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At least 163 records · Page 9Linked to original sources
PubMed2006

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.

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.

Borna disease virus↗
PubMed1998

Inhibition of Cellular RNA polymerase II transcription by delta antigen of hepatitis delta virus.

Hepatitis delta virus (HDV) contains a circular, viroid-like RNA and the hepatitis delta antigen (HDAg) protein. The viral RNA is replicated via RNA-dependent RNA synthesis, which is thought to be mediated by host DNA-dependent RNA polymerase II (pol II). The precise mechanism of HDV RNA replication using RNA as a template remains to be elucidated, though it is clear that HDAg is involved. We demonstrate here that both SP1-activated and basal pol II transcription are inhibited by HDAg. This inhibitory effect of HDAg was observed in vivo in transient cotransfection assays as well as in vitro in HeLa nuclear extracts with purified, recombinant HDAg. The in vitro inhibition of pol II transcription could be reversed with excess HeLa nuclear extracts. Furthermore, HDAg specifically inhibited pol II-mediated transcription but not pol I- or III-mediated transcription. These results provide support for the model in which HDAg participates in a complex with host cell pol II transcription factors to mediate pol II-dependent HDV RNA replication, concomitantly cellular pol II transcription.

Chloramphenicol O-Acetyltransferase↗
PubMed2005

Association of the influenza A virus RNA-dependent RNA polymerase with cellular RNA polymerase II.

Transcription by the influenza virus RNA-dependent RNA polymerase is dependent on cellular RNA processing activities that are known to be associated with cellular RNA polymerase II (Pol II) transcription, namely, capping and splicing. Therefore, it had been hypothesized that transcription by the viral RNA polymerase and Pol II might be functionally linked. Here, we demonstrate for the first time that the influenza virus RNA polymerase complex interacts with the large subunit of Pol II via its C-terminal domain. The viral polymerase binds hyperphosphorylated forms of Pol II, indicating that it targets actively transcribing Pol II. In addition, immunofluorescence analysis is consistent with a new model showing that influenza virus polymerase accumulates at Pol II transcription sites. The present findings provide a framework for further studies to elucidate the mechanistic principles of transcription by a viral RNA polymerase and have implications for the regulation of Pol II activities in infected cells.

Animals↗
PubMed1996

Repair of rDNA in Saccharomyces cerevisiae: RAD4-independent strand-specific nucleotide excision repair of RNA polymerase I transcribed genes.

Removal of UV-induced pyrimidine dimers from the individual strands of the rDNA locus in Saccharomyces cerevisiae was studied. Yeast rDNA, that is transcribed by RNA polymerase I(RNA pol I), is repaired efficiently, slightly strand-specific and independently of RAD26, which has been implicated in transcription-coupled repair of the RNA pol II transcribed RPB2 gene. No repair of rDNA is observed in rad1,2,3 and 14 mutants, demonstrating that dimer removal from this highly repetitive DNA is accomplished by nucleotide excision repair (NER). In rad7 and rad16 mutants, which are specifically deficient in repair of non-transcribed DNA, there is a clear preferential repair of the transcribed strand of rDNA, indicating that strand-specific and therefore probably transcription-coupled repair of RNA pol I transcribed genes does exist in yeast. Unexpectedly, the transcribed but not the non-transcribed strand of rDNA can be repaired in rad4 mutants, which seem otherwise completely NER-deficient.

DNA Repair↗
PubMed1993

In vivo regulation of rRNA transcription occurs rapidly in nondividing and dividing Drosophila cells in response to a phorbol ester and serum.

The synthesis of ribosomes is an essential cellular process which requires the transcription of the rRNA genes by RNA polymerase I (Pol I). The regulation of rRNA synthesis is known to be coupled to growth regulation. In nongrowing, slowly growing, and rapidly growing Drosophila cells, exposure to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) increases the synthesis of precursor and mature rRNAs. Using nuclear run-on assays, we show that TPA enhances transcription of the rRNA genes. These results suggest that TPA regulates expression of RNA genes transcribed by Pol I, irrespective of the growth state of the cells. In slowly dividing Drosophila cells, increasing the serum concentration rapidly alters the accumulation of rRNA by enhancing rDNA transcription within 1 h. Thus, TPA and serum are each able to rapidly regulate rRNA gene expression in Drosophila cells. These results indicate that the RNA Pol I transcription system can be regulated by agents which have previously been shown to effect specific genes transcribed by the RNA Pol II system.

Animals↗
PubMed2002

Coordination of PIC assembly and chromatin remodeling during differentiation-induced gene activation.

We analyzed the ordered recruitment of factors to the human alpha1 antitrypsin promoter around the initial activation of the gene during enterocyte differentiation. We found that a complete preinitiation complex, including phosphorylated RNA pol II, was assembled at the promoter long before transcriptional activation. The histone acetyltransferases CBP and P/CAF were recruited subsequently, but local histone hyperacetylation was delayed. After transient recruitment of the human Brahma homolog hBrm, remodeling of the neighboring nucleosome coincided with transcription initiation. The results suggest that, at this promoter, chromatin reconfiguration is a defining step of the initiation process, acting after the assembly of the Pol II machinery.

Acetylation↗
PubMed1999

GAL4 is regulated by the RNA polymerase II holoenzyme-associated cyclin-dependent protein kinase SRB10/CDK8.

Phosphorylation of the yeast transcription factor GAL4 at S699 is required for efficient galactose-inducible transcription. We demonstrate that this site is a substrate for the RNA polymerase holoenzyme-associated CDK SRB10. S699 phosphorylation requires SRB10 in vivo, and this site is phosphorylated by purified SRB10/ SRB11 CDK/cyclin in vitro. RNA Pol II holoenzymes purified from WT yeast phosphorylate GAL4 at sites observed in vivo whereas holoenzymes from srb10 yeast are incapable of phosphorylating GAL4 at S699. Mutations at GAL4 S699 and srb10 are epistatic for GAL induction, demonstrating that SRB10 regulates GAL4 activity through this phosphorylation in vivo. These results demonstrate a function for the SRB10/ CDK8 holoenzyme-associated CDK that involves regulation of transactivators by phosphorylation during transcriptional activation.

Animals↗
PubMed2001

A role for coactivators and histone acetylation in estrogen receptor alpha-mediated transcription initiation.

Transcriptional regulation by estrogen receptor alpha (ERalpha) involves protein-protein interactions among the receptor, its associated coactivators and the RNA polymerase II transcriptional machinery. We have used an in vitro chromatin assembly and transcription system to examine the biochemistry of interactions among ERalpha, the SRC proteins and p300/CBP. Using polypeptides designed to block specific receptor- cofactor or cofactor-cofactor interactions, we show that interactions among ERalpha, its coactivators and the RNA pol II machinery are all required for ERalpha- mediated transcription. Furthermore, we show that ERalpha-SRC-p300/CBP interactions are necessary and sufficient for the targeted acetylation of nucleosomal histones on estrogen-responsive promoters in the absence of transcription. The protein-protein interactions required for histone acetylation constitute a subset of the interactions required for transcriptional activation. Finally, we show that the major role of SRC-p300/CBP interactions is to enhance ERalpha- mediated transcription initiation, and they have little or no role in stimulating subsequent rounds of transcription. Together, our results indicate a specific role for the SRC and p300/CBP coactivators, as well as targeted histone acetylation, in ERalpha-mediated transcription.

Acetylation↗
PubMed1995

Phosphorylation of tyrosine residues of RNA polymerase II and other nuclear proteins by active chromatin tyrosine kinase(s).

We demonstrate here for the first time that protein tyrosine kinases are present in the active chromatin of nucleus. The presence of tyrosine kinase activity in the active chromatin was initially determined using poly (Glu,Na-Tyr;4:1) (PGT) as a tyrosine phosphorylatable substrate. Active chromatin in the presence of cofactors phosphorylated PGT at a rate of 40 pmol/min. The phosphorylation of PGT by active chromatin was inhibited by 41, 47 and approximately 95% with genistein, n-ethylmaleimide and quercetin (known inhibitors of tyrosine kinases), respectively. A Lineweaver-Burk plot revealed an apparent Km of 50 micrograms/ml and Vmax of 45 pmol/min for active chromatin tyrosine kinase(s). Analyses of phosphorylation of endogenous substrates by immunoprecipitation, western blotting and phosphoamino acids revealed that active chromatin protein tyrosine kinase(s) are able to phosphorylate tyrosine residues of the large subunit of RNA pol II and several other active chromatin proteins. The ability of AC-PTKs to phosphorylate many proteins of active chromatin components argues strongly for its role(s) in regulating transcription.

Animals↗
PubMed2005

Paracoccidioides brasiliensis RNA biogenesis apparatus revealed by functional genome analysis.

The RNA biogenesis machinery of Paracoccidioides brasiliensis was assessed by comparative analyses of PbAESTs (P. brasiliensis assembled expressed sequence tags (ESTs)) with sequences from Saccharomyces cerevisiae MIPS database. PbAESTs related to almost all categories of S. cerevisiae RNA biogenesis were found. Two of the 12 S. cerevisiae RNA Pol II core subunits, Rpb3 and Rpb7, were found, probably reflecting the growth phase from which the cDNA libraries used in ESTs generation were constructed, as well as the low abundance of some of these transcripts. We have also found orthologs to TATA-box-binding protein (TBP), and at least one subunit of each TBP-associated factors (TFII) in P. brasiliensis transcriptome, except TFIIB. Genes associated to the chromatin remodeling complex, as well as transcription factors probably involved in the control of genes associated to a sexual cycle and virulence, were also identified. With respect to the pre-mRNA processing, 65 PbAEST orthologs to S. cerevisiae basal splicing machinery and 21 orthologs of 5'- and 3'-end formation processes were found. Components involved in RNA interference were detected, suggesting that this gene expression regulation mechanism is probably used by P. brasiliensis. Twelve PbAESTs related to Pol I and Pol III machineries were assigned as S. cerevisiae orthologs. Finally, 25 and 10 PbAESTs associated to rRNA and tRNA processing, respectively, were detected. Taken together, our results enable us to depict, for the first time, a global view of transcription and RNA processing in P. brasiliensis.

Expressed Sequence Tags↗
PubMed2006

Protein kinase A regulates RNA polymerase III transcription through the nuclear localization of Maf1.

Maf1 is an essential and specific mediator of transcriptional repression in the RNA polymerase (pol) III system. Maf1-dependent repression occurs in response to a wide range of conditions, suggesting that the protein itself is targeted by the major nutritional and stress-signaling pathways. We show that Maf1 is a substrate for cAMP-dependent PKA in vitro and is differentially phosphorylated on PKA sites in vivo under normal versus repressing conditions. PKA activity negatively regulates Maf1 function because strains with unregulated high PKA activity block repression of pol III transcription in vivo, and strains lacking all PKA activity are hyperrepressible. Nuclear accumulation of Maf1 is required for transcriptional repression and is regulated by two nuclear localization sequences in the protein. An analysis of PKA phosphosite mutants shows that the localization of Maf1 is affected via the N-terminal nuclear localization sequence. In particular, mutations that prevent phosphorylation at PKA consensus sites promote nuclear accumulation of Maf1 without inducing repression. These results indicate that negative regulation of Maf1 by PKA is achieved by inhibiting its nuclear import and suggest that a PKA-independent activation step is required for nuclear Maf1 to function in the repression of pol III transcription. Finally, we report a previously undescribed phenotype for Maf1 in tRNA gene-mediated silencing of nearby RNA pol II transcription.

Active Transport, Cell Nucleus↗
PubMed2003

Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I.

The structure of the yeast DNA-dependent RNA polymerase I (RNA Pol I), prepared by cryo-negative staining, was studied by electron microscopy. A structural model of the enzyme at a resolution of 1.8 nm was determined from the analysis of isolated molecules and showed an excellent fit with the atomic structure of the RNA Pol II Delta4/7. The high signal-to-noise ratio (SNR) of the stained molecular images revealed a conformational flexibility within the image data set that could be recovered in three-dimensions after implementation of a novel strategy to sort the "open" and "closed" conformations in our heterogeneous data set. This conformational change mapped in the "wall/flap" domain of the second largest subunit (beta-like) and allows a better accessibility of the DNA-binding groove. This displacement of the wall/flap domain could play an important role in the transition between initiation and elongation state of the enzyme. Moreover, a protrusion was apparent in the cryo-negatively stained model, which was absent in the atomic structure and was not detected in previous 3D models of RNA Pol I. This structure could, however, be detected in unstained views of the enzyme obtained from frozen hydrated 2D crystals, indicating that this novel feature is not induced by the staining process. Unexpectedly, negatively charged molybdenum compounds were found to accumulate within the DNA-binding groove, which is best explained by the highly positive electrostatic potential of this region of the molecule, thus, suggesting that the stain distribution reflects the overall surface charge of the molecule.

Binding Sites↗
PubMed2004

Expression and localization of nuclear proteins in autosomal-dominant Emery-Dreifuss muscular dystrophy with LMNA R377H mutation.

BACKGROUND: The autosomal dominant form of Emery-Dreifuss muscular dystrophy (AD-EDMD) is caused by mutations in the gene encoding for the lamins A and C (LMNA). Lamins are intermediate filament proteins which form the nuclear lamina underlying the inner nuclear membrane. We have studied the expression and the localization of nuclear envelope proteins in three different cell types and muscle tissue of an AD-EDMD patient carrying a point mutation R377H in the lamin A/C gene. RESULTS: Lymphoblastoid cells, skin fibroblasts, primary myoblasts and muscle thin sections were studied by immunocytochemistry and electron microscopy. Cellular levels of A-type lamins were reduced compared to control cells. In contrast, the amount of emerin and lamin B appeared unaltered. Cell synchronization experiments showed that the reduction of the cellular level of A-type lamin was due to instability of lamin A. By electron microscopy, we identified a proportion of nuclei with morphological alterations in lymphoblastoid cells, fibroblasts and mature muscle fibres. Immunofluorescence microscopy showed that a major population of the lamin B receptor (LBR), an inner nuclear membrane protein, was recovered in the cytoplasm in association with the ER. In addition, the intranuclear organization of the active form of RNA polymerase II was markedly different in cells of this AD-EDMD patient. This aberrant intranuclear distribution was specifically observed in muscle cells where the pathology of EDMD predominates. CONCLUSIONS: From our results we conclude: Firstly, that structural alterations of the nuclei which are found only in a minor fraction of lymphoblastoid cells and mature muscle fibres are not sufficient to explain the clinical pathology of EDMD; Secondly, that wild type lamin A is required not only for the retention of LBR in the inner nuclear membrane but also for a correct localization of the transcriptionally active RNA pol II in muscle cells. We speculate that a rearrangement of the internal chromatin could lead to muscle-specific disease symptoms by interference with proper mRNA transcription.

Amino Acid Substitution↗
PubMed2005

The antisense strand of small interfering RNAs directs histone methylation and transcriptional gene silencing in human cells.

To determine mechanistically how siRNAs mediate transcriptional gene silencing (TGS) in human cells, we have measured histone methylation at targeted promoters, the dependency on active transcription, and whether or not both strands of the siRNA are required for siRNA-mediated TGS. We report here that siRNA treatment increases both H3K9 and H3K27 methylation of the targeted EF1A promoter and that this increase is dependent on nuclear specific delivery of the siRNA. We also find that TGS can be directed by the antisense strand alone, and requires active transcription by RNA polymerase II in human cells as evidenced by sensitivity to alpha-amanatin. The observation of antisense strand-specific siRNA-mediated TGS of EF1A was substantiated by targeting the U3 region of the HIV-1 LTR/promoter. Furthermore, we show that the antisense strand of siRNA EF52 associates with the transiently expressed Flag-tagged DNMT3A, the targeted EF1A promoter, and trimethylated H3K27. The observations reported here implicate a functional link between siRNA-mediated targeting of genomic regions (promoters), RNA Pol II function, histone methylation, and DNMT3A and support a paradigm in which the antisense strands of siRNAs alone can direct sequence-specific transcriptional gene silencing in human cells.

Amanitins↗
PubMed2001

Effect of deletion and the site of insertion in double copy anti-tat retroviral vectors: viral titres and production of anti-tat mRNA.

In attempts to further develop murine leukemia virus (MLV) based retroviral vectors for gene therapy, we investigated vector production and antisense expression from retroviral constructs with U3 deletions or insertions. Promoter elements in the U3 region of the 3' LTR of the vector pLXSN were deleted and replaced with DNA encoding the HIV anti-tat gene under control of the tRNAmet promoter to produce a double copy self inactivating vector (DC-SIN). DC-SIN constructs were compared to vectors containing the anti-tat cassette inserted at 5 different sites of the U3 region (DC-insertions). Titres of DC-SIN and DC-insertion vectors were similar but approximately 10 fold lower than parental pLXSN. Cells transduced with DC-SIN and DC-insertion vectors all expressed anti-tat mRNA. Transcripts from the MLV-LTR were detected in cells transduced with DC-insertion but not DC-SIN vectors or a vector with the anti-tat cassette between CAAT and TATA boxes of the promoter, indicating inactivation of the viral promoter in the latter vectors. Cells transduced with constructs of either design showed comparable efficacy of protection against HIV challenge. Thus, no U3 insertion site was preferred for virus production. Insertion of a tRNA promoter between CAAT and TATA boxes and the DC-SIN design which would not introduce an active RNA pol II promoter into the genome are attractive for further development of safe gene therapy agents.

Genes, tat↗
PubMed2002

Nucleocytoplasmic transport of proteins and poly(A)+ RNA in reconstituted Tpr-less nuclei in living mammalian cells.

BACKGROUND: It is known that Tpr is a component of an intranuclear long filament which extends from the nuclear pore complex (NPC) into the nucleoplasm. Since the over-expression of the full-length of or some fragments of Tpr in living cells leads to the accumulation of poly(A)+ RNA within the nuclei, it is generally thought that a relationship exists between Tpr and the nuclear export of mRNA in mammalian cells. In contrast, the nuclear export of poly(A)+ RNA was not inhibited in a double deletion mutant of yeast Tpr homologues (Mlp1p and Mlp2p). Therefore, the precise function of Tpr remains unknown. RESULTS: By microinjecting two types of polyclonal antibodies which are specific to Tpr into the cytoplasm of living mammalian interphase cells, we succeeded in reconstituting the Tpr-less nuclei. In the Tpr-less nuclei, the localization of the major components of the NPC, the nuclear import of SV40 T-NLS substrates and the nuclear export of HIV Rev NES-substrates were not affected. However poly(A)+ RNA accumulated in the non-snRNP splicing factor SC35-positive clusters, which became larger in size and fewer in number, compared with normal nuclei. CONCLUSION: These results indicate that Tpr plays a critical role in the intranuclear dynamics of RNA pol II transcripts, including the processing, intranuclear transport and targeting, as well as their translocation through the NPC in mammalian cells.

Antibodies↗
PubMed2005

Histone modifications as key regulators of transcription.

Covalent modifications of the amino-termini of the core histones in nucleosomes have been shown to be one of the key regulatory mechanisms in transcription regulation. Recently, new roles for histone modifications have been uncovered for the efficient functioning of RNA Pol II. Besides acetylation, which is the most characterized to date these modifications comprise phosphorylation, methylation, and ubiquitination. This review gives comprehensive view of all the major histone modifications and their effect on transcriptional regulation, in Saccharomyces cerevisiae.

Acetylation↗
PubMed2007

Human SSRP1 has Spt16-dependent and -independent roles in gene transcription.

The facilitating chromatin transcription (FACT) complex, a heterodimer of SSRP1 and Spt16, has been shown to regulate transcription elongation through a chromatin template in vitro and on specific genes in cells. However, its global role in transcription regulation in human cells remains largely elusive. We conducted spotted microarray analyses using arrays harboring 8308 human genes to assess the gene expression profile after knocking down SSRP1 or Spt16 levels in human non-small cell lung carcinoma (H1299) cells. Although the changes of these transcripts were surprisingly subtle, there were approximately 170 genes whose transcript levels were either reduced or induced >1.5-fold. Approximately 106 genes with >1.2-fold change at the level of transcripts were the common targets of both SSRP1 and Spt16 ( approximately 1.3%). A subset of genes was regulated by SSRP1 independent of Spt16. Further analyses of some of these genes not only verified this observation but also identified the serum-responsive gene, egr1, as a novel target for both SSRP1 and Spt16. We further showed that SSRP1 and Spt16 are important for the progression of elongation RNA pol II on the egr1 gene. These results suggest that SSRP1 has Spt16-dependent and -independent roles in regulating gene transcription in human cells.

Cell Cycle Proteins↗