scholarly journals RNA polymerase II condensate formation and association with Cajal and histone locus bodies in living human cells

2020 ◽  
Author(s):  
Takashi Imada ◽  
Takeshi Shimi ◽  
Ai Kaiho ◽  
Yasushi Saeki ◽  
Hiroshi Kimura
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Clusters ◽  
Nuclear Bodies ◽  
Histone Genes ◽  
Small Nuclear Rna ◽  
Pol Ii ◽  
Histone Locus Bodies ◽  
Dynamic Structures ◽  
Histone Locus

ABSTRACTIn eukaryotic nuclei, a number of phase-separated nuclear bodies (NBs) are present. RNA polymerase II (Pol II) is the main player in transcription and forms large condensates in addition to localizing at numerous transcription foci. Cajal bodies (CBs) and histone locus bodies (HLBs) are NBs that are involved in transcriptional and post-transcriptional regulation of small nuclear RNA and histone genes. By live-cell imaging using human HCT116 cells, we here show that Pol II condensates (PCs) nucleated near CBs and HLBs, and the number of PCs increased during S phase concomitantly with the activation period of histone genes. Ternary PC–CB– HLB associates were formed via three pathways: nucleation of PCs and HLBs near CBs, interaction between preformed PC–HLBs with CBs, and nucleation of PCs near preformed CB– HLBs. Coilin knockout increased the co-localization rate between PCs and HLBs, whereas the number, nucleation timing, and phosphorylation status of PCs remained unchanged. Depletion of PCs did not affect CBs and HLBs. Treatment with 1,6-hexanediol revealed that PCs were more liquid-like than CBs and HLBs. Thus, PCs are dynamic structures often nucleated following the activation of gene clusters associated with other NBs. (187 words)

scholarly journals Proximal sequence element-binding transcription factor (PTF) is a multisubunit complex required for transcription of both RNA polymerase II- and RNA polymerase III-dependent small nuclear RNA genes.

1995 ◽  
Vol 15 (4) ◽  
pp. 2019-2027 ◽  
Author(s):  
J B Yoon ◽  
S Murphy ◽  
L Bai ◽  
Z Wang ◽  
R G Roeder
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Small Nuclear Rna ◽  
Class Iii ◽  
Sequence Element ◽  
Pol Ii ◽  
Proximal Sequence Element ◽  
Nuclear Rna ◽  
Class Iii Genes ◽  
Pol Iii

The proximal sequence element (PSE), found in both RNA polymerase II (Pol II)- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes, is specifically bound by the PSE-binding transcription factor (PTF). We have purified PTF to near homogeneity from HeLa cell extracts by using a combination of conventional and affinity chromatographic methods. Purified PTF is composed of four polypeptides with apparent molecular masses of 180, 55, 45, and 44 kDa. A combination of preparative electrophoretic mobility shift and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses has conclusively identified these four polypeptides as subunits of human PTF, while UV cross-linking experiments demonstrate that the largest subunit of PTF is in close contact with the PSE. The purified PTF activates transcription from promoters of both Pol II- and Pol III-transcribed snRNA genes in a PSE-dependent manner. In addition, we have investigated factor requirements in transcription of Pol III-dependent snRNA genes. We show that in extracts that have been depleted of TATA-binding protein (TBP) and associated factors, recombinant TBP restores transcription from U6 and 7SK promoters but not from the VAI promoter, whereas the highly purified TBP-TBP-associated factor complex TFIIIB restores transcription from the VAI but not the U6 or 7SK promoter. Furthermore, by complementation of heat-treated extracts lacking TFIIIC activity, we show that TFIIIC1 is required for transcription of both the 7SK and VAI genes, whereas TFIIIC2 is required only for transcription of the VAI gene. From these observations, we conclude (i) that PTF and TFIIIC2 function as gene-specific as gene-specific factors for PSE-and B-box-containing Pol III genes, respectively, (ii) that the form of TBP used by class III genes with upstream promoter elements differs from the from used by class III genes with internal promoters, and (iii) that TFIIIC1 is required for both internal and external Pol III promoters.

scholarly journals Creb-Binding Protein (Cbp/P300) and RNA Polymerase II Colocalize in Transcriptionally Active Domains in the Nucleus

2000 ◽  
Vol 150 (1) ◽  
pp. 265-274 ◽  
Author(s):  
Anna von Mikecz ◽  
Suisheng Zhang ◽  
Marc Montminy ◽  
Eng M. Tan ◽  
Peter Hemmerich
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Active Sites ◽  
Spatial Organization ◽  
Binding Protein ◽  
Nuclear Bodies ◽  
Laser Scanning Microscopy ◽  
Creb Binding Protein ◽  
Pol Ii ◽  
Pml Bodies

The spatial organization of transcription- associated proteins is an important control mechanism of eukaryotic gene expression. Here we analyzed the nuclear distribution of the transcriptional coactivators CREB-binding protein (CBP)/p300 in situ by confocal laser scanning microscopy, and in vivo complex formation by coimmunoprecipitation. A subpopulation of CBP and p300 is targeted to active sites of transcription and partially colocalizes with hyper- and hypophosphorylated RNA polymerase II (pol II) in discrete regions of variable size throughout the nucleus. However, the coactivators were found in tight association with hypophosphorylated, but not hyperphosphorylated pol II. Transcriptional inhibition induced a relocation of CBP/p300 and pol II into speckles. Moreover, double and triple immunofluorescence analyses revealed the presence of CBP, p300, and pol II in a subset of promyelocytic leukemia (PML) bodies. Our results provide evidence for a dynamic spacial link between coactivators of transcription and the basal transcription machinery in discrete nuclear domains dependent upon the transcriptional activity of the cell. The identification of pol II in CBP/PML-containing nuclear bodies supports the idea that transcription takes place at PML bodies.

Role of the C-terminal domain of RNA polymerase II in expression of small nuclear RNA genes

2008 ◽  
Vol 36 (3) ◽  
pp. 537-539 ◽  
Author(s):  
Sylvain Egloff ◽  
Shona Murphy
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Repeat Unit ◽  
Small Nuclear Rna ◽  
Protein Coding ◽  
Pol Ii ◽  
Terminal Domain ◽  
Covalent Modifications

Pol II (RNA polymerase II) transcribes the genes encoding proteins and non-coding snRNAs (small nuclear RNAs). The largest subunit of Pol II contains a distinctive CTD (C-terminal domain) comprising a repetitive heptad amino acid sequence, Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. This domain is now known to play a major role in the processes of transcription and co-transcriptional RNA processing in expression of both snRNA and protein-coding genes. The heptapeptide repeat unit can be extensively modified in vivo and covalent modifications of the CTD during the transcription cycle result in the ordered recruitment of RNA-processing factors. The most studied modifications are the phosphorylation of the serine residues in position 2 and 5 (Ser2 and Ser5), which play an important role in the co-transcriptional processing of both mRNA and snRNA. An additional, recently identified CTD modification, phosphorylation of the serine residue in position 7 (Ser7) of the heptapeptide, is however specifically required for expression of snRNA genes. These findings provide interesting insights into the control of gene-specific Pol II function.

scholarly journals Coiled Bodies and U2 snRNA Genes Adjacent to Coiled Bodies Are Enriched in Factors Required for snRNA Transcription

1998 ◽  
Vol 9 (5) ◽  
pp. 1025-1036 ◽  
Author(s):  
Wouter Schul ◽  
Roel van Driel ◽  
Luitzen de Jong
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Gene Clusters ◽  
Small Nuclear Rna ◽  
U2 Snrna ◽  
Coiled Bodies ◽  
High Concentrations ◽  
Tata Box Binding Protein ◽  
Nuclear Domains

A significant percentage of the gene clusters that contain the human genes for U1 small nuclear RNA (snRNA) or for U2 snRNA have been found associated with small nuclear domains, known as coiled bodies. We show here, by immunofluorescent labeling of human cells, that coiled bodies are enriched in factors required for the transcription of these snRNA genes. The 45-kDa γ-subunit of the transcription factor, proximal element sequence-binding transcription factor (PTF), which is specific for the snRNA genes, was found in high concentrations in coiled bodies, along with the general transcription factor TATA-box binding protein and a subset of RNA polymerase II. We show that the transcription factors and RNA polymerase II are concentrated in irregularly shaped domains that not only overlap with coiled bodies but also extend to their immediate surroundings. Fluorescent in situ hybridization showed that these domains can overlap with U2 snRNA genes adjacent to coiled bodies. In addition, we found the domains to contain newly synthesized RNA, visualized by 5-bromo-uridine triphosphate labeling. Our data suggest that coiled bodies are involved in the expression of snRNA genes, which leads us to propose the model that coiled bodies are associated with snRNA genes to facilitate and regulate their transcription. These findings point to a general principle of higher order organization of gene expression in the nucleus.

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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