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 Noncoding Transcription Is a Driving Force for Nucleosome Instability inspt16Mutant Cells

2016 ◽  
Vol 36 (13) ◽  
pp. 1856-1867 ◽  
Author(s):  
Jianxun Feng ◽  
Haiyun Gan ◽  
Matthew L. Eaton ◽  
Hui Zhou ◽  
Shuqi Li ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Histone Modifications ◽  
Driving Force ◽  
Pol Ii ◽  
Transcriptional Inhibition ◽  
Small Subgroup ◽  
Position Shift ◽  
Nucleosome Stability ◽  
Noncoding Transcription

FACT (facilitateschromatintranscription) consists of two essential subunits, Spt16 and Pob3, and functions as a histone chaperone. Mutation ofspt16results in a global loss of nucleosomes as well as aberrant transcription. Here, we show that the majority of nucleosome changes upon Spt16 depletion are alterations in nucleosome fuzziness and position shift. Most nucleosomal changes are suppressed by the inhibition of RNA polymerase II (Pol II) activity. Surprisingly, a small subgroup of nucleosome changes is resistant to transcriptional inhibition. Notably, Spt16 and distinct histone modifications are enriched at this subgroup of nucleosomes. We also report 1,037Spt16-suppressednoncodingtranscripts (SNTs) and found that the SNT start sites are enriched with the subgroup of nucleosomes resistant to Pol II inhibition. Finally, the nucleosomes at genes overlapping SNTs are more susceptible to changes upon Spt16 depletion than those without SNTs. Taken together, our results support a model in which Spt16 has a role in maintaining local nucleosome stability to inhibit initiation of SNT transcription, which once initiated drives additional nucleosome loss upon Spt16 depletion.

scholarly journals Association of the Influenza A Virus RNA-Dependent RNA Polymerase with Cellular RNA Polymerase II

2005 ◽  
Vol 79 (9) ◽  
pp. 5812-5818 ◽  
Author(s):  
Othmar G. Engelhardt ◽  
Matt Smith ◽  
Ervin Fodor
Keyword(s):  
Influenza Virus ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Influenza A ◽  
Large Subunit ◽  
Viral Rna ◽  
Rna Dependent Rna Polymerase ◽  
Pol Ii ◽  
Virus Rna ◽  
Transcription Sites

ABSTRACT 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.

scholarly journals HIF-1 Interacts with TRIM28 and DNA-PK to release paused RNA polymerase II and activate target gene transcription in response to hypoxia

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Yongkang Yang ◽  
Haiquan Lu ◽  
Chelsey Chen ◽  
Yajing Lyu ◽  
Robert N. Cole ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Transcription ◽  
Target Genes ◽  
Large Subunit ◽  
Hypoxia Inducible Factor ◽  
Elongation Factor ◽  
Transcriptional Elongation ◽  
Pol Ii

AbstractHypoxia-inducible factor-1 (HIF-1) is a transcription factor that acts as a regulator of oxygen (O2) homeostasis in metazoan species by binding to hypoxia response elements (HREs) and activating the transcription of hundreds of genes in response to reduced O2 availability. RNA polymerase II (Pol II) initiates transcription of many HIF target genes under non-hypoxic conditions but pauses after approximately 30–60 nucleotides and requires HIF-1 binding for release. Here we report that in hypoxic breast cancer cells, HIF-1 recruits TRIM28 and DNA-dependent protein kinase (DNA-PK) to HREs to release paused Pol II. We show that HIF-1α and TRIM28 assemble the catalytically-active DNA-PK heterotrimer, which phosphorylates TRIM28 at serine-824, enabling recruitment of CDK9, which phosphorylates serine-2 of the Pol II large subunit C-terminal domain as well as the negative elongation factor to release paused Pol II, thereby stimulating productive transcriptional elongation. Our studies reveal a molecular mechanism by which HIF-1 stimulates gene transcription and reveal that the anticancer effects of drugs targeting DNA-PK in breast cancer may be due in part to their inhibition of HIF-dependent transcription.

scholarly journals Walleye Dermal Sarcoma Virus Cyclin Interacts with Components of the Mediator Complex and the RNA Polymerase II Holoenzyme

2002 ◽  
Vol 76 (16) ◽  
pp. 8031-8039 ◽  
Author(s):  
Joel Rovnak ◽  
Sandra L. Quackenbush
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Subunit ◽  
Mediator Complex ◽  
Pol Ii ◽  
Walleye Dermal Sarcoma Virus ◽  
Cyclin C ◽  
Sarcoma Virus ◽  
Virus Expression ◽  
Dermal Sarcoma

ABSTRACT Walleye dermal sarcoma virus (WDSV) encodes an accessory protein, OrfA, with sequence homology to cyclins (retrovirus cyclin). In cells transfected with an expression construct, OrfA was localized to the nucleus and was concentrated in interchromatin granule clusters (IGCs), sites where splicing factors are concentrated. Other proteins identified in IGCs include transcription factors, the large subunit of RNA polymerase II (Pol II), and cyclin-dependent kinase 8 (cdk8). cdk8 is the kinase partner of cyclin C and a component of the mediator complex, associated with the Pol II holoenzyme. cdk8 and cyclin C can regulate transcription via phosphorylation of cyclin H and the carboxy-terminal domain of Pol II. OrfA in transfected HeLa cells was found to colocalize and copurify with hyperphosphorylated forms of Pol II (Pol IIO) in IGCs, and OrfA was coimmunoprecipitated from lysates of transfected cells with an antibody against Pol IIO. Likewise, Pol IIO could be coprecipitated with an antibody against OrfA. A survey with antibodies against several different cdks resulted in coimmunoprecipitation of OrfA with anti-cdk8, and antiserum against OrfA was able to coprecipitate cdk8 from lysates of cells that express OrfA. Coprecipitation of OrfA with anti-cyclin C demonstrated that it was included in complexes with OrfA and cdk8. OrfA has sequence and structural similarities to cyclin C, and, functionally, OrfA appears to have the capacity to both enhance and inhibit the activity of promoters in a cell-specific manner, similar to functions of the mediator complex. These data suggest that WDSV OrfA functions through its interactions with these large, transcription complexes. Further investigations will clarify the role of the retrovirus cyclin in control of virus expression and transformation.

scholarly journals Structure of the p53/RNA polymerase II assembly

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shu-Hao Liou ◽  
Sameer K. Singh ◽  
Robert H. Singer ◽  
Robert A. Coleman ◽  
Wei-Li Liu
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Conformational Changes ◽  
P53 Protein ◽  
Binding Activity ◽  
Transactivation Domain ◽  
Pol Ii ◽  
Dna Binding Activity ◽  
Regulated Gene Expression

AbstractThe tumor suppressor p53 protein activates expression of a vast gene network in response to stress stimuli for cellular integrity. The molecular mechanism underlying how p53 targets RNA polymerase II (Pol II) to regulate transcription remains unclear. To elucidate the p53/Pol II interaction, we have determined a 4.6 Å resolution structure of the human p53/Pol II assembly via single particle cryo-electron microscopy. Our structure reveals that p53’s DNA binding domain targets the upstream DNA binding site within Pol II. This association introduces conformational changes of the Pol II clamp into a further-closed state. A cavity was identified between p53 and Pol II that could possibly host DNA. The transactivation domain of p53 binds the surface of Pol II’s jaw that contacts downstream DNA. These findings suggest that p53’s functional domains directly regulate DNA binding activity of Pol II to mediate transcription, thereby providing insights into p53-regulated gene expression.

scholarly journals RecQL5 Promotes Genome Stabilization through Two Parallel Mechanisms—Interacting with RNA Polymerase II and Acting as a Helicase

2010 ◽  
Vol 30 (10) ◽  
pp. 2460-2472 ◽  
Author(s):  
M. Nurul Islam ◽  
David Fox ◽  
Rong Guo ◽  
Takemi Enomoto ◽  
Weidong Wang
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Genome Stability ◽  
Transcriptional Regulators ◽  
Helicase Activity ◽  
Parallel Mechanisms ◽  
Recq Helicase ◽  
Pol Ii ◽  
Kix Domain ◽  
Genome Stabilization

ABSTRACT The RecQL5 helicase is essential for maintaining genome stability and reducing cancer risk. To elucidate its mechanism of action, we purified a RecQL5-associated complex and identified its major component as RNA polymerase II (Pol II). Bioinformatics and structural modeling-guided mutagenesis revealed two conserved regions in RecQL5 as KIX and SRI domains, already known in transcriptional regulators for Pol II. The RecQL5-KIX domain binds both initiation (Pol IIa) and elongation (Pol IIo) forms of the polymerase, whereas the RecQL5-SRI domain interacts only with the elongation form. Fully functional RecQL5 requires both helicase activity and associations with the initiation polymerase, because mutants lacking either activity are partially defective in the suppression of sister chromatid exchange and resistance to camptothecin-induced DNA damage, and mutants lacking both activities are completely defective. We propose that RecQL5 promotes genome stabilization through two parallel mechanisms: by participation in homologous recombination-dependent DNA repair as a RecQ helicase and by regulating the initiation of Pol II to reduce transcription-associated replication impairment and recombination.

scholarly journals Direct Interaction between the Subunit RAP30 of Transcription Factor IIF (TFIIF) and RNA Polymerase Subunit 5, Which Contributes to the Association between TFIIF and RNA Polymerase II

2001 ◽  
Vol 276 (15) ◽  
pp. 12266-12273 ◽  
Author(s):  
Wenxiang Wei ◽  
Dorjbal Dorjsuren ◽  
Yong Lin ◽  
Weiping Qin ◽  
Takahiro Nomura ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Middle Part ◽  
Wild Type ◽  
Binding Region ◽  
Pol Ii ◽  
Transcription Factor Iif ◽  
B Virus

The general transcription factor IIF (TFIIF) assembled in the initiation complex, and RAP30 of TFIIF, have been shown to associate with RNA polymerase II (pol II), although it remains unclear which pol II subunit is responsible for the interaction. We examined whether TFIIF interacts with RNA polymerase II subunit 5 (RPB5), the exposed domain of which binds transcriptional regulatory factors such as hepatitis B virus X protein and a novel regulatory protein, RPB5-mediating protein. The results demonstrated that RPB5 directly binds RAP30in vitrousing purified recombinant proteins andin vivoin COS1 cells transiently expressing recombinant RAP30 and RPB5. The RAP30-binding region was mapped to the central region (amino acids (aa) 47–120) of RPB5, which partly overlaps the hepatitis B virus X protein-binding region. Although the middle part (aa 101–170) and the N-terminus (aa 1–100) of RAP30 independently bound RPB5, the latter was not involved in the RPB5 binding when RAP30 was present in TFIIF complex. Scanning of the middle part of RAP30 by clustered alanine substitutions and then point alanine substitutions pinpointed two residues critical for the RPB5 binding inin vitroandin vivoassays. Wild type but not mutants Y124A and Q131A of RAP30 coexpressed with FLAG-RAP74 efficiently recovered endogenous RPB5 to the FLAG-RAP74-bound anti-FLAG M2 resin. The recovered endogenous RPB5 is assembled in pol II as demonstrated immunologically. Interestingly, coexpression of the central region of RPB5 and wild type RAP30 inhibited recovery of endogenous pol II to the FLAG-RAP74-bound M2 resin, strongly suggesting that the RAP30-binding region of RPB5 inhibited the association of TFIIF and pol II. The exposed domain of RPB5 interacts with RAP30 of TFIIF and is important for the association between pol II and TFIIF.

scholarly journals Transcription of Hepatitis Delta Virus RNA by RNA Polymerase II

2007 ◽  
Vol 82 (3) ◽  
pp. 1118-1127 ◽  
Author(s):  
Jinhong Chang ◽  
Xingcao Nie ◽  
Ho Eun Chang ◽  
Ziying Han ◽  
John Taylor
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Hepatitis Delta Virus ◽  
Cell System ◽  
Hepatitis Delta ◽  
Pol Ii ◽  
Delta Antigen ◽  
Nuclear Extracts ◽  
Virus Rna ◽  
Delta Virus

ABSTRACT Previous studies have indicated that the replication of the RNA genome of hepatitis delta virus (HDV) involves redirection of RNA polymerase II (Pol II), a host enzyme that normally uses DNA as a template. However, there has been some controversy about whether in one part of this HDV RNA transcription, a polymerase other than Pol II is involved. The present study applied a recently described cell system (293-HDV) of tetracycline-inducible HDV RNA replication to provide new data regarding the involvement of host polymerases in HDV transcription. The data generated with a nuclear run-on assay demonstrated that synthesis not only of genomic RNA but also of its complement, the antigenome, could be inhibited by low concentrations of amanitin specific for Pol II transcription. Subsequent studies used immunoprecipitation and rate-zonal sedimentation of nuclear extracts together with double immunostaining of 293-HDV cells, in order to examine the associations between Pol II and HDV RNAs, as well as the small delta antigen, an HDV-encoded protein known to be essential for replication. Findings include evidence that HDV replication is somehow able to direct the available delta antigen to sites in the nucleoplasm, almost exclusively colocalized with Pol II in what others have described as transcription factories.

Genome-wide regulations of the pre-initiation complex formation and elongating RNA polymerase II by an E3 ubiquitin ligase, San1.

2021 ◽  
Author(s):  
Priyanka Barman ◽  
Rwik Sen ◽  
Amala Kaja ◽  
Jannatul Ferdoush ◽  
Shalini Guha ◽  
...  
Keyword(s):  
Quality Control ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ubiquitin Ligase ◽  
Nuclear Protein ◽  
Protein Quality ◽  
Initiation Complex ◽  
Pol Ii ◽  
Intrinsically Disordered ◽  
Genome Wide

San1 ubiquitin ligase is involved in nuclear protein quality control via its interaction with intrinsically disordered proteins for ubiquitylation and proteasomal degradation. Since several transcription/chromatin regulatory factors contain intrinsically disordered domains and can be inhibitory to transcription when in excess, San1 might be involved in transcription regulation. To address this, we analyzed the role of San1 in genome-wide association of TBP [that nucleates pre-initiation complex (PIC) formation for transcription initiation] and RNA polymerase II (Pol II). Our results reveal the roles of San1 in regulating TBP recruitment to the promoters and Pol II association with the coding sequences, and hence PIC formation and coordination of elongating Pol II, respectively. Consistently, transcription is altered in the absence of San1. Such transcriptional alteration is associated with impaired ubiquitylation and proteasomal degradation of Spt16 and gene association of Paf1, but not the incorporation of centromeric histone, Cse4, into the active genes in Δsan1 . Collectively, our results demonstrate distinct functions of a nuclear protein quality control factor in regulating the genome-wide PIC formation and elongating Pol II (and hence transcription), thus unraveling new gene regulatory mechanisms.

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