scholarly journals Stress Induces Changes in the Phosphorylation of Trypanosoma cruzi RNA Polymerase II, Affecting Its Association with Chromatin and RNA Processing

2014 ◽  
Vol 13 (7) ◽  
pp. 855-865 ◽  
Author(s):  
Antônio Augusto Rocha ◽  
Nilmar Silvio Moretti ◽  
Sergio Schenkman
Keyword(s):  
Heat Shock ◽  
Trypanosoma Cruzi ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Transcription Control ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Content Type ◽  
Polycistronic Transcription

ABSTRACT The phosphorylation of the carboxy-terminal heptapeptide repeats of the largest subunit of RNA polymerase II (Pol II) controls several transcription-related events in eukaryotes. Trypanosomatids lack these typical repeats and display an unusual transcription control. RNA Pol II associates with the transcription site of the spliced leader (SL) RNA, which is used in the trans -splicing of all mRNAs transcribed on long polycistronic units. We found that Trypanosoma cruzi RNA Pol II associated with chromatin is highly phosphorylated. When transcription is inhibited by actinomycin D, the enzyme runs off from SL genes, remaining hyperphosphorylated and associated with polycistronic transcription units. Upon heat shock, the enzyme is dephosphorylated and remains associated with the chromatin. Transcription is partially inhibited with the accumulation of housekeeping precursor mRNAs, except for heat shock genes. DNA damage caused dephosphorylation and transcription arrest, with RNA Pol II dissociating from chromatin although staying at the SL. In the presence of calyculin A, the hyperphosphorylated form detached from chromatin, including the SL loci. These results indicate that in trypanosomes, the unusual RNA Pol II is phosphorylated during the transcription of SL and polycistronic operons. Different types of stresses modify its phosphorylation state, affecting pre-RNA processing.

scholarly journals Mechanism of RNA polymerase II stalling by DNA alkylation

2017 ◽  
Vol 114 (46) ◽  
pp. 12172-12177 ◽  
Author(s):  
Stefano Malvezzi ◽  
Lucas Farnung ◽  
Claudia M. N. Aloisi ◽  
Todor Angelov ◽  
Patrick Cramer ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Anticancer Agents ◽  
Excision Repair ◽  
Minor Groove ◽  
Dna Alkylation ◽  
Drug Induced ◽  
Rna Pol Ii ◽  
Pol Ii

Several anticancer agents that form DNA adducts in the minor groove interfere with DNA replication and transcription to induce apoptosis. Therapeutic resistance can occur, however, when cells are proficient in the removal of drug-induced damage. Acylfulvenes are a class of experimental anticancer agents with a unique repair profile suggesting their capacity to stall RNA polymerase (Pol) II and trigger transcription-coupled nucleotide excision repair. Here we show how different forms of DNA alkylation impair transcription by RNA Pol II in cells and with the isolated enzyme and unravel a mode of RNA Pol II stalling that is due to alkylation of DNA in the minor groove. We incorporated a model for acylfulvene adducts, the stable 3-deaza-3-methoxynaphtylethyl-adenosine analog (3d-Napht-A), and smaller 3-deaza-adenosine analogs, into DNA oligonucleotides to assess RNA Pol II transcription elongation in vitro. RNA Pol II was strongly blocked by a 3d-Napht-A analog but bypassed smaller analogs. Crystal structure analysis revealed that a DNA base containing 3d-Napht-A can occupy the +1 templating position and impair closing of the trigger loop in the Pol II active center and polymerase translocation into the next template position. These results show how RNA Pol II copes with minor-groove DNA alkylation and establishes a mechanism for drug resistance.

scholarly journals Structure of GPN-Loop GTPase Npa3 and Implications for RNA Polymerase II Assembly

2015 ◽  
Vol 36 (5) ◽  
pp. 820-831 ◽  
Author(s):  
Jürgen Niesser ◽  
Felix R. Wagner ◽  
Dirk Kostrewa ◽  
Wolfgang Mühlbacher ◽  
Patrick Cramer
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Protein Complexes ◽  
Hydrophobic Pocket ◽  
Pol Ii ◽  
Content Type ◽  
Open Conformation ◽  
Peptide Release ◽  
Hydrophobic Peptide

Biogenesis of the 12-subunit RNA polymerase II (Pol II) transcription complex requires so-called GPN-loop GTPases, but the function of these enzymes is unknown. Here we report the first crystal structure of a eukaryotic GPN-loop GTPase, theSaccharomyces cerevisiaeenzyme Npa3 (a homolog of human GPN1, also called RPAP4, XAB1, and MBDin), and analyze its catalytic mechanism. The enzyme was trapped in a GDP-bound closed conformation and in a novel GTP analog-bound open conformation displaying a conserved hydrophobic pocket distant from the active site. We show that Npa3 has chaperone activity and interacts with hydrophobic peptide regions of Pol II subunits that form interfaces in the assembled Pol II complex. Biochemical results are consistent with a model that the hydrophobic pocket binds peptides and that this can allosterically stimulate GTPase activity and subsequent peptide release. These results suggest that GPN-loop GTPases are assembly chaperones for Pol II and other protein complexes.

scholarly journals Increased processing of SINE B2 non coding RNAs unveils a novel type of transcriptome de-regulation underlying amyloid beta neuro-pathology

2020 ◽  
Author(s):  
Yubo Cheng ◽  
Babita Gollen ◽  
Luke Saville ◽  
Christopher Isaac ◽  
Jogender Mehla ◽  
...  
Keyword(s):  
Stress Response ◽  
Rna Polymerase Ii ◽  
Amyloid Beta ◽  
Transcriptional Activation ◽  
Rna Processing ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Amyloid Toxicity ◽  
B2 Rna ◽  
Non Coding Rnas

ABSTRACTMore than 97% of the mammalian genome is non-protein coding, and repetitive elements account for more than 50% of noncoding space. However, the functional importance of many non-coding RNAs generated by these elements and their connection with pathologic processes remains elusive. We have previously shown that B2 RNAs, a class of non-coding RNAs that belong to the B2 family of SINE repeats, mediate the transcriptional activation of stress response genes (SRGs) upon application of a stimulus. Notably, B2 RNAs bind RNA Polymerase II (RNA Pol II) and suppress SRG transcription during pro-stimulation state. Upon application of a stimulus, B2 RNAs are processed into fragments and degraded, which in turn releases RNA Pol II from suppression and upregulates SRGs. Here, we demonstrate a novel role for B2 RNAs in transcriptome response to amyloid beta toxicity and pathology in the mouse hippocampus. In healthy hippocampi, activation of SRGs is followed by a transient upregulation of pro-apoptotic factors, such as p53 and miRNA-34c, which target SRGs creating a negative feedback loop that facilitates transition to the pro-stimulation state. Using an integrative RNA genomics approach, we show that in mouse hippocampi of an amyloid precursor protein knock-in mouse model and in an in vitro cell culture model of amyloid beta toxicity, this regulatory loop is dysfunctional due to increased levels of B2 RNA processing, constitutively elevated SRG expression and high p53 levels. Evidence indicates that Hsf1, a master regulator of stress response, mediates B2 RNA processing in cells, and is upregulated during amyloid toxicity accelerating the processing of SINE RNAs and SRG hyper-activation. Our study reveals that in mouse, SINE RNAs constitute a novel pathway deregulated in amyloid beta pathology, with potential implications for similar cases in the human brain, such as Alzheimer’s disease (AD). This data attributes a role to SINE RNA processing in a pathological process as well as a new function to Hsf1 that is independent of its transcription factor activity.

scholarly journals Development, embryonic genome activity and mitochondrial characteristics of bovine–pig inter-family nuclear transfer embryos

Reproduction ◽  
2010 ◽  
Vol 140 (2) ◽  
pp. 273-285 ◽  
Author(s):  
Irina Lagutina ◽  
Helena Fulka ◽  
Tiziana A L Brevini ◽  
Stefania Antonini ◽  
Dario Brunetti ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Transfer ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Pol I ◽  
Embryonic Genome ◽  
Genome Activity

The best results of inter-species somatic cell nuclear transfer (iSCNT) in mammals were obtained using closely related species that can hybridise naturally. However, in the last years, many reports describing blastocyst development following iSCNT between species with distant taxonomical relations (inter-classes, inter-order and inter-family) have been published. This indicates that embryonic genome activation (EGA) in xeno-cytoplasm is possible, albeit very rarely. Using a bovine–pig (inter-family) iSCNT model, we studied the basic characteristics of EGA: expression and activity of RNA polymerase II (RNA Pol II), formation of nucleoli (as an indicator of RNA polymerase I (RNA Pol I) activity), expression of the key pluripotency gene NANOG and alteration of mitochondrial mass. In control embryos (obtained by IVF or iSCNT), EGA was characterised by RNA Pol II accumulation and massive production of poly-adenylated transcripts (detected with oligo dT probes) in blastomere nuclei, and formation of nucleoli as a result of RNA Pol I activity. Conversely, iSCNT embryos were characterised by the absence of accumulation and low activity of RNA Pol II and inability to form active mature nucleoli. Moreover, in iSCNT embryos, NANOG was not expressed, and mitochondria mass was significantly lower than in intra-species embryos. Finally, the complete developmental block at the 16–25-cell stage for pig–bovine iSCNT embryos and at the four-cell stage for bovine–pig iSCNT embryos strongly suggests that EGA is not taking place in iSCNT embryos. Thus, our experiments clearly demonstrate poor nucleus–cytoplasm compatibility between these animal species.

scholarly journals A role for the RNA pol II–associated PAF complex in AID-induced immune diversification

2012 ◽  
Vol 209 (11) ◽  
pp. 2099-2111 ◽  
Author(s):  
Katharina L. Willmann ◽  
Sara Milosevic ◽  
Siim Pauklin ◽  
Kerstin-Maike Schmitz ◽  
Gopinath Rangam ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Protein Complexes ◽  
Chromatin Modification ◽  
Cytosine Deamination ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Antibody Diversification ◽  
Dna Instability ◽  
Paf Complex

Antibody diversification requires the DNA deaminase AID to induce DNA instability at immunoglobulin (Ig) loci upon B cell stimulation. For efficient cytosine deamination, AID requires single-stranded DNA and needs to gain access to Ig loci, with RNA pol II transcription possibly providing both aspects. To understand these mechanisms, we isolated and characterized endogenous AID-containing protein complexes from the chromatin of diversifying B cells. The majority of proteins associated with AID belonged to RNA polymerase II elongation and chromatin modification complexes. Besides the two core polymerase subunits, members of the PAF complex, SUPT5H, SUPT6H, and FACT complex associated with AID. We show that AID associates with RNA polymerase-associated factor 1 (PAF1) through its N-terminal domain, that depletion of PAF complex members inhibits AID-induced immune diversification, and that the PAF complex can serve as a binding platform for AID on chromatin. A model is emerging of how RNA polymerase II elongation and pausing induce and resolve AID lesions.

scholarly journals HIV-1 Tat-associated RNA polymerase C-terminal domain kinase, CDK2, phosphorylates CDK7 and stimulates Tat-mediated transcription

2002 ◽  
Vol 364 (3) ◽  
pp. 649-657 ◽  
Author(s):  
Sergei NEKHAI ◽  
Meisheng ZHOU ◽  
Anne FERNANDEZ ◽  
William S. LANE ◽  
Ned J.C. LAMB ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Nuclear Extract ◽  
Viral Gene ◽  
Transcriptional Elongation ◽  
Rna Pol Ii ◽  
Hela Nuclear Extract ◽  
Pol Ii ◽  
Terminal Domain ◽  
Hiv 1

HIV-1 gene expression is regulated by a viral transactivator protein (Tat) which induces transcriptional elongation of HIV-1 long tandem repeat (LTR). This induction requires hyperphosphorylation of the C-terminal domain (CTD) repeats of RNA polymerase II (Pol II). To achieve CTD hyperphosphorylation, Tat stimulates CTD kinases associated with general transcription factors of the promoter complex, specifically TFIIH-associated CDK7 and positive transcription factor b-associated CDK9 (cyclin-dependent kinase 9). Other studies indicate that Tat may bind an additional CTD kinase that regulates the target-specific phosphorylation of RNA Pol II CTD. We previously reported that Tat-associated T-cell-derived kinase (TTK), purified from human primary T-cells, stimulates Tat-dependent transcription of HIV-1 LTR in vivo [Nekhai, Shukla, Fernandez, Kumar and Lamb (2000) Virology 266, 246–256]. In the work presented here, we characterized the components of TTK by biochemical fractionation and the function of TTK in transcription assays in vitro. TTK uniquely co-purified with CDK2 and not with either CDK9 or CDK7. Tat induced the TTK-associated CDK2 kinase to phosphorylate CTD, specifically at Ser-2 residues. The TTK fraction restored Tat-mediated transcription activation of HIV-1 LTR in a HeLa nuclear extract immunodepleted of CDK9, but not in the HeLa nuclear extract double-depleted of CDK9 and CDK7. Direct microinjection of the TTK fraction augmented Tat transactivation of HIV-1 LTR in human primary HS68 fibroblasts. The results argue that TTK-associated CDK2 may function to maintain target-specific phosphorylation of RNA Pol II that is essential for Tat transactivation of HIV-1 promoter. They are also consistent with the observed cell-cycle-specific induction of viral gene transactivation.

scholarly journals The Werner Syndrome Protein Is Involved in RNA Polymerase II Transcription

1999 ◽  
Vol 10 (8) ◽  
pp. 2655-2668 ◽  
Author(s):  
Adayabalam S. Balajee ◽  
Amrita Machwe ◽  
Alfred May ◽  
Matthew D. Gray ◽  
Junko Oshima ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Lines ◽  
Werner Syndrome ◽  
Molecular Defect ◽  
Rna Pol Ii ◽  
Vitro Assay ◽  
Pol Ii ◽  
Cell Extracts

Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)–dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40–60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II–dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid–protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype.

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 Emerging Views on the CTD Code

2012 ◽  
Vol 2012 ◽  
pp. 1-19 ◽  
Author(s):  
David W. Zhang ◽  
Juan B. Rodríguez-Molina ◽  
Joshua R. Tietjen ◽  
Corey M. Nemec ◽  
Aseem Z. Ansari
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Regulation ◽  
Chromatin Remodeling ◽  
Posttranslational Modifications ◽  
Rna Processing ◽  
Transcription Initiation ◽  
Protein Complexes ◽  
Pol Ii

The C-terminal domain (CTD) of RNA polymerase II (Pol II) consists of conserved heptapeptide repeats that function as a binding platform for different protein complexes involved in transcription, RNA processing, export, and chromatin remodeling. The CTD repeats are subject to sequential waves of posttranslational modifications during specific stages of the transcription cycle. These patterned modifications have led to the postulation of the “CTD code” hypothesis, where stage-specific patterns define a spatiotemporal code that is recognized by the appropriate interacting partners. Here, we highlight the role of CTD modifications in directing transcription initiation, elongation, and termination. We examine the major readers, writers, and erasers of the CTD code and examine the relevance of describing patterns of posttranslational modifications as a “code.” Finally, we discuss major questions regarding the function of the newly discovered CTD modifications and the fundamental insights into transcription regulation that will necessarily emerge upon addressing those challenges.

scholarly journals Heat Shock Causes a Reversible Increase in RNA Polymerase II Occupancy Downstream of mRNA Genes, Consistent with a Global Loss in Transcriptional Termination

2018 ◽  
Vol 38 (18) ◽  
Author(s):  
Joseph F. Cardiello ◽  
James A. Goodrich ◽  
Jennifer F. Kugel
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Repression ◽  
Normal Growth ◽  
Global Changes ◽  
Transcriptional Initiation ◽  
Pol Ii ◽  
Transcriptional Termination ◽  
Number Of Genes

ABSTRACT Cellular transcriptional programs are tightly controlled but can profoundly change in response to environmental challenges or stress. Here we describe global changes in mammalian RNA polymerase II (Pol II) occupancy at mRNA genes in response to heat shock and after recovery from the stress. After a short heat shock, Pol II occupancy across thousands of genes decreased, consistent with widespread transcriptional repression, whereas Pol II occupancy increased at a small number of genes in a manner consistent with activation. Most striking, however, was loss of the Pol II peak near the 3′ ends of mRNA genes, coupled to a gain in polymerase occupancy extending tens of kilobases downstream of 3′ ends. Typical patterns of 3′ end occupancy were largely restored 60 min after cells returned to normal growth temperatures. These changes in polymerase occupancy revealed a heat shock-induced loss of normal termination, which was potent, global, and reversible. The occupancy of the termination factor CPSF73 at the 3′ ends of representative genes was reduced after heat shock, suggesting a mechanism for impaired termination. The data support a model in which heat shock induces widespread repression of transcriptional initiation and loss of transcription termination, which reverses as cells return to homeostasis.

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