Transcriptional analysis of product-concentration driven changes in cellular programs of recombinant Clostridium acetobutylicumstrains.

PubMed

Tummala, Seshu B; Junne, Stefan G; Paredes, Carlos J; Papoutsakis, Eleftherios T

2003-12-30

Antisense RNA (asRNA) downregulation alters protein expression without changing the regulation of gene expression. Downregulation of primary metabolic enzymes possibly combined with overexpression of other metabolic enzymes may result in profound changes in product formation, and this may alter the large-scale transcriptional program of the cells. DNA-array based large-scale transcriptional analysis has the potential to elucidate factors that control cellular fluxes even in the absence of proteome data. These themes are explored in the study of large-scale transcriptional analysis programs and the in vivo primary-metabolism fluxes of several related recombinant C. acetobutylicum strains: C. acetobutylicum ATCC 824(pSOS95del) (plasmid control; produces high levels of butanol snd acetone), 824(pCTFB1AS) (expresses antisense RNA against CoA transferase (ctfb1-asRNA); produces very low levels of butanol and acetone), and 824(pAADB1) (expresses ctfb1-asRNA and the alcohol-aldehyde dahydrogenase gene (aad); produce high alcohol and low acetone levels). DNA-array based transcriptional analysis revealed that the large changes in product concentrations (snd notably butanol concentration) due to ctfb1-asRNA expression alone and in combination with aad overexpression resulted in dramatic changes of the cellular transcriptome. Cluster analysis and gene expression patterns of established and putative operons involved in stress response, motility, sporulation, and fatty-acid biosynthesis indicate that these simple genetic changes dramatically alter the cellular programs of C. acetobutylicum. Comparison of gene expression and flux analysis data may point to possible flux-controling steps and suggest unknown regulatory mechanisms. Copyright 2003; Wiley Periodicals, Inc.

DNA and RNA editing of retrotransposons accelerate mammalian genome evolution.

PubMed

Knisbacher, Binyamin A; Levanon, Erez Y

2015-04-01

Genome evolution is commonly viewed as a gradual process that is driven by random mutations that accumulate over time. However, DNA- and RNA-editing enzymes have been identified that can accelerate evolution by actively modifying the genomically encoded information. The apolipoprotein B mRNA editing enzymes, catalytic polypeptide-like (APOBECs) are potent restriction factors that can inhibit retroelements by cytosine-to-uridine editing of retroelement DNA after reverse transcription. In some cases, a retroelement may successfully integrate into the genome despite being hypermutated. Such events introduce unique sequences into the genome and are thus a source of genomic innovation. adenosine deaminases that act on RNA (ADARs) catalyze adenosine-to-inosine editing in double-stranded RNA, commonly formed by oppositely oriented retroelements. The RNA editing confers plasticity to the transcriptome by generating many transcript variants from a single genomic locus. If the editing produces a beneficial variant, the genome may maintain the locus that produces the RNA-edited transcript for its novel function. Here, we discuss how these two powerful editing mechanisms, which both target inserted retroelements, facilitate expedited genome evolution. © 2015 New York Academy of Sciences.

Global RNA association with the transcriptionally active chromosome of chloroplasts.

PubMed

Lehniger, Marie-Kristin; Finster, Sabrina; Melonek, Joanna; Oetke, Svenja; Krupinska, Karin; Schmitz-Linneweber, Christian

2017-10-01

Processed chloroplast RNAs are co-enriched with preparations of the chloroplast transcriptionally active chromosome. Chloroplast genomes are organized as a polyploid DNA-protein structure called the nucleoid. Transcriptionally active chloroplast DNA together with tightly bound protein factors can be purified by gel filtration as a functional entity called the transcriptionally active chromosome (TAC). Previous proteomics analyses of nucleoids and of TACs demonstrated a considerable overlap in protein composition including RNA binding proteins. Therefore the RNA content of TAC preparations from Nicotiana tabacum was determined using whole genome tiling arrays. A large number of chloroplast RNAs was found to be associated with the TAC. The pattern of RNAs attached to the TAC consists of RNAs produced by different chloroplast RNA polymerases and differs from the pattern of RNA found in input controls. An analysis of RNA splicing and RNA editing of selected RNA species demonstrated that TAC-associated RNAs are processed to a similar extent as the RNA in input controls. Thus, TAC fractions contain a specific subset of the processed chloroplast transcriptome.

High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme.

PubMed Central

Shields, T P; Mollova, E; Ste Marie, L; Hansen, M R; Pardi, A

1999-01-01

An improved method is presented for the preparation of milligram quantities of homogenous-length RNAs suitable for nuclear magnetic resonance or X-ray crystallographic structural studies. Heterogeneous-length RNA transcripts are processed with a hammerhead ribozyme to yield homogenous-length products that are then readily purified by anion exchange high-performance liquid chromatography. This procedure eliminates the need for denaturing polyacrylamide gel electrophoresis, which is the most laborious step in the standard procedure for large-scale production of RNA by in vitro transcription. The hammerhead processing of the heterogeneous-length RNA transcripts also substantially improves the overall yield and purity of the desired RNA product. PMID:10496226

The structure of an RNAi polymerase links RNA silencing and transcription.

PubMed

Salgado, Paula S; Koivunen, Minni R L; Makeyev, Eugene V; Bamford, Dennis H; Stuart, David I; Grimes, Jonathan M

2006-12-01

RNA silencing refers to a group of RNA-induced gene-silencing mechanisms that developed early in the eukaryotic lineage, probably for defence against pathogens and regulation of gene expression. In plants, protozoa, fungi, and nematodes, but apparently not insects and vertebrates, it involves a cell-encoded RNA-dependent RNA polymerase (cRdRP) that produces double-stranded RNA triggers from aberrant single-stranded RNA. We report the 2.3-A resolution crystal structure of QDE-1, a cRdRP from Neurospora crassa, and find that it forms a relatively compact dimeric molecule, each subunit of which comprises several domains with, at its core, a catalytic apparatus and protein fold strikingly similar to the catalytic core of the DNA-dependent RNA polymerases responsible for transcription. This evolutionary link between the two enzyme types suggests that aspects of RNA silencing in some organisms may recapitulate transcription/replication pathways functioning in the ancient RNA-based world.

Maximizing RNA yield from archival renal tumors and optimizing gene expression analysis.

PubMed

Glenn, Sean T; Head, Karen L; Teh, Bin T; Gross, Kenneth W; Kim, Hyung L

2010-01-01

Formalin-fixed, paraffin-embedded tissues are widely available for gene expression analysis using TaqMan PCR. Five methods, including 4 commercial kits, for recovering RNA from paraffin-embedded renal tumor tissue were compared. The MasterPure kit from Epicentre produced the highest RNA yield. However, the difference in RNA yield between the kit from Epicenter and Invitrogen's TRIzol method was not significant. Using the top 3 RNA isolation methods, the manufacturers' protocols were modified to include an overnight Proteinase K digestion. Overnight protein digestion resulted in a significant increase in RNA yield. To optimize the reverse transcription reaction, conventional reverse transcription with random oligonucleotide primers was compared to reverse transcription using primers specific for genes of interest. Reverse transcription using gene-specific primers significantly increased the quantity of cDNA detectable by TaqMan PCR. Therefore, expression profiling of formalin-fixed, paraffin-embedded tissue using TaqMan qPCR can be optimized by using the MasterPure RNA isolation kit modified to include an overnight Proteinase K digestion and gene-specific primers during the reverse transcription.

Forced Complementation between Subgenomic RNAs: Does Human Immunodeficiency Type 1 Virus Reverse Transcription Occur in Viral Core, Cytoplasm, or Early Endosome?

PubMed Central

Han, Weining; Li, Yuejin; Bagaya, Bernard S.; Tian, Meijuan; Chamanian, Mastooreh; Zhu, Chuanwu; Shen, Jie; Gao, Yong

2016-01-01

Although the process of reverse transcription is well elucidated, it remains unclear if viral core disruption provides a more cellular or viral milieu for HIV-1 reverse transcription. We have devised a method to require mixing of viral cores or core constituents to produce infectious progeny virus by a bipartite subgenomic RNA (sgRNA) system, in which HIV-1 cplt_R/U5/gag/Δpol and nfl sgRNAs are complementary to each other and when together can complete viral reverse transcription. Only the heterodiploid virus containing both the nfl and cplt_R/U5/gag/Δpol sgRNAs can complete reverse transcription and propagate infectious virus upon de novo infection. Dual exposure of U87.CD4.CXCR4 cells with high titers of the homodimeric nfl and cplt_R/U5/gag/Δpol virus particles did not result in productive virus infection. On the other hand, in early endosomes, the HIV-1 sgRNAs released from viral cores can retain function and complete the reverse transcription and result in productive infection. These findings confirm the assumptions that, in natural infection, HIV-1 cores, and likely other retrovirus cores, remain largely intact and do not mix/fuse in the cytoplasm during the reverse transcription process, and circulating cytoplasmic HIV-1 sgRNA (produced through transfection) could not help the complementary sgRNA in the viral core to complement the reverse transcription process. PMID:27239643

Pea chloroplast tRNA(Lys) (UUU) gene: transcription and analysis of an intron-containing gene.

PubMed

Boyer, S K; Mullet, J E

1988-07-01

The pea chloroplast trnK gene which encodes tRNA(Lys) (UUU) was sequenced. TrnK is located 210 bp upstream from the promoter of psbA and immediately downstream from the 3'-end of rbcL. The gene is transcribed from the same DNA strand as psbA and rbcL. A 2447 bp intron with class II features is located in the trnK anticodon loop. The intron contains a 506 amino acid open reading frame which could encode an RNA maturase. The primary transcript of trnK is 2.9 kb long; its 5'-end was identified as a site of transcription initiation by in vitro transcription experiments. The 5'-terminus is adjacent to DNA sequences previously identified as transcription promoter elements. The most abundant trnK transcript is 2.5 kb long with termini corresponding to the 5' and 3' ends of the trnK exons. Intron specific RNAs were not detected. This suggests that RNA processing which produces tRNA(Lys) leads to rapid degradation of intron sequences.

RNA polymerase activity is associated with viral particles isolated from Leishmania braziliensis subsp. guyanensis.

PubMed Central

Widmer, G; Keenan, M C; Patterson, J L

1990-01-01

Viral particles purified from species of the protozoan parasite Leishmania braziliensis subsp. guyanensis by centrifugation in CsCl gradients were examined for the presence of viral polymerase. We demonstrated that RNA-dependent RNA polymerase is associated with viral particles. Viral transcription was studied in vitro with pulse-chase experiments and by assaying the RNase sensitivity of the viral transcripts. Viral polymerase synthesized full-length transcripts within 1 h. Double-strained, genome-length, and single-stranded RNAs were produced in this system. The nature of the RNA extracted from virions was also tested by RNase protection assays; both single-stranded and double-stranded RNAs were found. Images PMID:2370680

Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products

PubMed Central

Ludwig, Linda B; Ambrus, Julian L; Krawczyk, Kristie A; Sharma, Sanjay; Brooks, Stephen; Hsiao, Chiu-Bin; Schwartz, Stanley A

2006-01-01

Background While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ) protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). Results Inspection of published sequences revealed a potential transcription initiator element (INR) situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR) suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s) could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s) were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK) sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP) sequences identified HAPs from HIV+ human peripheral blood lymphocytes. Conclusion HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The novel HAPs are encoded in a region of the LTR that has already been shown to be deleted in some HIV-infected long-term survivors and represent new potential targets for vaccine development. PMID:17090330

Sequencing the transcriptome of milk production: milk trumps mammary tissue.

PubMed

Lemay, Danielle G; Hovey, Russell C; Hartono, Stella R; Hinde, Katie; Smilowitz, Jennifer T; Ventimiglia, Frank; Schmidt, Kimberli A; Lee, Joyce W S; Islas-Trejo, Alma; Silva, Pedro Ivo; Korf, Ian; Medrano, Juan F; Barry, Peter A; German, J Bruce

2013-12-12

Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, this assay has not been adequately validated in primates. Thus, the objectives of the current study were to assess the suitability of lactating rhesus macaques as a model for lactating humans and to determine whether RNA extracted from milk fractions is representative of RNA extracted from mammary tissue for the purpose of studying the transcriptome of milk-producing cells. We confirmed that macaque milk contains cytoplasmic crescents and that ample high-quality RNA can be obtained for sequencing. Using RNA sequencing, RNA extracted from macaque milk fat and milk cell fractions more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from whole mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat, whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from milk-producing mammary epithelial cells. RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells in a non-human primate. However, this sample type clearly requires protocols that minimize RNA degradation. Overall, we validated the use of RNA extracted from human and macaque milk and provided evidence to support the use of lactating macaques as a model for human lactation.

Investigation of RNA Synthesis Using 5-Bromouridine Labelling and Immunoprecipitation.

PubMed

Kofoed, Rikke H; Betzer, Cristine; Lykke-Andersen, Søren; Molska, Ewa; Jensen, Poul H

2018-05-03

When steady state RNA levels are compared between two conditions, it is not possible to distinguish whether changes are caused by alterations in production or degradation of RNA. This protocol describes a method for measurement of RNA production, using 5-Bromouridine labelling of RNA followed by immunoprecipitation, which enables investigation of RNA synthesized within a short timeframe (e.g., 1 h). The advantage of 5-Bromouridine-labelling and immunoprecipitation over the use of toxic transcriptional inhibitors, such as α-amanitin and actinomycin D, is that there are no or very low effects on cell viability during short-term use. However, because 5-Bromouridine-immunoprecipitation only captures RNA produced within the short labelling time, slowly produced as well as rapidly degraded RNA can be difficult to measure by this method. The 5-Bromouridine-labelled RNA captured by 5-Bromouridine-immunoprecipitation can be analyzed by reverse transcription, quantitative polymerase chain reaction, and next generation sequencing. All types of RNA can be investigated, and the method is not limited to measuring mRNA as is presented in this example.

Circular RNAs (circRNAs) in Health and Disease

PubMed Central

Haque, Shahnaz; Harries, Lorna W.

2017-01-01

Splicing events do not always produce a linear transcript. Circular RNAs (circRNAs) are a class of RNA that are emerging as key new members of the gene regulatory milieu, which are produced by back-splicing events within genes. In circRNA formation, rather than being spliced in a linear fashion, exons can be circularised by use of the 3′ acceptor splice site of an upstream exon, leading to the formation of a circular RNA species. circRNAs have been demonstrated across species and have the potential to present genetic information in new orientations distinct from their parent transcript. The importance of these RNA players in gene regulation and normal cellular homeostasis is now beginning to be recognised. They have several potential modes of action, from serving as sponges for micro RNAs and RNA binding proteins, to acting as transcriptional regulators. In accordance with an important role in the normal biology of the cell, perturbations of circRNA expression are now being reported in association with disease. Furthermore, the inherent stability of circRNAs conferred by their circular structure and exonuclease resistance, and their expression in blood and other peripheral tissues in association with endosomes and microvesicles, renders them excellent candidates as disease biomarkers. In this review, we explore the state of knowledge on this exciting class of transcripts in regulating gene expression and discuss their emerging role in health and disease. PMID:29182528

The RNA synthesis machinery of negative-stranded RNA viruses

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

Ortín, Juan, E-mail: jortin@cnb.csic.es; Martín-Benito, Jaime, E-mail: jmartinb@cnb.csic.es

The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure ofmore » their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes.« less

Enzymes Involved in Post-transcriptional RNA Metabolism in Gram-negative bacteria

PubMed Central

Mohanty, Bijoy K.

2018-01-01

Gene expression in Gram-negative bacteria is regulated at many levels, including transcription initiation, RNA processing, RNA/RNA interactions, mRNA decay, and translational controls involving enzymes that alter translational efficiency. In this chapter we discuss the various enzymes that control transcription, translation and RNA stability through RNA processing and degradation. RNA processing is essential to generate functional RNAs, while degradation helps control the steady-state level of each individual transcript. For example, all the pre-tRNAs are transcribed with extra nucleotides at both their 5′ and 3′ termini, which are subsequently processed to produce mature tRNAs that can be aminoacylated. Similarly, rRNAs that are transcribed as part of a 30S polycistronic transcript, are matured to individual 16S, 23S and 5S rRNAs. Decay of mRNAs plays a key role in gene regulation through controlling the steady-state level of each transcript, which is essential for maintaining appropriate protein levels. In addition, degradation of both translated and non-translated RNAs recycles nucleotides to facilitate new RNA synthesis. To carry out all these reactions Gram-negative bacteria employ a large number of endonucleases, exonucleases, RNA helicases, and poly(A) polymerase as well as proteins that regulate the catalytic activity of particular ribonucleases. Under certain stress conditions an additional group of specialized endonucleases facilitate the cell’s ability to adapt and survive. Many of the enzymes, such as RNase E, RNase III, polynucleotide phosphorylase, RNase R, and poly(A) polymerase I participate in multiple RNA processing and decay pathways. PMID:29676246

The Role of CYP3A4 mRNA Transcript with Shortened 3′-Untranslated Region in Hepatocyte Differentiation, Liver Development, and Response to Drug InductionS⃞

PubMed Central

Li, Dan; Gaedigk, Roger; Hart, Steven N.; Leeder, J. Steven

2012-01-01

Cytochrome P450 3A4 (CYP3A4) metabolizes more than 50% of prescribed drugs. The expression of CYP3A4 changes during liver development and may be affected by the administration of some drugs. Alternative mRNA transcripts occur in more than 90% of human genes and are frequently observed in cells responding to developmental and environmental signals. Different mRNA transcripts may encode functionally distinct proteins or contribute to variability of mRNA stability or protein translation efficiency. The purpose of this study was to examine expression of alternative CYP3A4 mRNA transcripts in hepatocytes in response to developmental signals and drugs. cDNA cloning and RNA sequencing (RNA-Seq) were used to identify CYP3A4 mRNA transcripts. Three transcripts were found in HepaRG cells and liver tissues: one represented a canonical mRNA with full-length 3′-untranslated region (UTR), one had a shorter 3′-UTR, and one contained partial intron-6 retention. The alternative mRNA transcripts were validated by either rapid amplification of cDNA 3′-end or endpoint polymerase chain reaction (PCR). Quantification of the transcripts by RNA-Seq and real time quantitative PCR revealed that the CYP3A4 transcript with shorter 3′-UTR was preferentially expressed in developed livers, differentiated hepatocytes, and in rifampicin- and phenobarbital-induced hepatocytes. The CYP3A4 transcript with shorter 3′-UTR was more stable and produced more protein compared with the CYP3A4 transcript with canonical 3′-UTR. We conclude that the 3′-end processing of CYP3A4 contributes to the quantitative regulation of CYP3A4 gene expression through alternative polyadenylation, which may serve as a regulatory mechanism explaining changes of CYP3A4 expression and activity during hepatocyte differentiation and liver development and in response to drug induction. PMID:21998292

The allosteric HIV-1 integrase inhibitor BI-D affects virion maturation but does not influence packaging of a functional RNA genome.

PubMed

van Bel, Nikki; van der Velden, Yme; Bonnard, Damien; Le Rouzic, Erwann; Das, Atze T; Benarous, Richard; Berkhout, Ben

2014-01-01

The viral integrase (IN) is an essential protein for HIV-1 replication. IN inserts the viral dsDNA into the host chromosome, thereby aided by the cellular co-factor LEDGF/p75. Recently a new class of integrase inhibitors was described: allosteric IN inhibitors (ALLINIs). Although designed to interfere with the IN-LEDGF/p75 interaction to block HIV DNA integration during the early phase of HIV-1 replication, the major impact was surprisingly found on the process of virus maturation during the late phase, causing a reverse transcription defect upon infection of target cells. Virus particles produced in the presence of an ALLINI are misformed with the ribonucleoprotein located outside the virus core. Virus assembly and maturation are highly orchestrated and regulated processes in which several viral proteins and RNA molecules closely interact. It is therefore of interest to study whether ALLINIs have unpredicted pleiotropic effects on these RNA-related processes. We confirm that the ALLINI BI-D inhibits virus replication and that the produced virus is non-infectious. Furthermore, we show that the wild-type level of HIV-1 genomic RNA is packaged in virions and these genomes are in a dimeric state. The tRNAlys3 primer for reverse transcription was properly placed on this genomic RNA and could be extended ex vivo. In addition, the packaged reverse transcriptase enzyme was fully active when extracted from virions. As the RNA and enzyme components for reverse transcription are properly present in virions produced in the presence of BI-D, the inhibition of reverse transcription is likely to reflect the mislocalization of the components in the aberrant virus particle.

Suppressor of sable [Su(s)] and Wdr82 down-regulate RNA from heat-shock-inducible repetitive elements by a mechanism that involves transcription termination

PubMed Central

Brewer-Jensen, Paul; Wilson, Carrie B.; Abernethy, John; Mollison, Lonna; Card, Samantha

2016-01-01

Although RNA polymerase II (Pol II) productively transcribes very long genes in vivo, transcription through extragenic sequences often terminates in the promoter-proximal region and the nascent RNA is degraded. Mechanisms that induce early termination and RNA degradation are not well understood in multicellular organisms. Here, we present evidence that the suppressor of sable [su(s)] regulatory pathway of Drosophila melanogaster plays a role in this process. We previously showed that Su(s) promotes exosome-mediated degradation of transcripts from endogenous repeated elements at an Hsp70 locus (Hsp70-αβ elements). In this report, we identify Wdr82 as a component of this process and show that it works with Su(s) to inhibit Pol II elongation through Hsp70-αβ elements. Furthermore, we show that the unstable transcripts produced during this process are polyadenylated at heterogeneous sites that lack canonical polyadenylation signals. We define two distinct regions that mediate this regulation. These results indicate that the Su(s) pathway promotes RNA degradation and transcription termination through a novel mechanism. PMID:26577379

Diphosphates at the 5' end of the positive strand of yeast L-A double-stranded RNA virus as a molecular self-identity tag.

PubMed

Fujimura, Tsutomu; Esteban, Rosa

2016-10-01

The 5'end of RNA conveys important information on self-identity. In mammalian cells, double-stranded RNA (dsRNA) with 5'di- or triphosphates generated during virus infection is recognized as foreign and elicits the host innate immune response. Here, we analyze the 5' ends of the dsRNA genome of the yeast L-A virus. The positive strand has largely diphosphates with a minor amount of triphosphates, while the negative strand has only diphosphates. Although the virus can produce capped transcripts by cap snatching, neither strand carried a cap structure, suggesting that only non-capped transcripts serve as genomic RNA for encapsidation. We also found that the 5' diphosphates of the positive but not the negative strand within the dsRNA genome are crucial for transcription in vitro. Furthermore, the presence of a cap structure in the dsRNA abrogated its template activity. Given that the 5' diphosphates of the transcripts are also essential for cap acquisition and that host cytosolic RNAs (mRNA, rRNA, and tRNA) are uniformly devoid of 5' pp-structures, the L-A virus takes advantage of its 5' terminal diphosphates, using them as a self-identity tag to propagate in the host cytoplasm. © 2016 John Wiley & Sons Ltd.

Sequencing the transcriptome of milk production: milk trumps mammary tissue

PubMed Central

2013-01-01

Background Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, this assay has not been adequately validated in primates. Thus, the objectives of the current study were to assess the suitability of lactating rhesus macaques as a model for lactating humans and to determine whether RNA extracted from milk fractions is representative of RNA extracted from mammary tissue for the purpose of studying the transcriptome of milk-producing cells. Results We confirmed that macaque milk contains cytoplasmic crescents and that ample high-quality RNA can be obtained for sequencing. Using RNA sequencing, RNA extracted from macaque milk fat and milk cell fractions more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from whole mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat, whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from milk-producing mammary epithelial cells. Conclusions RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells in a non-human primate. However, this sample type clearly requires protocols that minimize RNA degradation. Overall, we validated the use of RNA extracted from human and macaque milk and provided evidence to support the use of lactating macaques as a model for human lactation. PMID:24330573

High serum levels of pregenomic RNA reflect frequently failing reverse transcription in hepatitis B virus particles.

PubMed

Prakash, Kasthuri; Rydell, Gustaf E; Larsson, Simon B; Andersson, Maria; Norkrans, Gunnar; Norder, Heléne; Lindh, Magnus

2018-05-15

Hepatocytes infected by hepatitis B virus (HBV) produce different HBV RNA species, including pregenomic RNA (pgRNA), which is reverse transcribed during replication. Particles containing HBV RNA are present in serum of infected individuals, and quantification of this HBV RNA could be clinically useful. In a retrospective study of 95 patients with chronic HBV infection, we characterised HBV RNA in serum in terms of concentration, particle association and sequence. HBV RNA was detected by real-time PCR at levels almost as high as HBV DNA. The HBV RNA was protected from RNase and it was found in particles of similar density as particles containing HBV DNA after fractionation on a Nycodenz gradient. Sequencing the epsilon region of the RNA did not reveal mutations that would preclude its binding to the viral polymerase before encapsidation. Specific quantification of precore RNA and pgRNA by digital PCR showed almost seven times lower ratio of precore RNA/pgRNA in serum than in liver tissue, which corresponds to poorer encapsidation of this RNA as compared with pgRNA. The serum ratio between HBV DNA and HBV RNA was higher in genotype D as compared with other genotypes. The results suggest that HBV RNA in serum is present in viral particles with failing reverse transcription activity, which are produced at almost as high rates as viral particles containing DNA. The results encourage further studies of the mechanisms by which these particles are produced, the impact of genotype, and the potential clinical utility of quantifying HBV RNA in serum.

High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay.

PubMed

Yucha, Robert W; Hobbs, Kristen S; Hanhauser, Emily; Hogan, Louise E; Nieves, Wildaliz; Ozen, Mehmet O; Inci, Fatih; York, Vanessa; Gibson, Erica A; Thanh, Cassandra; Shafiee, Hadi; El Assal, Rami; Kiselinova, Maja; Robles, Yvonne P; Bae, Helen; Leadabrand, Kaitlyn S; Wang, ShuQi; Deeks, Steven G; Kuritzkes, Daniel R; Demirci, Utkan; Henrich, Timothy J

2017-06-01

Reactivation of latent viral reservoirs is on the forefront of HIV-1 eradication research. However, it is unknown if latency reversing agents (LRAs) increase the level of viral transcription from cells producing HIV RNA or harboring transcriptionally-inactive (latent) infection. We therefore developed a microfluidic single-cell-in-droplet (scd)PCR assay to directly measure the number of CD4 + T cells that produce unspliced (us)RNA and multiply spliced (ms)RNA following ex vivo latency reversal with either an histone deacetylase inhibitor (romidepsin) or T cell receptor (TCR) stimulation. Detection of HIV-1 transcriptional activity can also be performed on hundreds of thousands of CD4+ T-cells in a single experiment. The scdPCR method was then applied to CD4 + T cells obtained from HIV-1-infected individuals on antiretroviral therapy. Overall, our results suggest that effects of LRAs on HIV-1 reactivation may be heterogeneous-increasing transcription from active cells in some cases and increasing the number of transcriptionally active cells in others. Genomic DNA and human mRNA isolated from HIV-1 reactivated cells could also be detected and quantified from individual cells. As a result, our assay has the potential to provide needed insight into various reservoir eradication strategies. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Genome improvement of the acarbose producer Actinoplanes sp. SE50/110 and annotation refinement based on RNA-seq analysis.

PubMed

Wolf, Timo; Schneiker-Bekel, Susanne; Neshat, Armin; Ortseifen, Vera; Wibberg, Daniel; Zemke, Till; Pühler, Alfred; Kalinowski, Jörn

2017-06-10

Actinoplanes sp. SE50/110 is the natural producer of acarbose, which is used in the treatment of diabetes mellitus type II. However, until now the transcriptional organization and regulation of the acarbose biosynthesis are only understood rudimentarily. The genome sequence of Actinoplanes sp. SE50/110 was known before, but was resequenced in this study to remove assembly artifacts and incorrect base callings. The annotation of the genome was refined in a multi-step approach, including modern bioinformatic pipelines, transcriptome and proteome data. A whole transcriptome RNA-seq library as well as an RNA-seq library enriched for primary 5'-ends were used for the detection of transcription start sites, to correct tRNA predictions, to identify novel transcripts like small RNAs and to improve the annotation through the correction of falsely annotated translation start sites. The transcriptome data sets were also applied to identify 31 cis-regulatory RNA structures, such as riboswitches or RNA thermometers as well as three leaderless transcribed short peptides found in putative attenuators upstream of genes for amino acid biosynthesis. The transcriptional organization of the acarbose biosynthetic gene cluster was elucidated in detail and fourteen novel biosynthetic gene clusters were suggested. The accurate genome sequence and precise annotation of the Actinoplanes sp. SE50/110 genome will be the foundation for future genetic engineering and systems biology studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Intra- and intermolecular nonenzymatic ligations occur within transcripts derived from the peach latent mosaic viroid.

PubMed

Lafontaine, D; Beaudry, D; Marquis, P; Perreault, J P

1995-10-01

We report here the nonenzymatic self-ligation of transcripts corresponding to the peach latent mosaic viroid (PLMVd). This is the first description of this process with viroid sequences, although it has been reported to occur with human hepatitis delta virus RNA. Self-ligation occurs when the 5'-hydroxyl and the 2',3'-cyclic phosphate termini produced by the hammerhead self-cleavage of the viroid RNA are juxtaposed by the viroid rod-like structure, and a phosphodiester bond is formed between the two following hydrolysis of the cyclic phosphate. Unit-length transcripts undergo intramolecular folding, and their subsequent self-ligation produces circular molecules. The self-ligation observed in vitro may contribute to PLMVd circularization during rolling circle replication; however, this does not exclude the possibility that a host RNA ligase catalyzes the ligation steps in vivo. Like self-cleavage, self-ligation is probably an ancestral reaction, and the enzyme-catalyzed ligation most likely evolved from this primitive mechanism. Furthermore, the intermolecular self-ligation of annealed transcripts derived from PLMVd is demonstrated, suggesting a possible mechanism for sequence reassortment in viroids.

SWI/SNF Associates with Nascent Pre-mRNPs and Regulates Alternative Pre-mRNA Processing

PubMed Central

Tyagi, Anu; Ryme, Jessica; Brodin, David; Östlund Farrants, Ann Kristin; Visa, Neus

2009-01-01

The SWI/SNF chromatin remodeling complexes regulate the transcription of many genes by remodeling nucleosomes at promoter regions. In Drosophila, SWI/SNF plays an important role in ecdysone-dependent transcription regulation. Studies in human cells suggest that Brahma (Brm), the ATPase subunit of SWI/SNF, regulates alternative pre-mRNA splicing by modulating transcription elongation rates. We describe, here, experiments that study the association of Brm with transcribed genes in Chironomus tentans and Drosophila melanogaster, the purpose of which was to further elucidate the mechanisms by which Brm regulates pre-mRNA processing. We show that Brm becomes incorporated into nascent Balbiani ring pre-mRNPs co-transcriptionally and that the human Brm and Brg1 proteins are associated with RNPs. We have analyzed the expression profiles of D. melanogaster S2 cells in which the levels of individual SWI/SNF subunits have been reduced by RNA interference, and we show that depletion of SWI/SNF core subunits changes the relative abundance of alternative transcripts from a subset of genes. This observation, and the fact that a fraction of Brm is not associated with chromatin but with nascent pre-mRNPs, suggest that SWI/SNF affects pre-mRNA processing by acting at the RNA level. Ontology enrichment tests indicate that the genes that are regulated post-transcriptionally by SWI/SNF are mostly enzymes and transcription factors that regulate postembryonic developmental processes. In summary, the data suggest that SWI/SNF becomes incorporated into nascent pre-mRNPs and acts post-transcriptionally to regulate not only the amount of mRNA synthesized from a given promoter but also the type of alternative transcript produced. PMID:19424417

Continuous in vitro evolution of bacteriophage RNA polymerase promoters

NASA Technical Reports Server (NTRS)

Breaker, R. R.; Banerji, A.; Joyce, G. F.

1994-01-01

Rapid in vitro evolution of bacteriophage T7, T3, and SP6 RNA polymerase promoters was achieved by a method that allows continuous enrichment of DNAs that contain functional promoter elements. This method exploits the ability of a special class of nucleic acid molecules to replicate continuously in the presence of both a reverse transcriptase and a DNA-dependent RNA polymerase. Replication involves the synthesis of both RNA and cDNA intermediates. The cDNA strand contains an embedded promoter sequence, which becomes converted to a functional double-stranded promoter element, leading to the production of RNA transcripts. Synthetic cDNAs, including those that contain randomized promoter sequences, can be used to initiate the amplification cycle. However, only those cDNAs that contain functional promoter sequences are able to produce RNA transcripts. Furthermore, each RNA transcript encodes the RNA polymerase promoter sequence that was responsible for initiation of its own transcription. Thus, the population of amplifying molecules quickly becomes enriched for those templates that encode functional promoters. Optimal promoter sequences for phage T7, T3, and SP6 RNA polymerase were identified after a 2-h amplification reaction, initiated in each case with a pool of synthetic cDNAs encoding greater than 10(10) promoter sequence variants.

Identification and characterization of a class of MALAT1 -like genomic loci

DOE PAGES

Zhang, Bin; Mao, Yuntao S.; Diermeier, Sarah D.; ...

2017-05-23

The MALAT1 (Metastasis-Associated Lung Adenocarcinoma Transcript 1) gene encodes a noncoding RNA that is processed into a long nuclear retained transcript ( MALAT1) and a small cytoplasmic tRNA-like transcript (mascRNA). Using an RNA sequence- and structure-based covariance model, we identified more than 130 genomic loci in vertebrate genomes containing the MALAT1 3' end triple-helix structure and its immediate downstream tRNA-like structure, including 44 in the green lizard Anolis carolinensis. Structural and computational analyses revealed a co-occurrence of components of the 3' end module. MALAT1-like genes in Anolis carolinensis are highly expressed in adult testis, thus we named them testis-abundant longmore » noncoding RNAs (tancRNAs). MALAT1-like loci also produce multiple small RNA species, including PIWI-interacting RNAs (piRNAs), from the antisense strand. The 3' ends of tancRNAs serve as potential targets for the PIWI-piRNA complex. Furthermore, we have identified an evolutionarily conserved class of long noncoding RNAs (lncRNAs) with similar structural constraints, post-transcriptional processing, and subcellular localization and a distinct function in spermatocytes.« less

Identification and characterization of a class of MALAT1 -like genomic loci

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

Zhang, Bin; Mao, Yuntao S.; Diermeier, Sarah D.

The MALAT1 (Metastasis-Associated Lung Adenocarcinoma Transcript 1) gene encodes a noncoding RNA that is processed into a long nuclear retained transcript ( MALAT1) and a small cytoplasmic tRNA-like transcript (mascRNA). Using an RNA sequence- and structure-based covariance model, we identified more than 130 genomic loci in vertebrate genomes containing the MALAT1 3' end triple-helix structure and its immediate downstream tRNA-like structure, including 44 in the green lizard Anolis carolinensis. Structural and computational analyses revealed a co-occurrence of components of the 3' end module. MALAT1-like genes in Anolis carolinensis are highly expressed in adult testis, thus we named them testis-abundant longmore » noncoding RNAs (tancRNAs). MALAT1-like loci also produce multiple small RNA species, including PIWI-interacting RNAs (piRNAs), from the antisense strand. The 3' ends of tancRNAs serve as potential targets for the PIWI-piRNA complex. Furthermore, we have identified an evolutionarily conserved class of long noncoding RNAs (lncRNAs) with similar structural constraints, post-transcriptional processing, and subcellular localization and a distinct function in spermatocytes.« less

Finding Order in Randomness: Single-Molecule Studies Reveal Stochastic RNA Processing | Center for Cancer Research

Cancer.gov

Producing a functional eukaryotic messenger RNA (mRNA) requires the coordinated activity of several large protein complexes to initiate transcription, elongate nascent transcripts, splice together exons, and cleave and polyadenylate the 3’ end. Kinetic competition between these various processes has been proposed to regulate mRNA maturation, but this model could lead to multiple, randomly determined, or stochastic, pathways or outcomes. Regulatory checkpoints have been suggested as a means of ensuring quality control. However, current methods have been unable to tease apart the contributions of these processes at a single gene or on a time scale that could provide mechanistic insight. To begin to investigate the kinetic relationship between transcription and splicing, Daniel Larson, Ph.D., of CCR’s Laboratory of Receptor Biology and Gene Expression, and his colleagues employed a single-molecule RNA imaging approach to monitor production and processing of a human β-globin reporter gene in living cells.

Combinations of ERK and p38 MAPK inhibitors ablate tumor necrosis factor-alpha (TNF-alpha ) mRNA induction. Evidence for selective destabilization of TNF-alpha transcripts.

PubMed

Rutault, K; Hazzalin, C A; Mahadevan, L C

2001-03-02

Tumor necrosis factor-alpha (TNF-alpha) is a potent proinflammatory cytokine whose synthesis and secretion are implicated in diverse pathologies. Hence, inhibition of TNF-alpha transcription or translation and neutralization of its protein product represent major pharmaceutical strategies to control inflammation. We have studied the role of ERK and p38 mitogen-activated protein (MAP) kinase in controlling TNF-alpha mRNA levels in differentiated THP-1 cells and in freshly purified human monocytes. We show here that it is possible to produce virtually complete inhibition of lipopolysaccharide-stimulated TNF-alpha mRNA accumulation by using a combination of ERK and p38 MAP kinase inhibitors. Furthermore, substantial inhibition is achievable using combinations of 1 microm of each inhibitor, whereas inhibitors used individually are incapable of producing complete inhibition even at high concentrations. Finally, addressing mechanisms involved, we show that inhibition of p38 MAP kinase selectively destabilizes TNF-alpha transcripts but does not affect degradation of c-jun transcripts. These results impinge on the controversy in the literature surrounding the mode of action of MAP kinase inhibitors on TNF-alpha mRNA and suggest the use of combinations of MAP kinase inhibitors as an effective anti-inflammatory strategy.

Enhancement of single guide RNA transcription for efficient CRISPR/Cas-based genomic engineering.

PubMed

Ui-Tei, Kumiko; Maruyama, Shohei; Nakano, Yuko

2017-06-01

Genomic engineering using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein is a promising approach for targeting the genomic DNA of virtually any organism in a sequence-specific manner. Recent remarkable advances in CRISPR/Cas technology have made it a feasible system for use in therapeutic applications and biotechnology. In the CRISPR/Cas system, a guide RNA (gRNA), interacting with the Cas protein, recognizes a genomic region with sequence complementarity, and the double-stranded DNA at the target site is cleaved by the Cas protein. A widely used gRNA is an RNA polymerase III (pol III)-driven single gRNA (sgRNA), which is produced by artificial fusion of CRISPR RNA (crRNA) and trans-activation crRNA (tracrRNA). However, we identified a TTTT stretch, known as a termination signal of RNA pol III, in the scaffold region of the sgRNA. Here, we revealed that sgRNA carrying a TTTT stretch reduces the efficiency of sgRNA transcription due to premature transcriptional termination, and decreases the efficiency of genome editing. Unexpectedly, it was also shown that the premature terminated sgRNA may have an adverse effect of inducing RNA interference. Such disadvantageous effects were avoided by substituting one base in the TTTT stretch.

The majority of total nuclear-encoded non-ribosomal RNA in a human cell is 'dark matter' un-annotated RNA.

PubMed

Kapranov, Philipp; St Laurent, Georges; Raz, Tal; Ozsolak, Fatih; Reynolds, C Patrick; Sorensen, Poul H B; Reaman, Gregory; Milos, Patrice; Arceci, Robert J; Thompson, John F; Triche, Timothy J

2010-12-21

Discovery that the transcriptional output of the human genome is far more complex than predicted by the current set of protein-coding annotations and that most RNAs produced do not appear to encode proteins has transformed our understanding of genome complexity and suggests new paradigms of genome regulation. However, the fraction of all cellular RNA whose function we do not understand and the fraction of the genome that is utilized to produce that RNA remain controversial. This is not simply a bookkeeping issue because the degree to which this un-annotated transcription is present has important implications with respect to its biologic function and to the general architecture of genome regulation. For example, efforts to elucidate how non-coding RNAs (ncRNAs) regulate genome function will be compromised if that class of RNAs is dismissed as simply 'transcriptional noise'. We show that the relative mass of RNA whose function and/or structure we do not understand (the so called 'dark matter' RNAs), as a proportion of all non-ribosomal, non-mitochondrial human RNA (mt-RNA), can be greater than that of protein-encoding transcripts. This observation is obscured in studies that focus only on polyA-selected RNA, a method that enriches for protein coding RNAs and at the same time discards the vast majority of RNA prior to analysis. We further show the presence of a large number of very long, abundantly-transcribed regions (100's of kb) in intergenic space and further show that expression of these regions is associated with neoplastic transformation. These overlap some regions found previously in normal human embryonic tissues and raises an interesting hypothesis as to the function of these ncRNAs in both early development and neoplastic transformation. We conclude that 'dark matter' RNA can constitute the majority of non-ribosomal, non-mitochondrial-RNA and a significant fraction arises from numerous very long, intergenic transcribed regions that could be involved in neoplastic transformation.

Transcription of exogenous and endogenous deoxyribonucleic acid templates in cold-shocked Bacillus subtilis.

PubMed Central

Kuhl, S J; Brown, L R

1980-01-01

Ribonucleic acid (RNA) synthesis was examined in cold-shocked Bacillus subtilis cells. The cells were grown to mid-log stage, harvested, and cold shocked. RNA synthesis was monitored by the incorporation of [3H]uridine triphosphate or [alpha 32P]adenosine triphosphate into trichloroacetic acid-precipitable material in the presence of all four nucleoside triphosphates. The inhibition of RNA synthesis in cold-shocked cells by lipiarmycin, ethidium bromide, rifampin. or streptolydigin was analyzed using mutant or wild-type cells. Also examined were the effects of temperature, salt concentration, and the addition of polyamines or highly phosphorylated nucleotides. In ultraviolet-irradiated and cold-shocked cells, RNA wynthesis decreased to low levels. The addition of exogenous phi 29 or TSP-1 template to these cells caused a 13- to 20-fold increase in RNA synthesis, as monitored by trichloroacetic acid-precipitable counts. RNA synthesized in the presence of phi 29 deoxyribonucleic acid (DNA) hybridizes mainly to EcoRI fragments A and C of phi 29 DBA, These two fragments direct transcription by purified RNA polymerase in vitro and hybridize to early phi 29 DNA produced in vivo. Our results with TSP-1 DNA in this system indicated that the RNA produced hybridizes to the same fragments as early RNA produced in vivo. Plasmic pUB110 DNA was not transcribed in this system. Images PMID:6157674

The point of no return: The poly(A)-associated elongation checkpoint.

PubMed

Tellier, Michael; Ferrer-Vicens, Ivan; Murphy, Shona

2016-01-01

Cyclin-dependent kinases play critical roles in transcription by RNA polymerase II (pol II) and processing of the transcripts. For example, CDK9 regulates transcription of protein-coding genes, splicing, and 3' end formation of the transcripts. Accordingly, CDK9 inhibitors have a drastic effect on the production of mRNA in human cells. Recent analyses indicate that CDK9 regulates transcription at the early-elongation checkpoint of the vast majority of pol II-transcribed genes. Our recent discovery of an additional CDK9-regulated elongation checkpoint close to poly(A) sites adds a new layer to the control of transcription by this critical cellular kinase. This novel poly(A)-associated checkpoint has the potential to powerfully regulate gene expression just before a functional polyadenylated mRNA is produced: the point of no return. However, many questions remain to be answered before the role of this checkpoint becomes clear. Here we speculate on the possible biological significance of this novel mechanism of gene regulation and the players that may be involved.

Human cytomegalovirus and Herpes Simplex type I virus can engage RNA polymerase I for transcription of immediate early genes

PubMed Central

Kostopoulou, Ourania N.; Wilhelmi, Vanessa; Raiss, Sina; Ananthaseshan, Sharan; Lindström, Mikael S.; Bartek, Jiri; Söderberg-Naucler, Cecilia

2017-01-01

Human cytomegalovirus (HCMV) utilizes RNA polymerase II to transcribe viral genes and produce viral mRNAs. It can specifically target the nucleolus to facilitate viral transcription and translation. As RNA polymerase I (Pol I)-mediated transcription is active in the nucleolus, we investigated the role of Pol I, along with relative contributions of the human Pol II and Pol III, to early phases of viral transcription in HCMV infected cells, compared with Herpes Simplex Virus-1 (HSV-1) and Murine cytomegalovirus (MCMV). Inhibition of Pol I with siRNA or the Pol I inhibitors CX-5461 or Actinomycin D (5nM) resulted in significantly decreased IE and pp65 mRNA and protein levels in human fibroblasts at early times post infection. This initially delayed replication was compensated for later during the replication process, at which stage it didn’t significantly affect virus production. Pol I inhibition also reduced HSV-1 ICP0 and gB transcripts, suggesting that some herpesviruses engage Pol I for their early transcription. In contrast, inhibition of Pol I failed to affect MCMV transcription. Collectively, our results contribute to better understanding of the functional interplay between RNA Pol I-mediated nucleolar events and the Herpes viruses, particularly HCMV whose pathogenic impact ranges from congenital malformations and potentially deadly infections among immunosuppressed patients, up to HCMV’s emerging oncomodulatory role in human tumors. PMID:29228551

RNA editing and regulation of Drosophila 4f-rnp expression by sas-10 antisense readthrough mRNA transcripts

PubMed Central

PETERS, NICK T.; ROHRBACH, JUSTIN A.; ZALEWSKI, BRIAN A.; BYRKETT, COLLEEN M.; VAUGHN, JACK C.

2003-01-01

We have previously described an example of extensively A-to-G edited cDNA derived from adult heads of the fruitfly Drosophila melanogaster. In that study, the source of the predicted antisense RNA pairing strand for template recognition by dADAR editase was not identified, and the biological significance of the observed hyperediting was not known. Here, we address each of these questions. 4f-rnp and sas-10 are closely adjacent X-linked genes located on opposite DNA strands that produce convergent transcripts. We show that developmentally regulated antisense sas-10 readthrough mRNA arises by activation of an upstream promoter P2 during the late embryo stage of fly development. The sas-10 readthrough transcripts pair with 4f-rnp mRNA to form double-stranded molecules, as indicated by A-to-G editing observed in both RNA strands. It would be predicted that perfect RNA duplexes would be targeted for modification/degradation by enzyme pathways that recognize double-stranded RNAs, leading to decline in 4f-rnp mRNA levels, and this is what we observe. The observation using quantitative RT-PCR that sas-10 readthrough and 4f-rnp transcript levels are inversely related suggests a role for the antisense RNA in posttranscriptional regulation of 4f-rnp gene expression during development. Potential molecular mechanisms that could lead to this result are discussed, one of which is targeted transcript degradation via the RNAi pathway. Insofar as the dADAR editase and RNAi pathways are known to be constitutive in this system, it is likely that control of antisense RNA transcription is the rate-limiting factor. The results provide insight into roles of naturally occurring antisense RNAs in regulation of eukaryotic gene expression. PMID:12756328

Position-specific binding of FUS to nascent RNA regulates mRNA length

PubMed Central

Masuda, Akio; Takeda, Jun-ichi; Okuno, Tatsuya; Okamoto, Takaaki; Ohkawara, Bisei; Ito, Mikako; Ishigaki, Shinsuke; Sobue, Gen

2015-01-01

More than half of all human genes produce prematurely terminated polyadenylated short mRNAs. However, the underlying mechanisms remain largely elusive. CLIP-seq (cross-linking immunoprecipitation [CLIP] combined with deep sequencing) of FUS (fused in sarcoma) in neuronal cells showed that FUS is frequently clustered around an alternative polyadenylation (APA) site of nascent RNA. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) of RNA polymerase II (RNAP II) demonstrated that FUS stalls RNAP II and prematurely terminates transcription. When an APA site is located upstream of an FUS cluster, FUS enhances polyadenylation by recruiting CPSF160 and up-regulates the alternative short transcript. In contrast, when an APA site is located downstream from an FUS cluster, polyadenylation is not activated, and the RNAP II-suppressing effect of FUS leads to down-regulation of the alternative short transcript. CAGE-seq (cap analysis of gene expression [CAGE] combined with deep sequencing) and PolyA-seq (a strand-specific and quantitative method for high-throughput sequencing of 3' ends of polyadenylated transcripts) revealed that position-specific regulation of mRNA lengths by FUS is operational in two-thirds of transcripts in neuronal cells, with enrichment in genes involved in synaptic activities. PMID:25995189

A combination of improved differential and global RNA-seq reveals pervasive transcription initiation and events in all stages of the life-cycle of functional RNAs in Propionibacterium acnes, a major contributor to wide-spread human disease

PubMed Central

2013-01-01

Background Sequencing of the genome of Propionibacterium acnes produced a catalogue of genes many of which enable this organism to colonise skin and survive exposure to the elements. Despite this platform, there was little understanding of the gene regulation that gives rise to an organism that has a major impact on human health and wellbeing and causes infections beyond the skin. To address this situation, we have undertaken a genome–wide study of gene regulation using a combination of improved differential and global RNA-sequencing and an analytical approach that takes into account the inherent noise within the data. Results We have produced nucleotide-resolution transcriptome maps that identify and differentiate sites of transcription initiation from sites of stable RNA processing and mRNA cleavage. Moreover, analysis of these maps provides strong evidence for ‘pervasive’ transcription and shows that contrary to initial indications it is not biased towards the production of antisense RNAs. In addition, the maps reveal an extensive array of riboswitches, leaderless mRNAs and small non-protein-coding RNAs alongside vegetative promoters and post-transcriptional events, which includes unusual tRNA processing. The identification of such features will inform models of complex gene regulation, as illustrated here for ribonucleotide reductases and a potential quorum-sensing, two-component system. Conclusions The approach described here, which is transferable to any bacterial species, has produced a step increase in whole-cell knowledge of gene regulation in P. acnes. Continued expansion of our maps to include transcription associated with different growth conditions and genetic backgrounds will provide a new platform from which to computationally model the gene expression that determines the physiology of P. acnes and its role in human disease. PMID:24034785

Convergent transcription in the butyrolactone regulon in Streptomyces coelicolor confers a bistable genetic switch for antibiotic biosynthesis.

PubMed

Chatterjee, Anushree; Drews, Laurie; Mehra, Sarika; Takano, Eriko; Kaznessis, Yiannis N; Hu, Wei-Shou

2011-01-01

cis-encoded antisense RNAs (cis asRNA) have been reported to participate in gene expression regulation in both eukaryotic and prokaryotic organisms. Its presence in Streptomyces coelicolor has also been reported recently; however, its role has yet to be fully investigated. Using mathematical modeling we explore the role of cis asRNA produced as a result of convergent transcription in scbA-scbR genetic switch. scbA and scbR gene pair, encoding repressor-amplifier proteins respectively, mediates the synthesis of a signaling molecule, the γ-butyrolactone SCB1 and controls the onset of antibiotic production. Our model considers that transcriptional interference caused by convergent transcription of two opposing RNA polymerases results in fatal collision and transcriptional termination, which suppresses transcription efficiency. Additionally, convergent transcription causes sense and antisense interactions between complementary sequences from opposing strands, rendering the full length transcript inaccessible for translation. We evaluated the role of transcriptional interference and the antisense effect conferred by convergent transcription on the behavior of scbA-scbR system. Stability analysis showed that while transcriptional interference affects the system, it is asRNA that confers scbA-scbR system the characteristics of a bistable switch in response to the signaling molecule SCB1. With its critical role of regulating the onset of antibiotic synthesis the bistable behavior offers this two gene system the needed robustness to be a genetic switch. The convergent two gene system with potential of transcriptional interference is a frequent feature in various genomes. The possibility of asRNA regulation in other such gene-pairs is yet to be examined.

Plant-specific multisubunit RNA polymerase in gene silencing.

PubMed

Lahmy, Sylvie; Bies-Etheve, Natacha; Lagrange, Thierry

2010-01-01

In recent years, a major breakthrough in the study of epigenetic silencing in eukaryotes came with the discovery that the RNA-interference pathway (RNAi) is generally implicated in heterochromatin assembly and gene silencing. An important and paradoxical feature of the RNAi-mediated heterochromatin pathways is their requirement for some form of transcription. In fission yeast, Schizosaccharomyces pombe, centromeric siRNAs have been shown to derive from chromatin-bound nascent transcripts produced by RNA polymerase II (PolII) at the site of heterochromatin formation. Likewise, chromatin-bound nascent transcripts generated by a PolII-related DNA-dependent RNA polymerase, known as PolIVb/PolV, have recently been implicated in RNA-directed DNA methylation (RdDM), the prominent RNAi-mediated chromatin pathway in plants. In this review we discuss recent work on the plant-specific PolII variant enzymes and discuss the mechanistic convergences that have been observed in the role of these enzymes in their respective siRNA-mediated heterochromatin formation pathways.

RNA-Seq profiling of single bovine oocyte transcript abundance and its modulation by cytoplasmic polyadenylation.

PubMed

Reyes, Juan M; Chitwood, James L; Ross, Pablo J

2015-02-01

Molecular changes occurring during mammalian oocyte maturation are partly regulated by cytoplasmic polyadenylation (CP) and affect oocyte quality, yet the extent of CP activity during oocyte maturation remains unknown. Single bovine oocyte RNA sequencing (RNA-Seq) was performed to examine changes in transcript abundance during in vitro oocyte maturation in cattle. Polyadenylated RNA from individual germinal-vesicle and metaphase-II oocytes was amplified and processed for Illumina sequencing, producing approximately 30 million reads per replicate for each sample type. A total of 10,494 genes were found to be expressed, of which 2,455 were differentially expressed (adjusted P < 0.05 and fold change >2) between stages, with 503 and 1,952 genes respectively increasing and decreasing in abundance. Differentially expressed genes with complete 3'-untranslated-region sequence (279 increasing and 918 decreasing in polyadenylated transcript abundance) were examined for the presence, position, and distribution of motifs mediating CP, revealing enrichment (85%) and lack thereof (18%) in up- and down-regulated genes, respectively. Examination of total and polyadenylated RNA abundance by quantitative PCR validated these RNA-Seq findings. The observed increases in polyadenylated transcript abundance within the RNA-Seq data are likely due to CP, providing novel insight into targeted transcripts and resultant differential gene expression profiles that contribute to oocyte maturation. © 2015 Wiley Periodicals, Inc.

Variable RNA expression from recently acquired, endogenous viral elements (EVE) of white spot syndrome virus (WSSV) in shrimp.

PubMed

Utari, Heny Budi; Soowannayan, Chumporn; Flegel, Timothy W; Whityachumnarnkul, Boonsirm; Kruatrachue, Maleeya

2017-11-01

The viral accommodation hypothesis proposes that endogenous viral elements (EVE) from both RNA and DNA viruses are being continually integrated into the shrimp genome by natural host processes and that they can result in tolerance to viral infection by fortuitous production of antisense, immunospecific RNA (imRNA). Thus, we hypothesized that previously reported microarray results for the presence of white spot syndrome virus (WSSV) open reading frames (ORFs) formerly called 151, 366 and 427 in a domesticated giant tiger shrimp (Penaeus monodon) breeding stock might have represented expression from EVE, since the stock had shown uninterrupted freedom from white spot disease (WSD) for many generations. To test this hypothesis, 128 specimens from a current stock generation were confirmed for freedom from WSSV infection using two nested PCR detection methods. Subsequent nested-PCR testing revealed 33/128 specimens (26%) positive for at least one of the ORF at very high sequence identity (95-99%) to extant WSSV. Positive results for ORF 366 (now known to be a fragment of the WSSV capsid protein gene) dominated (28/33 = 84.8%), so 9 arbitrarily selected 366-positive specimens were tested by strand-specific, nested RT-PCR using DNase-treated RNA templates. This revealed variable RNA expression in individual shrimp including no RNA transcripts (n = 1), sense transcripts only (n = 1), antisense transcripts only (n = 2) or transcripts of both sense (n = 5). The latter 7 expression products indicated specimens producing putative imRNA. The variable types and numbers of the EVE and the variable RNA expression (including potential imRNA) support predictions of the viral accommodation hypothesis that EVE are randomly produced and expressed. Positive nested PCR test results for EVE of ORF 366 using DNA templates derived from shrimp sperm (germ cells), indicated that they were heritable. Copyright © 2017 Elsevier Ltd. All rights reserved.

Strand-Specific RNA-Seq Analyses of Fruiting Body Development in Coprinopsis cinerea

DOE PAGES

Muraguchi, Hajime; Umezawa, Kiwamu; Niikura, Mai; ...

2015-10-28

We report that the basidiomycete fungus Coprinopsis cinerea is an important model system for multicellular development. Fruiting bodies of C. cinerea are typical mushrooms, which can be produced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq) was performed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues) with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs) between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC).more » To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovered both transcription factor candidates and antisense transcripts that are likely to be involved in developmental regulation for fruiting.« less

Strand-Specific RNA-Seq Analyses of Fruiting Body Development in Coprinopsis cinerea

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

Muraguchi, Hajime; Umezawa, Kiwamu; Niikura, Mai

We report that the basidiomycete fungus Coprinopsis cinerea is an important model system for multicellular development. Fruiting bodies of C. cinerea are typical mushrooms, which can be produced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq) was performed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues) with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs) between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC).more » To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovered both transcription factor candidates and antisense transcripts that are likely to be involved in developmental regulation for fruiting.« less

High-throughput annotation of full-length long noncoding RNAs with capture long-read sequencing.

PubMed

Lagarde, Julien; Uszczynska-Ratajczak, Barbara; Carbonell, Silvia; Pérez-Lluch, Sílvia; Abad, Amaya; Davis, Carrie; Gingeras, Thomas R; Frankish, Adam; Harrow, Jennifer; Guigo, Roderic; Johnson, Rory

2017-12-01

Accurate annotation of genes and their transcripts is a foundation of genomics, but currently no annotation technique combines throughput and accuracy. As a result, reference gene collections remain incomplete-many gene models are fragmentary, and thousands more remain uncataloged, particularly for long noncoding RNAs (lncRNAs). To accelerate lncRNA annotation, the GENCODE consortium has developed RNA Capture Long Seq (CLS), which combines targeted RNA capture with third-generation long-read sequencing. Here we present an experimental reannotation of the GENCODE intergenic lncRNA populations in matched human and mouse tissues that resulted in novel transcript models for 3,574 and 561 gene loci, respectively. CLS approximately doubled the annotated complexity of targeted loci, outperforming existing short-read techniques. Full-length transcript models produced by CLS enabled us to definitively characterize the genomic features of lncRNAs, including promoter and gene structure, and protein-coding potential. Thus, CLS removes a long-standing bottleneck in transcriptome annotation and generates manual-quality full-length transcript models at high-throughput scales.

Understanding Transcriptional Enhancement in Monoclonal Antibody-Producing Chinese Hamster Ovary Cells

NASA Astrophysics Data System (ADS)

Nicoletti, Sarah E.

With the demand for monoclonal antibody (mAB) therapeutics continually increasing, the need to better understand what makes a high productivity clone has gained substantial interest. Monoclonal antibody producing Chinese hamster ovary (CHO) cells with different productivities were provided by a biopharmaceutical company for investigation. Gene copy numbers, mRNA levels, and mAb productivities were previously determined for two low producing clones and their amplified progeny. These results showed an increase in mRNA copy number in amplified clones, which correlated to the observed increases in specific productivity of these clones. The presence of multiple copies of mRNA per one copy of DNA in the higher productivity clones has been coined as transcriptional enhancement. The methylation status of the CMV promoter as well as transcription factor/promoter interactions were evaluated to determine the cause of transcriptional enhancement. Methylation analysis via bisulfite sequencing revealed no significant difference in overall methylation status of the CMV promoter. These data did, however, reveal the possibility of differential interactions of transcription factors between the high and low productivity cell clones. This finding was further supported by chromatin immunoprecipitations previously performed in the lab, as well as literature studies. Transcription activator-like effector (TALE) binding proteins were constructed and utilized to selectively immunoprecipitate the CMV promoter along with its associated transcription factors in the different CHO cell clones. Cells were transfected with the TALE proteins, harvested and subjected to a ChIP-like procedure. Results obtained from the TALE ChIP demonstrated the lack of binding of the protein to the promoter and the need to redesign the TALE. Overall, results obtained from this study were unable to give a clear indication as to the causes of transcriptional enhancement in the amplified CHO cell clones. Further investigations into both epigenetic modifications and transcription factor interactions will help gain a better understanding of what characteristics make a higher producing CHO cell line. Discovery of these characteristics will aid in better genetic engineering strategies for future recombinant cell lines, improving productivity and shortening the clonal selection process.

Analysis of microRNA expression and function.

PubMed

Van Wynsberghe, Priscilla M; Chan, Shih-Peng; Slack, Frank J; Pasquinelli, Amy E

2011-01-01

Originally discovered in C. elegans, microRNAs (miRNAs) are small RNAs that regulate fundamental cellular processes in diverse organisms. MiRNAs are encoded within the genome and are initially transcribed as primary transcripts that can be several kilobases in length. Primary transcripts are successively cleaved by two RNase III enzymes, Drosha in the nucleus and Dicer in the cytoplasm, to produce ∼70 nucleotide (nt) long precursor miRNAs and 22 nt long mature miRNAs, respectively. Mature miRNAs regulate gene expression post-transcriptionally by imperfectly binding target mRNAs in association with the multiprotein RNA induced silencing complex (RISC). The conserved sequence, expression pattern, and function of some miRNAs across distinct species as well as the importance of specific miRNAs in many biological pathways have led to an explosion in the study of miRNA biogenesis, miRNA target identification, and miRNA target regulation. Many advances in our understanding of miRNA biology have come from studies in the powerful model organism C. elegans. This chapter reviews the current methods used in C. elegans to study miRNA biogenesis, small RNA populations, miRNA-protein complexes, and miRNA target regulation. Copyright © 2011 Elsevier Inc. All rights reserved.

Tudor-domain containing proteins act to make the piRNA pathways more robust in Drosophila.

PubMed

Sato, Kaoru; Iwasaki, Yuka W; Siomi, Haruhiko; Siomi, Mikiko C

2015-01-01

PIWI-interacting RNAs (piRNAs), a subset of small non-coding RNAs enriched in animal gonads, repress transposons by assembling with PIWI proteins to form potent gene-silencing RNP complexes, piRISCs. Accumulating evidence suggests that piRNAs are produced through three interdependent pathways; the de novo primary pathway, the ping-pong pathway, and the phased primary pathway. The de novo primary pathway in Drosophila ovaries produces primary piRNAs for two PIWI members, Piwi and Aub. Aub then initiates the ping-pong pathway to produce secondary piRNAs for AGO3. AGO3-slicer dependent cleavage subsequently produces secondary piRNAs for Aub. Trailer products of AGO3-slicer activity are consumed by the phased primary pathway to increase the Piwi-bound piRNA population. All these pathways are regulated by a number of piRNA factors in a highly coordinated fashion. Recent studies show that two Tudor-domain containing piRNA factors, Krimper (Krimp) and Qin/Kumo, play crucial roles in making Aub-AGO3 heterotypic ping-pong robust. This maintains the levels of piRNAs loaded onto Piwi and Aub to efficiently repress transposons at transcriptional and post-transcriptional levels, respectively.

Knockdown of Polyphenol Oxidase Gene Expression in Potato (Solanum tuberosum L.) with Artificial MicroRNAs.

PubMed

Chi, Ming; Bhagwat, Basdeo; Tang, Guiliang; Xiang, Yu

2016-01-01

It is of great importance and interest to develop crop varieties with low polyphenol oxidase (PPO) activity for the food industry because PPO-mediated oxidative browning is a main cause of post-harvest deterioration and quality loss of fresh produce and processed foods. We recently demonstrated that potato tubers with reduced browning phenotypes can be produced by inhibition of the expression of several PPO gene isoforms using artificial microRNA (amiRNA) technology. The approach introduces a single type of 21-nucleotide RNA population to guide silencing of the PPO gene transcripts in potato tissues. Some advantages of the technology are: small RNA molecules are genetically transformed, off-target gene silencing can be avoided or minimized at the stage of amiRNA designs, and accuracy and efficiency of the processes can be detected at every step using molecular biological techniques. Here we describe the methods for transformation and regeneration of potatoes with amiRNA vectors, detection of the expression of amiRNAs, identification of the cleaved product of the target gene transcripts, and assay of the expression level of PPO gene isoforms in potatoes.

DHX9 regulates production of hepatitis B virus-derived circular RNA and viral protein levels

PubMed Central

Sekiba, Kazuma; Otsuka, Motoyuki; Ohno, Motoko; Kishikawa, Takahiro; Yamagami, Mari; Suzuki, Tatsunori; Ishibashi, Rei; Seimiya, Takahiro; Tanaka, Eri; Koike, Kazuhiko

2018-01-01

Hepatitis B virus (HBV) infection, which is a major health concern worldwide, can lead to liver cirrhosis and hepatocellular carcinoma. Although current nucleos(t)ide analogs efficiently inhibit viral reverse transcription and viral DNA load clinically, episomal viral covalently closed circular DNA (cccDNA) minichromosomes and transcripts from cccDNA continue to be expressed over the long term. We hypothesized that, under these conditions, viral transcripts may have biological functions involved in pathogenesis. Here, we show that the host protein DExH-box helicase 9 (DXH9) is associated with viral RNAs. We also show that viral-derived circular RNA is produced during HBV replication, and the amount is increased by knockdown of the DHX9 protein, which, in turn, results in decreased viral protein levels but does not affect the levels of HBV DNA. These phenomena were observed in the HBV-producing cell culture model and HBV mini-circle model mimicking HBV cccDNA, as well as in human primary hepatocytes infected with HBV. Based on these results, we conclude that, in HBV infection, the RNA binding factor DHX9 is a novel regulator of viral circular RNA and viral protein levels. PMID:29765512

Infection of capilloviruses requires subgenomic RNAs whose transcription is controlled by promoter-like sequences conserved among flexiviruses.

PubMed

Komatsu, Ken; Hirata, Hisae; Fukagawa, Takako; Yamaji, Yasuyuki; Okano, Yukari; Ishikawa, Kazuya; Adachi, Tatsushi; Maejima, Kensaku; Hashimoto, Masayoshi; Namba, Shigetou

2012-07-01

The first open-reading frame (ORF) of apple stem grooving virus (ASGV), of the genus Capillovirus, encodes an apparently chimeric polyprotein containing conserved regions for replicase (Rep) and coat protein (CP). However, our previous study revealed that ASGV mutants with distinct and discontinuous Rep- and CP-coding regions successfully infect plants, indicating that CP expressed via a subgenomic RNA (sgRNA) is sufficient for viability of the virus. Here we identified a transcription start site of the CP sgRNA and revealed that CP translated from the sgRNA is essential for ASGV infection. We mapped the transcription start sites of both the CP and the movement protein (MP) sgRNAs of ASGV and found a hexanucleotide motif, UUAGGU, conserved upstream from both sgRNA transcription start sites. Mutational analysis of the putative CP initiation codon and of the UUAGGU sequence upstream from the transcription start site of CP sgRNA demonstrated their importance for ASGV accumulation. Our results also demonstrated that potato virus T (PVT), an unassigned species closely related to ASGV, produces two sgRNAs putatively deployed for the CP and MP expression and that the same hexanucleotide motif as found in ASGV is located upstream from the transcription start sites of both sgRNAs. This motif, which constituted putative core elements of the sgRNA promoter, is broadly conserved among viruses in the families Alphaflexiviridae and Betaflexiviridae, suggesting that the gene expression strategy of the viruses in both families has been conserved throughout evolution. Copyright © 2012 Elsevier B.V. All rights reserved.

Genome-Wide Spectra of Transcription Insertions and Deletions Reveal That Slippage Depends on RNA:DNA Hybrid Complementarity

PubMed Central

Traverse, Charles C.

2017-01-01

ABSTRACT Advances in sequencing technologies have enabled direct quantification of genome-wide errors that occur during RNA transcription. These errors occur at rates that are orders of magnitude higher than rates during DNA replication, but due to technical difficulties such measurements have been limited to single-base substitutions and have not yet quantified the scope of transcription insertions and deletions. Previous reporter gene assay findings suggested that transcription indels are produced exclusively by elongation complex slippage at homopolymeric runs, so we enumerated indels across the protein-coding transcriptomes of Escherichia coli and Buchnera aphidicola, which differ widely in their genomic base compositions and incidence of repeat regions. As anticipated from prior assays, transcription insertions prevailed in homopolymeric runs of A and T; however, transcription deletions arose in much more complex sequences and were rarely associated with homopolymeric runs. By reconstructing the relocated positions of the elongation complex as inferred from the sequences inserted or deleted during transcription, we show that continuation of transcription after slippage hinges on the degree of nucleotide complementarity within the RNA:DNA hybrid at the new DNA template location. PMID:28851848

ORMAN: optimal resolution of ambiguous RNA-Seq multimappings in the presence of novel isoforms.

PubMed

Dao, Phuong; Numanagić, Ibrahim; Lin, Yen-Yi; Hach, Faraz; Karakoc, Emre; Donmez, Nilgun; Collins, Colin; Eichler, Evan E; Sahinalp, S Cenk

2014-03-01

RNA-Seq technology is promising to uncover many novel alternative splicing events, gene fusions and other variations in RNA transcripts. For an accurate detection and quantification of transcripts, it is important to resolve the mapping ambiguity for those RNA-Seq reads that can be mapped to multiple loci: >17% of the reads from mouse RNA-Seq data and 50% of the reads from some plant RNA-Seq data have multiple mapping loci. In this study, we show how to resolve the mapping ambiguity in the presence of novel transcriptomic events such as exon skipping and novel indels towards accurate downstream analysis. We introduce ORMAN ( O ptimal R esolution of M ultimapping A mbiguity of R N A-Seq Reads), which aims to compute the minimum number of potential transcript products for each gene and to assign each multimapping read to one of these transcripts based on the estimated distribution of the region covering the read. ORMAN achieves this objective through a combinatorial optimization formulation, which is solved through well-known approximation algorithms, integer linear programs and heuristics. On a simulated RNA-Seq dataset including a random subset of transcripts from the UCSC database, the performance of several state-of-the-art methods for identifying and quantifying novel transcripts, such as Cufflinks, IsoLasso and CLIIQ, is significantly improved through the use of ORMAN. Furthermore, in an experiment using real RNA-Seq reads, we show that ORMAN is able to resolve multimapping to produce coverage values that are similar to the original distribution, even in genes with highly non-uniform coverage. ORMAN is available at http://orman.sf.net

Kc167, a widely used Drosophila cell line, contains an active primary piRNA pathway.

PubMed

Vrettos, Nicholas; Maragkakis, Manolis; Alexiou, Panagiotis; Mourelatos, Zissimos

2017-01-01

PIWI family proteins bind to small RNAs known as PIWI-interacting RNAs (piRNAs) and play essential roles in the germline by silencing transposons and by promoting germ cell specification and function. Here we report that the widely used Kc167 cell line, derived from Drosophila melanogaster embryos, expresses piRNAs that are loaded to Aub and Piwi. Kc167 piRNAs are produced by a canonical, primary piRNA biogenesis pathway, from phased processing of precursor transcripts by the Zuc endonuclease, Armi helicase, and dGasz mitochondrial scaffold protein. Kc167 piRNAs derive from cytoplasmic transcripts, notably tRNAs and mRNAs, and their abundance correlates with that of parent transcripts. The expression of Aub is robust in Kc167, that of Piwi is modest, while Ago3 is undetectable, explaining the lack of transposon-related piRNA amplification by the Aub-Ago3, ping-pong mechanism. We propose that the default state of the primary piRNA biogenesis machinery is random transcript sampling to allow generation of piRNAs from any transcript, including newly acquired retrotransposons. This state is unmasked in Kc167, likely because they do not express piRNA cluster transcripts in sufficient amounts and do not amplify transposon piRNAs. We use Kc167 to characterize an inactive isoform of Aub protein. Since most Kc167 piRNAs are genic, they can be mapped uniquely to the genome, facilitating computational analyses. Furthermore, because Kc167 is a widely used and well-characterized cell line that is easily amenable to experimental manipulations, we expect that it will serve as an excellent system to study piRNA biogenesis and piRNA-related factors. © 2016 Vrettos et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Integrative Analysis of Sex-Specific microRNA Networks Following Stress in Mouse Nucleus Accumbens.

PubMed

Pfau, Madeline L; Purushothaman, Immanuel; Feng, Jian; Golden, Sam A; Aleyasin, Hossein; Lorsch, Zachary S; Cates, Hannah M; Flanigan, Meghan E; Menard, Caroline; Heshmati, Mitra; Wang, Zichen; Ma'ayan, Avi; Shen, Li; Hodes, Georgia E; Russo, Scott J

2016-01-01

Adult women are twice as likely as men to suffer from affective and anxiety disorders, although the mechanisms underlying heightened female stress susceptibility are incompletely understood. Recent findings in mouse Nucleus Accumbens (NAc) suggest a role for DNA methylation-driven sex differences in genome-wide transcriptional profiles. However, the role of another epigenetic process-microRNA (miR) regulation-has yet to be explored. We exposed male and female mice to Subchronic Variable Stress (SCVS), a stress paradigm that produces depression-like behavior in female, but not male, mice, and performed next generation mRNA and miR sequencing on NAc tissue. We applied a combination of differential expression, miR-mRNA network and functional enrichment analyses to characterize the transcriptional and post-transcriptional landscape of sex differences in NAc stress response. We find that male and female mice exhibit largely non-overlapping miR and mRNA profiles following SCVS. The two sexes also show enrichment of different molecular pathways and functions. Collectively, our results suggest that males and females mount fundamentally different transcriptional and post-transcriptional responses to SCVS and engage sex-specific molecular processes following stress. These findings have implications for the pathophysiology and treatment of stress-related disorders in women.

Cassandra retrotransposons carry independently transcribed 5S RNA

PubMed Central

Kalendar, Ruslan; Tanskanen, Jaakko; Chang, Wei; Antonius, Kristiina; Sela, Hanan; Peleg, Ofer; Schulman, Alan H.

2008-01-01

We report a group of TRIMs (terminal-repeat retrotransposons in miniature), which are small nonautonomous retrotransposons. These elements, named Cassandra, universally carry conserved 5S RNA sequences and associated RNA polymerase (pol) III promoters and terminators in their long terminal repeats (LTRs). They were found in all vascular plants investigated. Uniquely for LTR retrotransposons, Cassandra produces noncapped, polyadenylated transcripts from the 5S pol III promoter. Capped, read-through transcripts containing Cassandra sequences can also be detected in RNA and in EST databases. The predicted Cassandra RNA 5S secondary structures resemble those for cellular 5S rRNA, with high information content specifically in the pol III promoter region. Genic integration sites are common for Cassandra, an unusual feature for abundant retrotransposons. The 5S in each LTR produces a tandem 5S arrangement with an inter-5S spacing resembling that of cellular 5S. The distribution of 5S genes is very variable in flowering plants and may be partially explained by Cassandra activity. Cassandra thus appears both to have adapted a ubiquitous cellular gene for ribosomal RNA for use as a promoter and to parasitize an as-yet-unidentified group of retrotransposons for the proteins needed in its lifecycle. PMID:18408163

Characterization of Two Cysteine Transfer RNA Genes from Xenopus Laevis

DTIC Science & Technology

1984-07-12

containing amino acids glycine, alanine and serine, are produced by the posterior silk gland of Bombyx mori and therefore high level of tRNAgly, tRNA^Ia...1979) Studies on tRNA adaptation, tRNA turnover, precursor tRNA and tRNA gene distribution in Bombyx mori by using two-dimensional polyacrylamlde gel...Nucleic Acids Research, 1^, 8537-8546. 26. Garber, R.L. and Gage, L.P. (1979) Transcription of a cloned Bombyx mori tRNA^2 gene: Nucleotide sequence of

Targeting siRNA Missiles to Her2+ Breast Cancer

DTIC Science & Technology

2009-06-01

that HerPBK10 protects siRNA from serum nuclease-mediated degradation, T7 transcribed siRNA is more cytotoxic than synthetic siRNA when delivered to...nuclease-mediated degradation, T7 transcribed siRNA is more cytotoxic than synthetic siRNA when delivered to HER2+ breast cancer cells by HerPBK10...produced either synthetically by a commercial vendor (Dharmacon), or from a T7 transcription kit (Ambion), and shRNA, which is reportedly a more effective

The point of no return: The poly(A)-associated elongation checkpoint

PubMed Central

Tellier, Michael; Ferrer-Vicens, Ivan; Murphy, Shona

2016-01-01

abstract Cyclin-dependent kinases play critical roles in transcription by RNA polymerase II (pol II) and processing of the transcripts. For example, CDK9 regulates transcription of protein-coding genes, splicing, and 3′ end formation of the transcripts. Accordingly, CDK9 inhibitors have a drastic effect on the production of mRNA in human cells. Recent analyses indicate that CDK9 regulates transcription at the early-elongation checkpoint of the vast majority of pol II-transcribed genes. Our recent discovery of an additional CDK9-regulated elongation checkpoint close to poly(A) sites adds a new layer to the control of transcription by this critical cellular kinase. This novel poly(A)-associated checkpoint has the potential to powerfully regulate gene expression just before a functional polyadenylated mRNA is produced: the point of no return. However, many questions remain to be answered before the role of this checkpoint becomes clear. Here we speculate on the possible biological significance of this novel mechanism of gene regulation and the players that may be involved. PMID:26853452

Metazoan tRNA introns generate stable circular RNAs in vivo

PubMed Central

Lu, Zhipeng; Filonov, Grigory S.; Noto, John J.; Schmidt, Casey A.; Hatkevich, Talia L.; Wen, Ying; Jaffrey, Samie R.; Matera, A. Gregory

2015-01-01

We report the discovery of a class of abundant circular noncoding RNAs that are produced during metazoan tRNA splicing. These transcripts, termed tRNA intronic circular (tric)RNAs, are conserved features of animal transcriptomes. Biogenesis of tricRNAs requires anciently conserved tRNA sequence motifs and processing enzymes, and their expression is regulated in an age-dependent and tissue-specific manner. Furthermore, we exploited this biogenesis pathway to develop an in vivo expression system for generating “designer” circular RNAs in human cells. Reporter constructs expressing RNA aptamers such as Spinach and Broccoli can be used to follow the transcription and subcellular localization of tricRNAs in living cells. Owing to the superior stability of circular vs. linear RNA isoforms, this expression system has a wide range of potential applications, from basic research to pharmaceutical science. PMID:26194134

Metazoan tRNA introns generate stable circular RNAs in vivo.

PubMed

Lu, Zhipeng; Filonov, Grigory S; Noto, John J; Schmidt, Casey A; Hatkevich, Talia L; Wen, Ying; Jaffrey, Samie R; Matera, A Gregory

2015-09-01

We report the discovery of a class of abundant circular noncoding RNAs that are produced during metazoan tRNA splicing. These transcripts, termed tRNA intronic circular (tric)RNAs, are conserved features of animal transcriptomes. Biogenesis of tricRNAs requires anciently conserved tRNA sequence motifs and processing enzymes, and their expression is regulated in an age-dependent and tissue-specific manner. Furthermore, we exploited this biogenesis pathway to develop an in vivo expression system for generating "designer" circular RNAs in human cells. Reporter constructs expressing RNA aptamers such as Spinach and Broccoli can be used to follow the transcription and subcellular localization of tricRNAs in living cells. Owing to the superior stability of circular vs. linear RNA isoforms, this expression system has a wide range of potential applications, from basic research to pharmaceutical science. © 2015 Lu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Polymerization of non-complementary RNA: systematic symmetric nucleotide exchanges mainly involving uracil produce mitochondrial RNA transcripts coding for cryptic overlapping genes.

PubMed

Seligmann, Hervé

2013-03-01

Usual DNA→RNA transcription exchanges T→U. Assuming different systematic symmetric nucleotide exchanges during translation, some GenBank RNAs match exactly human mitochondrial sequences (exchange rules listed in decreasing transcript frequencies): C↔U, A↔U, A↔U+C↔G (two nucleotide pairs exchanged), G↔U, A↔G, C↔G, none for A↔C, A↔G+C↔U, and A↔C+G↔U. Most unusual transcripts involve exchanging uracil. Independent measures of rates of rare replicational enzymatic DNA nucleotide misinsertions predict frequencies of RNA transcripts systematically exchanging the corresponding misinserted nucleotides. Exchange transcripts self-hybridize less than other gene regions, self-hybridization increases with length, suggesting endoribonuclease-limited elongation. Blast detects stop codon depleted putative protein coding overlapping genes within exchange-transcribed mitochondrial genes. These align with existing GenBank proteins (mainly metazoan origins, prokaryotic and viral origins underrepresented). These GenBank proteins frequently interact with RNA/DNA, are membrane transporters, or are typical of mitochondrial metabolism. Nucleotide exchange transcript frequencies increase with overlapping gene densities and stop densities, indicating finely tuned counterbalancing regulation of expression of systematic symmetric nucleotide exchange-encrypted proteins. Such expression necessitates combined activities of suppressor tRNAs matching stops, and nucleotide exchange transcription. Two independent properties confirm predicted exchanged overlap coding genes: discrepancy of third codon nucleotide contents from replicational deamination gradients, and codon usage according to circular code predictions. Predictions from both properties converge, especially for frequent nucleotide exchange types. Nucleotide exchanging transcription apparently increases coding densities of protein coding genes without lengthening genomes, revealing unsuspected functional DNA coding potential. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

NC10 bacteria in marine oxygen minimum zones

PubMed Central

Padilla, Cory C; Bristow, Laura A; Sarode, Neha; Garcia-Robledo, Emilio; Gómez Ramírez, Eddy; Benson, Catherine R; Bourbonnais, Annie; Altabet, Mark A; Girguis, Peter R; Thamdrup, Bo; Stewart, Frank J

2016-01-01

Bacteria of the NC10 phylum link anaerobic methane oxidation to nitrite denitrification through a unique O2-producing intra-aerobic methanotrophy pathway. A niche for NC10 in the pelagic ocean has not been confirmed. We show that NC10 bacteria are present and transcriptionally active in oceanic oxygen minimum zones (OMZs) off northern Mexico and Costa Rica. NC10 16S rRNA genes were detected at all sites, peaking in abundance in the anoxic zone with elevated nitrite and methane concentrations. Phylogenetic analysis of particulate methane monooxygenase genes further confirmed the presence of NC10. rRNA and mRNA transcripts assignable to NC10 peaked within the OMZ and included genes of the putative nitrite-dependent intra-aerobic pathway, with high representation of transcripts containing the unique motif structure of the nitric oxide (NO) reductase of NC10 bacteria, hypothesized to participate in O2-producing NO dismutation. These findings confirm pelagic OMZs as a niche for NC10, suggesting a role for this group in OMZ nitrogen, methane and oxygen cycling. PMID:26918666

Detection of non-coding RNA in bacteria and archaea using the DETR'PROK Galaxy pipeline.

PubMed

Toffano-Nioche, Claire; Luo, Yufei; Kuchly, Claire; Wallon, Claire; Steinbach, Delphine; Zytnicki, Matthias; Jacq, Annick; Gautheret, Daniel

2013-09-01

RNA-seq experiments are now routinely used for the large scale sequencing of transcripts. In bacteria or archaea, such deep sequencing experiments typically produce 10-50 million fragments that cover most of the genome, including intergenic regions. In this context, the precise delineation of the non-coding elements is challenging. Non-coding elements include untranslated regions (UTRs) of mRNAs, independent small RNA genes (sRNAs) and transcripts produced from the antisense strand of genes (asRNA). Here we present a computational pipeline (DETR'PROK: detection of ncRNAs in prokaryotes) based on the Galaxy framework that takes as input a mapping of deep sequencing reads and performs successive steps of clustering, comparison with existing annotation and identification of transcribed non-coding fragments classified into putative 5' UTRs, sRNAs and asRNAs. We provide a step-by-step description of the protocol using real-life example data sets from Vibrio splendidus and Escherichia coli. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Extensive Use of RNA-Binding Proteins in Drosophila Sensory Neuron Dendrite Morphogenesis

PubMed Central

Olesnicky, Eugenia C.; Killian, Darrell J.; Garcia, Evelyn; Morton, Mary C.; Rathjen, Alan R.; Sola, Ismail E.; Gavis, Elizabeth R.

2013-01-01

The large number of RNA-binding proteins and translation factors encoded in the Drosophila and other metazoan genomes predicts widespread use of post-transcriptional regulation in cellular and developmental processes. Previous studies identified roles for several RNA-binding proteins in dendrite branching morphogenesis of Drosophila larval sensory neurons. To determine the larger contribution of post-transcriptional gene regulation to neuronal morphogenesis, we conducted an RNA interference screen to identify additional Drosophila proteins annotated as either RNA-binding proteins or translation factors that function in producing the complex dendritic trees of larval class IV dendritic arborization neurons. We identified 88 genes encoding such proteins whose knockdown resulted in aberrant dendritic morphology, including alterations in dendritic branch number, branch length, field size, and patterning of the dendritic tree. In particular, splicing and translation initiation factors were associated with distinct and characteristic phenotypes, suggesting that different morphogenetic events are best controlled at specific steps in post-transcriptional messenger RNA metabolism. Many of the factors identified in the screen have been implicated in controlling the subcellular distributions and translation of maternal messenger RNAs; thus, common post-transcriptional regulatory strategies may be used in neurogenesis and in the generation of asymmetry in the female germline and embryo. PMID:24347626

Cytoplasmic Control of Sense-Antisense mRNA Pairs.

PubMed

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

2015-09-22

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

Double-stranded RNA transcribed from vector-based oligodeoxynucleotide acts as transcription factor decoy

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

Xiao, Xiao; Gang, Yi; Department of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, Shaanxi Province

2015-02-06

Highlights: • A shRNA vector based transcription factor decoy, VB-ODN, was designed. • VB-ODN for NF-κB inhibited cell viability in HEK293 cells. • VB-ODN inhibited expression of downstream genes of target transcription factors. • VB-ODN may enhance nuclear entry ratio for its feasibility of virus production. - Abstract: In this study, we designed a short hairpin RNA vector-based oligodeoxynucleotide (VB-ODN) carrying transcription factor (TF) consensus sequence which could function as a decoy to block TF activity. Specifically, VB-ODN for Nuclear factor-κB (NF-κB) could inhibit cell viability and decrease downstream gene expression in HEK293 cells without affecting expression of NF-κB itself.more » The specific binding between VB-ODN produced double-stranded RNA and NF-κB was evidenced by electrophoretic mobility shift assay. Moreover, similar VB-ODNs designed for three other TFs also inhibit their downstream gene expression but not that of themselves. Our study provides a new design of decoy for blocking TF activity.« less

Transcriptional mutagenesis: causes and involvement in tumor development

PubMed Central

Brégeon, Damien; Doetsch, Paul W.

2013-01-01

The majority of normal cells in a human do not multiply continuously but are quiescent and devote most of their energy to gene transcription. When DNA damages in the transcribed strand of an active gene are bypassed by an RNA polymerase, they can miscode at the damaged site and produce mutant transcripts. This process known as transcriptional mutagenesis can lead to the production of mutant proteins that could be important in tumor development. PMID:21346784

Activity-Dependent Human Brain Coding/Noncoding Gene Regulatory Networks

PubMed Central

Lipovich, Leonard; Dachet, Fabien; Cai, Juan; Bagla, Shruti; Balan, Karina; Jia, Hui; Loeb, Jeffrey A.

2012-01-01

While most gene transcription yields RNA transcripts that code for proteins, a sizable proportion of the genome generates RNA transcripts that do not code for proteins, but may have important regulatory functions. The brain-derived neurotrophic factor (BDNF) gene, a key regulator of neuronal activity, is overlapped by a primate-specific, antisense long noncoding RNA (lncRNA) called BDNFOS. We demonstrate reciprocal patterns of BDNF and BDNFOS transcription in highly active regions of human neocortex removed as a treatment for intractable seizures. A genome-wide analysis of activity-dependent coding and noncoding human transcription using a custom lncRNA microarray identified 1288 differentially expressed lncRNAs, of which 26 had expression profiles that matched activity-dependent coding genes and an additional 8 were adjacent to or overlapping with differentially expressed protein-coding genes. The functions of most of these protein-coding partner genes, such as ARC, include long-term potentiation, synaptic activity, and memory. The nuclear lncRNAs NEAT1, MALAT1, and RPPH1, composing an RNAse P-dependent lncRNA-maturation pathway, were also upregulated. As a means to replicate human neuronal activity, repeated depolarization of SY5Y cells resulted in sustained CREB activation and produced an inverse pattern of BDNF-BDNFOS co-expression that was not achieved with a single depolarization. RNAi-mediated knockdown of BDNFOS in human SY5Y cells increased BDNF expression, suggesting that BDNFOS directly downregulates BDNF. Temporal expression patterns of other lncRNA-messenger RNA pairs validated the effect of chronic neuronal activity on the transcriptome and implied various lncRNA regulatory mechanisms. lncRNAs, some of which are unique to primates, thus appear to have potentially important regulatory roles in activity-dependent human brain plasticity. PMID:22960213

Analysis of Post-transcriptional Gene Regulation of Nod-Like Receptors via the 3'UTR.

PubMed

Haneklaus, Moritz

2016-01-01

Innate immune signaling is the front line of defense against pathogens, leading to an appropriate response of immune cells upon activation of their pattern recognition receptors (PRRs) by microbial products, such as Toll-like receptors (TLRs). Apart from transcriptional control, gene expression in the innate immune system is also highly regulated at the post-transcriptional level. miRNA or RNA-binding protein can bind to the 3' untranslated region (UTR) of target mRNAs and affect their mRNA stability and translation efficiency, which ultimately affects the amount of protein that is produced. In recent years, a new group of PRRs, the Nod-like receptors (NLR) have been discovered. They often cooperate with TLR signaling to induce potent inflammatory responses. Many NLRs can form inflammasomes, which facilitate the production of the potent pro-inflammatory cytokine IL-1β and other inflammatory mediators. In contrast to TLRs, the importance of post-transcriptional regulators in the context of inflammasomes has not been well defined. This chapter describes a series of experimental approaches to determine the effect of post-transcriptional regulation for a gene of interest using the best-studied NLR, NLRP3, as an example. To start investigating post-transcriptional regulation, 3'UTR luciferase experiments can be performed to test if regulatory sequences in the 3'UTR are functional. An RNA pull-down approach followed by mass spectrometry provides an unbiased assay to identify RNA-binding proteins that target the 3'UTR. Candidate binding proteins can then be further validated by RNA immunoprecipitation (RNA-IP), where the candidate protein is isolated using a specific antibody and bound mRNAs are analyzed by qPCR.

Gene expression profiling of whole blood: Comparison of target preparation methods for accurate and reproducible microarray analysis

PubMed Central

Vartanian, Kristina; Slottke, Rachel; Johnstone, Timothy; Casale, Amanda; Planck, Stephen R; Choi, Dongseok; Smith, Justine R; Rosenbaum, James T; Harrington, Christina A

2009-01-01

Background Peripheral blood is an accessible and informative source of transcriptomal information for many human disease and pharmacogenomic studies. While there can be significant advantages to analyzing RNA isolated from whole blood, particularly in clinical studies, the preparation of samples for microarray analysis is complicated by the need to minimize artifacts associated with highly abundant globin RNA transcripts. The impact of globin RNA transcripts on expression profiling data can potentially be reduced by using RNA preparation and labeling methods that remove or block globin RNA during the microarray assay. We compared four different methods for preparing microarray hybridization targets from human whole blood collected in PAXGene tubes. Three of the methods utilized the Affymetrix one-cycle cDNA synthesis/in vitro transcription protocol but varied treatment of input RNA as follows: i. no treatment; ii. treatment with GLOBINclear; or iii. treatment with globin PNA oligos. In the fourth method cDNA targets were prepared with the Ovation amplification and labeling system. Results We find that microarray targets generated with labeling methods that reduce globin mRNA levels or minimize the impact of globin transcripts during hybridization detect more transcripts in the microarray assay compared with the standard Affymetrix method. Comparison of microarray results with quantitative PCR analysis of a panel of genes from the NF-kappa B pathway shows good correlation of transcript measurements produced with all four target preparation methods, although method-specific differences in overall correlation were observed. The impact of freezing blood collected in PAXGene tubes on data reproducibility was also examined. Expression profiles show little or no difference when RNA is extracted from either fresh or frozen blood samples. Conclusion RNA preparation and labeling methods designed to reduce the impact of globin mRNA transcripts can significantly improve the sensitivity of the DNA microarray expression profiling assay for whole blood samples. While blockage of globin transcripts during first strand cDNA synthesis with globin PNAs resulted in the best overall performance in this study, we conclude that selection of a protocol for expression profiling studies in blood should depend on several factors, including implementation requirements of the method and study design. RNA isolated from either freshly collected or frozen blood samples stored in PAXGene tubes can be used without altering gene expression profiles. PMID:19123946

Analysis of RDR1/RDR2/RDR6-independent small RNAs in Arabidopsis thaliana improves MIRNA annotations and reveals unexplained types of short interfering RNA loci.

PubMed

Polydore, Seth; Axtell, Michael J

2018-06-01

Plant small RNAs (sRNAs) modulate key physiological mechanisms through post-transcriptional and transcriptional silencing of gene expression. Small RNAs fall into two major categories: those are reliant on RNA-dependent RNA polymerases (RDRs) for biogenesis and those that are not. Known RDR1/2/6-dependent sRNAs include phased and repeat-associated short interfering RNAs, while known RDR1/2/6-independent sRNAs are primarily microRNAs (miRNA) and other hairpin-derived sRNAs. In this study we produced and analyzed sRNA-seq libraries from rdr1/rdr2/rdr6 triple mutant plants. We found 58 previously annotated miRNA loci that were reliant on RDR1, -2, or -6 function, casting doubt on their classification. We also found 38 RDR1/2/6-independent sRNA loci that are not MIRNAs or otherwise hairpin-derived, and did not fit into other known paradigms for sRNA biogenesis. These 38 sRNA-producing loci have as-yet-undescribed biogenesis mechanisms, and are frequently located in the vicinity of protein-coding genes. Altogether, our analysis suggests that these 38 loci represent one or more undescribed types of sRNA in Arabidopsis thaliana. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

Regulation of insulin preRNA splicing by glucose

PubMed Central

Wang, Juehu; Shen, Luping; Najafi, Habiba; Kolberg, Janice; Matschinsky, Franz M.; Urdea, Mickey; German, Michael

1997-01-01

Glucose tightly regulates the synthesis and secretion of insulin by β cells in the pancreatic islets of Langerhans. To investigate whether glucose regulates insulin synthesis at the level of insulin RNA splicing, we developed a method to detect and quantify a small amount of RNA by using the branched DNA (bDNA) signal-amplification technique. This assay is both sensitive and highly specific: mouse insulin II mRNA can be detected from a single β cell (βTC3 cells or mouse islets), whereas 1 million non-insulin-producing α cells (αTC1.6 cells) give no signal. By using intron and exon sequences, oligonucleotide probes were designed to distinguish the various unspliced and partially spliced insulin preRNAs from mature insulin mRNA. Insulin RNA splicing rates were estimated from the rate of disappearance of insulin preRNA signal from β cells treated with actinomycin D to block transcription. We found that the two introns in mouse insulin II are not spliced with the same efficiency. Intron 2 is spliced out more efficiently than intron 1. As a result, some mRNA retaining intron 1 enters the cytoplasm, making up ≈2-10% of insulin mRNA in the cell. This partially spliced cytoplasmic mRNA is quite stable, with a half-life similar to the completely spliced form. When islets grown in high glucose are shifted to low glucose medium, the level of insulin preRNA and the rate of splicing fall significantly. We conclude that glucose stimulates insulin gene transcription and insulin preRNA splicing. Previous estimates of insulin transcription rates based on insulin preRNA levels that did not consider the rate of splicing may have underestimated the effect of glucose on insulin gene transcription. PMID:9113994

Recombinant modified vaccinia virus Ankara generating excess early double-stranded RNA transiently activates protein kinase R and triggers enhanced innate immune responses.

PubMed

Wolferstätter, Michael; Schweneker, Marc; Späth, Michaela; Lukassen, Susanne; Klingenberg, Marieken; Brinkmann, Kay; Wielert, Ursula; Lauterbach, Henning; Hochrein, Hubertus; Chaplin, Paul; Suter, Mark; Hausmann, Jürgen

2014-12-01

Double-stranded RNA (dsRNA) is an important molecular pattern associated with viral infection and is detected by various extra- and intracellular recognition molecules. Poxviruses have evolved to avoid producing dsRNA early in infection but generate significant amounts of dsRNA late in infection due to convergent transcription of late genes. Protein kinase R (PKR) is activated by dsRNA and triggers major cellular defenses against viral infection, including protein synthesis shutdown, apoptosis, and type I interferon (IFN-I) production. The poxviral E3 protein binds and sequesters viral dsRNA and is a major antagonist of the PKR pathway. We found that the highly replication-restricted modified vaccinia virus Ankara (MVA) engineered to produce excess amounts of dsRNA early in infection showed enhanced induction of IFN-β in murine and human cells in the presence of an intact E3L gene. IFN-β induction required a minimum overlap length of 300 bp between early complementary transcripts and was strongly PKR dependent. Excess early dsRNA produced by MVA activated PKR early but transiently in murine cells and induced enhanced systemic levels of IFN-α, IFN-γ, and other cytokines and chemokines in mice in a largely PKR-dependent manner. Replication-competent chorioallantois vaccinia virus Ankara (CVA) generating excess early dsRNA also enhanced IFN-I production and was apathogenic in mice even at very high doses but showed no in vitro host range defect. Thus, genetically adjuvanting MVA and CVA to generate excess early dsRNA is an effective method to enhance innate immune stimulation by orthopoxvirus vectors and to attenuate replicating vaccinia virus in vivo. Efficient cellular sensing of pathogen-specific components, including double-stranded RNA (dsRNA), is an important prerequisite of an effective antiviral immune response. The prototype poxvirus vaccinia virus (VACV) and its derivative modified vaccinia virus Ankara (MVA) produce dsRNA as a by-product of viral transcription. We found that inhibition of cellular dsRNA recognition established by the virus-encoded proteins E3 and K3 can be overcome by directing viral overexpression of dsRNA early in infection without compromising replication of MVA in permissive cells. Early dsRNA induced transient activation of the cellular dsRNA sensor protein kinase R (PKR), resulting in enhanced production of interferons and cytokines in cells and mice. Enhancing the capacity of MVA to activate the innate immune system is an important approach to further improve the immunogenicity of this promising vaccine vector. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Differential accumulation of nif structural gene mRNA in Azotobacter vinelandii.

PubMed

Hamilton, Trinity L; Jacobson, Marty; Ludwig, Marcus; Boyd, Eric S; Bryant, Donald A; Dean, Dennis R; Peters, John W

2011-09-01

Northern analysis was employed to investigate mRNA produced by mutant strains of Azotobacter vinelandii with defined deletions in the nif structural genes and in the intergenic noncoding regions. The results indicate that intergenic RNA secondary structures effect the differential accumulation of transcripts, supporting the high Fe protein-to-MoFe protein ratio required for optimal diazotrophic growth.

Extensive cross-regulation of post-transcriptional regulatory networks in Drosophila

DOE PAGES

Stoiber, Marcus H.; Olson, Sara; May, Gemma E.; ...

2015-08-20

In eukaryotic cells, RNAs exist as ribonucleoprotein particles (RNPs). Despite the importance of these complexes in many biological processes, including splicing, polyadenylation, stability, transportation, localization, and translation, their compositions are largely unknown. We affinity-purified 20 distinct RNA-binding proteins (RBPs) from cultured Drosophila melanogaster cells under native conditions and identified both the RNA and protein compositions of these RNP complexes. We identified “high occupancy target” (HOT) RNAs that interact with the majority of the RBPs we surveyed. HOT RNAs encode components of the nonsense-mediated decay and splicing machinery, as well as RNA-binding and translation initiation proteins. The RNP complexes contain proteinsmore » and mRNAs involved in RNA binding and post-transcriptional regulation. Genes with the capacity to produce hundreds of mRNA isoforms, ultracomplex genes, interact extensively with heterogeneous nuclear ribonuclear proteins (hnRNPs). Our data are consistent with a model in which subsets of RNPs include mRNA and protein products from the same gene, indicating the widespread existence of auto-regulatory RNPs. Lastly, from the simultaneous acquisition and integrative analysis of protein and RNA constituents of RNPs, we identify extensive cross-regulatory and hierarchical interactions in post-transcriptional control.« less

Extensive cross-regulation of post-transcriptional regulatory networks in Drosophila

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

Stoiber, Marcus H.; Olson, Sara; May, Gemma E.

In eukaryotic cells, RNAs exist as ribonucleoprotein particles (RNPs). Despite the importance of these complexes in many biological processes, including splicing, polyadenylation, stability, transportation, localization, and translation, their compositions are largely unknown. We affinity-purified 20 distinct RNA-binding proteins (RBPs) from cultured Drosophila melanogaster cells under native conditions and identified both the RNA and protein compositions of these RNP complexes. We identified “high occupancy target” (HOT) RNAs that interact with the majority of the RBPs we surveyed. HOT RNAs encode components of the nonsense-mediated decay and splicing machinery, as well as RNA-binding and translation initiation proteins. The RNP complexes contain proteinsmore » and mRNAs involved in RNA binding and post-transcriptional regulation. Genes with the capacity to produce hundreds of mRNA isoforms, ultracomplex genes, interact extensively with heterogeneous nuclear ribonuclear proteins (hnRNPs). Our data are consistent with a model in which subsets of RNPs include mRNA and protein products from the same gene, indicating the widespread existence of auto-regulatory RNPs. Lastly, from the simultaneous acquisition and integrative analysis of protein and RNA constituents of RNPs, we identify extensive cross-regulatory and hierarchical interactions in post-transcriptional control.« less

Hypoxia regulates alternative splicing of HIF and non-HIF target genes.

PubMed

Sena, Johnny A; Wang, Liyi; Heasley, Lynn E; Hu, Cheng-Jun

2014-09-01

Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1α (HIF1α) and 2α (HIF2α/EPAS1) to activate gene transcription, which promotes tumor cell survival. The majority of human genes are alternatively spliced, producing RNA isoforms that code for functionally distinct proteins. Thus, an effective hypoxia response requires increased HIF target gene expression as well as proper RNA splicing of these HIF-dependent transcripts. However, it is unclear if and how hypoxia regulates RNA splicing of HIF targets. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in hepatocellular carcinoma cells and characterized the role of HIF in regulating AS of HIF-induced genes. The results indicate that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia-reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF targets, including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirm that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of a PDK1 minigene by other transcription factors in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing. This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. ©2014 American Association for Cancer Research.

Alternative polyadenylation of the gene transcripts encoding a rat DNA polymerase beta.

PubMed

Konopiński, R; Nowak, R; Siedlecki, J A

1996-10-17

Rat cells produce two different transcripts of DNA polymerase beta (beta-Pol). The low-molecular-weight transcript (1.4 kb) was already sequenced. We report here the cloning and sequencing of the full-length cDNA, corresponding to the high-molecular-weight (HMW) transcript (4.0 kb) of beta-Pol. Sequence data strongly suggest that both transcripts are produced from a single gene by alternative polyadenylation. The HMW transcript contains the entire 1.4 kb transcript sequence and additional 2.2 kb on the 3' end. The 3' UTR of the HMW transcript contains some regulatory sequences which are not present in the 1.4-kb transcript. The A + U-rich fragment and (GU)21 sequence are believed to influence the stability of the mRNA. The functional significance of the A-rich region locally destabilizing double-stranded secondary structure remains unknown.

Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides.

PubMed

Hia, Fabian; Chionh, Yok Hian; Pang, Yan Ling Joy; DeMott, Michael S; McBee, Megan E; Dedon, Peter C

2015-03-11

A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species. The reproducibility of the method with BCG was evident in bioanalyzer electrophoretic analysis of isolated RNA, which revealed quantitatively significant differences in the ncRNA profiles of exponentially growing and non-replicating hypoxic bacilli. The method also overcame an historical inconsistency in 5S rRNA isolation, with direct sequencing revealing a novel post-transcriptional processing of 5S rRNA to its functional form and with chemical analysis revealing seven post-transcriptional ribonucleoside modifications in the 5S rRNA. This optimized RNA isolation procedure thus provides a means to more rigorously explore the biology of ncRNA species in mycobacteria. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

RNA transcript sequencing reveals inorganic sulfur compound oxidation pathways in the acidophile Acidithiobacillus ferrivorans.

PubMed

Christel, Stephan; Fridlund, Jimmy; Buetti-Dinh, Antoine; Buck, Moritz; Watkin, Elizabeth L; Dopson, Mark

2016-04-01

Acidithiobacillus ferrivorans is an acidophile implicated in low-temperature biomining for the recovery of metals from sulfide minerals. Acidithiobacillus ferrivorans obtains its energy from the oxidation of inorganic sulfur compounds, and genes encoding several alternative pathways have been identified. Next-generation sequencing of At. ferrivorans RNA transcripts identified the genes coding for metabolic and electron transport proteins for energy conservation from tetrathionate as electron donor. RNA transcripts suggested that tetrathionate was hydrolyzed by the tetH1 gene product to form thiosulfate, elemental sulfur and sulfate. Despite two of the genes being truncated, RNA transcripts for the SoxXYZAB complex had higher levels than for thiosulfate quinone oxidoreductase (doxDAgenes). However, a lack of heme-binding sites in soxX suggested that DoxDA was responsible for thiosulfate metabolism. Higher RNA transcript counts also suggested that elemental sulfur was metabolized by heterodisulfide reductase (hdrgenes) rather than sulfur oxygenase reductase (sor). The sulfite produced as a product of heterodisulfide reductase was suggested to be oxidized by a pathway involving the sat gene product or abiotically react with elemental sulfur to form thiosulfate. Finally, several electron transport complexes were involved in energy conservation. This study has elucidated the previously unknown At. ferrivorans tetrathionate metabolic pathway that is important in biomining. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Comparison of RNA Isolation Methods From Insect Larvae

PubMed Central

Ridgeway, J. A.; Timm, A. E.

2014-01-01

Abstract Isolating RNA from insects is becoming increasingly important in molecular entomology. Four methods including three commercial kits RNeasy Mini Kit (Qiagen), SV Total RNA isolation system (Promega), TRIzol reagent (Invitrogen), and a cetyl trimethylammonium bromide (CTAB)-based method were compared regarding their ability to isolate RNA from whole-body larvae of Thaumatotibia leucotreta (Meyrick), Thanatophilus micans (F.), Plutella xylostella (L.), and Tenebrio molitor (L.). A difference was observed among the four methods regarding RNA quality but not quantity. However, RNA quality and quantity obtained was not dependent on the insect species. The CTAB-based method produced low-quality RNA and the Trizol reagent produced partially degraded RNA, whereas the RNeasy Mini Kit and SV Total RNA isolation system produced RNA of consistently high quality. However, after reverse transcription to cDNA, RNA produced using all four extraction methods could be used to successfully amplify a 708 bp fragment of the cytochrome oxidase I gene. Of the four methods, the SV Total RNA isolation system showed the least amount of DNA contamination with the highest RNA integrity number and is thus recommended for stringent applications where high-quality RNA is required. This is the first comparison of RNA isolation methods among different insect species and the first to compare RNA isolation methods in insects in the last 20 years. PMID:25527580

An essential role for the RNA-binding protein Smaug during the Drosophila maternal-to-zygotic transition.

PubMed

Benoit, Beatrice; He, Chun Hua; Zhang, Fan; Votruba, Sarah M; Tadros, Wael; Westwood, J Timothy; Smibert, Craig A; Lipshitz, Howard D; Theurkauf, William E

2009-03-01

Genetic control of embryogenesis switches from the maternal to the zygotic genome during the maternal-to-zygotic transition (MZT), when maternal mRNAs are destroyed, high-level zygotic transcription is initiated, the replication checkpoint is activated and the cell cycle slows. The midblastula transition (MBT) is the first morphological event that requires zygotic gene expression. The Drosophila MBT is marked by blastoderm cellularization and follows 13 cleavage-stage divisions. The RNA-binding protein Smaug is required for cleavage-independent maternal transcript destruction during the Drosophila MZT. Here, we show that smaug mutants also disrupt syncytial blastoderm stage cell-cycle delays, DNA replication checkpoint activation, cellularization, and high-level zygotic expression of protein coding and micro RNA genes. We also show that Smaug protein levels increase through the cleavage divisions and peak when the checkpoint is activated and zygotic transcription initiates, and that transgenic expression of Smaug in an anterior-to-posterior gradient produces a concomitant gradient in the timing of maternal transcript destruction, cleavage cell cycle delays, zygotic gene transcription, cellularization and gastrulation. Smaug accumulation thus coordinates progression through the MZT.

Diversity of transcripts and transcript processing forms in plastids of the dinoflagellate alga Karenia mikimotoi.

PubMed

Dorrell, Richard G; Hinksman, George A; Howe, Christopher J

2016-02-01

Plastids produce a vast diversity of transcripts. These include mature transcripts containing coding sequences, and their processing precursors, as well as transcripts that lack direct coding functions, such as antisense transcripts. Although plastid transcriptomes have been characterised for many plant species, less is known about the transcripts produced in other plastid lineages. We characterised the transcripts produced in the fucoxanthin-containing plastids of the dinoflagellate alga Karenia mikimotoi. This plastid lineage, acquired through tertiary endosymbiosis, utilises transcript processing pathways that are very different from those found in plants and green algae, including 3' poly(U) tail addition, and extensive substitutional editing of transcript sequences. We have sequenced the plastid transcriptome of K. mikimotoi, and have detected evidence for divergent evolution of fucoxanthin plastid genomes. We have additionally characterised polycistronic and monocistronic transcripts from two plastid loci, psbD-tRNA (Met)-ycf4 and rpl36-rps13-rps11. We find evidence for a range of transcripts produced from each locus that differ in terms of editing state, 5' end cleavage position, and poly(U) tail addition. Finally, we identify antisense transcripts in K. mikimotoi, which appear to undergo different processing events from the corresponding sense transcripts. Overall, our study provides insights into the diversity of transcripts and processing intermediates found in plastid lineages across the eukaryotes.

Enterovirus A71 DNA-Launched Infectious Clone as a Robust Reverse Genetic Tool

PubMed Central

Tan, Chee Wah; Tee, Han Kang; Lee, Michelle Hui Pheng; Sam, I-Ching; Chan, Yoke Fun

2016-01-01

Enterovirus A71 (EV-A71) causes major outbreaks of hand, foot and mouth disease, and is occasionally associated with neurological complications and death in children. Reverse genetics is widely used in the field of virology for functional study of viral genes. For EV-A71, such tools are limited to clones that are transcriptionally controlled by T7/SP6 bacteriophage promoter. This is often time-consuming and expensive. Here, we describe the development of infectious plasmid DNA-based EV-A71 clones, for which EV-A71 genome expression is under transcriptional control by the CMV-intermediate early promoter and SV40 transcriptional-termination signal. Transfection of this EV-A71 infectious DNA produces good virus yield similar to in vitro-transcribed EV-A71 infectious RNA, 6.4 and 5.8 log10PFU/ml, respectively. Infectious plasmid with enhanced green fluorescence protein and Nano luciferase reporter genes also produced good virus titers, with 4.3 and 5.0 log10 PFU/ml, respectively. Another infectious plasmid with both CMV and T7 promoters was also developed for easy manipulation of in vitro transcription or direct plasmid transfection. Transfection with either dual-promoter infectious plasmid DNA or infectious RNA derived from this dual-promoter clone produced infectious viral particles. Incorporation of hepatitis delta virus ribozyme, which yields precise 3’ ends of the DNA-launched EV-A71 genomic transcripts, increased infectious viral production. In contrast, the incorporation of hammerhead ribozyme in the DNA-launched EV-A71 resulted in lower virus yield, but improved the virus titers for T7 promoter-derived infectious RNA. This study describes rapid and robust reverse genetic tools for EV-A71. PMID:27617744

Rapid detection of biothreat agents based on cellular machinery.

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

Lane, Todd W.; Gantt, Richard W.

This research addresses rapid and sensitive identification of biological agents in a complex background. We attempted to devise a method by which the specificity of the cellular transcriptional machinery could be used to detect and identify bacterial bio-terror agents in a background of other organisms. Bacterial cells contain RNA polymerases and transcription factors that transcribe genes into mRNA for translation into proteins. RNA polymerases in conjunction with transcription factors recognize regulatory elements (promoters) upstream of the gene. These promoters are, in many cases, recognized by the polymerase and transcription factor combinations of one species only. We have engineered a plasmid,more » for Escherichia coli, containing the virA promoter from the target species Shigella flexneri. This promoter was fused to a reporter gene Green Fluorescent Protein (GFP). In theory the indicator strain (carrying the plasmid) is mixed with the target strain and the two are lysed. The cellular machinery from both cells mixes and the GFP is produced. This report details the results of testing this system.« less

Intragenic DNA methylation prevents spurious transcription initiation.

PubMed

Neri, Francesco; Rapelli, Stefania; Krepelova, Anna; Incarnato, Danny; Parlato, Caterina; Basile, Giulia; Maldotti, Mara; Anselmi, Francesca; Oliviero, Salvatore

2017-03-02

In mammals, DNA methylation occurs mainly at CpG dinucleotides. Methylation of the promoter suppresses gene expression, but the functional role of gene-body DNA methylation in highly expressed genes has yet to be clarified. Here we show that, in mouse embryonic stem cells, Dnmt3b-dependent intragenic DNA methylation protects the gene body from spurious RNA polymerase II entry and cryptic transcription initiation. Using different genome-wide approaches, we demonstrate that this Dnmt3b function is dependent on its enzymatic activity and recruitment to the gene body by H3K36me3. Furthermore, the spurious transcripts can either be degraded by the RNA exosome complex or capped, polyadenylated, and delivered to the ribosome to produce aberrant proteins. Elongating RNA polymerase II therefore triggers an epigenetic crosstalk mechanism that involves SetD2, H3K36me3, Dnmt3b and DNA methylation to ensure the fidelity of gene transcription initiation, with implications for intragenic hypomethylation in cancer.

Oncogenes and RNA splicing of human tumor viruses

PubMed Central

Ajiro, Masahiko; Zheng, Zhi-Ming

2014-01-01

Approximately 10.8% of human cancers are associated with infection by an oncogenic virus. These viruses include human papillomavirus (HPV), Epstein–Barr virus (EBV), Merkel cell polyomavirus (MCV), human T-cell leukemia virus 1 (HTLV-1), Kaposi's sarcoma-associated herpesvirus (KSHV), hepatitis C virus (HCV) and hepatitis B virus (HBV). These oncogenic viruses, with the exception of HCV, require the host RNA splicing machinery in order to exercise their oncogenic activities, a strategy that allows the viruses to efficiently export and stabilize viral RNA and to produce spliced RNA isoforms from a bicistronic or polycistronic RNA transcript for efficient protein translation. Infection with a tumor virus affects the expression of host genes, including host RNA splicing factors, which play a key role in regulating viral RNA splicing of oncogene transcripts. A current prospective focus is to explore how alternative RNA splicing and the expression of viral oncogenes take place in a cell- or tissue-specific manner in virus-induced human carcinogenesis. PMID:26038756

Hepatitis B virus pathogenesis: Fresh insights into hepatitis B virus RNA.

PubMed

Sekiba, Kazuma; Otsuka, Motoyuki; Ohno, Motoko; Yamagami, Mari; Kishikawa, Takahiro; Suzuki, Tatsunori; Ishibashi, Rei; Seimiya, Takahiro; Tanaka, Eri; Koike, Kazuhiko

2018-06-07

Hepatitis B virus (HBV) is still a worldwide health concern. While divergent factors are involved in its pathogenesis, it is now clear that HBV RNAs, principally templates for viral proteins and viral DNAs, have diverse biological functions involved in HBV pathogenesis. These functions include viral replication, hepatic fibrosis and hepatocarcinogenesis. Depending on the sequence similarities, HBV RNAs may act as sponges for host miRNAs and may deregulate miRNA functions, possibly leading to pathological consequences. Some parts of the HBV RNA molecule may function as viral-derived miRNA, which regulates viral replication. HBV DNA can integrate into the host genomic DNA and produce novel viral-host fusion RNA, which may have pathological functions. To date, elimination of HBV-derived covalently closed circular DNA has not been achieved. However, RNA transcription silencing may be an alternative practical approach to treat HBV-induced pathogenesis. A full understanding of HBV RNA transcription and the biological functions of HBV RNA may open a new avenue for the development of novel HBV therapeutics.

Structure and Function of the N-Terminal Domain of the Vesicular Stomatitis Virus RNA Polymerase

PubMed Central

Qiu, Shihong; Ogino, Minako; Luo, Ming

2015-01-01

ABSTRACT Viruses have various mechanisms to duplicate their genomes and produce virus-specific mRNAs. Negative-strand RNA viruses encode their own polymerases to perform each of these processes. For the nonsegmented negative-strand RNA viruses, the polymerase is comprised of the large polymerase subunit (L) and the phosphoprotein (P). L proteins from members of the Rhabdoviridae, Paramyxoviridae, and Filoviridae share sequence and predicted secondary structure homology. Here, we present the structure of the N-terminal domain (conserved region I) of the L protein from a rhabdovirus, vesicular stomatitis virus, at 1.8-Å resolution. The strictly and strongly conserved residues in this domain cluster in a single area of the protein. Serial mutation of these residues shows that many of the amino acids are essential for viral transcription but not for mRNA capping. Three-dimensional alignments show that this domain shares structural homology with polymerases from other viral families, including segmented negative-strand RNA and double-stranded RNA (dsRNA) viruses. IMPORTANCE Negative-strand RNA viruses include a diverse set of viral families that infect animals and plants, causing serious illness and economic impact. The members of this group of viruses share a set of functionally conserved proteins that are essential to their replication cycle. Among this set of proteins is the viral polymerase, which performs a unique set of reactions to produce genome- and subgenome-length RNA transcripts. In this article, we study the polymerase of vesicular stomatitis virus, a member of the rhabdoviruses, which has served in the past as a model to study negative-strand RNA virus replication. We have identified a site in the N-terminal domain of the polymerase that is essential to viral transcription and that shares sequence homology with members of the paramyxoviruses and the filoviruses. Newly identified sites such as that described here could prove to be useful targets in the design of new therapeutics against negative-strand RNA viruses. PMID:26512087

The nucleolus—guardian of cellular homeostasis and genome integrity.

PubMed

Grummt, Ingrid

2013-12-01

All organisms sense and respond to conditions that stress their homeostasis by downregulating the synthesis of rRNA and ribosome biogenesis, thus designating the nucleolus as the central hub in coordinating the cellular stress response. One of the most intriguing roles of the nucleolus, long regarded as a mere ribosome-producing factory, is its participation in monitoring cellular stress signals and transmitting them to the RNA polymerase I (Pol I) transcription machinery. As rRNA synthesis is a most energy-consuming process, switching off transcription of rRNA genes is an effective way of saving the energy required to maintain cellular homeostasis during acute stress. The Pol I transcription machinery is the key convergence point that collects and integrates a vast array of information from cellular signaling cascades to regulate ribosome production which, in turn, guides cell growth and proliferation. This review focuses on the mechanisms that link cell physiology to rDNA silencing, a prerequisite for nucleolar integrity and cell survival.

Comparison of human and monkey cells for the ability to attenuate transcripts that begin at the adenovirus major late promoter

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

Seiberg, M.; Aloni, Y.; Levine, A.J.

1989-09-01

Late transcription from the adenovirus major late promoter can terminate prematurely at a site 182 to 188 nucleotides downstream. Experiments have been designed, with run-on transcription in nuclei in vitro or riboprobe protection of RNA obtained both in vivo and in vitro, that demonstrate that the ratio of attenuator RNA to readthrough RNA is greater in monkey cells (CV-1) than in human cells (HeLa). This may explain, in part, why the human adenoviruses replicate more poorly in CV-1 cells than in HeLa cells. A mutant adenovirus that replicates better than wild-type virus in monkey cells produces less of the attenuatormore » RNA than wild-type adenovirus does in monkey cells. Monkey cell extracts have been shown to contain a factor that, when added to human cell extracts transcribing adenovirus DNA in vitro, increases the production of attenuator RNA in these reactions. These observations help to explain a portion of the block to the production of infectious adenoviruses in monkey cells.« less

Connecting the dots: chromatin and alternative splicing in EMT.

PubMed

Warns, Jessica A; Davie, James R; Dhasarathy, Archana

2016-02-01

Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases, and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process.

SEASTAR: systematic evaluation of alternative transcription start sites in RNA.

PubMed

Qin, Zhiyi; Stoilov, Peter; Zhang, Xuegong; Xing, Yi

2018-05-04

Alternative first exons diversify the transcriptomes of eukaryotes by producing variants of the 5' Untranslated Regions (5'UTRs) and N-terminal coding sequences. Accurate transcriptome-wide detection of alternative first exons typically requires specialized experimental approaches that are designed to identify the 5' ends of transcripts. We developed a computational pipeline SEASTAR that identifies first exons from RNA-seq data alone then quantifies and compares alternative first exon usage across multiple biological conditions. The exons inferred by SEASTAR coincide with transcription start sites identified directly by CAGE experiments and bear epigenetic hallmarks of active promoters. To determine if differential usage of alternative first exons can yield insights into the mechanism controlling gene expression, we applied SEASTAR to an RNA-seq dataset that tracked the reprogramming of mouse fibroblasts into induced pluripotent stem cells. We observed dynamic temporal changes in the usage of alternative first exons, along with correlated changes in transcription factor expression. Using a combined sequence motif and gene set enrichment analysis we identified N-Myc as a regulator of alternative first exon usage in the pluripotent state. Our results demonstrate that SEASTAR can leverage the available RNA-seq data to gain insights into the control of gene expression and alternative transcript variation in eukaryotic transcriptomes.

Stress-induced alterations in 5-HT1A receptor transcriptional modulators NUDR and Freud-1.

PubMed

Szewczyk, Bernadeta; Kotarska, Katarzyna; Daigle, Mireille; Misztak, Paulina; Sowa-Kucma, Magdalena; Rafalo, Anna; Curzytek, Katarzyna; Kubera, Marta; Basta-Kaim, Agnieszka; Nowak, Gabriel; Albert, Paul R

2014-11-01

The effect of stress on the mRNA and protein level of the 5-HT1A receptor and two of its key transcriptional modulators, NUDR and Freud-1, was examined in the prefrontal cortex (PFC) and hippocampus (Hp) using rodent models: olfactory bulbectomy (OB) and prenatal stress (PS) in male and female rats; chronic mild stress in male rats (CMS) and pregnancy stress. In PFC, CMS induced the most widespread changes, with significant reduction in both mRNA and protein levels of NUDR, 5-HT1A receptor and in Freud-1 mRNA; while in Hp 5-HT1A receptor and Freud-1 protein levels were also decreased. In male, but not female OB rats PFC Freud-1 and 5-HT1A receptor protein levels were reduced, while in Hp 5-HT1A receptor, Freud-1 and NUDR mRNA's but not protein were reduced. In PS rats PFC 5-HT1A receptor protein was reduced more in females than males; while in Hp Freud-1 protein was increased in females. In pregnancy stress, PFC NUDR, Freud-1 and 5-HT1A protein receptor levels were reduced, and in HP 5-HT1A receptor protein levels were also reduced; in HP only NUDR and Freud-1 mRNA levels were reduced. Overall, CMS and stress during pregnancy produced the most salient changes in 5-HT1A receptor and transcription factor expression, suggesting a primary role for altered transcription factor expression in chronic regulation of 5-HT1A receptor expression. By contrast, OB (in males) and PS (in females) produced gender-specific reductions in PFC 5-HT1A receptor protein levels, suggesting a role for post-transcriptional regulation. These and previous data suggest that chronic stress might be a key regulator of NUDR/Freud-1 gene expression.

Small RNAs: artificial piRNAs for transcriptional silencing.

PubMed

Hirano, Takamasa; Siomi, Haruhiko

2015-03-30

Technologies have been developed in animal germ cells that produce artificial piRNAs from transgenes in piRNA clusters to silence target genes by cleaving their transcripts. A new study provides a simple way to generate artificial piRNAs to direct de novo DNA methylation in mice. Copyright © 2015 Elsevier Ltd. All rights reserved.

RNA-seq analysis of antibiotic-producing Bacillus subtilis SC-8 in response to signal peptide PapR of Bacillus cereus.

PubMed

Yeo, In-Cheol; Lee, Nam Keun; Yang, Byung Wook; Hahm, Young Tae

2014-01-01

Bacillus subtilis SC-8 produces an antibiotic that has narrow antagonistic activity against bacteria in the Bacillus cereus group. In B. cereus group bacteria, peptide-activating PlcR (PapR) plays a significant role in regulating the transcription of virulence factors. When B. subtilis SC-8 and B. cereus are co-cultured, PapR is assumed to stimulate antibiotic production by B. subtilis SC-8. To better understand the effect of PapR on this interspecies interaction, the global transcriptome profile of B. subtilis SC-8 was analyzed in the presence of PapR. Significant changes were detected in 12.8 % of the total transcripts. Genes related to amino acid transport and metabolism (16.5 %) and transcription (15 %) were mainly upregulated, whereas genes involved in carbohydrate transport and metabolism (12.7 %) were markedly downregulated. The expression of genes related to transcription, including several transcriptional regulators and proteins involved in tRNA biosynthesis, was increased. The expression levels of genes associated with several transport systems, such as antibiotic, cobalt, and iron complex transporters, was also significantly altered. Among the downregulated genes were transcripts associated with spore formation, the subtilosin A gene cluster, and nitrogen metabolism.

Adenosine Deaminase Acting on RNA 1 (ADAR1) Suppresses the Induction of Interferon by Measles Virus

PubMed Central

Li, Zhiqun; Okonski, Kristina M.

2012-01-01

ADAR1, the interferon (IFN)-inducible adenosine deaminase acting on RNA, catalyzes the C-6 deamination of adenosine (A) to produce inosine (I) in RNA substrates with a double-stranded character. Because double-stranded RNA is a known inducer of IFN, we tested the role of ADAR1 in IFN induction following virus infection. HeLa cells made stably deficient in ADAR1 (ADAR1kd) were compared to vector control (CONkd) and protein kinase PKR-deficient (PKRkd) cells for IFN-β induction following infection with either parental (wild-type [WT]) recombinant Moraten vaccine strain measles virus (MV) or isogenic knockout mutants deficient for either V (Vko) or C (Cko) protein expression. We observed potent IFN-β transcript induction in ADAR1kd cells by all three viruses; in contrast, in ADAR1-sufficient CONkd cells, only the Cko mutant virus was an effective inducer and the IFN-β RNA induction was amplified by PKR. The enhanced IFN-β transcript-inducing capacity of the WT and Vko viruses seen in ADAR1-deficient cells correlated with the enhanced activation of PKR, IFN regulatory factor IRF3, and activator of transcription ATF2, reaching levels similar to those seen in Cko virus-infected cells. However, the level of IFN-β protein produced was not proportional to the level of IFN-β RNA but rather correlated inversely with the level of activated PKR. These results suggest that ADAR1 functions as an important suppressor of MV-mediated responses, including the activation of PKR and IRF3 and the induction of IFN-β RNA. Our findings further implicate a balanced interplay between PKR and ADAR1 in modulating IFN-β protein production following virus infection. PMID:22278222

T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures

PubMed Central

Schaffter, Samuel W; Green, Leopold N; Schneider, Joanna; Subramanian, Hari K K; Schulman, Rebecca

2018-01-01

Abstract The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases. PMID:29718412

T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures.

PubMed

Schaffter, Samuel W; Green, Leopold N; Schneider, Joanna; Subramanian, Hari K K; Schulman, Rebecca; Franco, Elisa

2018-06-01

The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases.

Noninfectious virus-like particles produced by Moloney murine leukemia virus-based retrovirus packaging cells deficient in viral envelope become infectious in the presence of lipofection reagents

PubMed Central

Sharma, Sanjai; Murai, Fukashi; Miyanohara, Atsushi; Friedmann, Theodore

1997-01-01

Retrovirus packaging cell lines expressing the Moloney murine leukemia virus gag and pol genes but lacking virus envelope genes produce virus-like particles constitutively, whether or not they express a transcript from an integrated retroviral provirus. In the absence of a proviral transcript, the assembled particles contain processed gag and reverse transcriptase, and particles made by cells expressing an integrated lacZ provirus also contain viral RNA. The virus-like particles from both cell types are enveloped and are secreted/budded into the extracellular space but are noninfectious. Their physicochemical properties are similar to those of mature retroviral particles. The noninfectious gag pol RNA particles can readily be made infectious by the addition of lipofection reagents to produce preparations with titers of up to 105 colony-forming units per ml. PMID:9380714

Characterization of the platelet transcriptome by RNA sequencing in patients with acute myocardial infarction

PubMed Central

Eicher, John D.; Wakabayashi, Yoshiyuki; Vitseva, Olga; Esa, Nada; Yang, Yanqin; Zhu, Jun; Freedman, Jane E.; McManus, David D.; Johnson, Andrew D.

2016-01-01

Transcripts in platelets are largely produced in precursor megakaryocytes but remain physiologically-active as platelets translate RNAs and regulate protein/RNA levels. Recent studies using transcriptome sequencing (RNA-seq) characterized the platelet transcriptome in limited numbers of non-diseased individuals. Here, we expand upon these RNA-seq studies by completing RNA-seq in platelets from 32 patients with acute myocardial infarction (MI). Our goals were to characterize the platelet transcriptome using a population of patients with acute MI and relate gene expression to platelet aggregation measures and ST-segment elevation MI (STEMI) (n=16) versus non-STEMI (NSTEMI) (n=16) subtypes. Similar to other studies, we detected 9,565 expressed transcripts, including several known platelet-enriched markers (e.g., PPBP, OST4). Our RNA-seq data strongly correlated with independently ascertained platelet expression data and showed enrichment for platelet-related pathways (e.g., wound response, hemostasis, and platelet activation), as well as actin-related and post-transcriptional processes. Several transcripts displayed suggestively higher (FBXL4, ECHDC3, KCNE1, TAOK2, AURKB, ERG, and FKBP5) and lower (MIAT, PVRL3and PZP) expression in STEMI platelets compared to NSTEMI. We also identified transcripts correlated with platelet aggregation to TRAP (ATP6V1G2, SLC2A3), collagen (CEACAM1, ITGA2), and ADP (PDGFB, PDGFC, ST3GAL6). Our study adds to current platelet gene expression resources by providing transcriptome-wide analyses in platelets isolated from patients with acute MI. In concert with prior studies, we identify various genes for further study in regards to platelet function and acute MI. Future platelet RNA-seq studies examining more diverse sets of healthy and diseased samples will add to our understanding of platelet thrombotic and non-thrombotic functions. PMID:26367242

Ultrastructural modifications and changes in the expression of hormonal genes produced by indomethacin in the anterior pituitary gland of the rat.

PubMed

Romano, L A; Rivolta, C; Machiavelli, G A; Burdman, J A

1995-10-01

Indomethacin decreases the level of prolactin (50%) and growth hormone (70%) mRNA in the anterior pituitary gland of the rat. Actin mRNA increases (59%). Ultrastructurally there is a decrease in the number of secretory granules. Indomethacin also prevents the increase in prolactin secretory granules produced by the administration of estradiol. The results indicate that indomethacin inhibits hormonal synthesis in the APG at a transcriptional level. This effect appears selective because mRNA level for actin synthesis in the pituitary gland was higher than in nontreated rats.

In Vivo Delivery of Cytoplasmic RNA Virus-derived miRNAs

PubMed Central

Langlois, Ryan A; Shapiro, Jillian S; Pham, Alissa M; tenOever, Benjamin R

2012-01-01

The discovery of microRNAs (miRNAs) revealed an unappreciated level of post-transcriptional control used by the cell to maintain optimal protein levels. This process has represented an attractive strategy for therapeutics that is currently limited by in vivo delivery constraints. Here, we describe the generation of a single-stranded, cytoplasmic virus of negative polarity capable of producing functional miRNAs. Cytoplasmic RNA virus-derived miRNAs accumulated to high levels in vitro, generated significant amounts of miRNA star strand, associated with the RNA-induced silencing complex (RISC), and conferred post transcriptional gene silencing in a sequence-specific manner. Furthermore, we demonstrate that these vectors could deliver miRNAs to a wide range of tissues, and sustain prolonged expression capable of achieving measurable knockdown of physiological targets in vivo. Taken together, these results validate noncanonical processing of cytoplasmic-derived miRNAs and provide a novel platform for small RNA delivery. PMID:22086233

In vivo modulation of androgen receptor by androgens.

PubMed

Kumar, V L; Majumder, P K; Kumar, V

2002-09-01

To study the effect of androgen and antiandrogen on the level of androgen receptor (AR) mRNA. The total RNA was extracted from the prostate and analyzed by slot blot analysis. The blots were hybridized with AR cDNA probe and 1A probe (internal control) and autoradiography was performed. The intensity of signal was measured with a densitometer and the ratio of AR RNA and 1A RNA was calculated. Androgenic deprivation produced by castration decreased the weight of the prostate and increased the levels of AR mRNA. Treatment of the castrated rats with testostrone increased the weight of prostate and decreased the levels of AR mRNA. Treatment of normal rats with flutamide decreased the weight of the gland and increased the levels of AR mRNA. Androgens produce proliferative effect on the prostate and negatively regulate the AR transcription.

mRNA Cap Methyltransferase, RNMT-RAM, Promotes RNA Pol II-Dependent Transcription.

PubMed

Varshney, Dhaval; Lombardi, Olivia; Schweikert, Gabriele; Dunn, Sianadh; Suska, Olga; Cowling, Victoria H

2018-05-01

mRNA cap addition occurs early during RNA Pol II-dependent transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA Pol II transcription independent of mRNA capping and translation. In cells, sublethal suppression of RNMT-RAM reduces RNA Pol II occupancy, net mRNA synthesis, and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independent of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription-associated factors including the RNA Pol II subunits SPT4, SPT6, and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2BK120 ubiquitination, H3K4 and H3K36 methylation, RNA Pol II CTD S5 and S2 phosphorylation, and PAFc recruitment. These findings suggest that multiple interactions among RNMT-RAM, RNA Pol II factors, and RNA along the transcription unit stimulate transcription. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Circadian transcription factor BMAL1 regulates innate immunity against select RNA viruses.

PubMed

Majumdar, Tanmay; Dhar, Jayeeta; Patel, Sonal; Kondratov, Roman; Barik, Sailen

2017-02-01

BMAL1 (brain and muscle ARNT-like protein 1, also known as MOP3 or ARNT3) belongs to the family of the basic helix-loop-helix (bHLH)-PAS domain-containing transcription factors, and is a key component of the molecular oscillator that generates circadian rhythms. Here, we report that BMAL1-deficient cells are significantly more susceptible to infection by two major respiratory viruses of the Paramyxoviridae family, namely RSV and PIV3. Embryonic fibroblasts from Bmal1 -/- mice produced nearly 10-fold more progeny virus than their wild type controls. These results were supported by animal studies whereby pulmonary infection of RSV produced a more severe disease and morbidity in Bmal1 -/- mice. These results show that BMAL1 can regulate cellular innate immunity against specific RNA viruses.

The 3D protein of duck hepatitis A virus type 1 binds to a viral genomic 3' UTR and shows RNA-dependent RNA polymerase activity.

PubMed

Zhang, Yu; Cao, Qianda; Wang, Mingshu; Jia, Renyong; Chen, Shun; Zhu, Dekang; Liu, Mafeng; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Zhao, Xinxin; Chen, Xiaoyue; Cheng, Anchun

2017-12-01

To explore the RNA-dependent RNA polymerase (RdRP) function of the 3D protein of duck hepatitis A virus type 1 (DHAV-1), the gene was cloned into the pET-32a(+) vector for prokaryotic expression. The 3' untranslated region (3' UTR) of DHAV-1 together with a T7 promoter was cloned into the pMD19-T vector for in vitro transcription of 3' UTR RNA, which was further used as a template in RNA-dependent RNA polymerization. In this study, three methods were applied to analyze the RdRP function of the 3D protein: (1) ammonium molybdate spectrophotometry to detect pyrophosphate produced during polymerization; (2) quantitative reverse transcription PCR (RT-qPCR) to investigate the changes in RNA quantity during polymerization; and (3) electrophoresis mobility shift assay to examine the interaction between the 3D protein and 3' UTR. The results showed the 3D protein was successfully expressed in bacteria culture supernatant in a soluble form, which could be purified by affinity chromatography. In 3D enzymatic activity assays, pyrophosphate and RNA were produced, the amounts of which increased based on approximative kinetics, and binding of the 3D protein to the 3' UTR was observed. These results indicate that prokaryotically expressed soluble DHAV-13D protein can bind to a viral genomic 3' UTR and exhibit RdRP activity.

Comparative analysis reveals genomic features of stress-induced transcriptional readthrough

PubMed Central

Vilborg, Anna; Sabath, Niv; Wiesel, Yuval; Nathans, Jenny; Levy-Adam, Flonia; Yario, Therese A.; Steitz, Joan A.; Shalgi, Reut

2017-01-01

Transcription is a highly regulated process, and stress-induced changes in gene transcription have been shown to play a major role in stress responses and adaptation. Genome-wide studies reveal prevalent transcription beyond known protein-coding gene loci, generating a variety of RNA classes, most of unknown function. One such class, termed downstream of gene-containing transcripts (DoGs), was reported to result from transcriptional readthrough upon osmotic stress in human cells. However, how widespread the readthrough phenomenon is, and what its causes and consequences are, remain elusive. Here we present a genome-wide mapping of transcriptional readthrough, using nuclear RNA-Seq, comparing heat shock, osmotic stress, and oxidative stress in NIH 3T3 mouse fibroblast cells. We observe massive induction of transcriptional readthrough, both in levels and length, under all stress conditions, with significant, yet not complete, overlap of readthrough-induced loci between different conditions. Importantly, our analyses suggest that stress-induced transcriptional readthrough is not a random failure process, but is rather differentially induced across different conditions. We explore potential regulators and find a role for HSF1 in the induction of a subset of heat shock-induced readthrough transcripts. Analysis of public datasets detected increases in polymerase II occupancy in DoG regions after heat shock, supporting our findings. Interestingly, DoGs tend to be produced in the vicinity of neighboring genes, leading to a marked increase in their antisense-generating potential. Finally, we examine genomic features of readthrough transcription and observe a unique chromatin signature typical of DoG-producing regions, suggesting that readthrough transcription is associated with the maintenance of an open chromatin state. PMID:28928151

Potential miRNA regulators of differential HPG axis gene expression between low egg producing and high egg producing turkey hens

USDA-ARS?s Scientific Manuscript database

Expression differences exist in key genes of the hypothalamo-pituitary-gonadal (HPG) axis in low egg producing hens (LEPH) and high egg producing hens (HEPH); however, regulation of these differences is unknown. MicroRNAs (miRNAs) are small non-coding RNAs that play a role in post-transcriptional re...

Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays

PubMed Central

Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Sethi, Himanshu; Liang, Shoudan; Nelson, David C.; Hegeman, Adrian; Nelson, Clark; Rancour, David; Bednarek, Sebastian; Ulrich, Eldon L.; Zhao, Qin; Wrobel, Russell L.; Newman, Craig S.; Fox, Brian G.; Phillips, George N.; Markley, John L.; Sussman, Michael R.

2005-01-01

Using a maskless photolithography method, we produced DNA oligonucleotide microarrays with probe sequences tiled throughout the genome of the plant Arabidopsis thaliana. RNA expression was determined for the complete nuclear, mitochondrial, and chloroplast genomes by tiling 5 million 36-mer probes. These probes were hybridized to labeled mRNA isolated from liquid grown T87 cells, an undifferentiated Arabidopsis cell culture line. Transcripts were detected from at least 60% of the nearly 26,330 annotated genes, which included 151 predicted genes that were not identified previously by a similar genome-wide hybridization study on four different cell lines. In comparison with previously published results with 25-mer tiling arrays produced by chromium masking-based photolithography technique, 36-mer oligonucleotide probes were found to be more useful in identifying intron–exon boundaries. Using two-dimensional HPLC tandem mass spectrometry, a small-scale proteomic analysis was performed with the same cells. A large amount of strongly hybridizing RNA was found in regions “antisense” to known genes. Similarity of antisense activities between the 25-mer and 36-mer data sets suggests that it is a reproducible and inherent property of the experiments. Transcription activities were also detected for many of the intergenic regions and the small RNAs, including tRNA, small nuclear RNA, small nucleolar RNA, and microRNA. Expression of tRNAs correlates with genome-wide amino acid usage. PMID:15755812

Dynamic Hsp83 RNA localization during Drosophila oogenesis and embryogenesis.

PubMed Central

Ding, D; Parkhurst, S M; Halsell, S R; Lipshitz, H D

1993-01-01

Hsp83 is the Drosophila homolog of the mammalian Hsp90 family of regulatory molecular chaperones. We show that maternally synthesized Hsp83 transcripts are localized to the posterior pole of the early Drosophila embryo by a novel mechanism involving a combination of generalized RNA degradation and local protection at the posterior. This protection of Hsp83 RNA occurs in wild-type embryos and embryos produced by females carrying the maternal effect mutations nanos and pumilio, which eliminate components of the posterior polar plasm without disrupting polar granule integrity. In contrast, Hsp83 RNA is not protected at the posterior pole of embryos produced by females carrying maternal mutations that disrupt the posterior polar plasm and the polar granules--cappuccino, oskar, spire, staufen, tudor, valois, and vasa. Mislocalization of oskar RNA to the anterior pole, which has been shown to result in induction of germ cells at the anterior, leads to anterior protection of maternal Hsp83 RNA. These results suggest that Hsp83 RNA is a component of the posterior polar plasm that might be associated with polar granules. In addition, we show that zygotic expression of Hsp83 commences in the anterior third of the embryo at the syncytial blastoderm stage and is regulated by the anterior morphogen, bicoid. We consider the possible developmental significance of this complex control of Hsp83 transcript distribution. Images PMID:7684502

Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays

NASA Technical Reports Server (NTRS)

Stolc, Viktor; Samanta, Manoj Pratim; Tongprasit, Waraporn; Sethi, Himanshu; Liang, Shoudan; Nelson, David C.; Hegeman, Adrian; Nelson, Clark; Rancour, David; Bednarek, Sebastian;

Microprocessor mediates transcriptional termination in long noncoding microRNA genes

PubMed Central

Dhir, Ashish; Dhir, Somdutta; Proudfoot, Nick J.; Jopling, Catherine L.

2015-01-01

MicroRNA (miRNA) play a major role in the post-transcriptional regulation of gene expression. Mammalian miRNA biogenesis begins with co-transcriptional cleavage of RNA polymerase II (Pol II) transcripts by the Microprocessor complex. While most miRNA are located within introns of protein coding genes, a substantial minority of miRNA originate from long non coding (lnc) RNA where transcript processing is largely uncharacterized. We show, by detailed characterization of liver-specific lnc-pri-miR-122 and genome-wide analysis in human cell lines, that most lnc-pri-miRNA do not use the canonical cleavage and polyadenylation (CPA) pathway, but instead use Microprocessor cleavage to terminate transcription. This Microprocessor inactivation leads to extensive transcriptional readthrough of lnc-pri-miRNA and transcriptional interference with downstream genes. Consequently we define a novel RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells. PMID:25730776

A long noncoding RNA contributes to neuropathic pain by silencing Kcna2 in primary afferent neurons

PubMed Central

Zhao, Xiuli; Tang, Zongxiang; Zhang, Hongkang; Atianjoh, Fidelis E.; Zhao, Jian-Yuan; Liang, Lingli; Wang, Wei; Guan, Xiaowei; Kao, Sheng-Chin; Tiwari, Vinod; Gao, Yong-Jing; Hoffman, Paul N.; Cui, Hengmi; Li, Min; Dong, Xinzhong; Tao, Yuan-Xiang

2013-01-01

Neuropathic pain is a refractory disease characterized by maladaptive changes in gene transcription and translation within the sensory pathway. Long noncoding RNAs (lncRNAs) are emerging as new players in gene regulation, but how lncRNAs operate in the development of neuropathic pain is unclear. Here we identify a conserved lncRNA for Kcna2 (named Kcna2 antisense RNA) in first-order sensory neurons of rat dorsal root ganglion (DRG). Peripheral nerve injury increases Kcna2 antisense RNA expression in injured DRG through activation of myeloid zinc finger protein 1, a transcription factor that binds to Kcna2 antisense RNA gene promoter. Mimicking this increase downregulates Kcna2, reduces total Kv current, increases excitability in DRG neurons, and produces neuropathic pain symptoms. Blocking this increase reverses nerve injury-induced downregulation of DRG Kcna2 and attenuates development and maintenance of neuropathic pain. These findings suggest native Kcna2 antisense RNA as a new therapeutic target for the treatment of neuropathic pain. PMID:23792947

A Herpesvirus Protein Selectively Inhibits Cellular mRNA Nuclear Export.

PubMed

Gong, Danyang; Kim, Yong Hoon; Xiao, Yuchen; Du, Yushen; Xie, Yafang; Lee, Kevin K; Feng, Jun; Farhat, Nisar; Zhao, Dawei; Shu, Sara; Dai, Xinghong; Chanda, Sumit K; Rana, Tariq M; Krogan, Nevan J; Sun, Ren; Wu, Ting-Ting

2016-11-09

Nuclear mRNA export is highly regulated to ensure accurate cellular gene expression. Viral inhibition of cellular mRNA export can enhance viral access to the cellular translation machinery and prevent anti-viral protein production but is generally thought to be nonselective. We report that ORF10 of Kaposi's sarcoma-associated herpesvirus (KSHV), a nuclear DNA virus, inhibits mRNA export in a transcript-selective manner to control cellular gene expression. Nuclear export inhibition by ORF10 requires an interaction with an RNA export factor, Rae1. Genome-wide analysis reveals a subset of cellular mRNAs whose nuclear export is blocked by ORF10 with the 3' UTRs of ORF10-targeted transcripts conferring sensitivity to export inhibition. The ORF10-Rae1 interaction is important for the virus to express viral genes and produce infectious virions. These results suggest that a nuclear DNA virus can selectively interfere with RNA export to restrict host gene expression for optimal replication. Published by Elsevier Inc.

Stochastic resetting in backtrack recovery by RNA polymerases

NASA Astrophysics Data System (ADS)

Roldán, Édgar; Lisica, Ana; Sánchez-Taltavull, Daniel; Grill, Stephan W.

2016-06-01

Transcription is a key process in gene expression, in which RNA polymerases produce a complementary RNA copy from a DNA template. RNA polymerization is frequently interrupted by backtracking, a process in which polymerases perform a random walk along the DNA template. Recovery of polymerases from the transcriptionally inactive backtracked state is determined by a kinetic competition between one-dimensional diffusion and RNA cleavage. Here we describe backtrack recovery as a continuous-time random walk, where the time for a polymerase to recover from a backtrack of a given depth is described as a first-passage time of a random walker to reach an absorbing state. We represent RNA cleavage as a stochastic resetting process and derive exact expressions for the recovery time distributions and mean recovery times from a given initial backtrack depth for both continuous and discrete-lattice descriptions of the random walk. We show that recovery time statistics do not depend on the discreteness of the DNA lattice when the rate of one-dimensional diffusion is large compared to the rate of cleavage.

Uptake of dietary milk microRNAs by adult humans: Rules for the game of hide and seek

USDA-ARS?s Scientific Manuscript database

Milk producers recently used a social media campaign to build public confidence in the health benefits of their product; however, it is not clear why they did not tout the abundant microRNAs (miRNAs), small non-coding RNA molecules that function in RNA silencing and post-transcriptional regulation o...

Synthetic in vitro transcriptional oscillators

PubMed Central

Kim, Jongmin; Winfree, Erik

2011-01-01

The construction of synthetic biochemical circuits from simple components illuminates how complex behaviors can arise in chemistry and builds a foundation for future biological technologies. A simplified analog of genetic regulatory networks, in vitro transcriptional circuits, provides a modular platform for the systematic construction of arbitrary circuits and requires only two essential enzymes, bacteriophage T7 RNA polymerase and Escherichia coli ribonuclease H, to produce and degrade RNA signals. In this study, we design and experimentally demonstrate three transcriptional oscillators in vitro. First, a negative feedback oscillator comprising two switches, regulated by excitatory and inhibitory RNA signals, showed up to five complete cycles. To demonstrate modularity and to explore the design space further, a positive-feedback loop was added that modulates and extends the oscillatory regime. Finally, a three-switch ring oscillator was constructed and analyzed. Mathematical modeling guided the design process, identified experimental conditions likely to yield oscillations, and explained the system's robust response to interference by short degradation products. Synthetic transcriptional oscillators could prove valuable for systematic exploration of biochemical circuit design principles and for controlling nanoscale devices and orchestrating processes within artificial cells. PMID:21283141

Synthetic transcripts of double-stranded Birnavirus genome are infectious.

PubMed Central

Mundt, E; Vakharia, V N

1996-01-01

We have developed a system for generation of infectious bursal disease virus (IBDV), a segmented double-stranded RNA virus of the Birnaviridae family, with the use of synthetic transcripts derived from cloned cDNA. Independent full-length cDNA clones were constructed that contained the entire coding and noncoding regions of RNA segments A and B of two distinguishable IBDV strains of serotype I. Segment A encodes all of the structural (VP2, VP4, and VP3) and nonstructural (VP5) proteins, whereas segment B encodes the RNA-dependent RNA polymerase (VP1). Synthetic RNAs of both segments were produced by in vitro transcription of linearized plasmids with T7 RNA polymerase. Transfection of Vero cells with combined plus-sense transcripts of both segments generated infectious virus as early as 36 hr after transfection. The infectivity and specificity of the recovered chimeric virus was ascertained by the appearance of cytopathic effect in chicken embryo cells, by immunofluorescence staining of infected Vero cells with rabbit anti-IBDV serum, and by nucleotide sequence analysis of the recovered virus, respectively. In addition, transfectant viruses containing genetically tagged sequences in either segment A or segment B of IBDV were generated to confirm the feasibility of this system. The development of a reverse genetics system for double-stranded RNA viruses will greatly facilitate studies of the regulation of viral gene expression, pathogenesis, and design of a new generation of live vaccines. Images Fig. 2 Fig. 3 Fig. 4 PMID:8855321

Identification of new TSGA10 transcript variants in human testis with conserved regulatory RNA elements in 5'untranslated region and distinct expression in breast cancer.

PubMed

Salehipour, Pouya; Nematzadeh, Mahsa; Mobasheri, Maryam Beigom; Afsharpad, Mandana; Mansouri, Kamran; Modarressi, Mohammad Hossein

2017-09-01

Testis specific gene antigen 10 (TSGA10) is a cancer testis antigen involved in the process of spermatogenesis. TSGA10 could also play an important role in the inhibition of angiogenesis by preventing nuclear localization of HIF-1α. Although it has been shown that TSGA10 messenger RNA (mRNA) is mainly expressed in testis and some tumors, the transcription pattern and regulatory mechanisms of this gene remain largely unknown. Here, we report that human TSGA10 comprises at least 22 exons and generates four different transcript variants. It was identified that using two distinct promoters and splicing of exons 4 and 7 produced these transcript variants, which have the same coding sequence, but the sequence of 5'untanslated region (5'UTR) is different between them. This is significant because conserved regulatory RNA elements like upstream open reading frame (uORF) and putative internal ribosome entry site (IRES) were found in this region which have different combinations in each transcript variant and it may influence translational efficiency of them in normal or unusual environmental conditions like hypoxia. To indicate the transcription pattern of TSGA10 in breast cancer, expression of identified transcript variants was analyzed in 62 breast cancer samples. We found that TSGA10 tends to express variants with shorter 5'UTR and fewer uORF elements in breast cancer tissues. Our study demonstrates for the first time the expression of different TSGA10 transcript variants in testis and breast cancer tissues and provides a first clue to a role of TSGA10 5'UTR in regulation of translation in unusual environmental conditions like hypoxia. Copyright © 2017. Published by Elsevier B.V.

Expression of Shigella flexneri gluQ-rs gene is linked to dksA and controlled by a transcriptional terminator.

PubMed

Caballero, Valeria C; Toledo, Viviana P; Maturana, Cristian; Fisher, Carolyn R; Payne, Shelley M; Salazar, Juan Carlos

2012-10-05

Glutamyl queuosine-tRNA(Asp) synthetase (GluQ-RS) is a paralog of the catalytic domain of glutamyl-tRNA synthetase and catalyzes the formation of glutamyl-queuosine on the wobble position of tRNA(Asp). Here we analyze the transcription of its gene in Shigella flexneri, where it is found downstream of dksA, which encodes a transcriptional regulator involved in stress responses. The genomic organization, dksA-gluQ-rs, is conserved in more than 40 bacterial species. RT-PCR assays show co-transcription of both genes without a significant change in transcript levels during growth of S. flexneri. However, mRNA levels of the intergenic region changed during growth, increasing at stationary phase, indicating an additional level of control over the expression of gluQ-rs gene. Transcriptional fusions with lacZ as a reporter gene only produced β-galactosidase activity when the constructs included the dksA promoter, indicating that gluQ-rs do not have a separate promoter. Using bioinformatics, we identified a putative transcriptional terminator between dksA and gluQ-rs. Deletion or alteration of the predicted terminator resulted in increased expression of the lacZ reporter compared with cells containing the wild type terminator sequence. Analysis of the phenotype of a gluQ-rs mutant suggested that it may play a role in some stress responses, since growth of the mutant was impaired in the presence of osmolytes. The results presented here, show that the expression of gluQ-rs depends on the dksA promoter, and strongly suggest the presence and the functionality of a transcriptional terminator regulating its expression. Also, the results indicate a link between glutamyl-queuosine synthesis and stress response in Shigella flexneri.

Control of transcription of the Bacillus subtilis spoIIIG gene, which codes for the forespore-specific transcription factor sigma G.

PubMed

Sun, D X; Cabrera-Martinez, R M; Setlow, P

1991-05-01

The Bacillus subtilis spoIIIG gene codes for a sigma factor termed sigma G which directs transcription of genes expressed only in the forespore compartment of the sporulating cell. Use of spoIIIG-lacZ transcriptional fusions showed that spoIIIG is cotranscribed with the spoIIG operon beginning at t0.5-1 of sporulation. However, this large mRNA produced little if any sigma G, and transferring the spoIIIG gene without the spoIIG promoter into the amyE locus resulted in a Spo+ phenotype. Significant translation of spoIIIG began at t2.5-3 with use of an mRNA whose 5' end is just upstream of the spoIIIG coding sequence. Synthesis of this spoIIIG-specific mRNA was not abolished by a deletion in spoIIIG itself. Similar results were obtained when a spoIIIG-lacZ translational fusion lacking the spoIIG promoter was integrated at the amyE locus. These data suggest that synthesis of sigma G is dependent neither on transcription from the spoIIG promoter nor on sigma G itself but can be due to another transcription factor. This transcription factor may be sigma F, the product of the spoIIAC locus, since a spoIIAC mutation blocked spoIIIG expression, and sequences upstream of the 5' end of the spoIIIG-specific mRNA agree well with the recognition sequence for sigma F. RNA polymerase containing sigma F (E sigma F) initiated transcription in vitro on a spoIIIG template at the 5' end found in vivo, as did E sigma G. However, E sigma F showed a greater than 20-fold preference for spoIIIG over a known sigma G-dependent gene compared with the activity of E sigma G.

Regulation of glucose-dependent gene expression by the RNA helicase Dbp2 in Saccharomyces cerevisiae.

PubMed

Beck, Zachary T; Cloutier, Sara C; Schipma, Matthew J; Petell, Christopher J; Ma, Wai Kit; Tran, Elizabeth J

2014-11-01

Cellular homeostasis requires a fine balance between energy uptake, utilization, and growth. Dbp2 is a member of the DEAD-box protein family in Saccharomyces cerevisiae with characterized ATPase and helicase activity in vitro. DEAD-box RNA helicases are a class of enzymes that utilize ATP hydrolysis to remodel RNA and/or RNA-protein (RNP) composition. Dbp2 has been proposed to utilize its helicase activity in vivo to promote RNA-protein complex assembly of both messenger (m)RNAs and long noncoding (lnc)RNAs. Previous work from our laboratory demonstrated that loss of DBP2 enhances the lncRNA-dependent transcriptional induction of the GAL genes by abolishing glucose-dependent repression. Herein, we report that either a carbon source switch or glucose deprivation results in rapid export of Dbp2 to the cytoplasm. Genome-wide RNA sequencing identified a new class of antisense hexose transporter transcripts that are specifically upregulated upon loss of DBP2. Further investigation revealed that both sense and antisense hexose transporter (HXT) transcripts are aberrantly expressed in DBP2-deficient cells and that this expression pathway can be partially mimicked in wild-type cells by glucose depletion. We also find that Dbp2 promotes ribosome biogenesis and represses alternative ATP-producing pathways, as loss of DBP2 alters the transcript levels of ribosome biosynthesis (snoRNAs and associated proteins) and respiration gene products. This suggests that Dbp2 is a key integrator of nutritional status and gene expression programs required for energy homeostasis. Copyright © 2014 by the Genetics Society of America.

Seed Dormancy in Arabidopsis Is Controlled by Alternative Polyadenylation of DOG1.

PubMed

Cyrek, Malgorzata; Fedak, Halina; Ciesielski, Arkadiusz; Guo, Yanwu; Sliwa, Aleksandra; Brzezniak, Lien; Krzyczmonik, Katarzyna; Pietras, Zbigniew; Kaczanowski, Szymon; Liu, Fuquan; Swiezewski, Szymon

2016-02-01

DOG1 (Delay of Germination 1) is a key regulator of seed dormancy in Arabidopsis (Arabidopsis thaliana) and other plants. Interestingly, the C terminus of DOG1 is either absent or not conserved in many plant species. Here, we show that in Arabidopsis, DOG1 transcript is subject to alternative polyadenylation. In line with this, mutants in RNA 3' processing complex display weakened seed dormancy in parallel with defects in DOG1 proximal polyadenylation site selection, suggesting that the short DOG1 transcript is functional. This is corroborated by the finding that the proximally polyadenylated short DOG1 mRNA is translated in vivo and complements the dog1 mutant. In summary, our findings indicate that the short DOG1 protein isoform produced from the proximally polyadenylated DOG1 mRNA is a key player in the establishment of seed dormancy in Arabidopsis and characterizes a set of mutants in RNA 3' processing complex required for production of proximally polyadenylated functional DOG1 transcript. © 2016 American Society of Plant Biologists. All Rights Reserved.

Reduced changes in protein compared to mRNA levels across non-proliferating tissues.

PubMed

Perl, Kobi; Ushakov, Kathy; Pozniak, Yair; Yizhar-Barnea, Ofer; Bhonker, Yoni; Shivatzki, Shaked; Geiger, Tamar; Avraham, Karen B; Shamir, Ron

2017-04-18

The quantitative relations between RNA and protein are fundamental to biology and are still not fully understood. Across taxa, it was demonstrated that the protein-to-mRNA ratio in steady state varies in a direction that lessens the change in protein levels as a result of changes in the transcript abundance. Evidence for this behavior in tissues is sparse. We tested this phenomenon in new data that we produced for the mouse auditory system, and in previously published tissue datasets. A joint analysis of the transcriptome and proteome was performed across four datasets: inner-ear mouse tissues, mouse organ tissues, lymphoblastoid primate samples and human cancer cell lines. We show that the protein levels are more conserved than the mRNA levels in all datasets, and that changes in transcription are associated with translational changes that exert opposite effects on the final protein level, in all tissues except cancer. Finally, we observe that some functions are enriched in the inner ear on the mRNA level but not in protein. We suggest that partial buffering between transcription and translation ensures that proteins can be made rapidly in response to a stimulus. Accounting for the buffering can improve the prediction of protein levels from mRNA levels.

ADARs, Viruses and Innate Immunity

PubMed Central

Samuel, Charles E.

2013-01-01

Double-stranded RNA (dsRNA) functions both as a substrate of ADARs and also as a molecular trigger of innate immune responses. ADARs, adenosine deaminases that act on RNA, catalyze the deamination of adenosine (A) to produce inosine (I) in dsRNA. ADARs thereby can destablize RNA structures, because the generated I:U mismatch pairs are less stable than A:U base pairs. Additionally, I is read as G instead of A by ribosomes during translation and by viral RNA-dependent RNA polymerases during RNA replication. Members of several virus families have the capacity to produce dsRNA during viral genome transcription and replication. Sequence changes (A-to-G, and U-to-C) characteristic of A-to-I editing occur during virus growth and persistence. Foreign viral dsRNA also mediates both the induction and the action of interferons. In this chapter our current understanding of the role and significance of ADARs in the context of innate immunity, and as determinants of the outcome of viral infection, will be considered. PMID:21809195

Connecting the dots: chromatin and alternative splicing in EMT

PubMed Central

Warns, Jessica A.; Davie, James R.; Dhasarathy, Archana

2015-01-01

Nature has devised sophisticated cellular machinery to process mRNA transcripts produced by RNA Polymerase II, removing intronic regions and connecting exons together, to produce mature RNAs. This process, known as splicing, is very closely linked to transcription. Alternative splicing, or the ability to produce different combinations of exons that are spliced together from the same genomic template, is a fundamental means of regulating protein complexity. Similar to transcription, both constitutive and alternative splicing can be regulated by chromatin and its associated factors in response to various signal transduction pathways activated by external stimuli. This regulation can vary between different cell types, and interference with these pathways can lead to changes in splicing, often resulting in aberrant cellular states and disease. The epithelial to mesenchymal transition (EMT), which leads to cancer metastasis, is influenced by alternative splicing events of chromatin remodelers and epigenetic factors such as DNA methylation and non-coding RNAs. In this review, we will discuss the role of epigenetic factors including chromatin, chromatin remodelers, DNA methyltransferases and microRNAs in the context of alternative splicing, and discuss their potential involvement in alternative splicing during the EMT process. PMID:26291837

Identification of an Enhancer That Increases miR-200b~200a~429 Gene Expression in Breast Cancer Cells

PubMed Central

Attema, Joanne L.; Bert, Andrew G.; Lim, Yat-Yuen; Kolesnikoff, Natasha; Lawrence, David M.; Pillman, Katherine A.; Smith, Eric; Drew, Paul A.; Khew-Goodall, Yeesim; Shannon, Frances; Goodall, Gregory J.

2013-01-01

The miR-200b~200a~429 gene cluster is a key regulator of EMT and cancer metastasis, however the transcription-based mechanisms controlling its expression during this process are not well understood. We have analyzed the miR-200b~200a~429 locus for epigenetic modifications in breast epithelial and mesenchymal cell lines using chromatin immunoprecipitation assays and DNA methylation analysis. We discovered a novel enhancer located approximately 5.1kb upstream of the miR-200b~200a~429 transcriptional start site. This region was associated with the active enhancer chromatin signature comprising H3K4me1, H3K27ac, RNA polymerase II and CpG dinucleotide hypomethylation. Luciferase reporter assays revealed the upstream enhancer stimulated the transcription of the miR-200b~200a~429 minimal promoter region approximately 27-fold in breast epithelial cells. Furthermore, we found that a region of the enhancer was transcribed, producing a short, GC-rich, mainly nuclear, non-polyadenylated RNA transcript designated miR-200b eRNA. Over-expression of miR-200b eRNA had little effect on miR-200b~200a~429 promoter activity and its production did not correlate with miR-200b~200a~429 gene expression. While additional investigations of miR-200b eRNA function will be necessary, it is possible that miR-200b eRNA may be involved in the regulation of miR-200b~200a~429 gene expression and silencing. Taken together, these findings reveal the presence of a novel enhancer, which contributes to miR-200b~200a~429 transcriptional regulation in epithelial cells. PMID:24086551

Saccharomyces cerevisiae Sen1 Helicase Domain Exhibits 5'- to 3'-Helicase Activity with a Preference for Translocation on DNA Rather than RNA.

PubMed

Martin-Tumasz, Stephen; Brow, David A

2015-09-18

In the yeast Saccharomyces cerevisiae, the essential nuclear helicase Sen1 is required for efficient termination of transcription of short noncoding RNA genes by RNA polymerase II. However, the mechanism by which Sen1 promotes transcription termination is not known. Prior biochemical studies on the Sen1 homolog from Schizosaccharomyces pombe showed that it can bind and unwind both DNA and RNA, but the S. pombe protein is not essential and has not been demonstrated to function in transcription. Furthermore, Sen1 from either yeast has not previously been expressed as a recombinant protein, due to its large molecular mass (252 kDa in S. cerevisiae). Here, we report the purification and characterization of the 89-kDa S. cerevisiae Sen1 helicase domain (Sen1-HD) produced in Escherichia coli. Sen1-HD binds single-stranded RNA and DNA with similar affinity in the absence of ATP, but it binds RNA more stably than DNA in the presence of ATP, apparently due to a slower translocation rate on RNA. Translocation occurs in the 5' to 3' direction, as for the S. pombe protein. When purified from E. coli at a moderate salt concentration, Sen1-HD was associated with short RNAs that are enriched for the trinucleotide repeat (CAN)4. We propose that Sen1 binds to RNAs and prevents their stable pairing with DNA, consistent with in vivo studies by others showing increased R-loop (RNA/DNA hybrid) formation when Sen1 activity is impaired by mutations. Our results are consistent with a model in which Sen1 promotes transcription termination by resolving R-loops. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Separation of replication and transcription domains in nucleoli.

PubMed

Smirnov, E; Borkovec, J; Kováčik, L; Svidenská, S; Schröfel, A; Skalníková, M; Švindrych, Z; Křížek, P; Ovesný, M; Hagen, G M; Juda, P; Michalová, K; Cardoso, M C; Cmarko, D; Raška, I

2014-12-01

In mammalian cells, active ribosomal genes produce the 18S, 5.8S and 28S RNAs of ribosomal particles. Transcription levels of these genes are very high throughout interphase, and the cell needs a special strategy to avoid collision of the DNA polymerase and RNA polymerase machineries. To investigate this problem, we measured the correlation of various replication and transcription signals in the nucleoli of HeLa, HT-1080 and NIH 3T3 cells using a specially devised software for analysis of confocal images. Additionally, to follow the relationship between nucleolar replication and transcription in living cells, we produced a stable cell line expressing GFP-RPA43 (subunit of RNA polymerase I, pol I) and RFP-PCNA (the sliding clamp protein) based on human fibrosarcoma HT-1080 cells. We found that replication and transcription signals are more efficiently separated in nucleoli than in the nucleoplasm. In the course of S phase, separation of PCNA and pol I signals gradually increased. During the same period, separation of pol I and incorporated Cy5-dUTP signals decreased. Analysis of single molecule localization microscopy (SMLM) images indicated that transcriptionally active FC/DFC units (i.e. fibrillar centers with adjacent dense fibrillar components) did not incorporate DNA nucleotides. Taken together, our data show that replication of the ribosomal genes is spatially separated from their transcription, and FC/DFC units may provide a structural basis for that separation. Copyright © 2014 Elsevier Inc. All rights reserved.

Contributions of in vitro transcription to the understanding of human RNA polymerase III transcription

PubMed Central

Dumay-Odelot, Hélène; Durrieu-Gaillard, Stéphanie; El Ayoubi, Leyla; Parrot, Camila; Teichmann, Martin

2014-01-01

Human RNA polymerase III transcribes small untranslated RNAs that contribute to the regulation of essential cellular processes, including transcription, RNA processing and translation. Analysis of this transcription system by in vitro transcription techniques has largely contributed to the discovery of its transcription factors and to the understanding of the regulation of human RNA polymerase III transcription. Here we review some of the key steps that led to the identification of transcription factors and to the definition of minimal promoter sequences for human RNA polymerase III transcription. PMID:25764111

Mitotic inheritance of mRNA facilitates translational activation of the osteogenic-lineage commitment factor Runx2 in progeny of osteoblastic cells

PubMed Central

Varela, Nelson; Aranguiz, Alejandra; Lizama, Carlos; Sepulveda, Hugo; Antonelli, Marcelo; Thaler, Roman; Moreno, Ricardo D.; Montecino, Martin; Stein, Gary S.; van Wijnen, Andre J.; Galindo, Mario

2017-01-01

Epigenetic mechanisms mediate the acquisition of specialized cellular phenotypes during tissue development, maintenance and repair. When phenotype-committed cells transit through mitosis, chromosomal condensation counteracts epigenetic activation of gene expression. Subsequent post-mitotic re-activation of transcription depends on epigenetic DNA and histone modifications, as well as other architecturally bound proteins that ‘bookmark’ the genome. Osteogenic lineage commitment, differentiation and progenitor proliferation require the bone-related runt-related transcription factor Runx2. Here, we characterized a non-genomic mRNA mediated mechanism by which osteoblast precursors retain their phenotype during self-renewal. We show that osteoblasts produce maximal levels of Runx2 mRNA, but not protein, prior to mitotic cell division. Runx2 mRNA partitions symmetrically between daughter cells in a non-chromosomal tubulin-containing compartment. Subsequently, transcription-independent de novo synthesis of Runx2 protein in early G1 phase results in increased functional interactions of Runx2 with a representative osteoblast-specific target gene (osteocalcin/BGLAP2) in chromatin. Somatic transmission of Runx2 mRNAs in osteoblasts and osteosarcoma cells represents a versatile mechanism for translational rather than transcriptional induction of this principal gene regulator to maintain osteoblast phenotype identity after mitosis. PMID:26381402

RNA sequencing of synaptic and cytoplasmic Upf1-bound transcripts supports contribution of nonsense-mediated decay to epileptogenesis

PubMed Central

Mooney, Claire M.; Jimenez-Mateos, Eva M.; Engel, Tobias; Mooney, Catherine; Diviney, Mairead; Venø, Morten T.; Kjems, Jørgen; Farrell, Michael A.; O’Brien, Donncha F.; Delanty, Norman; Henshall, David C.

2017-01-01

The nonsense mediated decay (NMD) pathway is a critical surveillance mechanism for identifying aberrant mRNA transcripts. It is unknown, however, whether the NMD system is affected by seizures in vivo and whether changes confer beneficial or maladaptive responses that influence long-term outcomes such the network alterations that produce spontaneous recurrent seizures. Here we explored the responses of the NMD pathway to prolonged seizures (status epilepticus) and investigated the effects of NMD inhibition on epilepsy in mice. Status epilepticus led to increased protein levels of Up-frameshift suppressor 1 homolog (Upf1) within the mouse hippocampus. Upf1 protein levels were also higher in resected hippocampus from patients with intractable temporal lobe epilepsy. Immunoprecipitation of Upf1-bound RNA from the cytoplasmic and synaptosomal compartments followed by RNA sequencing identified unique populations of NMD-associated transcripts and altered levels after status epilepticus, including known substrates such as Arc as well as novel targets including Inhba and Npas4. Finally, long-term video-EEG recordings determined that pharmacologic interference in the NMD pathway after status epilepticus reduced the later occurrence of spontaneous seizures in mice. These findings suggest compartment-specific recruitment and differential loading of transcripts by NMD pathway components may contribute to the process of epileptogenesis. PMID:28128343

De novo assembled expressed gene catalog of a fast-growing Eucalyptus tree produced by Illumina mRNA-Seq

PubMed Central

2010-01-01

Background De novo assembly of transcript sequences produced by short-read DNA sequencing technologies offers a rapid approach to obtain expressed gene catalogs for non-model organisms. A draft genome sequence will be produced in 2010 for a Eucalyptus tree species (E. grandis) representing the most important hardwood fibre crop in the world. Genome annotation of this valuable woody plant and genetic dissection of its superior growth and productivity will be greatly facilitated by the availability of a comprehensive collection of expressed gene sequences from multiple tissues and organs. Results We present an extensive expressed gene catalog for a commercially grown E. grandis × E. urophylla hybrid clone constructed using only Illumina mRNA-Seq technology and de novo assembly. A total of 18,894 transcript-derived contigs, a large proportion of which represent full-length protein coding genes were assembled and annotated. Analysis of assembly quality, length and diversity show that this dataset represent the most comprehensive expressed gene catalog for any Eucalyptus tree. mRNA-Seq analysis furthermore allowed digital expression profiling of all of the assembled transcripts across diverse xylogenic and non-xylogenic tissues, which is invaluable for ascribing putative gene functions. Conclusions De novo assembly of Illumina mRNA-Seq reads is an efficient approach for transcriptome sequencing and profiling in Eucalyptus and other non-model organisms. The transcriptome resource (Eucspresso, http://eucspresso.bi.up.ac.za/) generated by this study will be of value for genomic analysis of woody biomass production in Eucalyptus and for comparative genomic analysis of growth and development in woody and herbaceous plants. PMID:21122097

Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks

PubMed Central

Trapnell, Cole; Roberts, Adam; Goff, Loyal; Pertea, Geo; Kim, Daehwan; Kelley, David R; Pimentel, Harold; Salzberg, Steven L; Rinn, John L; Pachter, Lior

2012-01-01

Recent advances in high-throughput cDNA sequencing (RNA-seq) can reveal new genes and splice variants and quantify expression genome-wide in a single assay. The volume and complexity of data from RNA-seq experiments necessitate scalable, fast and mathematically principled analysis software. TopHat and Cufflinks are free, open-source software tools for gene discovery and comprehensive expression analysis of high-throughput mRNA sequencing (RNA-seq) data. Together, they allow biologists to identify new genes and new splice variants of known ones, as well as compare gene and transcript expression under two or more conditions. This protocol describes in detail how to use TopHat and Cufflinks to perform such analyses. It also covers several accessory tools and utilities that aid in managing data, including CummeRbund, a tool for visualizing RNA-seq analysis results. Although the procedure assumes basic informatics skills, these tools assume little to no background with RNA-seq analysis and are meant for novices and experts alike. The protocol begins with raw sequencing reads and produces a transcriptome assembly, lists of differentially expressed and regulated genes and transcripts, and publication-quality visualizations of analysis results. The protocol's execution time depends on the volume of transcriptome sequencing data and available computing resources but takes less than 1 d of computer time for typical experiments and ~1 h of hands-on time. PMID:22383036

Coupled Evolution of Transcription and mRNA Degradation

PubMed Central

Dori-Bachash, Mally; Shema, Efrat; Tirosh, Itay

2011-01-01

mRNA levels are determined by the balance between transcription and mRNA degradation, and while transcription has been extensively studied, very little is known regarding the regulation of mRNA degradation and its coordination with transcription. Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in mRNA degradation were coupled to transcriptional changes that exert opposite effects on mRNA levels. Analysis of mRNA degradation rates in an interspecific hybrid further suggests that opposite evolutionary changes in transcription and in mRNA degradation are mechanistically coupled and were generated by the same individual mutations. Coupled changes are associated with divergence of two complexes that were previously implicated both in transcription and in mRNA degradation (Rpb4/7 and Ccr4-Not), as well as with sequence divergence of transcription factor binding motifs. These results suggest that an opposite coupling between the regulation of transcription and that of mRNA degradation has shaped the evolution of gene regulation in yeast. PMID:21811398

Enhanced Upregulation of CRH mRNA Expression in the Nucleus Accumbens of Male Rats after a Second Injection of Methamphetamine Given Thirty Days Later

PubMed Central

Cadet, Jean Lud; Brannock, Christie; Ladenheim, Bruce; McCoy, Michael T.; Krasnova, Irina N.; Lehrmann, Elin; Becker, Kevin G.; Jayanthi, Subramaniam

2014-01-01

Methamphetamine (METH) is a widely abused amphetamine analog. Few studies have investigated the molecular effects of METH exposure in adult animals. Herein, we determined the consequences of an injection of METH (10 mg/kg) on transcriptional effects of a second METH (2.5 mg/kg) injection given one month later. We thus measured gene expression by microarray analyses in the nucleus accumbens (NAc) of 4 groups of rats euthanized 2 hours after the second injection: saline-pretreated followed by saline-challenged (SS) or METH-challenged (SM); and METH-pretreated followed by saline-challenged (MS) or METH-challenged (MM). Microarray analyses revealed that METH (2.5 mg/kg) produced acute changes (1.8-fold; P<0.01) in the expression of 412 (352 upregulated, 60 down-regulated) transcripts including cocaine and amphetamine regulated transcript, corticotropin-releasing hormone (Crh), oxytocin (Oxt), and vasopressin (Avp) that were upregulated. Injection of METH (10 mg/kg) altered the expression of 503 (338 upregulated, 165 down-regulated) transcripts measured one month later (MS group). These genes also included Cart and Crh. The MM group showed altered expression of 766 (565 upregulated, 201 down-regulated) transcripts including Avp, Cart, and Crh. The METH-induced increased Crh expression was enhanced in the MM group in comparison to SM and MS groups. Quantitative PCR confirmed the METH-induced changes in mRNA levels. Therefore, a single injection of METH produced long-lasting changes in gene expression in the rodent NAc. The long-term increases in Crh, Cart, and Avp mRNA expression suggest that METH exposure produced prolonged activation of the endogenous stress system. The METH-induced changes in oxytocin expression also suggest the possibility that this neuropeptide might play a significant role in the neuroplastic and affiliative effects of this drug. PMID:24475032

Peregrine: A rapid and unbiased method to produce strand-specific RNA-Seq libraries from small quantities of starting material.

PubMed

Langevin, Stanley A; Bent, Zachary W; Solberg, Owen D; Curtis, Deanna J; Lane, Pamela D; Williams, Kelly P; Schoeniger, Joseph S; Sinha, Anupama; Lane, Todd W; Branda, Steven S

2013-04-01

Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows.

miRvestigator: web application to identify miRNAs responsible for co-regulated gene expression patterns discovered through transcriptome profiling.

PubMed

Plaisier, Christopher L; Bare, J Christopher; Baliga, Nitin S

2011-07-01

Transcriptome profiling studies have produced staggering numbers of gene co-expression signatures for a variety of biological systems. A significant fraction of these signatures will be partially or fully explained by miRNA-mediated targeted transcript degradation. miRvestigator takes as input lists of co-expressed genes from Caenorhabditis elegans, Drosophila melanogaster, G. gallus, Homo sapiens, Mus musculus or Rattus norvegicus and identifies the specific miRNAs that are likely to bind to 3' un-translated region (UTR) sequences to mediate the observed co-regulation. The novelty of our approach is the miRvestigator hidden Markov model (HMM) algorithm which systematically computes a similarity P-value for each unique miRNA seed sequence from the miRNA database miRBase to an overrepresented sequence motif identified within the 3'-UTR of the query genes. We have made this miRNA discovery tool accessible to the community by integrating our HMM algorithm with a proven algorithm for de novo discovery of miRNA seed sequences and wrapping these algorithms into a user-friendly interface. Additionally, the miRvestigator web server also produces a list of putative miRNA binding sites within 3'-UTRs of the query transcripts to facilitate the design of validation experiments. The miRvestigator is freely available at http://mirvestigator.systemsbiology.net.

In silico methods for co-transcriptional RNA secondary structure prediction and for investigating alternative RNA structure expression.

PubMed

Meyer, Irmtraud M

2017-05-01

RNA transcripts are the primary products of active genes in any living organism, including many viruses. Their cellular destiny not only depends on primary sequence signals, but can also be determined by RNA structure. Recent experimental evidence shows that many transcripts can be assigned more than a single functional RNA structure throughout their cellular life and that structure formation happens co-transcriptionally, i.e. as the transcript is synthesised in the cell. Moreover, functional RNA structures are not limited to non-coding transcripts, but can also feature in coding transcripts. The picture that now emerges is that RNA structures constitute an additional layer of information that can be encoded in any RNA transcript (and on top of other layers of information such as protein-context) in order to exert a wide range of functional roles. Moreover, different encoded RNA structures can be expressed at different stages of a transcript's life in order to alter the transcript's behaviour depending on its actual cellular context. Similar to the concept of alternative splicing for protein-coding genes, where a single transcript can yield different proteins depending on cellular context, it is thus appropriate to propose the notion of alternative RNA structure expression for any given transcript. This review introduces several computational strategies that my group developed to detect different aspects of RNA structure expression in vivo. Two aspects are of particular interest to us: (1) RNA secondary structure features that emerge during co-transcriptional folding and (2) functional RNA structure features that are expressed at different times of a transcript's life and potentially mutually exclusive. Copyright © 2017. Published by Elsevier Inc.

Stress-induced alterations in 5-HT1A receptor transcriptional modulators NUDR and Freud-1

PubMed Central

Szewczyk, Bernadeta; Kotarska, Katarzyna; Daigle, Mireille; Misztak, Paulina; Sowa-Kucma, Magdalena; Rafalo, Anna; Curzytek, Katarzyna; Kubera, Marta; Basta-Kaim, Agnieszka; Nowak, Gabriel; Albert, Paul R

2015-01-01

The effect of stress on the mRNA and protein level of the 5-HT1A receptor and two of its key transcriptional modulators, NUDR and Freud-1, was examined in the prefrontal cortex (PFC) and hippocampus (Hp) using rodent models: olfactory bulbectomy (OB) and prenatal stress (PS) in male and female rats; chronic mild stress in male rats (CMS) and pregnancy stress. In PFC, CMS induced the most widespread changes, with significant reduction in both mRNA and protein levels of NUDR, 5-HT1A receptor and in Freud-1 mRNA; while in Hp 5-HT1A receptor and Freud-1 protein levels were also decreased. In male, but not female OB rats PFC Freud-1 and 5-HT1A receptor protein levels were reduced, while in Hp 5-HT1A receptor, Freud-1 and NUDR mRNA’s but not protein were reduced. In PS rats PFC 5-HT1A receptor protein was reduced more in females than males; while in Hp Freud-1 protein was increased in females. In pregnancy stress, PFC NUDR, Freud-1 and 5-HT1A protein receptor levels were reduced, and in HP 5-HT1A receptor protein levels were also reduced; in HP only NUDR and Freud-1 mRNA levels were reduced. Overall, CMS and stress during pregnancy produced the most salient changes in 5-HT1A receptor and transcription factor expression, suggesting a primary role for altered transcription factor expression in chronic regulation of 5-HT1A receptor expression. By contrast, OB (in males) and PS (in females) produced gender-specific reductions in PFC 5-HT1A receptor protein levels, suggesting a role for post-transcriptional regulation. These and previous data suggest that chronic stress might be a key regulator of NUDR/Freud-1 gene expression. PMID:24946016

Conifers have a unique small RNA silencing signature

PubMed Central

Dolgosheina, Elena V.; Morin, Ryan D.; Aksay, Gozde; Sahinalp, S. Cenk; Magrini, Vincent; Mardis, Elaine R.; Mattsson, Jim; Unrau, Peter J.

2008-01-01

Plants produce small RNAs to negatively regulate genes, viral nucleic acids, and repetitive elements at either the transcriptional or post-transcriptional level in a process that is referred to as RNA silencing. While RNA silencing has been extensively studied across the different phyla of the animal kingdom (e.g., mouse, fly, worm), similar studies in the plant kingdom have focused primarily on angiosperms, thus limiting evolutionary studies of RNA silencing in plants. Here we report on an unexpected phylogenetic difference in the size distribution of small RNAs among the vascular plants. By extracting total RNA from freshly growing shoot tissue, we conducted a survey of small RNAs in 24 vascular plant species. We find that conifers, which radiated from the other seed-bearing plants ∼260 million years ago, fail to produce significant amounts of 24-nucleotide (nt) RNAs that are known to guide DNA methylation and heterochromatin formation in angiosperms. Instead, they synthesize a diverse population of small RNAs that are exactly 21-nt long. This finding was confirmed by high-throughput sequencing of the small RNA sequences from a conifer, Pinus contorta. A conifer EST search revealed the presence of a novel Dicer-like (DCL) family, which may be responsible for the observed change in small RNA expression. No evidence for DCL3, an enzyme that matures 24-nt RNAs in angiosperms, was found. We hypothesize that the diverse class of 21-nt RNAs found in conifers may help to maintain organization of their unusually large genomes. PMID:18566193

Conifers have a unique small RNA silencing signature.

PubMed

Dolgosheina, Elena V; Morin, Ryan D; Aksay, Gozde; Sahinalp, S Cenk; Magrini, Vincent; Mardis, Elaine R; Mattsson, Jim; Unrau, Peter J

2008-08-01

Plants produce small RNAs to negatively regulate genes, viral nucleic acids, and repetitive elements at either the transcriptional or post-transcriptional level in a process that is referred to as RNA silencing. While RNA silencing has been extensively studied across the different phyla of the animal kingdom (e.g., mouse, fly, worm), similar studies in the plant kingdom have focused primarily on angiosperms, thus limiting evolutionary studies of RNA silencing in plants. Here we report on an unexpected phylogenetic difference in the size distribution of small RNAs among the vascular plants. By extracting total RNA from freshly growing shoot tissue, we conducted a survey of small RNAs in 24 vascular plant species. We find that conifers, which radiated from the other seed-bearing plants approximately 260 million years ago, fail to produce significant amounts of 24-nucleotide (nt) RNAs that are known to guide DNA methylation and heterochromatin formation in angiosperms. Instead, they synthesize a diverse population of small RNAs that are exactly 21-nt long. This finding was confirmed by high-throughput sequencing of the small RNA sequences from a conifer, Pinus contorta. A conifer EST search revealed the presence of a novel Dicer-like (DCL) family, which may be responsible for the observed change in small RNA expression. No evidence for DCL3, an enzyme that matures 24-nt RNAs in angiosperms, was found. We hypothesize that the diverse class of 21-nt RNAs found in conifers may help to maintain organization of their unusually large genomes.

APOBEC3G Inhibits HIV-1 RNA Elongation by Inactivating the Viral Trans-Activation Response Element

PubMed Central

Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

2014-01-01

Deamination of cytidine residues in viral DNA (vDNA) is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient HIV-1 replication. dC to dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here we demonstrate that A3G provides an additional layer of defence against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. Finally, we show that free ssDNA termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3′+5′ ends is sufficient for A3G deamination, identifying A3G as an efficient mutator, and that deamination of (−)SSDNA results in an early block of HIV-1 transcription. PMID:24859335

APOBEC3G inhibits HIV-1 RNA elongation by inactivating the viral trans-activation response element.

PubMed

Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

2014-07-29

Deamination of cytidine residues in viral DNA is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1 (HIV-1) replication. dC-to-dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here, we demonstrate that A3G provides an additional layer of defense against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. We further show that free single-stranded DNA (ssDNA) termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3'+5' ends is sufficient for A3G deamination. These results identify A3G as an efficient mutator and that deamination of (-)SSDNA results in an early block of HIV-1 transcription. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effect of Crc and Hfq proteins on the transcription, processing, and stability of the Pseudomonas putida CrcZ sRNA.

PubMed

Hernández-Arranz, Sofía; Sánchez-Hevia, Dione; Rojo, Fernando; Moreno, Renata

2016-12-01

In Pseudomonas putida, the Hfq and Crc proteins regulate the expression of many genes in response to nutritional and environmental cues, by binding to mRNAs that bear specific target motifs and inhibiting their translation. The effect of these two proteins is antagonized by the CrcZ and CrcY small RNAs (sRNAs), the levels of which vary greatly according to growth conditions. The crcZ and crcY genes are transcribed from promoters PcrcZ and PcrcY, respectively, a process that relies on the CbrB transcriptional activator and the RpoN σ factor. Here we show that crcZ can also be transcribed from the promoter of the immediate upstream gene, cbrB, a weak constitutive promoter. The cbrB-crcZ transcript was processed to render a sRNA very similar in size to the CrcZ produced from promoter PcrcZ The processed sRNA, termed CrcZ*, was able to antagonize Hfq/Crc because, when provided in trans, it relieved the deregulated Hfq/Crc-dependent hyperrepressing phenotype of a ΔcrcZΔcrcY strain. CrcZ* may help in attaining basal levels of CrcZ/CrcZ* that are sufficient to protect the cell from an excessive Hfq/Crc-dependent repression. Since a functional sRNA can be produced from PcrcZ, an inducible strong promoter, or by cleavage of the cbrB-crcZ mRNA, crcZ can be considered a 3'-untranslated region of the cbrB-crcZ mRNA. In the absence of Hfq, the processed form of CrcZ was not observed. In addition, we show that Crc and Hfq increase CrcZ stability, which supports the idea that these proteins can form a complex with CrcZ and protect it from degradation by RNases. © 2016 Hernández-Arranz et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Molecular Characterization of Global Regulatory RNA Species That Control Pathogenicity Factors in Erwinia amylovora and Erwinia herbicola pv. gypsophilae†‡

PubMed Central

Ma, Weilei; Cui, Yaya; Liu, Yang; Dumenyo, C. Korsi; Mukherjee, Asita; Chatterjee, Arun K.

2001-01-01

rsmBEcc specifies a nontranslatable RNA regulator that controls exoprotein production and pathogenicity in soft rot-causing Erwinia carotovora subsp. carotovora. This effect of rsmBEcc RNA is mediated mostly by neutralizing the function of RsmAEcc, an RNA-binding protein of E. carotovora subsp. carotovora, which acts as a global negative regulator. To determine the occurrence of functional homologs of rsmBEcc in non-soft-rot-causing Erwinia species, we cloned the rsmB genes of E. amylovora (rsmBEa) and E. herbicola pv. gypsophilae (rsmBEhg). We show that rsmBEa in E. amylovora positively regulates extracellular polysaccharide (EPS) production, motility, and pathogenicity. In E. herbicola pv. gypsophilae, rsmBEhg elevates the levels of transcripts of a cytokinin (etz) gene and stimulates the production of EPS and yellow pigment as well as motility. RsmAEa and RsmAEhg have more than 93% identity to RsmAEcc and, like the latter, function as negative regulators by affecting the transcript stability of the target gene. The rsmB genes reverse the negative effects of RsmAEa, RsmAEhg, and RsmAEcc, but the extent of reversal is highest with homologous combinations of rsm genes. These observations and findings that rsmBEa and rsmBEhg RNA bind RsmAEcc indicate that the rsmB effect is channeled via RsmA. Additional support for this conclusion comes from the observation that the rsmB genes are much more effective as positive regulators in a RsmA+ strain of E. carotovora subsp. carotovora than in its RsmA− derivative. E. herbicola pv. gypsophilae produces a 290-base rsmB transcript that is not subject to processing. By contrast, E. amylovora produces 430- and 300-base rsmB transcripts, the latter presumably derived by processing of the primary transcript as previously noted with the transcripts of rsmBEcc. Southern blot hybridizations revealed the presence of rsmB homologs in E. carotovora, E. chrysanthemi, E. amylovora, E. herbicola, E. stewartii and E. rhapontici, as well as in other enterobacteria such as Escherichia coli, Salmonella enterica serovar Typhimurium, Serratia marcescens, Shigella flexneri, Enterobacter aerogenes, Klebsiella pneumoniae, Yersinia enterocolitica, and Y. pseudotuberculosis. A comparison of rsmB sequences from several of these enterobacterial species revealed a highly conserved 34-mer region which is predicted to play a role in positive regulation by rsmB RNA. PMID:11222584

The RNAs of RNA-directed DNA methylation

PubMed Central

Wendte, Jered M.; Pikaard, Craig S.

2016-01-01

Summary RNA-directed chromatin modification that includes cytosine methylation silences transposable elements in both plants and mammals, contributing to genome defense and stability. In Arabidopsis thaliana, most RNA-directed DNA methylation (RdDM) is guided by small RNAs derived from double-stranded precursors synthesized at cytosine-methylated loci by nuclear multisubunit RNA Polymerase IV (Pol IV), in close partnership with the RNA-dependent RNA polymerase, RDR2. These small RNAs help keep transposons transcriptionally inactive. However, if transposons escape silencing, and are transcribed by multisubunit RNA polymerase II (Pol II), their mRNAs can be recognized and degraded, generating small RNAs that can also guide initial DNA methylation, thereby enabling subsequent Pol IV-RDR2 recruitment. In both pathways, the small RNAs find their target sites by interacting with longer noncoding RNAs synthesized by multisubunit RNA Polymerase V (Pol V). Despite a decade of progress, numerous questions remain concerning the initiation, synthesis, processing, size and features of the RNAs that drive RdDM. Here, we review recent insights, questions and controversies concerning RNAs produced by Pols IV and V, and their functions in RdDM. We also provide new data concerning Pol V transcript 5’ and 3’ ends. PMID:27521981

BLM helicase facilitates RNA polymerase I-mediated ribosomal RNA transcription

PubMed Central

Grierson, Patrick M.; Lillard, Kate; Behbehani, Gregory K.; Combs, Kelly A.; Bhattacharyya, Saumitri; Acharya, Samir; Groden, Joanna

2012-01-01

Bloom's syndrome (BS) is an autosomal recessive disorder that is invariably characterized by severe growth retardation and cancer predisposition. The Bloom's syndrome helicase (BLM), mutations of which lead to BS, localizes to promyelocytic leukemia protein bodies and to the nucleolus of the cell, the site of RNA polymerase I-mediated ribosomal RNA (rRNA) transcription. rRNA transcription is fundamental for ribosome biogenesis and therefore protein synthesis, cellular growth and proliferation; its inhibition limits cellular growth and proliferation as well as bodily growth. We report that nucleolar BLM facilitates RNA polymerase I-mediated rRNA transcription. Immunofluorescence studies demonstrate the dependance of BLM nucleolar localization upon ongoing RNA polymerase I-mediated rRNA transcription. In vivo protein co-immunoprecipitation demonstrates that BLM interacts with RPA194, a subunit of RNA polymerase I. 3H-uridine pulse-chase assays demonstrate that BLM expression is required for efficient rRNA transcription. In vitro helicase assays demonstrate that BLM unwinds GC-rich rDNA-like substrates that form in the nucleolus and normally inhibit progression of the RNA polymerase I transcription complex. These studies suggest that nucleolar BLM modulates rDNA structures in association with RNA polymerase I to facilitate RNA polymerase I-mediated rRNA transcription. Given the intricate relationship between rDNA metabolism and growth, our data may help in understanding the etiology of proportional dwarfism in BS. PMID:22106380

BLM helicase facilitates RNA polymerase I-mediated ribosomal RNA transcription.

PubMed

Grierson, Patrick M; Lillard, Kate; Behbehani, Gregory K; Combs, Kelly A; Bhattacharyya, Saumitri; Acharya, Samir; Groden, Joanna

2012-03-01

Bloom's syndrome (BS) is an autosomal recessive disorder that is invariably characterized by severe growth retardation and cancer predisposition. The Bloom's syndrome helicase (BLM), mutations of which lead to BS, localizes to promyelocytic leukemia protein bodies and to the nucleolus of the cell, the site of RNA polymerase I-mediated ribosomal RNA (rRNA) transcription. rRNA transcription is fundamental for ribosome biogenesis and therefore protein synthesis, cellular growth and proliferation; its inhibition limits cellular growth and proliferation as well as bodily growth. We report that nucleolar BLM facilitates RNA polymerase I-mediated rRNA transcription. Immunofluorescence studies demonstrate the dependance of BLM nucleolar localization upon ongoing RNA polymerase I-mediated rRNA transcription. In vivo protein co-immunoprecipitation demonstrates that BLM interacts with RPA194, a subunit of RNA polymerase I. (3)H-uridine pulse-chase assays demonstrate that BLM expression is required for efficient rRNA transcription. In vitro helicase assays demonstrate that BLM unwinds GC-rich rDNA-like substrates that form in the nucleolus and normally inhibit progression of the RNA polymerase I transcription complex. These studies suggest that nucleolar BLM modulates rDNA structures in association with RNA polymerase I to facilitate RNA polymerase I-mediated rRNA transcription. Given the intricate relationship between rDNA metabolism and growth, our data may help in understanding the etiology of proportional dwarfism in BS.

Effects of guanosine tetraphosphate on cell-free synthesis of Escherichia coli ribosomal RNA and other gene products.

PubMed Central

Reiness, G; Yang, H L; Zubay, G; Cashel, M

1975-01-01

A cell-free system derived from E. coli is described in which mature-sized 16S and 23S ribosomal RNAs (rRNA) are synthesized at a high relative rate, comprising 17-25% of the total transcription. The addition of guanosine tetraphosphate (ppGpp) to this system results in up to a 5-fold selective inhibition of rRNA accumulation. This effect is exerted at the level of synthesis rather than degradation. It is concluded that ppGpp, which is produced in large amounts by E. coli during amino-acid deprivation, could mediate the decrease in rRNA synthesis that accompanies such deprivation. The expression of other genes has also been investigated. No selective reduction of transfer RNA synthesis by ppGpp is observed. The trp and lac operons are found to be stimulated at the transcriptional level by the presence of this nucleotide. It is hypothesized that ppGpp interacts with the RNA polymerase in such a manner as to alter the affinity of the enzyme for promoters in an operon-specific fashion. PMID:1103124

Linear RNA amplification for the production of microarray hybridization probes.

PubMed

Klebes, Ansgar; Kornberg, Thomas B

2008-01-01

To understand Drosophila development and other genetically controlled processes, it is often desirable to identify differences in gene expression levels. An experimental approach to investigate these processes is to catalog the transcriptome by hybridization of mRNA to DNA microbar-rays. In these experiments mRNA-derived hybridization probes are produced and hybridized to an array of DNA spots on a solid support. The labeled cDNAs of the complex hybridization probe will bind to their complementary sequences and provide quantification of the relative concentration of the corresponding transcript in the starting material. However, such approaches are often limited by the scarcity of the experimental sample because standard methods of probe preparation require microgram quantities of mRNA template. Linear RNA amplification can alleviate such limitations to support the generation of microarray hybridization probes from a few 100 pg of mRNA. These smaller quantities can be isolated from a few 100 cells. Here, we present a linear amplification protocol designed to preserve both the relative abundance of transcripts as well as their sequence complexity.

Genome-Wide Characterization of Light-Regulated Genes in Neurospora crassa

PubMed Central

Wu, Cheng; Yang, Fei; Smith, Kristina M.; Peterson, Matthew; Dekhang, Rigzin; Zhang, Ying; Zucker, Jeremy; Bredeweg, Erin L.; Mallappa, Chandrashekara; Zhou, Xiaoying; Lyubetskaya, Anna; Townsend, Jeffrey P.; Galagan, James E.; Freitag, Michael; Dunlap, Jay C.; Bell-Pedersen, Deborah; Sachs, Matthew S.

2014-01-01

The filamentous fungus Neurospora crassa responds to light in complex ways. To thoroughly study the transcriptional response of this organism to light, RNA-seq was used to analyze capped and polyadenylated mRNA prepared from mycelium grown for 24 hr in the dark and then exposed to light for 0 (control) 15, 60, 120, and 240 min. More than three-quarters of all defined protein coding genes (79%) were expressed in these cells. The increased sensitivity of RNA-seq compared with previous microarray studies revealed that the RNA levels for 31% of expressed genes were affected two-fold or more by exposure to light. Additionally, a large class of mRNAs, enriched for transcripts specifying products involved in rRNA metabolism, showed decreased expression in response to light, indicating a heretofore undocumented effect of light on this pathway. Based on measured changes in mRNA levels, light generally increases cellular metabolism and at the same time causes significant oxidative stress to the organism. To deal with this stress, protective photopigments are made, antioxidants are produced, and genes involved in ribosome biogenesis are transiently repressed. PMID:25053707

A novel route for preparing 5′ cap mimics and capped RNAs: phosphate-modified cap analogues obtained via click chemistry† †Electronic supplementary information (ESI) available: Tables S1–S6 and Fig. S1–S10, experimental procedures, HPLC profiles, NMR and HRMS spectra. See DOI: 10.1039/c6sc02437h Click here for additional data file. Click here for additional data file.

PubMed Central

Walczak, Sylwia; Nowicka, Anna; Kubacka, Dorota; Fac, Kaja; Wanat, Przemyslaw; Mroczek, Seweryn; Kowalska, Joanna

2017-01-01

The significant biological role of the mRNA 5′ cap in translation initiation makes it an interesting subject for chemical modifications aimed at producing useful tools for the selective modulation of intercellular processes and development of novel therapeutic interventions. However, traditional approaches to the chemical synthesis of cap analogues are time-consuming and labour-intensive, which impedes the development of novel compounds and their applications. Here, we explore a different approach for synthesizing 5′ cap mimics, making use of click chemistry (CuAAC) to combine two mononucleotide units and yield a novel class of dinucleotide cap analogues containing a triazole ring within the oligophosphate chain. As a result, we synthesized a library of 36 mRNA cap analogues differing in the location of the triazole ring, the polyphosphate chain length, and the type of linkers joining the phosphate and the triazole moieties. After biochemical evaluation, we identified two analogues that, when incorporated into mRNA, produced transcripts translated with efficiency similar to compounds unmodified in the oligophosphate bridge obtained by traditional synthesis. Moreover, we demonstrated that the triazole-modified cap structures can be generated at the RNA 5′ end using two alternative capping strategies: either the typical co-transcriptional approach, or a new post-transcriptional approach based on CuAAC. Our findings open new possibilities for developing chemically modified mRNAs for research and therapeutic applications, including RNA-based vaccinations. PMID:28451173

The katG mRNA of Mycobacterium tuberculosis and Mycobacterium smegmatis is processed at its 5' end and is stabilized by both a polypurine sequence and translation initiation

PubMed Central

Sala, Claudia; Forti, Francesca; Magnoni, Francesca; Ghisotti, Daniela

2008-01-01

Background In Mycobacterium tuberculosis and in Mycobacterium smegmatis the furA-katG loci, encoding the FurA regulatory protein and the KatG catalase-peroxidase, are highly conserved. In M. tuberculosis furA-katG constitute a single operon, whereas in M. smegmatis a single mRNA covering both genes could not be found. In both species, specific 5' ends have been identified: the first one, located upstream of the furA gene, corresponds to transcription initiation from the furA promoter; the second one is the katG mRNA 5' end, located in the terminal part of furA. Results In this work we demonstrate by in vitro transcription and by RNA polymerase Chromatin immunoprecipitation that no promoter is present in the M. smegmatis region covering the latter 5' end, suggesting that it is produced by specific processing of longer transcripts. Several DNA fragments of M. tuberculosis and M. smegmatis were inserted in a plasmid between the sigA promoter and the lacZ reporter gene, and expression of the reporter gene was measured. A polypurine sequence, located four bp upstream of the katG translation start codon, increased beta-galactosidase activity and stabilized the lacZ transcript. Mutagenesis of this sequence led to destabilization of the mRNA. Analysis of constructs, in which the polypurine sequence of M. smegmatis was followed by an increasing number of katG codons, demonstrated that mRNA stability requires translation of at least 20 amino acids. In order to define the requirements for the 5' processing of the katG transcript, we created several mutations in this region and analyzed the 5' ends of the transcripts: the distance from the polypurine sequence does not seem to influence the processing, neither the sequence around the cutting point. Only mutations which create a double stranded region around the processing site prevented RNA processing. Conclusion This is the first reported case in mycobacteria, in which both a polypurine sequence and translation initiation are shown to contribute to mRNA stability. The furA-katG mRNA is transcribed from the furA promoter and immediately processed; this processing is prevented by a double stranded RNA at the cutting site, suggesting that the endoribonuclease responsible for the cleavage cuts single stranded RNA. PMID:18394163

CCAT2, a novel noncoding RNA mapping to 8q24, underlies metastatic progression and chromosomal instability in colon cancer

PubMed Central

Ling, Hui; Spizzo, Riccardo; Atlasi, Yaser; Nicoloso, Milena; Shimizu, Masayoshi; Redis, Roxana S.; Nishida, Naohiro; Gafà, Roberta; Song, Jian; Guo, Zhiyi; Ivan, Cristina; Barbarotto, Elisa; De Vries, Ingrid; Zhang, Xinna; Ferracin, Manuela; Churchman, Mike; van Galen, Janneke F.; Beverloo, Berna H.; Shariati, Maryam; Haderk, Franziska; Estecio, Marcos R.; Garcia-Manero, Guillermo; Patijn, Gijs A.; Gotley, David C.; Bhardwaj, Vikas; Shureiqi, Imad; Sen, Subrata; Multani, Asha S.; Welsh, James; Yamamoto, Ken; Taniguchi, Itsuki; Song, Min-Ae; Gallinger, Steven; Casey, Graham; Thibodeau, Stephen N.; Le Marchand, Loïc; Tiirikainen, Maarit; Mani, Sendurai A.; Zhang, Wei; Davuluri, Ramana V.; Mimori, Koshi; Mori, Masaki; Sieuwerts, Anieta M.; Martens, John W.M.; Tomlinson, Ian; Negrini, Massimo; Berindan-Neagoe, Ioana; Foekens, John A.; Hamilton, Stanley R.; Lanza, Giovanni; Kopetz, Scott; Fodde, Riccardo; Calin, George A.

2013-01-01

The functional roles of SNPs within the 8q24 gene desert in the cancer phenotype are not yet well understood. Here, we report that CCAT2, a novel long noncoding RNA transcript (lncRNA) encompassing the rs6983267 SNP, is highly overexpressed in microsatellite-stable colorectal cancer and promotes tumor growth, metastasis, and chromosomal instability. We demonstrate that MYC, miR–17–5p, and miR–20a are up-regulated by CCAT2 through TCF7L2-mediated transcriptional regulation. We further identify the physical interaction between CCAT2 and TCF7L2 resulting in an enhancement of WNT signaling activity. We show that CCAT2 is itself a WNT downstream target, which suggests the existence of a feedback loop. Finally, we demonstrate that the SNP status affects CCAT2 expression and the risk allele G produces more CCAT2 transcript. Our results support a new mechanism of MYC and WNT regulation by the novel lncRNA CCAT2 in colorectal cancer pathogenesis, and provide an alternative explanation of the SNP-conferred cancer risk. PMID:23796952

Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

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

Shin, Ilchung; Ray, Judhajeet; Gupta, Vinayak

2014-04-20

We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from themore » GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.« less

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

PubMed Central

Jablonski, Joseph; Clementz, Mark; Ryan, Kevin; Valente, Susana T.

2014-01-01

The 3’ end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages. Processing of the 3’ end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5’ cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3’ end processing of HIV-1 mRNAs. PMID:24835792

kdgREcc Negatively Regulates Genes for Pectinases, Cellulase, Protease, HarpinEcc, and a Global RNA Regulator in Erwinia carotovora subsp. carotovora†

PubMed Central

Liu, Yang; Jiang, Guoqiao; Cui, Yaya; Mukherjee, Asita; Ma, Wei Lei; Chatterjee, Arun K.

1999-01-01

Erwinia carotovora subsp. carotovora produces extracellular pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt). The concerted actions of these enzymes largely determine the virulence of this plant-pathogenic bacterium. E. carotovora subsp. carotovora also produces HarpinEcc, the elicitor of the hypersensitive reaction. We document here that KdgREcc (Kdg, 2-keto-3-deoxygluconate; KdgR, general repressor of genes involved in pectin and galacturonate catabolism), a homolog of the E. chrysanthemi repressor, KdgREch and the Escherichia coli repressor, KdgREco, negatively controls not only the pectinases, Pel and Peh, but also Cel, Prt, and HarpinEcc production in E. carotovora subsp. carotovora. The levels of pel-1, peh-1, celV, and hrpNEcc transcripts are markedly affected by KdgREcc. The KdgREcc− mutant is more virulent than the KdgREcc+ parent. Thus, our data for the first time establish a global regulatory role for KdgREcc in E. carotovora subsp. carotovora. Another novel observation is the negative effect of KdgREcc on the transcription of rsmB (previously aepH), which specifies an RNA regulator controlling exoenzyme and HarpinEcc production. The levels of rsmB RNA are higher in the KdgREcc− mutant than in the KdgREcc+ parent. Moreover, by DNase I protection assays we determined that purified KdgREcc protected three 25-bp regions within the transcriptional unit of rsmB. Alignment of the protected sequences revealed the 21-mer consensus sequence of the KdgREcc-binding site as 5′-G/AA/TA/TGAAA[N6]TTTCAG/TG/TA-3′. Two such KdgREcc-binding sites occur in rsmB DNA in a close proximity to each other within nucleotides +79 and +139 and the third KdgREcc-binding site within nucleotides +207 and +231. Analysis of lacZ transcriptional fusions shows that the KdgR-binding sites negatively affect the expression of rsmB. KdgREcc also binds the operator DNAs of pel-1 and peh-1 genes and represses expression of a pel1-lacZ and a peh1-lacZ transcriptional fusions. We conclude that KdgREcc affects extracellular enzyme production by two ways: (i) directly, by inhibiting the transcription of exoenzyme genes; and (ii) indirectly, by preventing the production of a global RNA regulator. Our findings support the idea that KdgREcc affects transcription by promoter occlusion, i.e., preventing the initiation of transcription, and by a roadblock mechanism, i.e., by affecting the elongation of transcription. PMID:10198003

RNA Nuclear Export: From Neurological Disorders to Cancer.

PubMed

Hautbergue, Guillaume M

2017-01-01

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

Transcription profiling suggests that mitochondrial topoisomerase IB acts as a topological barrier and regulator of mitochondrial DNA transcription.

PubMed

Dalla Rosa, Ilaria; Zhang, Hongliang; Khiati, Salim; Wu, Xiaolin; Pommier, Yves

2017-12-08

Mitochondrial DNA (mtDNA) is essential for cell viability because it encodes subunits of the respiratory chain complexes. Mitochondrial topoisomerase IB (TOP1MT) facilitates mtDNA replication by removing DNA topological tensions produced during mtDNA transcription, but it appears to be dispensable. To test whether cells lacking TOP1MT have aberrant mtDNA transcription, we performed mitochondrial transcriptome profiling. To that end, we designed and implemented a customized tiling array, which enabled genome-wide, strand-specific, and simultaneous detection of all mitochondrial transcripts. Our technique revealed that Top1mt KO mouse cells process the mitochondrial transcripts normally but that protein-coding mitochondrial transcripts are elevated. Moreover, we found discrete long noncoding RNAs produced by H-strand transcription and encompassing the noncoding regulatory region of mtDNA in human and murine cells and tissues. Of note, these noncoding RNAs were strongly up-regulated in the absence of TOP1MT. In contrast, 7S DNA, produced by mtDNA replication, was reduced in the Top1mt KO cells. We propose that the long noncoding RNA species in the D-loop region are generated by the extension of H-strand transcripts beyond their canonical stop site and that TOP1MT acts as a topological barrier and regulator for mtDNA transcription and D-loop formation.

Recognizing the enemy within: licensing RNA-guided genome defense

PubMed Central

Dumesic, Phillip A.; Madhani, Hiten D.

2014-01-01

How do cells distinguish normal genes from transposons? Although much has been learned about RNAi-related RNA silencing pathways responsible for genome defense, this fundamental question remains. The literature points to several classes of mechanisms. In some cases, double-stranded RNA structures produced by transposon inverted repeats or antisense integration trigger endo-siRNA biogenesis. In other instances, DNA features associated with transposons—such as their unusual copy number, chromosomal arrangement, and/or chromatin environment—license RNA silencing. Finally, recent studies have identified improper transcript processing events, such as stalled pre-mRNA splicing, as signals for siRNA production. Thus, the suboptimal gene expression properties of selfish elements can enable their identification by RNA silencing pathways. PMID:24280023

Coupling mRNA processing with transcription in time and space

PubMed Central

Bentley, David L.

2015-01-01

Maturation of mRNA precursors often occurs simultaneously with their synthesis by RNA polymerase II (Pol II). The co-transcriptional nature of mRNA processing has permitted the evolution of coupling mechanisms that coordinate transcription with mRNA capping, splicing, editing and 3′ end formation. Recent experiments using sophisticated new methods for analysis of nascent RNA have provided important insights into the relative amount of co-transcriptional and post-transcriptional processing, the relationship between mRNA elongation and processing, and the role of the Pol II carboxy-terminal domain (CTD) in regulating these processes. PMID:24514444

Divergent transcription is associated with promoters of transcriptional regulators

PubMed Central

2013-01-01

Background Divergent transcription is a wide-spread phenomenon in mammals. For instance, short bidirectional transcripts are a hallmark of active promoters, while longer transcripts can be detected antisense from active genes in conditions where the RNA degradation machinery is inhibited. Moreover, many described long non-coding RNAs (lncRNAs) are transcribed antisense from coding gene promoters. However, the general significance of divergent lncRNA/mRNA gene pair transcription is still poorly understood. Here, we used strand-specific RNA-seq with high sequencing depth to thoroughly identify antisense transcripts from coding gene promoters in primary mouse tissues. Results We found that a substantial fraction of coding-gene promoters sustain divergent transcription of long non-coding RNA (lncRNA)/mRNA gene pairs. Strikingly, upstream antisense transcription is significantly associated with genes related to transcriptional regulation and development. Their promoters share several characteristics with those of transcriptional developmental genes, including very large CpG islands, high degree of conservation and epigenetic regulation in ES cells. In-depth analysis revealed a unique GC skew profile at these promoter regions, while the associated coding genes were found to have large first exons, two genomic features that might enforce bidirectional transcription. Finally, genes associated with antisense transcription harbor specific H3K79me2 epigenetic marking and RNA polymerase II enrichment profiles linked to an intensified rate of early transcriptional elongation. Conclusions We concluded that promoters of a class of transcription regulators are characterized by a specialized transcriptional control mechanism, which is directly coupled to relaxed bidirectional transcription. PMID:24365181

Genome activation by raspberry bushy dwarf virus coat protein.

PubMed

Macfarlane, Stuart A; McGavin, Wendy J

2009-03-01

Two sets of infectious cDNA clones of raspberry bushy dwarf virus (RBDV) have been constructed, enabling either the synthesis of infectious RNA transcripts or the delivery of infectious binary plasmid DNA by infiltration of Agrobacterium tumefaciens. In whole plants and in protoplasts, inoculation of RBDV RNA1 and RNA2 transcripts led to a low level of infection, which was greatly increased by the addition of RNA3, a subgenomic RNA coding for the RBDV coat protein (CP). Agroinfiltration of RNA1 and RNA2 constructs did not produce a detectable infection but, again, inclusion of a construct encoding the CP led to high levels of infection. Thus, RBDV replication is greatly stimulated by the presence of the CP, a mechanism that also operates with ilarviruses and alfalfa mosaic virus, where it is referred to as genome activation. Mutation to remove amino acids from the N terminus of the CP showed that the first 15 RBDV CP residues are not required for genome activation. Other experiments, in which overlapping regions at the CP N terminus were fused to the monomeric red fluorescent protein, showed that sequences downstream of the first 48 aa are not absolutely required for genome activation.

Antisense Transcription Is Pervasive but Rarely Conserved in Enteric Bacteria

PubMed Central

Raghavan, Rahul; Sloan, Daniel B.; Ochman, Howard

2012-01-01

ABSTRACT Noncoding RNAs, including antisense RNAs (asRNAs) that originate from the complementary strand of protein-coding genes, are involved in the regulation of gene expression in all domains of life. Recent application of deep-sequencing technologies has revealed that the transcription of asRNAs occurs genome-wide in bacteria. Although the role of the vast majority of asRNAs remains unknown, it is often assumed that their presence implies important regulatory functions, similar to those of other noncoding RNAs. Alternatively, many antisense transcripts may be produced by chance transcription events from promoter-like sequences that result from the degenerate nature of bacterial transcription factor binding sites. To investigate the biological relevance of antisense transcripts, we compared genome-wide patterns of asRNA expression in closely related enteric bacteria, Escherichia coli and Salmonella enterica serovar Typhimurium, by performing strand-specific transcriptome sequencing. Although antisense transcripts are abundant in both species, less than 3% of asRNAs are expressed at high levels in both species, and only about 14% appear to be conserved among species. And unlike the promoters of protein-coding genes, asRNA promoters show no evidence of sequence conservation between, or even within, species. Our findings suggest that many or even most bacterial asRNAs are nonadaptive by-products of the cell’s transcription machinery. PMID:22872780

miRNA Enriched in Human Neuroblast Nuclei Bind the MAZ Transcription Factor and Their Precursors Contain the MAZ Consensus Motif.

PubMed

Goldie, Belinda J; Fitzsimmons, Chantel; Weidenhofer, Judith; Atkins, Joshua R; Wang, Dan O; Cairns, Murray J

2017-01-01

While the cytoplasmic function of microRNA (miRNA) as post-transcriptional regulators of mRNA has been the subject of significant research effort, their activity in the nucleus is less well characterized. Here we use a human neuronal cell model to show that some mature miRNA are preferentially enriched in the nucleus. These molecules were predominantly primate-specific and contained a sequence motif with homology to the consensus MAZ transcription factor binding element. Precursor miRNA containing this motif were shown to have affinity for MAZ protein in nuclear extract. We then used Ago1/2 RIP-Seq to explore nuclear miRNA-associated mRNA targets. Interestingly, the genes for Ago2-associated transcripts were also significantly enriched with MAZ binding sites and neural function, whereas Ago1-transcripts were associated with general metabolic processes and localized with SC35 spliceosomes. These findings suggest the MAZ transcription factor is associated with miRNA in the nucleus and may influence the regulation of neuronal development through Ago2-associated miRNA induced silencing complexes. The MAZ transcription factor may therefore be important for organizing higher order integration of transcriptional and post-transcriptional processes in primate neurons.

The Drosophila Tis11 protein and its effects on mRNA expression in flies.

PubMed

Choi, Youn-Jeong; Lai, Wi S; Fedic, Robert; Stumpo, Deborah J; Huang, Weichun; Li, Leping; Perera, Lalith; Brewer, Brandy Y; Wilson, Gerald M; Mason, James M; Blackshear, Perry J

2014-12-19

Members of the mammalian tristetraprolin family of CCCH tandem zinc finger proteins can bind to certain AU-rich elements (AREs) in mRNAs, leading to their deadenylation and destabilization. Mammals express three or four members of this family, but Drosophila melanogaster and other insects appear to contain a single gene, Tis11. We found that recombinant Drosophila Tis11 protein could bind to ARE-containing RNA oligonucleotides with low nanomolar affinity. Remarkably, co-expression in mammalian cells with "target" RNAs demonstrated that Tis11 could promote destabilization of ARE-containing mRNAs and that this was partially dependent on a conserved C-terminal sequence resembling the mammalian NOT1 binding domain. Drosophila Tis11 promoted both deadenylation and decay of a target transcript in this heterologous cell system. We used chromosome deletion/duplication and P element insertion to produce two types of Tis11 deficiency in adult flies, both of which were viable and fertile. To address the hypothesis that Tis11 deficiency would lead to the abnormal accumulation of potential target transcripts, we analyzed gene expression in adult flies by deep mRNA sequencing. We identified 69 transcripts from 56 genes that were significantly up-regulated more than 1.5-fold in both types of Tis11-deficient flies. Ten of the up-regulated transcripts encoded probable proteases, but many other functional classes of proteins were represented. Many of the up-regulated transcripts contained potential binding sites for tristetraprolin family member proteins that were conserved in other Drosophila species. Tis11 is thus an ARE-binding, mRNA-destabilizing protein that may play a role in post-transcriptional gene expression in Drosophila and other insects. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The Drosophila Tis11 Protein and Its Effects on mRNA Expression in Flies*

PubMed Central

Choi, Youn-Jeong; Lai, Wi S.; Fedic, Robert; Stumpo, Deborah J.; Huang, Weichun; Li, Leping; Perera, Lalith; Brewer, Brandy Y.; Wilson, Gerald M.; Mason, James M.; Blackshear, Perry J.

2014-01-01

Members of the mammalian tristetraprolin family of CCCH tandem zinc finger proteins can bind to certain AU-rich elements (AREs) in mRNAs, leading to their deadenylation and destabilization. Mammals express three or four members of this family, but Drosophila melanogaster and other insects appear to contain a single gene, Tis11. We found that recombinant Drosophila Tis11 protein could bind to ARE-containing RNA oligonucleotides with low nanomolar affinity. Remarkably, co-expression in mammalian cells with “target” RNAs demonstrated that Tis11 could promote destabilization of ARE-containing mRNAs and that this was partially dependent on a conserved C-terminal sequence resembling the mammalian NOT1 binding domain. Drosophila Tis11 promoted both deadenylation and decay of a target transcript in this heterologous cell system. We used chromosome deletion/duplication and P element insertion to produce two types of Tis11 deficiency in adult flies, both of which were viable and fertile. To address the hypothesis that Tis11 deficiency would lead to the abnormal accumulation of potential target transcripts, we analyzed gene expression in adult flies by deep mRNA sequencing. We identified 69 transcripts from 56 genes that were significantly up-regulated more than 1.5-fold in both types of Tis11-deficient flies. Ten of the up-regulated transcripts encoded probable proteases, but many other functional classes of proteins were represented. Many of the up-regulated transcripts contained potential binding sites for tristetraprolin family member proteins that were conserved in other Drosophila species. Tis11 is thus an ARE-binding, mRNA-destabilizing protein that may play a role in post-transcriptional gene expression in Drosophila and other insects. PMID:25342740

Pervasive, Coordinated Protein-Level Changes Driven by Transcript Isoform Switching during Meiosis.

PubMed

Cheng, Ze; Otto, George Maxwell; Powers, Emily Nicole; Keskin, Abdurrahman; Mertins, Philipp; Carr, Steven Alfred; Jovanovic, Marko; Brar, Gloria Ann

2018-02-22

To better understand the gene regulatory mechanisms that program developmental processes, we carried out simultaneous genome-wide measurements of mRNA, translation, and protein through meiotic differentiation in budding yeast. Surprisingly, we observed that the levels of several hundred mRNAs are anti-correlated with their corresponding protein products. We show that rather than arising from canonical forms of gene regulatory control, the regulation of at least 380 such cases, or over 8% of all measured genes, involves temporally regulated switching between production of a canonical, translatable transcript and a 5' extended isoform that is not efficiently translated into protein. By this pervasive mechanism for the modulation of protein levels through a natural developmental program, a single transcription factor can coordinately activate and repress protein synthesis for distinct sets of genes. The distinction is not based on whether or not an mRNA is induced but rather on the type of transcript produced. Copyright © 2018 Elsevier Inc. All rights reserved.

Transposon-driven transcription is a conserved feature of vertebrate spermatogenesis and transcript evolution.

PubMed

Davis, Matthew P; Carrieri, Claudia; Saini, Harpreet K; van Dongen, Stijn; Leonardi, Tommaso; Bussotti, Giovanni; Monahan, Jack M; Auchynnikava, Tania; Bitetti, Angelo; Rappsilber, Juri; Allshire, Robin C; Shkumatava, Alena; O'Carroll, Dónal; Enright, Anton J

2017-07-01

Spermatogenesis is associated with major and unique changes to chromosomes and chromatin. Here, we sought to understand the impact of these changes on spermatogenic transcriptomes. We show that long terminal repeats (LTRs) of specific mouse endogenous retroviruses (ERVs) drive the expression of many long non-coding transcripts (lncRNA). This process occurs post-mitotically predominantly in spermatocytes and round spermatids. We demonstrate that this transposon-driven lncRNA expression is a conserved feature of vertebrate spermatogenesis. We propose that transposon promoters are a mechanism by which the genome can explore novel transcriptional substrates, increasing evolutionary plasticity and allowing for the genesis of novel coding and non-coding genes. Accordingly, we show that a small fraction of these novel ERV-driven transcripts encode short open reading frames that produce detectable peptides. Finally, we find that distinct ERV elements from the same subfamilies act as differentially activated promoters in a tissue-specific context. In summary, we demonstrate that LTRs can act as tissue-specific promoters and contribute to post-mitotic spermatogenic transcriptome diversity. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Connections between Transcription Downstream of Genes and cis-SAGe Chimeric RNA.

PubMed

Chwalenia, Katarzyna; Qin, Fujun; Singh, Sandeep; Tangtrongstittikul, Panjapon; Li, Hui

2017-11-22

cis-Splicing between adjacent genes (cis-SAGe) is being recognized as one way to produce chimeric fusion RNAs. However, its detail mechanism is not clear. Recent study revealed induction of transcriptions downstream of genes (DoGs) under osmotic stress. Here, we investigated the influence of osmotic stress on cis-SAGe chimeric RNAs and their connection to DoGs. We found,the absence of induction of at least some cis-SAGe fusions and/or their corresponding DoGs at early time point(s). In fact, these DoGs and their cis-SAGe fusions are inversely correlated. This negative correlation was changed to positive at a later time point. These results suggest a direct competition between the two categories of transcripts when total pool of readthrough transcripts is limited at an early time point. At a later time point, DoGs and corresponding cis-SAGe fusions are both induced, indicating that total readthrough transcripts become more abundant. Finally, we observed overall enhancement of cis-SAGe chimeric RNAs in KCl-treated samples by RNA-Seq analysis.

The context of transcription start site regions is crucial for transcription of a plant tRNA(Lys)(UUU) gene group both in vitro and in vivo.

PubMed

Yukawa, Yasushi; Akama, Kazuhito; Noguchi, Kanta; Komiya, Masaaki; Sugiura, Masahiro

2013-01-10

Nuclear tRNA genes are transcribed by RNA polymerase III. The A- and B-boxes located within the transcribed regions are essential promoter elements for nuclear tRNA gene transcription. The Arabidopsis genome contains ten annotated genes encoding identical tRNA(Lys)(UUU) molecules, which are scattered on the five chromosomes. In this study, we prepared ten tDNA constructs including each of the tRNA(Lys)(UUU) coding sequences with their individual 5' and 3' flanking sequences, and assayed tRNA expression using an in vitro RNA polymerase III-dependent transcription system. Transcription levels differed significantly among the ten genes and two of the tRNA genes were transcribed at a very low level, despite possessing A- and B-boxes identical to those of the other tRNA genes. To examine whether the in vitro results were reproducible in vivo, the 5' flanking sequence of an amber suppressor tRNA gene was then replaced with those of the ten tRNA(Lys) genes. An in vivo experiment based on an amber suppressor tRNA that mediates suppression of a premature amber codon in a β-glucuronidase (GUS) reporter gene in plant tissues generated nearly identical results to those obtained in vitro. Analysis of mutated versions of the amber suppressor tRNA gene, which contained base substitutions around the transcription start site (TSS), showed that the context around the transcription start sites is a crucial determinant for transcription of plant tRNA(Lys)(UUU) both in vitro and in vivo. The above transcription regulation by context around TSS differed between tRNA genes and other Pol III-dependent genes. Copyright © 2012 Elsevier B.V. All rights reserved.

Two Genetic Determinants Acquired Late in Mus Evolution Regulate the Inclusion of Exon 5, which Alters Mouse APOBEC3 Translation Efficiency

PubMed Central

Li, Jun; Hakata, Yoshiyuki; Takeda, Eri; Liu, Qingping; Iwatani, Yasumasa; Kozak, Christine A.; Miyazawa, Masaaki

2012-01-01

Mouse apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like editing complex 3 (mA3), an intracellular antiviral factor, has 2 allelic variations that are linked with different susceptibilities to beta- and gammaretrovirus infections among various mouse strains. In virus-resistant C57BL/6 (B6) mice, mA3 transcripts are more abundant than those in susceptible BALB/c mice both in the spleen and bone marrow. These strains of mice also express mA3 transcripts with different splicing patterns: B6 mice preferentially express exon 5-deficient (Δ5) mA3 mRNA, while BALB/c mice produce exon 5-containing full-length mA3 mRNA as the major transcript. Although the protein product of the Δ5 mRNA exerts stronger antiretroviral activities than the full-length protein, how exon 5 affects mA3 antiviral activity, as well as the genetic mechanisms regulating exon 5 inclusion into the mA3 transcripts, remains largely uncharacterized. Here we show that mA3 exon 5 is indeed a functional element that influences protein synthesis at a post-transcriptional level. We further employed in vitro splicing assays using genomic DNA clones to identify two critical polymorphisms affecting the inclusion of exon 5 into mA3 transcripts: the number of TCCT repeats upstream of exon 5 and the single nucleotide polymorphism within exon 5 located 12 bases upstream of the exon 5/intron 5 boundary. Distribution of the above polymorphisms among different Mus species indicates that the inclusion of exon 5 into mA3 mRNA is a relatively recent event in the evolution of mice. The widespread geographic distribution of this exon 5-including genetic variant suggests that in some Mus populations the cost of maintaining an effective but mutagenic enzyme may outweigh its antiviral function. PMID:22275865

Dicer cleaves 5'-extended microRNA precursors originating from RNA polymerase II transcription start sites.

PubMed

Sheng, Peike; Fields, Christopher; Aadland, Kelsey; Wei, Tianqi; Kolaczkowski, Oralia; Gu, Tongjun; Kolaczkowski, Bryan; Xie, Mingyi

2018-05-09

MicroRNAs (miRNAs) are approximately 22 nucleotide (nt) long and play important roles in post-transcriptional regulation in both plants and animals. In animals, precursor (pre-) miRNAs are ∼70 nt hairpins produced by Drosha cleavage of long primary (pri-) miRNAs in the nucleus. Exportin-5 (XPO5) transports pre-miRNAs into the cytoplasm for Dicer processing. Alternatively, pre-miRNAs containing a 5' 7-methylguanine (m7G-) cap can be generated independently of Drosha and XPO5. Here we identify a class of m7G-capped pre-miRNAs with 5' extensions up to 39 nt long. The 5'-extended pre-miRNAs are transported by Exportin-1 (XPO1). Unexpectedly, a long 5' extension does not block Dicer processing. Rather, Dicer directly cleaves 5'-extended pre-miRNAs by recognizing its 3' end to produce mature 3p miRNA and extended 5p miRNA both in vivo and in vitro. The recognition of 5'-extended pre-miRNAs by the Dicer Platform-PAZ-Connector (PPC) domain can be traced back to ancestral animal Dicers, suggesting that this previously unrecognized Dicer reaction mode is evolutionarily conserved. Our work reveals additional genetic sources for small regulatory RNAs and substantiates Dicer's essential role in RNAi-based gene regulation.

The exosome component Rrp6 is required for RNA polymerase II termination at specific targets of the Nrd1-Nab3 pathway.

PubMed

Fox, Melanie J; Gao, Hongyu; Smith-Kinnaman, Whitney R; Liu, Yunlong; Mosley, Amber L

2015-01-01

The exosome and its nuclear specific subunit Rrp6 form a 3'-5' exonuclease complex that regulates diverse aspects of RNA biology including 3' end processing and degradation of a variety of noncoding RNAs (ncRNAs) and unstable transcripts. Known targets of the nuclear exosome include short (<1000 bp) RNAPII transcripts such as small noncoding RNAs (snRNAs), cryptic unstable transcripts (CUTs), and some stable unannotated transcripts (SUTs) that are terminated by an Nrd1, Nab3, and Sen1 (NNS) dependent mechanism. NNS-dependent termination is coupled to RNA 3' end processing and/or degradation by the Rrp6/exosome in yeast. Recent work suggests Nrd1 is necessary for transcriptome surveillance, regulating promoter directionality and suppressing antisense transcription independently of, or prior to, Rrp6 activity. It remains unclear whether Rrp6 is directly involved in termination; however, Rrp6 has been implicated in the 3' end processing and degradation of ncRNA transcripts including CUTs. To determine the role of Rrp6 in NNS termination globally, we performed RNA sequencing (RNA-Seq) on total RNA and perform ChIP-exo analysis of RNA Polymerase II (RNAPII) localization. Deletion of RRP6 promotes hyper-elongation of multiple NNS-dependent transcripts resulting from both improperly processed 3' RNA ends and faulty transcript termination at specific target genes. The defects in RNAPII termination cause transcriptome-wide changes in mRNA expression through transcription interference and/or antisense repression, similar to previously reported effects of depleting Nrd1 from the nucleus. Elongated transcripts were identified within all classes of known NNS targets with the largest changes in transcription termination occurring at CUTs. Interestingly, the extended transcripts that we have detected in our studies show remarkable similarity to Nrd1-unterminated transcripts at many locations, suggesting that Rrp6 acts with the NNS complex globally to promote transcription termination in addition to 3' end RNA processing and/or degradation at specific targets.

Effect of Crc and Hfq proteins on the transcription, processing, and stability of the Pseudomonas putida CrcZ sRNA

PubMed Central

Hernández-Arranz, Sofía; Sánchez-Hevia, Dione; Rojo, Fernando; Moreno, Renata

2016-01-01

In Pseudomonas putida, the Hfq and Crc proteins regulate the expression of many genes in response to nutritional and environmental cues, by binding to mRNAs that bear specific target motifs and inhibiting their translation. The effect of these two proteins is antagonized by the CrcZ and CrcY small RNAs (sRNAs), the levels of which vary greatly according to growth conditions. The crcZ and crcY genes are transcribed from promoters PcrcZ and PcrcY, respectively, a process that relies on the CbrB transcriptional activator and the RpoN σ factor. Here we show that crcZ can also be transcribed from the promoter of the immediate upstream gene, cbrB, a weak constitutive promoter. The cbrB-crcZ transcript was processed to render a sRNA very similar in size to the CrcZ produced from promoter PcrcZ. The processed sRNA, termed CrcZ*, was able to antagonize Hfq/Crc because, when provided in trans, it relieved the deregulated Hfq/Crc-dependent hyperrepressing phenotype of a ΔcrcZΔcrcY strain. CrcZ* may help in attaining basal levels of CrcZ/CrcZ* that are sufficient to protect the cell from an excessive Hfq/Crc-dependent repression. Since a functional sRNA can be produced from PcrcZ, an inducible strong promoter, or by cleavage of the cbrB-crcZ mRNA, crcZ can be considered a 3′-untranslated region of the cbrB-crcZ mRNA. In the absence of Hfq, the processed form of CrcZ was not observed. In addition, we show that Crc and Hfq increase CrcZ stability, which supports the idea that these proteins can form a complex with CrcZ and protect it from degradation by RNases. PMID:27777366

Microprocessor mediates transcriptional termination of long noncoding RNA transcripts hosting microRNAs.

PubMed

Dhir, Ashish; Dhir, Somdutta; Proudfoot, Nick J; Jopling, Catherine L

2015-04-01

MicroRNAs (miRNAs) play a major part in the post-transcriptional regulation of gene expression. Mammalian miRNA biogenesis begins with cotranscriptional cleavage of RNA polymerase II (Pol II) transcripts by the Microprocessor complex. Although most miRNAs are located within introns of protein-coding transcripts, a substantial minority of miRNAs originate from long noncoding (lnc) RNAs, for which transcript processing is largely uncharacterized. We show, by detailed characterization of liver-specific lnc-pri-miR-122 and genome-wide analysis in human cell lines, that most lncRNA transcripts containing miRNAs (lnc-pri-miRNAs) do not use the canonical cleavage-and-polyadenylation pathway but instead use Microprocessor cleavage to terminate transcription. Microprocessor inactivation leads to extensive transcriptional readthrough of lnc-pri-miRNA and transcriptional interference with downstream genes. Consequently we define a new RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells.

Quantifying circular RNA expression from RNA-seq data using model-based framework.

PubMed

Li, Musheng; Xie, Xueying; Zhou, Jing; Sheng, Mengying; Yin, Xiaofeng; Ko, Eun-A; Zhou, Tong; Gu, Wanjun

2017-07-15

Circular RNAs (circRNAs) are a class of non-coding RNAs that are widely expressed in various cell lines and tissues of many organisms. Although the exact function of many circRNAs is largely unknown, the cell type-and tissue-specific circRNA expression has implicated their crucial functions in many biological processes. Hence, the quantification of circRNA expression from high-throughput RNA-seq data is becoming important to ascertain. Although many model-based methods have been developed to quantify linear RNA expression from RNA-seq data, these methods are not applicable to circRNA quantification. Here, we proposed a novel strategy that transforms circular transcripts to pseudo-linear transcripts and estimates the expression values of both circular and linear transcripts using an existing model-based algorithm, Sailfish. The new strategy can accurately estimate transcript expression of both linear and circular transcripts from RNA-seq data. Several factors, such as gene length, amount of expression and the ratio of circular to linear transcripts, had impacts on quantification performance of circular transcripts. In comparison to count-based tools, the new computational framework had superior performance in estimating the amount of circRNA expression from both simulated and real ribosomal RNA-depleted (rRNA-depleted) RNA-seq datasets. On the other hand, the consideration of circular transcripts in expression quantification from rRNA-depleted RNA-seq data showed substantial increased accuracy of linear transcript expression. Our proposed strategy was implemented in a program named Sailfish-cir. Sailfish-cir is freely available at https://github.com/zerodel/Sailfish-cir . tongz@medicine.nevada.edu or wanjun.gu@gmail.com. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

Plant polycistronic precursors containing non-homologous microRNAs target transcripts encoding functionally related proteins

PubMed Central

2009-01-01

Background MicroRNAs (miRNAs) are endogenous single-stranded small RNAs that regulate the expression of specific mRNAs involved in diverse biological processes. In plants, miRNAs are generally encoded as a single species in independent transcriptional units, referred to as MIRNA genes, in contrast to animal miRNAs, which are frequently clustered. Results We performed a comparative genomic analysis in three model plants (rice, poplar and Arabidopsis) and characterized miRNA clusters containing two to eight miRNA species. These clusters usually encode miRNAs of the same family and certain share a common evolutionary origin across monocot and dicot lineages. In addition, we identified miRNA clusters harboring miRNAs with unrelated sequences that are usually not evolutionarily conserved. Strikingly, non-homologous miRNAs from the same cluster were predicted to target transcripts encoding related proteins. At least four Arabidopsis non-homologous clusters were expressed as single transcriptional units. Overexpression of one of these polycistronic precursors, producing Ath-miR859 and Ath-miR774, led to the DCL1-dependent accumulation of both miRNAs and down-regulation of their different mRNA targets encoding F-box proteins. Conclusions In addition to polycistronic precursors carrying related miRNAs, plants also contain precursors allowing coordinated expression of non-homologous miRNAs to co-regulate functionally related target transcripts. This mechanism paves the way for using polycistronic MIRNA precursors as a new molecular tool for plant biologists to simultaneously control the expression of different genes. PMID:19951405

RNA polymerase II transcriptional fidelity control and its functional interplay with DNA modifications

PubMed Central

Xu, Liang; Wang, Wei; Chong, Jenny; Shin, Ji Hyun; Xu, Jun; Wang, Dong

2016-01-01

Accurate genetic information transfer is essential for life. As a key enzyme involved in the first step of gene expression, RNA polymerase II (Pol II) must maintain high transcriptional fidelity while it reads along DNA template and synthesizes RNA transcript in a stepwise manner during transcription elongation. DNA lesions or modifications may lead to significant changes in transcriptional fidelity or transcription elongation dynamics. In this review, we will summarize recent progress towards understanding the molecular basis of RNA Pol II transcriptional fidelity control and impacts of DNA lesions and modifications on Pol II transcription elongation. PMID:26392149

Transcriptional and phylogenetic analysis of five complete ambystomatid salamander mitochondrial genomes.

PubMed

Samuels, Amy K; Weisrock, David W; Smith, Jeramiah J; France, Katherine J; Walker, John A; Putta, Srikrishna; Voss, S Randal

2005-04-11

We report on a study that extended mitochondrial transcript information from a recent EST project to obtain complete mitochondrial genome sequence for 5 tiger salamander complex species (Ambystoma mexicanum, A. t. tigrinum, A. andersoni, A. californiense, and A. dumerilii). We describe, for the first time, aspects of mitochondrial transcription in a representative amphibian, and then use complete mitochondrial sequence data to examine salamander phylogeny at both deep and shallow levels of evolutionary divergence. The available mitochondrial ESTs for A. mexicanum (N=2481) and A. t. tigrinum (N=1205) provided 92% and 87% coverage of the mitochondrial genome, respectively. Complete mitochondrial sequences for all species were rapidly obtained by using long distance PCR and DNA sequencing. A number of genome structural characteristics (base pair length, base composition, gene number, gene boundaries, codon usage) were highly similar among all species and to other distantly related salamanders. Overall, mitochondrial transcription in Ambystoma approximated the pattern observed in other vertebrates. We inferred from the mapping of ESTs onto mtDNA that transcription occurs from both heavy and light strand promoters and continues around the entire length of the mtDNA, followed by post-transcriptional processing. However, the observation of many short transcripts corresponding to rRNA genes indicates that transcription may often terminate prematurely to bias transcription of rRNA genes; indeed an rRNA transcription termination signal sequence was observed immediately following the 16S rRNA gene. Phylogenetic analyses of salamander family relationships consistently grouped Ambystomatidae in a clade containing Cryptobranchidae and Hynobiidae, to the exclusion of Salamandridae. This robust result suggests a novel alternative hypothesis because previous studies have consistently identified Ambystomatidae and Salamandridae as closely related taxa. Phylogenetic analyses of tiger salamander complex species also produced robustly supported trees. The D-loop, used in previous molecular phylogenetic studies of the complex, was found to contain a relatively low level of variation and we identified mitochondrial regions with higher rates of molecular evolution that are more useful in resolving relationships among species. Our results show the benefit of using complete genome mitochondrial information in studies of recently and rapidly diverged taxa.

Cyclin-dependent Kinase 9 Links RNA Polymerase II Transcription to Processing of Ribosomal RNA*

PubMed Central

Burger, Kaspar; Mühl, Bastian; Rohrmoser, Michaela; Coordes, Britta; Heidemann, Martin; Kellner, Markus; Gruber-Eber, Anita; Heissmeyer, Vigo; Strässer, Katja; Eick, Dirk

2013-01-01

Ribosome biogenesis is a process required for cellular growth and proliferation. Processing of ribosomal RNA (rRNA) is highly sensitive to flavopiridol, a specific inhibitor of cyclin-dependent kinase 9 (Cdk9). Cdk9 has been characterized as the catalytic subunit of the positive transcription elongation factor b (P-TEFb) of RNA polymerase II (RNAPII). Here we studied the connection between RNAPII transcription and rRNA processing. We show that inhibition of RNAPII activity by α-amanitin specifically blocks processing of rRNA. The block is characterized by accumulation of 3′ extended unprocessed 47 S rRNAs and the entire inhibition of other 47 S rRNA-specific processing steps. The transcription rate of rRNA is moderately reduced after inhibition of Cdk9, suggesting that defective 3′ processing of rRNA negatively feeds back on RNAPI transcription. Knockdown of Cdk9 caused a strong reduction of the levels of RNAPII-transcribed U8 small nucleolar RNA, which is essential for 3′ rRNA processing in mammalian cells. Our data demonstrate a pivotal role of Cdk9 activity for coupling of RNAPII transcription with small nucleolar RNA production and rRNA processing. PMID:23744076

Upstream regulatory elements are necessary and sufficient for transcription of a U6 RNA gene by RNA polymerase III.

PubMed Central

Das, G; Henning, D; Wright, D; Reddy, R

1988-01-01

Whereas the genes coding for trimethyl guanosine-capped snRNAs are transcribed by RNA polymerase II, the U6 RNA genes are transcribed by RNA polymerase III. In this study, we have analyzed the cis-regulatory elements involved in the transcription of a mouse U6 snRNA gene in vitro and in frog oocytes. Transcriptional analysis of mutant U6 gene constructs showed that, unlike most known cases of polymerase III transcription, intragenic sequences except the initiation nucleotide are dispensable for efficient and accurate transcription of U6 gene in vitro. Transcription of 5' deletion mutants in vitro and in frog oocytes showed that the upstream region, within 79 bp from the initiation nucleotide, contains elements necessary for U6 gene transcription. Transcription studies were carried out in frog oocytes with U6 genes containing 5' distal sequence; these studies revealed that the distal element acts as an orientation-dependent enhancer when present upstream to the gene, while it is orientation-independent but distance-dependent enhancer when placed down-stream to the U6 gene. Analysis of 3' deletion mutants showed that the transcription termination of U6 RNA is dependent on a T cluster present on the 3' end of the gene, thus providing further support to other lines of evidence that U6 genes are transcribed by RNA polymerase III. These observations suggest the involvement of a composite of components of RNA polymerase II and III transcription machineries in the transcription of U6 genes by RNA polymerase III. Images PMID:3366121

Transcript levels, alternative splicing and proteolytic cleavage of TFIIIA control 5S rRNA accumulation during Arabidopsis thaliana development.

PubMed

Layat, Elodie; Cotterell, Sylviane; Vaillant, Isabelle; Yukawa, Yasushi; Tutois, Sylvie; Tourmente, Sylvette

2012-07-01

Ribosome biogenesis is critical for eukaryotic cells and requires coordinated synthesis of the protein and rRNA moieties of the ribosome, which are therefore highly regulated. 5S ribosomal RNA, an essential component of the large ribosomal subunit, is transcribed by RNA polymerase III and specifically requires transcription factor IIIA (TFIIIA). To obtain insight into the regulation of 5S rRNA transcription, we have investigated the expression of 5S rRNA and the exon-skipped (ES) and exon-including (EI) TFIIIA transcripts, two transcript isoforms that result from alternative splicing of the TFIIIA gene, and TFIIIA protein amounts with respect to requirements for 5S rRNA during development. We show that 5S rRNA quantities are regulated through distinct but complementary mechanisms operating through transcriptional and post-transcriptional control of TFIIIA transcripts as well as at the post-translational level through proteolytic cleavage of the TFIIIA protein. During the reproductive phase, high expression of the TFIIIA gene together with low proteolytic cleavage contributes to accumulation of functional, full-length TFIIIA protein, and results in 5S rRNA accumulation in the seed. In contrast, just after germination, the levels of TFIIIA-encoding transcripts are low and stable. Full-length TFIIIA protein is undetectable, and the level of 5S rRNA stored in the embryo progressively decreases. After day 4, in correlation with the reorganization of 5S rDNA chromatin to a mature state, full-length TFIIIA protein with transcriptional activity accumulates and permits de novo transcription of 5S rRNA. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

A Mechanistic Model for Cooperative Behavior of Co-transcribing RNA Polymerases

PubMed Central

Heberling, Tamra; Davis, Lisa; Gedeon, Jakub; Morgan, Charles; Gedeon, Tomáš

2016-01-01

In fast-transcribing prokaryotic genes, such as an rrn gene in Escherichia coli, many RNA polymerases (RNAPs) transcribe the DNA simultaneously. Active elongation of RNAPs is often interrupted by pauses, which has been observed to cause RNAP traffic jams; yet some studies indicate that elongation seems to be faster in the presence of multiple RNAPs than elongation by a single RNAP. We propose that an interaction between RNAPs via the torque produced by RNAP motion on helically twisted DNA can explain this apparent paradox. We have incorporated the torque mechanism into a stochastic model and simulated transcription both with and without torque. Simulation results illustrate that the torque causes shorter pause durations and fewer collisions between polymerases. Our results suggest that the torsional interaction of RNAPs is an important mechanism in maintaining fast transcription times, and that transcription should be viewed as a cooperative group effort by multiple polymerases. PMID:27517607

The RNA-induced transcriptional silencing complex targets chromatin exclusively via interacting with nascent transcripts.

PubMed

Shimada, Yukiko; Mohn, Fabio; Bühler, Marc

2016-12-01

Small RNAs regulate chromatin modification and transcriptional gene silencing across the eukaryotic kingdom. Although these processes have been well studied, fundamental mechanistic aspects remain obscure. Specifically, it is unclear exactly how small RNA-loaded Argonaute protein complexes target chromatin to mediate silencing. Here, using fission yeast, we demonstrate that transcription of the target locus is essential for RNA-directed formation of heterochromatin. However, high transcriptional activity is inhibitory; thus, a transcriptional window exists that is optimal for silencing. We further found that pre-mRNA splicing is compatible with RNA-directed heterochromatin formation. However, the kinetics of pre-mRNA processing is critical. Introns close to the 5' end of a transcript that are rapidly spliced result in a bistable response whereby the target either remains euchromatic or becomes fully silenced. Together, our results discount siRNA-DNA base pairing in RNA-mediated heterochromatin formation, and the mechanistic insights further reveal guiding paradigms for the design of small RNA-directed chromatin silencing studies in multicellular organisms. © 2016 Shimada et al.; Published by Cold Spring Harbor Laboratory Press.

Widespread promoter-mediated coordination of transcription and mRNA degradation

PubMed Central

2012-01-01

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

Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape.

PubMed

Cecere, Germano; Hoersch, Sebastian; O'Keeffe, Sean; Sachidanandam, Ravi; Grishok, Alla

2014-04-01

Argonaute proteins and their small RNA cofactors short interfering RNAs are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) that are antisense to germline transcripts. However, its role in gene expression regulation remains controversial. Here we used genome-wide profiling of nascent RNA transcripts and found that the CSR-1 RNA interference pathway promoted sense-oriented RNA polymerase II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. On the basis of these findings, we propose that the CSR-1 pathway helps maintain the directionality of active transcription, thereby propagating the distinction between transcriptionally active and silent genomic regions.

The 5'-end heterogeneity of adenovirus virus-associated RNAI contributes to the asymmetric guide strand incorporation into the RNA-induced silencing complex.

PubMed

Xu, Ning; Gkountela, Sofia; Saeed, Khalid; Akusjärvi, Göran

2009-11-01

Human Adenovirus type 5 encodes two short RNA polymerase III transcripts, the virus-associated (VA) RNAI and VA RNAII, which can adopt stable hairpin structures that resemble micro-RNA precursors. The terminal stems of the VA RNAs are processed into small RNAs (mivaRNAs) that are incorporated into RISC. It has been reported that VA RNAI has two transcription initiation sites, which produce two VA RNAI species; a major species, VA RNAI(G), which accounts for 75% of the VA RNAI pool, and a minor species, VA RNAI(A), which initiates transcription three nucleotides upstream compared to VA RNAI(G). We show that this 5'-heterogeneity results in a dramatic difference in RISC assembly. Thus, both VA RNAI(G) and VA RNAI(A) are processed by Dicer at the same position in the terminal stem generating the same 3'-strand mivaRNA. This mivaRNA is incorporated into RISC with 200-fold higher efficiency compared to the 5'-strand of mivaRNAI. Of the small number of 5'-strands used in RISC assembly only VA RNAI(A) generated active RISC complexes. We also show that the 3'-strand of mivaRNAI, although being the preferred substrate for RISC assembly, generates unstable RISC complexes with a low in vitro cleavage activity, only around 2% compared to RISC assembled on the VA RNAI(A) 5'-strand.

RNAi screening of subtracted transcriptomes reveals tumor suppression by taurine-activated GABAA receptors involved in volume regulation

PubMed Central

van Nierop, Pim; Vormer, Tinke L.; Foijer, Floris; Verheij, Joanne; Lodder, Johannes C.; Andersen, Jesper B.; Mansvelder, Huibert D.; te Riele, Hein

2018-01-01

To identify coding and non-coding suppressor genes of anchorage-independent proliferation by efficient loss-of-function screening, we have developed a method for enzymatic production of low complexity shRNA libraries from subtracted transcriptomes. We produced and screened two LEGO (Low-complexity by Enrichment for Genes shut Off) shRNA libraries that were enriched for shRNA vectors targeting coding and non-coding polyadenylated transcripts that were reduced in transformed Mouse Embryonic Fibroblasts (MEFs). The LEGO shRNA libraries included ~25 shRNA vectors per transcript which limited off-target artifacts. Our method identified 79 coding and non-coding suppressor transcripts. We found that taurine-responsive GABAA receptor subunits, including GABRA5 and GABRB3, were induced during the arrest of non-transformed anchor-deprived MEFs and prevented anchorless proliferation. We show that taurine activates chloride currents through GABAA receptors on MEFs, causing seclusion of cell volume in large membrane protrusions. Volume seclusion from cells by taurine correlated with reduced proliferation and, conversely, suppression of this pathway allowed anchorage-independent proliferation. In human cholangiocarcinomas, we found that several proteins involved in taurine signaling via GABAA receptors were repressed. Low GABRA5 expression typified hyperproliferative tumors, and loss of taurine signaling correlated with reduced patient survival, suggesting this tumor suppressive mechanism operates in vivo. PMID:29787571

Post-transcriptional inducible gene regulation by natural antisense RNA.

PubMed

Nishizawa, Mikio; Ikeya, Yukinobu; Okumura, Tadayoshi; Kimura, Tominori

2015-01-01

Accumulating data indicate the existence of natural antisense transcripts (asRNAs), frequently transcribed from eukaryotic genes and do not encode proteins in many cases. However, their importance has been overlooked due to their heterogeneity, low expression level, and unknown function. Genes induced in responses to various stimuli are transcriptionally regulated by the activation of a gene promoter and post-transcriptionally regulated by controlling mRNA stability and translatability. A low-copy-number asRNA may post-transcriptionally regulate gene expression with cis-controlling elements on the mRNA. The asRNA itself may act as regulatory RNA in concert with trans-acting factors, including various RNA-binding proteins that bind to cis-controlling elements, microRNAs, and drugs. A novel mechanism that regulates mRNA stability includes the interaction of asRNA with mRNA by hybridization to loops in secondary structures. Furthermore, recent studies have shown that the functional network of mRNAs, asRNAs, and microRNAs finely tunes the levels of mRNA expression. The post-transcriptional mechanisms via these RNA-RNA interactions may play pivotal roles to regulate inducible gene expression and present the possibility of the involvement of asRNAs in various diseases.

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics.

PubMed

Zambenedetti, Miriam Ribas; Pavoni, Daniela Parada; Dallabona, Andreia Cristine; Dominguez, Alejandro Correa; Poersch, Celina de Oliveira; Fragoso, Stenio Perdigão; Krieger, Marco Aurélio

2017-05-01

Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

PubMed Central

Zambenedetti, Miriam Ribas; Pavoni, Daniela Parada; Dallabona, Andreia Cristine; Dominguez, Alejandro Correa; Poersch, Celina de Oliveira; Fragoso, Stenio Perdigão; Krieger, Marco Aurélio

2017-01-01

BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses. PMID:28403327

Developmental expression of a regulatory gene is programmed at the level of splicing.

PubMed Central

Chou, T B; Zachar, Z; Bingham, P M

1987-01-01

We report sequence and transcript structures for a 6191-base chromosomal segment containing the presumptive regulatory gene from Drosophila, suppressor-of-white-apricot [su(wa)]. Our results indicate that su(wa) expression is controlled by regulating occurrence of specific splices. Seven introns are removed from the su(wa) primary transcript during precellular blastoderm development. The sequence of this mature RNA indicates that it is a conventional messenger RNA. In contrast, after cellular blastoderm the first two of these introns cease to be efficiently removed. The mature RNAs resulting from this failure to remove the first two introns have structures quite unexpected of mRNAs. We propose that postcellular blastoderm su(wa) expression is repressed by preventing splices necessary to produce a functional mRNA. Implications and mechanisms are discussed. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2832151

Establishment of an in vitro transcription system for Peste des petits ruminant virus.

PubMed

Yunus, Mohammad; Shaila, Melkote S

2012-12-05

Peste-des-petits ruminants virus (PPRV) is a non segmented negative strand RNA virus of the genus Morbillivirus within Paramyxoviridae family. Negative strand RNA viruses are known to carry nucleocapsid (N) protein, phospho (P) protein and RNA polymerase (L protein) packaged within the virion which possess all activities required for transcription, post-transcriptional modification of mRNA and replication. In order to understand the mechanism of transcription and replication of the virus, an in vitro transcription reconstitution system is required. In the present work, an in vitro transcription system has been developed with ribonucleoprotein (RNP) complex purified from virus infected cells as well as partially purified recombinant polymerase (L-P) complex from insect cells along with N-RNA (genomic RNA encapsidated by N protein) template isolated from virus infected cells. RNP complex isolated from virus infected cells and recombinant L-P complex purified from insect cells was used to reconstitute transcription on N-RNA template. The requirement for this transcription reconstitution has been defined. Transcription of viral genes in the in vitro system was confirmed by PCR amplification of cDNAs corresponding to individual transcripts using gene specific primers. In order to measure the relative expression level of viral transcripts, real time PCR analysis was carried out. qPCR analysis of the transcription products made in vitro showed a gradient of polarity of transcription from 3' end to 5' end of the genome similar to that exhibited by the virus in infected cells. This report describes for the first time, the development of an in vitro transcription reconstitution system for PPRV with RNP complex purified from infected cells and recombinant L-P complex expressed in insect cells. Both the complexes were able to synthesize all the mRNA species in vitro, exhibiting a gradient of polarity in transcription.

De novo piRNA cluster formation in the Drosophila germ line triggered by transgenes containing a transcribed transposon fragment.

PubMed

Olovnikov, Ivan; Ryazansky, Sergei; Shpiz, Sergey; Lavrov, Sergey; Abramov, Yuri; Vaury, Chantal; Jensen, Silke; Kalmykova, Alla

2013-06-01

PIWI-interacting RNAs (piRNAs) provide defence against transposable element (TE) expansion in the germ line of metazoans. piRNAs are processed from the transcripts encoded by specialized heterochromatic clusters enriched in damaged copies of transposons. How these regions are recognized as a source of piRNAs is still elusive. The aim of this study is to determine how transgenes that contain a fragment of the Long Interspersed Nuclear Elements (LINE)-like I transposon lead to an acquired TE resistance in Drosophila. We show that such transgenes, being inserted in unique euchromatic regions that normally do not produce small RNAs, become de novo bidirectional piRNA clusters that silence I-element activity in the germ line. Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences--not only from the transposon fragment. Chromatin immunoprecipitation analysis shows that in ovaries, the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes producing piRNAs. We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs.

Promoter for Sindbis virus RNA-dependent subgenomic RNA transcription.

PubMed

Levis, R; Schlesinger, S; Huang, H V

1990-04-01

Sindbis virus is a positive-strand RNA enveloped virus, a member of the Alphavirus genus of the Togaviridae family. Two species of mRNA are synthesized in cells infected with Sindbis virus; one, the 49S RNA, is the genomic RNA; the other, the 26S RNA, is a subgenomic RNA that is identical in sequence to the 3' one-third of the genomic RNA. Ou et al. (J.-H. Ou, C. M. Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79:5235-5239, 1982) identified a highly conserved region 19 nucleotides upstream and 2 nucleotides downstream from the start of the 26S RNA and proposed that in the negative-strand template, these nucleotides compose the promoter for directing the synthesis of the subgenomic RNA. Defective interfering (DI) RNAs of Sindbis virus were used to test this proposal. A 227-nucleotide sequence encompassing 98 nucleotides upstream and 117 nucleotides downstream from the start site of the Sindbis virus subgenomic RNA was inserted into a DI genome. The DI RNA containing the insert was replicated and packaged in the presence of helper virus, and cells infected with these DI particles produced a subgenomic RNA of the size and sequence expected if the promoter was functional. The initiating nucleotide was identical to that used for Sindbis virus subgenomic mRNA synthesis. Deletion analysis showed that the minimal region required to detect transcription of a subgenomic RNA from the negative-strand template of a DI RNA was 18 or 19 nucleotides upstream and 5 nucleotides downstream from the start of the subgenomic RNA.

The agents of natural genome editing.

PubMed

Witzany, Guenther

2011-06-01

The DNA serves as a stable information storage medium and every protein which is needed by the cell is produced from this blueprint via an RNA intermediate code. More recently it was found that an abundance of various RNA elements cooperate in a variety of steps and substeps as regulatory and catalytic units with multiple competencies to act on RNA transcripts. Natural genome editing on one side is the competent agent-driven generation and integration of meaningful DNA nucleotide sequences into pre-existing genomic content arrangements, and the ability to (re-)combine and (re-)regulate them according to context-dependent (i.e. adaptational) purposes of the host organism. Natural genome editing on the other side designates the integration of all RNA activities acting on RNA transcripts without altering DNA-encoded genes. If we take the genetic code seriously as a natural code, there must be agents that are competent to act on this code because no natural code codes itself as no natural language speaks itself. As code editing agents, viral and subviral agents have been suggested because there are several indicators that demonstrate viruses competent in both RNA and DNA natural genome editing.

Sequence analysis of RNase MRP RNA reveals its origination from eukaryotic RNase P RNA

PubMed Central

Zhu, Yanglong; Stribinskis, Vilius; Ramos, Kenneth S.; Li, Yong

2006-01-01

RNase MRP is a eukaryote-specific endoribonuclease that generates RNA primers for mitochondrial DNA replication and processes precursor rRNA. RNase P is a ubiquitous endoribonuclease that cleaves precursor tRNA transcripts to produce their mature 5′ termini. We found extensive sequence homology of catalytic domains and specificity domains between their RNA subunits in many organisms. In Candida glabrata, the internal loop of helix P3 is 100% conserved between MRP and P RNAs. The helix P8 of MRP RNA from microsporidia Encephalitozoon cuniculi is identical to that of P RNA. Sequence homology can be widely spread over the whole molecule of MRP RNA and P RNA, such as those from Dictyostelium discoideum. These conserved nucleotides between the MRP and P RNAs strongly support the hypothesis that the MRP RNA is derived from the P RNA molecule in early eukaryote evolution. PMID:16540690

Reverse Transcription of a Self-Primed Retrotransposon Requires an RNA Structure Similar to the U5-IR Stem-Loop of Retroviruses

PubMed Central

Lin, Jia-Hwei; Levin, Henry L.

1998-01-01

An inverted repeat (IR) within the U5 region of the Rous sarcoma virus (RSV) mRNA forms a structure composed of a 7-bp stem and a 5-nucleotide (nt) loop. This U5-IR structure has been shown to be required for the initiation of reverse transcription. The mRNA of Tf1, long terminal repeat-containing retrotransposon from fission yeast (Schizosaccharomyces pombe) contains nucleotides with the potential to form a U5-IR stem-loop that is strikingly similar to that of RSV. The putative U5-IR stem-loop of Tf1 consists of a 7-bp stem and a 25-nt loop. Results from mutagenesis studies indicate that the U5-IR stem-loop in the mRNA of Tf1 does form and that it is required for Tf1 transposition. Although the loop is required for transposition, we were surprised that the specific sequence of the nucleotides within the loop was unimportant for function. Additional investigation indicates that the loss of transposition activity due to a reduction in the loop size to 6 nt could be rescued by increasing the GC content of the stem. This result indicates that the large loop in the Tf1 mRNA relative to that of the RSV allows the formation of the relatively weak U5-IR stem. The levels of Tf1 proteins expressed and the amounts of Tf1 RNA packaged into the virus-like particles were not affected by mutations in the U5-IR structure. However, all of the mutations in the U5-IR structure that caused defects in transposition produced low amounts of reverse transcripts. A unique feature in the initiation of Tf1 reverse transcription is that, instead of a tRNA, the first 11 nt of the Tf1 mRNA serve as the minus-strand primer. Analysis of the 5′ end of Tf1 mRNA revealed that the mutations in the U5-IR stem-loop that resulted in defects in reverse transcription caused a reduction in the cleavage activity required to generate the Tf1 primer. Our results indicate that the U5-IR stems of Tf1 and RSV are conserved in size, position, and function. PMID:9774699

Circular RNAs: analysis, expression and potential functions

PubMed Central

Salzman, Julia

2016-01-01

Just a few years ago, it had been assumed that the dominant RNA isoforms produced from eukaryotic genes were variants of messenger RNA, functioning as intermediates in gene expression. In early 2012, however, a surprising discovery was made: circular RNA (circRNA) was shown to be a transcriptional product in thousands of human and mouse genes and in hundreds of cases constituted the dominant RNA isoform. Subsequent studies revealed that the expression of circRNAs is developmentally regulated, tissue and cell-type specific, and shared across the eukaryotic tree of life. These features suggest important functions for these molecules. Here, we describe major advances in the field of circRNA biology, focusing on the regulation of and functional roles played by these molecules. PMID:27246710

Cloning and expression of porcine Dicer and the impact of developmental stage and culture conditions on MicroRNA expression in porcine embryos.

PubMed

Stowe, Heather M; Curry, Erin; Calcatera, Samantha M; Krisher, Rebecca L; Paczkowski, Melissa; Pratt, Scott L

2012-06-15

MicroRNA (miRNA) is a class of small, single-stranded ribonucleic acids that regulate gene expression post-transcriptionally and are involved in somatic cell, germ cell, and embryonic development. As the enzyme responsible for producing mature miRNA, Dicer is crucial to miRNA production. Characterization of Dicer and its expression at the nucleotide level, as well as the identification of miRNA expression in reproductive tissues, have yet to be reported for the domestic pig (Sus scrofa), a species important for disease modeling, biomedical research, and food production. In this study we determined the primary cDNA sequence of porcine Dicer (pDicer), confirmed its expression in porcine oocytes and early stage embryos, and evaluated the expression of specific miRNA during early embryonic development and between in vivo (IVO) and in vitro (IVF) produced embryos. Total cellular RNA (tcRNA) was isolated and subjected to end point RT-PCR, subcloning, and sequencing. The pDicer coding sequence was found to be highly conserved, and phylogenetic analysis showed that pDicer is more highly conserved to human Dicer (hDicer) than the mouse homolog. Expression of pDicer mRNA was detected in oocytes and in IVO produced blastocyst embryos. Two RT-PCR procedures were conducted to identify and quantitate miRNA expressed in metaphase II oocytes (MII) and embryos. RT-PCR array was conducted using primers designed for human miRNA, and 86 putative porcine miRNA in MII and early embryos were detected. Fewer miRNAs were detected in 8-cell (8C) embryos compared to MII and blastocysts (B) (P=0.026 and P<0.0001, respectively). Twenty-one miRNA (of 88 examined) were differentially expressed between MII and 8C, 8C and B, or MII and B. Transcripts targeted by the differentially expressed miRNA were enriched in gene ontology (GO) categories associated with cellular development and differentiation. Further, we evaluated the effects of IVF culture on the expression of specific miRNA at the blastocyst stage. Quantitative RT-PCR was conducted on blastocyst tcRNA isolated from individual IVO and IVF produced embryos for miR-18a, -21, and -24. Only the expression level of miR-24 differed due to culture conditions, with lower levels detected in the IVO embryos. These data show that pDicer and miRNA are present in porcine oocytes and embryos. In addition, specific miRNA levels are altered due to stage of embryonic development and, in the case of miR-24, due to culture conditions, making this miRNA a candidate for screening of embryo quality. Additional studies characterizing Dicer and miRNA expression during early embryonic development from IVO and IVF sources are required to further examine and evaluate the use of miRNA as a marker for embryo quality. Copyright © 2012 Elsevier B.V. All rights reserved.

RNA polymerase I transcription in a Brassica interspecific hybrid and its progenitors: Tests of transcription factor involvement in nucleolar dominance.

PubMed Central

Frieman, M; Chen, Z J; Saez-Vasquez, J; Shen, L A; Pikaard, C S

1999-01-01

In interspecific hybrids or allopolyploids, often one parental set of ribosomal RNA genes is transcribed and the other is silent, an epigenetic phenomenon known as nucleolar dominance. Silencing is enforced by cytosine methylation and histone deacetylation, but the initial discrimination mechanism is unknown. One hypothesis is that a species-specific transcription factor is inactivated, thereby silencing one set of rRNA genes. Another is that dominant rRNA genes have higher binding affinities for limiting transcription factors. A third suggests that selective methylation of underdominant rRNA genes blocks transcription factor binding. We tested these hypotheses using Brassica napus (canola), an allotetraploid derived from B. rapa and B. oleracea in which only B. rapa rRNA genes are transcribed. B. oleracea and B. rapa rRNA genes were active when transfected into protoplasts of the other species, which argues against the species-specific transcription factor model. B. oleracea and B. rapa rRNA genes also competed equally for the pol I transcription machinery in vitro and in vivo. Cytosine methylation had no effect on rRNA gene transcription in vitro, which suggests that transcription factor binding was unimpaired. These data are inconsistent with the prevailing models and point to discrimination mechanisms that are likely to act at a chromosomal level. PMID:10224274

Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts

PubMed Central

Komoda, Keisuke; Naito, Satoshi; Ishikawa, Masayuki

2004-01-01

The replication of eukaryotic positive-strand RNA virus genomes occurs through a complex process involving multiple viral and host proteins and intracellular membranes. Here we report a cell-free system that reproduces this process in vitro. This system uses a membrane-containing extract of uninfected plant protoplasts from which the vacuoles had been removed by Percoll gradient centrifugation. We demonstrate that the system supported translation, negative-strand RNA synthesis, genomic RNA replication, and subgenomic RNA transcription of tomato mosaic virus and two other plant positive-strand RNA viruses. The RNA synthesis, which depended on translation of the genomic RNA, produced virus-related RNA species similar to those that are generated in vivo. This system will aid in the elucidation of the mechanisms of genome replication in these viruses. PMID:14769932

Altered minor-groove hydrogen bonds in DNA block transcription elongation by T7 RNA polymerase.

PubMed

Tanasova, Marina; Goeldi, Silvan; Meyer, Fabian; Hanawalt, Philip C; Spivak, Graciela; Sturla, Shana J

2015-05-26

DNA transcription depends upon the highly efficient and selective function of RNA polymerases (RNAPs). Modifications in the template DNA can impact the progression of RNA synthesis, and a number of DNA adducts, as well as abasic sites, arrest or stall transcription. Nonetheless, data are needed to understand why certain modifications to the structure of DNA bases stall RNA polymerases while others are efficiently bypassed. In this study, we evaluate the impact that alterations in dNTP/rNTP base-pair geometry have on transcription. T7 RNA polymerase was used to study transcription over modified purines and pyrimidines with altered H-bonding capacities. The results suggest that introducing wobble base-pairs into the DNA:RNA heteroduplex interferes with transcriptional elongation and stalls RNA polymerase. However, transcriptional stalling is not observed if mismatched base-pairs do not H-bond. Together, these studies show that RNAP is able to discriminate mismatches resulting in wobble base-pairs, and suggest that, in cases of modifications with minor steric impact, DNA:RNA heteroduplex geometry could serve as a controlling factor for initiating transcription-coupled DNA repair. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detergents with different chemical properties induce variable degree of cytotoxicity and mRNA expression of lipid-metabolizing enzymes and differentiation markers in cultured keratinocytes.

PubMed

Wei, Tianling; Geijer, Sophia; Lindberg, Magnus; Berne, Berit; Törmä, Hans

2006-12-01

The knowledge how detergents with different chemical properties influence epidermal keratinocytes is sparse. In the present study, the effects of five detergents were examined with respect to cell-toxicity and mRNA expression of key-enzymes in barrier lipid production and keratinocyte differentiation markers. First, the LD(50) for each detergent were determined. Secondly, keratinocytes were exposed to sub-toxic concentrations and the mRNA expression was analysed by real-time PCR after 24 h exposure to the detergents. SLS and CAPB induced a concentration-dependent increase in the expression of enzymes producing cholesterol and ceramides, while transcripts of enzymes producing fatty acids were unaffected. SLES and cocoglucoside increased the expression of certain enzymes involved in cholesterol and fatty acid synthesis while sodium cocoamphoacetate (SCAA) stimulated expression of transcripts involved in fatty acid synthesis. The expression of differentiation markers were increased by SLS, SLES and CAPB, while SCAA and cocoglucoside exhibited no effect. The present findings show that detergents have variable effects on lipid synthesis and keratinocyte differentiation, which could partly explain their barrier destruction potential in vivo.

Transcription boundaries of U1 small nuclear RNA.

PubMed Central

Kunkel, G R; Pederson, T

1985-01-01

Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly. Images PMID:2942763

Expression of mRNA encoding leukaemia inhibitory factor (LIF) and its receptor (LIFRβ) in buffalo preimplantation embryos produced in vitro: markers of successful embryo implantation.

PubMed

Eswari, S; Sai Kumar, G; Sharma, G Taru

2013-05-01

Summary The objective of this study was to evaluate the effect of supplementation of recombinant leukaemia inhibitory factor (LIF) in culture media on blastocyst development, total cell number and blastocyst hatching rates and the reverse transcription-polymerase chain reaction analysis of preimplantation buffalo embryos to determine whether they contain the LIF-encoding mRNA and its beta receptor (LIFRβ) genes in different stages of preimplantation buffalo embryos. Cumulus-oocyte complexes retrieved from slaughterhouse buffalo ovaries were matured in vitro and fertilized using frozen buffalo semen. After 18 h of co-incubation with sperm, the presumptive zygotes were cultured in modified synthetic oviductal fluid without (control) or with rhLIF (100 ng/ml). There was no significant difference in the overall cleavage rate up to morula stage however the development of blastocysts, hatching rate and total cell numbers were significantly higher in the LIF-treated group than control. Transcripts for LIFRβ were detected from immature, in vitro-matured oocytes and in the embryos up to blastocyst stage, while transcripts for the LIF were detected from 8-16-cell stage up to blastocyst, which indicated that embryo-derived LIF can act in an autocrine manner on differentiation process and blastocyst formation. This study indicated that the addition of LIF to the embryo culture medium improved development of blastocysts, functional (hatching) and morphological (number of cells) quality of the blastocysts produced in vitro. The stage-specific expression pattern of LIF and LIFRβ mRNA transcripts in buffalo embryos indicated that LIF might play an important role in the preimplantation development and subsequent implantation of buffalo embryos.

Effects of RNA integrity on transcript quantification by total RNA sequencing of clinically collected human placental samples.

PubMed

Reiman, Mario; Laan, Maris; Rull, Kristiina; Sõber, Siim

2017-08-01

RNA degradation is a ubiquitous process that occurs in living and dead cells, as well as during handling and storage of extracted RNA. Reduced RNA quality caused by degradation is an established source of uncertainty for all RNA-based gene expression quantification techniques. RNA sequencing is an increasingly preferred method for transcriptome analyses, and dependence of its results on input RNA integrity is of significant practical importance. This study aimed to characterize the effects of varying input RNA integrity [estimated as RNA integrity number (RIN)] on transcript level estimates and delineate the characteristic differences between transcripts that differ in degradation rate. The study used ribodepleted total RNA sequencing data from a real-life clinically collected set ( n = 32) of human solid tissue (placenta) samples. RIN-dependent alterations in gene expression profiles were quantified by using DESeq2 software. Our results indicate that small differences in RNA integrity affect gene expression quantification by introducing a moderate and pervasive bias in expression level estimates that significantly affected 8.1% of studied genes. The rapidly degrading transcript pool was enriched in pseudogenes, short noncoding RNAs, and transcripts with extended 3' untranslated regions. Typical slowly degrading transcripts (median length, 2389 nt) represented protein coding genes with 4-10 exons and high guanine-cytosine content.-Reiman, M., Laan, M., Rull, K., Sõber, S. Effects of RNA integrity on transcript quantification by total RNA sequencing of clinically collected human placental samples. © FASEB.

Arm-specific cleavage and mutation during reverse transcription of 2΄,5΄-branched RNA by Moloney murine leukemia virus reverse transcriptase

PubMed Central

Döring, Jessica

2017-01-01

Abstract Branchpoint nucleotides of intron lariats induce pausing of DNA synthesis by reverse transcriptases (RTs), but it is not known yet how they direct RT RNase H activity on branched RNA (bRNA). Here, we report the effects of the two arms of bRNA on branchpoint-directed RNA cleavage and mutation produced by Moloney murine leukemia virus (M-MLV) RT during DNA polymerization. We constructed a long-chained bRNA template by splinted-ligation. The bRNA oligonucleotide is chimeric and contains DNA to identify RNA cleavage products by probe hybridization. Unique sequences surrounding the branchpoint facilitate monitoring of bRNA purification by terminal-restriction fragment length polymorphism analysis. We evaluate the M-MLV RT-generated cleavage and mutational patterns. We find that cleavage of bRNA and misprocessing of the branched nucleotide proceed arm-specifically. Bypass of the branchpoint from the 2΄-arm causes single-mismatch errors, whereas bypass from the 3΄-arm leads to deletion mutations. The non-template arm is cleaved when reverse transcription is primed from the 3΄-arm but not from the 2΄-arm. This suggests that RTs flip ∼180° at branchpoints and RNases H cleave the non-template arm depending on its accessibility. Our observed interplay between M-MLV RT and bRNA would be compatible with a bRNA-mediated control of retroviral and related retrotransposon replication. PMID:28160599

Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks.

PubMed

Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

2016-05-10

RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks.

Dissecting the expression relationships between RNA-binding proteins and their cognate targets in eukaryotic post-transcriptional regulatory networks

NASA Astrophysics Data System (ADS)

Nishtala, Sneha; Neelamraju, Yaseswini; Janga, Sarath Chandra

2016-05-01

RNA-binding proteins (RBPs) are pivotal in orchestrating several steps in the metabolism of RNA in eukaryotes thereby controlling an extensive network of RBP-RNA interactions. Here, we employed CLIP (cross-linking immunoprecipitation)-seq datasets for 60 human RBPs and RIP-ChIP (RNP immunoprecipitation-microarray) data for 69 yeast RBPs to construct a network of genome-wide RBP- target RNA interactions for each RBP. We show in humans that majority (~78%) of the RBPs are strongly associated with their target transcripts at transcript level while ~95% of the studied RBPs were also found to be strongly associated with expression levels of target transcripts when protein expression levels of RBPs were employed. At transcript level, RBP - RNA interaction data for the yeast genome, exhibited a strong association for 63% of the RBPs, confirming the association to be conserved across large phylogenetic distances. Analysis to uncover the features contributing to these associations revealed the number of target transcripts and length of the selected protein-coding transcript of an RBP at the transcript level while intensity of the CLIP signal, number of RNA-Binding domains, location of the binding site on the transcript, to be significant at the protein level. Our analysis will contribute to improved modelling and prediction of post-transcriptional networks.

Transcriptional mapping of the ribosomal RNA region of mouse L-cell mitochondrial DNA.

PubMed Central

Nagley, P; Clayton, D A

1980-01-01

The map positions in mouse mitochondrial DNA of the two ribosomal RNA genes and adjacent genes coding several small transcripts have been determined precisely by application of a procedure in which DNA-RNA hybrids have been subjected to digestion by S1 nuclease under conditions of varying severity. Digestion of the DNA-RNA hybrids with S1 nuclease yielded a series of species which were shown to contain ribosomal RNA molecules together with adjacent transcripts hybridized conjointly to a continuous segment of mitochondrial DNA. There is one small transcript about 60 bases long whose gene adjoins the sequences coding the 5'-end of the small ribosomal RNA (950 bases) and which lies approximately 200 nucleotides from the D-loop origin of heavy strand mitochondrial DNA synthesis. An 80-base transcript lies between the small and large ribosomal RNA genes, and genes for two further short transcript (each about 80 bases in length) abut the sequences coding the 3'-end of the large ribosomal RNA (approximately 1500 bases). The ability to isolate a discrete DNA-RNA hybrid species approximately 2700 base pairs in length containing all these transcripts suggests that there can be few nucleotides in this region of mouse mitochondrial DNA which are not represented as stable RNA species. Images PMID:6253898

X-linked Alport syndrome caused by splicing mutations in COL4A5.

PubMed

Nozu, Kandai; Vorechovsky, Igor; Kaito, Hiroshi; Fu, Xue Jun; Nakanishi, Koichi; Hashimura, Yuya; Hashimoto, Fusako; Kamei, Koichi; Ito, Shuichi; Kaku, Yoshitsugu; Imasawa, Toshiyuki; Ushijima, Katsumi; Shimizu, Junya; Makita, Yoshio; Konomoto, Takao; Yoshikawa, Norishige; Iijima, Kazumoto

2014-11-07

X-linked Alport syndrome is caused by mutations in the COL4A5 gene. Although many COL4A5 mutations have been detected, the mutation detection rate has been unsatisfactory. Some men with X-linked Alport syndrome show a relatively mild phenotype, but molecular basis investigations have rarely been conducted to clarify the underlying mechanism. In total, 152 patients with X-linked Alport syndrome who were suspected of having Alport syndrome through clinical and pathologic investigations and referred to the hospital for mutational analysis between January of 2006 and January of 2013 were genetically diagnosed. Among those patients, 22 patients had suspected splice site mutations. Transcripts are routinely examined when suspected splice site mutations for abnormal transcripts are detected; 11 of them showed expected exon skipping, but others showed aberrant splicing patterns. The mutation detection strategy had two steps: (1) genomic DNA analysis using PCR and direct sequencing and (2) mRNA analysis using RT-PCR to detect RNA processing abnormalities. Six splicing consensus site mutations resulting in aberrant splicing patterns, one exonic mutation leading to exon skipping, and four deep intronic mutations producing cryptic splice site activation were identified. Interestingly, one case produced a cryptic splice site with a single nucleotide substitution in the deep intron that led to intronic exonization containing a stop codon; however, the patient showed a clearly milder phenotype for X-linked Alport syndrome in men with a truncating mutation. mRNA extracted from the kidney showed both normal and abnormal transcripts, with the normal transcript resulting in the milder phenotype. This novel mechanism leads to mild clinical characteristics. This report highlights the importance of analyzing transcripts to enhance the mutation detection rate and provides insight into genotype-phenotype correlations. This approach can clarify the cause of atypically mild phenotypes in X-linked Alport syndrome. Copyright © 2014 by the American Society of Nephrology.

A high-throughput pipeline for the production of synthetic antibodies for analysis of ribonucleoprotein complexes

PubMed Central

Na, Hong; Laver, John D.; Jeon, Jouhyun; Singh, Fateh; Ancevicius, Kristin; Fan, Yujie; Cao, Wen Xi; Nie, Kun; Yang, Zhenglin; Luo, Hua; Wang, Miranda; Rissland, Olivia; Westwood, J. Timothy; Kim, Philip M.; Smibert, Craig A.; Lipshitz, Howard D.; Sidhu, Sachdev S.

2016-01-01

Post-transcriptional regulation of mRNAs plays an essential role in the control of gene expression. mRNAs are regulated in ribonucleoprotein (RNP) complexes by RNA-binding proteins (RBPs) along with associated protein and noncoding RNA (ncRNA) cofactors. A global understanding of post-transcriptional control in any cell type requires identification of the components of all of its RNP complexes. We have previously shown that these complexes can be purified by immunoprecipitation using anti-RBP synthetic antibodies produced by phage display. To develop the large number of synthetic antibodies required for a global analysis of RNP complex composition, we have established a pipeline that combines (i) a computationally aided strategy for design of antigens located outside of annotated domains, (ii) high-throughput antigen expression and purification in Escherichia coli, and (iii) high-throughput antibody selection and screening. Using this pipeline, we have produced 279 antibodies against 61 different protein components of Drosophila melanogaster RNPs. Together with those produced in our low-throughput efforts, we have a panel of 311 antibodies for 67 RNP complex proteins. Tests of a subset of our antibodies demonstrated that 89% immunoprecipitate their endogenous target from embryo lysate. This panel of antibodies will serve as a resource for global studies of RNP complexes in Drosophila. Furthermore, our high-throughput pipeline permits efficient production of synthetic antibodies against any large set of proteins. PMID:26847261

MYC Mediates mRNA Cap Methylation of Canonical Wnt/β-catenin Signaling Transcripts by Recruiting CDK7 and RNA Methyltransferase

PubMed Central

Posternak, Valeriya; Ung, Matthew H.; Cheng, Chao; Cole, Michael D.

2016-01-01

MYC is a pleiotropic transcription factor that activates and represses a wide range of target genes and is frequently deregulated in human tumors. While much is known about the role of MYC in transcriptional activation and repression, MYC can also regulate mRNA cap methylation through a mechanism that has remained poorly understood. Here it is reported that MYC enhances mRNA cap methylation of transcripts globally, specifically increasing mRNA cap methylation of genes involved in Wnt/β-catenin signaling. Elevated mRNA cap methylation of Wnt signaling transcripts in response to MYC leads to augmented translational capacity, elevated protein levels, and enhanced Wnt signaling activity. Mechanistic evidence indicates that MYC promotes recruitment of RNA methyltransferase (RNMT) to Wnt signaling gene promoters by enhancing phosphorylation of serine 5 on the RNA Polymerase II Carboxy-Terminal Domain, mediated in part through an interaction between the TIP60 acetyltransferase complex and TFIIH. Implications MYC enhances mRNA cap methylation above and beyond transcriptional induction. PMID:27899423

Controlling nuclear RNA levels.

PubMed

Schmid, Manfred; Jensen, Torben Heick

2018-05-10

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

Shiga Toxin 2 Subtypes of Enterohemorrhagic E. coli O157:H- E32511 Analyzed by RT-qPCR and Top-Down Proteomics Using MALDI-TOF-TOF-MS

NASA Astrophysics Data System (ADS)

Fagerquist, Clifton K.; Zaragoza, William J.

2015-05-01

We have measured the relative abundance of the B-subunits and mRNA transcripts of two Stx2 subtypes present in Shiga toxin-producing Escherichia coli (STEC) O157:H- strain E32511 using matrix-assisted laser desorption/ionization time-of-flight-time-of-flight tandem mass spectrometry (MALDI-TOF-TOF-MS/MS) with post source decay (PSD) and real time-quantitative polymerase chain reaction (RT-qPCR). Stx2a and Stx2c in STEC strain E32511 were quantified from the integrated peak area of their singly charged disulfide-intact B-subunit ions at m/z ~7819 and m/z ~7774, respectively. We found that the Stx2a subtype was 21-fold more abundant than the Stx2c subtype. The two amino acid substitutions (16D ↔ 16 N and 24D ↔ 24A) that distinguish Stx2a from Stx2c not only result in a mass difference of 45 Da between their respective B-subunits but also result in distinctly different fragmentation channels by MS/MS-PSD because both substitutions involve an aspartic acid (D) residue. Importantly, these two substitutions have also been linked to differences in subtype toxicity. We measured the relative abundances of mRNA transcripts using RT-qPCR and determined that the stx2a transcript is 13-fold more abundant than stx2c transcript. In silico secondary structure analysis of the full mRNA operons of stx2a and stx2c suggest that transcript structural differences may also contribute to a relative increase of Stx2a over Stx2c. In consequence, toxin expression may be under both transcriptional and post-transcriptional control.

Stress induction of Bm1 RNA in silkworm larvae: SINEs, an unusual class of stress genes

PubMed Central

Kimura, Richard H.; Choudary, Prabhakara V.; Stone, Koni K.; Schmid, Carl W.

2001-01-01

This study surveys the induction of RNA polymerase III (Pol III)–directed expression of short interspersed element (SINE) transcripts by various stresses in an animal model, silkworm larvae. Sublethal heat shock and exposure to several toxic compounds increase the level of Bm1 RNA, the silkworm SINE transcript, while also transiently increasing expression of a well-characterized stress-induced transcript, Hsp70 messenger RNA (mRNA). In certain cases, the Bm1 RNA response coincides with that of Hsp70 mRNA, but more often Bm1 RNA responds later in recovery. Baculovirus infection and exposure to certain toxic compounds increase Bm1 RNA but not Hsp70 mRNA, showing that SINE induction is not necessarily coupled to transcription of this particular heat shock gene. SINEs behave as an additional class of stress-inducible genes in living animals but are unusual as stress genes because of their high copy number, genomic dispersion, and Pol III–directed transcription. PMID:11599568

TGF-β Suppression of HBV RNA through AID-Dependent Recruitment of an RNA Exosome Complex

PubMed Central

Kitamura, Kouichi; Wang, Zhe; Chowdhury, Sajeda; Monjurul, Ahasan Md; Wakae, Kousho; Koura, Miki; Shimadu, Miyuki; Kinoshita, Kazuo; Muramatsu, Masamichi

2015-01-01

Transforming growth factor (TGF)-β inhibits hepatitis B virus (HBV) replication although the intracellular effectors involved are not determined. Here, we report that reduction of HBV transcripts by TGF-β is dependent on AID expression, which significantly decreases both HBV transcripts and viral DNA, resulting in inhibition of viral replication. Immunoprecipitation reveals that AID physically associates with viral P protein that binds to specific virus RNA sequence called epsilon. AID also binds to an RNA degradation complex (RNA exosome proteins), indicating that AID, RNA exosome, and P protein form an RNP complex. Suppression of HBV transcripts by TGF-β was abrogated by depletion of either AID or RNA exosome components, suggesting that AID and the RNA exosome involve in TGF-β mediated suppression of HBV RNA. Moreover, AID-mediated HBV reduction does not occur when P protein is disrupted or when viral transcription is inhibited. These results suggest that induced expression of AID by TGF-β causes recruitment of the RNA exosome to viral RNP complex and the RNA exosome degrades HBV RNA in a transcription-coupled manner. PMID:25836330

Low-level laser irradiation modulates brain-derived neurotrophic factor mRNA transcription through calcium-dependent activation of the ERK/CREB pathway.

PubMed

Yan, Xiaodong; Liu, Juanfang; Zhang, Zhengping; Li, Wenhao; Sun, Siguo; Zhao, Jian; Dong, Xin; Qian, Jixian; Sun, Honghui

2017-01-01

Low-level laser (LLL) irradiation has been reported to promote neuronal differentiation, but the mechanism remains unclear. Brain-derived neurotrophic factor (BDNF) has been confirmed to be one of the most important neurotrophic factors because it is critical for the differentiation and survival of neurons during development. Thus, this study aimed to investigate the effects of LLL irradiation on Bdnf messenger RNA (mRNA) transcription and the molecular pathway involved in LLL-induced Bdnf mRNA transcription in cultured dorsal root ganglion neurons (DRGNs) using Ca 2+ imaging, pharmacological detections, RNA interference, immunocytochemistry assay, Western blot, and qPCR analysis. We show here that LLL induced increases in the [Ca 2+ ] i level, Bdnf mRNA transcription, cAMP-response element-binding protein (CREB) phosphorylation, and extracellular signal-regulated kinase (ERK) phosphorylation, mediated by Ca 2+ release via inositol triphosphate receptor (IP3R)-sensitive calcium (Ca 2+ ) stores. Blockade of Ca 2+ increase suppressed Bdnf mRNA transcription, CREB phosphorylation, and ERK phosphorylation. Downregulation of phosphorylated (p)-CREB reduced Bdnf mRNA transcription triggered by LLL. Furthermore, blockade of ERK using PD98059 inhibitor reduced p-CREB and Bdnf mRNA transcription induced by LLL. Taken together, these findings establish the Ca 2+ -ERK-CREB cascade as a potential signaling pathway involved in LLL-induced Bdnf mRNA transcription. To our knowledge, this is the first report of the mechanisms of Ca 2+ -dependent Bdnf mRNA transcription triggered by LLL. These findings may help further explore the complex molecular signaling networks in LLL-triggered nerve regeneration in vivo and may also provide experimental evidence for the development of LLL for clinical applications.

Small interfering RNA-producing loci in the ancient parasitic eukaryote Trypanosoma brucei

PubMed Central

2012-01-01

Background At the core of the RNA interference (RNAi) pathway in Trypanosoma brucei is a single Argonaute protein, TbAGO1, with an established role in controlling retroposon and repeat transcripts. Recent evidence from higher eukaryotes suggests that a variety of genomic sequences with the potential to produce double-stranded RNA are sources for small interfering RNAs (siRNAs). Results To test whether such endogenous siRNAs are present in T. brucei and to probe the individual role of the two Dicer-like enzymes, we affinity purified TbAGO1 from wild-type procyclic trypanosomes, as well as from cells deficient in the cytoplasmic (TbDCL1) or nuclear (TbDCL2) Dicer, and subjected the bound RNAs to Illumina high-throughput sequencing. In wild-type cells the majority of reads originated from two classes of retroposons. We also considerably expanded the repertoire of trypanosome siRNAs to encompass a family of 147-bp satellite-like repeats, many of the regions where RNA polymerase II transcription converges, large inverted repeats and two pseudogenes. Production of these newly described siRNAs is strictly dependent on the nuclear DCL2. Notably, our data indicate that putative centromeric regions, excluding the CIR147 repeats, are not a significant source for endogenous siRNAs. Conclusions Our data suggest that endogenous RNAi targets may be as evolutionarily old as the mechanism itself. PMID:22925482

Direct Regulation of Mitochondrial RNA Synthesis by Thyroid Hormone

PubMed Central

Enríquez, José A.; Fernández-Silva, Patricio; Garrido-Pérez, Nuria; López-Pérez, Manuel J.; Pérez-Martos, Acisclo; Montoya, Julio

1999-01-01

We have analyzed the influence of in vivo treatment and in vitro addition of thyroid hormone on in organello mitochondrial DNA (mtDNA) transcription and, in parallel, on the in organello footprinting patterns at the mtDNA regions involved in the regulation of transcription. We found that thyroid hormone modulates mitochondrial RNA levels and the mRNA/rRNA ratio by influencing the transcriptional rate. In addition, we found conspicuous differences between the mtDNA dimethyl sulfate footprinting patterns of mitochondria derived from euthyroid and hypothyroid rats at the transcription initiation sites but not at the mitochondrial transcription termination factor (mTERF) binding region. Furthermore, direct addition of thyroid hormone to the incubation medium of mitochondria isolated from hypothyroid rats restored the mRNA/rRNA ratio found in euthyroid rats as well as the mtDNA footprinting patterns at the transcription initiation area. Therefore, we conclude that the regulatory effect of thyroid hormone on mitochondrial transcription is partially exerted by a direct influence of the hormone on the mitochondrial transcription machinery. Particularly, the influence on the mRNA/rRNA ratio is achieved by selective modulation of the alternative H-strand transcription initiation sites and does not require the previous activation of nuclear genes. These results provide the first functional demonstration that regulatory signals, such as thyroid hormone, that modify the expression of nuclear genes can also act as primary signals for the transcriptional apparatus of mitochondria. PMID:9858589

A meiotic gene regulatory cascade driven by alternative fates for newly synthesized transcripts

PubMed Central

Cremona, Nicole; Potter, Kristine; Wise, Jo Ann

2011-01-01

To determine the relative importance of transcriptional regulation versus RNA processing and turnover during the transition from proliferation to meiotic differentiation in the fission yeast Schizosaccharomyces pombe, we analyzed temporal profiles and effects of RNA surveillance factor mutants on expression of 32 meiotic genes. A comparison of nascent transcription with steady-state RNA accumulation reveals that the vast majority of these genes show a lag between maximal RNA synthesis and peak RNA accumulation. During meiosis, total RNA levels parallel 3′ processing, which occurs in multiple, temporally distinct waves that peak from 3 to 6 h after meiotic induction. Most early genes and one middle gene, mei4, share a regulatory mechanism in which a specialized RNA surveillance factor targets newly synthesized transcripts for destruction. Mei4p, a member of the forkhead transcription factor family, in turn regulates a host of downstream genes. Remarkably, a spike in transcription is observed for less than one-third of the genes surveyed, and even these show evidence of RNA-level regulation. In aggregate, our findings lead us to propose that a regulatory cascade driven by changes in processing and stability of newly synthesized transcripts operates alongside the well-known transcriptional cascade as fission yeast cells enter meiosis. PMID:21148298

Mechanical Properties of Transcription

NASA Astrophysics Data System (ADS)

Sevier, Stuart A.; Levine, Herbert

2017-06-01

The mechanical properties of transcription have recently been shown to play a central role in gene expression. However, a full physical characterization of this central biological process is lacking. In this Letter, we introduce a simple description of the basic physical elements of transcription where RNA elongation, RNA polymerase rotation, and DNA supercoiling are coupled. The resulting framework describes the relative amount of RNA polymerase rotation and DNA supercoiling that occurs during RNA elongation. Asymptotic behavior is derived and can be used to experimentally extract unknown mechanical parameters of transcription. Mechanical limits to transcription are incorporated through the addition of a DNA supercoiling-dependent RNA polymerase velocity. This addition can lead to transcriptional stalling and resulting implications for gene expression, chromatin structure and genome organization are discussed.

Maf1 Protein, Repressor of RNA Polymerase III, Indirectly Affects tRNA Processing*

PubMed Central

Karkusiewicz, Iwona; Turowski, Tomasz W.; Graczyk, Damian; Towpik, Joanna; Dhungel, Nripesh; Hopper, Anita K.; Boguta, Magdalena

2011-01-01

Maf1 is negative regulator of RNA polymerase III in yeast. We observed high levels of both primary transcript and end-matured, intron-containing pre-tRNAs in the maf1Δ strain. This pre-tRNA accumulation could be overcome by transcription inhibition, arguing against a direct role of Maf1 in tRNA maturation and suggesting saturation of processing machinery by the increased amounts of primary transcripts. Saturation of the tRNA exportin, Los1, is one reason why end-matured intron-containing pre-tRNAs accumulate in maf1Δ cells. However, it is likely possible that other components of the processing pathway are also limiting when tRNA transcription is increased. According to our model, Maf1-mediated transcription control and nuclear export by Los1 are two major stages of tRNA biosynthesis that are regulated by environmental conditions in a coordinated manner. PMID:21940626

Maf1 protein, repressor of RNA polymerase III, indirectly affects tRNA processing.

PubMed

Karkusiewicz, Iwona; Turowski, Tomasz W; Graczyk, Damian; Towpik, Joanna; Dhungel, Nripesh; Hopper, Anita K; Boguta, Magdalena

2011-11-11

Maf1 is negative regulator of RNA polymerase III in yeast. We observed high levels of both primary transcript and end-matured, intron-containing pre-tRNAs in the maf1Δ strain. This pre-tRNA accumulation could be overcome by transcription inhibition, arguing against a direct role of Maf1 in tRNA maturation and suggesting saturation of processing machinery by the increased amounts of primary transcripts. Saturation of the tRNA exportin, Los1, is one reason why end-matured intron-containing pre-tRNAs accumulate in maf1Δ cells. However, it is likely possible that other components of the processing pathway are also limiting when tRNA transcription is increased. According to our model, Maf1-mediated transcription control and nuclear export by Los1 are two major stages of tRNA biosynthesis that are regulated by environmental conditions in a coordinated manner.

Expression of different functional isoforms in haematopoiesis.

PubMed

Grech, Godfrey; Pollacco, Joel; Portelli, Mark; Sacco, Keith; Baldacchino, Shawn; Grixti, Justine; Saliba, Christian

2014-01-01

Haematopoiesis is a complex process regulated at various levels facilitating rapid responses to external factors including stress, modulation of lineage commitment and terminal differentiation of progenitors. Although the transcription program determines the RNA pool of a cell, various mRNA strands can be obtained from the same template, giving rise to multiple protein isoforms. The majority of variants and isoforms co-occur in normal haematopoietic cells or are differentially expressed at various maturity stages of progenitor maturation and cellular differentiation within the same lineage or across lineages. Genetic aberrations or specific cellular states result in the predominant expression of abnormal isoforms leading to deregulation and disease. The presence of upstream open reading frames (uORF) in 5' untranslated regions (UTRs) of a transcript, couples the utilization of start codons with the cellular status and availability of translation initiation factors (eIFs). In addition, tissue-specific and cell lineage-specific alternative promoter use, regulates several transcription factors producing transcript variants with variable 5' exons. In this review, we propose to give a detailed account of the differential isoform formation, causing haematological malignancies.

A single miR390 targeting event is sufficient for triggering TAS3-tasiRNA biogenesis in Arabidopsis

PubMed Central

Marchais, Antonin; Sarazin, Alexis; Oberlin, Stefan; Voinnet, Olivier

2017-01-01

Abstract In plants, tasiRNAs form a class of endogenous secondary siRNAs produced through the action of RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6) upon microRNA-mediated cleavage of non-coding TAS RNAs. In Arabidopsis thaliana, TAS1, TAS2 and TAS4 tasiRNA production proceeds via a single cleavage event mediated by 22nt-long or/and asymmetric miRNAs in an ARGONAUTE-1 (AGO1)-dependent manner. By contrast, tasiRNA production from TAS3 seems to follow the so-called ‘two-hit’ process, where dual targeting of TAS3, specifically mediated by the 21nt-long, symmetric miR390, initiates AGO7-dependent tasiRNA production. Interestingly, features for TAS3 tasiRNA production differ in other plant species and we show here that such features also enable TAS3 tasiRNA biogenesis in Arabidopsis, and that a single miR390 targeting event is, in fact, sufficient for this process, suggesting that the ‘one-hit’ model underpins all the necessary rudiments of secondary siRNA biogenesis from plant TAS transcripts. Further results suggest that the two-hit configuration likely enhances the fidelity of tasiRNA production and, hence, the accuracy of downstream gene regulation. Finally, we show that a ‘non-cleavable one-hit’ process allows tasiRNA production from both TAS1 and TAS3 transcripts, indicating that RDR6 recruitment does not require miRNA cleavage, nor does the recruitment, as we further show, of SUPRRESSOR-OF-GENE-SILENCING-3, indispensable for tasiRNA generation. PMID:28334969

Genome-Wide Spectra of Transcription Insertions and Deletions Reveal That Slippage Depends on RNA:DNA Hybrid Complementarity.

PubMed

Traverse, Charles C; Ochman, Howard

2017-08-29

Advances in sequencing technologies have enabled direct quantification of genome-wide errors that occur during RNA transcription. These errors occur at rates that are orders of magnitude higher than rates during DNA replication, but due to technical difficulties such measurements have been limited to single-base substitutions and have not yet quantified the scope of transcription insertions and deletions. Previous reporter gene assay findings suggested that transcription indels are produced exclusively by elongation complex slippage at homopolymeric runs, so we enumerated indels across the protein-coding transcriptomes of Escherichia coli and Buchnera aphidicola , which differ widely in their genomic base compositions and incidence of repeat regions. As anticipated from prior assays, transcription insertions prevailed in homopolymeric runs of A and T; however, transcription deletions arose in much more complex sequences and were rarely associated with homopolymeric runs. By reconstructing the relocated positions of the elongation complex as inferred from the sequences inserted or deleted during transcription, we show that continuation of transcription after slippage hinges on the degree of nucleotide complementarity within the RNA:DNA hybrid at the new DNA template location. IMPORTANCE The high level of mistakes generated during transcription can result in the accumulation of malfunctioning and misfolded proteins which can alter global gene regulation and in the expenditure of energy to degrade these nonfunctional proteins. The transcriptome-wide occurrence of base substitutions has been elucidated in bacteria, but information on transcription insertions and deletions-errors that potentially have more dire effects on protein function-is limited to reporter gene constructs. Here, we capture the transcriptome-wide spectrum of insertions and deletions in Escherichia coli and Buchnera aphidicola and show that they occur at rates approaching those of base substitutions. Knowledge of the full extent of sequences subject to transcription indels supports a new model of bacterial transcription slippage, one that relies on the number of complementary bases between the transcript and the DNA template to which it slipped. Copyright © 2017 Traverse and Ochman.

piRNA-guided slicing of transposon transcripts enforces their transcriptional silencing via specifying the nuclear piRNA repertoire.

PubMed

Senti, Kirsten-André; Jurczak, Daniel; Sachidanandam, Ravi; Brennecke, Julius

2015-08-15

PIWI clade Argonaute proteins silence transposon expression in animal gonads. Their target specificity is defined by bound ∼23- to 30-nucleotide (nt) PIWI-interacting RNAs (piRNAs) that are processed from single-stranded precursor transcripts via two distinct pathways. Primary piRNAs are defined by the endonuclease Zucchini, while biogenesis of secondary piRNAs depends on piRNA-guided transcript cleavage and results in piRNA amplification. Here, we analyze the interdependencies between these piRNA biogenesis pathways in developing Drosophila ovaries. We show that secondary piRNA-guided target slicing is the predominant mechanism that specifies transcripts—including those from piRNA clusters—as primary piRNA precursors and defines the spectrum of Piwi-bound piRNAs in germline cells. Post-transcriptional silencing in the cytoplasm therefore enforces nuclear transcriptional target silencing, which ensures the tight suppression of transposons during oogenesis. As target slicing also defines the nuclear piRNA pool during mouse spermatogenesis, our findings uncover an unexpected conceptual similarity between the mouse and fly piRNA pathways. © 2015 Senti et al.; Published by Cold Spring Harbor Laboratory Press.

The cellular growth rate controls overall mRNA turnover, and modulates either transcription or degradation rates of particular gene regulons.

PubMed

García-Martínez, José; Delgado-Ramos, Lidia; Ayala, Guillermo; Pelechano, Vicent; Medina, Daniel A; Carrasco, Fany; González, Ramón; Andrés-León, Eduardo; Steinmetz, Lars; Warringer, Jonas; Chávez, Sebastián; Pérez-Ortín, José E

2016-05-05

We analyzed 80 different genomic experiments, and found a positive correlation between both RNA polymerase II transcription and mRNA degradation with growth rates in yeast. Thus, in spite of the marked variation in mRNA turnover, the total mRNA concentration remained approximately constant. Some genes, however, regulated their mRNA concentration by uncoupling mRNA stability from the transcription rate. Ribosome-related genes modulated their transcription rates to increase mRNA levels under fast growth. In contrast, mitochondria-related and stress-induced genes lowered mRNA levels by reducing mRNA stability or the transcription rate, respectively. We also detected these regulations within the heterogeneity of a wild-type cell population growing in optimal conditions. The transcriptomic analysis of sorted microcolonies confirmed that the growth rate dictates alternative expression programs by modulating transcription and mRNA decay.The regulation of overall mRNA turnover keeps a constant ratio between mRNA decay and the dilution of [mRNA] caused by cellular growth. This regulation minimizes the indiscriminate transmission of mRNAs from mother to daughter cells, and favors the response capacity of the latter to physiological signals and environmental changes. We also conclude that, by uncoupling mRNA synthesis from decay, cells control the mRNA abundance of those gene regulons that characterize fast and slow growth. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The effects of potato virus Y-derived virus small interfering RNAs of three biologically distinct strains on potato (Solanum tuberosum) transcriptome.

PubMed

Moyo, Lindani; Ramesh, Shunmugiah V; Kappagantu, Madhu; Mitter, Neena; Sathuvalli, Vidyasagar; Pappu, Hanu R

2017-07-17

Potato virus Y (PVY) is one of the most economically important pathogen of potato that is present as biologically distinct strains. The virus-derived small interfering RNAs (vsiRNAs) from potato cv. Russet Burbank individually infected with PVY-N, PVY-NTN and PVY-O strains were recently characterized. Plant defense RNA-silencing mechanisms deployed against viruses produce vsiRNAs to degrade homologous viral transcripts. Based on sequence complementarity, the vsiRNAs can potentially degrade host RNA transcripts raising the prospect of vsiRNAs as pathogenicity determinants in virus-host interactions. This study investigated the global effects of PVY vsiRNAs on the host potato transcriptome. The strain-specific vsiRNAs of PVY, expressed in high copy number, were analyzed in silico for their proclivity to target potato coding and non-coding RNAs using psRobot and psRNATarget algorithms. Functional annotation of target coding transcripts was carried out to predict physiological effects of the vsiRNAs on the potato cv. Russet Burbank. The downregulation of selected target coding transcripts was further validated using qRT-PCR. The vsiRNAs derived from biologically distinct strains of PVY displayed diversity in terms of absolute number, copy number and hotspots for siRNAs on their respective genomes. The vsiRNAs populations were derived with a high frequency from 6 K1, P1 and Hc-Pro for PVY-N, P1, Hc-Pro and P3 for PVY-NTN, and P1, 3' UTR and NIa for PVY-O genomic regions. The number of vsiRNAs that displayed interaction with potato coding transcripts and number of putative coding target transcripts were comparable between PVY-N and PVY-O, and were relatively higher for PVY-NTN. The most abundant target non-coding RNA transcripts for the strain specific PVY-derived vsiRNAs were found to be MIR821, 28S rRNA,18S rRNA, snoR71, tRNA-Met and U5. Functional annotation and qRT-PCR validation suggested that the vsiRNAs target genes involved in plant hormone signaling, genetic information processing, plant-pathogen interactions, plant defense and stress response processes in potato. The findings suggested that the PVY-derived vsiRNAs could act as a pathogenicity determinant and as a counter-defense strategy to host RNA silencing in PVY-potato interactions. The broad range of host genes targeted by PVY vsiRNAs in infected potato suggests a diverse role for vsiRNAs that includes suppression of host stress responses and developmental processes. The interactome scenario is the first report on the interaction between one of the most important Potyvirus genome-derived siRNAs and the potato transcripts.

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

PubMed

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

2003-10-03

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

Interdependence between transcription and mRNP processing and export, and its impact on genetic stability.

PubMed

Luna, Rosa; Jimeno, Sonia; Marín, Mercedes; Huertas, Pablo; García-Rubio, María; Aguilera, Andrés

2005-06-10

The conserved eukaryotic THO-TREX complex acts at the interface between transcription and mRNA export and affects transcription-associated recombination. To investigate the interdependence of nuclear mRNA processes and their impact on genomic integrity, we analyzed transcript accumulation and recombination of 40 selected mutants covering representative steps of the biogenesis and export of the messenger ribonucleoprotein particle (mRNP). None of the mutants analyzed shared the strong transcript-accumulation defect and hyperrecombination of THO mutants. Nevertheless, mutants in 3' end cleavage/polyadenylation, nuclear exosome, and mRNA export showed a weak but significant effect on recombination and transcript accumulation. Mutants of the nuclear exosome (rrp6) and 3' end processing factors (rna14 and rna15) showed inefficient transcription elongation and genetic interactions with THO. The results suggest a tight interdependence among mRNP biogenesis steps and transcription and an unexpected effect of the nuclear exosome and the cleavage/polyadenylation factors on transcription elongation and genetic integrity.

A Molecular Titration System Coordinates Ribosomal Protein Gene Transcription with Ribosomal RNA Synthesis.

PubMed

Albert, Benjamin; Knight, Britta; Merwin, Jason; Martin, Victoria; Ottoz, Diana; Gloor, Yvonne; Bruzzone, Maria Jessica; Rudner, Adam; Shore, David

2016-11-17

Cell growth potential is determined by the rate of ribosome biogenesis, a complex process that requires massive and coordinated transcriptional output. In the yeast Saccharomyces cerevisiae, ribosome biogenesis is highly regulated at the transcriptional level. Although evidence for a system that coordinates ribosomal RNA (rRNA) and ribosomal protein gene (RPG) transcription has been described, the molecular mechanisms remain poorly understood. Here we show that an interaction between the RPG transcriptional activator Ifh1 and the rRNA processing factor Utp22 serves to coordinate RPG transcription with that of rRNA. We demonstrate that Ifh1 is rapidly released from RPG promoters by a Utp22-independent mechanism following growth inhibition, but that its long-term dissociation requires Utp22. We present evidence that RNA polymerase I activity inhibits the ability of Utp22 to titrate Ifh1 from RPG promoters and propose that a dynamic Ifh1-Utp22 interaction fine-tunes RPG expression to coordinate RPG and rRNA transcription. Copyright © 2016 Elsevier Inc. All rights reserved.

DNA replication initiator Cdc6 also regulates ribosomal DNA transcription initiation.

PubMed

Huang, Shijiao; Xu, Xiaowei; Wang, Guopeng; Lu, Guoliang; Xie, Wenbing; Tao, Wei; Zhang, Hongyin; Jiang, Qing; Zhang, Chuanmao

2016-04-01

RNA-polymerase-I-dependent ribosomal DNA (rDNA) transcription is fundamental to rRNA processing, ribosome assembly and protein synthesis. However, how this process is initiated during the cell cycle is not fully understood. By performing a proteomic analysis of transcription factors that bind RNA polymerase I during rDNA transcription initiation, we identified that the DNA replication initiator Cdc6 interacts with RNA polymerase I and its co-factors, and promotes rDNA transcription in G1 phase in an ATPase-activity-dependent manner. We further showed that Cdc6 is targeted to the nucleolus during late mitosis and G1 phase in a manner that is dependent on B23 (also known as nucleophosmin, NPM1), and preferentially binds to the rDNA promoter through its ATP-binding domain. Overexpression of Cdc6 increases rDNA transcription, whereas knockdown of Cdc6 results in a decreased association of both RNA polymerase I and the RNA polymerase I transcription factor RRN3 with rDNA, and a reduction of rDNA transcription. Furthermore, depletion of Cdc6 impairs the interaction between RRN3 and RNA polymerase I. Taken together, our data demonstrate that Cdc6 also serves as a regulator of rDNA transcription initiation, and indicate a mechanism by which initiation of rDNA transcription and DNA replication can be coordinated in cells. © 2016. Published by The Company of Biologists Ltd.

Promoter for Sindbis virus RNA-dependent subgenomic RNA transcription.

PubMed Central

Levis, R; Schlesinger, S; Huang, H V

1990-01-01

Sindbis virus is a positive-strand RNA enveloped virus, a member of the Alphavirus genus of the Togaviridae family. Two species of mRNA are synthesized in cells infected with Sindbis virus; one, the 49S RNA, is the genomic RNA; the other, the 26S RNA, is a subgenomic RNA that is identical in sequence to the 3' one-third of the genomic RNA. Ou et al. (J.-H. Ou, C. M. Rice, L. Dalgarno, E. G. Strauss, and J. H. Strauss, Proc. Natl. Acad. Sci. USA 79:5235-5239, 1982) identified a highly conserved region 19 nucleotides upstream and 2 nucleotides downstream from the start of the 26S RNA and proposed that in the negative-strand template, these nucleotides compose the promoter for directing the synthesis of the subgenomic RNA. Defective interfering (DI) RNAs of Sindbis virus were used to test this proposal. A 227-nucleotide sequence encompassing 98 nucleotides upstream and 117 nucleotides downstream from the start site of the Sindbis virus subgenomic RNA was inserted into a DI genome. The DI RNA containing the insert was replicated and packaged in the presence of helper virus, and cells infected with these DI particles produced a subgenomic RNA of the size and sequence expected if the promoter was functional. The initiating nucleotide was identical to that used for Sindbis virus subgenomic mRNA synthesis. Deletion analysis showed that the minimal region required to detect transcription of a subgenomic RNA from the negative-strand template of a DI RNA was 18 or 19 nucleotides upstream and 5 nucleotides downstream from the start of the subgenomic RNA. Images PMID:2319651

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

PubMed Central

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

2011-01-01

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

Genome-wide mapping of infection-induced SINE RNAs reveals a role in selective mRNA export.

PubMed

Karijolich, John; Zhao, Yang; Alla, Ravi; Glaunsinger, Britt

2017-06-02

Short interspersed nuclear elements (SINEs) are retrotransposons evolutionarily derived from endogenous RNA Polymerase III RNAs. Though SINE elements have undergone exaptation into gene regulatory elements, how transcribed SINE RNA impacts transcriptional and post-transcriptional regulation is largely unknown. This is partly due to a lack of information regarding which of the loci have transcriptional potential. Here, we present an approach (short interspersed nuclear element sequencing, SINE-seq), which selectively profiles RNA Polymerase III-derived SINE RNA, thereby identifying transcriptionally active SINE loci. Applying SINE-seq to monitor murine B2 SINE expression during a gammaherpesvirus infection revealed transcription from 28 270 SINE loci, with ∼50% of active SINE elements residing within annotated RNA Polymerase II loci. Furthermore, B2 RNA can form intermolecular RNA-RNA interactions with complementary mRNAs, leading to nuclear retention of the targeted mRNA via a mechanism involving p54nrb. These findings illuminate a pathway for the selective regulation of mRNA export during stress via retrotransposon activation. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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

PubMed

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.

The maize stripe virus major noncapsid protein messenger RNA transcripts contain heterogeneous leader sequences at their 5' termini.

PubMed

Huiet, L; Feldstein, P A; Tsai, J H; Falk, B W

1993-12-01

Primer extension analyses and a PCR-based cloning strategy were used to identify and characterize 5' nucleotide sequences on the maize stripe virus (MStV) RNA4 mRNA transcripts encoding the major noncapsid protein (NCP). Direct RNA sequence analysis by primer extension showed that the NCP mRNA transcripts had 10-15 nucleotides beyond the 5' terminus of the MStV RNA4 nucleotide sequence. MStV genomic RNAs isolated from ribonucleoprotein particles (RNPs) lacked the additional 5' nucleotides. cDNA clones representing the 5' region of the mRNA transcripts were constructed, and the nucleotide sequences of the 5' regions were determined for 16 clones. Each was found to have a distinct 10-15 nucleotide sequence immediately 5' of the MStV RNA4 sequence. Eleven of 16 clones had the correct MStV RNA4 5' nucleotide sequence, while five showed minor variations at or near the 5' most MStV RNA4 nucleotide. These characteristics show strong similarities to other viral mRNA transcripts which are synthesized by cap snatching.

[In vitro generation of insulin-producing cells from the neonatal rat bone marrow mesenchymal stem cells].

PubMed

Li, Xiaohu; Huang, Haiyan; Liu, Xirong; Xia, Hongxia; Li, Mincai

2015-03-01

To observe the differentiation of the neonatal rat bone marrow mesenchymal stem cells (MSCs) into insulin-producing cells and detect the expressions of insulin, pancreatic duodenal homebox-1 (PDX-1) and nestin. MSCs were isolated from the neonatal rats and cultured in the modified medium composed of 10 μg/L human epidermal growth factor (EGF), 10 μg/L basic fibroblast growth factor (bFGF), 10 μg/L hepatocyte growth factor (HGF), 10 μg/L human B cell regulin, 20 mmol/L nicotinamide and 20 g/L B27. After the induction, the mRNA expressions of insulin, PDX-1 and nestin were examined by reverse transcription-PCR, and the insulin, PDX-1 and nestin protein levels were detected by immunocytochemistry. The insulin and PDX-1 mRNA expressions increased and the nestin mRNA expression decreased in the differentiation of the neonatal rat MSCs into insulin-producing cells. The nestin, PDX-1 and insulin proteins were co-expressed in insulin-producing cells. MSCs can be induced to differentiate into insulin-producing cells.

Protection of germline gene expression by the C. elegans Argonaute CSR-1.

PubMed

Wedeles, Christopher J; Wu, Monica Z; Claycomb, Julie M

2013-12-23

In Caenorhabditis elegans, the Piwi-interacting small RNA (piRNA)-mediated germline surveillance system encodes more than 30,000 unique 21-nucleotide piRNAs, which silence a variety of foreign nucleic acids. What mechanisms allow endogenous germline-expressed transcripts to evade silencing by the piRNA pathway? One likely candidate in a protective mechanism is the Argonaute CSR-1, which interacts with 22G-small RNAs that are antisense to nearly all germline-expressed genes. Here, we use an in vivo RNA tethering assay to demonstrate that the recruitment of CSR-1 to a transcript licenses expression of the transcript, protecting it from piRNA-mediated silencing. Licensing occurs mainly at the level of transcription, as we observe changes in pre-mRNA levels consistent with transcriptional activation when CSR-1 is tethered. Furthermore, the recruitment of CSR-1 to a previously silenced locus transcriptionally activates its expression. Together, these results demonstrate a rare positive role for an endogenous Argonaute pathway in heritably licensing and protecting germline transcripts.

Identification of MicroRNA Targets of Capsicum spp. Using MiRTrans—a Trans-Omics Approach

PubMed Central

Zhang, Lu; Qin, Cheng; Mei, Junpu; Chen, Xiaocui; Wu, Zhiming; Luo, Xirong; Cheng, Jiaowen; Tang, Xiangqun; Hu, Kailin; Li, Shuai C.

2017-01-01

The microRNA (miRNA) can regulate the transcripts that are involved in eukaryotic cell proliferation, differentiation, and metabolism. Especially for plants, our understanding of miRNA targets, is still limited. Early attempts of prediction on sequence alignments have been plagued by enormous false positives. It is helpful to improve target prediction specificity by incorporating the other data sources such as the dependency between miRNA and transcript expression or even cleaved transcripts by miRNA regulations, which are referred to as trans-omics data. In this paper, we developed MiRTrans (Prediction of MiRNA targets by Trans-omics data) to explore miRNA targets by incorporating miRNA sequencing, transcriptome sequencing, and degradome sequencing. MiRTrans consisted of three major steps. First, the target transcripts of miRNAs were predicted by scrutinizing their sequence characteristics and collected as an initial potential targets pool. Second, false positive targets were eliminated if the expression of miRNA and its targets were weakly correlated by lasso regression. Third, degradome sequencing was utilized to capture the miRNA targets by examining the cleaved transcripts that regulated by miRNAs. Finally, the predicted targets from the second and third step were combined by Fisher's combination test. MiRTrans was applied to identify the miRNA targets for Capsicum spp. (i.e., pepper). It can generate more functional miRNA targets than sequence-based predictions by evaluating functional enrichment. MiRTrans identified 58 miRNA-transcript pairs with high confidence from 18 miRNA families conserved in eudicots. Most of these targets were transcription factors; this lent support to the role of miRNA as key regulator in pepper. To our best knowledge, this work is the first attempt to investigate the miRNA targets of pepper, as well as their regulatory networks. Surprisingly, only a small proportion of miRNA-transcript pairs were shared between degradome sequencing and expression dependency predictions, suggesting that miRNA targets predicted by a single technology alone may be prone to report false negatives. PMID:28443105

Coupled transcription and processing of mouse ribosomal RNA in a cell-free system.

PubMed Central

Mishima, Y; Mitsuma, T; Ogata, K

1985-01-01

An in vitro processing system of mouse rRNA was achieved using an RNA polymerase I-specific transcription system, (S100) and recombinant plasmids consisting of mouse rRNA gene (rDNA) segments containing the transcription initiation and 5'-terminal region of 18S (or 41S) rRNA. Pulse-chase experiments showed that a specific processing occurred with transcripts of the plasmid DNAs when the direction of transcription was the correct orientation relative to the 18S rRNA coding sequence, but not with transcripts of the DNA templates in which this coding sequence was in the opposite orientation. From the S1 nuclease protection analyses, we concluded that there are several steps of endonucleolytic cleavage including one 105 nucleotides upstream from the 5' end of 18S rRNA. Intermediates cleaved at this site were identified in in vivo processing of rRNA. This result indicates that endonucleolytic cleavage takes place 105 nucleotides upstream from the 5' terminus of 18S rRNA prior to the formation of mature 18S rRNA. Trimming or cleavage of the 105 nucleotides may be involved in the formation of the 5' terminus of mature 18S rRNA. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:3004977

Grape RNA-Seq analysis pipeline environment

PubMed Central

Knowles, David G.; Röder, Maik; Merkel, Angelika; Guigó, Roderic

2013-01-01

Motivation: The avalanche of data arriving since the development of NGS technologies have prompted the need for developing fast, accurate and easily automated bioinformatic tools capable of dealing with massive datasets. Among the most productive applications of NGS technologies is the sequencing of cellular RNA, known as RNA-Seq. Although RNA-Seq provides similar or superior dynamic range than microarrays at similar or lower cost, the lack of standard and user-friendly pipelines is a bottleneck preventing RNA-Seq from becoming the standard for transcriptome analysis. Results: In this work we present a pipeline for processing and analyzing RNA-Seq data, that we have named Grape (Grape RNA-Seq Analysis Pipeline Environment). Grape supports raw sequencing reads produced by a variety of technologies, either in FASTA or FASTQ format, or as prealigned reads in SAM/BAM format. A minimal Grape configuration consists of the file location of the raw sequencing reads, the genome of the species and the corresponding gene and transcript annotation. Grape first runs a set of quality control steps, and then aligns the reads to the genome, a step that is omitted for prealigned read formats. Grape next estimates gene and transcript expression levels, calculates exon inclusion levels and identifies novel transcripts. Grape can be run on a single computer or in parallel on a computer cluster. It is distributed with specific mapping and quantification tools, but given its modular design, any tool supporting popular data interchange formats can be integrated. Availability: Grape can be obtained from the Bioinformatics and Genomics website at: http://big.crg.cat/services/grape. Contact: david.gonzalez@crg.eu or roderic.guigo@crg.eu PMID:23329413

Crystal Structures of the E. coli Transcription Initiation Complexes with a Complete Bubble

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

Zuo, Yuhong; Steitz, Thomas A.

2015-05-01

During transcription initiation, RNA polymerase binds to promoter DNA to form an initiation complex containing a DNA bubble and enters into abortive cycles of RNA synthesis before escaping the promoter to transit into the elongation phase for processive RNA synthesis. Here we present the crystal structures of E. coli transcription initiation complexes containing a complete transcription bubble and de novo synthesized RNA oligonucleotides at about 6-Å resolution. The structures show how RNA polymerase recognizes DNA promoters that contain spacers of different lengths and reveal a bridging interaction between the 5'-triphosphate of the nascent RNA and the σ factor that maymore » function to stabilize the short RNA-DNA hybrids during the early stage of transcription initiation. The conformation of the RNA oligonucleotides and the paths of the DNA strands in the complete initiation complexes provide insights into the mechanism that controls both the abortive and productive RNA synthesis.« less

Influence of 5'-flanking sequence on 4.5SI RNA gene transcription by RNA polymerase III.

PubMed

Gogolevskaya, Irina K; Stasenko, Danil V; Tatosyan, Karina A; Kramerov, Dmitri A

2018-05-01

Short nuclear 4.5SI RNA can be found in three related rodent families. Its function remains unknown. The genes of 4.5SI RNA contain an internal promoter of RNA polymerase III composed of the boxes A and B. Here, the effect of the sequence immediately upstream of the mouse 4.5SI RNA gene on its transcription was studied. The gene with deletions and substitutions in the 5'-flanking sequence was used to transfect HeLa cells and its transcriptional activity was evaluated from the cellular level of 4.5SI RNA. Single-nucleotide substitutions in the region adjacent to the transcription start site (positions -2 to -8) decreased the expression activity of the gene down to 40%-60% of the control. The substitution of the conserved pentanucleotide AGAAT (positions -14 to -18) could either decrease (43%-56%) or increase (134%) the gene expression. A TATA-like box (TACATGA) was found at positions -24 to -30 of the 4.5SI RNA gene. Its replacement with a polylinker fragment of the vector did not decrease the transcription level, while its replacement with a GC-rich sequence almost completely (down to 2%-5%) suppressed the transcription of the 4.5SI RNA gene. The effect of plasmid sequences bordering the gene on its transcription by RNA polymerase III is discussed.

Differential utilization of TATA box-binding protein (TBP) and TBP-related factor 1 (TRF1) at different classes of RNA polymerase III promoters.

PubMed

Verma, Neha; Hung, Ko-Hsuan; Kang, Jin Joo; Barakat, Nermeen H; Stumph, William E

2013-09-20

In the fruit fly Drosophila melanogaster, RNA polymerase III transcription was found to be dependent not upon the canonical TATA box-binding protein (TBP) but instead upon the TBP-related factor 1 (TRF1) (Takada, S., Lis, J. T., Zhou, S., and Tjian, R. (2000) Cell 101, 459-469). Here we confirm that transcription of fly tRNA genes requires TRF1. However, we unexpectedly find that U6 snRNA gene promoters are occupied primarily by TBP in cells and that knockdown of TBP, but not TRF1, inhibits U6 transcription in cells. Moreover, U6 transcription in vitro effectively utilizes TBP, whereas TBP cannot substitute for TRF1 to promote tRNA transcription in vitro. Thus, in fruit flies, different classes of RNA polymerase III promoters differentially utilize TBP and TRF1 for the initiation of transcription.

RNA-Seq-Based Transcript Structure Analysis with TrBorderExt.

PubMed

Wang, Yejun; Sun, Ming-An; White, Aaron P

2018-01-01

RNA-Seq has become a routine strategy for genome-wide gene expression comparisons in bacteria. Despite lower resolution in transcript border parsing compared with dRNA-Seq, TSS-EMOTE, Cappable-seq, Term-seq, and others, directional RNA-Seq still illustrates its advantages: low cost, quantification and transcript border analysis with a medium resolution (±10-20 nt). To facilitate mining of directional RNA-Seq datasets especially with respect to transcript structure analysis, we developed a tool, TrBorderExt, which can parse transcript start sites and termination sites accurately in bacteria. A detailed protocol is described in this chapter for how to use the software package step by step to identify bacterial transcript borders from raw RNA-Seq data. The package was developed with Perl and R programming languages, and is accessible freely through the website: http://www.szu-bioinf.org/TrBorderExt .

A simplified strategy for studying the etiology of viral diseases: Apple stem grooving virus as a case study.

PubMed

Dhir, Sunny; Walia, Yashika; Zaidi, A A; Hallan, Vipin

2015-03-01

A simple method to amplify infective, complete genomes of single stranded RNA viruses by long distance PCR (LD PCR) from woody plant tissues is described in detail. The present protocol eliminates partial purification of viral particles and the amplification is achieved in three steps: (i) easy preparation of template RNA by incorporating a pre processing step before loading onto the column (ii) reverse transcription by AMV or Superscript reverse transcriptase and (iii) amplification of cDNA by LD PCR using LA or Protoscript Taq DNA polymerase. Incorporation of a preprocessing step helped to isolate consistent quality RNA from recalcitrant woody tissues such as apple, which was critical for efficient amplification of the complete genomes of Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV). Complete genome of ASGV was cloned under T7 RNA polymerase promoter and was confirmed to be infectious through transcript inoculation producing symptoms similar to the wild type virus. This is the first report for the largest RNA virus genome amplified by PCR from total nucleic acid extracts of woody plant tissues. Copyright © 2014 Elsevier B.V. All rights reserved.

Use of armored RNA as a standard to construct a calibration curve for real-time RT-PCR.

PubMed

Donia, D; Divizia, M; Pana', A

2005-06-01

Armored Enterovirus RNA was used to standardize a real-time reverse transcription (RT)-PCR for environmental testing. Armored technology is a system to produce a robust and stable RNA standard, trapped into phage proteins, to be used as internal control. The Armored Enterovirus RNA protected sequence includes 263 bp of highly conserved sequences in 5' UTR region. During these tests, Armored RNA has been used to produce a calibration curve, comparing three different fluorogenic chemistry: TaqMan system, Syber Green I and Lux-primers. The effective evaluation of three amplifying commercial reagent kits, in use to carry out real-time RT-PCR, and several extraction procedures of protected viral RNA have been carried out. The highest Armored RNA recovery was obtained by heat treatment while chemical extraction may decrease the quantity of RNA. The best sensitivity and specificity was obtained using the Syber Green I technique since it is a reproducible test, easy to use and the cheapest one. TaqMan and Lux-primer assays provide good RT-PCR efficiency in relationship to the several extraction methods used, since labelled probe or primer request in these chemistry strategies, increases the cost of testing.

An empirical strategy to detect bacterial transcript structure from directional RNA-seq transcriptome data.

PubMed

Wang, Yejun; MacKenzie, Keith D; White, Aaron P

2015-05-07

As sequencing costs are being lowered continuously, RNA-seq has gradually been adopted as the first choice for comparative transcriptome studies with bacteria. Unlike microarrays, RNA-seq can directly detect cDNA derived from mRNA transcripts at a single nucleotide resolution. Not only does this allow researchers to determine the absolute expression level of genes, but it also conveys information about transcript structure. Few automatic software tools have yet been established to investigate large-scale RNA-seq data for bacterial transcript structure analysis. In this study, 54 directional RNA-seq libraries from Salmonella serovar Typhimurium (S. Typhimurium) 14028s were examined for potential relationships between read mapping patterns and transcript structure. We developed an empirical method, combined with statistical tests, to automatically detect key transcript features, including transcriptional start sites (TSSs), transcriptional termination sites (TTSs) and operon organization. Using our method, we obtained 2,764 TSSs and 1,467 TTSs for 1331 and 844 different genes, respectively. Identification of TSSs facilitated further discrimination of 215 putative sigma 38 regulons and 863 potential sigma 70 regulons. Combining the TSSs and TTSs with intergenic distance and co-expression information, we comprehensively annotated the operon organization in S. Typhimurium 14028s. Our results show that directional RNA-seq can be used to detect transcriptional borders at an acceptable resolution of ±10-20 nucleotides. Technical limitations of the RNA-seq procedure may prevent single nucleotide resolution. The automatic transcript border detection methods, statistical models and operon organization pipeline that we have described could be widely applied to RNA-seq studies in other bacteria. Furthermore, the TSSs, TTSs, operons, promoters and unstranslated regions that we have defined for S. Typhimurium 14028s may constitute valuable resources that can be used for comparative analyses with other Salmonella serotypes.

Transcriptional silencing of a transgene by RNAi in the soma of C. elegans.

PubMed

Grishok, Alla; Sinskey, Jina L; Sharp, Phillip A

2005-03-15

The silencing of transgene expression at the level of transcription in the soma of Caenorhabditis elegans through an RNAi-dependent pathway has not been previously characterized. Most gene silencing due to RNAi in C. elegans occurs at the post-transcriptional level. We observed transcriptional silencing when worms containing the elt-2::gfp/LacZ transgene were fed RNA produced from the commonly used L4440 vector. The transgene and the vector share plasmid backbone sequences. This transgene silencing depends on multiple RNAi pathway genes, including dcr-1, rde-1, rde-4, and rrf-1. Unlike post-transcriptional gene silencing in worms, elt-2::gfp/LacZ silencing is dependent on the PAZ-PIWI protein Alg-1 and on the HP1 homolog Hpl-2. The latter is a chromatin silencing factor, and expression of the transgene is inhibited at the level of intron-containing precursor mRNA. This inhibition is accompanied by a decrease in the acetylation of histones associated with the transgene. This transcriptional silencing in the soma can be distinguished from transgene silencing in the germline by its inability to be transmitted across generations and its dependence on the rde-1 gene. We therefore define this type of silencing as RNAi-induced Transcriptional Gene Silencing (RNAi-TGS). Additional chromatin-modifying components affecting RNAi-TGS were identified in a candidate RNAi screen.

Mitochondrial run-on transcription assay using biotin labeling.

PubMed

Kühn, Kristina

2015-01-01

RNA synthesis and different posttranscriptional processes shape the transcriptome of plant mitochondria. It is believed that mitochondrial transcription in plants is not stringently controlled, and that RNA degradation has a major impact on mitochondrial steady-state transcript levels. Nevertheless, the presence of two RNA polymerases with different gene specificities in mitochondria of dicotyledonous species indicates that transcriptional mechanisms may provide a means to control mitochondrial steady-state RNA pools and gene expression. To experimentally assess transcriptional activities in mitochondria, run-on transcription assays have been developed. These assays measure elongation rates for endogenous transcripts in freshly prepared mitochondrial extracts. The mitochondrial run-on transcription protocol described here has been optimized for the model plant Arabidopsis (Arabidopsis thaliana). It uses mitochondria prepared from soil-grown Arabidopsis plants and employs nonradioactive labeling for the subsequent detection of run-on transcripts.

mTORC1 Directly Phosphorylates and Regulates Human MAF1▿

PubMed Central

Michels, Annemieke A.; Robitaille, Aaron M.; Buczynski-Ruchonnet, Diane; Hodroj, Wassim; Reina, Jaime H.; Hall, Michael N.; Hernandez, Nouria

2010-01-01

mTORC1 is a central regulator of growth in response to nutrient availability, but few direct targets have been identified. RNA polymerase (pol) III produces a number of essential RNA molecules involved in protein synthesis, RNA maturation, and other processes. Its activity is highly regulated, and deregulation can lead to cell transformation. The human phosphoprotein MAF1 becomes dephosphorylated and represses pol III transcription after various stresses, but neither the significance of the phosphorylations nor the kinase involved is known. We find that human MAF1 is absolutely required for pol III repression in response to serum starvation or TORC1 inhibition by rapamycin or Torin1. The protein is phosphorylated mainly on residues S60, S68, and S75, and this inhibits its pol III repression function. The responsible kinase is mTORC1, which phosphorylates MAF1 directly. Our results describe molecular mechanisms by which mTORC1 controls human MAF1, a key repressor of RNA polymerase III transcription, and add a new branch to the signal transduction cascade immediately downstream of TORC1. PMID:20516213

mTORC1 directly phosphorylates and regulates human MAF1.

PubMed

Michels, Annemieke A; Robitaille, Aaron M; Buczynski-Ruchonnet, Diane; Hodroj, Wassim; Reina, Jaime H; Hall, Michael N; Hernandez, Nouria

2010-08-01

mTORC1 is a central regulator of growth in response to nutrient availability, but few direct targets have been identified. RNA polymerase (pol) III produces a number of essential RNA molecules involved in protein synthesis, RNA maturation, and other processes. Its activity is highly regulated, and deregulation can lead to cell transformation. The human phosphoprotein MAF1 becomes dephosphorylated and represses pol III transcription after various stresses, but neither the significance of the phosphorylations nor the kinase involved is known. We find that human MAF1 is absolutely required for pol III repression in response to serum starvation or TORC1 inhibition by rapamycin or Torin1. The protein is phosphorylated mainly on residues S60, S68, and S75, and this inhibits its pol III repression function. The responsible kinase is mTORC1, which phosphorylates MAF1 directly. Our results describe molecular mechanisms by which mTORC1 controls human MAF1, a key repressor of RNA polymerase III transcription, and add a new branch to the signal transduction cascade immediately downstream of TORC1.

Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression.

PubMed

Lim, Hyoun-Sub; Vaira, Anna Maria; Domier, Leslie L; Lee, Sung Chul; Kim, Hong Gi; Hammond, John

2010-06-20

We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein expression, based on Alternanthera mosaic virus (AltMV), for infection of a wide range of host plants including Nicotiana benthamiana and Arabidopsis thaliana by either mechanical inoculation of in vitro transcripts or via agroinfiltration. In vivo transcripts produced by co-agroinfiltration of bacteriophage T7 RNA polymerase resulted in T7-driven AltMV infection from a binary vector in the absence of the Cauliflower mosaic virus 35S promoter. An artificial bipartite viral vector delivery system was created by separating the AltMV RNA-dependent RNA polymerase and Triple Gene Block (TGB)123-Coat protein (CP) coding regions into two constructs each bearing the AltMV 5' and 3' non-coding regions, which recombined in planta to generate a full-length AltMV genome. Substitution of TGB1 L(88)P, and equivalent changes in other potexvirus TGB1 proteins, affected RNA silencing suppression efficacy and suitability of the vectors from protein expression to VIGS. Published by Elsevier Inc.

Rift valley fever virus nonstructural protein NSs promotes viral RNA replication and transcription in a minigenome system.

PubMed

Ikegami, Tetsuro; Peters, C J; Makino, Shinji

2005-05-01

Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, has a tripartite negative-strand genome (S, M, and L segments) and is an important mosquito-borne pathogen for domestic animals and humans. We established an RVFV T7 RNA polymerase-driven minigenome system in which T7 RNA polymerase from an expression plasmid drove expression of RNA transcripts for viral proteins and minigenome RNA transcripts carrying a reporter gene between both termini of the M RNA segment in 293T cells. Like other viruses of the Bunyaviridae family, replication and transcription of the RVFV minigenome required expression of viral N and L proteins. Unexpectedly, the coexpression of an RVFV nonstructural protein, NSs, with N and L proteins resulted in a significant enhancement of minigenome RNA replication. Coexpression of NSs protein with N and L proteins also enhanced minigenome mRNA transcription in the cells expressing viral-sense minigenome RNA transcripts. NSs protein expression increased the RNA replication of minigenomes that originated from S and L RNA segments. Enhancement of minigenome RNA synthesis by NSs protein occurred in cells lacking alpha/beta interferon (IFN-alpha/beta) genes, indicating that the effect of NSs protein on minigenome RNA replication was unrelated to a putative NSs protein-induced inhibition of IFN-alpha/beta production. Our finding that RVFV NSs protein augmented minigenome RNA synthesis was in sharp contrast to reports that Bunyamwera virus (genus Bunyavirus) NSs protein inhibits viral minigenome RNA synthesis, suggesting that RVFV NSs protein and Bunyamwera virus NSs protein have distinctly different biological roles in viral RNA synthesis.

Genome-wide mapping of infection-induced SINE RNAs reveals a role in selective mRNA export

PubMed Central

Zhao, Yang; Alla, Ravi

2017-01-01

Abstract Short interspersed nuclear elements (SINEs) are retrotransposons evolutionarily derived from endogenous RNA Polymerase III RNAs. Though SINE elements have undergone exaptation into gene regulatory elements, how transcribed SINE RNA impacts transcriptional and post-transcriptional regulation is largely unknown. This is partly due to a lack of information regarding which of the loci have transcriptional potential. Here, we present an approach (short interspersed nuclear element sequencing, SINE-seq), which selectively profiles RNA Polymerase III-derived SINE RNA, thereby identifying transcriptionally active SINE loci. Applying SINE-seq to monitor murine B2 SINE expression during a gammaherpesvirus infection revealed transcription from 28 270 SINE loci, with ∼50% of active SINE elements residing within annotated RNA Polymerase II loci. Furthermore, B2 RNA can form intermolecular RNA–RNA interactions with complementary mRNAs, leading to nuclear retention of the targeted mRNA via a mechanism involving p54nrb. These findings illuminate a pathway for the selective regulation of mRNA export during stress via retrotransposon activation. PMID:28334904

A complex structure in the mRNA of Tf1 is recognized and cleaved to generate the primer of reverse transcription.

PubMed

Lin, J H; Levin, H L

1997-01-15

All retroviruses and LTR-containing retrotransposons are thought to require specific tRNA molecules to serve as primers of reverse transcription. An exception is the LTR-containing retrotransposon Tf1, isolated from Schizosaccharomyces pombe. Instead of requiring a tRNA, the reverse transcriptase of Tf1 uses the first 11 bases of the Tf1 transcript as the primer for reverse transcription. The primer is generated by a cleavage that occurs between bases 11 and 12 of the Tf1 mRNA. Sequence analysis of the 5' untranslated region of the Tf1 mRNA resulted in the identification of a region with the potential to form an RNA structure of 89 bases that included the primer binding site and the first 11 bases of the Tf1 mRNA. Systematic mutagenesis of this region revealed 34 single-point mutants in the structure that resulted in reduced transposition activity. The defects in transposition correlated with reduced level of Tf1 reverse transcripts as determined by DNA blot analysis. Evidence that the RNA structure did form in vivo included the result that strains with second site mutations that restored complementarity resulted in increased levels of reverse transcripts and Tf1 transposition. The majority of the mutants defective for reverse transcription were unable to cleave the Tf1 mRNA between bases 11 and 12. These data indicate that formation of an extensive RNA structure was required for the cleavage reaction that generated the primer for Tf1 reverse transcription.

Multiple Transcript Properties Related to Translation Affect mRNA Degradation Rates in Saccharomyces cerevisiae

PubMed Central

Neymotin, Benjamin; Ettorre, Victoria; Gresham, David

2016-01-01

Degradation of mRNA contributes to variation in transcript abundance. Studies of individual mRNAs have shown that both cis and trans factors affect mRNA degradation rates. However, the factors underlying transcriptome-wide variation in mRNA degradation rates are poorly understood. We investigated the contribution of different transcript properties to transcriptome-wide degradation rate variation in the budding yeast, Saccharomyces cerevisiae, using multiple regression analysis. We find that multiple transcript properties are significantly associated with variation in mRNA degradation rates, and that a model incorporating these properties explains ∼50% of the genome-wide variance. Predictors of mRNA degradation rates include transcript length, ribosome density, biased codon usage, and GC content of the third position in codons. To experimentally validate these factors, we studied individual transcripts expressed from identical promoters. We find that decreasing ribosome density by mutating the first translational start site of a transcript increases its degradation rate. Using coding sequence variants of green fluorescent protein (GFP) that differ only at synonymous sites, we show that increased GC content of the third position of codons results in decreased rates of mRNA degradation. Thus, in steady-state conditions, a large fraction of genome-wide variation in mRNA degradation rates is determined by inherent properties of transcripts, many of which are related to translation, rather than specific regulatory mechanisms. PMID:27633789

Next generation sequencing analysis reveals that the ribonucleases RNase II, RNase R and PNPase affect bacterial motility and biofilm formation in E. coli.

PubMed

Pobre, Vânia; Arraiano, Cecília M

2015-02-14

The RNA steady-state levels in the cell are a balance between synthesis and degradation rates. Although transcription is important, RNA processing and turnover are also key factors in the regulation of gene expression. In Escherichia coli there are three main exoribonucleases (RNase II, RNase R and PNPase) involved in RNA degradation. Although there are many studies about these exoribonucleases not much is known about their global effect in the transcriptome. In order to study the effects of the exoribonucleases on the transcriptome, we sequenced the total RNA (RNA-Seq) from wild-type cells and from mutants for each of the exoribonucleases (∆rnb, ∆rnr and ∆pnp). We compared each of the mutant transcriptome with the wild-type to determine the global effects of the deletion of each exoribonucleases in exponential phase. We determined that the deletion of RNase II significantly affected 187 transcripts, while deletion of RNase R affects 202 transcripts and deletion of PNPase affected 226 transcripts. Surprisingly, many of the transcripts are actually down-regulated in the exoribonuclease mutants when compared to the wild-type control. The results obtained from the transcriptomic analysis pointed to the fact that these enzymes were changing the expression of genes related with flagellum assembly, motility and biofilm formation. The three exoribonucleases affected some stable RNAs, but PNPase was the main exoribonuclease affecting this class of RNAs. We confirmed by qPCR some fold-change values obtained from the RNA-Seq data, we also observed that all the exoribonuclease mutants were significantly less motile than the wild-type cells. Additionally, RNase II and RNase R mutants were shown to produce more biofilm than the wild-type control while the PNPase mutant did not form biofilms. In this work we demonstrate how deep sequencing can be used to discover new and relevant functions of the exoribonucleases. We were able to obtain valuable information about the transcripts affected by each of the exoribonucleases and compare the roles of the three enzymes. Our results show that the three exoribonucleases affect cell motility and biofilm formation that are two very important factors for cell survival, especially for pathogenic cells.

HC-Pro silencing suppressor significantly alters the gene expression profile in tobacco leaves and flowers

PubMed Central

2011-01-01

Background RNA silencing is used in plants as a major defence mechanism against invasive nucleic acids, such as viruses. Accordingly, plant viruses have evolved to produce counter defensive RNA-silencing suppressors (RSSs). These factors interfere in various ways with the RNA silencing machinery in cells, and thereby disturb the microRNA (miRNA) mediated endogene regulation and induce developmental and morphological changes in plants. In this study we have explored these effects using previously characterized transgenic tobacco plants which constitutively express (under CaMV 35S promoter) the helper component-proteinase (HC-Pro) derived from a potyviral genome. The transcript levels of leaves and flowers of these plants were analysed using microarray techniques (Tobacco 4 × 44 k, Agilent). Results Over expression of HC-Pro RSS induced clear phenotypic changes both in growth rate and in leaf and flower morphology of the tobacco plants. The expression of 748 and 332 genes was significantly changed in the leaves and flowers, respectively, in the HC-Pro expressing transgenic plants. Interestingly, these transcriptome alterations in the HC-Pro expressing tobacco plants were similar as those previously detected in plants infected with ssRNA-viruses. Particularly, many defense-related and hormone-responsive genes (e.g. ethylene responsive transcription factor 1, ERF1) were differentially regulated in these plants. Also the expression of several stress-related genes, and genes related to cell wall modifications, protein processing, transcriptional regulation and photosynthesis were strongly altered. Moreover, genes regulating circadian cycle and flowering time were significantly altered, which may have induced a late flowering phenotype in HC-Pro expressing plants. The results also suggest that photosynthetic oxygen evolution, sugar metabolism and energy levels were significantly changed in these transgenic plants. Transcript levels of S-adenosyl-L-methionine (SAM) were also decreased in these plants, apparently leading to decreased transmethylation capacity. The proteome analysis using 2D-PAGE indicated significantly altered proteome profile, which may have been both due to altered transcript levels, decreased translation, and increased proteosomal/protease activity. Conclusion Expression of the HC-Pro RSS mimics transcriptional changes previously shown to occur in plants infected with intact viruses (e.g. Tobacco etch virus, TEV). The results indicate that the HC-Pro RSS contributes a significant part of virus-plant interactions by changing the levels of multiple cellular RNAs and proteins. PMID:21507209

Possible linkage of SP6 transcriptional activity with amelogenesis by protein stabilization.

PubMed

Utami, Trianna W; Miyoshi, Keiko; Hagita, Hiroko; Yanuaryska, Ryna Dwi; Horiguchi, Taigo; Noma, Takafumi

2011-01-01

Ameloblasts produce enamel matrix proteins such as amelogenin, ameloblastin, and amelotin during tooth development. The molecular mechanisms of ameloblast differentiation (amelogenesis) are currently not well understood. SP6 is a transcription factor of the Sp/KLF family that was recently found to regulate cell proliferation in a cell-type-specific manner. Sp6-deficient mice demonstrate characteristic tooth anomalies such as delayed eruption of the incisors and supernumerary teeth with disorganized amelogenesis. However, it remains unclear how Sp6 controls amelogenesis. In this study, we used SP6 high producer cells to identify SP6 target genes. Based on the observations that long-term culture of SP6 high producer cells reduced SP6 protein expression but not Sp6 mRNA expression, we found that SP6 is short lived and specifically degraded through a proteasome pathway. We established an in vitro inducible SP6 expression system coupled with siRNA knockdown and found a possible linkage between SP6 and amelogenesis through the regulation of amelotin and Rock1 gene expression by microarray analysis. Our findings suggest that the regulation of SP6 protein stability is one of the crucial steps in amelogenesis.

Possible Linkage of SP6 Transcriptional Activity with Amelogenesis by Protein Stabilization

PubMed Central

Utami, Trianna W.; Miyoshi, Keiko; Hagita, Hiroko; Yanuaryska, Ryna Dwi; Horiguchi, Taigo; Noma, Takafumi

2011-01-01

Ameloblasts produce enamel matrix proteins such as amelogenin, ameloblastin, and amelotin during tooth development. The molecular mechanisms of ameloblast differentiation (amelogenesis) are currently not well understood. SP6 is a transcription factor of the Sp/KLF family that was recently found to regulate cell proliferation in a cell-type-specific manner. Sp6-deficient mice demonstrate characteristic tooth anomalies such as delayed eruption of the incisors and supernumerary teeth with disorganized amelogenesis. However, it remains unclear how Sp6 controls amelogenesis. In this study, we used SP6 high producer cells to identify SP6 target genes. Based on the observations that long-term culture of SP6 high producer cells reduced SP6 protein expression but not Sp6 mRNA expression, we found that SP6 is short lived and specifically degraded through a proteasome pathway. We established an in vitro inducible SP6 expression system coupled with siRNA knockdown and found a possible linkage between SP6 and amelogenesis through the regulation of amelotin and Rock1 gene expression by microarray analysis. Our findings suggest that the regulation of SP6 protein stability is one of the crucial steps in amelogenesis. PMID:22046099

Evidence for the packaging of multiple copies of Tf1 mRNA into particles and the trans priming of reverse transcription.

PubMed

Haag, A L; Lin, J H; Levin, H L

2000-08-01

Long terminal repeat (LTR)-containing retrotransposons and retroviruses are close relatives that possess similar mechanisms of reverse transcription. The particles of retroviruses package two copies of viral mRNA that both function as templates for the reverse transcription of the element. We studied the LTR-retrotransposon Tf1 of Schizosaccharomyces pombe to test whether multiple copies of transposon mRNA participate in the production of cDNA. Using the unique self-priming property of Tf1, we obtained evidence that multiple copies of Tf1 mRNA were packaged into virus-like particles. By coexpressing two distinct versions of Tf1, we found that the bulk of reverse transcription that was initiated on one mRNA template was subsequently transferred to others. In addition, the first 11 nucleotides of one mRNA were able to prime, in trans, the reverse transcription of another mRNA.

Accurate, Streamlined Analysis of mRNA Translation by Sucrose Gradient Fractionation

PubMed Central

Aboulhouda, Soufiane; Di Santo, Rachael; Therizols, Gabriel; Weinberg, David

2017-01-01

The efficiency with which proteins are produced from mRNA molecules can vary widely across transcripts, cell types, and cellular states. Methods that accurately assay the translational efficiency of mRNAs are critical to gaining a mechanistic understanding of post-transcriptional gene regulation. One way to measure translational efficiency is to determine the number of ribosomes associated with an mRNA molecule, normalized to the length of the coding sequence. The primary method for this analysis of individual mRNAs is sucrose gradient fractionation, which physically separates mRNAs based on the number of bound ribosomes. Here, we describe a streamlined protocol for accurate analysis of mRNA association with ribosomes. Compared to previous protocols, our method incorporates internal controls and improved buffer conditions that together reduce artifacts caused by non-specific mRNA–ribosome interactions. Moreover, our direct-from-fraction qRT-PCR protocol eliminates the need for RNA purification from gradient fractions, which greatly reduces the amount of hands-on time required and facilitates parallel analysis of multiple conditions or gene targets. Additionally, no phenol waste is generated during the procedure. We initially developed the protocol to investigate the translationally repressed state of the HAC1 mRNA in S. cerevisiae, but we also detail adapted procedures for mammalian cell lines and tissues. PMID:29170751

N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

PubMed Central

Tirumuru, Nagaraja; Zhao, Boxuan Simen; Lu, Wuxun; Lu, Zhike; He, Chuan; Wu, Li

2016-01-01

The internal N6-methyladenosine (m6A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1–3) bind to m6A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1–3 proteins recognize m6A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4+ T-cells. We further mapped the YTHDF1–3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1–3 in cells had the opposite effects. Moreover, silencing the m6A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m6A erasers increased Gag expression. Our findings suggest an important role of m6A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis. DOI: http://dx.doi.org/10.7554/eLife.15528.001 PMID:27371828

RNA-seq analysis of an nsdC mutant in Aspergillus flavus

USDA-ARS?s Scientific Manuscript database

The C2H2-type transcription factor NsdC (Never in Sexual Development C) has been shown to play a role in asexual development and secondary metabolite production in Aspergillus flavus, an agriculturally relevant, aflatoxin-producing species. The nsdC knoackout mutant demonstrates perturbed morphologi...

Backtracking dynamics of RNA polymerase: pausing and error correction.

PubMed

Sahoo, Mamata; Klumpp, Stefan

2013-09-18

Transcription by RNA polymerases is frequently interrupted by pauses. One mechanism of such pauses is backtracking, where the RNA polymerase translocates backward with respect to both the DNA template and the RNA transcript, without shortening the transcript. Backtracked RNA polymerases move in a diffusive fashion and can return to active transcription either by diffusive return to the position where backtracking was initiated or by cleaving the transcript. The latter process also provides a mechanism for proofreading. Here we present some exact results for a kinetic model of backtracking and analyse its impact on the speed and the accuracy of transcription. We show that proofreading through backtracking is different from the classical (Hopfield-Ninio) scheme of kinetic proofreading. Our analysis also suggests that, in addition to contributing to the accuracy of transcription, backtracking may have a second effect: it attenuates the slow down of transcription that arises as a side effect of discriminating between correct and incorrect nucleotides based on the stepping rates.

Backtracking dynamics of RNA polymerase: pausing and error correction

NASA Astrophysics Data System (ADS)

Sahoo, Mamata; Klumpp, Stefan

2013-09-01

Transcription by RNA polymerases is frequently interrupted by pauses. One mechanism of such pauses is backtracking, where the RNA polymerase translocates backward with respect to both the DNA template and the RNA transcript, without shortening the transcript. Backtracked RNA polymerases move in a diffusive fashion and can return to active transcription either by diffusive return to the position where backtracking was initiated or by cleaving the transcript. The latter process also provides a mechanism for proofreading. Here we present some exact results for a kinetic model of backtracking and analyse its impact on the speed and the accuracy of transcription. We show that proofreading through backtracking is different from the classical (Hopfield-Ninio) scheme of kinetic proofreading. Our analysis also suggests that, in addition to contributing to the accuracy of transcription, backtracking may have a second effect: it attenuates the slow down of transcription that arises as a side effect of discriminating between correct and incorrect nucleotides based on the stepping rates.

Organization and transient expression of the gene for human U11 snRNA

PubMed Central

Clemens, Suter-Crazzolara; Walter, Keller

1991-01-01

The nucleotide sequence of U11 small nuclear RNA, a minor U RNA from HeLa cells, was determined. Computer analysis of the sequence (135 residues) predicts two strong hairpin loops which are separated by seventeen nucleotides containing an Sm binding site (AAUUUUUUGG). A synthetic gene was constructed in which the coding region of U11 RNA is under the control of a T7 promoter. This vector can be used to produce U11 RNA in vitro. Southern hybridization and PCR analysis of HeLa genomic DNA suggest that U11 RNA is encoded by a single copy gene, and that at least three genomic regions could be U11 RNA pseudogenes. A HeLa genomic copy of a U11 gene was isolated by inverted PCR. This gene contains the U11 RNA coding sequence and several sequence elements unique for the U RNA genes. These include a Distal Sequence Element (DSE, ATTTGCATA) present between positions −215 and −223 relative to the start of transcription; a Proximal Sequence Element (PSE, TTCACCTTTACCAAAAATG) located between positions −43 and −63 ; and a 3′box (GTTAGGCGAAATATTA) between positions +150 and +166. Transfection of HeLa cells with this gene revealed that it is functioning in vivo and can produce U11 RNA. PMID:1820214

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

PubMed Central

LeBlanc, Jason; Weil, Jason; Beemon, Karen

2013-01-01

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

Peregrine

PubMed Central

Langevin, Stanley A.; Bent, Zachary W.; Solberg, Owen D.; Curtis, Deanna J.; Lane, Pamela D.; Williams, Kelly P.; Schoeniger, Joseph S.; Sinha, Anupama; Lane, Todd W.; Branda, Steven S.

2013-01-01

Use of second generation sequencing (SGS) technologies for transcriptional profiling (RNA-Seq) has revolutionized transcriptomics, enabling measurement of RNA abundances with unprecedented specificity and sensitivity and the discovery of novel RNA species. Preparation of RNA-Seq libraries requires conversion of the RNA starting material into cDNA flanked by platform-specific adaptor sequences. Each of the published methods and commercial kits currently available for RNA-Seq library preparation suffers from at least one major drawback, including long processing times, large starting material requirements, uneven coverage, loss of strand information and high cost. We report the development of a new RNA-Seq library preparation technique that produces representative, strand-specific RNA-Seq libraries from small amounts of starting material in a fast, simple and cost-effective manner. Additionally, we have developed a new quantitative PCR-based assay for precisely determining the number of PCR cycles to perform for optimal enrichment of the final library, a key step in all SGS library preparation workflows. PMID:23558773

The Human RNA Polymerase I Transcription Terminator Complex Acts as a Replication Fork Barrier That Coordinates the Progress of Replication with rRNA Transcription Activity.

PubMed

Akamatsu, Yufuko; Kobayashi, Takehiko

2015-05-01

In S phase, the replication and transcription of genomic DNA need to accommodate each other, otherwise their machineries collide, with chromosomal instability as a possible consequence. Here, we characterized the human replication fork barrier (RFB) that is present downstream from the 47S pre-rRNA gene (ribosomal DNA [rDNA]). We found that the most proximal transcription terminator, Sal box T1, acts as a polar RFB, while the other, Sal box T4/T5, arrests replication forks bidirectionally. The fork-arresting activity at these sites depends on polymerase I (Pol I) transcription termination factor 1 (TTF-1) and a replisome component, TIMELESS (TIM). We also found that the RFB activity was linked to rDNA copies with hypomethylated CpG and coincided with the time that actively transcribed rRNA genes are replicated. Failed fork arrest at RFB sites led to a slowdown of fork progression moving in the opposite direction to rRNA transcription. Chemical inhibition of transcription counteracted this deceleration of forks, indicating that rRNA transcription impedes replication in the absence of RFB activity. Thus, our results reveal a role of RFB for coordinating the progression of replication and transcription activity in highly transcribed rRNA genes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Stimulation of nuclear receptor REV-ERBs regulates tumor necrosis factor-induced expression of proinflammatory molecules in C6 astroglial cells

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

Morioka, Norimitsu, E-mail: mnori@hiroshima-u.ac.jp; Tomori, Mizuki; Zhang, Fang Fang

Under physiological conditions, astrocytes maintain homeostasis in the CNS. Following inflammation and injury to the CNS, however, activated astrocytes produce neurotoxic molecules such as cytokines and chemokines, amplifying the initial molecular-cellular events evoked by inflammation and injury. Nuclear receptors REV-ERBα and REV-ERBβ (REV-ERBs) are crucial in the regulation of inflammation- and metabolism-related gene transcription. The current study sought to elucidate a role of REV-ERBs in rat C6 astroglial cells on the expression of inflammatory molecules following stimulation with the neuroinflammatory cytokine tumor necrosis factor (TNF). Stimulation of C6 cells with TNF (10 ng/ml) significantly increased the mRNA expression of CCL2, interleukin-6more » (IL-6), inducible nitric oxide synthase (iNOS), and matrix metalloprotease (MMP)-9, but not fibroblast growth factor-2 (FGF-2), cyclooxygenase-2 (COX-2) and MMP-2. Treatment with either REV-ERB agonists GSK4112 or SR9009 significantly blocked TNF-induced upregulation of CCL2 mRNA and MMP-9 mRNA, but not IL-6 mRNA and iNOS mRNA expression. Furthermore, treatment with RGFP966, a selective histone deacetylase 3 (HDAC3) inhibitor, potently reversed the inhibitory effects of GSK4112 on TNF-induced expression of MMP-9 mRNA, but not CCL2 mRNA. Expression of Rev-erbs mRNA in C6 astroglial cells, primary cultured rat cortical and spinal astrocytes was confirmed by reverse transcription polymerase chain reaction. Together, the findings demonstrate an anti-inflammatory effect, downregulating of MMP-9 and CCL2 transcription, of astroglial REV-ERBs activation through HDAC3-dependent and HDAC3-independent mechanisms. - Highlights: • Rev-erbα mRNA and Rev-erbβ mRNA are expressed in C6 astroglial cells. • TNF increases the expression of CCL2, IL-6, MMP-9 and iNOS mRNA. • REV-ERB activation inhibits CCL2 mRNA and MMP-9 mRNA expression. • HDAC3 activity is involved in the inhibitory effect of REV-ERB on MMP-9 induction.« less

Maternal noncoding transcripts antagonize the targeting of DNA elimination by scanRNAs in Paramecium tetraurelia

PubMed Central

Lepère, Gersende; Bétermier, Mireille; Meyer, Eric; Duharcourt, Sandra

2008-01-01

The germline genome of ciliates is extensively rearranged during the development of a new somatic macronucleus from the germline micronucleus, after sexual events. In Paramecium tetraurelia, single-copy internal eliminated sequences (IESs) are precisely excised from coding sequences and intergenic regions. For a subset of IESs, introduction of the IES sequence into the maternal macronucleus specifically inhibits excision of the homologous IES in the developing zygotic macronucleus, suggesting that epigenetic regulation of excision involves a global comparison of germline and somatic genomes. ScanRNAs (scnRNAs) produced during micronuclear meiosis by a developmentally regulated RNAi pathway have been proposed to mediate this transnuclear cross-talk. In this study, microinjection experiments provide direct evidence that 25-nucleotide (nt) scnRNAs promote IES excision. We further show that noncoding RNAs are produced from the somatic maternal genome, both during vegetative growth and during sexual events. Maternal inhibition of IES excision is abolished when maternal somatic transcripts containing an IES are targeted for degradation by a distinct RNAi pathway involving 23-nt siRNAs. The results strongly support a scnRNA/macronuclear RNA scanning model in which a natural genomic subtraction, occurring during meiosis between deletion-inducing scnRNAs and antagonistic transcripts from the maternal macronucleus, regulates rearrangements of the zygotic genome. PMID:18519642

Massively parallel single-molecule and single-cell emulsion reverse transcription polymerase chain reaction using agarose droplet microfluidics.

PubMed

Zhang, Huifa; Jenkins, Gareth; Zou, Yuan; Zhu, Zhi; Yang, Chaoyong James

2012-04-17

A microfluidic device for performing single copy, emulsion Reverse Transcription Polymerase Chain Reaction (RT-PCR) within agarose droplets is presented. A two-aqueous-inlet emulsion droplet generator was designed and fabricated to produce highly uniform monodisperse picoliter agarose emulsion droplets with RT-PCR reagents in carrier oil. Template RNA or cells were delivered from one inlet with RT-PCR reagents/cell lysis buffer delivered separately from the other. Efficient RNA/cell encapsulation and RT-PCR at the single copy level was achieved in agarose-in-oil droplets, which, after amplification, can be solidified into agarose beads for further analysis. A simple and efficient method to graft primer to the polymer matrix using 5'-acrydite primer was developed to ensure highly efficient trapping of RT-PCR products in agarose. High-throughput single RNA molecule/cell RT-PCR was demonstrated in stochastically diluted solutions. Our results indicate that single-molecule RT-PCR can be efficiently carried out in agarose matrix. Single-cell RT-PCR was successfully performed which showed a clear difference in gene expression level of EpCAM, a cancer biomarker gene, at the single-cell level between different types of cancer cells. This work clearly demonstrates for the first time, single-copy RT-PCR in agarose droplets. We believe this will open up new possibilities for viral RNA detection and single-cell transcription analysis.

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

PubMed Central

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

2018-01-01

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

Posttranscriptional mRNA processing as a mechanism for regulation of human A1 adenosine receptor expression.

PubMed Central

Ren, H; Stiles, G L

1994-01-01

The human A1 adenosine receptor gene contains six exons with exons 1, 2, 3, 4, and part of 5 representing 5' untranslated regions. Reverse transcription-PCR with exon-specific primers showed two distinct transcripts containing either exons 3, 5, and 6 or exons 4, 5, and 6, with exons 3 and 4 being mutually exclusive. No mature mRNAs containing exons 1 and 2 have been detected. All human tissues that express any A1 receptors contain mRNA with exons 4, 5, and 6. Tissues which express high levels of A1 receptors contain mRNA with exons 3, 5, and 6. Exon 4 contains two upstream ATG codons whereas exon 3 contains none. COS cells transfected with expression vectors containing exon 4 (exons 1-6, 3-6, or Ex4-6) express much lower levels of A1 receptors than vectors without exon 4 (exons 3, 5, and 6). Mutation of upstream ATG codons in exon 4 leads to 3- to 7-fold increased A1 receptor expression, up to the level seen with the construct containing exons 3, 5, and 6. Thus, in human tissues "basal" levels of A1 receptors can be expressed by use of mRNA containing exons 4, 5, and 6, but when high levels are needed, alternative transcripts with exons 3, 5, and 6 are produced. Images PMID:8197148

Action of boron at the molecular level: effects on transcription and translation in an acellular system.

PubMed

Dzondo-Gadet, M; Mayap-Nzietchueng, R; Hess, K; Nabet, P; Belleville, F; Dousset, B

2002-01-01

It has been shown that boric acid has well-defined biological effects such as stimulation of wound healing in vivo, release of growth factors and cytokines, and increase of the extracellular matrice turnover. We examined its action at the molecular level, using cell-free systems of transcription (isolated placenta nuclei) and translation (wheat germ extract). We found that 10 mM boric acid greatly increased RNA synthesis, measured by absorbance at 260 nm (x 6.4) or by [3H]-UTP uptake (x 11). Full-length functional mRNA was produced because proteins of 14-80 kDa were translated. Among these proteins, factors involved in angiogenesis and, subsequently, in wound healing (VEGF and TGFbeta) were identified by slot blot, whereas growth factors such as FGF1 and TNFalpha were not detected. These results demonstrate that boron may contribute to biological cell activities at both the transcription and translation levels. However, the mechanism of action is still not known.

Preparation of Small RNAs Using Rolling Circle Transcription and Site-Specific RNA Disconnection.

PubMed

Wang, Xingyu; Li, Can; Gao, Xiaomeng; Wang, Jing; Liang, Xingguo

2015-01-13

A facile and robust RNA preparation protocol was developed by combining rolling circle transcription (RCT) with RNA cleavage by RNase H. Circular DNA with a complementary sequence was used as the template for promoter-free transcription. With the aid of a 2'-O-methylated DNA, the RCT-generated tandem repeats of the desired RNA sequence were disconnected at the exact end-to-end position to harvest the desired RNA oligomers. Compared with the template DNA, more than 4 × 10(3) times the amount of small RNA products were obtained when modest cleavage was carried out during transcription. Large amounts of RNA oligomers could easily be obtained by simply increasing the reaction volume.

Alternative splicing of the tyrosinase gene transcript in normal human melanocytes and lymphocytes.

PubMed

Fryer, J P; Oetting, W S; Brott, M J; King, R A

2001-11-01

We have identified and isolated ectopically expressed tyrosinase transcripts in normal human melanocytes and lymphocytes and in a human melanoma (MNT-1) cell line to establish a baseline for the expression pattern of this gene in normal tissue. Tyrosinase mRNA from human lymphoblastoid cell lines was reverse transcribed and amplified using specific "nested" primers. This amplification yielded eight identifiable transcripts; five that resulted from alternative splicing patterns arising from the utilization of normal and alternative splice sequences. Identical splicing patterns were found in transcripts from human primary melanocytes in culture and a melanoma cell line, indicating that lymphoblastoid cell lines provide an accurate reflection of transcript processing in melanocytes. Similar splicing patterns have also been found with murine melanocyte tyrosinase transcripts. Our results demonstrate that alternative splicing of human tyrosinase gene transcript produces a number of predictable and identifiable transcripts, and that human lymphoblastoid cell lines provide a source of ectopically expressed transcripts that can be used to study the biology of tyrosinase gene expression in humans.

Transcriptional bursting is intrinsically caused by interplay between RNA polymerases on DNA

NASA Astrophysics Data System (ADS)

Fujita, Keisuke; Iwaki, Mitsuhiro; Yanagida, Toshio

2016-12-01

Cell-to-cell variability plays a critical role in cellular responses and decision-making in a population, and transcriptional bursting has been broadly studied by experimental and theoretical approaches as the potential source of cell-to-cell variability. Although molecular mechanisms of transcriptional bursting have been proposed, there is little consensus. An unsolved key question is whether transcriptional bursting is intertwined with many transcriptional regulatory factors or is an intrinsic characteristic of RNA polymerase on DNA. Here we design an in vitro single-molecule measurement system to analyse the kinetics of transcriptional bursting. The results indicate that transcriptional bursting is caused by interplay between RNA polymerases on DNA. The kinetics of in vitro transcriptional bursting is quantitatively consistent with the gene-nonspecific kinetics previously observed in noisy gene expression in vivo. Our kinetic analysis based on a cellular automaton model confirms that arrest and rescue by trailing RNA polymerase intrinsically causes transcriptional bursting.

Simultaneous sequencing of coding and noncoding RNA reveals a human transcriptome dominated by a small number of highly expressed noncoding genes.

PubMed

Boivin, Vincent; Deschamps-Francoeur, Gabrielle; Couture, Sonia; Nottingham, Ryan M; Bouchard-Bourelle, Philia; Lambowitz, Alan M; Scott, Michelle S; Abou-Elela, Sherif

2018-07-01

Comparing the abundance of one RNA molecule to another is crucial for understanding cellular functions but most sequencing techniques can target only specific subsets of RNA. In this study, we used a new fragmented ribodepleted TGIRT sequencing method that uses a thermostable group II intron reverse transcriptase (TGIRT) to generate a portrait of the human transcriptome depicting the quantitative relationship of all classes of nonribosomal RNA longer than 60 nt. Comparison between different sequencing methods indicated that FRT is more accurate in ranking both mRNA and noncoding RNA than viral reverse transcriptase-based sequencing methods, even those that specifically target these species. Measurements of RNA abundance in different cell lines using this method correlate with biochemical estimates, confirming tRNA as the most abundant nonribosomal RNA biotype. However, the single most abundant transcript is 7SL RNA, a component of the signal recognition particle. S tructured n on c oding RNAs (sncRNAs) associated with the same biological process are expressed at similar levels, with the exception of RNAs with multiple functions like U1 snRNA. In general, sncRNAs forming RNPs are hundreds to thousands of times more abundant than their mRNA counterparts. Surprisingly, only 50 sncRNA genes produce half of the non-rRNA transcripts detected in two different cell lines. Together the results indicate that the human transcriptome is dominated by a small number of highly expressed sncRNAs specializing in functions related to translation and splicing. © 2018 Boivin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Myelin basic protein-messenger RNA (MBP-mRNA) expression during triethyltin-induced myelin edema.

PubMed

Veronesi, B; Jones, K; Gupta, S; Pringle, J; Mezei, C

1991-01-01

Triethyltin (TET) is a neurotoxicant that produces severe but transient cerebral edema, characterized ultrastructurally by vacuolation of the intraperiod line of central nervous system (CNS) myelin. TET has been reported to depress levels of myelin basic protein (MBP), a protein thought to play a critical role in myelin compaction. In the present study, the genomic expression (i.e., mRNA) of MBP was monitored throughout the pathogenesis of TET-induced myelin edema and recovery in Sprague-Dawley rats given a single injection of a neuropathic (8.0 mg/kg) or non-neuropathic (0.8 mg/kg) dose of TET-bromide. Levels of MBP-mRNA from the anterior and posterior brain were collected 1 hr, 3 hr, 2d, and 7d, postexposure. The optic nerve and caudal brainstem, representing anterior and posterior brain sites, respectively, were examined at the same time-points for ultrastructural evidence of edema and recovery. Our data indicate that neuropathic doses (8.0 mg/kg) of TET significantly stimulated MBP transcript throughout the brain at all exposure time-points. The magnitude and time-course of this stimulation differed in the anterior and posterior brain, with the latter region showing higher levels of MBP-mRNA. In the posterior brain, the highest levels of mRNA correlated with the appearance of edema in the caudal brainstem. In the anterior brain, MBP-mRNA levels were only marginally increased over controls. Ultrastructural evidence of myelin edema was confined to the brainstem in rats treated with neuropathic dose of TET. Intralamellar vacuolation appeared at 3 hr and 2d postexposure and could be correlated with peak levels of MBP transcript, whereas, recompacted myelin, which appeared by 7d postexposure, was associated with declining levels of the mRNA. Ultrastructural changes in the oligodendroglia were suggestive of metabolic stimulation and correlated with high MBP-mRNA levels. In summary, these data indicate that an initial genomic event in TET-induced myelin edema is stimulation of MBP transcript.

MicroRNA-142-3p and let-7g Negatively Regulates Augmented IL-6 Production in Neonatal Polymorphonuclear Leukocytes

PubMed Central

Huang, Hsin-Chun; Yu, Hong-Ren; Hsu, Te-Yao; Chen, I-Lun; Huang, Hui-Chen; Chang, Jen-Chieh; Yang, Kuender D.

2017-01-01

Neonatal PMN are qualitatively impaired in functions, yet they frequently reveal augmented inflammatory reactions during sepsis. Here, we hypothesized that PMN from newborns produce more IL-6 than those from adults under LPS stimulation, in which transcriptional or posttranscriptional regulation is involved in the altered expression. We found that neonatal PMN produced significantly higher IL-6 mRNA and protein than adult PMN. The higher IL-6 expression was not related to transcriptional but posttranscriptional regulation as the IL-6 expression was affected by the addition of cycloheximide but not actinomycin. To examine whether miRNA was involved in the IL-6 regulation of neonatal PMN, we surveyed differential displays of miRNAs that could potentially regulate IL-6 expression before and after LPS stimulation. Four miRNAs: hsa-miR-26a, hsa-miR-26b, hsa-miR-142-3p and hsa-let 7g decreased or increased after LPS treatment for 4 h. Further validation by qRT-PCR identified miR-26b, miR-142-3p and let-7g significantly changed in neonatal PMN after LPS stimulation. The functional verification by transfection of miR-142-3p and let-7g precursors into neonatal PMN significantly repressed the IL-6 mRNA and protein expression, suggesting that miR-142-3p and let-7g negatively regulate IL-6 expression in neonatal PMN. Modulation of miRNA expression may be used to regulate IL-6 production in newborns with altered inflammatory reactions. PMID:28655995

Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors

DOE PAGES

Carbonell, Alberto; Fahlgren, Noah; Mitchell, Skyler; ...

2015-05-20

Artificial microRNAs (amiRNAs) are used for selective gene silencing in plants. However, current methods to produce amiRNA constructs for silencing transcripts in monocot species are not suitable for simple, cost-effective and large-scale synthesis. Here, a series of expression vectors based on Oryza sativa MIR390 (OsMIR390) precursor was developed for high-throughput cloning and high expression of amiRNAs in monocots. Four different amiRNA sequences designed to target specifically endogenous genes and expressed from OsMIR390-based vectors were validated in transgenic Brachypodium distachyon plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric OsMIR390-based precursors that include distalmore » stem-loop sequences from Arabidopsis thaliana MIR390a (AtMIR390a). In all cases, transgenic plants displayed the predicted phenotypes induced by target gene repression, and accumulated high levels of amiRNAs and low levels of the corresponding target transcripts. Genome-wide transcriptome profiling combined with 5-RLM-RACE analysis in transgenic plants confirmed that amiRNAs were highly specific. Finally, significance Statement A series of amiRNA vectors based on Oryza sativa MIR390 (OsMIR390) precursor were developed for simple, cost-effective and large-scale synthesis of amiRNA constructs to silence genes in monocots. Unexpectedly, amiRNAs produced from chimeric OsMIR390-based precursors including Arabidopsis thaliana MIR390a distal stem-loop sequences accumulated elevated levels of highly effective and specific amiRNAs in transgenic Brachypodium distachyon plants.« less

Higher miRNA Tolerance in Immortal Li-Fraumeni Fibroblasts with Abrogated Interferon Signaling Pathway

PubMed Central

Li, Qunfang; Tainsky, Michael A.

2013-01-01

The IFN pathway is abrogated in fibroblasts from Li-Fraumeni syndrome (LFS) patients during spontaneous cellular immortalization, a necessary step in carcinogenesis. Microarray profiling of differentially expressed microRNAs (miRNA) revealed that most miRNAs were upregulated in IFN pathway–defective MDAH087-10 fibroblasts compared with MDAH087-N cells with relatively normal IFN signaling. Overexpression of Dicer, a critical enzyme in miRNA biogenesis, promoted cell growth and colony formation in MDAH087-10 cells. However, double-stranded miRNA produced by Dicer enhanced the expression of IFN-stimulated genes in MDAH087-N cells resulting in significant cell death and reduced cell growth. Furthermore, manipulation of the IFN pathway in immortal LFS fibroblasts through transcription factor IRF7 reversed their response to Dicer overexpression due to changed IFN pathway activity. Dicer overexpressing MDAH087-N cells contained lower levels of miRNA than vector control, and conversely much higher miRNA expression was detected in Dicertransfected MDAH087-10 cells. Therefore, cells with a defective IFN pathway have a higher miRNA tolerance than cells with normal IFN pathway. This work indicates for the first time that the IFN pathway as mediated through the transcription factor IRF7 must be disrupted to permit miRNA upregulation to occur in early carcinogenesis. The IFN pathway appears to provide a checkpoint for miRNA level tolerance and its abrogation leads to cellular immortalization. PMID:21199806

Higher miRNA tolerance in immortal Li-Fraumeni fibroblasts with abrogated interferon signaling pathway.

PubMed

Li, Qunfang; Tainsky, Michael A

2011-01-01

The IFN pathway is abrogated in fibroblasts from Li-Fraumeni syndrome (LFS) patients during spontaneous cellular immortalization, a necessary step in carcinogenesis. Microarray profiling of differentially expressed microRNAs (miRNA) revealed that most miRNAs were upregulated in IFN pathway-defective MDAH087-10 fibroblasts compared with MDAH087-N cells with relatively normal IFN signaling. Overexpression of Dicer, a critical enzyme in miRNA biogenesis, promoted cell growth and colony formation in MDAH087-10 cells. However, double-stranded miRNA produced by Dicer enhanced the expression of IFN-stimulated genes in MDAH087-N cells resulting in significant cell death and reduced cell growth. Furthermore, manipulation of the IFN pathway in immortal LFS fibroblasts through transcription factor IRF7 reversed their response to Dicer overexpression due to changed IFN pathway activity. Dicer overexpressing MDAH087-N cells contained lower levels of miRNA than vector control, and conversely much higher miRNA expression was detected in Dicer-transfected MDAH087-10 cells. Therefore, cells with a defective IFN pathway have a higher miRNA tolerance than cells with normal IFN pathway. This work indicates for the first time that the IFN pathway as mediated through the transcription factor IRF7 must be disrupted to permit miRNA upregulation to occur in early carcinogenesis. The IFN pathway appears to provide a checkpoint for miRNA level tolerance and its abrogation leads to cellular immortalization. © 2011 AACR.

Noncoding transcripts in sense and antisense orientation regulate the epigenetic state of ribosomal RNA genes.

PubMed

Bierhoff, H; Schmitz, K; Maass, F; Ye, J; Grummt, I

2010-01-01

Alternative transcription of the same gene in sense and antisense orientation regulates expression of protein-coding genes. Here we show that noncoding RNA (ncRNA) in sense and antisense orientation also controls transcription of rRNA genes (rDNA). rDNA exists in two types of chromatin--a euchromatic conformation that is permissive to transcription and a heterochromatic conformation that is transcriptionally silent. Silencing of rDNA is mediated by NoRC, a chromatin-remodeling complex that triggers heterochromatin formation. NoRC function requires RNA that is complementary to the rDNA promoter (pRNA). pRNA forms a DNA:RNA triplex with a regulatory element in the rDNA promoter, and this triplex structure is recognized by DNMT3b. The results imply that triplex-mediated targeting of DNMT3b to specific sequences may be a common pathway in epigenetic regulation. We also show that rDNA is transcribed in antisense orientation. The level of antisense RNA (asRNA) is down-regulated in cancer cells and up-regulated in senescent cells. Ectopic asRNA triggers trimethylation of histone H4 at lysine 20 (H4K20me3), suggesting that antisense transcripts guide the histone methyltransferase Suv4-20 to rDNA. The results reveal that noncoding RNAs in sense and antisense orientation are important determinants of the epigenetic state of rDNA.

Directional RNA-seq reveals highly complex condition-dependent transcriptomes in E. coli K12 through accurate full-length transcripts assembling.

PubMed

Li, Shan; Dong, Xia; Su, Zhengchang

2013-07-30

Although prokaryotic gene transcription has been studied over decades, many aspects of the process remain poorly understood. Particularly, recent studies have revealed that transcriptomes in many prokaryotes are far more complex than previously thought. Genes in an operon are often alternatively and dynamically transcribed under different conditions, and a large portion of genes and intergenic regions have antisense RNA (asRNA) and non-coding RNA (ncRNA) transcripts, respectively. Ironically, similar studies have not been conducted in the model bacterium E coli K12, thus it is unknown whether or not the bacterium possesses similar complex transcriptomes. Furthermore, although RNA-seq becomes the major method for analyzing the complexity of prokaryotic transcriptome, it is still a challenging task to accurately assemble full length transcripts using short RNA-seq reads. To fill these gaps, we have profiled the transcriptomes of E. coli K12 under different culture conditions and growth phases using a highly specific directional RNA-seq technique that can capture various types of transcripts in the bacterial cells, combined with a highly accurate and robust algorithm and tool TruHMM (http://bioinfolab.uncc.edu/TruHmm_package/) for assembling full length transcripts. We found that 46.9 ~ 63.4% of expressed operons were utilized in their putative alternative forms, 72.23 ~ 89.54% genes had putative asRNA transcripts and 51.37 ~ 72.74% intergenic regions had putative ncRNA transcripts under different culture conditions and growth phases. As has been demonstrated in many other prokaryotes, E. coli K12 also has a highly complex and dynamic transcriptomes under different culture conditions and growth phases. Such complex and dynamic transcriptomes might play important roles in the physiology of the bacterium. TruHMM is a highly accurate and robust algorithm for assembling full-length transcripts in prokaryotes using directional RNA-seq short reads.

Directional RNA-seq reveals highly complex condition-dependent transcriptomes in E. coli K12 through accurate full-length transcripts assembling

PubMed Central

2013-01-01

Background Although prokaryotic gene transcription has been studied over decades, many aspects of the process remain poorly understood. Particularly, recent studies have revealed that transcriptomes in many prokaryotes are far more complex than previously thought. Genes in an operon are often alternatively and dynamically transcribed under different conditions, and a large portion of genes and intergenic regions have antisense RNA (asRNA) and non-coding RNA (ncRNA) transcripts, respectively. Ironically, similar studies have not been conducted in the model bacterium E coli K12, thus it is unknown whether or not the bacterium possesses similar complex transcriptomes. Furthermore, although RNA-seq becomes the major method for analyzing the complexity of prokaryotic transcriptome, it is still a challenging task to accurately assemble full length transcripts using short RNA-seq reads. Results To fill these gaps, we have profiled the transcriptomes of E. coli K12 under different culture conditions and growth phases using a highly specific directional RNA-seq technique that can capture various types of transcripts in the bacterial cells, combined with a highly accurate and robust algorithm and tool TruHMM (http://bioinfolab.uncc.edu/TruHmm_package/) for assembling full length transcripts. We found that 46.9 ~ 63.4% of expressed operons were utilized in their putative alternative forms, 72.23 ~ 89.54% genes had putative asRNA transcripts and 51.37 ~ 72.74% intergenic regions had putative ncRNA transcripts under different culture conditions and growth phases. Conclusions As has been demonstrated in many other prokaryotes, E. coli K12 also has a highly complex and dynamic transcriptomes under different culture conditions and growth phases. Such complex and dynamic transcriptomes might play important roles in the physiology of the bacterium. TruHMM is a highly accurate and robust algorithm for assembling full-length transcripts in prokaryotes using directional RNA-seq short reads. PMID:23899370

Inhibition of host cell RNA polymerase III-mediated transcription by poliovirus: Inactivation of specific transcription factors

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

Fradkin, L.G.; Yoshinaga, S.K.; Berk, A.J.

1987-11-01

The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription ofmore » RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoted, however, was not altered by infection of cells with the virus. The authors conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirtus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.« less

MicroRNA-Dependent Transcriptional Silencing of Transposable Elements in Drosophila Follicle Cells.

PubMed

Mugat, Bruno; Akkouche, Abdou; Serrano, Vincent; Armenise, Claudia; Li, Blaise; Brun, Christine; Fulga, Tudor A; Van Vactor, David; Pélisson, Alain; Chambeyron, Séverine

2015-05-01

RNA interference-related silencing mechanisms concern very diverse and distinct biological processes, from gene regulation (via the microRNA pathway) to defense against molecular parasites (through the small interfering RNA and the Piwi-interacting RNA pathways). Small non-coding RNAs serve as specificity factors that guide effector proteins to ribonucleic acid targets via base-pairing interactions, to achieve transcriptional or post-transcriptional regulation. Because of the small sequence complementarity required for microRNA-dependent post-transcriptional regulation, thousands of microRNA (miRNA) putative targets have been annotated in Drosophila. In Drosophila somatic ovarian cells, genomic parasites, such as transposable elements (TEs), are transcriptionally repressed by chromatin changes induced by Piwi-interacting RNAs (piRNAs) that prevent them from invading the germinal genome. Here we show, for the first time, that a functional miRNA pathway is required for the piRNA-mediated transcriptional silencing of TEs in this tissue. Global miRNA depletion, caused by tissue- and stage-specific knock down of drosha (involved in miRNA biogenesis), AGO1 or gawky (both responsible for miRNA activity), resulted in loss of TE-derived piRNAs and chromatin-mediated transcriptional de-silencing of TEs. This specific TE de-repression was also observed upon individual titration (by expression of the complementary miRNA sponge) of two miRNAs (miR-14 and miR-34) as well as in a miR-14 loss-of-function mutant background. Interestingly, the miRNA defects differentially affected TE- and 3' UTR-derived piRNAs. To our knowledge, this is the first indication of possible differences in the biogenesis or stability of TE- and 3' UTR-derived piRNAs. This work is one of the examples of detectable phenotypes caused by loss of individual miRNAs in Drosophila and the first genetic evidence that miRNAs have a role in the maintenance of genome stability via piRNA-mediated TE repression.

Comparison of transcriptional profiles of Clostridium thermocellum grown on cellobiose and pretreated yellow poplar using RNA-Seq

PubMed Central

Wei, Hui; Fu, Yan; Magnusson, Lauren; Baker, John O.; Maness, Pin-Ching; Xu, Qi; Yang, Shihui; Bowersox, Andrew; Bogorad, Igor; Wang, Wei; Tucker, Melvin P.; Himmel, Michael E.; Ding, Shi-You

2014-01-01

The anaerobic, thermophilic bacterium, Clostridium thermocellum, secretes multi-protein enzyme complexes, termed cellulosomes, which synergistically interact with the microbial cell surface and efficiently disassemble plant cell wall biomass. C. thermocellum has also been considered a potential consolidated bioprocessing (CBP) organism due to its ability to produce the biofuel products, hydrogen, and ethanol. We found that C. thermocellum fermentation of pretreated yellow poplar (PYP) produced 30 and 39% of ethanol and hydrogen product concentrations, respectively, compared to fermentation of cellobiose. RNA-seq was used to analyze the transcriptional profiles of these cells. The PYP-grown cells taken for analysis at the late stationary phase showed 1211 genes up-regulated and 314 down-regulated by more than two-fold compared to the cellobiose-grown cells. These affected genes cover a broad spectrum of specific functional categories. The transcriptional analysis was further validated by sub-proteomics data taken from the literature; as well as by quantitative reverse transcription-PCR (qRT-PCR) analyses of selected genes. Specifically, 47 cellulosomal protein-encoding genes, genes for 4 pairs of SigI-RsgI for polysaccharide sensing, 7 cellodextrin ABC transporter genes, and a set of NAD(P)H hydogenase and alcohol dehydrogenase genes were up-regulated for cells growing on PYP compared to cellobiose. These genes could be potential candidates for future studies aimed at gaining insight into the regulatory mechanism of this organism as well as for improvement of C. thermocellum in its role as a CBP organism. PMID:24782837

Pervasive, Genome-Wide Transcription in the Organelle Genomes of Diverse Plastid-Bearing Protists.

PubMed

Sanitá Lima, Matheus; Smith, David Roy

2017-11-06

Organelle genomes are among the most sequenced kinds of chromosome. This is largely because they are small and widely used in molecular studies, but also because next-generation sequencing technologies made sequencing easier, faster, and cheaper. However, studies of organelle RNA have not kept pace with those of DNA, despite huge amounts of freely available eukaryotic RNA-sequencing (RNA-seq) data. Little is known about organelle transcription in nonmodel species, and most of the available eukaryotic RNA-seq data have not been mined for organelle transcripts. Here, we use publicly available RNA-seq experiments to investigate organelle transcription in 30 diverse plastid-bearing protists with varying organelle genomic architectures. Mapping RNA-seq data to organelle genomes revealed pervasive, genome-wide transcription, regardless of the taxonomic grouping, gene organization, or noncoding content. For every species analyzed, transcripts covered ≥85% of the mitochondrial and/or plastid genomes (all of which were ≤105 kb), indicating that most of the organelle DNA-coding and noncoding-is transcriptionally active. These results follow earlier studies of model species showing that organellar transcription is coupled and ubiquitous across the genome, requiring significant downstream processing of polycistronic transcripts. Our findings suggest that noncoding organelle DNA can be transcriptionally active, raising questions about the underlying function of these transcripts and underscoring the utility of publicly available RNA-seq data for recovering complete genome sequences. If pervasive transcription is also found in bigger organelle genomes (>105 kb) and across a broader range of eukaryotes, this could indicate that noncoding organelle RNAs are regulating fundamental processes within eukaryotic cells. Copyright © 2017 Sanitá Lima and Smith.

lncRNA requirements for mouse acute myeloid leukemia and normal differentiation

PubMed Central

Knott, Simon RV; Munera Maravilla, Ester; Jackson, Benjamin T; Wild, Sophia A; Kovacevic, Tatjana; Stork, Eva Maria; Zhou, Meng; Erard, Nicolas; Lee, Emily; Kelley, David R; Roth, Mareike; Barbosa, Inês AM; Zuber, Johannes; Rinn, John L

2017-01-01

A substantial fraction of the genome is transcribed in a cell-type-specific manner, producing long non-coding RNAs (lncRNAs), rather than protein-coding transcripts. Here, we systematically characterize transcriptional dynamics during hematopoiesis and in hematological malignancies. Our analysis of annotated and de novo assembled lncRNAs showed many are regulated during differentiation and mis-regulated in disease. We assessed lncRNA function via an in vivo RNAi screen in a model of acute myeloid leukemia. This identified several lncRNAs essential for leukemia maintenance, and found that a number act by promoting leukemia stem cell signatures. Leukemia blasts show a myeloid differentiation phenotype when these lncRNAs were depleted, and our data indicates that this effect is mediated via effects on the MYC oncogene. Bone marrow reconstitutions showed that a lncRNA expressed across all progenitors was required for the myeloid lineage, whereas the other leukemia-induced lncRNAs were dispensable in the normal setting. PMID:28875933

lncRNA requirements for mouse acute myeloid leukemia and normal differentiation.

PubMed

Delás, M Joaquina; Sabin, Leah R; Dolzhenko, Egor; Knott, Simon Rv; Munera Maravilla, Ester; Jackson, Benjamin T; Wild, Sophia A; Kovacevic, Tatjana; Stork, Eva Maria; Zhou, Meng; Erard, Nicolas; Lee, Emily; Kelley, David R; Roth, Mareike; Barbosa, Inês Am; Zuber, Johannes; Rinn, John L; Smith, Andrew D; Hannon, Gregory J

2017-09-06

A substantial fraction of the genome is transcribed in a cell-type-specific manner, producing long non-coding RNAs (lncRNAs), rather than protein-coding transcripts. Here, we systematically characterize transcriptional dynamics during hematopoiesis and in hematological malignancies. Our analysis of annotated and de novo assembled lncRNAs showed many are regulated during differentiation and mis-regulated in disease. We assessed lncRNA function via an in vivo RNAi screen in a model of acute myeloid leukemia. This identified several lncRNAs essential for leukemia maintenance, and found that a number act by promoting leukemia stem cell signatures. Leukemia blasts show a myeloid differentiation phenotype when these lncRNAs were depleted, and our data indicates that this effect is mediated via effects on the MYC oncogene. Bone marrow reconstitutions showed that a lncRNA expressed across all progenitors was required for the myeloid lineage, whereas the other leukemia-induced lncRNAs were dispensable in the normal setting.

RNA editing produces glycine receptor alpha3(P185L), resulting in high agonist potency.

PubMed

Meier, Jochen C; Henneberger, Christian; Melnick, Igor; Racca, Claudia; Harvey, Robert J; Heinemann, Uwe; Schmieden, Volker; Grantyn, Rosemarie

2005-06-01

The function of supramedullary glycine receptors (GlyRs) is still unclear. Using Wistar rat collicular slices, we demonstrate GlyR-mediated inhibition of spike discharge elicited by low glycine (10 microM). Searching for the molecular basis of this phenomenon, we identified a new GlyR isoform. GlyR alpha3(P185L), a result of cytidine 554 deamination, confers high glycine sensitivity (EC50 approximately 5 microM) to neurons and thereby promotes the generation of sustained chloride conductances associated with tonic inhibition. The level of GlyR alpha3-C554U RNA editing is sensitive to experimentally induced brain lesion, inhibition of cytidine deamination by zebularine and inhibition of mRNA transcription by actinomycin D, but not to blockade of protein synthesis by cycloheximide. Conditional regulation of GlyR alpha3(P185L) is thus likely to be part of a post-transcriptional adaptive mechanism in neurons with enhanced excitability.

Construction of in vitro transcription system for Corynebacterium glutamicum and its use in the recognition of promoters of different classes.

PubMed

Holátko, Jiří; Silar, Radoslav; Rabatinová, Alžbeta; Sanderová, Hana; Halada, Petr; Nešvera, Jan; Krásný, Libor; Pátek, Miroslav

2012-10-01

To facilitate transcription studies in Corynebacterium glutamicum, we have developed an in vitro transcription system for this bacterium used as an industrial producer of amino acids and a model organism for actinobacteria. This system consists of C. glutamicum RNA polymerase (RNAP) core (α2, β, β'), a sigma factor and a promoter-carrying DNA template, that is specifically recognized by the RNAP holoenzyme formed. The RNAP core was purified from the C. glutamicum strain with the modified rpoC gene, which produced His-tagged β' subunit. The C. glutamicum sigA and sigH genes were cloned and overexpressed using the Escherichia coli plasmid vector, and the sigma subunits σ(A) and σ(H) were purified by affinity chromatography. Using the reconstituted C. glutamicum holo-RNAPs, recognition of the σ(A)- and σ(H)-dependent promoters and synthesis of the specific transcripts was demonstrated. The developed in vitro transcription system is a novel tool that can be used to directly prove the specific recognition of a promoter by the particular σ factor(s) and to analyze transcriptional control by various regulatory proteins in C. glutamicum.

Rapid transient isoform-specific neuregulin1 transcription in motor neurons is regulated by neurotrophic factors and axon-target interactions.

PubMed

Wang, Jiajing; Hmadcha, Abdelkrim; Zakarian, Vaagn; Song, Fei; Loeb, Jeffrey A

2015-09-01

The neuregulins (NRGs) are a family of alternatively spliced factors that play important roles in nervous system development and disease. In motor neurons, NRG1 expression is regulated by activity and neurotrophic factors, however, little is known about what controls isoform-specific transcription. Here we show that NRG1 expression in the chick embryo increases in motor neurons that have extended their axons and that limb bud ablation before motor axon outgrowth prevents this induction, suggesting a trophic role from the developing limb. Consistently, NRG1 induction after limb bud ablation can be rescued by adding back the neurotrophic factors BDNF and GDNF. Mechanistically, BDNF induces a rapid and transient increase in type I and type III NRG1 mRNAs that peak at 4h in rat embryonic ventral spinal cord cultures. Blocking MAPK or PI3K signaling or blocking transcription with Actinomycin D blocks BDNF induced NRG1 gene induction. BDNF had no effect on mRNA degradation, suggesting that transcriptional activation rather than message stability is important. Furthermore, BDNF activates a reporter construct that includes 700bp upstream of the type I NRG1 start site. Protein synthesis is also required for type I NRG1 mRNA transcription as cycloheximide produced a super-induction of type I, but not type III NRG1 mRNA, possibly through a mechanism involving sustained activation of MAPK and PI3K. These results reveal the existence of highly responsive, transient transcriptional regulatory mechanisms that differentially modulate NRG1 isoform expression as a function of extracellular and intracellular signaling cascades and mediated by neurotrophic factors and axon-target interactions. Copyright © 2015 Elsevier Inc. All rights reserved.

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

PubMed

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

1989-01-01

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

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

PubMed

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

2017-12-08

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

Heterochromatic siRNAs and DDM1 Independently Silence Aberrant 5S rDNA Transcripts in Arabidopsis

PubMed Central

Blevins, Todd; Pontes, Olga; Pikaard, Craig S.; Meins, Frederick

2009-01-01

5S ribosomal RNA gene repeats are arranged in heterochromatic arrays (5S rDNA) situated near the centromeres of Arabidopsis chromosomes. The chromatin remodeling factor DDM1 is known to maintain 5S rDNA methylation patterns while silencing transcription through 5S rDNA intergenic spacers (IGS). We mapped small-interfering RNAs (siRNA) to a composite 5S rDNA repeat, revealing a high density of siRNAs matching silenced IGS transcripts. IGS transcript repression requires proteins of the heterochromatic siRNA pathway, including RNA polymerase IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Using molecular and cytogenetic approaches, we show that the DDM1 and siRNA-dependent silencing effects are genetically independent. DDM1 suppresses production of the siRNAs, however, thereby limiting RNA-directed DNA methylation at 5S rDNA repeats. We conclude that DDM1 and siRNA-dependent silencing are overlapping processes that both repress aberrant 5S rDNA transcription and contribute to the heterochromatic state of 5S rDNA arrays. PMID:19529764

Probing the structure of Nun transcription arrest factor bound to RNA polymerase

PubMed Central

Mustaev, Arkady; Vitiello, Christal L.; Gottesman, Max E.

2016-01-01

The coliphage HK022 protein Nun transcription elongation arrest factor inhibits RNA polymerase translocation. In vivo, Nun acts specifically to block transcription of the coliphage λ chromosome. Using in vitro assays, we demonstrate that Nun cross-links RNA in an RNA:DNA hybrid within a ternary elongation complex (TEC). Both the 5′ and the 3′ ends of the RNA cross-link Nun, implying that Nun contacts RNA polymerase both at the upstream edge of the RNA:DNA hybrid and in the vicinity of the catalytic center. This finding suggests that Nun may inhibit translocation by more than one mechanism. Transcription elongation factor GreA efficiently blocked Nun cross-linking to the 3′ end of the transcript, whereas the highly homologous GreB factor did not. Surprisingly, both factors strongly suppressed Nun cross-linking to the 5′ end of the RNA, suggesting that GreA and GreB can enter the RNA exit channel as well as the secondary channel, where they are known to bind. These findings extend the known action mechanism for these ubiquitous cellular factors. PMID:27436904

Capturing in vivo RNA transcriptional dynamics from the malaria parasite Plasmodium falciparum

PubMed Central

Painter, Heather J.; Carrasquilla, Manuela; Llinás, Manuel

2017-01-01

To capture the transcriptional dynamics within proliferating cells, methods to differentiate nascent transcription from preexisting mRNAs are desired. One approach is to label newly synthesized mRNA transcripts in vivo through the incorporation of modified pyrimidines. However, the human malaria parasite, Plasmodium falciparum, is incapable of pyrimidine salvage for mRNA biogenesis. To capture cellular mRNA dynamics during Plasmodium development, we engineered parasites that can salvage pyrimidines through the expression of a single bifunctional yeast fusion gene, cytosine deaminase/uracil phosphoribosyltransferase (FCU). We show that expression of FCU allows for the direct incorporation of thiol-modified pyrimidines into nascent mRNAs. Using developmental stage-specific promoters to express FCU-GFP enables the biosynthetic capture and in-depth analysis of mRNA dynamics from subpopulations of cells undergoing differentiation. We demonstrate the utility of this method by examining the transcriptional dynamics of the sexual gametocyte stage transition, a process that is essential to malaria transmission between hosts. Using the pfs16 gametocyte-specific promoter to express FCU-GFP in 3D7 parasites, we found that sexual stage commitment is governed by transcriptional reprogramming and stabilization of a subset of essential gametocyte transcripts. We also measured mRNA dynamics in F12 gametocyte-deficient parasites and demonstrate that the transcriptional program required for sexual commitment and maturation is initiated but likely aborted due to the absence of the PfAP2-G transcriptional regulator and a lack of gametocyte-specific mRNA stabilization. Biosynthetic labeling of Plasmodium mRNAs is incredibly versatile, can be used to measure transcriptional dynamics at any stage of parasite development, and will allow for future applications to comprehensively measure RNA-protein interactions in the malaria parasite. PMID:28416533

Quantification of Yeast and Bacterial Gene Transcripts in Retail Cheeses by Reverse Transcription-Quantitative PCR

PubMed Central

Straub, Cécile; Castellote, Jessie; Onesime, Djamila; Bonnarme, Pascal; Irlinger, Françoise

2013-01-01

The cheese microbiota contributes to a large extent to the development of the typical color, flavor, and texture of the final product. Its composition is not well defined in most cases and varies from one cheese to another. The aim of the present study was to establish procedures for gene transcript quantification in cheeses by reverse transcription-quantitative PCR. Total RNA was extracted from five smear-ripened cheeses purchased on the retail market, using a method that does not involve prior separation of microbial cells. 16S rRNA and malate:quinone oxidoreductase gene transcripts of Corynebacterium casei, Brevibacterium aurantiacum, and Arthrobacter arilaitensis and 26S rRNA and beta tubulin gene transcripts of Geotrichum candidum and Debaryomyces hansenii could be detected and quantified in most of the samples. Three types of normalization were applied: against total RNA, against the amount of cheese, and against a reference gene. For the first two types of normalization, differences of reverse transcription efficiencies from one sample to another were taken into account by analysis of exogenous control mRNA. No good correlation was found between the abundances of target mRNA or rRNA transcripts and the viable cell concentration of the corresponding species. However, in most cases, no mRNA transcripts were detected for species that did not belong to the dominant species. The applications of gene expression measurement in cheeses containing an undefined microbiota, as well as issues concerning the strategy of normalization and the assessment of amplification specificity, are discussed. PMID:23124230

A Land Plant-Specific Transcription Factor Directly Enhances Transcription of a Pathogenic Noncoding RNA Template by DNA-Dependent RNA Polymerase II[OPEN

PubMed Central

Qu, Jie; Ji, Shaoyi; Wallace, Andrew J.; Wu, Jian; Li, Yi; Gopalan, Venkat; Ding, Biao

2016-01-01

Some DNA-dependent RNA polymerases (DdRPs) possess RNA-dependent RNA polymerase activity, as was first discovered in the replication of Potato spindle tuber viroid (PSTVd) RNA genome in tomato (Solanum lycopersicum). Recent studies revealed that this activity in bacteria and mammals is important for transcriptional and posttranscriptional regulatory mechanisms. Here, we used PSTVd as a model to uncover auxiliary factors essential for RNA-templated transcription by DdRP. PSTVd replication in the nucleoplasm generates (−)-PSTVd intermediates and (+)-PSTVd copies. We found that the Nicotiana benthamiana canonical 9-zinc finger (ZF) Transcription Factor IIIA (TFIIIA-9ZF) as well as its variant TFIIIA-7ZF interacted with (+)-PSTVd, but only TFIIIA-7ZF interacted with (−)-PSTVd. Suppression of TFIIIA-7ZF reduced PSTVd replication, and overexpression of TFIIIA-7ZF enhanced PSTVd replication in planta. Consistent with the locale of PSTVd replication, TFIIIA-7ZF was found in the nucleoplasm and nucleolus, in contrast to the strictly nucleolar localization of TFIIIA-9ZF. Footprinting assays revealed that only TFIIIA-7ZF bound to a region of PSTVd critical for initiating transcription. Furthermore, TFIIIA-7ZF strongly enhanced the in vitro transcription of circular (+)-PSTVd by partially purified Pol II. Together, our results identify TFIIIA-7ZF as a dedicated cellular transcription factor that acts in DdRP-catalyzed RNA-templated transcription, highlighting both the extraordinary evolutionary adaptation of viroids and the potential of DdRPs for a broader role in cellular processes. PMID:27113774

RNA-directed DNA methylation involves co-transcriptional small-RNA-guided slicing of polymerase V transcripts in Arabidopsis.

PubMed

Liu, Wanlu; Duttke, Sascha H; Hetzel, Jonathan; Groth, Martin; Feng, Suhua; Gallego-Bartolome, Javier; Zhong, Zhenhui; Kuo, Hsuan Yu; Wang, Zonghua; Zhai, Jixian; Chory, Joanne; Jacobsen, Steven E

2018-03-01

Small RNAs regulate chromatin modifications such as DNA methylation and gene silencing across eukaryotic genomes. In plants, RNA-directed DNA methylation (RdDM) requires 24-nucleotide small interfering RNAs (siRNAs) that bind to ARGONAUTE 4 (AGO4) and target genomic regions for silencing. RdDM also requires non-coding RNAs transcribed by RNA polymerase V (Pol V) that probably serve as scaffolds for binding of AGO4-siRNA complexes. Here, we used a modified global nuclear run-on protocol followed by deep sequencing to capture Pol V nascent transcripts genome-wide. We uncovered unique characteristics of Pol V RNAs, including a uracil (U) common at position 10. This uracil was complementary to the 5' adenine found in many AGO4-bound 24-nucleotide siRNAs and was eliminated in a siRNA-deficient mutant as well as in the ago4/6/9 triple mutant, suggesting that the +10 U signature is due to siRNA-mediated co-transcriptional slicing of Pol V transcripts. Expression of wild-type AGO4 in ago4/6/9 mutants was able to restore slicing of Pol V transcripts, but a catalytically inactive AGO4 mutant did not correct the slicing defect. We also found that Pol V transcript slicing required SUPPRESSOR OF TY INSERTION 5-LIKE (SPT5L), an elongation factor whose function is not well understood. These results highlight the importance of Pol V transcript slicing in RNA-mediated transcriptional gene silencing, which is a conserved process in many eukaryotes.

An RNA motif advances transcription by preventing Rho-dependent termination

PubMed Central

Sevostyanova, Anastasia; Groisman, Eduardo A.

2015-01-01

The transcription termination factor Rho associates with most nascent bacterial RNAs as they emerge from RNA polymerase. However, pharmacological inhibition of Rho derepresses only a small fraction of these transcripts. What, then, determines the specificity of Rho-dependent transcription termination? We now report the identification of a Rho-antagonizing RNA element (RARE) that hinders Rho-dependent transcription termination. We establish that RARE traps Rho in an inactive complex but does not prevent Rho binding to its recruitment sites. Although translating ribosomes normally block Rho access to an mRNA, inefficient translation of an open reading frame in the leader region of the Salmonella mgtCBR operon actually enables transcription of its associated coding region by favoring an RNA conformation that sequesters RARE. The discovery of an RNA element that inactivates Rho signifies that the specificity of nucleic-acid binding proteins is defined not only by the sequences that recruit these proteins but also by sequences that antagonize their activity. PMID:26630006

A global transcriptional analysis of Plasmodium falciparum malaria reveals a novel family of telomere-associated lncRNAs

PubMed Central

2011-01-01

Background Mounting evidence suggests a major role for epigenetic feedback in Plasmodium falciparum transcriptional regulation. Long non-coding RNAs (lncRNAs) have recently emerged as a new paradigm in epigenetic remodeling. We therefore set out to investigate putative roles for lncRNAs in P. falciparum transcriptional regulation. Results We used a high-resolution DNA tiling microarray to survey transcriptional activity across 22.6% of the P. falciparum strain 3D7 genome. We identified 872 protein-coding genes and 60 putative P. falciparum lncRNAs under developmental regulation during the parasite's pathogenic human blood stage. Further characterization of lncRNA candidates led to the discovery of an intriguing family of lncRNA telomere-associated repetitive element transcripts, termed lncRNA-TARE. We have quantified lncRNA-TARE expression at 15 distinct chromosome ends and mapped putative transcriptional start and termination sites of lncRNA-TARE loci. Remarkably, we observed coordinated and stage-specific expression of lncRNA-TARE on all chromosome ends tested, and two dominant transcripts of approximately 1.5 kb and 3.1 kb transcribed towards the telomere. Conclusions We have characterized a family of 22 telomere-associated lncRNAs in P. falciparum. Homologous lncRNA-TARE loci are coordinately expressed after parasite DNA replication, and are poised to play an important role in P. falciparum telomere maintenance, virulence gene regulation, and potentially other processes of parasite chromosome end biology. Further study of lncRNA-TARE and other promising lncRNA candidates may provide mechanistic insight into P. falciparum transcriptional regulation. PMID:21689454

Temperature sensing in Yersinia pestis: translation of the LcrF activator protein is thermally regulated.

PubMed Central

Hoe, N P; Goguen, J D

1993-01-01

The lcrF gene of Yersinia pestis encodes a transcription activator responsible for inducing expression of several virulence-related proteins in response to temperature. The mechanism of this thermoregulation was investigated. An lcrF clone was found to produce much lower levels of LcrF protein at 26 than at 37 degrees C in Y. pestis, although it was transcribed at similar levels at both temperatures. High-level T7 polymerase-directed transcription of the lcrF gene in Escherichia coli also resulted in temperature-dependent production of the LcrF protein. Pulse-chase experiments showed that the LcrF protein was stable at 26 and 37 degrees C, suggesting that translation rate or message degradation is thermally controlled. The lcrF mRNA appears to be highly unstable and could not be reliably detected in Y. pestis. Insertion of the lcrF gene into plasmid pET4a, which produces high levels of plasmid-length RNA, aided detection of lcrF-specific message in E. coli. Comparison of the amount of LcrF protein produced per unit of message at 26 and 37 degrees C indicated that the efficiency of translation of lcrF message increased with temperature. mRNA secondary structure predictions suggest that the lcrF Shine-Dalgarno sequence is sequestered in a stem-loop. A model in which decreased stability of this stem-loop with increasing temperature leads to increased efficiency of translation initiation of lcrF message is presented. Images PMID:7504666

Global effects of the CSR-1 RNA interference pathway on transcriptional landscape

PubMed Central

Cecere, Germano; Hoersch, Sebastian; O’Keeffe, Sean; Sachidanandam, Ravi; Grishok, Alla

2014-01-01

Argonaute proteins and their small RNA co-factors short interfering RNAs (siRNAs) are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) antisense to germline transcripts and associates with chromatin in a siRNA-dependent manner. However, its role in gene expression regulation remains controversial. Here, we used a genome-wide profiling of nascent RNA transcripts to demonstrate that the CSR-1 RNAi pathway promotes sense-oriented Pol II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. Based on these findings, we propose that the CSR-1 pathway has a role in maintaining the directionality of active transcription thereby propagating the distinction between transcriptionally active and silent genomic regions. PMID:24681887

The Potential Organ Involved in Cantharidin Biosynthesis in Epicauta chinensis Laporte (Coleoptera: Meloidae)

PubMed Central

Jiang, Ming; Lü, Shumin

2017-01-01

Cantharidin, a terpenoid defensive toxin mainly produced by blister beetles, is among the most widely known insect natural products in the world. However, little is known about the site of cantharidin biosynthesis in vivo. Our previous research showed that 3-hydroxy-3-methylglutary-CoA reductase (HMGR) is an essential enzyme in cantharidin biosynthesis. In this report, we further investigated cantharidin titer and HMGR mRNA expression levels in different tissues of male and female Epicauta chinensis, and performed a comparative analysis of HMGR transcript levels in male Tenebrio molitor, a Tenebrionidae beetle that cannot produce cantharidin. HMGR transcripts had a positive correlation with cantharidin production. Furthermore, the specifically high amounts of HMGR transcript and abundant cantharidin production in fat body of male E. chinensis indicated the process of cantharidin synthesis may occur in the fat body. PMID:28423415

RNA Sequencing Identifies New RNase III Cleavage Sites in Escherichia coli and Reveals Increased Regulation of mRNA

DOE PAGES

Gordon, Gina C.; Cameron, Jeffrey C.; Pfleger, Brian F.

2017-03-28

Ribonucleases facilitate rapid turnover of RNA, providing cells with another mechanism to adjust transcript and protein levels in response to environmental conditions. While many examples have been documented, a comprehensive list of RNase targets is not available. To address this knowledge gap, we compared levels of RNA sequencing coverage of Escherichia coli and a corresponding RNase III mutant to expand the list of known RNase III targets. RNase III is a widespread endoribonuclease that binds and cleaves double-stranded RNA in many critical transcripts. RNase III cleavage at novel sites found in aceEF, proP, tnaC, dctA, pheM, sdhC, yhhQ, glpT, aceK,more » and gluQ accelerated RNA decay, consistent with previously described targets wherein RNase III cleavage initiates rapid degradation of secondary messages by other RNases. In contrast, cleavage at three novel sites in the ahpF, pflB, and yajQ transcripts led to stabilized secondary transcripts. Two other novel sites in hisL and pheM overlapped with transcriptional attenuators that likely serve to ensure turnover of these highly structured RNAs. Many of the new RNase III target sites are located on transcripts encoding metabolic enzymes. For instance, two novel RNase III sites are located within transcripts encoding enzymes near a key metabolic node connecting glycolysis and the tricarboxylic acid (TCA) cycle. Pyruvate dehydrogenase activity was increased in an rnc deletion mutant compared to the wild-type (WT) strain in early stationary phase, confirming the novel link between RNA turnover and regulation of pathway activity. Identification of these novel sites suggests that mRNA turnover may be an underappreciated mode of regulating metabolism. IMPORTANCE: The concerted action and overlapping functions of endoribonucleases, exoribonucleases, and RNA processing enzymes complicate the study of global RNA turnover and recycling of specific transcripts. More information about RNase specificity and activity is needed to make predictions of transcript half-life and to design synthetic transcripts with optimal stability. RNase III does not have a conserved target sequence but instead recognizes RNA secondary structure. Prior to this study, only a few RNase III target sites in E. coli were known, so we used RNA sequencing to provide a more comprehensive list of cleavage sites and to examine the impact of RNase III on transcript degradation. Finally, with this added information on how RNase III participates in transcript regulation and recycling, a more complete picture of RNA turnover can be developed for E. coli. Similar approaches could be used to augment our understanding of RNA turnover in other bacteria.« less

RNA Sequencing Identifies New RNase III Cleavage Sites in Escherichia coli and Reveals Increased Regulation of mRNA

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

Gordon, Gina C.; Cameron, Jeffrey C.; Pfleger, Brian F.

Ribonucleases facilitate rapid turnover of RNA, providing cells with another mechanism to adjust transcript and protein levels in response to environmental conditions. While many examples have been documented, a comprehensive list of RNase targets is not available. To address this knowledge gap, we compared levels of RNA sequencing coverage of Escherichia coli and a corresponding RNase III mutant to expand the list of known RNase III targets. RNase III is a widespread endoribonuclease that binds and cleaves double-stranded RNA in many critical transcripts. RNase III cleavage at novel sites found in aceEF, proP, tnaC, dctA, pheM, sdhC, yhhQ, glpT, aceK,more » and gluQ accelerated RNA decay, consistent with previously described targets wherein RNase III cleavage initiates rapid degradation of secondary messages by other RNases. In contrast, cleavage at three novel sites in the ahpF, pflB, and yajQ transcripts led to stabilized secondary transcripts. Two other novel sites in hisL and pheM overlapped with transcriptional attenuators that likely serve to ensure turnover of these highly structured RNAs. Many of the new RNase III target sites are located on transcripts encoding metabolic enzymes. For instance, two novel RNase III sites are located within transcripts encoding enzymes near a key metabolic node connecting glycolysis and the tricarboxylic acid (TCA) cycle. Pyruvate dehydrogenase activity was increased in an rnc deletion mutant compared to the wild-type (WT) strain in early stationary phase, confirming the novel link between RNA turnover and regulation of pathway activity. Identification of these novel sites suggests that mRNA turnover may be an underappreciated mode of regulating metabolism. IMPORTANCE: The concerted action and overlapping functions of endoribonucleases, exoribonucleases, and RNA processing enzymes complicate the study of global RNA turnover and recycling of specific transcripts. More information about RNase specificity and activity is needed to make predictions of transcript half-life and to design synthetic transcripts with optimal stability. RNase III does not have a conserved target sequence but instead recognizes RNA secondary structure. Prior to this study, only a few RNase III target sites in E. coli were known, so we used RNA sequencing to provide a more comprehensive list of cleavage sites and to examine the impact of RNase III on transcript degradation. Finally, with this added information on how RNase III participates in transcript regulation and recycling, a more complete picture of RNA turnover can be developed for E. coli. Similar approaches could be used to augment our understanding of RNA turnover in other bacteria.« less

Moving messages in the developing brain—emerging roles for mRNA transport and local translation in neural stem cells

PubMed Central

Pilaz, Louis-Jan; Silver, Debra L.

2017-01-01

The mammalian cerebral cortex is a complex brain structure integral to our higher cognition. During embryonic cortical development, radial glial progenitors (RGCs) produce neurons and serve as physical structures for migrating neurons. Recent discoveries highlight new roles for RNA localization and local translation in RGCs, both at the cell body and at distal structures called basal endfeet. By implementing technologies from the field of RNA research to brain development, investigators can manipulate RNA-binding proteins as well as visualize single-molecule RNAs, live movement of mRNAs and their binding proteins, and translation. Going forward, these studies establish a framework for investigating how post-transcriptional RNA regulation helps shape RGC function and triggers neurodevelopmental diseases. PMID:28304078

RNA processing in Neurospora crassa mitochondria: use of transfer RNA sequences as signals.

PubMed Central

Breitenberger, C A; Browning, K S; Alzner-DeWeerd, B; RajBhandary, U L

1985-01-01

We have used RNA gel transfer hybridization, S1 nuclease mapping and primer extension to analyze transcripts derived from several genes in Neurospora crassa mitochondria. The transcripts studied include those for cytochrome oxidase subunit III, 17S rRNA and an unidentified open reading frame. In all three cases, initial transcripts are long, include tRNA sequences, and are subsequently processed to generate the mature RNAs. We find that endpoints of the most abundant transcripts generally coincide with those of tRNA sequences. We therefore conclude that tRNA sequences in long transcripts act as primary signals for RNA processing in N. crassa mitochondria. The situation is somewhat analogous to that observed in mammalian mitochondrial systems. The difference, however, is that in mammalian mitochondria, noncoding spacers between tRNA, rRNA and protein genes are very short and in many cases non-existent, allowing no room for intergenic RNA processing signals whereas, in N. crassa mtDNA, intergenic non-coding sequences are usually several hundred nucleotides long and contain highly conserved GC-rich palindromic sequences. Since these GC-rich palindromic sequences are retained in the processed mature RNAs, we conclude that they do not serve as signals for RNA processing. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2990893

A screen for nuclear transcripts identifies two linked noncoding RNAs associated with SC35 splicing domains

PubMed Central

Hutchinson, John N; Ensminger, Alexander W; Clemson, Christine M; Lynch, Christopher R; Lawrence, Jeanne B; Chess, Andrew

2007-01-01

Background Noncoding RNA species play a diverse set of roles in the eukaryotic cell. While much recent attention has focused on smaller RNA species, larger noncoding transcripts are also thought to be highly abundant in mammalian cells. To search for large noncoding RNAs that might control gene expression or mRNA metabolism, we used Affymetrix expression arrays to identify polyadenylated RNA transcripts displaying nuclear enrichment. Results This screen identified no more than three transcripts; XIST, and two unique noncoding nuclear enriched abundant transcripts (NEAT) RNAs strikingly located less than 70 kb apart on human chromosome 11: NEAT1, a noncoding RNA from the locus encoding for TncRNA, and NEAT2 (also known as MALAT-1). While the two NEAT transcripts share no significant homology with each other, each is conserved within the mammalian lineage, suggesting significant function for these noncoding RNAs. NEAT2 is extraordinarily well conserved for a noncoding RNA, more so than even XIST. Bioinformatic analyses of publicly available mouse transcriptome data support our findings from human cells as they confirm that the murine homologs of these noncoding RNAs are also nuclear enriched. RNA FISH analyses suggest that these noncoding RNAs function in mRNA metabolism as they demonstrate an intimate association of these RNA species with SC35 nuclear speckles in both human and mouse cells. These studies show that one of these transcripts, NEAT1 localizes to the periphery of such domains, whereas the neighboring transcript, NEAT2, is part of the long-sought polyadenylated component of nuclear speckles. Conclusion Our genome-wide screens in two mammalian species reveal no more than three abundant large non-coding polyadenylated RNAs in the nucleus; the canonical large noncoding RNA XIST and NEAT1 and NEAT2. The function of these noncoding RNAs in mRNA metabolism is suggested by their high levels of conservation and their intimate association with SC35 splicing domains in multiple mammalian species. PMID:17270048

Quantifying Intrinsic and Extrinsic Variability in Stochastic Gene Expression Models

PubMed Central

Singh, Abhyudai; Soltani, Mohammad

2013-01-01

Genetically identical cell populations exhibit considerable intercellular variation in the level of a given protein or mRNA. Both intrinsic and extrinsic sources of noise drive this variability in gene expression. More specifically, extrinsic noise is the expression variability that arises from cell-to-cell differences in cell-specific factors such as enzyme levels, cell size and cell cycle stage. In contrast, intrinsic noise is the expression variability that is not accounted for by extrinsic noise, and typically arises from the inherent stochastic nature of biochemical processes. Two-color reporter experiments are employed to decompose expression variability into its intrinsic and extrinsic noise components. Analytical formulas for intrinsic and extrinsic noise are derived for a class of stochastic gene expression models, where variations in cell-specific factors cause fluctuations in model parameters, in particular, transcription and/or translation rate fluctuations. Assuming mRNA production occurs in random bursts, transcription rate is represented by either the burst frequency (how often the bursts occur) or the burst size (number of mRNAs produced in each burst). Our analysis shows that fluctuations in the transcription burst frequency enhance extrinsic noise but do not affect the intrinsic noise. On the contrary, fluctuations in the transcription burst size or mRNA translation rate dramatically increase both intrinsic and extrinsic noise components. Interestingly, simultaneous fluctuations in transcription and translation rates arising from randomness in ATP abundance can decrease intrinsic noise measured in a two-color reporter assay. Finally, we discuss how these formulas can be combined with single-cell gene expression data from two-color reporter experiments for estimating model parameters. PMID:24391934

Quantifying intrinsic and extrinsic variability in stochastic gene expression models.

PubMed

Singh, Abhyudai; Soltani, Mohammad

2013-01-01

Genetically identical cell populations exhibit considerable intercellular variation in the level of a given protein or mRNA. Both intrinsic and extrinsic sources of noise drive this variability in gene expression. More specifically, extrinsic noise is the expression variability that arises from cell-to-cell differences in cell-specific factors such as enzyme levels, cell size and cell cycle stage. In contrast, intrinsic noise is the expression variability that is not accounted for by extrinsic noise, and typically arises from the inherent stochastic nature of biochemical processes. Two-color reporter experiments are employed to decompose expression variability into its intrinsic and extrinsic noise components. Analytical formulas for intrinsic and extrinsic noise are derived for a class of stochastic gene expression models, where variations in cell-specific factors cause fluctuations in model parameters, in particular, transcription and/or translation rate fluctuations. Assuming mRNA production occurs in random bursts, transcription rate is represented by either the burst frequency (how often the bursts occur) or the burst size (number of mRNAs produced in each burst). Our analysis shows that fluctuations in the transcription burst frequency enhance extrinsic noise but do not affect the intrinsic noise. On the contrary, fluctuations in the transcription burst size or mRNA translation rate dramatically increase both intrinsic and extrinsic noise components. Interestingly, simultaneous fluctuations in transcription and translation rates arising from randomness in ATP abundance can decrease intrinsic noise measured in a two-color reporter assay. Finally, we discuss how these formulas can be combined with single-cell gene expression data from two-color reporter experiments for estimating model parameters.

The transcriptional stress response of Candida albicans to weak organic acids.

PubMed

Cottier, Fabien; Tan, Alrina Shin Min; Chen, Jinmiao; Lum, Josephine; Zolezzi, Francesca; Poidinger, Michael; Pavelka, Norman

2015-01-29

Candida albicans is the most important fungal pathogen of humans, causing severe infections, especially in nosocomial and immunocompromised settings. However, it is also the most prevalent fungus of the normal human microbiome, where it shares its habitat with hundreds of trillions of other microbial cells. Despite weak organic acids (WOAs) being among the most abundant metabolites produced by bacterial microbiota, little is known about their effect on C. albicans. Here we used a sequencing-based profiling strategy to systematically investigate the transcriptional stress response of C. albicans to lactic, acetic, propionic, and butyric acid at several time points after treatment. Our data reveal a complex transcriptional response, with individual WOAs triggering unique gene expression profiles and with important differences between acute and chronic exposure. Despite these dissimilarities, we found significant overlaps between the gene expression changes induced by each WOA, which led us to uncover a core transcriptional response that was largely unrelated to other previously published C. albicans transcriptional stress responses. Genes commonly up-regulated by WOAs were enriched in several iron transporters, which was associated with an overall decrease in intracellular iron concentrations. Moreover, chronic exposure to any WOA lead to down-regulation of RNA synthesis and ribosome biogenesis genes, which resulted in significant reduction of total RNA levels and of ribosomal RNA in particular. In conclusion, this study suggests that gastrointestinal microbiota might directly influence C. albicans physiology via production of WOAs, with possible implications of how this fungus interacts with its host in both health and disease. Copyright © 2015 Cottier et al.

The Transcriptional Stress Response of Candida albicans to Weak Organic Acids

PubMed Central

Cottier, Fabien; Tan, Alrina Shin Min; Chen, Jinmiao; Lum, Josephine; Zolezzi, Francesca; Poidinger, Michael; Pavelka, Norman

2015-01-01

Candida albicans is the most important fungal pathogen of humans, causing severe infections, especially in nosocomial and immunocompromised settings. However, it is also the most prevalent fungus of the normal human microbiome, where it shares its habitat with hundreds of trillions of other microbial cells. Despite weak organic acids (WOAs) being among the most abundant metabolites produced by bacterial microbiota, little is known about their effect on C. albicans. Here we used a sequencing-based profiling strategy to systematically investigate the transcriptional stress response of C. albicans to lactic, acetic, propionic, and butyric acid at several time points after treatment. Our data reveal a complex transcriptional response, with individual WOAs triggering unique gene expression profiles and with important differences between acute and chronic exposure. Despite these dissimilarities, we found significant overlaps between the gene expression changes induced by each WOA, which led us to uncover a core transcriptional response that was largely unrelated to other previously published C. albicans transcriptional stress responses. Genes commonly up-regulated by WOAs were enriched in several iron transporters, which was associated with an overall decrease in intracellular iron concentrations. Moreover, chronic exposure to any WOA lead to down-regulation of RNA synthesis and ribosome biogenesis genes, which resulted in significant reduction of total RNA levels and of ribosomal RNA in particular. In conclusion, this study suggests that gastrointestinal microbiota might directly influence C. albicans physiology via production of WOAs, with possible implications of how this fungus interacts with its host in both health and disease. PMID:25636313

KDM1A triggers androgen-induced miRNA transcription via H3K4me2 demethylation and DNA oxidation.

PubMed

Yang, Shu; Zhang, Jiyuan; Zhang, Yalong; Wan, Xuechao; Zhang, Congzhe; Huang, Xiaohui; Huang, Wenhua; Pu, Honglei; Pei, Chaohan; Wu, Hai; Huang, Yan; Huang, Shengdong; Li, Yao

2015-06-15

Androgen receptor (AR) is a ligand dependent transcription factor that regulates the transcription of target genes. AR activity is closely involved in the maintenance and progression of prostate cancer. After the binding with androgen, AR moves into nucleus and binds to DNA sequence containing androgen response elements (ARE). Flavin-dependent monoamine oxidase KDM1A is necessary for AR driven transcription while the mechanism remains unclear. The association between androgen-dependent transcription and oxidation was tested through pharmaceutical inhibitions and siRNA knockdown of DNA oxidation repair components in prostate cancer cells. The recruitment of involved proteins and the histone methylation dynamics on ARE region was explored by chromatin immunoprecipitation (ChIP). Oxidation inhibition reduced AR dependent expression of KLK3, TMPRSS2, hsa-miR-125b2, and hsa-miR-133b. And such reduction could be restored by H2 O2 treatment. KDM1A recruitment and H3K4me2 demethylation on ARE regions, which produce H2 O2 , are associated with AR targets transcription. AR targets transcription and coupled oxidation recruit 8-oxoguanine-DNA glycosylase (OGG1) and the nuclease APEX1 to ARE regions. Such recruitment depends on KDM1A, and is necessary for AR targets transcription. Our work underlined the importance of histone demethylation and DNA oxidation/repairing machinery in androgen-dependent transcription. The present finds have implications for research into new druggable targets for prostate cancer relying on the cascade of AR activity regulation. © 2015 Wiley Periodicals, Inc.

An integrated approach to characterize transcription factor and microRNA regulatory networks involved in Schwann cell response to peripheral nerve injury

PubMed Central

2013-01-01

Background The regenerative response of Schwann cells after peripheral nerve injury is a critical process directly related to the pathophysiology of a number of neurodegenerative diseases. This SC injury response is dependent on an intricate gene regulatory program coordinated by a number of transcription factors and microRNAs, but the interactions among them remain largely unknown. Uncovering the transcriptional and post-transcriptional regulatory networks governing the Schwann cell injury response is a key step towards a better understanding of Schwann cell biology and may help develop novel therapies for related diseases. Performing such comprehensive network analysis requires systematic bioinformatics methods to integrate multiple genomic datasets. Results In this study we present a computational pipeline to infer transcription factor and microRNA regulatory networks. Our approach combined mRNA and microRNA expression profiling data, ChIP-Seq data of transcription factors, and computational transcription factor and microRNA target prediction. Using mRNA and microRNA expression data collected in a Schwann cell injury model, we constructed a regulatory network and studied regulatory pathways involved in Schwann cell response to injury. Furthermore, we analyzed network motifs and obtained insights on cooperative regulation of transcription factors and microRNAs in Schwann cell injury recovery. Conclusions This work demonstrates a systematic method for gene regulatory network inference that may be used to gain new information on gene regulation by transcription factors and microRNAs. PMID:23387820

Splice-Site Mutations Cause Rrp6-Mediated Nuclear Retention of the Unspliced RNAs and Transcriptional Down-Regulation of the Splicing-Defective Genes

PubMed Central

Eberle, Andrea B.; Hessle, Viktoria; Helbig, Roger; Dantoft, Widad; Gimber, Niclas; Visa, Neus

2010-01-01

Background Eukaryotic cells have developed surveillance mechanisms to prevent the expression of aberrant transcripts. An early surveillance checkpoint acts at the transcription site and prevents the release of mRNAs that carry processing defects. The exosome subunit Rrp6 is required for this checkpoint in Saccharomyces cerevisiae, but it is not known whether Rrp6 also plays a role in mRNA surveillance in higher eukaryotes. Methodology/Principal Findings We have developed an in vivo system to study nuclear mRNA surveillance in Drosophila melanogaster. We have produced S2 cells that express a human β-globin gene with mutated splice sites in intron 2 (mut β-globin). The transcripts encoded by the mut β-globin gene are normally spliced at intron 1 but retain intron 2. The levels of the mut β-globin transcripts are much lower than those of wild type (wt) ß-globin mRNAs transcribed from the same promoter. We have compared the expression of the mut and wt β-globin genes to investigate the mechanisms that down-regulate the production of defective mRNAs. Both wt and mut β-globin transcripts are processed at the 3′, but the mut β-globin transcripts are less efficiently cleaved than the wt transcripts. Moreover, the mut β-globin transcripts are less efficiently released from the transcription site, as shown by FISH, and this defect is restored by depletion of Rrp6 by RNAi. Furthermore, transcription of the mut β-globin gene is significantly impaired as revealed by ChIP experiments that measure the association of the RNA polymerase II with the transcribed genes. We have also shown that the mut β-globin gene shows reduced levels of H3K4me3. Conclusions/Significance Our results show that there are at least two surveillance responses that operate cotranscriptionally in insect cells and probably in all metazoans. One response requires Rrp6 and results in the inefficient release of defective mRNAs from the transcription site. The other response acts at the transcription level and reduces the synthesis of the defective transcripts through a mechanism that involves histone modifications. PMID:20634951

An Annotation Agnostic Algorithm for Detecting Nascent RNA Transcripts in GRO-Seq.

PubMed

Azofeifa, Joseph G; Allen, Mary A; Lladser, Manuel E; Dowell, Robin D

2017-01-01

We present a fast and simple algorithm to detect nascent RNA transcription in global nuclear run-on sequencing (GRO-seq). GRO-seq is a relatively new protocol that captures nascent transcripts from actively engaged polymerase, providing a direct read-out on bona fide transcription. Most traditional assays, such as RNA-seq, measure steady state RNA levels which are affected by transcription, post-transcriptional processing, and RNA stability. GRO-seq data, however, presents unique analysis challenges that are only beginning to be addressed. Here, we describe a new algorithm, Fast Read Stitcher (FStitch), that takes advantage of two popular machine-learning techniques, hidden Markov models and logistic regression, to classify which regions of the genome are transcribed. Given a small user-defined training set, our algorithm is accurate, robust to varying read depth, annotation agnostic, and fast. Analysis of GRO-seq data without a priori need for annotation uncovers surprising new insights into several aspects of the transcription process.

Dampening DNA binding: a common mechanism of transcriptional repression for both ncRNAs and protein domains.

PubMed

Goodrich, James A; Kugel, Jennifer F

2010-01-01

With eukaryotic non-coding RNAs (ncRNAs) now established as critical regulators of cellular transcription, the true diversity with which they can elicit biological effects is beginning to be appreciated. Two ncRNAs, mouse B2 RNA and human Alu RNA, have been found to repress mRNA transcription in response to heat shock. They do so by binding directly to RNA polymerase II, assembling into complexes on promoter DNA, and disrupting contacts between the polymerase and the DNA. Such a mechanism of repression had not previously been observed for a eukaryotic ncRNA; however, there are examples of eukaryotic protein domains that repress transcription by blocking essential protein-DNA interactions. Comparing the mechanism of transcriptional repression utilized by these protein domains to that used by B2 and Alu RNAs raises intriguing questions regarding transcriptional control, and how B2 and Alu RNAs might themselves be regulated.

A Conserved Nuclear Cyclophilin Is Required for Both RNA Polymerase II Elongation and Co-transcriptional Splicing in Caenorhabditis elegans

PubMed Central

Ahn, Jeong H.; Rechsteiner, Andreas; Strome, Susan; Kelly, William G.

2016-01-01

The elongation phase of transcription by RNA Polymerase II (Pol II) involves numerous events that are tightly coordinated, including RNA processing, histone modification, and chromatin remodeling. RNA splicing factors are associated with elongating Pol II, and the interdependent coupling of splicing and elongation has been documented in several systems. Here we identify a conserved, multi-domain cyclophilin family member, SIG-7, as an essential factor for both normal transcription elongation and co-transcriptional splicing. In embryos depleted for SIG-7, RNA levels for over a thousand zygotically expressed genes are substantially reduced, Pol II becomes significantly reduced at the 3’ end of genes, marks of transcription elongation are reduced, and unspliced mRNAs accumulate. Our findings suggest that SIG-7 plays a central role in both Pol II elongation and co-transcriptional splicing and may provide an important link for their coordination and regulation. PMID:27541139

Strategic Approach To Produce Low-Cost, Efficient, and Stable Competitive Internal Controls for Detection of RNA Viruses by Use of Reverse Transcription-PCR▿

PubMed Central

Villanova, Gabriela V.; Gardiol, Daniela; Taborda, Miguel A.; Reggiardo, Virginia; Tanno, Hugo; Rivadeneira, Emilia D.; Perez, Germán R.; Giri, Adriana A.

2007-01-01

Molecular diagnostics based on reverse transcription (RT)-PCR are routinely complicated by the lack of stable internal controls, leading to falsely negative results. We describe a strategy to produce a stable competitive internal control (CIC) based on a Qβ phage derivative (recombinant Qβ [rQβ]) bearing primers KY78 and KY80, which are widely used in the detection of hepatitis C virus (HCV). rQβ was RNase resistant and stable at 4°C for 452 days in SM medium (0.1 M NaCl, 8 mM MgSO4·7H2O, 50 mM Tris HCl [pH 7.5], 2% gelatin) and for 125 days after lyophilization and reconstitution. rQβ performance as a CIC was evaluated. rQβ was added to HCV-positive samples, followed by RNA extraction and a CIC-HCV RT-PCR assay. This method combines RT-PCR, liquid hybridization with nonradioactive probes, and enzyme immunoanalysis. No influence of the CIC on qualitative HCV detection was observed independently of viral load, and results had high concordance with those of commercial kits. In conclusion, we describe a versatile, low-cost alternative strategy to armored RNA technology that can be adapted for detection or real-time applications of any RNA target. Moreover, the CIC reported here is an essential reagent for HCV screening in blood banks in resource-limited settings. PMID:17699653

Strategic approach to produce low-cost, efficient, and stable competitive internal controls for detection of RNA viruses by use of reverse transcription-PCR.

PubMed

Villanova, Gabriela V; Gardiol, Daniela; Taborda, Miguel A; Reggiardo, Virginia; Tanno, Hugo; Rivadeneira, Emilia D; Perez, Germán R; Giri, Adriana A

2007-11-01

Molecular diagnostics based on reverse transcription (RT)-PCR are routinely complicated by the lack of stable internal controls, leading to falsely negative results. We describe a strategy to produce a stable competitive internal control (CIC) based on a Qbeta phage derivative (recombinant Qbeta [rQbeta]) bearing primers KY78 and KY80, which are widely used in the detection of hepatitis C virus (HCV). rQbeta was RNase resistant and stable at 4 degrees C for 452 days in SM medium (0.1 M NaCl, 8 mM MgSO(4).7H(2)O, 50 mM Tris HCl [pH 7.5], 2% gelatin) and for 125 days after lyophilization and reconstitution. rQbeta performance as a CIC was evaluated. rQbeta was added to HCV-positive samples, followed by RNA extraction and a CIC-HCV RT-PCR assay. This method combines RT-PCR, liquid hybridization with nonradioactive probes, and enzyme immunoanalysis. No influence of the CIC on qualitative HCV detection was observed independently of viral load, and results had high concordance with those of commercial kits. In conclusion, we describe a versatile, low-cost alternative strategy to armored RNA technology that can be adapted for detection or real-time applications of any RNA target. Moreover, the CIC reported here is an essential reagent for HCV screening in blood banks in resource-limited settings.

Nascent RNA kinetics: Transient and steady state behavior of models of transcription

NASA Astrophysics Data System (ADS)

Choubey, Sandeep

2018-02-01

Regulation of transcription is a vital process in cells, but mechanistic details of this regulation still remain elusive. The dominant approach to unravel the dynamics of transcriptional regulation is to first develop mathematical models of transcription and then experimentally test the predictions these models make for the distribution of mRNA and protein molecules at the individual cell level. However, these measurements are affected by a multitude of downstream processes which make it difficult to interpret the measurements. Recent experimental advancements allow for counting the nascent mRNA number of a gene as a function of time at the single-inglr cell level. These measurements closely reflect the dynamics of transcription. In this paper, we consider a general mechanism of transcription with stochastic initiation and deterministic elongation and probe its impact on the temporal behavior of nascent RNA levels. Using techniques from queueing theory, we derive exact analytical expressions for the mean and variance of the nascent RNA distribution as functions of time. We apply these analytical results to obtain the mean and variance of nascent RNA distribution for specific models of transcription. These models of initiation exhibit qualitatively distinct transient behaviors for both the mean and variance which further allows us to discriminate between them. Stochastic simulations confirm these results. Overall the analytical results presented here provide the necessary tools to connect mechanisms of transcription initiation to single-cell measurements of nascent RNA.

Mediator directs co-transcriptional heterochromatin assembly by RNA interference-dependent and -independent pathways.

PubMed

Oya, Eriko; Kato, Hiroaki; Chikashige, Yuji; Tsutsumi, Chihiro; Hiraoka, Yasushi; Murakami, Yota

2013-01-01

Heterochromatin at the pericentromeric repeats in fission yeast is assembled and spread by an RNAi-dependent mechanism, which is coupled with the transcription of non-coding RNA from the repeats by RNA polymerase II. In addition, Rrp6, a component of the nuclear exosome, also contributes to heterochromatin assembly and is coupled with non-coding RNA transcription. The multi-subunit complex Mediator, which directs initiation of RNA polymerase II-dependent transcription, has recently been suggested to function after initiation in processes such as elongation of transcription and splicing. However, the role of Mediator in the regulation of chromatin structure is not well understood. We investigated the role of Mediator in pericentromeric heterochromatin formation and found that deletion of specific subunits of the head domain of Mediator compromised heterochromatin structure. The Mediator head domain was required for Rrp6-dependent heterochromatin nucleation at the pericentromere and for RNAi-dependent spreading of heterochromatin into the neighboring region. In the latter process, Mediator appeared to contribute to efficient processing of siRNA from transcribed non-coding RNA, which was required for efficient spreading of heterochromatin. Furthermore, the head domain directed efficient transcription in heterochromatin. These results reveal a pivotal role for Mediator in multiple steps of transcription-coupled formation of pericentromeric heterochromatin. This observation further extends the role of Mediator to co-transcriptional chromatin regulation.

Single-molecule RNA observation in vivo reveals dynamics of co-transcriptional splicing

NASA Astrophysics Data System (ADS)

Ferguson, M. L.; Coulon, A.; de Turris, V.; Palangat, M.; Chow, C. C.; Singer, R. H.; Larson, D. R.

2013-03-01

The synthesis of pre-mRNA and the splicing of that pre-mRNA to form completed transcripts requires coordination between two large multi-subunit complexes (the transcription elongation complex and the spliceosome). How this coordination occurs in vivo is unknown. Here we report the first experimental observation of transcription and splicing occurring at the same gene in living cells. By utilizing the PP7/MS2 fluorescent RNA reporter system, we can directly observe two distinct regions of the nascent RNA, allowing us to measure the rise and fall time of the intron and exon of a reporter gene stably integrated into a human cell line. The reporter gene consists of a beta globin gene where we have inserted a 24 RNA hairpin cassette into the intron/exon. Upon synthesis, the RNA hairpins are tightly bound by fluorescently-labeled PP7/MS2 bacteriophage coat proteins. After gene induction, a single locus of active transcription in the nucleus shows fluorescence intensity changes characteristic of the synthesis and excision of the intron/exon. Using fluctuation analysis, we determine the elongation rate to be 1.5 kb/min. From the temporal cross correlation function, we determine that splicing of this gene must be co-transcriptional with a splicing time of ~100 seconds before termination and a ~200 second pause at termination. We propose that dual-color RNA imaging may be extended to investigate other mechanisms of transcription, gene regulation, and RNA processing.

A novel RNA species from the turtle mitochondrial genome: induction and regulation of transcription and processing under anoxic and freezing stresses.

PubMed

Cai, Q; Storey, K B

1997-08-01

The present study identifies a previously cloned cDNA, pBTaR914, as homologous to the mitochondrial WANCY (tryptophan, alanine, asparagine, cysteine, and tyrosine) tRNA gene cluster. This cDNA clone has a 304-bp sequence and its homologue, pBTaR09, has a 158-bp sequence with a long poly(A)+ tail (more than 60 adenosines). RNA blotting analysis using pBTaR914 probe against the total RNA from the tissues of adult and hatchling turtles revealed five bands: 540, 1800, 2200, 3200, and 3900 nucleotides (nt). The 540-nt transcript is considered to be an intact mtRNA unit from a novel mtDNA gene designated WANCYHP that overlaps the WANCY tRNA gene cluster region. This transcript was highly induced by both anoxic and freezing stresses in turtle heart. The other transcripts are considered to be the processed intermediates of mtRNA transcripts with WANCYHP sequence. All these transcripts were differentially regulated by anoxia and freezing in different organs. The data suggest that mtRNA processing is sensitive to regulation by external stresses, oxygen deprivation, and freezing. Furthermore, the fact that the WANCYHP transcript is highly induced during anoxic exposure suggests that it may play an important role in the regulation of mitochondrial activities to coordinate the physiological adaptation to anoxia.

Construction and characterization of poliovirus subgenomic replicons.

PubMed

Kaplan, G; Racaniello, V R

1988-05-01

Poliovirus RNAs containing in-frame deletions within the capsid-coding region were produced by in vitro transcription of altered poliovirus type 1 cDNA by using bacteriophage T7 RNA polymerase. Three RNAs were transcribed that contained deletions of 2,317 nucleotides (bases 747 to 3064), 1,781 nucleotides (bases 1,175 to 2,956), and 1,295 nucleotides (bases 1,175 to 2,470). All three subgenomic RNAs replicated after transfection into HeLa cells, demonstrating that sequences encoding the capsid polypeptides are not essential for viral RNA replication in vivo. Viral RNA containing the largest deletion (R1) replicated approximately three times better than full-length RNA produced in vitro. Northern blot (RNA blot) hybridization analysis of total cellular RNA from HeLa cells at different times after transfection with R1 demonstrated the presence of increasing amounts of the expected 5.1-kilobase subgenomic RNA. Analysis by immunoprecipitation of viral proteins induced after transfection of R1 RNA into HeLa cells revealed the presence of proteins 2Apro, 2C, and 3Dpol and its precursors, suggesting that the polyprotein cleavages are similar to those occurring in virus-infected cells. Replication of P2/Lansing virion RNA was inhibited by cotransfection with the R1 replicon, as demonstrated by hybridization analysis with a serotype-specific oligonucleotide probe. A higher level of inhibition of RNA replication was observed when P2/Lansing RNA was cotransfected into HeLa cells with truncated R1 transcripts (R1-PvuII) that were missing 395 3' nucleotides and a poly(A) tail. These internally and terminally deleted RNAs inhibited the replication of subgenomic replicons R1, R2, and R3 and caused a reduction in plaque size when cotransfected with P1/Mahoney or P2/Lansing viral RNA, suggesting that individual cells had received both RNAs. No inhibition of plaque size was observed when replicon RNAs were used that were missing 1,384 or 1,839 3' nucleotides or contained plasmid-derived sequences downstream of the 3' poly(A). The trans-acting inhibitory effect of R1-PvuII on the replication of poliovirus P2/Lansing RNA did not involve entry of RNA into cells and appeared to reduce viral translation and RNA synthesis late in the infection cycle.

Evidence for the Packaging of Multiple Copies of Tf1 mRNA into Particles and the trans Priming of Reverse Transcription

PubMed Central

Haag, Amanda Leigh; Lin, Jia-Hwei; Levin, Henry L.

2000-01-01

Long terminal repeat (LTR)-containing retrotransposons and retroviruses are close relatives that possess similar mechanisms of reverse transcription. The particles of retroviruses package two copies of viral mRNA that both function as templates for the reverse transcription of the element. We studied the LTR-retrotransposon Tf1 of Schizosaccharomyces pombe to test whether multiple copies of transposon mRNA participate in the production of cDNA. Using the unique self-priming property of Tf1, we obtained evidence that multiple copies of Tf1 mRNA were packaged into virus-like particles. By coexpressing two distinct versions of Tf1, we found that the bulk of reverse transcription that was initiated on one mRNA template was subsequently transferred to others. In addition, the first 11 nucleotides of one mRNA were able to prime, in trans, the reverse transcription of another mRNA. PMID:10888658

Chronic nicotine treatment leads to induction of tyrosine hydroxylase in locus ceruleus neurons: the role of transcriptional activation.

PubMed

Sun, Baoyong; Chen, Xiqun; Xu, Lu; Sterling, Carol; Tank, A William

2004-10-01

Chronic nicotine treatment (two daily subcutaneous injections administered approximately 12 h apart for 14 days) is associated with long-term inductions of tyrosine hydroxylase (TH) protein and TH mRNA in locus ceruleus (LC) neurons. These increases persist for at least 3 days after the final nicotine injection in LC cell bodies and for at least 7 to 10 days in LC nerve terminal regions. We tested whether this long-term response is due to sustained stimulation of TH gene transcription rate. A semiquantitative reverse transcription-polymerase chain reaction assay was developed to assess changes in the levels of TH RNA primary transcripts; these changes are an indirect measurement of changes in TH gene transcription rate. TH RNA primary transcript levels increase rapidly in the LC after a single nicotine administration and return to basal levels by 24 h. A similar rapid and transient induction of LC TH RNA primary transcripts occurs after chronic nicotine administration. In contrast, TH RNA primary transcript levels remain elevated for a sustained period of time (at least 1 day) in the adrenal medulla after chronic nicotine administration. Similar rapid, but transient changes in LC TH RNA primary transcript levels are observed after repeated immobilization stress. These results suggest that TH gene transcription rate in the LC is stimulated rapidly after each nicotine injection; however, in contrast to the adrenal medulla, there is no sustained transcriptional response elicited by chronic nicotine treatment or repeated immobilization stress in the LC, suggesting that post-transcriptional mechanisms may also play a role in these long-term responses.

Folate deficiency facilitates recruitment of upstream binding factor to hot spots of DNA double-strand breaks of rRNA genes and promotes its transcription.

PubMed

Xie, Qiu; Li, Caihua; Song, Xiaozhen; Wu, Lihua; Jiang, Qian; Qiu, Zhiyong; Cao, Haiyan; Yu, Kaihui; Wan, Chunlei; Li, Jianting; Yang, Feng; Huang, Zebing; Niu, Bo; Jiang, Zhengwen; Zhang, Ting

2017-03-17

The biogenesis of ribosomes in vivo is an essential process for cellular functions. Transcription of ribosomal RNA (rRNA) genes is the rate-limiting step in ribosome biogenesis controlled by environmental conditions. Here, we investigated the role of folate antagonist on changes of DNA double-strand breaks (DSBs) landscape in mouse embryonic stem cells. A significant DSB enhancement was detected in the genome of these cells and a large majority of these DSBs were found in rRNA genes. Furthermore, spontaneous DSBs in cells under folate deficiency conditions were located exclusively within the rRNA gene units, representing a H3K4me1 hallmark. Enrichment H3K4me1 at the hot spots of DSB regions enhanced the recruitment of upstream binding factor (UBF) to rRNA genes, resulting in the increment of rRNA genes transcription. Supplement of folate resulted in a restored UBF binding across DNA breakage sites of rRNA genes, and normal rRNA gene transcription. In samples from neural tube defects (NTDs) with low folate level, up-regulation of rRNA gene transcription was observed, along with aberrant UBF level. Our results present a new view by which alterations in folate levels affects DNA breakage through epigenetic control leading to the regulation of rRNA gene transcription during the early stage of development. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Stimulation of Pol III-dependent 5S rRNA and U6 snRNA gene expression by AP-1 transcription factors.

PubMed

Ahuja, Richa; Kumar, Vijay

2017-07-01

RNA polymerase III transcribes structurally diverse group of essential noncoding RNAs including 5S ribosomal RNA (5SrRNA) and U6 snRNA. These noncoding RNAs are involved in RNA processing and ribosome biogenesis, thus, coupling Pol III activity to the rate of protein synthesis, cell growth, and proliferation. Even though a few Pol II-associated transcription factors have been reported to participate in Pol III-dependent transcription, its activation by activator protein 1 (AP-1) factors, c-Fos and c-Jun, has remained unexplored. Here, we show that c-Fos and c-Jun bind to specific sites in the regulatory regions of 5S rRNA (type I) and U6 snRNA (type III) gene promoters and stimulate their transcription. Our chromatin immunoprecipitation studies suggested that endogenous AP-1 factors bind to their cognate promoter elements during the G1/S transition of cell cycle apparently synchronous with Pol III transcriptional activity. Furthermore, the interaction of c-Jun with histone acetyltransferase p300 promoted the recruitment of p300/CBP complex on the promoters and facilitated the occupancy of Pol III transcriptional machinery via histone acetylation and chromatin remodeling. The findings of our study, together, suggest that AP-1 factors are novel regulators of Pol III-driven 5S rRNA and U6 snRNA expression with a potential role in cell proliferation. © 2017 Federation of European Biochemical Societies.

Flavonoids from Theobroma cacao down-regulate inflammatory mediators.

PubMed

Ramiro, Emma; Franch, Angels; Castellote, Cristina; Pérez-Cano, Francisco; Permanyer, Joan; Izquierdo-Pulido, Maria; Castell, Margarida

2005-11-02

In the present study, we report the effects of a cocoa extract on the secretion and RNA expression of various proinflammatory mediators by macrophages. Monocyte chemoattractant protein 1 and tumor necrosis factor alpha (TNFalpha) were significantly and dose-dependently diminished by cocoa extract, and this effect was higher than that produced by equivalent concentrations of epicatechin but was lower than that produced by isoquercitrin. Interestingly, cocoa extract added prior to cell activation resulted in a significantly greater inhibition of TNFalpha secretion. Both cocoa extract and epicatechin decreased TNFalpha, interleukin (IL) 1alpha, and IL-6 mRNA expression, suggesting that their inhibitory effect on cytokine secretion is produced, in part, at the transcriptional level. Cocoa extract also significantly decreased NO secretion in a dose-dependent manner and with a greater effect than that produced by epicatechin. In conclusion, our study shows that cocoa flavonoids not only inhibit NO release from macrophages but also down-regulate inflammatory cytokines and chemokines.

New insights into the promoterless transcription of DNA coligo templates by RNA polymerase III.

PubMed

Lama, Lodoe; Seidl, Christine I; Ryan, Kevin

2014-01-01

Chemically synthesized DNA can carry small RNA sequence information but converting that information into small RNA is generally thought to require large double-stranded promoters in the context of plasmids, viruses and genes. We previously found evidence that circularized oligodeoxynucleotides (coligos) containing certain sequences and secondary structures can template the synthesis of small RNA by RNA polymerase III in vitro and in human cells. By using immunoprecipitated RNA polymerase III we now report corroborating evidence that this enzyme is the sole polymerase responsible for coligo transcription. The immobilized polymerase enabled experiments showing that coligo transcripts can be formed through transcription termination without subsequent 3' end trimming. To better define the determinants of productive transcription, a structure-activity relationship study was performed using over 20 new coligos. The results show that unpaired nucleotides in the coligo stem facilitate circumtranscription, but also that internal loops and bulges should be kept small to avoid secondary transcription initiation sites. A polymerase termination sequence embedded in the double-stranded region of a hairpin-encoding coligo stem can antagonize transcription. Using lessons learned from new and old coligos, we demonstrate how to convert poorly transcribed coligos into productive templates. Our findings support the possibility that coligos may prove useful as chemically synthesized vectors for the ectopic expression of small RNA in human cells.

Transcription and translation in an RNA world

PubMed Central

Taylor, William R

2006-01-01

The RNA world hypothesis requires a ribozyme that was an RNA-directed RNA polymerase (ribopolymerase). If such a replicase makes a reverse complementary copy of any sequence (including itself), in a simple RNA world, there is no mechanism to prevent self-hybridization. It is proposed that this can be avoided through the synthesis of a parallel complementary copy. The logical consequences of this are pursued and developed in a computer simulation, where the behaviour of the parallel copy is compared to the conventional reverse complementary copy. It is found that the parallel copy is more efficient at higher temperatures (up to 90°C). A model for the ribopolymerase, based on the core of the large subunit (LSU) of the ribosome, is described. The geometry of a potential active site for this ribopolymerase suggests that it contained a cavity (now occupied by the aminoacyl-tRNA) and that an amino acid binding in this might have ‘poisoned’ the ribopolymerase by cross-reacting with the nucleoside-triphosphate before polymerization could occur. Based on a similarity to the active site components of the class-I tRNA synthetase enzymes, it is proposed that the amino acid could become attached to the nascent RNA transcript producing a variety of aminoacylated tRNA-like products. Using base-pairing interactions, some of these molecules might cross-link two ribopolymerases, giving rise to a precursor of the modern ribosome. A hybrid dimer, half polymerase and half proto-ribosome, could account for mRNA translocation before the advent of protein elongation factors. PMID:17008216

Fail-safe transcription termination: Because one is never enough.

PubMed

Lemay, Jean-François; Bachand, François

2015-01-01

Termination of RNA polymerase II (RNAPII) transcription is a fundamental step of gene expression that involves the release of the nascent transcript and dissociation of RNAPII from the DNA template. As transcription termination is intimately linked to RNA 3' end processing, termination pathways have a key decisive influence on the fate of the transcribed RNA. Quite remarkably, when reaching the 3' end of genes, a substantial fraction of RNAPII fail to terminate transcription, requiring the contribution of alternative or "fail-safe" mechanisms of termination to release the polymerase. This point of view covers redundant mechanisms of transcription termination and how they relate to conventional termination models. In particular, we expand on recent findings that propose a reverse torpedo model of termination, in which the 3'5' exonucleolytic activity of the RNA exosome targets transcription events associated with paused and backtracked RNAPII.

Infectious Maize rayado fino virus from cloned cDNA

USDA-ARS?s Scientific Manuscript database

Maize rayado fino virus (MRFV) is the type member of the marafiviruses within the family Tymoviridae. A cDNA clone from which infectious RNA can be transcribed was produced from a US isolate of MRFV (MRFV-US). Infectivity of transcripts derived from cDNA clones was demonstrated by infection of mai...

Structural insights into the rhabdovirus transcription/replication complex.

PubMed

Ivanov, Ivan; Yabukarski, Filip; Ruigrok, Rob W H; Jamin, Marc

2011-12-01

The rhabdoviruses have a non-segmented single stranded negative-sense RNA genome. Their multiplication in a host cell requires three viral proteins in addition to the viral RNA genome. The nucleoprotein (N) tightly encapsidates the viral RNA, and the N-RNA complex serves as the template for both transcription and replication. The viral RNA-dependent RNA polymerase is a two subunit complex that consists of a large subunit, L, and a non-catalytic cofactor, the phosphoprotein, P. P also acts as a chaperone of nascent RNA-free N by forming a N(0)-P complex that prevents N from binding to cellular RNAs and from polymerizing in the absence of RNA. Here, we discuss the recent molecular and structural studies of individual components and multi-molecular complexes that are involved in the transcription/replication complex of these viruses with regard to their implication in viral transcription and replication. Copyright © 2011 Elsevier B.V. All rights reserved.

Protein Interaction Profile Sequencing (PIP-seq).

PubMed

Foley, Shawn W; Gregory, Brian D

2016-10-10

Every eukaryotic RNA transcript undergoes extensive post-transcriptional processing from the moment of transcription up through degradation. This regulation is performed by a distinct cohort of RNA-binding proteins which recognize their target transcript by both its primary sequence and secondary structure. Here, we describe protein interaction profile sequencing (PIP-seq), a technique that uses ribonuclease-based footprinting followed by high-throughput sequencing to globally assess both protein-bound RNA sequences and RNA secondary structure. PIP-seq utilizes single- and double-stranded RNA-specific nucleases in the absence of proteins to infer RNA secondary structure. These libraries are also compared to samples that undergo nuclease digestion in the presence of proteins in order to find enriched protein-bound sequences. Combined, these four libraries provide a comprehensive, transcriptome-wide view of RNA secondary structure and RNA protein interaction sites from a single experimental technique. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

A parallel strategy for predicting the secondary structure of polycistronic microRNAs.

PubMed

Han, Dianwei; Tang, Guiliang; Zhang, Jun

2013-01-01

The biogenesis of a functional microRNA is largely dependent on the secondary structure of the microRNA precursor (pre-miRNA). Recently, it has been shown that microRNAs are present in the genome as the form of polycistronic transcriptional units in plants and animals. It will be important to design efficient computational methods to predict such structures for microRNA discovery and its applications in gene silencing. In this paper, we propose a parallel algorithm based on the master-slave architecture to predict the secondary structure from an input sequence. We conducted some experiments to verify the effectiveness of our parallel algorithm. The experimental results show that our algorithm is able to produce the optimal secondary structure of polycistronic microRNAs.

HALO--a Java framework for precise transcript half-life determination.

PubMed

Friedel, Caroline C; Kaufmann, Stefanie; Dölken, Lars; Zimmer, Ralf

2010-05-01

Recent improvements in experimental technologies now allow measurements of de novo transcription and/or RNA decay at whole transcriptome level and determination of precise transcript half-lives. Such transcript half-lives provide important insights into the regulation of biological processes and the relative contributions of RNA decay and de novo transcription to differential gene expression. In this article, we present HALO (Half-life Organizer), the first software for the precise determination of transcript half-lives from measurements of RNA de novo transcription or decay determined with microarrays or RNA-seq. In addition, methods for quality control, filtering and normalization are supplied. HALO provides a graphical user interface, command-line tools and a well-documented Java application programming interface (API). Thus, it can be used both by biologists to determine transcript half-lives fast and reliably with the provided user interfaces as well as software developers integrating transcript half-life analysis into other gene expression profiling pipelines. Source code, executables and documentation are available at http://www.bio.ifi.lmu.de/software/halo.

DLEU2 encodes an antisense RNA for the putative bicistronic RFP2/LEU5 gene in humans and mouse.

PubMed

Corcoran, Martin M; Hammarsund, Marianne; Zhu, Chaoyong; Lerner, Mikael; Kapanadze, Bagrat; Wilson, Bill; Larsson, Catharina; Forsberg, Lars; Ibbotson, Rachel E; Einhorn, Stefan; Oscier, David G; Grandér, Dan; Sangfelt, Olle

2004-08-01

Our group previously identified two novel genes, RFP2/LEU5 and DLEU2, within a 13q14.3 genomic region of loss seen in various malignancies. However, no specific inactivating mutations were found in these or other genes in the vicinity of the deletion, suggesting that a nonclassical tumor-suppressor mechanism may be involved. Here, we present data showing that the DLEU2 gene encodes a putative noncoding antisense RNA, with one exon directly overlapping the first exon of the RFP2/LEU5 gene in the opposite orientation. In addition, the RFP2/LEU5 transcript can be alternatively spliced to produce either several monocistronic transcripts or a putative bicistronic transcript encoding two separate open-reading frames, adding to the complexity of the locus. The finding that these gene structures are conserved in the mouse, including the putative bicistronic RFP2/LEU5 transcript as well as the antisense relationship with DLEU2, further underlines the significance of this unusual organization and suggests a biological function for DLEU2 in the regulation of RFP2/LEU5. Copyright 2004 Wiley-Liss, Inc.

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

PubMed

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

2013-01-01

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

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

PubMed Central

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

2013-01-01

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

Regulating RNA polymerase pausing and transcription elongation in embryonic stem cells

PubMed Central

Min, Irene M.; Waterfall, Joshua J.; Core, Leighton J.; Munroe, Robert J.; Schimenti, John; Lis, John T.

2011-01-01

Transitions between pluripotent stem cells and differentiated cells are executed by key transcription regulators. Comparative measurements of RNA polymerase distribution over the genome's primary transcription units in different cell states can identify the genes and steps in the transcription cycle that are regulated during such transitions. To identify the complete transcriptional profiles of RNA polymerases with high sensitivity and resolution, as well as the critical regulated steps upon which regulatory factors act, we used genome-wide nuclear run-on (GRO-seq) to map the density and orientation of transcriptionally engaged RNA polymerases in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). In both cell types, progression of a promoter-proximal, paused RNA polymerase II (Pol II) into productive elongation is a rate-limiting step in transcription of ∼40% of mRNA-encoding genes. Importantly, quantitative comparisons between cell types reveal that transcription is controlled frequently at paused Pol II's entry into elongation. Furthermore, “bivalent” ESC genes (exhibiting both active and repressive histone modifications) bound by Polycomb group complexes PRC1 (Polycomb-repressive complex 1) and PRC2 show dramatically reduced levels of paused Pol II at promoters relative to an average gene. In contrast, bivalent promoters bound by only PRC2 allow Pol II pausing, but it is confined to extremely 5′ proximal regions. Altogether, these findings identify rate-limiting targets for transcription regulation during cell differentiation. PMID:21460038

U6 small nuclear RNA is transcribed by RNA polymerase III.

PubMed Central

Kunkel, G R; Maser, R L; Calvet, J P; Pederson, T

1986-01-01

A DNA fragment homologous to U6 small nuclear RNA was isolated from a human genomic library and sequenced. The immediate 5'-flanking region of the U6 DNA clone had significant homology with a potential mouse U6 gene, including a "TATA box" at a position 26-29 nucleotides upstream from the transcription start site. Although this sequence element is characteristic of RNA polymerase II promoters, the U6 gene also contained a polymerase III "box A" intragenic control region and a typical run of five thymines at the 3' terminus (noncoding strand). The human U6 DNA clone was accurately transcribed in a HeLa cell S100 extract lacking polymerase II activity. U6 RNA transcription in the S100 extract was resistant to alpha-amanitin at 1 microgram/ml but was completely inhibited at 200 micrograms/ml. A comparison of fingerprints of the in vitro transcript and of U6 RNA synthesized in vivo revealed sequence congruence. U6 RNA synthesis in isolated HeLa cell nuclei also displayed low sensitivity to alpha-amanitin, in contrast to U1 and U2 RNA transcription, which was inhibited greater than 90% at 1 microgram/ml. In addition, U6 RNA synthesized in isolated nuclei was efficiently immunoprecipitated by an antibody against the La antigen, a protein known to bind most other RNA polymerase III transcripts. These results establish that, in contrast to the polymerase II-directed transcription of mammalian genes for U1-U5 small nuclear RNAs, human U6 RNA is transcribed by RNA polymerase III. Images PMID:3464970

The Role of E6 Spliced Isoforms (E6*) in Human Papillomavirus-Induced Carcinogenesis

PubMed Central

Olmedo-Nieva, Leslie; Muñoz-Bello, J. Omar; Contreras-Paredes, Adriana

2018-01-01

Persistent infections with High Risk Human Papillomaviruses (HR-HPVs) are the main cause of cervical cancer development. The E6 and E7 oncoproteins of HR-HPVs are derived from a polycistronic pre-mRNA transcribed from an HPV early promoter. Through alternative splicing, this pre-mRNA produces a variety of E6 spliced transcripts termed E6*. In pre-malignant lesions and HPV-related cancers, different E6/E6* transcriptional patterns have been found, although they have not been clearly associated to cancer development. Moreover, there is a controversy about the participation of E6* proteins in cancer progression. This review addresses the regulation of E6 splicing and the different functions that have been found for E6* proteins, as well as their possible role in HPV-induced carcinogenesis. PMID:29346309

TRAP binding to the Bacillus subtilis trp leader region RNA causes efficient transcription termination at a weak intrinsic terminator

PubMed Central

Potter, Kristine D.; Merlino, Natalie M.; Jacobs, Timothy; Gollnick, Paul

2011-01-01

The Bacillus subtilis trpEDCFBA operon is regulated by a transcription attenuation mechanism controlled by the trp RNA-binding attenuation protein (TRAP). TRAP binds to 11 (G/U)AG repeats in the trp leader transcript and prevents formation of an antiterminator, which allows formation of an intrinsic terminator (attenuator). Previously, formation of the attenuator RNA structure was believed to be solely responsible for signaling RNA polymerase (RNAP) to halt transcription. However, base substitutions that prevent formation of the antiterminator, and thus allow the attenuator structure to form constitutively, do not result in efficient transcription termination. The observation that the attenuator requires the presence of TRAP bound to the nascent RNA to cause efficient transcription termination suggests TRAP has an additional role in causing termination at the attenuator. We show that the trp attenuator is a weak intrinsic terminator due to low GC content of the hairpin stem and interruptions in the U-stretch following the hairpin. We also provide evidence that termination at the trp attenuator requires forward translocation of RNA polymerase and that TRAP binding to the nascent transcript can induce this activity. PMID:21097886

TRAP binding to the Bacillus subtilis trp leader region RNA causes efficient transcription termination at a weak intrinsic terminator.

PubMed

Potter, Kristine D; Merlino, Natalie M; Jacobs, Timothy; Gollnick, Paul

2011-03-01

The Bacillus subtilis trpEDCFBA operon is regulated by a transcription attenuation mechanism controlled by the trp RNA-binding attenuation protein (TRAP). TRAP binds to 11 (G/U)AG repeats in the trp leader transcript and prevents formation of an antiterminator, which allows formation of an intrinsic terminator (attenuator). Previously, formation of the attenuator RNA structure was believed to be solely responsible for signaling RNA polymerase (RNAP) to halt transcription. However, base substitutions that prevent formation of the antiterminator, and thus allow the attenuator structure to form constitutively, do not result in efficient transcription termination. The observation that the attenuator requires the presence of TRAP bound to the nascent RNA to cause efficient transcription termination suggests TRAP has an additional role in causing termination at the attenuator. We show that the trp attenuator is a weak intrinsic terminator due to low GC content of the hairpin stem and interruptions in the U-stretch following the hairpin. We also provide evidence that termination at the trp attenuator requires forward translocation of RNA polymerase and that TRAP binding to the nascent transcript can induce this activity.

Design and application of cotranscriptional non-enzymatic RNA circuits and signal transducers

PubMed Central

Bhadra, Sanchita; Ellington, Andrew D.

2014-01-01

Nucleic acid circuits are finding increasing real-life applications in diagnostics and synthetic biology. Although DNA has been the main operator in most nucleic acid circuits, transcriptionally produced RNA circuits could provide powerful alternatives for reagent production and their use in cells. Towards these goals, we have implemented a particular nucleic acid circuit, catalytic hairpin assembly, using RNA for both information storage and processing. Our results demonstrated that the design principles developed for DNA circuits could be readily translated to engineering RNA circuits that operated with similar kinetics and sensitivities of detection. Not only could purified RNA hairpins perform amplification reactions but RNA hairpins transcribed in vitro also mediated amplification, even without purification. Moreover, we could read the results of the non-enzymatic amplification reactions using a fluorescent RNA aptamer ‘Spinach’ that was engineered to undergo sequence-specific conformational changes. These advances were applied to the end-point and real-time detection of the isothermal strand displacement amplification reaction that produces single-stranded DNAs as part of its amplification cycle. We were also able to readily engineer gate structures with RNA similar to those that have previously formed the basis of DNA circuit computations. Taken together, these results validate an entirely new chemistry for the implementation of nucleic acid circuits. PMID:24493736

The adenovirus L4-22K protein regulates transcription and RNA splicing via a sequence-specific single-stranded RNA binding.

PubMed

Lan, Susan; Kamel, Wael; Punga, Tanel; Akusjärvi, Göran

2017-02-28

The adenovirus L4-22K protein both activates and suppresses transcription from the adenovirus major late promoter (MLP) by binding to DNA elements located downstream of the MLP transcriptional start site: the so-called DE element (positive) and the R1 region (negative). Here we show that L4-22K preferentially binds to the RNA form of the R1 region, both to the double-stranded RNA and the single-stranded RNA of the same polarity as the nascent MLP transcript. Further, L4-22K binds to a 5΄-CAAA-3΄ motif in the single-stranded RNA, which is identical to the sequence motif characterized for L4-22K DNA binding. L4-22K binding to single-stranded RNA results in an enhancement of U1 snRNA recruitment to the major late first leader 5΄ splice site. This increase in U1 snRNA binding results in a suppression of MLP transcription and a concurrent stimulation of major late first intron splicing. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

In Vitro Synthesized RNA Generated from cDNA Clones of Both Genomic Components of Cucurbit yellow stunting disorder virus Replicates in Cucumber Protoplasts

PubMed Central

Owen, Carolyn A.; Moukarzel, Romy; Huang, Xiao; Kassem, Mona A.; Eliasco, Eleonora; Aranda, Miguel A.; Coutts, Robert H. A.; Livieratos, Ioannis C.

2016-01-01

Cucurbit yellow stunting disorder virus (CYSDV), a bipartite whitefly-transmitted virus, constitutes a major threat to commercial cucurbit production worldwide. Here, construction of full-length CYSDV RNA1 and RNA2 cDNA clones allowed the in vitro synthesis of RNA transcripts able to replicate in cucumber protoplasts. CYSDV RNA1 proved competent for replication; transcription of both polarities of the genomic RNA was detectable 24 h post inoculation. Hybridization of total RNA extracted from transfected protoplasts or from naturally CYSDV-infected cucurbits revealed high-level transcription of the p22 subgenomic RNA species. Replication of CYSDV RNA2 following co-transfection with RNA1 was also observed, with similar transcription kinetics. A CYSDV RNA2 cDNA clone (T3CM8Δ) comprising the 5′- and 3′-UTRs plus the 3′-terminal gene, generated a 2.8 kb RNA able to replicate to high levels in protoplasts in the presence of CYSDV RNA1. The clone T3CM8Δ will facilitate reverse genetics studies of CYSDV gene function and RNA replication determinants. PMID:27314380

An Enumerative Combinatorics Model for Fragmentation Patterns in RNA Sequencing Provides Insights into Nonuniformity of the Expected Fragment Starting-Point and Coverage Profile.

PubMed

Prakash, Celine; Haeseler, Arndt Von

2017-03-01

RNA sequencing (RNA-seq) has emerged as the method of choice for measuring the expression of RNAs in a given cell population. In most RNA-seq technologies, sequencing the full length of RNA molecules requires fragmentation into smaller pieces. Unfortunately, the issue of nonuniform sequencing coverage across a genomic feature has been a concern in RNA-seq and is attributed to biases for certain fragments in RNA-seq library preparation and sequencing. To investigate the expected coverage obtained from fragmentation, we develop a simple fragmentation model that is independent of bias from the experimental method and is not specific to the transcript sequence. Essentially, we enumerate all configurations for maximal placement of a given fragment length, F, on transcript length, T, to represent every possible fragmentation pattern, from which we compute the expected coverage profile across a transcript. We extend this model to incorporate general empirical attributes such as read length, fragment length distribution, and number of molecules of the transcript. We further introduce the fragment starting-point, fragment coverage, and read coverage profiles. We find that the expected profiles are not uniform and that factors such as fragment length to transcript length ratio, read length to fragment length ratio, fragment length distribution, and number of molecules influence the variability of coverage across a transcript. Finally, we explore a potential application of the model where, with simulations, we show that it is possible to correctly estimate the transcript copy number for any transcript in the RNA-seq experiment.

An Enumerative Combinatorics Model for Fragmentation Patterns in RNA Sequencing Provides Insights into Nonuniformity of the Expected Fragment Starting-Point and Coverage Profile

PubMed Central

Haeseler, Arndt Von

2017-01-01

Abstract RNA sequencing (RNA-seq) has emerged as the method of choice for measuring the expression of RNAs in a given cell population. In most RNA-seq technologies, sequencing the full length of RNA molecules requires fragmentation into smaller pieces. Unfortunately, the issue of nonuniform sequencing coverage across a genomic feature has been a concern in RNA-seq and is attributed to biases for certain fragments in RNA-seq library preparation and sequencing. To investigate the expected coverage obtained from fragmentation, we develop a simple fragmentation model that is independent of bias from the experimental method and is not specific to the transcript sequence. Essentially, we enumerate all configurations for maximal placement of a given fragment length, F, on transcript length, T, to represent every possible fragmentation pattern, from which we compute the expected coverage profile across a transcript. We extend this model to incorporate general empirical attributes such as read length, fragment length distribution, and number of molecules of the transcript. We further introduce the fragment starting-point, fragment coverage, and read coverage profiles. We find that the expected profiles are not uniform and that factors such as fragment length to transcript length ratio, read length to fragment length ratio, fragment length distribution, and number of molecules influence the variability of coverage across a transcript. Finally, we explore a potential application of the model where, with simulations, we show that it is possible to correctly estimate the transcript copy number for any transcript in the RNA-seq experiment. PMID:27661099

The Battle of RNA Synthesis: Virus versus Host.

PubMed

Harwig, Alex; Landick, Robert; Berkhout, Ben

2017-10-21

Transcription control is the foundation of gene regulation. Whereas a cell is fully equipped for this task, viruses often depend on the host to supply tools for their transcription program. Over the course of evolution and adaptation, viruses have found diverse ways to optimally exploit cellular host processes such as transcription to their own benefit. Just as cells are increasingly understood to employ nascent RNAs in transcription regulation, recent discoveries are revealing how viruses use nascent RNAs to benefit their own gene expression. In this review, we first outline the two different transcription programs used by viruses, i.e., transcription (DNA-dependent) and RNA-dependent RNA synthesis. Subsequently, we use the distinct stages (initiation, elongation, termination) to describe the latest insights into nascent RNA-mediated regulation in the context of each relevant stage.

Interleukin-6 production by human monocytes treated with granulocyte-macrophage colony-stimulating factor in the presence of lipopolysaccharide of oral microorganisms.

PubMed

Baqui, A A; Meiller, T F; Chon, J J; Turng, B F; Falkler, W A

1998-06-01

This study focused on the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide of the putative periodontal pathogens Porphyromonas gingivalis or Fusobacterium nucleatum on IL-6 production by THP-1 cells (a human monocytic cell line). Resting THP-1 cells were alternatively treated with GM-CSF (50 IU/ml) and lipopolysaccharide of P. gingivalis or F. nucleatum, in varying concentrations for varying time periods. IL-6 production in supernatant fluids of treated cells was evaluated by an enzyme-linked immunosorbent assay (ELISA) and a reverse transcription polymerase chain reaction (RT-PCR) was used to evaluate gene expression. Untreated THP-1 cells did not produce IL-6 as determined by ELISA. RT-PCR also failed to detect IL-6 mRNA in untreated THP-1 cells, indicating that IL-6 was not constitutively produced. After stimulation of THP-1 cells with lipopolysaccharide of F. nucleatum or P. gingivalis, IL-6 was produced, peaking at 4 h (200-300 pg/ml) and thereafter sharply declining by 8 h. When GM-CSF was added together with lipopolysaccharide of P. gingivalis or F. nucleatum, there was a synergistic quantitative increase in production of IL-6 as measured by ELISA as compared with lipopolysaccharide alone. IL-6 mRNA was detected by RT-PCR, 15 min after stimulation with lipopolysaccharide of either P. gingivalis or F. nucleatum. GM-CSF supplementation with lipopolysaccharide of P. gingivalis shortened the transcription of IL-6 mRNA to 5 min, a shift which was not observed with lipopolysaccharide of F. nucleatum, possibly indicating a different mechanism of initiation of transcription. Production of IL-6 by GM-CSF-treated THP-1 cells in the presence of lipopolysaccharide of oral microorganisms may provide a model for studying the role of macrophages in acute and chronic periodontal diseases, including the clinical periodontal exacerbation as observed in chemotherapy patients receiving GM-CSF for bone marrow recovery.

Transcriptional profiling of murine osteoblast differentiation based on RNA-seq expression analyses.

PubMed

Khayal, Layal Abo; Grünhagen, Johannes; Provazník, Ivo; Mundlos, Stefan; Kornak, Uwe; Robinson, Peter N; Ott, Claus-Eric

2018-04-11

Osteoblastic differentiation is a multistep process characterized by osteogenic induction of mesenchymal stem cells, which then differentiate into proliferative pre-osteoblasts that produce copious amounts of extracellular matrix, followed by stiffening of the extracellular matrix, and matrix mineralization by hydroxylapatite deposition. Although these processes have been well characterized biologically, a detailed transcriptional analysis of murine primary calvaria osteoblast differentiation based on RNA sequencing (RNA-seq) analyses has not previously been reported. Here, we used RNA-seq to obtain expression values of 29,148 genes at four time points as murine primary calvaria osteoblasts differentiate in vitro until onset of mineralization was clearly detectable by microscopic inspection. Expression of marker genes confirmed osteogenic differentiation. We explored differential expression of 1386 protein-coding genes using unsupervised clustering and GO analyses. 100 differentially expressed lncRNAs were investigated by co-expression with protein-coding genes that are localized within the same topologically associated domain. Additionally, we monitored expression of 237 genes that are silent or active at distinct time points and compared differential exon usage. Our data represent an in-depth profiling of murine primary calvaria osteoblast differentiation by RNA-seq and contribute to our understanding of genetic regulation of this key process in osteoblast biology. Copyright © 2018 Elsevier Inc. All rights reserved.

Global impact of RNA splicing on transcriptome remodeling in the heart.

PubMed

Gao, Chen; Wang, Yibin

2012-08-01

In the eukaryotic transcriptome, both the numbers of genes and different RNA species produced by each gene contribute to the overall complexity. These RNA species are generated by the utilization of different transcriptional initiation or termination sites, or more commonly, from different messenger RNA (mRNA) splicing events. Among the 30,000+ genes in human genome, it is estimated that more than 95% of them can generate more than one gene product via alternative RNA splicing. The protein products generated from different RNA splicing variants can have different intracellular localization, activity, or tissue-distribution. Therefore, alternative RNA splicing is an important molecular process that contributes to the overall complexity of the genome and the functional specificity and diversity among different cell types. In this review, we will discuss current efforts to unravel the full complexity of the cardiac transcriptome using a deep-sequencing approach, and highlight the potential of this technology to uncover the global impact of RNA splicing on the transcriptome during development and diseases of the heart.

Changing genetic information through RNA editing

NASA Technical Reports Server (NTRS)

Maas, S.; Rich, A.

2000-01-01

RNA editing, the post-transcriptional alteration of a gene-encoded sequence, is a widespread phenomenon in eukaryotes. As a consequence of RNA editing, functionally distinct proteins can be produced from a single gene. The molecular mechanisms involved include single or multiple base insertions or deletions as well as base substitutions. In mammals, one type of substitutional RNA editing, characterized by site-specific base-modification, was shown to modulate important physiological processes. The underlying reaction mechanism of substitutional RNA editing involves hydrolytic deamination of cytosine or adenosine bases to uracil or inosine, respectively. Protein factors have been characterized that are able to induce RNA editing in vitro. A supergene family of RNA-dependent deaminases has emerged with the recent addition of adenosine deaminases specific for tRNA. Here we review the developments that have substantially increased our understanding of base-modification RNA editing over the past few years, with an emphasis on mechanistic differences, evolutionary aspects and the first insights into the regulation of editing activity.

A long antisense RNA in plant chloroplasts.

PubMed

Georg, J; Honsel, A; Voss, B; Rennenberg, H; Hess, W R

2010-05-01

Based on computational prediction of RNA secondary structures, a long antisense RNA (asRNA) was found in chloroplasts of Arabidopsis, Nicotiana tabacum and poplar, which occurs in two to three major transcripts. Mapping of primary 5' ends, northern hybridizations and quantitative real-time reverse transcription polymerase chain reaction (qPCR) experiments demonstrated that these transcripts originate from a promoter that is typical for the plastid-encoded RNA polymerase and are over their full length in antisense orientation to the gene ndhB and therefore were designated asRNA_ndhB. The asRNA_ndhB transcripts predominantly accumulate in young leaves and at physiological growth temperatures. Two nucleotide positions in the mRNA that are subject to C-to-U RNA editing and which were previously found to be sensitive to elevated temperatures are covered by asRNA_ndhB. Nevertheless, the correlation between the accumulation of asRNA_ndhB and RNA editing appeared weak in a temperature shift experiment. With asRNA_ndhB, we describe the first asRNA of plant chloroplasts that covers RNA editing sites, as well as a group II intron splice acceptor site, and that is under developmental control, raising the possibility that long asRNAs could be involved in RNA maturation or the control of RNA stability.

EMSAR: estimation of transcript abundance from RNA-seq data by mappability-based segmentation and reclustering.

PubMed

Lee, Soohyun; Seo, Chae Hwa; Alver, Burak Han; Lee, Sanghyuk; Park, Peter J

2015-09-03

RNA-seq has been widely used for genome-wide expression profiling. RNA-seq data typically consists of tens of millions of short sequenced reads from different transcripts. However, due to sequence similarity among genes and among isoforms, the source of a given read is often ambiguous. Existing approaches for estimating expression levels from RNA-seq reads tend to compromise between accuracy and computational cost. We introduce a new approach for quantifying transcript abundance from RNA-seq data. EMSAR (Estimation by Mappability-based Segmentation And Reclustering) groups reads according to the set of transcripts to which they are mapped and finds maximum likelihood estimates using a joint Poisson model for each optimal set of segments of transcripts. The method uses nearly all mapped reads, including those mapped to multiple genes. With an efficient transcriptome indexing based on modified suffix arrays, EMSAR minimizes the use of CPU time and memory while achieving accuracy comparable to the best existing methods. EMSAR is a method for quantifying transcripts from RNA-seq data with high accuracy and low computational cost. EMSAR is available at https://github.com/parklab/emsar.

The Helicase Aquarius/EMB-4 Is Required to Overcome Intronic Barriers to Allow Nuclear RNAi Pathways to Heritably Silence Transcription.

PubMed

Akay, Alper; Di Domenico, Tomas; Suen, Kin M; Nabih, Amena; Parada, Guillermo E; Larance, Mark; Medhi, Ragini; Berkyurek, Ahmet C; Zhang, Xinlian; Wedeles, Christopher J; Rudolph, Konrad L M; Engelhardt, Jan; Hemberg, Martin; Ma, Ping; Lamond, Angus I; Claycomb, Julie M; Miska, Eric A

2017-08-07

Small RNAs play a crucial role in genome defense against transposable elements and guide Argonaute proteins to nascent RNA transcripts to induce co-transcriptional gene silencing. However, the molecular basis of this process remains unknown. Here, we identify the conserved RNA helicase Aquarius/EMB-4 as a direct and essential link between small RNA pathways and the transcriptional machinery in Caenorhabditis elegans. Aquarius physically interacts with the germline Argonaute HRDE-1. Aquarius is required to initiate small-RNA-induced heritable gene silencing. HRDE-1 and Aquarius silence overlapping sets of genes and transposable elements. Surprisingly, removal of introns from a target gene abolishes the requirement for Aquarius, but not HRDE-1, for small RNA-dependent gene silencing. We conclude that Aquarius allows small RNA pathways to compete for access to nascent transcripts undergoing co-transcriptional splicing in order to detect and silence transposable elements. Thus, Aquarius and HRDE-1 act as gatekeepers coordinating gene expression and genome defense. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Transcriptional and Posttranscriptional Control of Phaseolin and Phytohemagglutinin Gene Expression in Developing Cotyledons of Phaseolus vulgaris.

PubMed

Chappell, J; Chrispeels, M J

1986-05-01

The expression of phaseolin and phytohemagglutinin (PHA) in the developing cotyledons of a normal (Greensleeves) and a PHA-deficient (Pinto 111) cultivar of Phaseolus vulgaris was investigated. Phaseolin mRNA translational activity and abundance were present at similar levels in both cultivars. In contrast, PHA mRNA translational activity and abundance in Pinto 111 were less than 1% of the levels measured in Greensleeves. Using nuclear runoff assays, the transcription rate of phaseolin gene sequences was similar in both cultivars. The transcription rate of PHA gene sequences in Pinto 111 was only 20% of that measured in Greensleeves. Comparison of the transcription rates with the relative mRNA amounts measured in RNA blot hybridizations indicated that the normally expressed storage protein gene mRNAs were very stable with half-lives greater than several days. Because a low level of PHA gene transcription in Pinto 111 was measurable but no PHA mRNA accumulated, these results suggest that the PHA deficiency in Pinto 111 is due to a reduced transcription rate and possibly an instability of the mRNA.

In vitro transcription activities of Pol IV, Pol V and RDR2 reveal coupling of Pol IV and RDR2 for dsRNA synthesis in plant RNA silencing

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

Haag, Jeremy R.; Ream, Thomas S.; Marasco, Michelle

2012-12-14

In Arabidopsis, RNA-dependent DNA methylation and transcriptional silencing involves three nuclear RNA polymerases that are biochemically undefined: the presumptive DNA-dependent RNA polymerases, Pol IV and Pol V and the putative RNA-dependent RNA polymerase, RDR2. Here, we demonstrate their RNA polymerase activities in vitro. Unlike Pol II, Pols IV and V require an RNA primer, are insensitive to alpha-amanitin and differ in their ability to displace non-template DNA during transcription. Biogenesis of 24 nt small interfering RNAs (siRNAs) requires both Pol IV and RDR2, which physically associate in vivo. Pol IV does not require RDR2 for activity, but RDR2 is nonfunctionalmore » in the absence of associated Pol IV, suggesting that their coupling explains the channeling of Pol IV transcripts into double-stranded RNAs that are then diced into 24 nt siRNAs.« less

Analysis of hairpin RNA transgene-induced gene silencing in Fusarium oxysporum

PubMed Central

2013-01-01

Background Hairpin RNA (hpRNA) transgenes can be effective at inducing RNA silencing and have been exploited as a powerful tool for gene function analysis in many organisms. However, in fungi, expression of hairpin RNA transcripts can induce post-transcriptional gene silencing, but in some species can also lead to transcriptional gene silencing, suggesting a more complex interplay of the two pathways at least in some fungi. Because many fungal species are important pathogens, RNA silencing is a powerful technique to understand gene function, particularly when gene knockouts are difficult to obtain. We investigated whether the plant pathogenic fungus Fusarium oxysporum possesses a functional gene silencing machinery and whether hairpin RNA transcripts can be employed to effectively induce gene silencing. Results Here we show that, in the phytopathogenic fungus F. oxysporum, hpRNA transgenes targeting either a β-glucuronidase (Gus) reporter transgene (hpGus) or the endogenous gene Frp1 (hpFrp) did not induce significant silencing of the target genes. Expression analysis suggested that the hpRNA transgenes are prone to transcriptional inactivation, resulting in low levels of hpRNA and siRNA production. However, the hpGus RNA can be efficiently transcribed by promoters acquired either by recombination with a pre-existing, actively transcribed Gus transgene or by fortuitous integration near an endogenous gene promoter allowing siRNA production. These siRNAs effectively induced silencing of a target Gus transgene, which in turn appeared to also induce secondary siRNA production. Furthermore, our results suggested that hpRNA transcripts without poly(A) tails are efficiently processed into siRNAs to induce gene silencing. A convergent promoter transgene, designed to express poly(A)-minus sense and antisense Gus RNAs, without an inverted-repeat DNA structure, induced consistent Gus silencing in F. oxysporum. Conclusions These results indicate that F. oxysporum possesses functional RNA silencing machineries for siRNA production and target mRNA cleavage, but hpRNA transgenes may induce transcriptional self-silencing due to its inverted-repeat structure. Our results suggest that F. oxysporum possesses a similar gene silencing pathway to other fungi like fission yeast, and indicate a need for developing more effective RNA silencing technology for gene function studies in this fungal pathogen. PMID:23819794

In vitro transcription in E. coli crude lysates prepared on cellophane discs.

PubMed Central

Valla, S; Lindqvist, B H

1978-01-01

An in vitro RNA-synthesizing system consisting of gently lysed E. coli cells on cellophane discs is described. The system has been optimalized with respect to total RNA synthesis. Under certain standard conditions DNA dependent RNA polymerase (EC 2.7.7.6) is responsible for the majority of the RNA synthesis. The extensive rifampicin sensitivity of the synthesis indicates that most of the transcripts are initiated in vitro. The RNA synthesizing system described here has been developed with the aim of studying phage transcription in vitro. We show here that lysates of a P4 infected P2 lysogen support initiation and propagation of transcription from the P2 prophage. PMID:27767

Transcriptome analysis of thermophilic methylotrophic Bacillus methanolicus MGA3 using RNA-sequencing provides detailed insights into its previously uncharted transcriptional landscape.

PubMed

Irla, Marta; Neshat, Armin; Brautaset, Trygve; Rückert, Christian; Kalinowski, Jörn; Wendisch, Volker F

2015-02-14

Bacillus methanolicus MGA3 is a thermophilic, facultative ribulose monophosphate (RuMP) cycle methylotroph. Together with its ability to produce high yields of amino acids, the relevance of this microorganism as a promising candidate for biotechnological applications is evident. The B. methanolicus MGA3 genome consists of a 3,337,035 nucleotides (nt) circular chromosome, the 19,174 nt plasmid pBM19 and the 68,999 nt plasmid pBM69. 3,218 protein-coding regions were annotated on the chromosome, 22 on pBM19 and 82 on pBM69. In the present study, the RNA-seq approach was used to comprehensively investigate the transcriptome of B. methanolicus MGA3 in order to improve the genome annotation, identify novel transcripts, analyze conserved sequence motifs involved in gene expression and reveal operon structures. For this aim, two different cDNA library preparation methods were applied: one which allows characterization of the whole transcriptome and another which includes enrichment of primary transcript 5'-ends. Analysis of the primary transcriptome data enabled the detection of 2,167 putative transcription start sites (TSSs) which were categorized into 1,642 TSSs located in the upstream region (5'-UTR) of known protein-coding genes and 525 TSSs of novel antisense, intragenic, or intergenic transcripts. Firstly, 14 wrongly annotated translation start sites (TLSs) were corrected based on primary transcriptome data. Further investigation of the identified 5'-UTRs resulted in the detailed characterization of their length distribution and the detection of 75 hitherto unknown cis-regulatory RNA elements. Moreover, the exact TSSs positions were utilized to define conserved sequence motifs for translation start sites, ribosome binding sites and promoters in B. methanolicus MGA3. Based on the whole transcriptome data set, novel transcripts, operon structures and mRNA abundances were determined. The analysis of the operon structures revealed that almost half of the genes are transcribed monocistronically (940), whereas 1,164 genes are organized in 381 operons. Several of the genes related to methylotrophy had highly abundant transcripts. The extensive insights into the transcriptional landscape of B. methanolicus MGA3, gained in this study, represent a valuable foundation for further comparative quantitative transcriptome analyses and possibly also for the development of molecular biology tools which at present are very limited for this organism.

Modeling RNA polymerase interaction in mitochondria of chordates

PubMed Central

2012-01-01

Background In previous work, we introduced a concept, a mathematical model and its computer realization that describe the interaction between bacterial and phage type RNA polymerases, protein factors, DNA and RNA secondary structures during transcription, including transcription initiation and termination. The model accurately reproduces changes of gene transcription level observed in polymerase sigma-subunit knockout and heat shock experiments in plant plastids. The corresponding computer program and a user guide are available at http://lab6.iitp.ru/en/rivals. Here we apply the model to the analysis of transcription and (partially) translation processes in the mitochondria of frog, rat and human. Notably, mitochondria possess only phage-type polymerases. We consider the entire mitochondrial genome so that our model allows RNA polymerases to complete more than one circle on the DNA strand. Results Our model of RNA polymerase interaction during transcription initiation and elongation accurately reproduces experimental data obtained for plastids. Moreover, it also reproduces evidence on bulk RNA concentrations and RNA half-lives in the mitochondria of frog, human with or without the MELAS mutation, and rat with normal (euthyroid) or hyposecretion of thyroid hormone (hypothyroid). The transcription characteristics predicted by the model include: (i) the fraction of polymerases terminating at a protein-dependent terminator in both directions (the terminator polarization), (ii) the binding intensities of the regulatory protein factor (mTERF) with the termination site and, (iii) the transcription initiation intensities (initiation frequencies) of all promoters in all five conditions (frog, healthy human, human with MELAS syndrome, healthy rat, and hypothyroid rat with aberrant mtDNA methylation). Using the model, absolute levels of all gene transcription can be inferred from an arbitrary array of the three transcription characteristics, whereas, for selected genes only relative RNA concentrations have been experimentally determined. Conversely, these characteristics and absolute transcription levels can be obtained using relative RNA concentrations and RNA half-lives known from various experimental studies. In this case, the “inverse problem” is solved with multi-objective optimization. Conclusions In this study, we demonstrate that our model accurately reproduces all relevant experimental data available for plant plastids, as well as the mitochondria of chordates. Using experimental data, the model is applied to estimate binding intensities of phage-type RNA polymerases to their promoters as well as predicting terminator characteristics, including polarization. In addition, one can predict characteristics of phage-type RNA polymerases and the transcription process that are difficult to measure directly, e.g., the association between the promoter’s nucleotide composition and the intensity of polymerase binding. To illustrate the application of our model in functional predictions, we propose a possible mechanism for MELAS syndrome development in human involving a decrease of Phe-tRNA, Val-tRNA and rRNA concentrations in the cell. In addition, we describe how changes in methylation patterns of the mTERF binding site and three promoters in hypothyroid rat correlate with changes in intensities of the mTERF binding and transcription initiations. Finally, we introduce an auxiliary model to describe the interaction between polysomal mRNA and ribonucleases. PMID:22873568

Elongated Hypocotyl 5-Homolog (HYH) Negatively Regulates Expression of the Ambient Temperature-Responsive MicroRNA Gene MIR169

PubMed Central

Serivichyaswat, Phanu T.; Susila, Hendry; Ahn, Ji Hoon

2017-01-01

Arabidopsis microRNA169 (miR169) is an ambient temperature-responsive microRNA that plays an important role in stress responses and the floral transition. However, the transcription factors that regulate the expression of MIR169 have remained unknown. In this study, we show that Elongated Hypocotyl 5-Homolog (HYH) directly binds to the promoter of MIR169a and negatively regulates its expression. Absolute quantification identified MIR169a as the major locus producing miR169. GUS reporter assays revealed that the deletion of a 498-bp fragment (–1,505 to –1,007, relative to the major transcriptional start site) of MIR169a abolished its ambient temperature-responsive expression. DNA-affinity chromatography followed by liquid chromatography-mass spectrometry analysis identified transcription factor HYH as a trans-acting factor that binds to the 498-bp promoter fragment of pri-miR169a. Electrophoretic mobility shift assays and chromatin immunoprecipitation–quantitative PCR demonstrated that the HYH.2 protein, a predominant isoform of HYH, directly associated with a G-box-like motif in the 498-bp fragment of pri-miR169a. Higher enrichment of HYH.2 protein on the promoter region of MIR169a was seen at 23°C, consistent with the presence of more HYH.2 protein in the cell at the temperature. Transcript levels of pri-miR169a increased in hyh mutants and decreased in transgenic plants overexpressing HYH. Consistent with the negative regulation of MIR169a by HYH, the diurnal levels of HYH mRNA and pri-miR169a showed opposite patterns. Taken together, our results suggest that HYH is a transcription factor that binds to a G-box-like motif in the MIR169a promoter and negatively regulates ambient temperature-responsive expression of MIR169a at higher temperatures in Arabidopsis. PMID:29270188

Fail-safe transcription termination: Because one is never enough

PubMed Central

Lemay, Jean-François; Bachand, François

2015-01-01

Termination of RNA polymerase II (RNAPII) transcription is a fundamental step of gene expression that involves the release of the nascent transcript and dissociation of RNAPII from the DNA template. As transcription termination is intimately linked to RNA 3′ end processing, termination pathways have a key decisive influence on the fate of the transcribed RNA. Quite remarkably, when reaching the 3′ end of genes, a substantial fraction of RNAPII fail to terminate transcription, requiring the contribution of alternative or “fail-safe” mechanisms of termination to release the polymerase. This point of view covers redundant mechanisms of transcription termination and how they relate to conventional termination models. In particular, we expand on recent findings that propose a reverse torpedo model of termination, in which the 3′5′ exonucleolytic activity of the RNA exosome targets transcription events associated with paused and backtracked RNAPII. PMID:26273910

Bacterial antisense RNAs are mainly the product of transcriptional noise.

PubMed

Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I; Serrano, Luis; Lluch-Senar, Maria

2016-03-01

cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome.

Bacterial antisense RNAs are mainly the product of transcriptional noise

PubMed Central

Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I.; Serrano, Luis; Lluch-Senar, Maria

2016-01-01

cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome. PMID:26973873

Short intronic repeat sequences facilitate circular RNA production

PubMed Central

Liang, Dongming

2014-01-01

Recent deep sequencing studies have revealed thousands of circular noncoding RNAs generated from protein-coding genes. These RNAs are produced when the precursor messenger RNA (pre-mRNA) splicing machinery “backsplices” and covalently joins, for example, the two ends of a single exon. However, the mechanism by which the spliceosome selects only certain exons to circularize is largely unknown. Using extensive mutagenesis of expression plasmids, we show that miniature introns containing the splice sites along with short (∼30- to 40-nucleotide) inverted repeats, such as Alu elements, are sufficient to allow the intervening exons to circularize in cells. The intronic repeats must base-pair to one another, thereby bringing the splice sites into close proximity to each other. More than simple thermodynamics is clearly at play, however, as not all repeats support circularization, and increasing the stability of the hairpin between the repeats can sometimes inhibit circular RNA biogenesis. The intronic repeats and exonic sequences must collaborate with one another, and a functional 3′ end processing signal is required, suggesting that circularization may occur post-transcriptionally. These results suggest detailed and generalizable models that explain how the splicing machinery determines whether to produce a circular noncoding RNA or a linear mRNA. PMID:25281217

Genome-wide association between DNA methylation and alternative splicing in an invertebrate

PubMed Central

2012-01-01

Background Gene bodies are the most evolutionarily conserved targets of DNA methylation in eukaryotes. However, the regulatory functions of gene body DNA methylation remain largely unknown. DNA methylation in insects appears to be primarily confined to exons. Two recent studies in Apis mellifera (honeybee) and Nasonia vitripennis (jewel wasp) analyzed transcription and DNA methylation data for one gene in each species to demonstrate that exon-specific DNA methylation may be associated with alternative splicing events. In this study we investigated the relationship between DNA methylation, alternative splicing, and cross-species gene conservation on a genome-wide scale using genome-wide transcription and DNA methylation data. Results We generated RNA deep sequencing data (RNA-seq) to measure genome-wide mRNA expression at the exon- and gene-level. We produced a de novo transcriptome from this RNA-seq data and computationally predicted splice variants for the honeybee genome. We found that exons that are included in transcription are higher methylated than exons that are skipped during transcription. We detected enrichment for alternative splicing among methylated genes compared to unmethylated genes using fisher’s exact test. We performed a statistical analysis to reveal that the presence of DNA methylation or alternative splicing are both factors associated with a longer gene length and a greater number of exons in genes. In concordance with this observation, a conservation analysis using BLAST revealed that each of these factors is also associated with higher cross-species gene conservation. Conclusions This study constitutes the first genome-wide analysis exhibiting a positive relationship between exon-level DNA methylation and mRNA expression in the honeybee. Our finding that methylated genes are enriched for alternative splicing suggests that, in invertebrates, exon-level DNA methylation may play a role in the construction of splice variants by positively influencing exon inclusion during transcription. The results from our cross-species homology analysis suggest that DNA methylation and alternative splicing are genetic mechanisms whose utilization could contribute to a longer gene length and a slower rate of gene evolution. PMID:22978521

Synthesis and RNA polymerase incorporation of the degenerate ribonucleotide analogue rPTP.

PubMed Central

Moriyama, K; Negishi, K; Briggs, M S; Smith, C L; Hill, F; Churcher, M J; Brown, D M; Loakes, D

1998-01-01

The synthesis and enzymatic incorporation into RNA of the hydrogen bond degenerate nucleoside analogue 6-(beta-d-ribofuranosyl)-3, 4-dihydro-8H-pyrimido[4,5-c]-[1,2]oxazin-7-one (P) is described. The 5'-triphosphate of this analogue is readily incorporated by T3, T7 and SP6 RNA polymerases into RNA transcripts, being best incorporated in place of UTP, but also in place of CTP. When all the uridine residues in an HIV-1 TAR RNA transcript are replaced by P the transcript has similar characteristics to the wild-type TAR RNA, as demonstrated by similar melting temperatures and CD spectra. The P-substituted TAR transcript binds to the Tat peptide ADP-1 with only 4-fold lowered efficiency compared with wild-type TAR. PMID:9547267

Synthesis and RNA polymerase incorporation of the degenerate ribonucleotide analogue rPTP.

PubMed

Moriyama, K; Negishi, K; Briggs, M S; Smith, C L; Hill, F; Churcher, M J; Brown, D M; Loakes, D

1998-05-01

The synthesis and enzymatic incorporation into RNA of the hydrogen bond degenerate nucleoside analogue 6-(beta-d-ribofuranosyl)-3, 4-dihydro-8H-pyrimido[4,5-c]-[1,2]oxazin-7-one (P) is described. The 5'-triphosphate of this analogue is readily incorporated by T3, T7 and SP6 RNA polymerases into RNA transcripts, being best incorporated in place of UTP, but also in place of CTP. When all the uridine residues in an HIV-1 TAR RNA transcript are replaced by P the transcript has similar characteristics to the wild-type TAR RNA, as demonstrated by similar melting temperatures and CD spectra. The P-substituted TAR transcript binds to the Tat peptide ADP-1 with only 4-fold lowered efficiency compared with wild-type TAR.

The Regulation of Transcription in Memory Consolidation

PubMed Central

Alberini, Cristina M.; Kandel, Eric R.

2015-01-01

De novo transcription of DNA is a fundamental requirement for the formation of long-term memory. It is required during both consolidation and reconsolidation, the posttraining and postreactivation phases that change the state of the memory from a fragile into a stable and long-lasting form. Transcription generates both mRNAs that are translated into proteins, which are necessary for the growth of new synaptic connections, as well as noncoding RNA transcripts that have regulatory or effector roles in gene expression. The result is a cascade of events that ultimately leads to structural changes in the neurons that mediate long-term memory storage. The de novo transcription, critical for synaptic plasticity and memory formation, is orchestrated by chromatin and epigenetic modifications. The complexity of transcription regulation, its temporal progression, and the effectors produced all contribute to the flexibility and persistence of long-term memory formation. In this article, we provide an overview of the mechanisms contributing to this transcriptional regulation underlying long-term memory formation. PMID:25475090

A simplified approach to construct infectious cDNA clones of a tobamovirus in a binary vector.

PubMed

Junqueira, Bruna Rayane Teodoro; Nicolini, Cícero; Lucinda, Natalia; Orílio, Anelise Franco; Nagata, Tatsuya

2014-03-01

Infectious cDNA clones of RNA viruses are important tools to study molecular processes such as replication and host-virus interactions. However, the cloning steps necessary for construction of cDNAs of viral RNA genomes in binary vectors are generally laborious. In this study, a simplified method of producing an agro-infectious Pepper mild mottle virus (PMMoV) clone is described in detail. Initially, the complete genome of PMMoV was amplified by a single-step RT-PCR, cloned, and subcloned into a small plasmid vector under the T7 RNA polymerase promoter to confirm the infectivity of the cDNA clone through transcript inoculation. The complete genome was then transferred to a binary vector using a single-step, overlap-extension PCR. The selected clones were agro-infiltrated to Nicotiana benthamiana plants and showed to be infectious, causing typical PMMoV symptoms. No differences in host responses were observed when the wild-type PMMoV isolate, the T7 RNA polymerase-derived transcripts and the agroinfiltration-derived viruses were inoculated to N. benthamiana, Capsicum chinense PI 159236 and Capsicum annuum plants. Copyright © 2013 Elsevier B.V. All rights reserved.

The RNA m6A Reader YTHDF2 Is Essential for the Post-transcriptional Regulation of the Maternal Transcriptome and Oocyte Competence.

PubMed

Ivanova, Ivayla; Much, Christian; Di Giacomo, Monica; Azzi, Chiara; Morgan, Marcos; Moreira, Pedro N; Monahan, Jack; Carrieri, Claudia; Enright, Anton J; O'Carroll, Dónal

2017-09-21

YTHDF2 binds and destabilizes N 6 -methyladenosine (m 6 A)-modified mRNA. The extent to which this branch of m 6 A RNA-regulatory pathway functions in vivo and contributes to mammalian development remains unknown. Here we find that YTHDF2 deficiency is partially permissive in mice and results in female-specific infertility. Using conditional mutagenesis, we demonstrate that YTHDF2 is autonomously required within the germline to produce MII oocytes that are competent to sustain early zygotic development. Oocyte maturation is associated with a wave of maternal RNA degradation, and the resulting relative changes to the MII transcriptome are integral to oocyte quality. The loss of YTHDF2 results in the failure to regulate transcript dosage of a cohort of genes during oocyte maturation, with enrichment observed for the YTHDF2-binding consensus and evidence of m 6 A in these upregulated genes. In summary, the m 6 A-reader YTHDF2 is an intrinsic determinant of mammalian oocyte competence and early zygotic development. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Long-read sequencing of nascent RNA reveals coupling among RNA processing events.

PubMed

Herzel, Lydia; Straube, Korinna; Neugebauer, Karla M

2018-06-14

Pre-mRNA splicing is accomplished by the spliceosome, a megadalton complex that assembles de novo on each intron. Because spliceosome assembly and catalysis occur cotranscriptionally, we hypothesized that introns are removed in the order of their transcription in genomes dominated by constitutive splicing. Remarkably little is known about splicing order and the regulatory potential of nascent transcript remodeling by splicing, due to the limitations of existing methods that focus on analysis of mature splicing products (mRNAs) rather than substrates and intermediates. Here, we overcome this obstacle through long-read RNA sequencing of nascent, multi-intron transcripts in the fission yeast Schizosaccharomyces pombe Most multi-intron transcripts were fully spliced, consistent with rapid cotranscriptional splicing. However, an unexpectedly high proportion of transcripts were either fully spliced or fully unspliced, suggesting that splicing of any given intron is dependent on the splicing status of other introns in the transcript. Supporting this, mild inhibition of splicing by a temperature-sensitive mutation in prp2 , the homolog of vertebrate U2AF65, increased the frequency of fully unspliced transcripts. Importantly, fully unspliced transcripts displayed transcriptional read-through at the polyA site and were degraded cotranscriptionally by the nuclear exosome. Finally, we show that cellular mRNA levels were reduced in genes with a high number of unspliced nascent transcripts during caffeine treatment, showing regulatory significance of cotranscriptional splicing. Therefore, overall splicing of individual nascent transcripts, 3' end formation, and mRNA half-life depend on the splicing status of neighboring introns, suggesting crosstalk among spliceosomes and the polyA cleavage machinery during transcription elongation. © 2018 Herzel et al.; Published by Cold Spring Harbor Laboratory Press.

An Unsolved Mystery: The Target-Recognizing RNA Species of MicroRNA Genes

PubMed Central

Chen, Chang-Zheng

2013-01-01

MicroRNAs (miRNAs) are an abundant class of endogenous ~ 21-nucleotide (nt) RNAs. These small RNAs are produced from long primary miRNA transcripts — pri-miRNAs — through sequential endonucleolytic maturation steps that yield precursor miRNA (pre-miRNA) intermediates and then the mature miRNAs. The mature miRNAs are loaded into the RNA-induced silencing complexes (RISC), and guide RISC to target mRNAs for cleavage and/or translational repression. This paradigm, which represents one of major discoveries of modern molecular biology, is built on the assumption that mature miRNAs are the only species produced from miRNA genes that recognize targets. This assumption has guided the miRNA field for more than a decade and has led to our current understanding of the mechanisms of target recognition and repression by miRNAs. Although progress has been made, fundamental questions remain unanswered with regard to the principles of target recognition and mechanisms of repression. Here I raise questions about the assumption that mature miRNAs are the only target-recognizing species produced from miRNA genes and discuss the consequences of working under an incomplete or incorrect assumption. Moreover, I present evolution-based and experimental evidence that support the roles of pri-/pre-miRNAs in target recognition and repression. Finally, I propose a conceptual framework that integrates the functions of pri-/pre-miRNAs and mature miRNAs in target recognition and repression. The integrated framework opens experimental enquiry and permits interpretation of fundamental problems that have so far been precluded. PMID:23685275

Facilitated recycling protects human RNA polymerase III from repression by Maf1 in vitro.

PubMed

Cabart, Pavel; Lee, JaeHoon; Willis, Ian M

2008-12-26

Yeast cells synthesize approximately 3-6 million molecules of tRNA every cell cycle at a rate of approximately 2-4 transcripts/gene/s. This high rate of transcription is achieved through many rounds of reinitiation by RNA polymerase (pol) III on stable DNA-bound complexes of the initiation factor TFIIIB. Studies in yeast have shown that the rate of reinitiation is increased by facilitated recycling, a process that involves the repeated reloading of the polymerase on the same transcription unit. However, when nutrients become limiting or stress conditions are encountered, RNA pol III transcription is rapidly repressed through the action of the conserved Maf1 protein. Here we examine the relationship between Maf1-mediated repression and facilitated recycling in a human RNA pol III in vitro system. Using an immobilized template transcription assay, we demonstrate that facilitated recycling is conserved from yeast to humans. We assessed the ability of recombinant human Maf1 to inhibit different steps in transcription before and after preinitiation complex assembly. We show that recombinant Maf1 can inhibit the recruitment of TFIIIB and RNA pol III to immobilized templates. However, RNA pol III bound to preinitiation complexes or in elongation complexes is protected from repression by Maf1 and can undergo several rounds of initiation. This indicates that recombinant Maf1 is unable to inhibit facilitated recycling. The data suggest that additional biochemical steps may be necessary for rapid Maf1-dependent repression of RNA pol III transcription.

Kaposi's Sarcoma-Associated Herpesvirus Hijacks RNA Polymerase II To Create a Viral Transcriptional Factory

PubMed Central

Chen, Christopher Phillip; Lyu, Yuanzhi; Chuang, Frank; Nakano, Kazushi; Izumiya, Chie; Jin, Di; Campbell, Mel

2017-01-01

ABSTRACT Locally concentrated nuclear factors ensure efficient binding to DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable preinitiation complexes. We have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around Kaposi's sarcoma-associated herpesvirus (KSHV) genomes in the host cell nucleus. Using immunofluorescence labeling of latent nuclear antigen (LANA) protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At the single-cell level, we found that not all episomes were uniformly transcribed following reactivation stimuli. However, those episomes that were being transcribed would spontaneously aggregate to form transcriptional “factories,” which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of “viral transcriptional factories” decreased the pool of cellular RNA polymerase II available for cellular gene transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an “all-in-one” factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells. IMPORTANCE B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. Three-dimensional imaging of viral gene expression in the nucleus allows us to study interactions and changes in the physical distribution of these episomes following stimulation. The results showed heterogeneity in the responses of individual KSHV episomes to stimuli within a single reactivating cell; those episomes that did respond to stimulation, aggregated within large domains that appear to function as viral transcription factories. A significant portion of cellular RNA polymerase II was trapped in these factories and served to transcribe viral genomes, which coincided with an overall decrease in cellular gene expression. Our findings uncover a strategy of KSHV gene regulation through focal assembly of KSHV episomes and a molecular mechanism of late gene expression. PMID:28331082

A 5' UTR-Overlapping LncRNA Activates the Male-Determining Gene doublesex1 in the Crustacean Daphnia magna.

PubMed

Kato, Yasuhiko; Perez, Christelle Alexa G; Mohamad Ishak, Nur Syafiqah; Nong, Quang D; Sudo, Yuumi; Matsuura, Tomoaki; Wada, Tadashi; Watanabe, Hajime

2018-06-04

Long noncoding RNAs (lncRNAs) are pervasively transcribed in the eukaryotic genome [1] and are important for the control of master regulatory genes that are involved in cell differentiation and development [2, 3]. Here, we show that a 5' UTR-overlapping lncRNA regulates the male-specific expression of the DM-domain gene doublesex1 (dsx1) in the crustacean Daphnia magna, which produces males in response to environmental stimuli. This lncRNA, named doublesex1 alpha promoter-associated long RNA (DAPALR), is transcribed upstream the transcription start site (TSS) in a sense orientation and subjected to 5' end capping and 3' end processing at a stem-loop structure before the dsx1 coding exon. Similar to dsx1, its expression is only activated in males by the juvenile hormone (JH) and basic-leucine zipper (bZIP) transcription factor Vrille (Vri) and is maintained during embryogenesis. Knockdown of DAPALR in males silenced dsx1 and led to feminization, including egg production, whereas ectopic expression of DAPALR in dsx1-silenced females resulted in the de-repression of dsx1. We further demonstrate that the DAPALR transcript overlaps the dsx1 5'-UTR, and this overlapping region is required for dsx1 activation. Our results suggest that DAPALR can transactivate and possibly maintain dsx1 expression. This might be important for converting transient environmental signals into stable male development, controlled by the continuous expression of dsx1. Copyright © 2018 Elsevier Ltd. All rights reserved.

The orphan nuclear receptor Nur77 regulates decidual prolactin expression in human endometrial stromal cells

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

Jiang, Yue; Hu, Yali; Zhao, Jing

2011-01-14

Research highlights: {yields} Decidually produced PRL plays a key role during pregnancy. {yields} Overexpression of Nur77 increased PRL mRNA expression and enhanced decidual PRL promoter activity. {yields} Knockdown of Nur77 decreased decidual PRL secretion induced by 8-Br-cAMP and MPA. {yields} Nur77 is a novel transcription factor that plays an active role in decidual prolactin expression. -- Abstract: Prolactin (PRL) is synthesized and released by several extrapituitary tissues, including decidualized stromal cells. Despite the important role of decidual PRL during pregnancy, little is understood about the factors involved in the proper regulation of decidual PRL expression. Here we present evidence thatmore » the transcription factor Nur77 plays an active role in decidual prolactin expression in human endometrial stromal cells (hESCs). Nur77 mRNA expression in hESCs was significantly increased after decidualization stimulated by 8-Br-cAMP and medroxyprogesterone acetate (MPA). Adenovirus-mediated overexpression of Nur77 in hESCs markedly increased PRL mRNA expression and enhanced decidual PRL promoter (dPRL/-332Luc) activity in a concentration-dependent manner. Furthermore, knockdown of Nur77 in hESCs significantly decreased decidual PRL promoter activation and substantially attenuated PRL mRNA expression and PRL secretion (P < 0.01) induced by 8-Br-cAMP and MPA. These results demonstrate that Nur77 is a novel transcription factor that contributes significantly to the regulation of prolactin gene expression in human endometrial stromal cells.« less

Circular RNAs: diversity of form and function.

PubMed

Lasda, Erika; Parker, Roy

2014-12-01

It is now clear that there is a diversity of circular RNAs in biological systems. Circular RNAs can be produced by the direct ligation of 5' and 3' ends of linear RNAs, as intermediates in RNA processing reactions, or by "backsplicing," wherein a downstream 5' splice site (splice donor) is joined to an upstream 3' splice site (splice acceptor). Circular RNAs have unique properties including the potential for rolling circle amplification of RNA, the ability to rearrange the order of genomic information, protection from exonucleases, and constraints on RNA folding. Circular RNAs can function as templates for viroid and viral replication, as intermediates in RNA processing reactions, as regulators of transcription in cis, as snoRNAs, and as miRNA sponges. Herein, we review the breadth of circular RNAs, their biogenesis and metabolism, and their known and anticipated functions. © 2014 Lasda and Parker; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

PubMed Central

Tang, Grace Y.; Pribisko, Melanie A.; Henning, Ryan K.; Lim, Punnajit; Termini, John; Gray, Harry B.; Grubbs, Robert H.

2015-01-01

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy. Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action. Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control. The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide. This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates. PMID:25867444

An in vitro enzymatic assay to measure transcription inhibition by gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles.

PubMed

Tang, Grace Y; Pribisko, Melanie A; Henning, Ryan K; Lim, Punnajit; Termini, John; Gray, Harry B; Grubbs, Robert H

2015-03-18

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic macrocycles that exhibit differential cytostatic and cytotoxic properties in specific cell lines, depending on the identities of the chelated metal and functional groups. The unique behavior of functionalized corroles towards specific cell lines introduces the possibility of targeted chemotherapy. Many anticancer drugs are evaluated by their ability to inhibit RNA transcription. Here we present a step-by-step protocol for RNA transcription in the presence of known and potential inhibitors. The evaluation of the RNA products of the transcription reaction by gel electrophoresis and UV-Vis spectroscopy provides information on inhibitive properties of potential anticancer drug candidates and, with modifications to the assay, more about their mechanism of action. Little is known about the molecular mechanism of action of corrole cytotoxicity. In this experiment, we consider two corrole compounds: gallium(III) 5,10,15-(tris)pentafluorophenylcorrole (Ga(tpfc)) and freebase analogue 5,10,15-(tris)pentafluorophenylcorrole (tpfc). An RNA transcription assay was used to examine the inhibitive properties of the corroles. Five transcription reactions were prepared: DNA treated with Actinomycin D, triptolide, Ga(tpfc), tpfc at a [complex]:[template DNA base] ratio of 0.01, respectively, and an untreated control. The transcription reactions were analyzed after 4 hr using agarose gel electrophoresis and UV-Vis spectroscopy. There is clear inhibition by Ga(tpfc), Actinomycin D, and triptolide. This RNA transcription assay can be modified to provide more mechanistic detail by varying the concentrations of the anticancer complex, DNA, or polymerase enzyme, or by incubating the DNA or polymerase with the complexes prior to RNA transcription; these modifications would differentiate between an inhibition mechanism involving the DNA or the enzyme. Adding the complex after RNA transcription can be used to test whether the complexes degrade or hydrolyze the RNA. This assay can also be used to study additional anticancer candidates.

The Conserved Foot Domain of RNA Pol II Associates with Proteins Involved in Transcriptional Initiation and/or Early Elongation

PubMed Central

García-López, M. Carmen; Pelechano, Vicent; Mirón-García, M. Carmen; Garrido-Godino, Ana I.; García, Alicia; Calvo, Olga; Werner, Michel; Pérez-Ortín, José E.; Navarro, Francisco

2011-01-01

RNA polymerase (pol) II establishes many protein–protein interactions with transcriptional regulators to coordinate different steps of transcription. Although some of these interactions have been well described, little is known about the existence of RNA pol II regions involved in contact with transcriptional regulators. We hypothesize that conserved regions on the surface of RNA pol II contact transcriptional regulators. We identified such an RNA pol II conserved region that includes the majority of the “foot” domain and identified interactions of this region with Mvp1, a protein required for sorting proteins to the vacuole, and Spo14, a phospholipase D. Deletion of MVP1 and SPO14 affects the transcription of their target genes and increases phosphorylation of Ser5 in the carboxy-terminal domain (CTD). Genetic, phenotypic, and functional analyses point to a role for these proteins in transcriptional initiation and/or early elongation, consistent with their genetic interactions with CEG1, a guanylyltransferase subunit of the Saccharomyces cerevisiae capping enzyme. PMID:21954159

Identification of hundreds of novel UPF1 target transcripts by direct determination of whole transcriptome stability

PubMed Central

Tani, Hidenori; Imamachi, Naoto; Salam, Kazi Abdus; Mizutani, Rena; Ijiri, Kenichi; Irie, Takuma; Yada, Tetsushi; Suzuki, Yutaka; Akimitsu, Nobuyoshi

2012-01-01

UPF1 eliminates aberrant mRNAs harboring premature termination codons, and regulates the steady-state levels of normal physiological mRNAs. Although genome-wide studies of UPF1 targets performed, previous studies did not distinguish indirect UPF1 targets because they could not determine UPF1-dependent altered RNA stabilities. Here, we measured the decay rates of the whole transcriptome in UPF1-depleted HeLa cells using BRIC-seq, an inhibitor-free method for directly measuring RNA stability. We determined the half-lives and expression levels of 9,229 transcripts. An amount of 785 transcripts were stabilized in UPF1-depleted cells. Among these, the expression levels of 76 transcripts were increased, but those of the other 709 transcripts were not altered. RNA immunoprecipitation showed UPF1 bound to the stabilized transcripts, suggesting that UPF1 directly degrades the 709 transcripts. Many UPF1 targets in this study were newly identified. This study clearly demonstrates that direct determination of RNA stability is a powerful approach for identifying targets of RNA degradation factors. PMID:23064114

Genetic and mechanistic diversity of piRNA 3' end formation

PubMed Central

Handler, Dominik; Mohn, Fabio; Ameres, Stefan L.; Brennecke, Julius

2016-01-01

Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and thereby play important roles in eukaryotic gene silencing1. Of the three small RNA classes, microRNAs and siRNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. piRNAs—the 22-30 nt long guides for PIWI-clade Ago proteins that silence transposons in animal gonads—are generated Dicer-independently from single-stranded precursors2,3. piRNA 5' ends are defined either by Zucchini, a mitochondria-anchored endonuclease4,5, or by piRNA-guided target cleavage6,7. Formation of piRNA 3' ends is poorly understood. Here, we find that two genetically and mechanistically distinct pathways generate piRNA 3' ends in Drosophila. The initiating nucleases are either Zucchini or the PIWI-clade proteins Aubergine (Aub)/Ago3. While Zucchini-mediated cleavages directly define mature piRNA 3' ends8,9, Aub/Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3'-to-5' exoribonuclease Nibbler/Mut-710–13. The relative activity of these two pathways dictates the extent to which piRNAs are fueled into cytoplasmic or nuclear PIWI-clade proteins and thereby sets the balance between post-transcriptional and transcriptional silencing. Strikingly, loss of both Zucchini and Nibbler reveals a minimal, Argonaute-driven small RNA biogenesis pathway where piRNA 5' and 3' ends are directly produced by closely spaced Aub/Ago3-mediated cleavage events. Our data establish a coherent blueprint for piRNA biogenesis, and set the stage for the mechanistic dissection of the processes that govern piRNA 3' end formation. PMID:27851737

New insights into the promoterless transcription of DNA coligo templates by RNA polymerase III

PubMed Central

Lama, Lodoe; Seidl, Christine I; Ryan, Kevin

2014-01-01

Chemically synthesized DNA can carry small RNA sequence information but converting that information into small RNA is generally thought to require large double-stranded promoters in the context of plasmids, viruses and genes. We previously found evidence that circularized oligodeoxynucleotides (coligos) containing certain sequences and secondary structures can template the synthesis of small RNA by RNA polymerase III in vitro and in human cells. By using immunoprecipitated RNA polymerase III we now report corroborating evidence that this enzyme is the sole polymerase responsible for coligo transcription. The immobilized polymerase enabled experiments showing that coligo transcripts can be formed through transcription termination without subsequent 3′ end trimming. To better define the determinants of productive transcription, a structure-activity relationship study was performed using over 20 new coligos. The results show that unpaired nucleotides in the coligo stem facilitate circumtranscription, but also that internal loops and bulges should be kept small to avoid secondary transcription initiation sites. A polymerase termination sequence embedded in the double-stranded region of a hairpin-encoding coligo stem can antagonize transcription. Using lessons learned from new and old coligos, we demonstrate how to convert poorly transcribed coligos into productive templates. Our findings support the possibility that coligos may prove useful as chemically synthesized vectors for the ectopic expression of small RNA in human cells. PMID:25764216

Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts

PubMed Central

Burel, Sebastien A.; Hart, Christopher E.; Cauntay, Patrick; Hsiao, Jill; Machemer, Todd; Katz, Melanie; Watt, Andy; Bui, Huynh-hoa; Younis, Husam; Sabripour, Mahyar; Freier, Susan M.; Hung, Gene; Dan, Amy; Prakash, T.P.; Seth, Punit P.; Swayze, Eric E.; Bennett, C. Frank; Crooke, Stanley T.; Henry, Scott P.

2016-01-01

High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation. PMID:26553810

MiRNA-Target Interaction Reveals Cell-Specific Post-Transcriptional Regulation in Mammalian Cell Lines

PubMed Central

Kulkarni, Varun; Naqvi, Afsar Raza; Uttamani, Juhi Raju; Nares, Salvador

2016-01-01

MicroRNAs are 18–22 nucleotides long, non-coding RNAs that bind transcripts with complementary sequences leading to either mRNA degradation or translational suppression. However, the inherent differences in preferred mode of miRNA regulation among cells of different origin have not been examined. In our previous transcriptome profiling studies, we observed that post-transcriptional regulation can differ substantially depending on the cell in context. Here we examined mechanistic differences in the regulation of a let-7a targeted (wild type) or resistant (mutant) engineered renilla transcript across various mammalian cell lines of diverse origin. Dual luciferase assays show that compared to mutant (mut), the reporter gene containing wild type (wt) let-7a binding sites was efficiently suppressed upon transfection in various cell lines. Importantly, the strength of miRNA regulation varied across the cell lines. Total RNA analysis demonstrates that wt renilla mRNA was expressed to similar or higher levels compared to mut suggesting that translation repression is a predominant mode of miRNA regulation. Nonetheless, transcript degradation was observed in some cell lines. Ago-2 immunoprecipitation show that miRNA repressed renilla mRNA are associated with functional mi-RISC (miRNA-RNA induced silencing complex). Given the immense potential of miRNA as a therapeutic option, these findings highlight the necessity to thoroughly examine the mode of mRNA regulation in order to achieve the beneficial effects in targeting cells. PMID:26761000

NMR Structural Profiling of Transcriptional Intermediates Reveals Riboswitch Regulation by Metastable RNA Conformations.

PubMed

Helmling, Christina; Wacker, Anna; Wolfinger, Michael T; Hofacker, Ivo L; Hengesbach, Martin; Fürtig, Boris; Schwalbe, Harald

2017-02-22

Gene repression induced by the formation of transcriptional terminators represents a prime example for the coupling of RNA synthesis, folding, and regulation. In this context, mapping the changes in available conformational space of transcription intermediates during RNA synthesis is important to understand riboswitch function. A majority of riboswitches, an important class of small metabolite-sensing regulatory RNAs, act as transcriptional regulators, but the dependence of ligand binding and the subsequent allosteric conformational switch on mRNA transcript length has not yet been investigated. We show a strict fine-tuning of binding and sequence-dependent alterations of conformational space by structural analysis of all relevant transcription intermediates at single-nucleotide resolution for the I-A type 2'dG-sensing riboswitch from Mesoplasma florum by NMR spectroscopy. Our results provide a general framework to dissect the coupling of synthesis and folding essential for riboswitch function, revealing the importance of metastable states for RNA-based gene regulation.

Distinct Mechanisms of Transcription Initiation by RNA Polymerases I and II.

PubMed

Engel, Christoph; Neyer, Simon; Cramer, Patrick

2018-05-20

RNA polymerases I and II (Pol I and Pol II) are the eukaryotic enzymes that catalyze DNA-dependent synthesis of ribosomal RNA and messenger RNA, respectively. Recent work shows that the transcribing forms of both enzymes are similar and the fundamental mechanisms of RNA chain elongation are conserved. However, the mechanisms of transcription initiation and its regulation differ between Pol I and Pol II. Recent structural studies of Pol I complexes with transcription initiation factors provided insights into how the polymerase recognizes its specific promoter DNA, how it may open DNA, and how initiation may be regulated. Comparison with the well-studied Pol II initiation system reveals a distinct architecture of the initiation complex and visualizes promoter- and gene-class-specific aspects of transcription initiation. On the basis of new structural studies, we derive a model of the Pol I transcription cycle and provide a molecular movie of Pol I transcription that can be used for teaching.

Purification of Xenopus laevis mitochondrial RNA polymerase and identification of a dissociable factor required for specific transcription

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

Bogenhagen, D.F.; Insdorf, N.F.

1988-07-01

The Xenopus laevis mitochondrial RNA (mtRNA) polymerase was purified to near homogeneity with an overall yield approaching 50%. The major polypeptides in the final fraction were a doublet of proteins of approximately 140 kilodaltons that copurified with the mtRNA polymerase activity. It appeared likely that the smaller polypeptide is a breakdown product of the larger one. The highly purified polymerase was active in nonspecific transcription but required a dissociable factor for specific transcription of X. laevis mtDNA. The factor could be resolved from mtRNA polymerase by hydrophobic chromatography and had a sedimentation coefficient of 3.0 S. The transcription factor elutedmore » from both the hydrophobic column and a Mono Q anion-exchange column as a single symmetrical peak. The mtRNA polymerase and this factor together are necessary and sufficient for active transcription from four promoters located in a noncoding region of the mtDNA genome between the gene for tRNA/sup Phe/ and the displacement loop.« less

saRNA-guided Ago2 targets the RITA complex to promoters to stimulate transcription.

PubMed

Portnoy, Victoria; Lin, Szu Hua Sharon; Li, Kathy H; Burlingame, Alma; Hu, Zheng-Hui; Li, Hao; Li, Long-Cheng

2016-03-01

Small activating RNAs (saRNAs) targeting specific promoter regions are able to stimulate gene expression at the transcriptional level, a phenomenon known as RNA activation (RNAa). It is known that RNAa depends on Ago2 and is associated with epigenetic changes at the target promoters. However, the precise molecular mechanism of RNAa remains elusive. Using human CDKN1A (p21) as a model gene, we characterized the molecular nature of RNAa. We show that saRNAs guide Ago2 to and associate with target promoters. saRNA-loaded Ago2 facilitates the assembly of an RNA-induced transcriptional activation (RITA) complex, which, in addition to saRNA-Ago2 complex, includes RHA and CTR9, the latter being a component of the PAF1 complex. RITA interacts with RNA polymerase II to stimulate transcription initiation and productive elongation, accompanied by monoubiquitination of histone 2B. Our results establish the existence of a cellular RNA-guided genome-targeting and transcriptional activation mechanism and provide important new mechanistic insights into the RNAa process.

Fructose bisphosphate aldolase is involved in the control of RNA polymerase III-directed transcription.

PubMed

Cieśla, Małgorzata; Mierzejewska, Jolanta; Adamczyk, Małgorzata; Farrants, Ann-Kristin Östlund; Boguta, Magdalena

2014-06-01

Yeast Fba1 (fructose 1,6-bisphosphate aldolase) is a glycolytic enzyme essential for viability. The overproduction of Fba1 enables overcoming of a severe growth defect caused by a missense mutation rpc128-1007 in a gene encoding the C128 protein, the second largest subunit of the RNA polymerase III complex. The suppression of the growth phenotype by Fba1 is accompanied by enhanced de novo tRNA transcription in rpc128-1007 cells. We inactivated residues critical for the catalytic activity of Fba1. Overproduction of inactive aldolase still suppressed the rpc128-1007 phenotype, indicating that the function of this glycolytic enzyme in RNA polymerase III transcription is independent of its catalytic activity. Yeast Fba1 was determined to interact with the RNA polymerase III complex by coimmunoprecipitation. Additionally, a role of aldolase in control of tRNA transcription was confirmed by ChIP experiments. The results indicate a novel direct relationship between RNA polymerase III transcription and aldolase. Copyright © 2014 Elsevier B.V. All rights reserved.

The molecular dynamics of long noncoding RNA control of transcription in PTEN and its pseudogene

PubMed Central

Lister, Nicholas; Shevchenko, Galina; Walshe, James L.; Groen, Jessica; Johnsson, Per; Vidarsdóttir, Linda; Grander, Dan; Ataide, Sandro F.; Morris, Kevin V.

2017-01-01

RNA has been found to interact with chromatin and modulate gene transcription. In human cells, little is known about how long noncoding RNAs (lncRNAs) interact with target loci in the context of chromatin. We find here, using the phosphatase and tensin homolog (PTEN) pseudogene as a model system, that antisense lncRNAs interact first with a 5′ UTR-containing promoter-spanning transcript, which is then followed by the recruitment of DNA methyltransferase 3a (DNMT3a), ultimately resulting in the transcriptional and epigenetic control of gene expression. Moreover, we find that the lncRNA and promoter-spanning transcript interaction are based on a combination of structural and sequence components of the antisense lncRNA. These observations suggest, on the basis of this one example, that evolutionary pressures may be placed on RNA structure more so than sequence conservation. Collectively, the observations presented here suggest a much more complex and vibrant RNA regulatory world may be operative in the regulation of gene expression. PMID:28847966

Mapping the subgenomic RNA promoter of the Citrus leaf blotch virus coat protein gene by Agrobacterium-mediated inoculation.

PubMed

Renovell, Agueda; Gago, Selma; Ruiz-Ruiz, Susana; Velázquez, Karelia; Navarro, Luis; Moreno, Pedro; Vives, Mari Carmen; Guerri, José

2010-10-25

Citrus leaf blotch virus has a single-stranded positive-sense genomic RNA (gRNA) of 8747 nt organized in three open reading frames (ORFs). The ORF1, encoding a polyprotein involved in replication, is translated directly from the gRNA, whereas ORFs encoding the movement (MP) and coat (CP) proteins are expressed via 3' coterminal subgenomic RNAs (sgRNAs). We characterized the minimal promoter region critical for the CP-sgRNA expression in infected cells by deletion analyses using Agrobacterium-mediated infection of Nicotiana benthamiana plants. The minimal CP-sgRNA promoter was mapped between nucleotides -67 and +50 nt around the transcription start site. Surprisingly, larger deletions in the region between the CP-sgRNA transcription start site and the CP translation initiation codon resulted in increased CP-sgRNA accumulation, suggesting that this sequence could modulate the CP-sgRNA transcription. Site-specific mutational analysis of the transcription start site revealed that the +1 guanylate and the +2 adenylate are important for CP-sgRNA synthesis. Copyright © 2010 Elsevier Inc. All rights reserved.

Differential gene expression in dentate granule cells in mesial temporal lobe epilepsy with and without hippocampal sclerosis.

PubMed

Griffin, Nicole G; Wang, Yu; Hulette, Christine M; Halvorsen, Matt; Cronin, Kenneth D; Walley, Nicole M; Haglund, Michael M; Radtke, Rodney A; Skene, J H Pate; Sinha, Saurabh R; Heinzen, Erin L

2016-03-01

Hippocampal sclerosis is the most common neuropathologic finding in cases of medically intractable mesial temporal lobe epilepsy. In this study, we analyzed the gene expression profiles of dentate granule cells of patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis to show that next-generation sequencing methods can produce interpretable genomic data from RNA collected from small homogenous cell populations, and to shed light on the transcriptional changes associated with hippocampal sclerosis. RNA was extracted, and complementary DNA (cDNA) was prepared and amplified from dentate granule cells that had been harvested by laser capture microdissection from surgically resected hippocampi from patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis. Sequencing libraries were sequenced, and the resulting sequencing reads were aligned to the reference genome. Differential expression analysis was used to ascertain expression differences between patients with and without hippocampal sclerosis. Greater than 90% of the RNA-Seq reads aligned to the reference. There was high concordance between transcriptional profiles obtained for duplicate samples. Principal component analysis revealed that the presence or absence of hippocampal sclerosis was the main determinant of the variance within the data. Among the genes up-regulated in the hippocampal sclerosis samples, there was significant enrichment for genes involved in oxidative phosphorylation. By analyzing the gene expression profiles of dentate granule cells from surgically resected hippocampal specimens from patients with mesial temporal lobe epilepsy with and without hippocampal sclerosis, we have demonstrated the utility of next-generation sequencing methods for producing biologically relevant results from small populations of homogeneous cells, and have provided insight on the transcriptional changes associated with this pathology. Wiley Periodicals, Inc. © 2016 International League Against Epilepsy.

Expression of a non-coding RNA in ectromelia virus is required for normal plaque formation.

PubMed

Esteban, David J; Upton, Chris; Bartow-McKenney, Casey; Buller, R Mark L; Chen, Nanhai G; Schriewer, Jill; Lefkowitz, Elliot J; Wang, Chunlin

2014-02-01

Poxviruses are dsDNA viruses with large genomes. Many genes in the genome remain uncharacterized, and recent studies have demonstrated that the poxvirus transcriptome includes numerous so-called anomalous transcripts not associated with open reading frames. Here, we characterize the expression and role of an apparently non-coding RNA in orthopoxviruses, which we call viral hairpin RNA (vhRNA). Using a bioinformatics approach, we predicted expression of a transcript not associated with an open reading frame that is likely to form a stem-loop structure due to the presence of a 21 nt palindromic sequence. Expression of the transcript as early as 2 h post-infection was confirmed by northern blot and analysis of publicly available vaccinia virus infected cell transcriptomes. The transcription start site was determined by RACE PCE and transcriptome analysis, and early and late promoter sequences were identified. Finally, to test the function of the transcript we generated an ectromelia virus knockout, which failed to form plaques in cell culture. The important role of the transcript in viral replication was further demonstrated using siRNA. Although the function of the transcript remains unknown, our work contributes to evidence of an increasingly complex poxvirus transcriptome, suggesting that transcripts such as vhRNA not associated with an annotated open reading frame can play an important role in viral replication.

Zinc Finger-Containing Cellular Transcription Corepressor ZBTB25 Promotes Influenza Virus RNA Transcription and Is a Target for Zinc Ejector Drugs.

PubMed

Chen, Shu-Chuan; Jeng, King-Song; Lai, Michael M C

2017-10-15

Influenza A virus (IAV) replication relies on an intricate interaction between virus and host cells. How the cellular proteins are usurped for IAV replication remains largely obscure. The aim of this study was to search for novel and potential cellular factors that participate in IAV replication. ZBTB25, a transcription repressor of a variety of cellular genes, was identified by an RNA interference (RNAi) genomic library screen. Depletion of ZBTB25 significantly reduced IAV production. Conversely, overexpression of ZBTB25 enhanced it. ZBTB25 interacted with the viral RNA-dependent RNA polymerase (RdRp) protein and modulated its transcription activity. In addition, ZBTB25 also functioned as a viral RNA (vRNA)-binding protein, binding preferentially to the U-rich sequence within the 5' untranslated region (UTR) of vRNA. Both protein-protein and protein-RNA interactions involving ZBTB25 facilitated viral RNA transcription and replication. In addition, ZBTB25 suppressed interferon production, further enhancing viral replication. ZBTB25-associated functions required an intact zinc finger domain and posttranslational SUMO-1 modification of ZBTB25. Furthermore, treatment with disulfiram (a zinc ejector) of ZBTB25-overexpressing cells showed significantly reduced IAV production as a result of reduced RNA synthesis. Our findings indicate that IAV usurps ZBTB25 for IAV RNA synthesis and serves as a novel and potential therapeutic antiviral target. IMPORTANCE IAV-induced seasonal influenza causes severe illness and death in high-risk populations. However, IAV has developed resistance to current antiviral drugs due to its high mutation rate. Therefore, development of drugs targeting cellular factors required for IAV replication is an attractive alternative for IAV therapy. Here, we discovered a cellular protein, ZBTB25, that enhances viral RdRp activity by binding to both viral RdRp and viral RNA to stimulate viral RNA synthesis. A unique feature of ZBTB25 in the regulation of viral replication is its dual transcription functions, namely, promoting viral RNA transcription through binding to the U-rich region of vRNA and suppressing cellular interferon production. ZBTB25 contains a zinc finger domain that is required for RNA-inhibitory activity by chelating zinc ions. Disulfiram treatment disrupts the zinc finger functions, effectively repressing IAV replication. Based on our findings, we demonstrate that ZBTB25 regulates IAV RNA transcription and replication and serves as a promising antiviral target for IAV treatment. Copyright © 2017 American Society for Microbiology.

Zinc Finger-Containing Cellular Transcription Corepressor ZBTB25 Promotes Influenza Virus RNA Transcription and Is a Target for Zinc Ejector Drugs

PubMed Central

Chen, Shu-Chuan; Jeng, King-Song

2017-01-01

ABSTRACT Influenza A virus (IAV) replication relies on an intricate interaction between virus and host cells. How the cellular proteins are usurped for IAV replication remains largely obscure. The aim of this study was to search for novel and potential cellular factors that participate in IAV replication. ZBTB25, a transcription repressor of a variety of cellular genes, was identified by an RNA interference (RNAi) genomic library screen. Depletion of ZBTB25 significantly reduced IAV production. Conversely, overexpression of ZBTB25 enhanced it. ZBTB25 interacted with the viral RNA-dependent RNA polymerase (RdRp) protein and modulated its transcription activity. In addition, ZBTB25 also functioned as a viral RNA (vRNA)-binding protein, binding preferentially to the U-rich sequence within the 5′ untranslated region (UTR) of vRNA. Both protein-protein and protein-RNA interactions involving ZBTB25 facilitated viral RNA transcription and replication. In addition, ZBTB25 suppressed interferon production, further enhancing viral replication. ZBTB25-associated functions required an intact zinc finger domain and posttranslational SUMO-1 modification of ZBTB25. Furthermore, treatment with disulfiram (a zinc ejector) of ZBTB25-overexpressing cells showed significantly reduced IAV production as a result of reduced RNA synthesis. Our findings indicate that IAV usurps ZBTB25 for IAV RNA synthesis and serves as a novel and potential therapeutic antiviral target. IMPORTANCE IAV-induced seasonal influenza causes severe illness and death in high-risk populations. However, IAV has developed resistance to current antiviral drugs due to its high mutation rate. Therefore, development of drugs targeting cellular factors required for IAV replication is an attractive alternative for IAV therapy. Here, we discovered a cellular protein, ZBTB25, that enhances viral RdRp activity by binding to both viral RdRp and viral RNA to stimulate viral RNA synthesis. A unique feature of ZBTB25 in the regulation of viral replication is its dual transcription functions, namely, promoting viral RNA transcription through binding to the U-rich region of vRNA and suppressing cellular interferon production. ZBTB25 contains a zinc finger domain that is required for RNA-inhibitory activity by chelating zinc ions. Disulfiram treatment disrupts the zinc finger functions, effectively repressing IAV replication. Based on our findings, we demonstrate that ZBTB25 regulates IAV RNA transcription and replication and serves as a promising antiviral target for IAV treatment. PMID:28768860

Developmental and sex-specific differences in expression of neuropeptides derived from allatotropin gene in the silkmoth Bombyx mori.

PubMed

Bednár, Branislav; Roller, Ladislav; Čižmár, Daniel; Mitrová, Diana; Žitňan, Dušan

2017-05-01

Allatotropin (AT) and related neuropeptides are widespread bioactive molecules that regulate development, food intake and muscle contractions in insects and other invertebrates. In moths, alternative splicing of the at gene generates three mRNA precursors encoding AT with different combinations of three structurally similar AT-like peptides (ATLI-III). We used in situ hybridization and immunohistochemistry to map the differential expression of these transcripts during the postembryonic development of Bombyx mori. Transcript encoding AT alone was expressed in numerous neurons of the central nervous system and frontal ganglion, whereas transcripts encoding AT with ATLs were produced by smaller specific subgroups of neurons in larval stages. Metamorphosis was associated with considerable developmental changes and sex-specific differences in the expression of all transcripts. The most notable was the appearance of AT/ATL transcripts (1) in the brain lateral neurosecretory cells producing prothoracicotropic hormone; (2) in the male-specific cluster of about 20 neurons in the posterior region of the terminal abdominal ganglion; (3) in the female-specific medial neurons in the abdominal ganglia AG2-7. Immunohistochemical staining showed that these neurons produced a mixture of various neuropeptides and innervated diverse peripheral organs. Our data suggest that AT/ATL neuropeptides are involved in multiple stage- and sex-specific functions during the development of B. mori.

MITIE: Simultaneous RNA-Seq-based transcript identification and quantification in multiple samples.

PubMed

Behr, Jonas; Kahles, André; Zhong, Yi; Sreedharan, Vipin T; Drewe, Philipp; Rätsch, Gunnar

2013-10-15

High-throughput sequencing of mRNA (RNA-Seq) has led to tremendous improvements in the detection of expressed genes and reconstruction of RNA transcripts. However, the extensive dynamic range of gene expression, technical limitations and biases, as well as the observed complexity of the transcriptional landscape, pose profound computational challenges for transcriptome reconstruction. We present the novel framework MITIE (Mixed Integer Transcript IdEntification) for simultaneous transcript reconstruction and quantification. We define a likelihood function based on the negative binomial distribution, use a regularization approach to select a few transcripts collectively explaining the observed read data and show how to find the optimal solution using Mixed Integer Programming. MITIE can (i) take advantage of known transcripts, (ii) reconstruct and quantify transcripts simultaneously in multiple samples, and (iii) resolve the location of multi-mapping reads. It is designed for genome- and assembly-based transcriptome reconstruction. We present an extensive study based on realistic simulated RNA-Seq data. When compared with state-of-the-art approaches, MITIE proves to be significantly more sensitive and overall more accurate. Moreover, MITIE yields substantial performance gains when used with multiple samples. We applied our system to 38 Drosophila melanogaster modENCODE RNA-Seq libraries and estimated the sensitivity of reconstructing omitted transcript annotations and the specificity with respect to annotated transcripts. Our results corroborate that a well-motivated objective paired with appropriate optimization techniques lead to significant improvements over the state-of-the-art in transcriptome reconstruction. MITIE is implemented in C++ and is available from http://bioweb.me/mitie under the GPL license.

Transcription termination and RNA processing in the 3'-end spacer of mouse ribosomal RNA genes.

PubMed Central

Miwa, T; Kominami, R; Yoshikura, H; Sudo, K; Muramatsu, M

1987-01-01

The 3' termini of ribosomal RNA precursors from mouse FM3A cultured cells are mapped to eight sites within 625 bp downstream from the 3' terminus of 28 S rRNA. Three additional sites are mapped in liver RNA from C3H/He strain mice. Two of them, the sites at 570 bp and 625 bp are assumed to be termination sites in vivo, because they correspond to in vitro termination sites of RNA polymerase I, and 45 S RNAs having these 3' termini decay with kinetics distinct from others. The amount of 45 S RNA having the 3' terminus at other sites is variable among several mouse strains, despite their having the same DNA sequence in these regions. The ability to produce 3' termini in these sites seems to follow Mendel's law of inheritance. Therefore, we postulate that these nine sites are RNA processing sites which are controlled genetically. Images PMID:3031586

Vitamin D and alternative splicing of RNA

PubMed Central

Zhou, Rui; Chun, Rene F.; Lisse, Thomas S.; Garcia, Alejandro J.; Xu, Jianzhong; Adams, John S.; Hewison, Martin

2014-01-01

The active form of vitamin D (1α,25-dihydroxyvitamin D, 1,25(OH)2D) exerts its genomic effects via binding to a nuclear high-affinity vitamin D receptor (VDR). Recent deep sequencing analysis of VDR binding locations across the complete genome has significantly expanded our understanding of the actions of vitamin D and VDR on gene transcription. However, these studies have also promoted appreciation of the extra-transcriptional impact of vitamin D on gene expression. It is now clear that vitamin D interacts with the epigenome via effects on DNA methylation, histone acetylation, and microRNA generation to maintain normal biological functions. There is also increasing evidence that vitamin D can influence pre-mRNA constitutive splicing and alternative splicing, although the mechanism for this remains unclear. Pre-mRNA splicing has long been thought to be a post-transcription RNA processing event, but current data indicate that this occurs co-transcriptionally. Several steroid hormones have been recognized to coordinately control gene transcription and pre-mRNA splicing through the recruitment of nuclear receptor co-regulators that can both control gene transcription and splicing. The current review will discuss this concept with specific reference to vitamin D, and the potential role of heterogeneous nuclear ribonucleoprotein C (hnRNPC), a nuclear factor with an established function in RNA splicing. hnRNPC, has been shown to be involved in the VDR transcriptional complex as a vitamin D-response element-binding protein (VDRE-BP), and may act as a coupling factor linking VDR-directed gene transcription with RNA splicing. In this way hnRNPC may provide an additional mechanism for the fine-tuning of vitamin D-regulated target gene expression. PMID:25447737

The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

PubMed

Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

2016-04-26

Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

HnRNP-like proteins as post-transcriptional regulators.

PubMed

Yeap, Wan-Chin; Namasivayam, Parameswari; Ho, Chai-Ling

2014-10-01

Plant cells contain a diverse repertoire of RNA-binding proteins (RBPs) that coordinate a network of post-transcriptional regulation. RBPs govern diverse developmental processes by modulating the gene expression of specific transcripts. Recent gene annotation and RNA sequencing clearly showed that heterogeneous nuclear ribonucleoprotein (hnRNP)-like proteins which form a family of RBPs, are also expressed in higher plants and serve specific plant functions. In addition to their involvement in post-transcriptional regulation from mRNA capping to translation, they are also involved in telomere regulation, gene silencing and regulation in chloroplast. Here, we review the involvement of plant hnRNP-like proteins in post-transcription regulation of RNA processes and their functional roles in control of plant developmental processes especially plant-specific functions including flowering, chloroplastic-specific mRNA regulation, long-distance phloem transportation and plant responses to environmental stresses. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Distributed biotin–streptavidin transcription roadblocks for mapping cotranscriptional RNA folding

PubMed Central

Strobel, Eric J.; Nedialkov, Yuri; Artsimovitch, Irina

2017-01-01

Abstract RNA folding during transcription directs an order of folding that can determine RNA structure and function. However, the experimental study of cotranscriptional RNA folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structure at nucleotide resolution. To address this, we previously developed cotranscriptional selective 2΄-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) to simultaneously probe all intermediate RNA transcripts during transcription by stalling elongation complexes at catalytically dead EcoRIE111Q roadblocks. While effective, the distribution of elongation complexes using EcoRIE111Q requires laborious PCR using many different oligonucleotides for each sequence analyzed. Here, we improve the broad applicability of cotranscriptional SHAPE-Seq by developing a sequence-independent biotin–streptavidin (SAv) roadblocking strategy that simplifies the preparation of roadblocking DNA templates. We first determine the properties of biotin–SAv roadblocks. We then show that randomly distributed biotin–SAv roadblocks can be used in cotranscriptional SHAPE-Seq experiments to identify the same RNA structural transitions related to a riboswitch decision-making process that we previously identified using EcoRIE111Q. Lastly, we find that EcoRIE111Q maps nascent RNA structure to specific transcript lengths more precisely than biotin–SAv and propose guidelines to leverage the complementary strengths of each transcription roadblock in cotranscriptional SHAPE-Seq. PMID:28398514

Distributed biotin-streptavidin transcription roadblocks for mapping cotranscriptional RNA folding.

PubMed

Strobel, Eric J; Watters, Kyle E; Nedialkov, Yuri; Artsimovitch, Irina; Lucks, Julius B

2017-07-07

RNA folding during transcription directs an order of folding that can determine RNA structure and function. However, the experimental study of cotranscriptional RNA folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structure at nucleotide resolution. To address this, we previously developed cotranscriptional selective 2΄-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) to simultaneously probe all intermediate RNA transcripts during transcription by stalling elongation complexes at catalytically dead EcoRIE111Q roadblocks. While effective, the distribution of elongation complexes using EcoRIE111Q requires laborious PCR using many different oligonucleotides for each sequence analyzed. Here, we improve the broad applicability of cotranscriptional SHAPE-Seq by developing a sequence-independent biotin-streptavidin (SAv) roadblocking strategy that simplifies the preparation of roadblocking DNA templates. We first determine the properties of biotin-SAv roadblocks. We then show that randomly distributed biotin-SAv roadblocks can be used in cotranscriptional SHAPE-Seq experiments to identify the same RNA structural transitions related to a riboswitch decision-making process that we previously identified using EcoRIE111Q. Lastly, we find that EcoRIE111Q maps nascent RNA structure to specific transcript lengths more precisely than biotin-SAv and propose guidelines to leverage the complementary strengths of each transcription roadblock in cotranscriptional SHAPE-Seq. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Global Transcriptional Start Site Mapping Using Differential RNA Sequencing Reveals Novel Antisense RNAs in Escherichia coli

PubMed Central

Thomason, Maureen K.; Bischler, Thorsten; Eisenbart, Sara K.; Förstner, Konrad U.; Zhang, Aixia; Herbig, Alexander; Nieselt, Kay

2014-01-01

While the model organism Escherichia coli has been the subject of intense study for decades, the full complement of its RNAs is only now being examined. Here we describe a survey of the E. coli transcriptome carried out using a differential RNA sequencing (dRNA-seq) approach, which can distinguish between primary and processed transcripts, and an automated prediction algorithm for transcriptional start sites (TSS). With the criterion of expression under at least one of three growth conditions examined, we predicted 14,868 TSS candidates, including 5,574 internal to annotated genes (iTSS) and 5,495 TSS corresponding to potential antisense RNAs (asRNAs). We examined expression of 14 candidate asRNAs by Northern analysis using RNA from wild-type E. coli and from strains defective for RNases III and E, two RNases reported to be involved in asRNA processing. Interestingly, nine asRNAs detected as distinct bands by Northern analysis were differentially affected by the rnc and rne mutations. We also compared our asRNA candidates with previously published asRNA annotations from RNA-seq data and discuss the challenges associated with these cross-comparisons. Our global transcriptional start site map represents a valuable resource for identification of transcription start sites, promoters, and novel transcripts in E. coli and is easily accessible, together with the cDNA coverage plots, in an online genome browser. PMID:25266388

Silencing of ecdysone receptor, insect intestinal mucin and sericotropin genes by bacterially produced double-stranded RNA affects larval growth and development in Plutella xylostella and Helicoverpa armigera.

PubMed

Israni, B; Rajam, M V

2017-04-01

RNA interference mediated gene silencing, which is triggered by double-stranded RNA (dsRNA), has become a important tool for functional genomics studies in various systems, including insects. Bacterially produced dsRNA employs the use of a bacterial strain lacking in RNaseIII activity and harbouring a vector with dual T7 promoter sites, which allow the production of intact dsRNA molecules. Here, we report an assessment of the functional relevance of the ecdysone receptor, insect intestinal mucin and sericotropin genes through silencing by dsRNA in two lepidopteran insect pests, Helicoverpa armigera and Plutella xylostella, both of which cause serious crop losses. Oral feeding of dsRNA led to significant reduction in transcripts of the target insect genes, which caused significant larval mortality with various moulting anomalies and an overall developmental delay. We also found a significant decrease in reproductive potential in female moths, with a drop in egg laying and compromised egg hatching from treated larvae as compared to controls. dsRNA was stable in the insect gut and was efficiently processed into small interfering RNAs (siRNAs), thus accounting for the phenotypes observed in the present work. The study revealed the importance of these genes in core insect processes, which are essential for insect development and survival. © 2016 The Royal Entomological Society.

SKI2 mediates degradation of RISC 5′-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis

PubMed Central

Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

2015-01-01

Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20–24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5′, but not 3′-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5′ to the cleavage site, but several examples of 3′-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5′-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5′-cleavage fragments. PMID:26464441

One nuclear calcium transient induced by a single burst of action potentials represents the minimum signal strength in activity-dependent transcription in hippocampal neurons.

PubMed

Yu, Yan; Oberlaender, Kristin; Bengtson, C Peter; Bading, Hilmar

2017-07-01

Neurons undergo dramatic changes in their gene expression profiles in response to synaptic stimulation. The coupling of neuronal excitation to gene transcription is well studied and is mediated by signaling pathways activated by cytoplasmic and nuclear calcium transients. Despite this, the minimum synaptic activity required to induce gene expression remains unknown. To address this, we used cultured hippocampal neurons and cellular compartment analysis of temporal activity by fluorescence in situ hybridization (catFISH) that allows detection of nascent transcripts in the cell nucleus. We found that a single burst of action potentials, consisting of 24.4±5.1 action potentials during a 6.7±1.9s depolarization of 19.5±2.0mV causing a 9.3±0.9s somatic calcium transient, is sufficient to activate transcription of the immediate early gene arc (also known as Arg3.1). The total arc mRNA yield produced after a single burst-induced nuclear calcium transient was very small and, compared to unstimulated control neurons, did not lead to a significant increase in arc mRNA levels measured using quantitative reverse transcriptase PCR (qRT-PCR) of cell lysates. Significantly increased arc mRNA levels became detectable in hippocampal neurons that had undergone 5-8 consecutive burst-induced nuclear calcium transients at 0.05-0.15Hz. These results indicate that a single burst-induced nuclear calcium transient can activate gene expression and that transcription is rapidly shut off after synaptic stimulation has ceased. Copyright © 2017 Elsevier Ltd. All rights reserved.

An operon encoding three glycolytic enzymes in Lactobacillus delbrueckii subsp. bulgaricus: glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase.

PubMed

Branny, P; de la Torre, F; Garel, J R

1998-04-01

The structural genes gap, pgk and tpi encoding three glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK) and triosephosphate isomerase (TPI), respectively, have been cloned and sequenced from Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus). The genes were isolated after screening genomic sublibraries with specific gap and pgk probes obtained by PCR amplification of chromosomal DNA with degenerate primers corresponding to amino acid sequences highly conserved in GAPDHs and PGKs. Nucleotide sequencing revealed that the three genes were organized in the order gap-pgk-tpi. The translation start codons of the three genes were identified by alignment of the N-terminal sequences. These genes predicted polypeptide chains of 338, 403 and 252 amino acids for GAPDH, PGK and TPI, respectively, and they were separated by 96 bp between gap and pgk, and by only 18 bp between pgk and tpi. The codon usage in gap, pgk, tpi and three other glycolytic genes from L. bulgaricus differed, noticeably from that in other chromosomal genes. The site of transcriptional initiation was located by primer extension, and a probable promoter was identified for the gap-pgk-tpi operon. Northern hybridization of total RNA with specific probes showed two transcripts, an mRNA of 1.4 kb corresponding to the gap gene, and a less abundant mRNA of 3.4 kb corresponding to the gap-pgk-tpi cluster. The absence of a visible terminator in the 3'-end of the shorter transcript and the location of this 3'-end inside the pgk gene indicated that this shorter transcript was produced by degradation of the longer one, rather than by an early termination of transcription after the gap gene.

Radiation-induced alternative transcripts as detected in total and polysome-bound mRNA.

PubMed

Wahba, Amy; Ryan, Michael C; Shankavaram, Uma T; Camphausen, Kevin; Tofilon, Philip J

2018-01-02

Alternative splicing is a critical event in the posttranscriptional regulation of gene expression. To investigate whether this process influences radiation-induced gene expression we defined the effects of ionizing radiation on the generation of alternative transcripts in total cellular mRNA (the transcriptome) and polysome-bound mRNA (the translatome) of the human glioblastoma stem-like cell line NSC11. For these studies, RNA-Seq profiles from control and irradiated cells were compared using the program SpliceSeq to identify transcripts and splice variations induced by radiation. As compared to the transcriptome (total RNA) of untreated cells, the radiation-induced transcriptome contained 92 splice events suggesting that radiation induced alternative splicing. As compared to the translatome (polysome-bound RNA) of untreated cells, the radiation-induced translatome contained 280 splice events of which only 24 were overlapping with the radiation-induced transcriptome. These results suggest that radiation not only modifies alternative splicing of precursor mRNA, but also results in the selective association of existing mRNA isoforms with polysomes. Comparison of radiation-induced alternative transcripts to radiation-induced gene expression in total RNA revealed little overlap (about 3%). In contrast, in the radiation-induced translatome, about 38% of the induced alternative transcripts corresponded to genes whose expression level was affected in the translatome. This study suggests that whereas radiation induces alternate splicing, the alternative transcripts present at the time of irradiation may play a role in the radiation-induced translational control of gene expression and thus cellular radioresponse.

Using in-cell SHAPE-Seq and simulations to probe structure–function design principles of RNA transcriptional regulators

PubMed Central

Takahashi, Melissa K.; Watters, Kyle E.; Gasper, Paul M.; Abbott, Timothy R.; Carlson, Paul D.; Chen, Alan A.

2016-01-01

Antisense RNA-mediated transcriptional regulators are powerful tools for controlling gene expression and creating synthetic gene networks. RNA transcriptional repressors derived from natural mechanisms called attenuators are particularly versatile, though their mechanistic complexity has made them difficult to engineer. Here we identify a new structure–function design principle for attenuators that enables the forward engineering of new RNA transcriptional repressors. Using in-cell SHAPE-Seq to characterize the structures of attenuator variants within Escherichia coli, we show that attenuator hairpins that facilitate interaction with antisense RNAs require interior loops for proper function. Molecular dynamics simulations of these attenuator variants suggest these interior loops impart structural flexibility. We further observe hairpin flexibility in the cellular structures of natural RNA mechanisms that use antisense RNA interactions to repress translation, confirming earlier results from in vitro studies. Finally, we design new transcriptional attenuators in silico using an interior loop as a structural requirement and show that they function as desired in vivo. This work establishes interior loops as an important structural element for designing synthetic RNA gene regulators. We anticipate that the coupling of experimental measurement of cellular RNA structure and function with computational modeling will enable rapid discovery of structure–function design principles for a diverse array of natural and synthetic RNA regulators. PMID:27103533

Modulation of yeast genome expression in response to defective RNA polymerase III-dependent transcription.

PubMed

Conesa, Christine; Ruotolo, Roberta; Soularue, Pascal; Simms, Tiffany A; Donze, David; Sentenac, André; Dieci, Giorgio

2005-10-01

We used genome-wide expression analysis in Saccharomyces cerevisiae to explore whether and how the expression of protein-coding, RNA polymerase (Pol) II-transcribed genes is influenced by a decrease in RNA Pol III-dependent transcription. The Pol II transcriptome was characterized in four thermosensitive, slow-growth mutants affected in different components of the RNA Pol III transcription machinery. Unexpectedly, we found only a modest correlation between altered expression of Pol II-transcribed genes and their proximity to class III genes, a result also confirmed by the analysis of single tRNA gene deletants. Instead, the transcriptome of all of the four mutants was characterized by increased expression of genes known to be under the control of the Gcn4p transcriptional activator. Indeed, GCN4 was found to be translationally induced in the mutants, and deleting the GCN4 gene eliminated the response. The Gcn4p-dependent expression changes did not require the Gcn2 protein kinase and could be specifically counteracted by an increased gene dosage of initiator tRNA(Met). Initiator tRNA(Met) depletion thus triggers a GCN4-dependent reprogramming of genome expression in response to decreased Pol III transcription. Such an effect might represent a key element in the coordinated transcriptional response of yeast cells to environmental changes.

RNA-DNA and DNA-DNA base-pairing at the upstream edge of the transcription bubble regulate translocation of RNA polymerase and transcription rate.

PubMed

KIreeva, Maria; Trang, Cyndi; Matevosyan, Gayane; Turek-Herman, Joshua; Chasov, Vitaly; Lubkowska, Lucyna; Kashlev, Mikhail

2018-06-20

Translocation of RNA polymerase (RNAP) along DNA may be rate-limiting for transcription elongation. The Brownian ratchet model posits that RNAP rapidly translocates back and forth until the post-translocated state is stabilized by NTP binding. An alternative model suggests that RNAP translocation is slow and poorly reversible. To distinguish between these two models, we take advantage of an observation that pyrophosphorolysis rates directly correlate with the abundance of the pre-translocated fraction. Pyrophosphorolysis by RNAP stabilized in the pre-translocated state by bacteriophage HK022 protein Nun was used as a reference point to determine the pre-translocated fraction in the absence of Nun. The stalled RNAP preferentially occupies the post-translocated state. The forward translocation rate depends, among other factors, on melting of the RNA-DNA base pair at the upstream edge of the transcription bubble. DNA-DNA base pairing immediately upstream from the RNA-DNA hybrid stabilizes the post-translocated state. This mechanism is conserved between E. coli RNAP and S. cerevisiae RNA polymerase II and is partially dependent on the lid domain of the catalytic subunit. Thus, the RNA-DNA hybrid and DNA reannealing at the upstream edge of the transcription bubble emerge as targets for regulation of the transcription elongation rate.

Mod5 protein binds to tRNA gene complexes and affects local transcriptional silencing

PubMed Central

Pratt-Hyatt, Matthew; Pai, Dave A.; Haeusler, Rebecca A.; Wozniak, Glenn G.; Good, Paul D.; Miller, Erin L.; McLeod, Ian X.; Yates, John R.; Hopper, Anita K.; Engelke, David R.

2013-01-01

The tRNA gene-mediated (tgm) silencing of RNA polymerase II promoters is dependent on subnuclear clustering of the tRNA genes, but genetic analysis shows that the silencing requires additional mechanisms. We have identified proteins that bind tRNA gene transcription complexes and are required for tgm silencing but not required for gene clustering. One of the proteins, Mod5, is a tRNA modifying enzyme that adds an N6-isopentenyl adenosine modification at position 37 on a small number of tRNAs in the cytoplasm, although a subpopulation of Mod5 is also found in the nucleus. Recent publications have also shown that Mod5 has tumor suppressor characteristics in humans as well as confers drug resistance through prion-like misfolding in yeast. Here, we show that a subpopulation of Mod5 associates with tRNA gene complexes in the nucleolus. This association occurs and is required for tgm silencing regardless of whether the pre-tRNA transcripts are substrates for Mod5 modification. In addition, Mod5 is bound to nuclear pre-tRNA transcripts, although they are not substrates for the A37 modification. Lastly, we show that truncation of the tRNA transcript to remove the normal tRNA structure also alleviates silencing, suggesting that synthesis of intact pre-tRNAs is required for the silencing mechanism. These results are discussed in light of recent results showing that silencing near tRNA genes also requires chromatin modification. PMID:23898186

The Mediator Complex: At the Nexus of RNA Polymerase II Transcription.

PubMed

Jeronimo, Célia; Robert, François

2017-10-01

Mediator is an essential, large, multisubunit, transcriptional co-activator highly conserved across eukaryotes. Mediator interacts with gene-specific transcription factors at enhancers as well as with the RNA polymerase II (RNAPII) transcription machinery bound at promoters. It also interacts with several other factors involved in various aspects of transcription, chromatin regulation, and mRNA processing. Hence, Mediator is at the nexus of RNAPII transcription, regulating its many steps and connecting transcription with co-transcriptional events. To achieve this flexible role, Mediator, which is divided into several functional modules, reorganizes its conformation and composition while making transient contacts with other components. Here, we review the mechanisms of action of Mediator and propose a unifying model for its function. Copyright © 2017 Elsevier Ltd. All rights reserved.

Chromatin-associated RNA sequencing (ChAR-seq) maps genome-wide RNA-to-DNA contacts

PubMed Central

Jukam, David; Teran, Nicole A; Risca, Viviana I; Smith, Owen K; Johnson, Whitney L; Skotheim, Jan M; Greenleaf, William James

2018-01-01

RNA is a critical component of chromatin in eukaryotes, both as a product of transcription, and as an essential constituent of ribonucleoprotein complexes that regulate both local and global chromatin states. Here, we present a proximity ligation and sequencing method called Chromatin-Associated RNA sequencing (ChAR-seq) that maps all RNA-to-DNA contacts across the genome. Using Drosophila cells, we show that ChAR-seq provides unbiased, de novo identification of targets of chromatin-bound RNAs including nascent transcripts, chromosome-specific dosage compensation ncRNAs, and genome-wide trans-associated RNAs involved in co-transcriptional RNA processing. PMID:29648534

The Choice of Alternative 5' Splice Sites in Influenza Virus M1 mRNA is Regulated by the Viral Polymerase Complex

NASA Astrophysics Data System (ADS)

Shih, Shin-Ru; Nemeroff, Martin E.; Krug, Robert M.

1995-07-01

The influenza virus M1 mRNA has two alternative 5' splice sites: a distal 5' splice site producing mRNA_3 that has the coding potential for 9 amino acids and a proximal 5' splice site producing M2 mRNA encoding the essential M2 ion-channel protein. Only mRNA_3 was made in uninfected cells transfected with DNA expressing M1 mRNA. Similarly, using nuclear extracts from uninfected cells, in vitro splicing of M1 mRNA yielded only mRNA_3. Only when the mRNA_3 5' splice site was inactivated by mutation was M2 mRNA made in uninfected cells and in uninfected cell extracts. In influenza virus-infected cells, M2 mRNA was made, but only after a delay, suggesting that newly synthesized viral gene product(s) were needed to activate the M2 5' splice site. We present strong evidence that these gene products are the complex of the three polymerase proteins, the same complex that functions in the transcription and replication of the viral genome. Gel shift experiments showed that the viral polymerase complex bound to the 5' end of the viral M1 mRNA in a sequence-specific and cap-dependent manner. During in vitro splicing catalyzed by uninfected cell extracts, the binding of the viral polymerase complex blocked the mRNA_3 5' splice site, resulting in the switch to the M2 mRNA 5' splice site and the production of M2 mRNA.

Transcription profile of boar spermatozoa as revealed by RNA-sequencing

USDA-ARS?s Scientific Manuscript database

High-throughput RNA sequencing (RNA-Seq) overcomes the limitations of the current hybridization-based techniques to detect the actual pool of RNA transcripts in spermatozoa. The application of this technology in livestock can speed the discovery of potential predictors of male fertility. As a first ...

microRNA in Cerebral Spinal Fluid as Biomarkers of Alzheimer’s Disease Risk After Brain Injury

DTIC Science & Technology

2016-08-01

protein processing is a key feature of AD. MiRNAs are small non- coding RNA that regulate mRNA transcription, and may be a significant cause of protein...non- coding RNA that regulate mRNA transcription, and may be a significant cause of protein dysregulation. Our investigative team has generated

In vitro transcription of two human rotaviruses.

PubMed Central

Flores, J; Myslinski, J; Kalica, A R; Greenberg, H B; Wyatt, R G; Kapikian, A Z; Chanock, R M

1982-01-01

The RNA polymerase activities of a cultivatable (Wa) and a noncultivatable (DS-1) strain of human rotavirus were studied. Under optimal conditions, transcription of all of their RNA segments occurred, as evidenced by the hybridization of labeled transcripts to genomic RNA. Cross-hybridization between the two viruses showed that none of their 11 genes were completely homologous. The transcription products could be translated in vitro, yielding proteins with an electrophoretic pattern resembling that obtained with proteins labeled in vivo during infection with the Wa virus. Images PMID:6292446

RNAi Functions in Adaptive Reprogramming of the Genome | Center for Cancer Research

Cancer.gov

The regulation of transcribing DNA into RNA, including the production, processing, and degradation of RNA transcripts, affects the expression and the regulation of the genome in ways that are just beginning to be unraveled. A surprising discovery in recent years is that the vast majority of the genome is transcribed to yield an abundance of RNA transcripts. Many transcripts

Gene Expression in Archaea: Studies of Transcriptional Promoters, Messenger RNA Processing, and Five Prime Untranslated Regions in "Methanocaldococcus Jannashchii"

ERIC Educational Resources Information Center

Zhang, Jian

2009-01-01

Gene expression in Archaea is less understood than those in Bacteria and Eucarya. In general, three steps are involved in gene expression--transcription, RNA processing, and translation. To expand our knowledge of these processes in Archaea, I have studied transcriptional promoters, messenger RNA processing, and 5'-untranslated regions in…

Using in-cell SHAPE-Seq and simulations to probe structure-function design principles of RNA transcriptional regulators.

PubMed

Takahashi, Melissa K; Watters, Kyle E; Gasper, Paul M; Abbott, Timothy R; Carlson, Paul D; Chen, Alan A; Lucks, Julius B

2016-06-01

Antisense RNA-mediated transcriptional regulators are powerful tools for controlling gene expression and creating synthetic gene networks. RNA transcriptional repressors derived from natural mechanisms called attenuators are particularly versatile, though their mechanistic complexity has made them difficult to engineer. Here we identify a new structure-function design principle for attenuators that enables the forward engineering of new RNA transcriptional repressors. Using in-cell SHAPE-Seq to characterize the structures of attenuator variants within Escherichia coli, we show that attenuator hairpins that facilitate interaction with antisense RNAs require interior loops for proper function. Molecular dynamics simulations of these attenuator variants suggest these interior loops impart structural flexibility. We further observe hairpin flexibility in the cellular structures of natural RNA mechanisms that use antisense RNA interactions to repress translation, confirming earlier results from in vitro studies. Finally, we design new transcriptional attenuators in silico using an interior loop as a structural requirement and show that they function as desired in vivo. This work establishes interior loops as an important structural element for designing synthetic RNA gene regulators. We anticipate that the coupling of experimental measurement of cellular RNA structure and function with computational modeling will enable rapid discovery of structure-function design principles for a diverse array of natural and synthetic RNA regulators. © 2016 Takahashi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Genome-Wide RNA Polymerase II Profiles and RNA Accumulation Reveal Kinetics of Transcription and Associated Epigenetic Changes During Diurnal Cycles

PubMed Central

Gilardi, Federica; Liechti, Robin; Martin, Olivier; Harshman, Keith; Delorenzi, Mauro; Desvergne, Béatrice; Herr, Winship; Deplancke, Bart; Schibler, Ueli; Rougemont, Jacques; Guex, Nicolas; Hernandez, Nouria; Naef, Felix

2012-01-01

Interactions of cell-autonomous circadian oscillators with diurnal cycles govern the temporal compartmentalization of cell physiology in mammals. To understand the transcriptional and epigenetic basis of diurnal rhythms in mouse liver genome-wide, we generated temporal DNA occupancy profiles by RNA polymerase II (Pol II) as well as profiles of the histone modifications H3K4me3 and H3K36me3. We used these data to quantify the relationships of phases and amplitudes between different marks. We found that rhythmic Pol II recruitment at promoters rather than rhythmic transition from paused to productive elongation underlies diurnal gene transcription, a conclusion further supported by modeling. Moreover, Pol II occupancy preceded mRNA accumulation by 3 hours, consistent with mRNA half-lives. Both methylation marks showed that the epigenetic landscape is highly dynamic and globally remodeled during the 24-hour cycle. While promoters of transcribed genes had tri-methylated H3K4 even at their trough activity times, tri-methylation levels reached their peak, on average, 1 hour after Pol II. Meanwhile, rhythms in tri-methylation of H3K36 lagged transcription by 3 hours. Finally, modeling profiles of Pol II occupancy and mRNA accumulation identified three classes of genes: one showing rhythmicity both in transcriptional and mRNA accumulation, a second class with rhythmic transcription but flat mRNA levels, and a third with constant transcription but rhythmic mRNAs. The latter class emphasizes widespread temporally gated posttranscriptional regulation in the mouse liver. PMID:23209382

Transcriptional and post-transcriptional regulation of the ionizing radiation response by ATM and p53

PubMed Central

Venkata Narayanan, Ishwarya; Paulsen, Michelle T.; Bedi, Karan; Berg, Nathan; Ljungman, Emily A.; Francia, Sofia; Veloso, Artur; Magnuson, Brian; di Fagagna, Fabrizio d’Adda; Wilson, Thomas E.; Ljungman, Mats

2017-01-01

In response to ionizing radiation (IR), cells activate a DNA damage response (DDR) pathway to re-program gene expression. Previous studies using total cellular RNA analyses have shown that the stress kinase ATM and the transcription factor p53 are integral components required for induction of IR-induced gene expression. These studies did not distinguish between changes in RNA synthesis and RNA turnover and did not address the role of enhancer elements in DDR-mediated transcriptional regulation. To determine the contribution of synthesis and degradation of RNA and monitor the activity of enhancer elements following exposure to IR, we used the recently developed Bru-seq, BruChase-seq and BruUV-seq techniques. Our results show that ATM and p53 regulate both RNA synthesis and stability as well as enhancer element activity following exposure to IR. Importantly, many genes in the p53-signaling pathway were coordinately up-regulated by both increased synthesis and RNA stability while down-regulated genes were suppressed either by reduced synthesis or stability. Our study is the first of its kind that independently assessed the effects of ionizing radiation on transcription and post-transcriptional regulation in normal human cells. PMID:28256581

Post-transcriptional bursting in genes regulated by small RNA molecules

NASA Astrophysics Data System (ADS)

Rodrigo, Guillermo

2018-03-01

Gene expression programs in living cells are highly dynamic due to spatiotemporal molecular signaling and inherent biochemical stochasticity. Here we study a mechanism based on molecule-to-molecule variability at the RNA level for the generation of bursts of protein production, which can lead to heterogeneity in a cell population. We develop a mathematical framework to show numerically and analytically that genes regulated post transcriptionally by small RNA molecules can exhibit such bursts due to different states of translation activity (on or off), mostly revealed in a regime of few molecules. We exploit this framework to compare transcriptional and post-transcriptional bursting and also to illustrate how to tune the resulting protein distribution with additional post-transcriptional regulations. Moreover, because RNA-RNA interactions are predictable with an energy model, we define the kinetic constants of on-off switching as functions of the two characteristic free-energy differences of the system, activation and formation, with a nonequilibrium scheme. Overall, post-transcriptional bursting represents a distinctive principle linking gene regulation to gene expression noise, which highlights the importance of the RNA layer beyond the simple information transfer paradigm and significantly contributes to the understanding of the intracellular processes from a first-principles perspective.

RNA Pol IV and V in Gene Silencing: Rebel Polymerases Evolving Away From Pol II’s Rules

PubMed Central

Zhou, Ming; Law, Julie A.

2015-01-01

Noncoding RNAs regulate gene expression at both the transcriptional and post-transcriptional levels, and play critical roles in development, imprinting and the maintenance of genome integrity in eukaryotic organisms [1–3]. Therefore, it is important to understand how the production of such RNAs are controlled. In addition to the three canonical DNA dependent RNA polymerases (Pol) Pol I, II and III, two non-redundant plant-specific RNA polymerases, Pol IV and Pol V, have been identified and shown to generate noncoding RNAs that are required for transcriptional gene silencing via the RNA-directed DNA methylation (RdDM) pathway. Thus, somewhat paradoxically, transcription is required for gene silencing. This paradox extends beyond plants, as silencing pathways in yeast, fungi, flies, worms, and mammals also require transcriptional machinery [4,5]. As plants have evolved specialized RNA polymerases to carry out gene silencing in a manner that is separate from the essential roles of Pol II, their characterization offers unique insight into how RNA polymerases facilitate gene silencing. In this review, we focus on the mechanisms of Pol IV and Pol V function, including their compositions, their transcripts, and their modes of recruitment to chromatin. PMID:26344361

Transfer RNA Post-Transcriptional Processing, Turnover, and Subcellular Dynamics in the Yeast Saccharomyces cerevisiae

PubMed Central

Hopper, Anita K.

2013-01-01

Transfer RNAs (tRNAs) are essential for protein synthesis. In eukaryotes, tRNA biosynthesis employs a specialized RNA polymerase that generates initial transcripts that must be subsequently altered via a multitude of post-transcriptional steps before the tRNAs beome mature molecules that function in protein synthesis. Genetic, genomic, biochemical, and cell biological approaches possible in the powerful Saccharomyces cerevisiae system have led to exciting advances in our understandings of tRNA post-transcriptional processing as well as to novel insights into tRNA turnover and tRNA subcellular dynamics. tRNA processing steps include removal of transcribed leader and trailer sequences, addition of CCA to the 3′ mature sequence and, for tRNAHis, addition of a 5′ G. About 20% of yeast tRNAs are encoded by intron-containing genes. The three-step splicing process to remove the introns surprisingly occurs in the cytoplasm in yeast and each of the splicing enzymes appears to moonlight in functions in addition to tRNA splicing. There are 25 different nucleoside modifications that are added post-transcriptionally, creating tRNAs in which ∼15% of the residues are nucleosides other than A, G, U, or C. These modified nucleosides serve numerous important functions including tRNA discrimination, translation fidelity, and tRNA quality control. Mature tRNAs are very stable, but nevertheless yeast cells possess multiple pathways to degrade inappropriately processed or folded tRNAs. Mature tRNAs are also dynamic in cells, moving from the cytoplasm to the nucleus and back again to the cytoplasm; the mechanism and function of this retrograde process is poorly understood. Here, the state of knowledge for tRNA post-transcriptional processing, turnover, and subcellular dynamics is addressed, highlighting the questions that remain. PMID:23633143

Polymerase III transcription factor B activity is reduced in extracts of growth-restricted cells.

PubMed Central

Tower, J; Sollner-Webb, B

1988-01-01

Extracts of cells that are down-regulated for transcription by RNA polymerase I and RNA polymerase III exhibit a reduced in vitro transcriptional capacity. We have recently demonstrated that the down-regulation of polymerase I transcription in extracts of cycloheximide-treated and stationary-phase cells results from a lack of an activated subform of RNA polymerase I which is essential for rDNA transcription. To examine whether polymerase III transcriptional down-regulation occurs by a similar mechanism, the polymerase III transcription factors were isolated and added singly and in pairs to control cell extracts and to extracts of cells that had reduced polymerase III transcriptional activity due to cycloheximide treatment or growth into stationary phase. These down-regulations result from a specific reduction in TFIIIB; TFIIIC and polymerase III activities remain relatively constant. Thus, although transcription by both polymerase III and polymerase I is substantially decreased in extracts of growth-arrested cells, this regulation is brought about by reduction of different kinds of activities: a component of the polymerase III stable transcription complex in the former case and the activated subform of RNA polymerase I in the latter. Images PMID:3352599

Imaging the Directed Transport of Single Engineered RNA Transcripts in Real-Time Using Ratiometric Bimolecular Beacons

PubMed Central

Zhang, Xuemei; Zajac, Allison L.; Huang, Lingyan; Behlke, Mark A.; Tsourkas, Andrew

2014-01-01

The relationship between RNA expression and cell function can often be difficult to decipher due to the presence of both temporal and sub-cellular processing of RNA. These intricacies of RNA regulation can often be overlooked when only acquiring global measurements of RNA expression. This has led to development of several tools that allow for the real-time imaging of individual engineered RNA transcripts in living cells. Here, we describe a new technique that utilizes an oligonucleotide-based probe, ratiometric bimolecular beacon (RBMB), to image RNA transcripts that were engineered to contain 96-tandem repeats of the RBMB target sequence in the 3′-untranslated region. Binding of RBMBs to the target RNA resulted in discrete bright fluorescent spots, representing individual transcripts, that could be imaged in real-time. Since RBMBs are a synthetic probe, the use of photostable, bright, and red-shifted fluorophores led to a high signal-to-background. RNA motion was readily characterized by both mean squared displacement and moment scaling spectrum analyses. These analyses revealed clear examples of directed, Brownian, and subdiffusive movements. PMID:24454933

Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs.

PubMed

Hayashi, Tetsutaro; Ozaki, Haruka; Sasagawa, Yohei; Umeda, Mana; Danno, Hiroki; Nikaido, Itoshi

2018-02-12

Total RNA sequencing has been used to reveal poly(A) and non-poly(A) RNA expression, RNA processing and enhancer activity. To date, no method for full-length total RNA sequencing of single cells has been developed despite the potential of this technology for single-cell biology. Here we describe random displacement amplification sequencing (RamDA-seq), the first full-length total RNA-sequencing method for single cells. Compared with other methods, RamDA-seq shows high sensitivity to non-poly(A) RNA and near-complete full-length transcript coverage. Using RamDA-seq with differentiation time course samples of mouse embryonic stem cells, we reveal hundreds of dynamically regulated non-poly(A) transcripts, including histone transcripts and long noncoding RNA Neat1. Moreover, RamDA-seq profiles recursive splicing in >300-kb introns. RamDA-seq also detects enhancer RNAs and their cell type-specific activity in single cells. Taken together, we demonstrate that RamDA-seq could help investigate the dynamics of gene expression, RNA-processing events and transcriptional regulation in single cells.

Structure and mechanism of the T-box riboswitches

PubMed Central

Zhang, Jinwei

2015-01-01

In most Gram-positive bacteria, including many clinically devastating pathogens from genera such as Bacillus, Clostridium, Listeria and Staphylococcus, T-box riboswitches sense and regulate intracellular availability of amino acids through a multipartite mRNA-tRNA interaction. The T-box mRNA leaders respond to nutrient starvation by specifically binding cognate tRNAs and sensing whether the bound tRNA is aminoacylated, as a proxy for amino acid availability. Based on this readout, T-boxes direct a transcriptional or translational switch to control the expression of downstream genes involved in various aspects of amino acid metabolism: biosynthesis, transport, aminoacylation, transamidation, etc. Two decades after its discovery, the structural and mechanistic underpinnings of the T-box riboswitch were recently elucidated, producing a wealth of insights into how two structured RNAs can recognize each other with robust affinity and exquisite selectivity. The T-box paradigm exemplifies how natural non-coding RNAs can interact not just through sequence complementarity, but can add molecular specificity by precisely juxtaposing RNA structural motifs, exploiting inherently flexible elements and the biophysical properties of post-transcriptional modifications, ultimately achieving a high degree of shape complementarity through mutually induced fit. The T-box also provides a proof-of-principle that compact RNA domains can recognize minute chemical changes (such as tRNA aminoacylation) on another RNA. The unveiling of the structure and mechanism of the T-box system thus expands our appreciation of the range of capabilities and modes of action of structured non-coding RNAs, and hints at the existence of networks of non-coding RNAs that communicate through both, structural and sequence specificity. PMID:25959893

A sense oligonucleotide to inducible nitric oxide synthase mRNA increases the survival rate of rats in septic shock.

PubMed

Okuyama, Tetsuya; Nakatake, Richi; Kaibori, Masaki; Okumura, Tadayoshi; Kon, Masanori; Nishizawa, Mikio

2018-01-30

Natural antisense transcripts (asRNAs) that do not encode proteins are transcribed from rat, mouse, and human genes, encoding inducible nitric oxide synthase (iNOS), which catalyzes the production of the inflammatory mediator nitric oxide (NO). In septic shock, NO is excessively produced in hepatocytes and macrophages. The iNOS asRNA interacts with and stabilizes iNOS mRNA. We found that single-stranded 'sense' oligonucleotides corresponding to the iNOS mRNA sequence reduced iNOS mRNA levels by interfering with the mRNA-asRNA interactions in rat hepatocytes. The iNOS sense oligonucleotides that were substituted with phosphorothioate bonds and locked nucleic acids efficiently decreased the levels of iNOS mRNA and iNOS protein. In this study, the gene expression patterns in the livers of two endotoxemia model rats with acute liver failure were compared. Next, we optimized the sequence and modification of the iNOS sense oligonucleotides in interleukin 1β-treated rat hepatocytes. When a sense oligonucleotide was simultaneously administered with d-galactosamine and bacterial lipopolysaccharide (LPS) to rats, their survival rate significantly increased compared to the rats administered d-galactosamine and LPS alone. In the livers of the sense oligonucleotide-administered rats, apoptosis in the hepatocytes markedly decreased. These results suggest that natural antisense transcript-targeted regulation technology using iNOS sense oligonucleotides may be used to treat human inflammatory diseases, such as sepsis and septic shock. Copyright © 2017 Elsevier Inc. All rights reserved.

Global assessment of small RNAs reveals a non-coding transcript involved in biofilm formation and attachment in Acinetobacter baumannii ATCC 17978

PubMed Central

Pérez, Astrid; Gómez, Manuel J.; Gayoso, Carmen; Vallejo, Juan A.; Ohneck, Emily J.; Valle, Jaione; Actis, Luis A.; Beceiro, Alejandro; Bou, Germán

2017-01-01

Many strains of Acinetobacter baumannii have been described as being able to form biofilm. Small non-coding RNAs (sRNAs) control gene expression in many regulatory circuits in bacteria. The aim of the present work was to provide a global description of the sRNAs produced both by planktonic and biofilm-associated (sessile) cells of A. baumannii ATCC 17978, and to compare the corresponding gene expression profiles to identify sRNAs molecules associated to biofilm formation and virulence. sRNA was extracted from both planktonic and sessile cells and reverse transcribed. cDNA was subjected to 454-pyrosequencing using the GS-FLX Titanium chemistry. The global analysis of the small RNA transcriptome revealed different sRNA expression patterns in planktonic and biofilm associated cells, with some of the transcripts only expressed or repressed in sessile bacteria. A total of 255 sRNAs were detected, with 185 of them differentially expressed in the different types of cells. A total of 9 sRNAs were expressed only in biofilm cells, while the expression of other 21 coding regions were repressed only in biofilm cells. Strikingly, the expression level of the sRNA 13573 was 120 times higher in biofilms than in planktonic cells, an observation that prompted us to further investigate the biological role of this non-coding transcript. Analyses of an isogenic mutant and over-expressing strains revealed that the sRNA 13573 gene is involved in biofilm formation and attachment to A549 human alveolar epithelial cells. The present work serves as a basis for future studies examining the complex regulatory network that regulate biofilm biogenesis and attachment to eukaryotic cells in A. baumannii ATCC 17978. PMID:28763494

Coordinated Pulses of mRNA and of Protein Translation or Degradation Produce EGF-Induced Protein Bursts.

PubMed

Golan-Lavi, Roni; Giacomelli, Chiara; Fuks, Garold; Zeisel, Amit; Sonntag, Johanna; Sinha, Sanchari; Köstler, Wolfgang; Wiemann, Stefan; Korf, Ulrike; Yarden, Yosef; Domany, Eytan

2017-03-28

Protein responses to extracellular cues are governed by gene transcription, mRNA degradation and translation, and protein degradation. In order to understand how these time-dependent processes cooperate to generate dynamic responses, we analyzed the response of human mammary cells to the epidermal growth factor (EGF). Integrating time-dependent transcript and protein data into a mathematical model, we inferred for several proteins their pre-and post-stimulus translation and degradation coefficients and found that they exhibit complex, time-dependent variation. Specifically, we identified strategies of protein production and degradation acting in concert to generate rapid, transient protein bursts in response to EGF. Remarkably, for some proteins, for which the response necessitates rapidly decreased abundance, cells exhibit a transient increase in the corresponding degradation coefficient. Our model and analysis allow inference of the kinetics of mRNA translation and protein degradation, without perturbing cells, and open a way to understanding the fundamental processes governing time-dependent protein abundance profiles. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

NASA Astrophysics Data System (ADS)

Kallehauge, Thomas Beuchert; Li, Shangzhong; Pedersen, Lasse Ebdrup; Ha, Tae Kwang; Ley, Daniel; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup; Lee, Gyun Min; Lewis, Nathan E.

2017-01-01

Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production.

Alternative splicing of natriuretic peptide A and B receptor transcripts in the rat brain.

PubMed

Francoeur, F; Gossard, F; Hamet, P; Tremblay, J

1995-12-01

1. In the present study we searched for variants of alternative splicing of guanylyl cyclase A and B mRNA in rats in vivo. 2. Guanylyl cyclase A2 and guanylyl cyclase B2 isoforms of guanylyl cyclase produced by alternative splicing leading to the deletion of exon 9 of both transcripts were quantified in several rat organs. 3. Only one alternative splicing was found in the regulatory domain, encoded by exons 8-15. 4. Quantification of the guanylyl cyclase B2 isoform in different rat organs and in cultured aortic smooth muscle cells showed that this alternative splicing was tissue-specific and occurred predominantly in the central nervous system where the alternatively spliced variant represented more than 50% of the guanylyl cyclase B mRNA. 5. The same alternative splicing existed for guanylyl cyclase A mRNA but at very low levels in the organs studied. 6. Alternative splicing of guanylyl cyclase B exon 9 in the brain may play an important role in signal transduction, since the expressed protein possesses a constitutionally active guanylyl cyclase acting independently of C-type natriuretic peptide regulation.

Opposite GC skews at the 5' and 3' ends of genes in unicellular fungi

PubMed Central

2011-01-01

Background GC-skews have previously been linked to transcription in some eukaryotes. They have been associated with transcription start sites, with the coding strand G-biased in mammals and C-biased in fungi and invertebrates. Results We show a consistent and highly significant pattern of GC-skew within genes of almost all unicellular fungi. The pattern of GC-skew is asymmetrical: the coding strand of genes is typically C-biased at the 5' ends but G-biased at the 3' ends, with intermediate skews at the middle of genes. Thus, the initiation, elongation, and termination phases of transcription are associated with different skews. This pattern influences the encoded proteins by generating differential usage of amino acids at the 5' and 3' ends of genes. These biases also affect fourfold-degenerate positions and extend into promoters and 3' UTRs, indicating that skews cannot be accounted by selection for protein function or translation. Conclusions We propose two explanations, the mutational pressure hypothesis, and the adaptive hypothesis. The mutational pressure hypothesis is that different co-factors bind to RNA pol II at different phases of transcription, producing different mutational regimes. The adaptive hypothesis is that cytidine triphosphate deficiency may lead to C-avoidance at the 3' ends of transcripts to control the flow of RNA pol II molecules and reduce their frequency of collisions. PMID:22208287

Saccharomyces cerevisiae RNA Polymerase I Terminates Transcription at the Reb1 Terminator In Vivo

PubMed Central

Reeder, Ronald H.; Guevara, Palmira; Roan, Judith G.

1999-01-01

We have mapped transcription termination sites for RNA polymerase I in the yeast Saccharomyces cerevisiae. S1 nuclease mapping shows that the primary terminator is the Reb1p terminator located at +93 downstream of the 3′ end of 25S rRNA. Reverse transcription coupled with quantitative PCR shows that approximately 90% of all transcripts terminate at this site. Transcripts which read through the +93 site quantitatively terminate at a fail-safe terminator located further downstream at +250. Inactivation of Rnt1p (an RNase III involved in processing the 3′ end of 25S rRNA) greatly stabilizes transcripts extending to both sites and increases readthrough at the +93 site. In vivo assay of mutants of the Reb1p terminator shows that this site operates in vivo by the same mechanism as has previously been delineated through in vitro studies. PMID:10523625

The conserved N-terminal basic residues and zinc-finger motifs of HIV-1 nucleocapsid restrict the viral cDNA synthesis during virus formation and maturation

PubMed Central

Didierlaurent, Ludovic; Houzet, Laurent; Morichaud, Zakia; Darlix, Jean-Luc; Mougel, Marylène

2008-01-01

Reverse transcription of the genomic RNA by reverse transcriptase occurs soon after HIV-1 infection of target cells. The viral nucleocapsid (NC) protein chaperones this process via its nucleic acid annealing activities and its interactions with the reverse transcriptase enzyme. To function, NC needs its two conserved zinc fingers and flanking basic residues. We recently reported a new role for NC, whereby it negatively controls reverse transcription in the course of virus formation. Indeed, deleting its zinc fingers causes reverse transcription activation in virus producer cells. To investigate this new NC function, we used viruses with subtle mutations in the conserved zinc fingers and its flanking domains. We monitored by quantitative PCR the HIV-1 DNA content in producer cells and in produced virions. Results showed that the two intact zinc-finger structures are required for the temporal control of reverse transcription by NC throughout the virus replication cycle. The N-terminal basic residues also contributed to this new role of NC, while Pro-31 residue between the zinc fingers and Lys-59 in the C-terminal region did not. These findings further highlight the importance of NC as a major target for anti-HIV-1 drugs. PMID:18641038

Development of recombinant adeno-associated virus vectors carrying small interfering RNA (shHec1)-mediated depletion of kinetochore Hec1 protein in tumor cells.

PubMed

Li, L; Yang, L; Scudiero, D A; Miller, S A; Yu, Z-X; Stukenberg, P T; Shoemaker, R H; Kotin, R M

2007-05-01

Transcript depletion using small interfering RNA (siRNA) technology represents a potentially valuable technique for the treatment of cancer. However, delivering therapeutic quantities of siRNA into solid tumors by chemical transfection is not feasible, whereas viral vectors efficiently transduce many human tumor cell lines. Yet producing sufficient quantities of viral vectors that elicit acute and selective cytotoxicity remains a major obstacle for preclinical and clinical trials. Using the invertebrate Spodoptera frugiperda (Sf9) cell line, we were able to produce high titer stocks of cytotoxic recombinant adeno-associated virus (rAAV) that express short hairpin RNA (shRNA) and that efficiently deplete Hec1 (highly expressed in cancer 1), or Kntc2 (kinetochore-associated protein 2), a kinetochore protein directly involved in kinetochore microtubule interactions, chromosome congression and spindle checkpoint signaling. Depletion of Hec1 protein results in persistent spindle checkpoint activation followed by cell death. Because Hec1 expression and activity are only present in mitotic cells, non-dividing cells were not affected by rAAV treatment. On the basis of the results of screening 56 human tumor cell lines with three different serotype vectors, we used a tumor xenograft model to test the effects in vivo. The effects of the shHec1 vector were evident in sectioned and stained tumors. The experiments with rAAV-shRNA vectors demonstrate the utility of producing vectors in invertebrate cells to obtain sufficient concentrations and quantities for solid tumor therapy. This addresses an important requirement for cancer gene therapy, to produce cytotoxic vectors in sufficient quantities and concentrations to enable quantitative transduction and selective killing of solid tumor cells.

A deep learning method for lincRNA detection using auto-encoder algorithm.

PubMed

Yu, Ning; Yu, Zeng; Pan, Yi

2017-12-06

RNA sequencing technique (RNA-seq) enables scientists to develop novel data-driven methods for discovering more unidentified lincRNAs. Meantime, knowledge-based technologies are experiencing a potential revolution ignited by the new deep learning methods. By scanning the newly found data set from RNA-seq, scientists have found that: (1) the expression of lincRNAs appears to be regulated, that is, the relevance exists along the DNA sequences; (2) lincRNAs contain some conversed patterns/motifs tethered together by non-conserved regions. The two evidences give the reasoning for adopting knowledge-based deep learning methods in lincRNA detection. Similar to coding region transcription, non-coding regions are split at transcriptional sites. However, regulatory RNAs rather than message RNAs are generated. That is, the transcribed RNAs participate the biological process as regulatory units instead of generating proteins. Identifying these transcriptional regions from non-coding regions is the first step towards lincRNA recognition. The auto-encoder method achieves 100% and 92.4% prediction accuracy on transcription sites over the putative data sets. The experimental results also show the excellent performance of predictive deep neural network on the lincRNA data sets compared with support vector machine and traditional neural network. In addition, it is validated through the newly discovered lincRNA data set and one unreported transcription site is found by feeding the whole annotated sequences through the deep learning machine, which indicates that deep learning method has the extensive ability for lincRNA prediction. The transcriptional sequences of lincRNAs are collected from the annotated human DNA genome data. Subsequently, a two-layer deep neural network is developed for the lincRNA detection, which adopts the auto-encoder algorithm and utilizes different encoding schemes to obtain the best performance over intergenic DNA sequence data. Driven by those newly annotated lincRNA data, deep learning methods based on auto-encoder algorithm can exert their capability in knowledge learning in order to capture the useful features and the information correlation along DNA genome sequences for lincRNA detection. As our knowledge, this is the first application to adopt the deep learning techniques for identifying lincRNA transcription sequences.

Influenza Virus Mounts a Two-Pronged Attack on Host RNA Polymerase II Transcription.

PubMed

Bauer, David L V; Tellier, Michael; Martínez-Alonso, Mónica; Nojima, Takayuki; Proudfoot, Nick J; Murphy, Shona; Fodor, Ervin

2018-05-15

Influenza virus intimately associates with host RNA polymerase II (Pol II) and mRNA processing machinery. Here, we use mammalian native elongating transcript sequencing (mNET-seq) to examine Pol II behavior during viral infection. We show that influenza virus executes a two-pronged attack on host transcription. First, viral infection causes decreased Pol II gene occupancy downstream of transcription start sites. Second, virus-induced cellular stress leads to a catastrophic failure of Pol II termination at poly(A) sites, with transcription often continuing for tens of kilobases. Defective Pol II termination occurs independently of the ability of the viral NS1 protein to interfere with host mRNA processing. Instead, this termination defect is a common effect of diverse cellular stresses and underlies the production of previously reported downstream-of-gene transcripts (DoGs). Our work has implications for understanding not only host-virus interactions but also fundamental aspects of mammalian transcription. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Transcription start site associated RNAs (TSSaRNAs) are ubiquitous in all domains of life.

PubMed

Zaramela, Livia S; Vêncio, Ricardo Z N; ten-Caten, Felipe; Baliga, Nitin S; Koide, Tie

2014-01-01

A plethora of non-coding RNAs has been discovered using high-resolution transcriptomics tools, indicating that transcriptional and post-transcriptional regulation is much more complex than previously appreciated. Small RNAs associated with transcription start sites of annotated coding regions (TSSaRNAs) are pervasive in both eukaryotes and bacteria. Here, we provide evidence for existence of TSSaRNAs in several archaeal transcriptomes including: Halobacterium salinarum, Pyrococcus furiosus, Methanococcus maripaludis, and Sulfolobus solfataricus. We validated TSSaRNAs from the model archaeon Halobacterium salinarum NRC-1 by deep sequencing two independent small-RNA enriched (RNA-seq) and a primary-transcript enriched (dRNA-seq) strand-specific libraries. We identified 652 transcripts, of which 179 were shown to be primary transcripts (∼7% of the annotated genome). Distinct growth-associated expression patterns between TSSaRNAs and their cognate genes were observed, indicating a possible role in environmental responses that may result from RNA polymerase with varying pausing rhythms. This work shows that TSSaRNAs are ubiquitous across all domains of life.

Widespread anti-sense transcription in apple is correlated with siRNA production and indicates a large potential for transcriptional and/or post-transcriptional control.

PubMed

Celton, Jean-Marc; Gaillard, Sylvain; Bruneau, Maryline; Pelletier, Sandra; Aubourg, Sébastien; Martin-Magniette, Marie-Laure; Navarro, Lionel; Laurens, François; Renou, Jean-Pierre

2014-07-01

Characterizing the transcriptome of eukaryotic organisms is essential for studying gene regulation and its impact on phenotype. The realization that anti-sense (AS) and noncoding RNA transcription is pervasive in many genomes has emphasized our limited understanding of gene transcription and post-transcriptional regulation. Numerous mechanisms including convergent transcription, anti-correlated expression of sense and AS transcripts, and RNAi remain ill-defined. Here, we have combined microarray analysis and high-throughput sequencing of small RNAs (sRNAs) to unravel the complexity of transcriptional and potential post-transcriptional regulation in eight organs of apple (Malus × domestica). The percentage of AS transcript expression is higher than that identified in annual plants such as rice and Arabidopsis thaliana. Furthermore, we show that a majority of AS transcripts are transcribed beyond 3'UTR regions, and may cover a significant portion of the predicted sense transcripts. Finally we demonstrate at a genome-wide scale that anti-sense transcript expression is correlated with the presence of both short (21-23 nt) and long (> 30 nt) siRNAs, and that the sRNA coverage depth varies with the level of AS transcript expression. Our study provides a new insight on the functional role of anti-sense transcripts at the genome-wide level, and a new basis for the understanding of sRNA biogenesis in plants. © 2014 INRA. New Phytologist © 2014 New Phytologist Trust.

Non-canonical transcription initiation: the expanding universe of transcription initiating substrates.

PubMed

Barvík, Ivan; Rejman, Dominik; Panova, Natalya; Šanderová, Hana; Krásný, Libor

2017-03-01

RNA polymerase (RNAP) is the central enzyme of transcription of the genetic information from DNA into RNA. RNAP recognizes four main substrates: ATP, CTP, GTP and UTP. Experimental evidence from the past several years suggests that, besides these four NTPs, other molecules can be used to initiate transcription: (i) ribooligonucleotides (nanoRNAs) and (ii) coenzymes such as NAD+, NADH, dephospho-CoA and FAD. The presence of these molecules at the 5΄ ends of RNAs affects the properties of the RNA. Here, we discuss the expanding portfolio of molecules that can initiate transcription, their mechanism of incorporation, effects on RNA and cellular processes, and we present an outlook toward other possible initiation substrates. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Transcriptional role of androgen receptor in the expression of long non-coding RNA Sox2OT in neurogenesis

PubMed Central

Tosetti, Valentina; Sassone, Jenny; Ferri, Anna L. M.; Taiana, Michela; Bedini, Gloria; Nava, Sara; Brenna, Greta; Di Resta, Chiara; Pareyson, Davide; Di Giulio, Anna Maria; Carelli, Stephana

2017-01-01

The complex architecture of adult brain derives from tightly regulated migration and differentiation of precursor cells generated during embryonic neurogenesis. Changes at transcriptional level of genes that regulate migration and differentiation may lead to neurodevelopmental disorders. Androgen receptor (AR) is a transcription factor that is already expressed during early embryonic days. However, AR role in the regulation of gene expression at early embryonic stage is yet to be determinate. Long non-coding RNA (lncRNA) Sox2 overlapping transcript (Sox2OT) plays a crucial role in gene expression control during development but its transcriptional regulation is still to be clearly defined. Here, using Bicalutamide in order to pharmacologically inactivated AR, we investigated whether AR participates in the regulation of the transcription of the lncRNASox2OTat early embryonic stage. We identified a new DNA binding region upstream of Sox2 locus containing three androgen response elements (ARE), and found that AR binds such a sequence in embryonic neural stem cells and in mouse embryonic brain. Our data suggest that through this binding, AR can promote the RNA polymerase II dependent transcription of Sox2OT. Our findings also suggest that AR participates in embryonic neurogenesis through transcriptional control of the long non-coding RNA Sox2OT. PMID:28704421

Transcriptional role of androgen receptor in the expression of long non-coding RNA Sox2OT in neurogenesis.

PubMed

Tosetti, Valentina; Sassone, Jenny; Ferri, Anna L M; Taiana, Michela; Bedini, Gloria; Nava, Sara; Brenna, Greta; Di Resta, Chiara; Pareyson, Davide; Di Giulio, Anna Maria; Carelli, Stephana; Parati, Eugenio A; Gorio, Alfredo

2017-01-01

The complex architecture of adult brain derives from tightly regulated migration and differentiation of precursor cells generated during embryonic neurogenesis. Changes at transcriptional level of genes that regulate migration and differentiation may lead to neurodevelopmental disorders. Androgen receptor (AR) is a transcription factor that is already expressed during early embryonic days. However, AR role in the regulation of gene expression at early embryonic stage is yet to be determinate. Long non-coding RNA (lncRNA) Sox2 overlapping transcript (Sox2OT) plays a crucial role in gene expression control during development but its transcriptional regulation is still to be clearly defined. Here, using Bicalutamide in order to pharmacologically inactivated AR, we investigated whether AR participates in the regulation of the transcription of the lncRNASox2OTat early embryonic stage. We identified a new DNA binding region upstream of Sox2 locus containing three androgen response elements (ARE), and found that AR binds such a sequence in embryonic neural stem cells and in mouse embryonic brain. Our data suggest that through this binding, AR can promote the RNA polymerase II dependent transcription of Sox2OT. Our findings also suggest that AR participates in embryonic neurogenesis through transcriptional control of the long non-coding RNA Sox2OT.

Antennally mediated negative feedback regulation of pheromone production in the pine engraver beetle, Ips pini

NASA Astrophysics Data System (ADS)

Ginzel, Matthew D.; Bearfield, Jeremy C.; Keeling, Christopher I.; McCormack, Colin C.; Blomquist, Gary J.; Tittiger, Claus

2007-01-01

Bark beetles use monoterpenoid aggregation pheromones to coordinate host colonization and mating. These chemical signals are produced de novo in midgut cells via the mevalonate pathway, and pheromone production may be regulated by a negative feedback system mediated through the antennae. In this study, we explored the effect of antennectomy on pheromone production and transcript levels of key mevalonate pathway genes in juvenile hormone III-treated male pine engraver beetles, Ips pini (Say). Antennectomized males produced significantly greater amounts of pheromone than podectomized males and those with intact antennae. Likewise, mRNA levels of three mevalonate pathway genes important in pheromone biosynthesis were measured by quantitative real-time PCR and found to be induced to a greater extent with antennectomy, suggesting a transcriptional regulation of pheromone production.

Design of a Temperature-Responsive Transcription Terminator.

PubMed

Roßmanith, Johanna; Weskamp, Mareen; Narberhaus, Franz

2018-02-16

RNA structures regulate various steps in gene expression. Transcription in bacteria is typically terminated by stable hairpin structures. Translation initiation can be modulated by metabolite- or temperature-sensitive RNA structures, called riboswitches or RNA thermometers (RNATs), respectively. RNATs control translation initiation by occlusion of the ribosome binding site at low temperatures. Increasing temperatures destabilize the RNA structure and facilitate ribosome access. In this study, we exploited temperature-responsive RNAT structures to design regulatory elements that control transcription termination instead of translation initiation in Escherichia coli. In order to mimic the structure of factor-independent intrinsic terminators, naturally occurring RNAT hairpins were genetically engineered to be followed by a U-stretch. Functional temperature-responsive terminators (thermoterms) prevented mRNA synthesis at low temperatures but resumed transcription after a temperature upshift. The successful design of temperature-controlled terminators highlights the potential of RNA structures as versatile gene expression control elements.

Systematically frameshifting by deletion of every 4th or 4th and 5th nucleotides during mitochondrial transcription: RNA self-hybridization regulates delRNA expression.

PubMed

Seligmann, Hervé

2016-01-01

In mitochondria, secondary structures punctuate post-transcriptional RNA processing. Recently described transcripts match the human mitogenome after systematic deletions of every 4th, respectively every 4th and 5th nucleotides, called delRNAs. Here I explore predicted stem-loop hairpin formation by delRNAs, and their associations with delRNA transcription and detected peptides matching their translation. Despite missing 25, respectively 40% of the nucleotides in the original sequence, del-transformed sequences form significantly more secondary structures than corresponding randomly shuffled sequences, indicating biological function, independently of, and in combination with, previously detected delRNA and thereof translated peptides. Self-hybridization decreases delRNA abundances, indicating downregulation. Systematic deletions of the human mitogenome reveal new, unsuspected coding and structural informations. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The association of TIF-IA and polymerase I mediates promoter recruitment and regulation of ribosomal RNA transcription in Acanthamoeba castellanii.

PubMed

Gogain, Joseph C; Paule, Marvin R

2005-01-01

Large amounts of energy are expended for the construction of the ribosome during both transcription and processing, so it is of utmost importance for the cell to efficiently regulate ribosome production. Understanding how this regulation occurs will provide important insights into cellular growth control and into the coordination of gene expression mediated by all three transcription systems. Ribosomal RNA (rRNA) transcription rates closely parallel the need for protein synthesis; as a cell approaches stationary phase or encounters conditions that negatively affect either growth rate or protein synthesis, rRNA transcription is decreased. In eukaryotes, the interaction of RNA polymerase I (pol I) with the essential transcription initiation factor IA (TIF-IA) has been implicated in this downregulation of transcription. In agreement with the first observation that rRNA transcription is regulated by altering recruitment of pol I to the promoter in Acanthamoeba castellanii, we show here that pol I and an 80-kDa homologue of TIF-IA are found tightly associated in pol I fractions competent for specific transcription. Disruption of the pol I-TIF-IA complex is mediated by a specific dephosphorylation of either pol I or TIF-IA. Phosphatase treatment of TIF-IA-containing A. castellanii pol I fractions results in a downregulation of both transcriptional activity and promoter binding, reminiscent of the inactive pol I fractions purified from encysted cells. The fraction of pol I competent for promoter recruitment is enriched in TIF-IA relative to that not bound by immobilized promoter DNA. This downregulation coincides with an altered electrophoretic mobility of TIF-IA, suggesting at least it is phosphorylated.

A pseudogene long noncoding RNA network regulates PTEN transcription and translation in human cells

PubMed Central

Johnsson, Per; Ackley, Amanda; Vidarsdottir, Linda; Lui, Weng-Onn; Corcoran, Martin; Grandér, Dan; Morris, Kevin V.

2013-01-01

PTEN is a tumor suppressor gene that has been shown to be under the regulatory control of a PTEN pseudogene expressed noncoding RNA, PTENpg1. Here, we characterize a previously unidentified PTENpg1 encoded antisense RNA (asRNA), which regulates PTEN transcription and PTEN mRNA stability. We find two PTENpg1 asRNA isoforms, alpha and beta. The alpha isoform functions in trans, localizes to the PTEN promoter, and epigenetically modulates PTEN transcription by the recruitment of DNMT3a and EZH2. In contrast, the beta isoform interacts with PTENpg1 through an RNA:RNA pairing interaction, which affects PTEN protein output via changes of PTENpg1 stability and microRNA sponge activity. Disruption of this asRNA-regulated network induces cell cycle arrest and sensitizes cells to doxorubicin, suggesting a biological function for the respective PTENpg1 expressed asRNAs. PMID:23435381

A pseudogene long-noncoding-RNA network regulates PTEN transcription and translation in human cells.

PubMed

Johnsson, Per; Ackley, Amanda; Vidarsdottir, Linda; Lui, Weng-Onn; Corcoran, Martin; Grandér, Dan; Morris, Kevin V

2013-04-01

PTEN is a tumor-suppressor gene that has been shown to be under the regulatory control of a PTEN pseudogene expressed noncoding RNA, PTENpg1. Here, we characterize a previously unidentified PTENpg1-encoded antisense RNA (asRNA), which regulates PTEN transcription and PTEN mRNA stability. We find two PTENpg1 asRNA isoforms, α and β. The α isoform functions in trans, localizes to the PTEN promoter and epigenetically modulates PTEN transcription by the recruitment of DNA methyltransferase 3a and Enhancer of Zeste. In contrast, the β isoform interacts with PTENpg1 through an RNA-RNA pairing interaction, which affects PTEN protein output through changes of PTENpg1 stability and microRNA sponge activity. Disruption of this asRNA-regulated network induces cell-cycle arrest and sensitizes cells to doxorubicin, which suggests a biological function for the respective PTENpg1 expressed asRNAs.

Shift from posttranscriptional to predominant transcriptional control of the expression of the GM-CSF gene during activation of human Jurkat cells.

PubMed

Razanajaona, D; Maroc, C; Lopez, M; Mannoni, P; Gabert, J

1992-05-01

The expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is differentially regulated in various cell types. We investigated the mechanisms controlling its expression in 12-O-tetradecanoylphorbol-13-acetate plus phytohemagglutinin-stimulated Jurkat cells, a human T-cell line. In unstimulated cells, GM-CSF mRNA was undetectable by Northern blot. Upon activation, it was detected from 3 h onward, with a progressive increase in the levels of the transcript up to 24 h of stimulation. Whereas cycloheximide treatment at the time of stimulation blocked mRNA induction, its addition at later times resulted in a marked increase in transcript levels. Run-on analysis showed that transcription of the GM-CSF gene was low to undetectable in unstimulated cells; stimulation led to transcriptional activation, which was weak at 6 h but had increased 16-fold at 24 h. In addition, the mRNA half-life decreased during activation, from 2.5 h at 6 h down to 45 min at 24 h. Cycloheximide treatment increased GM-CSF mRNA half-life (3- and 4-fold, respectively). Our results show: (a) both transcriptional and posttranscriptional signals regulate GM-CSF mRNA levels in activated Jurkat cells, (b) de novo protein synthesis is required for mRNA induction, whereas destabilizing labile proteins control the transcript stability, and (c) a shift from a posttranscriptional to a predominant transcriptional control of GM-CSF gene expression occurs during activation.

The acute salinity changes activate the dual pathways of endocrine responses in the brain and pituitary of tilapia.

PubMed

Aruna, Adimoolam; Nagarajan, Ganesan; Chang, Ching-Fong

2015-01-15

To analyze and compare the stress and osmoregulatory hormones and receptors in pituitary during acute salinity changes, the expression patterns of corticotropin releasing hormone (crh) in hypothalamus, prolactin (prl) releasing peptide (pRrp) in telencephalon and diencephalon, glucocorticoid receptors 2 (gr2), and mineralocorticoid receptor (mr), crh-r, pro-opiomelanocorticotropin (pomc), pRrp, prl, dopamine 2 receptor (d2-r), growth hormone (gh), gh-receptor (gh-r) and insulin-like growth hormone (igf-1) transcripts in pituitary were characterized in euryhaline tilapia. The results indicate that the crh transcripts increased in the hypothalamus and rostral pars distalis of the pituitary after the transfer of fish to SW. Similarly, the pRrp transcripts were more abundant in SW acclimated tilapia forebrain and hypothalamus. The crh-r, gr2 and mr transcripts were more expressed in rostral pars distalis and pars intermedia of pituitary at SW than FW tilapia. The data indicate that the SW acclimation stimulates these transcripts in the specific regions of the brain and pituitary which may be related to the activation of the hypothalamic-pituitary-interrenal (HPI)-axis. The results of dual in situ hybridization reveal that the transcripts of crh-r, gr2 and mr with pomc are highly co-localized in corticotrophs of pituitary. Furthermore, we demonstrate high expression of pRrp in the brain and low expression of pRrp and prl transcripts in the pituitary of SW fish. No crh-r and corticosteroid receptors were co-localized with prl transcripts in the pituitary. The gh-r and igf-1 mRNA levels were significantly increased in SW acclimated tilapia pituitary whereas there was no difference in the gh mRNA levels. The data suggest that the locally produced pRrp and d2-r may control and regulate the expression of prl mRNA in pituitary. Therefore, the dual roles of pRrp are involved in the stress (via brain-pituitary) and osmoregulatory (via pituitary) pathways in tilapia exposed to acute salinity changes. Copyright © 2014 Elsevier Inc. All rights reserved.

Transcription Profiling of Bacillus subtilis Cells Infected with AR9, a Giant Phage Encoding Two Multisubunit RNA Polymerases.

PubMed

Lavysh, Daria; Sokolova, Maria; Slashcheva, Marina; Förstner, Konrad U; Severinov, Konstantin

2017-02-14

Bacteriophage AR9 is a recently sequenced jumbo phage that encodes two multisubunit RNA polymerases. Here we investigated the AR9 transcription strategy and the effect of AR9 infection on the transcription of its host, Bacillus subtilis Analysis of whole-genome transcription revealed early, late, and continuously expressed AR9 genes. Alignment of sequences upstream of the 5' ends of AR9 transcripts revealed consensus sequences that define early and late phage promoters. Continuously expressed AR9 genes have both early and late promoters in front of them. Early AR9 transcription is independent of protein synthesis and must be determined by virion RNA polymerase injected together with viral DNA. During infection, the overall amount of host mRNAs is significantly decreased. Analysis of relative amounts of host transcripts revealed notable differences in the levels of some mRNAs. The physiological significance of up- or downregulation of host genes for AR9 phage infection remains to be established. AR9 infection is significantly affected by rifampin, an inhibitor of host RNA polymerase transcription. The effect is likely caused by the antibiotic-induced killing of host cells, while phage genome transcription is solely performed by viral RNA polymerases. IMPORTANCE Phages regulate the timing of the expression of their own genes to coordinate processes in the infected cell and maximize the release of viral progeny. Phages also alter the levels of host transcripts. Here we present the results of a temporal analysis of the host and viral transcriptomes of Bacillus subtilis infected with a giant phage, AR9. We identify viral promoters recognized by two virus-encoded RNA polymerases that are a unique feature of the phiKZ-related group of phages to which AR9 belongs. Our results set the stage for future analyses of highly unusual RNA polymerases encoded by AR9 and other phiKZ-related phages. Copyright © 2017 Lavysh et al.

Acetyl Coenzyme A Stimulates RNA Polymerase II Transcription and Promoter Binding by Transcription Factor IID in the Absence of Histones

PubMed Central

Galasinski, Shelly K.; Lively, Tricia N.; Grebe de Barron, Alexandra; Goodrich, James A.

2000-01-01

Protein acetylation has emerged as a means of controlling levels of mRNA synthesis in eukaryotic cells. Here we report that acetyl coenzyme A (acetyl-CoA) stimulates RNA polymerase II transcription in vitro in the absence of histones. The effect of acetyl-CoA on basal and activated transcription was studied in a human RNA polymerase II transcription system reconstituted from recombinant and highly purified transcription factors. Both basal and activated transcription were stimulated by the addition of acetyl-CoA to transcription reaction mixtures. By varying the concentrations of general transcription factors in the reaction mixtures, we found that acetyl-CoA decreased the concentration of TFIID required to observe transcription. Electrophoretic mobility shift assays and DNase I footprinting revealed that acetyl-CoA increased the affinity of the general transcription factor TFIID for promoter DNA in a TBP-associated factor (TAF)-dependent manner. Interestingly, acetyl-CoA also caused a conformational change in the TFIID-TFIIA-promoter complex as assessed by DNase I footprinting. These results show that acetyl-CoA alters the DNA binding activity of TFIID and indicate that this biologically important cofactor functions at multiple levels to control gene expression. PMID:10688640