RNA helicase A acts as a bridging factor linking nuclear β-actin with RNA polymerase II

Actin, the major component of the cytoplasmic skeleton, has been shown to exist in the nucleus. Nuclear actin functions in several steps of the transcription process, including chromatin remodelling and transcription initiation and elongation. However, as a part of PICs (pre-initiation complexes), the role of actin remains to be elucidated. In the present study, we identified RHA (RNA helicase A) as an actin-interacting protein in PICs. Using immunoprecipitation and immunofluorescence techniques, we have shown that RHA associates with β-actin in the nucleus. A GST (glutathione transferase) pulldown assay using different deletion mutants revealed that the RGG (Arg-Gly-Gly) region of RHA was responsible for the interaction with β-actin, and this dominant-negative mutant reduced the recruitment of Pol II (RNA polymerase II) into PICs. Moreover, overexpression or depletion of RHA could influence the interaction of Pol II with β-actin and β-actin-involved gene transcription regulation. These results suggest that RHA acts as a bridging factor linking nuclear β-actin with Pol II.

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Subnuclear Localization of Ku Protein: Functional Association with RNA Polymerase II Elongation Sites

Molecular and Cellular Biology ◽

10.1128/mcb.22.22.8088-8099.2002 ◽

2002 ◽

Vol 22 (22) ◽

pp. 8088-8099 ◽

Cited By ~ 44

Author(s):

Xianming Mo ◽

William S. Dynan

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Genome Stability ◽

Strand Break ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Rnap Ii

ABSTRACT Ku is an abundant nuclear protein with an essential function in the repair of DNA double-strand breaks. Various observations suggest that Ku also interacts with the cellular transcription machinery, although the mechanism and significance of this interaction are not well understood. In the present study, we investigated the subnuclear distribution of Ku in normally growing human cells by using confocal microscopy, chromatin immunoprecipitation, and protein immunoprecipitation. All three approaches indicated association of Ku with RNA polymerase II (RNAP II) elongation sites. This association occurred independently of the DNA-dependent protein kinase catalytic subunit and was highly selective. There was no detectable association with the initiating isoform of RNAP II or with the general transcription initiation factors. In vitro protein-protein interaction assays demonstrated that the association of Ku with elongation proteins is mediated, in part, by a discrete C-terminal domain in the Ku80 subunit. Functional disruption of this interaction with a dominant-negative mutant inhibited transcription in vitro and in vivo and suppressed cell growth. These results suggest that association of Ku with transcription sites is important for maintenance of global transcription levels. Tethering of double-strand break repair proteins to defined subnuclear structures may also be advantageous in maintenance of genome stability.

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A Functional Interaction between the Survival Motor Neuron Complex and RNA Polymerase II

The Journal of Cell Biology ◽

10.1083/jcb.152.1.75 ◽

2001 ◽

Vol 152 (1) ◽

pp. 75-86 ◽

Cited By ~ 154

Author(s):

Livio Pellizzoni ◽

Bernard Charroux ◽

Juri Rappsilber ◽

Matthias Mann ◽

Gideon Dreyfuss

Keyword(s):

Rna Polymerase ◽

Motor Neuron ◽

Rna Polymerase Ii ◽

Rna Helicase ◽

Survival Motor Neuron ◽

Dominant Negative Mutant ◽

Pol Ii ◽

Smn Complex

The survival motor neuron (SMN) protein, the protein product of the spinal muscular atrophy (SMA) disease gene, plays a role in the assembly and regeneration of small nuclear ribonucleoproteins (snRNPs) and spliceosomes. By nanoelectrospray mass spectrometry, we identified RNA helicase A (RHA) as an SMN complex–associated protein. RHA is a DEAH box RNA helicase which binds RNA polymerase II (pol II) and reportedly functions in transcription. SMN interacts with RHA in vitro, and this interaction is impaired in mutant SMNs found in SMA patients. Coimmunoprecipitation demonstrated that the SMN complex is associated with pol II, snRNPs, and RHA in vivo. In vitro experiments suggest that RHA mediates the association of SMN with the COOH-terminal domain of pol II. Moreover, transfection of cells with a dominant negative mutant of SMN, SMNΔN27, causes accumulation of pol II, snRNPs, and RHA in nuclear structures that contain the known markers of gems and coiled bodies, and inhibits RNA pol I and pol II transcription in vivo. These findings indicate a functional as well as physical association of the SMN complex with pol II and suggest a role for the SMN complex in the assembly of the pol II transcription/processing machinery.

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

Nucleic Acids Research ◽

10.1093/nar/gkaa322 ◽

2020 ◽

Vol 48 (11) ◽

pp. 6068-6080 ◽

Cited By ~ 1

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.

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Structural Biology of RNA Polymerase II Transcription: 20 Years On

Annual Review of Cell and Developmental Biology ◽

10.1146/annurev-cellbio-042020-021954 ◽

2020 ◽

Vol 36 (1) ◽

pp. 1-34 ◽

Cited By ~ 1

Author(s):

Sara Osman ◽

Patrick Cramer

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Focal Point ◽

Dna Lesions ◽

Cellular Regulation ◽

Pol Ii ◽

Associated Proteins ◽

Rna Polymerase Ii Transcription

Gene transcription by RNA polymerase II (Pol II) is the first step in the expression of the eukaryotic genome and a focal point for cellular regulation during development, differentiation, and responses to the environment. Two decades after the determination of the structure of Pol II, the mechanisms of transcription have been elucidated with studies of Pol II complexes with nucleic acids and associated proteins. Here we provide an overview of the nearly 200 available Pol II complex structures and summarize how these structures have elucidated promoter-dependent transcription initiation, promoter-proximal pausing and release of Pol II into active elongation, and the mechanisms that Pol II uses to navigate obstacles such as nucleosomes and DNA lesions. We predict that future studies will focus on how Pol II transcription is interconnected with chromatin transitions, RNA processing, and DNA repair.

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Sir2 Silences Gene Transcription by Targeting the Transition between RNA Polymerase II Initiation and Elongation

Molecular and Cellular Biology ◽

10.1128/mcb.00019-08 ◽

2008 ◽

Vol 28 (12) ◽

pp. 3979-3994 ◽

Cited By ~ 34

Author(s):

Lu Gao ◽

David S. Gross

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Transcriptional Silencing ◽

Pol Ii ◽

Initiation Factors ◽

Lysine Deacetylase ◽

Evolutionarily Conserved ◽

Mrna Capping ◽

Capping Enzyme

ABSTRACT It is well accepted that for transcriptional silencing in budding yeast, the evolutionarily conserved lysine deacetylase Sir2, in concert with its partner proteins Sir3 and Sir4, establishes a chromatin structure that prevents RNA polymerase II (Pol II) transcription. However, the mechanism of repression remains controversial. Here, we show that the recruitment of Pol II, as well as that of the general initiation factors TBP and TFIIH, occurs unimpeded to the silent HMR a 1 and HMLα1/HMLα2 mating promoters. This, together with the fact that Pol II is Ser5 phosphorylated, implies that SIR-mediated silencing is permissive to both preinitiation complex (PIC) assembly and transcription initiation. In contrast, the occupancy of factors critical to both mRNA capping and Pol II elongation, including Cet1, Abd1, Spt5, Paf1C, and TFIIS, is virtually abolished. In agreement with this, efficiency of silencing correlates not with a restriction in Pol II promoter occupancy but with a restriction in capping enzyme recruitment. These observations pinpoint the transition between polymerase initiation and elongation as the step targeted by Sir2 and indicate that transcriptional silencing is achieved through the differential accessibility of initiation and capping/elongation factors to chromatin. We compare Sir2-mediated transcriptional silencing to a second repression mechanism, mediated by Tup1. In contrast to Sir2, Tup1 prevents TBP, Pol II, and TFIIH recruitment to the HMLα1 promoter, thereby abrogating PIC formation.

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A Nonconserved Surface of the TFIIB Zinc Ribbon Domain Plays a Direct Role in RNA Polymerase II Recruitment

Molecular and Cellular Biology ◽

10.1128/mcb.24.7.2863-2874.2004 ◽

2004 ◽

Vol 24 (7) ◽

pp. 2863-2874 ◽

Cited By ~ 17

Author(s):

Thomas C. Tubon ◽

William P. Tansey ◽

Winship Herr

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Terminal Region ◽

General Role ◽

Pol Ii ◽

Amino Terminal ◽

Zinc Ribbon

ABSTRACT The general transcription factor TFIIB is a highly conserved and essential component of the eukaryotic RNA polymerase II (pol II) transcription initiation machinery. It consists of a single polypeptide with two conserved structural domains: an amino-terminal zinc ribbon structure (TFIIBZR) and a carboxy-terminal core (TFIIBCORE). We have analyzed the role of the amino-terminal region of human TFIIB in transcription in vivo and in vitro. We identified a small nonconserved surface of the TFIIBZR that is required for pol II transcription in vivo and for different types of basal pol II transcription in vitro. Consistent with a general role in transcription, this TFIIBZR surface is directly involved in the recruitment of pol II to a TATA box-containing promoter. Curiously, although the amino-terminal human TFIIBZR domain can recruit both human pol II and yeast (Saccharomyces cerevisiae) pol II, the yeast TFIIB amino-terminal region recruits yeast pol II but not human pol II. Thus, a critical process in transcription from many different promoters—pol II recruitment—has changed in sequence specificity during eukaryotic evolution.

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The Myc Transactivation Domain Promotes Global Phosphorylation of the RNA Polymerase II Carboxy-Terminal Domain Independently of Direct DNA Binding

Molecular and Cellular Biology ◽

10.1128/mcb.01828-06 ◽

2007 ◽

Vol 27 (6) ◽

pp. 2059-2073 ◽

Cited By ~ 85

Author(s):

Victoria H. Cowling ◽

Michael D. Cole

Keyword(s):

Dna Binding ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Target Genes ◽

Dependent Manner ◽

Transactivation Domain ◽

Pol Ii ◽

Terminal Domain ◽

Ctd Phosphorylation

ABSTRACT Myc is a transcription factor which is dependent on its DNA binding domain for transcriptional regulation of target genes. Here, we report the surprising finding that Myc mutants devoid of direct DNA binding activity and Myc target gene regulation can rescue a substantial fraction of the growth defect in myc −/− fibroblasts. Expression of the Myc transactivation domain alone induces a transcription-independent elevation of the RNA polymerase II (Pol II) C-terminal domain (CTD) kinases cyclin-dependent kinase 7 (CDK7) and CDK9 and a global increase in CTD phosphorylation. The Myc transactivation domain binds to the transcription initiation sites of these promoters and stimulates TFIIH binding in an MBII-dependent manner. Expression of the Myc transactivation domain increases CDK mRNA cap methylation, polysome loading, and the rate of translation. We find that some traditional Myc transcriptional target genes are also regulated by this Myc-driven translation mechanism. We propose that Myc transactivation domain-driven RNA Pol II CTD phosphorylation has broad effects on both transcription and mRNA metabolism.

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Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction

eLife ◽

10.7554/elife.55667 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 5

Author(s):

Anand Ranjan ◽

Vu Q Nguyen ◽

Sheng Liu ◽

Jan Wisniewski ◽

Jee Min Kim ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Single Molecule ◽

Transcription Initiation ◽

General Mechanism ◽

Pol Ii ◽

Promoter Escape ◽

Genome Wide ◽

A Genome ◽

Particle Imaging

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

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RNA polymerase II clustering through CTD phase separation

10.1101/316372 ◽

2018 ◽

Cited By ~ 8

Author(s):

M. Boehning ◽

C. Dugast-Darzacq ◽

M. Rankovic ◽

A. S. Hansen ◽

T. Yu ◽

...

Keyword(s):

Phase Separation ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Low Complexity ◽

Initiation Factor ◽

Published Data ◽

Pol Ii ◽

Intrinsically Disordered ◽

Ctd Phosphorylation

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation at increasing protein concentration, with the shorter yeast CTD forming less stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters/hubs at active genes through interactions between CTDs and with activators, and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.

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