scholarly journals RNA polymerase II transcription is concentrated outside replication domains throughout S-phase

1994 ◽  
Vol 107 (6) ◽  
pp. 1449-1456 ◽  
Author(s):  
D.G. Wansink ◽  
E.E. Manders ◽  
I. van der Kraan ◽  
J.A. Aten ◽  
R. van Driel ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Spatial Relationship ◽  
S Phase ◽  
Double Labelling ◽  
Confocal Immunofluorescence Microscopy ◽  
Nuclear Domain ◽  
Confocal Immunofluorescence ◽  
Nuclear Domains ◽  
Transcription And Replication

Transcription and replication are, like many other nuclear functions and components, concentrated in nuclear domains. Transcription domains and replication domains may play an important role in the coordination of gene expression and gene duplication in S-phase. We have investigated the spatial relationship between transcription and replication in S-phase nuclei after fluorescent labelling of nascent RNA and nascent DNA, using confocal immunofluorescence microscopy. Permeabilized human bladder carcinoma cells were labelled with 5-bromouridine 5′-triphosphate and digoxigenin-11-deoxyuridine 5′-triphosphate to visualize sites of RNA synthesis and DNA synthesis, respectively. Transcription by RNA polymerase II was localized in several hundreds of domains scattered throughout the nucleoplasm in all stages of S-phase. This distribution resembled that of nascent DNA in early S-phase. In contrast, replication patterns in late S-phase consisted of fewer, larger replication domains. In double-labelling experiments we found that transcription domains did not colocalize with replication domains in late S-phase nuclei. This is in agreement with the notion that late replicating DNA is generally not actively transcribed. Also in early S-phase nuclei, transcription domains and replication domains did not colocalize. We conclude that nuclear domains exist, large enough to be resolved by light microscopy, that are characterized by a high activity of either transcription or replication, but never both at the same time. This probably means that as soon as the DNA in a nuclear domain is being replicated, transcription of that DNA essentially stops until replication in the entire domain is completed.

scholarly journals Nuclear distribution of transcription factors in relation to sites of transcription and RNA polymerase II

1997 ◽  
Vol 110 (15) ◽  
pp. 1781-1791 ◽  
Author(s):  
M.A. Grande ◽  
I. van der Kraan ◽  
L. de Jong ◽  
R. van Driel
Keyword(s):  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Glucocorticoid Receptors ◽  
Cultured Cells ◽  
Spatial Relationship ◽  
Immunofluorescence Labelling ◽  
Transcription Sites ◽  
Nuclear Domains

We have investigated the spatial relationship between sites containing newly synthesized RNA and domains containing proteins involved in transcription, such as RNA polymerase II and the transcription factors TFIIH, Oct1, BRG1, E2F-1 and glucocorticoid receptors, using dual immunofluorescence labelling followed by confocal microscopy on cultured cells. As expected, a high degree of colocalisation between the RNA polymerase II and sites containing newly synthesised RNA was observed. Like the newly synthesised RNA and the RNA polymerase II, we found that all the transcription factors that we studied are distributed more or less homogeneously throughout the nucleoplasm, occupying numerous small domains. In addition to these small domains, TFIIH was found concentrated in coiled bodies and Oct1 in a single large domain of about 1.5 microm in 30% of the cells in an asynchronous HeLa cell culture. Remarkably, we found little or no relationship between the spatial distribution of the glucocorticoid receptor, Oct1 and E2F-1 on the one hand and RNA polymerase II and transcription sites on the other hand. In contrast, a significant but incomplete overlap was observed between the spatial distributions of transcription sites and BRG1 and TFIIH. These results indicate that many of the transcription factor-rich nuclear domains are not actively involved in transcription. They may represent incomplete transcription initiation complexes, inhibitory complexes, or storage sites.

scholarly journals Dynamic Nature of Cleavage Bodies and Their Spatial Relationship to DDX1 Bodies, Cajal Bodies, and Gems

2006 ◽  
Vol 17 (3) ◽  
pp. 1126-1140 ◽  
Author(s):  
Lei Li ◽  
Ken Roy ◽  
Sachin Katyal ◽  
Xuejun Sun ◽  
Stacey Bléoo ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Actin Polymerization ◽  
Spatial Relationship ◽  
S Phase ◽  
Nuclear Bodies ◽  
Cajal Bodies ◽  
Rna Transcription ◽  
Nuclear Structures ◽  
The Relationship ◽  
Striking Effect

DDX1 bodies, cleavage bodies, Cajal bodies (CBs), and gems are nuclear suborganelles that contain factors involved in RNA transcription and/or processing. Although all four nuclear bodies can exist as distinct entities, they often colocalize or overlap with each other. To better understand the relationship between these four nuclear bodies, we examined their spatial distribution as a function of the cell cycle. Here, we report that whereas DDX1 bodies, CBs and gems are present throughout interphase, CPSF-100-containing cleavage bodies are predominantly found during S and G2 phases, whereas CstF-64-containing cleavage bodies are primarily observed during S phase. All four nuclear bodies associate with each other during S phase, with cleavage bodies colocalizing with DDX1 bodies, and cleavage bodies/DDX1 bodies residing adjacent to gems and CBs. Although inhibitors of RNA transcription had no effect on DDX1 bodies or cleavage bodies, inhibitors of DNA replication resulted in loss of CstF-64-containing cleavage bodies. A striking effect on nuclear structures was observed with latrunculin B, an inhibitor of actin polymerization, resulting in the formation of needlelike nuclear spicules made up of CstF-64, CPSF-100, RNA, and RNA polymerase II. Our results suggest that cleavage body components are highly dynamic in nature.

scholarly journals Transcription-dependent redistribution of the large subunit of RNA polymerase II to discrete nuclear domains.

1995 ◽  
Vol 129 (2) ◽  
pp. 287-298 ◽  
Author(s):  
D B Bregman ◽  
L Du ◽  
S van der Zee ◽  
S L Warren
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Activity ◽  
Speckle Pattern ◽  
Large Subunit ◽  
Temperature Dependent ◽  
Pol Ii ◽  
Transcriptional Inhibition ◽  
Tight Association ◽  
Nuclear Domains

A subpopulation of the largest subunit of RNA polymerase II (Pol II LS) is located in 20-50 discrete subnuclear domains that are closely linked to speckle domains, which store splicing proteins. The speckle-associated fraction of Pol II LS is hyperphosphorylated on the COOH-terminal domain (CTD), and it is highly resistant to extraction by detergents. A diffuse nucleoplasmic fraction of Pol II LS is relatively hypophosphorylated on the CTD, and it is easily extracted by detergents. In transcriptionally active nuclei, speckle bound hyperphosphorylated Pol II LS molecules are distributed in irregularly shaped speckle domains, which appear to be interconnected via a reticular network. When transcription is inhibited, hyperphosphorylated Pol II LS and splicing protein SC35 accumulate in speckle domains, which are transformed into enlarged, dot-like structures lacking interconnections. When cells are released from transcriptional inhibition, Pol IIO and SC35 redistribute back to the interconnected speckle pattern of transcriptionally active cells. The redistribution of Pol II and SC35 is synchronous, reversible, and temperature dependent. It is concluded that: (a) hyperphosphorylation of Pol II LS's CTD is a better indicator of its tight association to discrete subnuclear domains than its transcriptional activity; (b) during states of transcriptional inhibition, hyperphosphorylated Pol II LS can be stored in enlarged speckle domains, which under the light microscope appear to coincide with the storage sites for splicing proteins; and (c) Pol II and splicing proteins redistribute simultaneously according to the overall transcriptional activity of the nucleus.

scholarly journals Splicing Speckles Are Not Reservoirs of RNA Polymerase II, but Contain an Inactive Form, Phosphorylated on Serine2 Residues of the C-Terminal Domain

2006 ◽  
Vol 17 (4) ◽  
pp. 1723-1733 ◽  
Author(s):  
Sheila Q. Xie ◽  
Sonya Martin ◽  
Pascale V. Guillot ◽  
David L. Bentley ◽  
Ana Pombo
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Optical Resolution ◽  
Polymerase Activity ◽  
Terminal Domain ◽  
Transcriptional Inhibition ◽  
Inactive Form ◽  
Splicing Speckles ◽  
Nuclear Domains ◽  
Specific Mrna

“Splicing speckles” are major nuclear domains rich in components of the splicing machinery and polyA+ RNA. Although speckles contain little detectable transcriptional activity, they are found preferentially associated with specific mRNA-coding genes and gene-rich R bands, and they accumulate some unspliced pre-mRNAs. RNA polymerase II transcribes mRNAs and is required for splicing, with some reports suggesting that the inactive complexes are stored in splicing speckles. Using ultrathin cryosections to improve optical resolution and preserve nuclear structure, we find that all forms of polymerase II are present, but not enriched, within speckles. Inhibition of polymerase activity shows that speckles do not act as major storage sites for inactive polymerase II complexes but that they contain a stable pool of polymerase II phosphorylated on serine2 residues of the C-terminal domain, which is transcriptionally inactive and may have roles in spliceosome assembly or posttranscriptional splicing of pre-mRNAs. Paraspeckle domains lie adjacent to speckles, but little is known about their protein content or putative roles in the expression of the speckle-associated genes. We find that paraspeckles are transcriptionally inactive but contain polymerase II, which remains stably associated upon transcriptional inhibition, when paraspeckles reorganize around nucleoli in the form of caps.

scholarly journals Synthesis of histone messenger RNAs by RNA polymerase II in nuclei from S phase HeLa S3cells

1978 ◽  
Vol 5 (5) ◽  
pp. 1515-1528 ◽  
Author(s):  
S. Detke ◽  
J.L. Stein ◽  
G.S. Stein
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
S Phase ◽  
Messenger Rnas

scholarly journals Identifying functional neighborhoods within the cell nucleus: Proximity analysis of early S-phase replicating chromatin domains to sites of transcription, RNA polymerase II, HP1γ, matrin 3 and SAF-A

2008 ◽  
Vol 105 (2) ◽  
pp. 391-403 ◽  
Author(s):  
Kishore S. Malyavantham ◽  
Sambit Bhattacharya ◽  
Marcos Barbeitos ◽  
Lopamudra Mukherjee ◽  
Jinhui Xu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Nucleus ◽  
S Phase ◽  
Chromatin Domains ◽  
Matrin 3 ◽  
Proximity Analysis

scholarly journals Role of the Virus Nucleoprotein in the Regulation of Lymphocytic Choriomeningitis Virus Transcription and RNA Replication

2003 ◽  
Vol 77 (6) ◽  
pp. 3882-3887 ◽  
Author(s):  
Daniel D. Pinschewer ◽  
Mar Perez ◽  
Juan Carlos de la Torre
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Helper Virus ◽  
Lymphocytic Choriomeningitis ◽  
Lymphocytic Choriomeningitis Virus ◽  
Rna Structures ◽  
Virus Transcription ◽  
Infected Cells ◽  
Polymerase I ◽  
Transcription And Replication

ABSTRACT The prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) has a bisegmented negative-strand RNA genome. Each segment carries two viral genes in opposite orientation and separated by an intergenic region (IGR). The RNA-dependent RNA polymerase (RdRp) L of LCMV produces subgenomic mRNA and full-length genomic and antigenomic RNA species in two different processes termed transcription and replication, respectively. It is widely accepted that intracellular nucleoprotein (NP) levels regulate these two processes. Intracellular NP levels increase during the course of the infection, resulting in the unfolding of secondary RNA structures within the IGR. Structure-dependent transcription termination at the IGR is thereby attenuated, promoting replication of genome and antigenome RNA species. To test this hypothesis, we established a helper-virus-free minigenome (MG) system where intracellular synthesis of an S segment analogue from a plasmid is driven by RNA polymerase I. Cotransfection with two additional plasmids expressing the minimal viral trans-acting factors L and NP under control of RNA polymerase II allowed for RNA synthesis mediated by the intracellularly reconstituted LCMV polymerase. Both processes, transcription and replication, were strictly dependent on NP. However, both were equally enhanced by incrementally increasing amounts of NP up to levels in the range of those in LCMV-infected cells. Our data are consistent with a central role for NP in transcription and replication of the LCMV genome, but they do not support the participation of NP levels in balancing the two processes.

scholarly journals Nuclear Distribution of RNA Polymerase II and mRNA Processing Machinery in Early Mammalian Embryos

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Irina O. Bogolyubova ◽  
Dmitry S. Bogolyubov
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Activation ◽  
Preimplantation Embryos ◽  
Similar Data ◽  
Nuclear Antigens ◽  
Mammalian Embryos ◽  
Zygotic Gene ◽  
Major Factors ◽  
Nuclear Domains

Spatial distribution of components of nuclear metabolism provides a significant impact on regulation of the processes of gene expression. While distribution of the key nuclear antigens and their association with the defined nuclear domains were thoroughly traced in mammalian somatic cells, similar data for the preimplantation embryos are scanty and fragmental. However, the period of cleavage is characterized by the most drastic and dynamic nuclear reorganizations accompanying zygotic gene activation. In this minireview, we try to summarize the results of studies concerning distribution of major factors involved in RNA polymerase II-dependent transcription, pre-mRNA splicing mRNA export that have been carried out on early embryos of mammals.

scholarly journals BRD4 Prevents R-Loop Formation and Transcription-Replication Conflicts by Ensuring Efficient Transcription Elongation

2019 ◽  
Author(s):  
Drake Edwards ◽  
Rohin Maganti ◽  
Jarred P. Tanksley ◽  
Jie Luo ◽  
James J.H. Park ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Rna Polymerase Ii ◽  
Protein Function ◽  
S Phase ◽  
Transcriptional Elongation ◽  
Loop Formation ◽  
Replication Machinery ◽  
Spatio Temporal ◽  
Transcription And Replication

ABSTRACTEffective spatio-temporal control of transcription and replication during S-phase is paramount to maintaining genomic integrity and cell survival. Dysregulation of these systems can lead to conflicts between the transcription and replication machinery causing DNA damage and cell death. BRD4, a BET bromodomain protein and known transcriptional regulator, interacts with P-TEFb to ensure efficient transcriptional elongation by stimulating phosphorylation of RNA Polymerase II (RNAPII). Here we report that disruption of BET bromodomain protein function causes RNAPII pausing on the chromatin and DNA damage affecting cells in S-phase. We find that this persistent, RNAPII-dependent pausing leads to accumulation of RNA:DNA hybrids (R-loops), which are known to lead to transcription-replication conflicts (TRCs), DNA damage, and cell death. Furthermore, we show that resolution of R-loops abrogates BET bromodomain inhibitor-induced DNA damage, and that BET bromodomain inhibition induces both R-loops and DNA damage at sites of BRD4 occupancy. Finally, we see that the BRD4 C-terminal domain, which interacts with P-TEFb, is required to prevent R-loop formation and DNA damage caused by BET bromodomain inhibition and that oncogenes which promote transcription and replication exacerbate BET bromodomain inhibitor-induced DNA damage. Together, these findings demonstrate that BET bromodomain inhibitors can damage DNA via induction of R-loops and TRCs in highly replicative cells.

Sign in / Sign up

Export Citation Format

Share Document