scholarly journals Development, embryonic genome activity and mitochondrial characteristics of bovine–pig inter-family nuclear transfer embryos

Reproduction ◽  
2010 ◽  
Vol 140 (2) ◽  
pp. 273-285 ◽  
Author(s):  
Irina Lagutina ◽  
Helena Fulka ◽  
Tiziana A L Brevini ◽  
Stefania Antonini ◽  
Dario Brunetti ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Transfer ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Pol I ◽  
Embryonic Genome ◽  
Genome Activity

The best results of inter-species somatic cell nuclear transfer (iSCNT) in mammals were obtained using closely related species that can hybridise naturally. However, in the last years, many reports describing blastocyst development following iSCNT between species with distant taxonomical relations (inter-classes, inter-order and inter-family) have been published. This indicates that embryonic genome activation (EGA) in xeno-cytoplasm is possible, albeit very rarely. Using a bovine–pig (inter-family) iSCNT model, we studied the basic characteristics of EGA: expression and activity of RNA polymerase II (RNA Pol II), formation of nucleoli (as an indicator of RNA polymerase I (RNA Pol I) activity), expression of the key pluripotency gene NANOG and alteration of mitochondrial mass. In control embryos (obtained by IVF or iSCNT), EGA was characterised by RNA Pol II accumulation and massive production of poly-adenylated transcripts (detected with oligo dT probes) in blastomere nuclei, and formation of nucleoli as a result of RNA Pol I activity. Conversely, iSCNT embryos were characterised by the absence of accumulation and low activity of RNA Pol II and inability to form active mature nucleoli. Moreover, in iSCNT embryos, NANOG was not expressed, and mitochondria mass was significantly lower than in intra-species embryos. Finally, the complete developmental block at the 16–25-cell stage for pig–bovine iSCNT embryos and at the four-cell stage for bovine–pig iSCNT embryos strongly suggests that EGA is not taking place in iSCNT embryos. Thus, our experiments clearly demonstrate poor nucleus–cytoplasm compatibility between these animal species.

scholarly journals Mechanism of RNA polymerase II stalling by DNA alkylation

2017 ◽  
Vol 114 (46) ◽  
pp. 12172-12177 ◽  
Author(s):  
Stefano Malvezzi ◽  
Lucas Farnung ◽  
Claudia M. N. Aloisi ◽  
Todor Angelov ◽  
Patrick Cramer ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Anticancer Agents ◽  
Excision Repair ◽  
Minor Groove ◽  
Dna Alkylation ◽  
Drug Induced ◽  
Rna Pol Ii ◽  
Pol Ii

Several anticancer agents that form DNA adducts in the minor groove interfere with DNA replication and transcription to induce apoptosis. Therapeutic resistance can occur, however, when cells are proficient in the removal of drug-induced damage. Acylfulvenes are a class of experimental anticancer agents with a unique repair profile suggesting their capacity to stall RNA polymerase (Pol) II and trigger transcription-coupled nucleotide excision repair. Here we show how different forms of DNA alkylation impair transcription by RNA Pol II in cells and with the isolated enzyme and unravel a mode of RNA Pol II stalling that is due to alkylation of DNA in the minor groove. We incorporated a model for acylfulvene adducts, the stable 3-deaza-3-methoxynaphtylethyl-adenosine analog (3d-Napht-A), and smaller 3-deaza-adenosine analogs, into DNA oligonucleotides to assess RNA Pol II transcription elongation in vitro. RNA Pol II was strongly blocked by a 3d-Napht-A analog but bypassed smaller analogs. Crystal structure analysis revealed that a DNA base containing 3d-Napht-A can occupy the +1 templating position and impair closing of the trigger loop in the Pol II active center and polymerase translocation into the next template position. These results show how RNA Pol II copes with minor-groove DNA alkylation and establishes a mechanism for drug resistance.

61 REVERSIBLE INHIBITION OF BOVINE MINOR EMBRYONIC GENOME ACTIVATION IMPAIRS PRE-IMPLANTATION DEVELOPMENT

2017 ◽  
Vol 29 (1) ◽  
pp. 138
Author(s):  
R. P. Nociti ◽  
R. V. Sampaio ◽  
V. F. M. H. de Lima ◽  
R. M. Schultz ◽  
P. J. Ross
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Reversible Inhibitor ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Developmental Potential ◽  
Embryonic Genome Activation ◽  
Transcriptional Inhibition ◽  
Embryonic Genome ◽  
Genome Activation

Bovine pre-implantation embryos can develop in the absence of gene expression up to the 8/16-cell stage, the time when the major embryonic genome activation (EGA) occurs. Some embryonic genes, however, are transcribed before EGA (minor EGA). This study used a reversible inhibitor of RNA Polymerase II (5,6 dichlorobenzimidazole 1-β-D-ribofuranoside; DRB) to assess the importance of minor EGA for development to the blastocyst stage. Oocytes were matured and inseminated in vitro, and the fertilized eggs were cultured in supplemented KSOMaa and allocated to different treatments 16 h post-insemination (hpi). Development was recorded at 44 and 72 hpi, and the incidence of blastocyst formation on Day 7 (IVF = Day 0) was recorded. Data were analysed by ANOVA followed by Duncan test. First, we tested different DRB concentrations [50 μM (D50), 75 μM (D75), 100 μM (D100), and dimethyl sulfoxide vehicle control (CTRL)] to block development to blastocyst when continuously present. Only embryos in CTRL produced blastocysts (45.0 ± 5.8%; 4 replicates with a total of 391 oocytes examined). No difference in development was observed at 44 h (57.9 ± 16.5, 53.3 ± 10.5, 60.5 ± 19.0, and 52.3 ± 5.8% for D50, D75, D100, and CTRL, respectively) and 72 h (78.9 ± 8.8, 66.1 ± 11.7, 71.5 ± 16.5, and 70.8 ± 5.6% for D50, D75, D100, and CTRL, respectively). Next, in 7 replicates (751 oocytes) we determined the effect of blocking transcription (50 μM DRB) spanning 2 embryo stages (periods of 28 h), initiated at 16 hpi (1&2C), 30 hpi (2&4C), and 44 hpi (4&8C). Controls included DRB treatment from 16 to 72 hpi (1–8C) and CTRL. There was no difference in development at 44 and 72 h. The incidence of blastocyst formation, however, was significantly decreased in all treatment groups compared with CTRL (27.7 ± 4.7; 15.1 ± 3.5; 23.3 ± 3.1; 20.5 ± 1.9; and 42.1 ± 3.2% for 1&2C, 2&4C, 4&8C, 1–8C, and CTRL, respectively). Finally, in 12 replicates (1499 oocytes), the effect of blocking transcription for 14-h periods, spanning mostly a unique cleavage stage, was evaluated. The DRB treatment (50 μM) started at 16 hpi (1C), 30 hpi (2C), 44 hpi (4C), and 58 hpi (8C). Furthermore, 1–16C and CTRL treatments were included. No difference in development at 44 and 72 h were observed. Development to the blastocyst was significantly lower from CTRL (46.0 ± 3.2%) in 2C, 4C, 8C, and 1–16C (28.9 ± 3.9, 26.1 ± 4.2, 30.1 ± 4.8, and 18.9 ± 3.2%, respectively) but not in 1C (34.7 ± 4.4%). In summary, continuous transcriptional inhibition using DRB resulted in a developmental block at the time of major EGA, similar to α-amanitin treatment (an irreversible RNA Polymerase II inhibitor). Transcriptional inhibition during single cleavage stages was sufficient to decrease the developmental potential of the embryo. We conclude that minor EGA has an important role for bovine development. This work was funded by NIH-NICHD R01HD070044 to P. J. Ross. R. P. Nociti was sponsored by CNPQ; R. V. Sampaio was sponsored by FAPESP.

scholarly journals Regulation of rRNA Synthesis by TATA-Binding Protein-Associated Factor Mot1

2007 ◽  
Vol 27 (8) ◽  
pp. 2886-2896 ◽  
Author(s):  
Arindam Dasgupta ◽  
Rebekka O. Sprouse ◽  
Sarah French ◽  
Pavel Aprikian ◽  
Robert Hontz ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Tata Binding Protein ◽  
Rrna Synthesis ◽  
Direct Role ◽  
Pol Ii ◽  
Associated Factor ◽  
Pol I

ABSTRACT Mot1 is an essential, conserved, TATA-binding protein (TBP)-associated factor in Saccharomyces cerevisiae with well-established roles in the global control of RNA polymerase II (Pol II) transcription. Previous results have suggested that Mot1 functions exclusively in Pol II transcription, but here we report a novel role for Mot1 in regulating transcription by RNA polymerase I (Pol I). In vivo, Mot1 is associated with the ribosomal DNA, and loss of Mot1 results in decreased rRNA synthesis. Consistent with a direct role for Mot1 in Pol I transcription, Mot1 also associates with the Pol I promoter in vitro in a reaction that depends on components of the Pol I general transcription machinery. Remarkably, in addition to Mot1's role in initiation, rRNA processing is delayed in mot1 cells. Taken together, these results support a model in which Mot1 affects the rate and efficiency of rRNA synthesis by both direct and indirect mechanisms, with resulting effects on transcription activation and the coupling of rRNA synthesis to processing.

scholarly journals RNA Polymerase I Transcribes Procyclin Genes and Variant Surface Glycoprotein Gene Expression Sites in Trypanosoma brucei

2003 ◽  
Vol 2 (3) ◽  
pp. 542-551 ◽  
Author(s):  
Arthur Günzl ◽  
Thomas Bruderer ◽  
Gabriele Laufer ◽  
Bernd Schimanski ◽  
Lan-Chun Tu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Trypanosoma Brucei ◽  
Protein C ◽  
Gene Promoter ◽  
Surface Glycoprotein ◽  
Variant Surface Glycoprotein ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Pol I

ABSTRACT In eukaryotes, RNA polymerase (pol) I exclusively transcribes the large rRNA gene unit (rDNA) and mRNA is synthesized by RNA pol II. The African trypanosome, Trypanosoma brucei, represents an exception to this rule. In this organism, transcription of genes encoding the variant surface glycoprotein (VSG) and the procyclins is resistant to α-amanitin, indicating that it is mediated by RNA pol I, while other protein-coding genes are transcribed by RNA pol II. To obtain firm proof for this concept, we generated a T. brucei cell line which exclusively expresses protein C epitope-tagged RNA pol I. Using an anti-protein C immunoaffinity matrix, we specifically depleted RNA pol I from transcriptionally active cell extracts. The depletion of RNA pol I impaired in vitro transcription initiated at the rDNA promoter, the GPEET procyclin gene promoter, and a VSG gene expression site promoter but did not affect transcription from the spliced leader (SL) RNA gene promoter. Fittingly, induction of RNA interference against the RNA pol I largest subunit in insect-form trypanosomes significantly reduced the relative transcriptional efficiency of rDNA, procyclin genes, and VSG expression sites in vivo whereas that of SL RNA, αβ-tubulin, and heat shock protein 70 genes was not affected. Our studies unequivocally show that T. brucei harbors a multifunctional RNA pol I which, in addition to transcribing rDNA, transcribes procyclin genes and VSG gene expression sites.

scholarly journals A role for the RNA pol II–associated PAF complex in AID-induced immune diversification

2012 ◽  
Vol 209 (11) ◽  
pp. 2099-2111 ◽  
Author(s):  
Katharina L. Willmann ◽  
Sara Milosevic ◽  
Siim Pauklin ◽  
Kerstin-Maike Schmitz ◽  
Gopinath Rangam ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Protein Complexes ◽  
Chromatin Modification ◽  
Cytosine Deamination ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Antibody Diversification ◽  
Dna Instability ◽  
Paf Complex

Antibody diversification requires the DNA deaminase AID to induce DNA instability at immunoglobulin (Ig) loci upon B cell stimulation. For efficient cytosine deamination, AID requires single-stranded DNA and needs to gain access to Ig loci, with RNA pol II transcription possibly providing both aspects. To understand these mechanisms, we isolated and characterized endogenous AID-containing protein complexes from the chromatin of diversifying B cells. The majority of proteins associated with AID belonged to RNA polymerase II elongation and chromatin modification complexes. Besides the two core polymerase subunits, members of the PAF complex, SUPT5H, SUPT6H, and FACT complex associated with AID. We show that AID associates with RNA polymerase-associated factor 1 (PAF1) through its N-terminal domain, that depletion of PAF complex members inhibits AID-induced immune diversification, and that the PAF complex can serve as a binding platform for AID on chromatin. A model is emerging of how RNA polymerase II elongation and pausing induce and resolve AID lesions.

scholarly journals HIV-1 Tat-associated RNA polymerase C-terminal domain kinase, CDK2, phosphorylates CDK7 and stimulates Tat-mediated transcription

2002 ◽  
Vol 364 (3) ◽  
pp. 649-657 ◽  
Author(s):  
Sergei NEKHAI ◽  
Meisheng ZHOU ◽  
Anne FERNANDEZ ◽  
William S. LANE ◽  
Ned J.C. LAMB ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
Viral Gene ◽  
Transcriptional Elongation ◽  
Rna Pol Ii ◽  
Hela Nuclear Extract ◽  
Pol Ii ◽  
Terminal Domain ◽  
Hiv 1

HIV-1 gene expression is regulated by a viral transactivator protein (Tat) which induces transcriptional elongation of HIV-1 long tandem repeat (LTR). This induction requires hyperphosphorylation of the C-terminal domain (CTD) repeats of RNA polymerase II (Pol II). To achieve CTD hyperphosphorylation, Tat stimulates CTD kinases associated with general transcription factors of the promoter complex, specifically TFIIH-associated CDK7 and positive transcription factor b-associated CDK9 (cyclin-dependent kinase 9). Other studies indicate that Tat may bind an additional CTD kinase that regulates the target-specific phosphorylation of RNA Pol II CTD. We previously reported that Tat-associated T-cell-derived kinase (TTK), purified from human primary T-cells, stimulates Tat-dependent transcription of HIV-1 LTR in vivo [Nekhai, Shukla, Fernandez, Kumar and Lamb (2000) Virology 266, 246–256]. In the work presented here, we characterized the components of TTK by biochemical fractionation and the function of TTK in transcription assays in vitro. TTK uniquely co-purified with CDK2 and not with either CDK9 or CDK7. Tat induced the TTK-associated CDK2 kinase to phosphorylate CTD, specifically at Ser-2 residues. The TTK fraction restored Tat-mediated transcription activation of HIV-1 LTR in a HeLa nuclear extract immunodepleted of CDK9, but not in the HeLa nuclear extract double-depleted of CDK9 and CDK7. Direct microinjection of the TTK fraction augmented Tat transactivation of HIV-1 LTR in human primary HS68 fibroblasts. The results argue that TTK-associated CDK2 may function to maintain target-specific phosphorylation of RNA Pol II that is essential for Tat transactivation of HIV-1 promoter. They are also consistent with the observed cell-cycle-specific induction of viral gene transactivation.

scholarly journals RNA Polymerase II-Controlled Expression of Antigenomic RNA Enhances the Rescue Efficacies of Two Different Members of the Mononegavirales Independently of the Site of Viral Genome Replication

2006 ◽  
Vol 80 (12) ◽  
pp. 5708-5715 ◽  
Author(s):  
Arnold Martin ◽  
Peter Staeheli ◽  
Urs Schneider
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Measles Virus ◽  
De Novo ◽  
Hammerhead Ribozyme ◽  
Genome Replication ◽  
Pol Ii ◽  
Pol I ◽  
Replication Phase ◽  
Viral Genome Replication

ABSTRACT De novo generation of negative-strand RNA viruses depends on the efficient expression of antigenomic RNA (cRNA) from cDNA. To improve the rescue system of Borna disease virus (BDV), a member of the Mononegavirales with a nuclear replication phase, we evaluated different RNA polymerase (Pol) promoters for viral cRNA expression. Human and mouse Pol I promoters did not increase the recovery rate of infectious BDV from cDNA compared to the originally employed T7 RNA polymerase system. In contrast, expression of viral cRNA under the control of an RNA Pol II promoter increased the rescue efficacy by nearly 20-fold. Similarly, rescue of measles virus (MV), a member of the Mononegavirales with a cytoplasmic replication phase, was strongly improved by Pol II-controlled expression of viral cRNA. Analysis of transcription levels derived from different promoters suggested that the rescue-enhancing function of the Pol II promoter was due mainly to enhanced cRNA synthesis from the plasmid. Remarkably, correct 5′-terminal processing of Pol II-transcribed cRNA by a hammerhead ribozyme was not necessary for efficient rescue of BDV or MV. The correct 5′ termini were reconstituted during replication of the artificially prolonged cRNA, indicating that the BDV and MV replicase complexes are able to recognize internal viral replication signals.

scholarly journals The Werner Syndrome Protein Is Involved in RNA Polymerase II Transcription

1999 ◽  
Vol 10 (8) ◽  
pp. 2655-2668 ◽  
Author(s):  
Adayabalam S. Balajee ◽  
Amrita Machwe ◽  
Alfred May ◽  
Matthew D. Gray ◽  
Junko Oshima ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Lines ◽  
Werner Syndrome ◽  
Molecular Defect ◽  
Rna Pol Ii ◽  
Vitro Assay ◽  
Pol Ii ◽  
Cell Extracts

Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)–dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40–60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II–dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid–protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype.

scholarly journals Stress Induces Changes in the Phosphorylation of Trypanosoma cruzi RNA Polymerase II, Affecting Its Association with Chromatin and RNA Processing

2014 ◽  
Vol 13 (7) ◽  
pp. 855-865 ◽  
Author(s):  
Antônio Augusto Rocha ◽  
Nilmar Silvio Moretti ◽  
Sergio Schenkman
Keyword(s):  
Heat Shock ◽  
Trypanosoma Cruzi ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Transcription Control ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Content Type ◽  
Polycistronic Transcription

ABSTRACT The phosphorylation of the carboxy-terminal heptapeptide repeats of the largest subunit of RNA polymerase II (Pol II) controls several transcription-related events in eukaryotes. Trypanosomatids lack these typical repeats and display an unusual transcription control. RNA Pol II associates with the transcription site of the spliced leader (SL) RNA, which is used in the trans -splicing of all mRNAs transcribed on long polycistronic units. We found that Trypanosoma cruzi RNA Pol II associated with chromatin is highly phosphorylated. When transcription is inhibited by actinomycin D, the enzyme runs off from SL genes, remaining hyperphosphorylated and associated with polycistronic transcription units. Upon heat shock, the enzyme is dephosphorylated and remains associated with the chromatin. Transcription is partially inhibited with the accumulation of housekeeping precursor mRNAs, except for heat shock genes. DNA damage caused dephosphorylation and transcription arrest, with RNA Pol II dissociating from chromatin although staying at the SL. In the presence of calyculin A, the hyperphosphorylated form detached from chromatin, including the SL loci. These results indicate that in trypanosomes, the unusual RNA Pol II is phosphorylated during the transcription of SL and polycistronic operons. Different types of stresses modify its phosphorylation state, affecting pre-RNA processing.

scholarly journals TFIIF Facilitates Dissociation of RNA Polymerase II from Noncoding RNAs That Lack a Repression Domain

2009 ◽  
Vol 30 (1) ◽  
pp. 91-97 ◽  
Author(s):  
Stacey D. Wagner ◽  
Jennifer F. Kugel ◽  
James A. Goodrich
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Repression ◽  
Noncoding Rnas ◽  
Mrna Transcription ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
General Transcription Factors ◽  
B2 Rna ◽  
Repression Domain

ABSTRACT Noncoding RNAs (ncRNAs) have recently been found to regulate multiple steps in mammalian mRNA transcription. Mouse B2 RNA and human Alu RNA bind RNA polymerase II (Pol II) and repress mRNA transcription, using regions of the ncRNAs referred to as repression domains. Two other ncRNAs, mouse B1 RNA and human small cytoplasmic Alu (scAlu) RNA, bind Pol II with high affinity but lack repression domains and hence do not inhibit transcription. To better understand the interplay between ncRNAs that bind Pol II and their functions in transcription, we studied how Pol II binding and transcriptional repression are controlled by general transcription factors. We found that TFIIF associates with B1 RNA/Pol II and scAlu RNA/Pol II complexes and decreases their kinetic stability. Both subunits of TFIIF are required for this activity. Importantly, fusing a repression domain to B1 RNA stabilizes its interaction with Pol II in the presence of TFIIF. These results suggest a new role for TFIIF in regulating the interaction of ncRNAs with Pol II; specifically, it destabilizes interactions with ncRNAs that are not transcriptional repressors. These studies also identify a new function for ncRNA repression domains: they stabilize interactions of ncRNAs with Pol II in the presence of TFIIF.

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