RNA polymerase II large subunit is cleaved by caspases during DNA damage-induced apoptosis

2002 ◽  
Vol 296 (4) ◽  
pp. 954-961 ◽  
Author(s):  
Yi Lu ◽  
Zhonghui Luo ◽  
David B Bregman
Keyword(s):  
Dna Damage ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Subunit ◽  
Induced Apoptosis

scholarly journals GSK-3 is an RNA polymerase II phospho-CTD kinase

2020 ◽  
Vol 48 (11) ◽  
pp. 6068-6080 ◽  
Author(s):  
Nicolás Nieto Moreno ◽  
Florencia Villafañez ◽  
Luciana E Giono ◽  
Carmen Cuenca ◽  
Gastón Soria ◽  
...  
Keyword(s):  
Dna Damage ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Glycogen Synthase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Transcriptional Elongation ◽  
Induced Apoptosis ◽  
Carboxy Terminal

Abstract We have previously found that UV-induced DNA damage causes hyperphosphorylation of the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), inhibition of transcriptional elongation and changes in alternative splicing (AS) due to kinetic coupling between transcription and splicing. In an unbiased search for protein kinases involved in the AS response to DNA damage, we have identified glycogen synthase kinase 3 (GSK-3) as an unforeseen participant. Unlike Cdk9 inhibition, GSK-3 inhibition only prevents CTD hyperphosphorylation triggered by UV but not basal phosphorylation. This effect is not due to differential degradation of the phospho-CTD isoforms and can be reproduced, at the AS level, by overexpression of a kinase-dead GSK-3 dominant negative mutant. GSK-3 inhibition abrogates both the reduction in RNAPII elongation and changes in AS elicited by UV. We show that GSK-3 phosphorylates the CTD in vitro, but preferentially when the substrate is previously phosphorylated, consistently with the requirement of a priming phosphorylation reported for GSK-3 efficacy. In line with a role for GSK-3 in the response to DNA damage, GSK-3 inhibition prevents UV-induced apoptosis. In summary, we uncover a novel role for a widely studied kinase in key steps of eukaryotic transcription and pre-mRNA processing.

scholarly journals Budding Yeast CTDK-I Is Required for DNA Damage-Induced Transcription

2003 ◽  
Vol 2 (2) ◽  
pp. 274-283 ◽  
Author(s):  
Denis Ostapenko ◽  
Mark J. Solomon
Keyword(s):  
Dna Damage ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Scale ◽  
Large Subunit ◽  
Growth Conditions ◽  
Dna Damaging Agents ◽  
Irradiation Treatment ◽  
Mutant Cells

ABSTRACT CTDK-I phosphorylates the C-terminal domain (CTD) of the large subunit of yeast RNA polymerase II in a reaction that stimulates transcription elongation. Mutations in CTDK-I subunits—Ctk1p, Ctk2p, and Ctk3p—confer conditional phenotypes. In this study, we examined the role of CTDK-I in the DNA damage response. We found that mutation of individual CTDK-I subunits rendered yeast sensitive to hydroxyurea (HU) and UV irradiation. Treatment with DNA-damaging agents increased phosphorylation of Ser2 within the CTD repeats in wild-type but not in ctk1Δ mutant cells. Using microarray hybridization, we identified genes whose transcription following DNA damage is Ctk1p dependent, including several DNA repair and stress response genes. Following HU treatment, the level of Ser2-phosphorylated RNA polymerase II increased both globally and on the CTDK-I-regulated genes. The pleiotropic phenotypes of ctk mutants suggest that CTDK-I activity is essential during large-scale transcriptional repatterning under stress and unfavorable growth conditions.

scholarly journals Ibulocydine Is a Novel Prodrug Cdk Inhibitor That Effectively Induces Apoptosis in Hepatocellular Carcinoma Cells

2011 ◽  
Vol 286 (22) ◽  
pp. 19662-19671 ◽  
Author(s):  
Seung-Ju Cho ◽  
Young-Jong Kim ◽  
Young-Joon Surh ◽  
B. Moon Kim ◽  
Seung-Ki Lee
Keyword(s):  
Hepatocellular Carcinoma ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Subunit ◽  
Cancer Drug ◽  
Cdk Inhibitors ◽  
Cdk Inhibitor ◽  
Anti Cancer ◽  
Induced Apoptosis ◽  
Hcc Cells

Hepatocellular carcinoma (HCC) is frequently associated with abnormalities in cell cycle regulation, leading to increased activity of cyclin-dependent kinases (Cdks) due to the loss, or low expression of, Cdk inhibitors. In this study, we showed that ibulocydine (an isobutyrate prodrug of the specific Cdk inhibitor, BMK-Y101) is a candidate anti-cancer drug for HCC. Ibulocydine has high activity against Cdk7/cyclin H/Mat1 and Cdk9/cyclin T. Ibulocydine inhibited the growth of HCC cells more effectively than other Cdk inhibitors, including olomoucine and roscovitine, whereas ibulocydine as well as the other Cdk inhibitors and BMK-Y101 minimally influenced the growth of normal hepatocyte cells. Ibulocydine induced apoptosis in HCC cells, most likely by inhibiting Cdk7 and Cdk9. In vitro treatment of HCC cells with ibulocydine rapidly blocked phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II, a process mediated by Cdk7/9. Anti-apoptotic gene products such as Mcl-1, survivin, and X-linked IAP (XIAP) are crucial for the survival of many cell types, including HCC. Following the inhibition of RNA polymerase II phosphorylation, ibulocydine caused rapid down-regulation of Mcl-1, survivin, and XIAP, thus inducing apoptosis. Furthermore, ibulocydine effectively induced apoptosis in HCC xenografts with no toxic side effects. These results suggest that ibulocydine is a strong candidate anti-cancer drug for the treatment of HCC.

scholarly journals Rsp5 Ubiquitin-Protein Ligase Mediates DNA Damage-Induced Degradation of the Large Subunit of RNA Polymerase II in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (10) ◽  
pp. 6972-6979 ◽  
Author(s):  
Sylvie L. Beaudenon ◽  
Maria R. Huacani ◽  
Guangli Wang ◽  
Donald P. McDonnell ◽  
Jon M. Huibregtse
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Damage ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Subunit ◽  
26S Proteasome ◽  
Regulatory Subunit ◽  
Ubiquitin Protein Ligase

ABSTRACT Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 proteins. We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation ofSEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW domain-containing hect (WW-hect) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.

scholarly journals Wwp2-Mediated Ubiquitination of the RNA Polymerase II Large Subunit in Mouse Embryonic Pluripotent Stem Cells

2007 ◽  
Vol 27 (15) ◽  
pp. 5296-5305 ◽  
Author(s):  
Hui Li ◽  
Zhihong Zhang ◽  
Beibei Wang ◽  
Junmei Zhang ◽  
Yingming Zhao ◽  
...  
Keyword(s):  
Dna Damage ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammalian Cells ◽  
Large Subunit ◽  
Terminal Domain ◽  
Ubiquitin E3 Ligase ◽  
Lysine Residues

ABSTRACT Ubiquitination and the degradation of the large subunit of RNA polymerase II, Rpb1, is not only involved in DNA damage-induced arrest but also in other transcription-obstructing events. However, the ubiquitin ligases responsible for DNA damage-independent processes in mammalian cells remain to be identified. Here, we identified Wwp2, a mouse HECT domain ubiquitin E3 ligase, as a novel ubiquitin ligase of Rpb1. We found that Wwp2 specifically interacted with mouse Rpb1 and targeted it for ubiquitination both in vitro and in vivo. Interestingly, the interaction with and ubiquitination of Rpb1 was dependent neither on its phosphorylation state nor on DNA damage. However, the enzymatic activity of Wwp2 was absolutely required for its ubiquitin modification of Rpb1. Furthermore, our study indicates that the interaction between Wwp2 and Rpb1 was mediated through WW domain of Wwp2 and C-terminal domain of Rpb1, respectively. Strikingly, downregulation of Wwp2 expression compromised Rpb1 ubiquitination and elevated its intracellular steady-state protein level significantly. Importantly, we identified six lysine residues in the C-terminal domain of Rpb1 as ubiquitin acceptor sites mediated by Wwp2. These results indicate that Wwp2 plays an important role in regulating expression of Rpb1 in normal physiological conditions.

scholarly journals Sub1 Globally Regulates RNA Polymerase II C-Terminal Domain Phosphorylation

2010 ◽  
Vol 30 (21) ◽  
pp. 5180-5193 ◽  
Author(s):  
Alicia García ◽  
Emanuel Rosonina ◽  
James L. Manley ◽  
Olga Calvo
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Large Subunit ◽  
Terminal Domain ◽  
Mrna Metabolism ◽  
Genes Encoding ◽  
The One ◽  
Ctd Phosphorylation ◽  
Transcription Cycle

ABSTRACT The transcriptional coactivator Sub1 has been implicated in several aspects of mRNA metabolism in yeast, such as activation of transcription, termination, and 3′-end formation. Here, we present evidence that Sub1 plays a significant role in controlling phosphorylation of the RNA polymerase II large subunit C-terminal domain (CTD). We show that SUB1 genetically interacts with the genes encoding all four known CTD kinases, SRB10, KIN28, BUR1, and CTK1, suggesting that Sub1 acts to influence CTD phosphorylation at more than one step of the transcription cycle. To address this directly, we first used in vitro kinase assays, and we show that, on the one hand, SUB1 deletion increased CTD phosphorylation by Kin28, Bur1, and Ctk1 but, on the other, it decreased CTD phosphorylation by Srb10. Second, chromatin immunoprecipitation assays revealed that SUB1 deletion decreased Srb10 chromatin association on the inducible GAL1 gene but increased Kin28 and Ctk1 chromatin association on actively transcribed genes. Taken together, our data point to multiple roles for Sub1 in the regulation of CTD phosphorylation throughout the transcription cycle.

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 Multi-gene phylogeny and taxonomy of Amauroderma s. lat. (Ganodermataceae)

2020 ◽  
Vol 44 (1) ◽  
pp. 206-239 ◽  
Author(s):  
Y.-F. Sun ◽  
D.H. Costa-Rezende ◽  
J.-H. Xing ◽  
J.-L. Zhou ◽  
B. Zhang ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Phylogenetic Analyses ◽  
Large Subunit ◽  
Elongation Factor ◽  
Morphological Examination ◽  
Translation Elongation ◽  
Multiple Loci ◽  
Phylogenetic Studies ◽  
Molecular Phylogenetic

Amauroderma s.lat. has been defined mainly by the morphological features of non-truncate and double-walled basidiospores with a distinctly ornamented endospore wall. In this work, taxonomic and phylogenetic studies on species of Amauroderma s.lat. are carried out by morphological examination together with ultrastructural observations, and molecular phylogenetic analyses of multiple loci including the internal transcribed spacer regions (ITS), the large subunit of nuclear ribosomal RNA gene (nLSU), the largest subunit of RNA polymerase II (RPB1) and the second largest subunit of RNA polymerase II (RPB2), the translation elongation factor 1-α gene (TEF) and the β-tubulin gene (TUB). The results demonstrate that species of Ganodermataceae formed ten clades. Species previously placed in Amauroderma s.lat. are divided into four clades: Amauroderma s.str., Foraminispora, Furtadoa and a new genus Sanguinoderma. The classification of Amauroderma s. lat. is thus revised, six new species are described and illustrated, and eight new combinations are proposed. SEM micrographs of basidiospores of Foraminispora and Sanguinoderma are provided, and the importance of SEM in delimitation of taxa in this study is briefly discussed. Keys to species of Amauroderma s.str., Foraminispora, Furtadoa, and Sanguinoderma are also provided.

scholarly journals Human Cyclin K, a Novel RNA Polymerase II-Associated Cyclin Possessing Both Carboxy-Terminal Domain Kinase and Cdk-Activating Kinase Activity

1998 ◽  
Vol 18 (7) ◽  
pp. 4291-4300 ◽  
Author(s):  
Michael C. Edwards ◽  
Calvin Wong ◽  
Stephen J. Elledge
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Cycle Progression ◽  
Large Subunit ◽  
Terminal Domain ◽  
Gene Coding ◽  
Acid Protein ◽  
Rnap Ii ◽  
Carboxy Terminal

ABSTRACT The gene coding for human cyclin K was isolated as aCPR (cell-cycle progression restoration) gene by virtue of its ability to impart a Far− phenotype to the budding yeast Saccharomyces cerevisiae and to rescue the lethality of a deletion of the G1 cyclin genes CLN1,CLN2, and CLN3. The cyclin K gene encodes a 357-amino-acid protein most closely related to human cyclins C and H, which have been proposed to play a role in regulating basal transcription through their association with and activation of cyclin-dependent kinases (Cdks) that phosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II). Murine and Drosophila melanogaster homologs of cyclin K have also been identified. Cyclin K mRNA is ubiquitously expressed in adult mouse and human tissues, but is most abundant in the developing germ cells of the adult testis and ovaries. Cyclin K is associated with potent CTD kinase and Cdk kinase (CAK) activity in vitro and coimmunoprecipitates with the large subunit of RNAP II. Thus, cyclin K represents a new member of the “transcription” cyclin family which may play a dual role in regulating Cdk and RNAP II activity.

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