts. In contrast, PDR5, a gene regulated by the transcription coactivator complex SAGA, is expressed using transcription bursts, resulting in larger variation. These data directly demonstrate the existence of multiple expression modes used to modulate the transcriptome.

Journal ArticleDOI
02 Jul 2008-PLOS ONE
TL;DR: Analysis of miRNA array analyses for age-matched normal cervix and cervical cancer tissues and cloning and sequencing of a HPV16+ CaSki cell small RNA library indicate that downregulation ofmiR-143 and miR-145 and upregulation ofMiR-146a play a role in cervical carcinogenesis.
Abstract: MicroRNAs (miRNAs) play important roles in cancer development. By cloning and sequencing of a HPV16+ CaSki cell small RNA library, we isolated 174 miRNAs (including the novel miR-193c) which could be grouped into 46 different miRNA species, with miR-21, miR-24, miR-27a, and miR-205 being most abundant. We chose for further study 10 miRNAs according to their cloning frequency and associated their levels in 10 cervical cancer- or cervical intraepithelial neoplasia-derived cell lines. No correlation was observed between their expression with the presence or absence of an integrated or episomal HPV genome. All cell lines examined contained no detectable miR-143 and miR-145. HPV-infected cell lines expressed a different set of miRNAs when grown in organotypic raft cultured as compared to monolayer cell culture, including expression of miR-143 and miR-145. This suggests a correlation between miRNA expression and tissue differentiation. Using miRNA array analyses for age-matched normal cervix and cervical cancer tissues, in combination with northern blot verification, we identified significantly deregulated miRNAs in cervical cancer tissues, with miR-126, miR-143, and miR-145 downregulation and miR-15b, miR-16, miR-146a, and miR-155 upregulation. Functional studies showed that both miR-143 and miR-145 are suppressive to cell growth. When introduced into cell lines, miR-146a was found to promote cell proliferation. Collectively, our data indicate that downregulation of miR-143 and miR-145 and upregulation of miR-146a play a role in cervical carcinogenesis.

Journal ArticleDOI
TL;DR: The data suggest that miRNA antagonists comprised of LNA are valuable tools for identifying miRNA targets in vivo and for studying the biological role of miRNAs and miRNA-associated gene-regulatory networks in a physiological context.
Abstract: MicroRNA-122 (miR-122) is an abundant liver-specific miRNA, implicated in fatty acid and cholesterol metabolism as well as hepatitis C viral replication. Here, we report that a systemically administered 16-nt, unconjugated LNA (locked nucleic acid)-antimiR oligonucleotide complementary to the 5′ 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 de-repression within 24 h of 199 liver mRNAs showing significant enrichment for miR-122 seed matches in their 3′ UTRs. Expression profiling extended to 3 weeks after the last LNA-antimiR dose revealed that most of the changes in liver gene expression were normalized to saline control levels coinciding with normalized miR-122 and plasma cholesterol levels. Combined, these data suggest that miRNA antagonists comprised of LNA are valuable tools for identifying miRNA targets in vivo and for studying the biological role of miRNAs and miRNA-associated gene-regulatory networks in a physiological context.

Journal ArticleDOI
TL;DR: The findings suggest that miR‐15b and miR-16 could play a role in the development of MDR in gastric cancer cells at least in part by modulation of apoptosis via targeting BCL2.
Abstract: microRNAs are endogenous small noncoding RNAs that regulate gene expression negatively at posttranscriptional level. This latest addition to the complex gene regulatory circuitry revolutionizes our way to understanding physiological and pathological processes in the human body. Here we investigated the possible role of microRNAs in the development of multidrug resistance (MDR) in gastric cancer cells. microRNA expression profiling revealed a limited set of microRNAs with altered expression in multidrug- resistant gastric cancer cell line SGC7901/VCR compared to its parental SGC7901 cell line. Among the downregulated microRNAs are miR-15b and miR-16, members of miR-15/16 family, whose expression was further validated by qRT-PCR. In vitro drug sensitivity assay demonstrated that overexpression of miR-15b or miR-16 sensitized SGC7901/VCR cells to anticancer drugs whereas inhibition of them using antisense oligonucleotides conferred SGC7901 cells MDR. The downregulation of miR-15b and miR-16 in SGC7901/VCR cells was concurrent with the upregulation of Bcl-2 protein. Enforced mir-15b or miR-16 expression reduced Bcl-2 protein level and the luciferase activity of a BCL2 3′ untranslated region-based reporter construct in SGC7901/VCR cells, suggesting that BCL2 is a direct target of miR-15b and miR-16. Moreover, overexpression of miR-15b or miR-16 could sensitize SGC7901/VCR cells to VCR-induced apoptosis. Taken together, our findings suggest that miR-15b and miR-16 could play a role in the development of MDR in gastric cancer cells at least in part by modulation of apoptosis via targeting BCL2. © 2008 Wiley-Liss, Inc.

Journal ArticleDOI
07 Aug 2008-Nature
TL;DR: HSV-1 expresses at least two primary miRNA precursors in latently infected neurons that may facilitate the establishment and maintenance of viral latency by post-transcriptionally regulating viral gene expression.
Abstract: Herpesviruses are characterized by their ability to maintain life-long latent infections in their animal hosts. However, the mechanisms that allow establishment and maintenance of the latent state remain poorly understood. Herpes simplex virus 1 (HSV-1) establishes latency in neurons of sensory ganglia, where the only abundant viral gene product is a non-coding RNA, the latency associated transcript (LAT). Here we show that LAT functions as a primary microRNA (miRNA) precursor that encodes four distinct miRNAs in HSV-1 infected cells. One of these miRNAs, miR-H2-3p, is transcribed in an antisense orientation to ICP0-a viral immediate-early transcriptional activator that is important for productive HSV-1 replication and thought to have a role in reactivation from latency. We show that miR-H2-3p is able to reduce ICP0 protein expression, but does not significantly affect ICP0 messenger RNA levels. We also identified a fifth HSV-1 miRNA in latently infected trigeminal ganglia, miR-H6, which derives from a previously unknown transcript distinct from LAT. miR-H6 shows extended seed complementarity to the mRNA encoding a second HSV-1 transcription factor, ICP4, and inhibits expression of ICP4, which is required for expression of most HSV-1 genes during productive infection. These results may explain the reported ability of LAT to promote latency. Thus, HSV-1 expresses at least two primary miRNA precursors in latently infected neurons that may facilitate the establishment and maintenance of viral latency by post-transcriptionally regulating viral gene expression.

Journal ArticleDOI
TL;DR: It is demonstrated that microRNA expression in rheumatoid arthritis peripheral blood mononuclear cells mimics that of synovial tissue/fibroblasts and suggested that normal miR-146a function is critical for the regulation of tumor necrosis factor-α production.
Abstract: Introduction MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression via degradation or translational repression of their targeted mRNAs. It is known that aberrant microRNA expression can play important roles in cancer, but the role of microRNAs in autoimmune diseases is only beginning to emerge. In this study, the expression of selected microRNAs is examined in rheumatoid arthritis.

Journal ArticleDOI
TL;DR: The results presented in this article reveal the existence of a NAT capable of activating Zeb2 expression, explain the mechanism involved in this activation, and demonstrate that this NAT regulates E-cadherin expression.
Abstract: Expression of Snail1 in epithelial cells triggers an epithelial–mesenchymal transition (EMT). Here, we demonstrate that the synthesis of Zeb2, a transcriptional repressor of E-cadherin, is up-regulated after Snail1-induced EMT. Snail1 does not affect the synthesis of Zeb2 mRNA, but prevents the processing of a large intron located in its 5′-untranslated region (UTR). This intron contains an internal ribosome entry site (IRES) necessary for the expression of Zeb2. Maintenance of 5′-UTR Zeb2 intron is dependent on the expression of a natural antisense transcript (NAT) that overlaps the 5′ splice site in the intron. Ectopic overexpression of this NAT in epithelial cells prevents splicing of the Zeb2 5′-UTR, increases the levels of Zeb2 protein, and consequently down-regulates E-cadherin mRNA and protein. The relevance of these results is demonstrated by the strong association between NAT presence and conservation of the 5′-UTR intron in cells that have undergone EMT or in human tumors with low E-cadherin expression. Therefore, the results presented in this article reveal the existence of a NAT capable of activating Zeb2 expression, explain the mechanism involved in this activation, and demonstrate that this NAT regulates E-cadherin expression.

Journal ArticleDOI
TL;DR: Results challenge the proposal that SREBF1 is central for milk fat synthesis regulation and highlight a pivotal role for a concerted action among PPARG, PPARGC1A, and INSIG1.
Abstract: The molecular events associated with regulation of milk fat synthesis in the bovine mammary gland remain largely unknown. Our objective was to study mammary tissue mRNA expression via quantitative PCR of 45 genes associated with lipid synthesis (triacylglycerol and phospholipids) and secretion from the late pre-partum/non-lactating period through the end of subsequent lactation. mRNA expression was coupled with milk fatty acid (FA) composition and calculated indexes of FA desaturation and de novo synthesis by the mammary gland. Marked up-regulation and/or % relative mRNA abundance during lactation were observed for genes associated with mammary FA uptake from blood (LPL, CD36), intracellular FA trafficking (FABP3), long-chain (ACSL1) and short-chain (ACSS2) intracellular FA activation, de novo FA synthesis (ACACA, FASN), desaturation (SCD, FADS1), triacylglycerol synthesis (AGPAT6, GPAM, LPIN1), lipid droplet formation (BTN1A1, XDH), ketone body utilization (BDH1), and transcription regulation (INSIG1, PPARG, PPARGC1A). Change in SREBF1 mRNA expression during lactation, thought to be central for milk fat synthesis regulation, was ≤2-fold in magnitude, while expression of INSIG1, which negatively regulates SREBP activation, was >12-fold and had a parallel pattern of expression to PPARGC1A. Genes involved in phospholipid synthesis had moderate up-regulation in expression and % relative mRNA abundance. The mRNA abundance and up-regulation in expression of ABCG2 during lactation was markedly high, suggesting a biological role of this gene in milk synthesis/secretion. Weak correlations were observed between both milk FA composition and desaturase indexes (i.e., apparent SCD activity) with mRNA expression pattern of genes measured. A network of genes participates in coordinating milk fat synthesis and secretion. Results challenge the proposal that SREBF1 is central for milk fat synthesis regulation and highlight a pivotal role for a concerted action among PPARG, PPARGC1A, and INSIG1. Expression of SCD, the most abundant gene measured, appears to be key during milk fat synthesis. The lack of correlation between gene expression and calculated desaturase indexes does not support their use to infer mRNA expression or enzyme activity (e.g., SCD). Longitudinal mRNA expression allowed development of transcriptional regulation networks and an updated model of milk fat synthesis regulation.

Journal ArticleDOI
25 Sep 2008-Neuron
TL;DR: Interestingly, many components of the activity-dependent gene expression program are mutated in human cognitive disorders, which suggest that this program is essential for proper brain development and function.

Journal ArticleDOI
01 May 2008-RNA
TL;DR: It is demonstrated that the coding sequence of Dnmt3b mediates regulation by the miR-148 family, providing evidence that coding regions of human genes can be targeted by miRNAs, and that such a mechanism might play a role in determining the relative abundance of different splice variants.
Abstract: MicroRNAs (miRNAs) are small noncoding RNA molecules of 20–24 nucleotides that regulate gene expression. In animals, miRNAs form imperfect interactions with sequences in the 3′ Untranslated region (3′UTR) of mRNAs, causing translational inhibition and mRNA decay. In contrast, plant miRNAs mostly associate with protein coding regions. Here we show that human miR-148 represses DNA methyltransferase 3b (Dnmt3b) gene expression through a region in its coding sequence. This region is evolutionary conserved and present in the Dnmt3b splice variants Dnmt3b1, Dnmt3b2, and Dnmt3b4, but not in the abundantly expressed Dnmt3b3. Whereas overexpression of miR-148 results in decreased DNMT3b1 expression, short-hairpin RNA-mediated miR-148 repression leads to an increase in DNMT3b1 expression. Interestingly, mutating the putative miR-148 target site in Dnmt3b1 abolishes regulation by miR-148. Moreover, endogenous Dnmt3b3 mRNA, which lacks the putative miR-148 target site, is resistant to miR-148-mediated regulation. Thus, our results demonstrate that the coding sequence of Dnmt3b mediates regulation by the miR-148 family. More generally, we provide evidence that coding regions of human genes can be targeted by miRNAs, and that such a mechanism might play a role in determining the relative abundance of different splice variants.

Journal ArticleDOI
TL;DR: This work found that the mRNAs of hilD (master regulator of the SPI-1 invasion genes) and flhDC (flagellar master regulator) were bound by Hfq, and presented a generic strategy that is ideal for the systems-level analysis of the post-transcriptional regulons of RNA-binding proteins and for sRNA discovery in a wide range of bacteria.
Abstract: Recent advances in high-throughput pyrosequencing (HTPS) technology now allow a thorough analysis of RNA bound to cellular proteins, and, therefore, of post-transcriptional regulons. We used HTPS to discover the Salmonella RNAs that are targeted by the common bacterial Sm-like protein, Hfq. Initial transcriptomic analysis revealed that Hfq controls the expression of almost a fifth of all Salmonella genes, including several horizontally acquired pathogenicity islands (SPI-1, -2, -4, -5), two sigma factor regulons, and the flagellar gene cascade. Subsequent HTPS analysis of 350,000 cDNAs, derived from RNA co-immunoprecipitation (coIP) with epitope-tagged Hfq or control coIP, identified 727 mRNAs that are Hfq-bound in vivo. The cDNA analysis discovered new, small noncoding RNAs (sRNAs) and more than doubled the number of sRNAs known to be expressed in Salmonella to 64; about half of these are associated with Hfq. Our analysis explained aspects of the pleiotropic effects of Hfq loss-of-function. Specifically, we found that the mRNAs of hilD (master regulator of the SPI-1 invasion genes) and flhDC (flagellar master regulator) were bound by Hfq. We predicted that defective SPI-1 secretion and flagellar phenotypes of the hfq mutant would be rescued by overexpression of HilD and FlhDC, and we proved this to be correct. The combination of epitope-tagging and HTPS of immunoprecipitated RNA detected the expression of many intergenic chromosomal regions of Salmonella. Our approach overcomes the limited availability of high-density microarrays that have impeded expression-based sRNA discovery in microorganisms. We present a generic strategy that is ideal for the systems-level analysis of the post-transcriptional regulons of RNA-binding proteins and for sRNA discovery in a wide range of bacteria.

Journal ArticleDOI
TL;DR: The data show that the radial position within the nucleus can influence the expression of some, but not all, genes, compatible with the suggestion that re-localisation of genes relative to the peripheral zone of the nucleus could be used by metazoans to modulate theexpression of selected genes during development and differentiation.
Abstract: The spatial organisation of the genome in the nucleus has a role in the regulation of gene expression. In vertebrates, chromosomal regions with low gene-density are located close to the nuclear periphery. Correlations have also been made between the transcriptional state of some genes and their location near the nuclear periphery. However, a crucial issue is whether this level of nuclear organisation directly affects gene function, rather than merely reflecting it. To directly investigate whether proximity to the nuclear periphery can influence gene expression in mammalian cells, here we relocate specific human chromosomes to the nuclear periphery by tethering them to a protein of the inner nuclear membrane. We show that this can reversibly suppress the expression of some endogenous human genes located near the tethering sites, and even genes further away. However, the expression of many other genes is not detectably reduced and we show that location at the nuclear periphery is not incompatible with active transcription. The dampening of gene expression around the nuclear periphery is dependent on the activity of histone deacetylases. Our data show that the radial position within the nucleus can influence the expression of some, but not all, genes. This is compatible with the suggestion that re-localisation of genes relative to the peripheral zone of the nucleus could be used by metazoans to modulate the expression of selected genes during development and differentiation.

Journal ArticleDOI
TL;DR: This study evaluated the expression of eight commonly used housekeeping genes as a function of stage and hormone/toxicant exposure during development, and by tissue type and sex in adult fish, and revealed sex differences that are gene- and tissue-specific, and treatment effects that are Gene-, vehicle- and ligand-specific.
Abstract: Research using the zebrafish model has experienced a rapid growth in recent years. Although real-time reverse transcription PCR (QPCR), normalized to an internal reference ("housekeeping") gene, is a frequently used method for quantifying gene expression changes in zebrafish, many commonly used housekeeping genes are known to vary with experimental conditions. To identify housekeeping genes that are stably expressed under different experimental conditions, and thus suitable as normalizers for QPCR in zebrafish, the present study evaluated the expression of eight commonly used housekeeping genes as a function of stage and hormone/toxicant exposure during development, and by tissue type and sex in adult fish. QPCR analysis was used to quantify mRNA levels of bactin1, tubulin alpha 1(tuba1), glyceraldehyde-3-phosphate dehydrogenase (gapdh), glucose-6-phosphate dehydrogenase (g6pd), TATA-box binding protein (tbp), beta-2-microglobulin (b2m), elongation factor 1 alpha (elfa), and 18s ribosomal RNA (18s) during development (2 – 120 hr postfertilization, hpf); in different tissue types (brain, eye, liver, heart, muscle, gonads) of adult males and females; and after treatment of embryos/larvae (24 – 96 hpf) with commonly used vehicles for administration and agents that represent known environmental endocrine disruptors. All genes were found to have some degree of variability under the conditions tested here. Rank ordering of expression stability using geNorm analysis identified 18s, b2m, and elfa as most stable during development and across tissue types, while gapdh, tuba1, and tpb were the most variable. Following chemical treatment, tuba1, bactin1, and elfa were the most stably expressed whereas tbp, 18s, and b2m were the least stable. Data also revealed sex differences that are gene- and tissue-specific, and treatment effects that are gene-, vehicle- and ligand-specific. When the accuracy of QPCR analysis was tested using different reference genes to measure suppression of cyp19a1b by an estrogen receptor antagonist and induction of cyp1a by an arylhydrocarbon receptor agonist, the direction and magnitude of effects with stable and unstable genes differed. This study provides data that can be expected to aid zebrafish researchers in their initial choice of housekeeping genes for future studies, but underlines the importance of further validating housekeeping genes for each new experimental paradigm and fish species.

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TL;DR: It is shown that microRNA 132 (miR132) is an activity-dependent rapid response gene regulated by the cAMP response element-binding (CREB) protein pathway and proposed that the miR132–p250GAP pathway plays a key role in activity- dependent structural and functional plasticity.
Abstract: Activity-regulated gene expression is believed to play a key role in the development and refinement of neuronal circuitry. Nevertheless, the transcriptional networks that regulate synapse growth and plasticity remain largely uncharacterized. Here, we show that microRNA 132 (miR132) is an activity-dependent rapid response gene regulated by the cAMP response element-binding (CREB) protein pathway. Introduction of miR132 into hippocampal neurons enhanced dendrite morphogenesis whereas inhibition of miR132 by 2'O-methyl RNA antagonists blocked these effects. Furthermore, neuronal activity inhibited translation of p250GAP, a miR132 target, and siRNA-mediated knockdown of p250GAP mimicked miR132-induced dendrite growth. Experiments using dominant-interfering mutants suggested that Rac signaling is downstream of miR132 and p250GAP. We propose that the miR132-p250GAP pathway plays a key role in activity-dependent structural and functional plasticity.

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TL;DR: An improved northern blot method that enhances detection of small RNA molecules including regulatory species such as microRNA (miRNA), short-interfering RNA (siRNA) and Piwi-interacting RNA by up to 50-fold is described.
Abstract: This protocol describes an improved northern blot method that enhances detection of small RNA molecules (<40 nt) including regulatory species such as microRNA (miRNA), short-interfering RNA (siRNA) and Piwi-interacting RNA. Northern blot analysis involves the separation of RNA molecules by denaturing gel electrophoresis followed by transfer and cross-linking of the separated molecules to nylon membrane. RNA of interest is then detected by hybridization with labeled complementary nucleic acid probes. We have replaced conventional UV-cross-linking of RNA to nylon membranes with a novel, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated, chemical cross-linking step that enhances detection of small RNA by up to 50-fold. This requires no specialized equipment, is relatively inexpensive and is technically straightforward. Northern blotting can be done in 2 d, but detection of a specific RNA can vary from minutes to days. Although chemical cross-linking takes longer (15 min to 2 h) than UV cross-linking, improved sensitivity means shorter periods of exposure are required to detect signal after hybridization.