scholarly journals Yeast PAF1 complex restricts the accumulation of RNA polymerase III and counters the replication stress on the transcribed genes

2018 ◽  
Author(s):  
Pratibha Bhalla ◽  
Dipti Vernekar ◽  
Ashutosh Shukla ◽  
Benoit Gilquin ◽  
Yohann Couté ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Associated Factors ◽  
Rna Polymerase Iii ◽  
Replication Stress ◽  
Regulatory Proteins ◽  
Pol Ii ◽  
Adverse Conditions ◽  
Pol I ◽  
Paf1 Complex ◽  
Pol Iii

AbstractMany regulatory proteins and complexes influence transcription by RNA polymerase (pol) II. In comparison, only a few regulatory proteins are known for pol III, which transcribes mostly house-keeping and non-coding genes. Yet, pol III transcription is precisely regulated under various stress conditions like starvation. We used pol III transcription complex components TFIIIC (Tfc6), pol III (Rpc128) and TFIIIB (Brf1) as baits to identify potential interactors through mass spectrometry-based proteomics. A large interactome constituting known chromatin modifiers, factors and regulators of transcription by pol I and pol II revealed the possibility of a large number of signaling cues for pol III transcription against adverse conditions. We found one of the pol II-associated factors, Paf1 complex (PAF1C) interacts with the three baits. Its occupancy on the pol III-transcribed genes is low and not correlated with pol III occupancy. Paf1 deletion leads to higher occupancy of pol III, γ-H2A and DNA pol2 but no change in nucleosome positions. Genotoxins exposure causes pol III but not Paf1 loss from the genes. PAF1C promotes the pol III pausing and restricts its accumulation on the genes, which reduces the replication stress caused by the pol III barrier and transcription-replication conflict on these highly transcribed genes.

scholarly journals Functional mRNA can be generated by RNA polymerase III.

1995 ◽  
Vol 15 (7) ◽  
pp. 3597-3607 ◽  
Author(s):  
S Gunnery ◽  
M B Mathews
Keyword(s):  
Human Immunodeficiency Virus ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Chloramphenicol Acetyltransferase ◽  
Pol Ii ◽  
Pol I ◽  
Immunodeficiency Virus ◽  
Pol Iii

Eukaryotic cellular mRNA is believed to be synthesized exclusively by RNA polymerase II (pol II), whereas pol I produces long rRNAs and pol III produces 5S rRNA, tRNA, and other small RNAs. To determine whether this functional differentiation is obligatory, we examined the translational potential of an artificial pol III transcript. The coding region of the human immunodeficiency virus type 1 tat gene was placed under the control of a strong pol III promoter from the adenovirus type 2 VA RNAI gene. The resultant chimera, pVA-Tat, was transcribed accurately in vivo and in vitro and gave rise to Tat protein, which transactivated a human immunodeficiency virus-driven chloramphenicol acetyltransferase reporter construct in transfected HeLa cells. pol III-specific mutations down-regulated VA-Tat RNA production in vivo and in vitro and dramatically reduced chloramphenicol acetyltransferase transactivation. As expected for a pol III transcript, VA-Tat RNA was not detectably capped at its 5' end or polyadenylated at its 3' end, but, like mRNA, it was associated with polysomes in a salt-stable manner. Mutational analysis of a short open reading frame upstream of the Tat-coding sequence implicates scanning in the initiation of VA-Tat RNA translation despite the absence of a cap. In comparison with tat mRNA generated by pol II, VA-Tat RNA was present on smaller polysomes and was apparently translated less efficiently, which is consistent with a relatively low initiation rate. Evidently, human cells are capable of utilizing pol III transcripts as functional mRNAs, and neither a cap nor a poly(A) tail is essential for translation, although they may be stimulatory. These findings raise the possibility that some cellular mRNAs are made by pol I or pol III.

scholarly journals Structural insights into RNA polymerase III-mediated transcription termination through trapping poly-deoxythymidine

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Haifeng Hou ◽  
Yan Li ◽  
Mo Wang ◽  
Aijun Liu ◽  
Zishuo Yu ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Termination ◽  
Transcription Elongation ◽  
Rna Polymerase Iii ◽  
Pol Ii ◽  
Template Strand ◽  
Pol I ◽  
Exit Tunnel ◽  
Pol Iii ◽  
Dna Template

AbstractTermination of the RNA polymerase III (Pol III)-mediated transcription requires the conversion of an elongation complex (EC) to a pre-termination complex (PTC) on poly-deoxythymidine (dT)-containing non-template strand, a mechanism distinct from Pol I and Pol II. Here, our in vitro transcription elongation assay showed that 5-7 dT-containing DNA template led to transcription termination of Pol III, but not Pol I or Pol II. We assembled human Pol III PTC on a 7 dT-containing DNA template and determined the structure at 3.6 Å resolution. The structure reveals that poly-dT are trapped in a narrow exit tunnel formed by RPC2. A hydrophobic gate of the exit tunnel separates the bases of two connected deoxythymidines and may prevent translocation of the non-template strand. The fork loop 2 stabilizes both template and non-template strands around the transcription fork, and may further prevent strand translocation. Our study shows that the Pol III-specific exit tunnel and FL2 allow for efficient translocation of non-poly-dT sequence during transcription elongation but trap poly-dT to promote DNA retention of Pol III, revealing molecular mechanism of poly-dT-dependent transcription termination of Pol III.

scholarly journals RNA polymerase I (Pol I) passage through nucleosomes depends on Pol I subunits binding its lobe structure

2020 ◽  
Vol 295 (15) ◽  
pp. 4782-4795 ◽  
Author(s):  
Philipp E. Merkl ◽  
Michael Pilsl ◽  
Tobias Fremter ◽  
Katrin Schwank ◽  
Christoph Engel ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Dna Templates ◽  
Pol Ii ◽  
Naked Dna ◽  
Pol I ◽  
Intrinsic Capacity ◽  
Pol Iii ◽  
Polymerase I

RNA polymerase I (Pol I) is a highly efficient enzyme specialized in synthesizing most ribosomal RNAs. After nucleosome deposition at each round of rDNA replication, the Pol I transcription machinery has to deal with nucleosomal barriers. It has been suggested that Pol I–associated factors facilitate chromatin transcription, but it is unknown whether Pol I has an intrinsic capacity to transcribe through nucleosomes. Here, we used in vitro transcription assays to study purified WT and mutant Pol I variants from the yeast Saccharomyces cerevisiae and compare their abilities to pass a nucleosomal barrier with those of yeast Pol II and Pol III. Under identical conditions, purified Pol I and Pol III, but not Pol II, could transcribe nucleosomal templates. Pol I mutants lacking either the heterodimeric subunit Rpa34.5/Rpa49 or the C-terminal part of the specific subunit Rpa12.2 showed a lower processivity on naked DNA templates, which was even more reduced in the presence of a nucleosome. Our findings suggest that the lobe-binding subunits Rpa34.5/Rpa49 and Rpa12.2 facilitate passage through nucleosomes, suggesting possible cooperation among these subunits. We discuss the contribution of Pol I–specific subunit domains to efficient Pol I passage through nucleosomes in the context of transcription rate and processivity.

scholarly journals Manipulation of RNA Polymerase III by Herpes Simplex Virus-1

2021 ◽  
Author(s):  
Sarah E Dremel ◽  
Frances L Sivrich ◽  
Jessica M Tucker ◽  
Britt A Glaunsinger ◽  
Neal A DeLuca
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Rna Polymerase ◽  
Viral Infection ◽  
Rna Polymerase Iii ◽  
Herpes Simplex Virus 1 ◽  
Pol Ii ◽  
Simplex Virus ◽  
Pol Iii ◽  
Hsv 1

RNA Polymerase III (Pol III) transcribes noncoding RNA, including transfer RNA (tRNA), and acts as a pathogen sensor during the innate immune response. To promote enhanced proliferation, the Pol III machinery is commonly targeted during cancer and viral infection. Herein we employ DM-RNA-Seq, 4SU-Seq, ChIP-Seq, and ATAC-Seq to characterize how Herpes Simplex Virus-1 (HSV-1) perturbs the Pol III landscape. We find that HSV-1 stimulates tRNA expression 10-fold, with mature tRNAs exhibiting a 2-fold increase within 12 hours of infection. Perturbation of host tRNA synthesis requires nuclear viral entry, but not synthesis of specific viral transcripts, nascent viral genomes, or viral progeny. Host tRNA with a specific codon bias were not targeted, rather increased transcription was observed from euchromatic, actively transcribed loci. tRNA upregulation is linked to unique crosstalk between the Pol II and III transcriptional machinery. While viral infection is known to mediate host transcriptional shut off and lead to a depletion of Pol II on host mRNA promoters, we find that Pol II binding to tRNA loci increases. Finally, we report Pol III and associated factors bind the HSV genome, which suggests a previously unrecognized role in HSV-1 gene expression. These data provide insight into novel mechanisms by which HSV-1 alters the host nuclear environment, shifting key processes in favor of the pathogen.

scholarly journals Engineered mini H1 promoters with dedicated RNA Polymerase II or III activity

2020 ◽  
pp. jbc.RA120.015386
Author(s):  
Zongliang Gao ◽  
Yme Ubeles van der Velden ◽  
Minghui Fan ◽  
Cynthia Alyssa van der Linden ◽  
Monique Vink ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Polymerase Activity ◽  
Attractive Alternative ◽  
Pol Ii ◽  
Guide Rnas ◽  
Non Coding Rna ◽  
Pol Iii ◽  
Short Hairpin Rnas

RNA polymerase III promoters such as 7SK, U6 and H1 are widely used for the expression of small non-coding RNAs, including short hairpin RNAs for RNAi experiments and guide RNAs for CRISPR-mediated genome editing. We previously reported dual RNA polymerase activity (Pol II/III) for the human H1 promoter and demonstrated that this promiscuous RNA polymerase use can be exploited for the simultaneous expression of both a non-coding RNA and an mRNA. However, this combination is not a desired feature in other experimental and therapeutic settings. To overcome this limitation of the H1 promoter we engineered a miniature H1/7SK hybrid promoter with minimal Pol II activity, thereby boosting the Pol III activity to a level that is higher than that of either parental promoter. In parallel, we also engineered small Pol II-specific H1 promoter variants and explored their use as general Pol II promoters for protein expression. The newly engineered promoter variants form an attractive alternative to the commonly-used H1 promoter in terms of activity and small promoter size, but also concerning safety by exclusive expression of the desired therapeutic transcript (either Pol II or Pol III, but not both).

scholarly journals Modulation of Yeast Genome Expression in Response to Defective RNA Polymerase III-Dependent Transcription

2005 ◽  
Vol 25 (19) ◽  
pp. 8631-8642 ◽  
Author(s):  
Christine Conesa ◽  
Roberta Ruotolo ◽  
Pascal Soularue ◽  
Tiffany A. Simms ◽  
David Donze ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Gene Dosage ◽  
Rna Polymerase Iii ◽  
Yeast Cells ◽  
Yeast Genome ◽  
Altered Expression ◽  
Pol Ii ◽  
Initiator Trna ◽  
Genome Expression ◽  
Pol Iii

ABSTRACT We used genome-wide expression analysis in Saccharomyces cerevisiae to explore whether and how the expression of protein-coding, RNA polymerase (Pol) II-transcribed genes is influenced by a decrease in RNA Pol III-dependent transcription. The Pol II transcriptome was characterized in four thermosensitive, slow-growth mutants affected in different components of the RNA Pol III transcription machinery. Unexpectedly, we found only a modest correlation between altered expression of Pol II-transcribed genes and their proximity to class III genes, a result also confirmed by the analysis of single tRNA gene deletants. Instead, the transcriptome of all of the four mutants was characterized by increased expression of genes known to be under the control of the Gcn4p transcriptional activator. Indeed, GCN4 was found to be translationally induced in the mutants, and deleting the GCN4 gene eliminated the response. The Gcn4p-dependent expression changes did not require the Gcn2 protein kinase and could be specifically counteracted by an increased gene dosage of initiator tRNAMet. Initiator tRNAMet depletion thus triggers a GCN4-dependent reprogramming of genome expression in response to decreased Pol III transcription. Such an effect might represent a key element in the coordinated transcriptional response of yeast cells to environmental changes.

scholarly journals RNA Polymerase I Transcription Silences Noncoding RNAs at the Ribosomal DNA Locus in Saccharomyces cerevisiae

2009 ◽  
Vol 9 (2) ◽  
pp. 325-335 ◽  
Author(s):  
Elisa Cesarini ◽  
Francesca Romana Mariotti ◽  
Francesco Cioci ◽  
Giorgio Camilloni
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Ribosomal Dna ◽  
Rna Polymerase Iii ◽  
Noncoding Rnas ◽  
Rdna Locus ◽  
Rna Polymerase I ◽  
Pol Ii ◽  
Pol I ◽  
Polymerase I

ABSTRACT In Saccharomyces cerevisiae the repeated units of the ribosomal locus, transcribed by RNA polymerase I (Pol I), are interrupted by nontranscribed spacers (NTSs). These NTS regions are transcribed by RNA polymerase III to synthesize 5S RNA and by RNA polymerase II (Pol II) to synthesize, at low levels, noncoding RNAs (ncRNAs). While transcription of both RNA polymerase I and III is highly characterized, at the ribosomal DNA (rDNA) locus only a few studies have been performed on Pol II, whose repression correlates with the SIR2-dependent silencing. The involvement of both chromatin organization and Pol I transcription has been proposed, and peculiar chromatin structures might justify “ribosomal” Pol II silencing. Reporter genes inserted within the rDNA units have been employed for these studies. We studied, in the natural context, yeast mutants differing in Pol I transcription in order to find whether correlations exist between Pol I transcription and Pol II ncRNA production. Here, we demonstrate that silencing at the rDNA locus represses ncRNAs with a strength inversely proportional to Pol I transcription. Moreover, localized regions of histone hyperacetylation appear in cryptic promoter elements when Pol II is active and in the coding region when Pol I is functional; in addition, DNA topoisomerase I site-specific activity follows RNA polymerase I transcription. The repression of ncRNAs at the rDNA locus, in response to RNA polymerase I transcription, could represent a physiological circuit control whose mechanism involves modification of histone acetylation.

scholarly journals Defective RNA Polymerase III is negatively regulated by the SUMO-Ubiquitin-Cdc48 Pathway

2018 ◽  
Author(s):  
Zheng Wang ◽  
Catherine Wu ◽  
Aaron Aslanian ◽  
John R. Yates ◽  
Tony Hunter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Neurodegenerative Disease ◽  
Regulatory Mechanism ◽  
Rna Polymerase Iii ◽  
Post Translational Modification ◽  
Pol Ii ◽  
Polymerase Iii ◽  
Defective Rna ◽  
Pol Iii

ABSTRACTTranscription by RNA polymerase III (Pol III) is an essential cellular process, and mutations in Pol III can cause neurodegenerative disease in humans. However, in contrast to Pol II transcription, which has been extensively studied, the knowledge of how Pol III is regulated is very limited. We report here that in budding yeast, Saccharomyces cerevisiae, Pol III is negatively regulated by the Small Ubiquitin-like MOdifier (SUMO), an essential post-translational modification pathway. Besides sumoylation, Pol III is also targeted by ubiquitylation and the Cdc48/p97 segregase, the three of which likely act in a sequential manner and eventually lead to proteasomal degradation of Pol III subunits, thereby repressing Pol III transcription. This study not only uncovered a regulatory mechanism for Pol III, but also suggests that the SUMO and ubiquitin modification pathways and the Cdc48/p97 segregase can be potential therapeutic targets for Pol III-related human diseases.

Small nuclear RNA genes transcribed by either RNA polymerase II or RNA polymerase III in monocot plants share three promoter elements and use a strategy to regulate gene expression different from that used by their dicot plant counterparts

1994 ◽  
Vol 14 (9) ◽  
pp. 5910-5919
Author(s):  
S Connelly ◽  
C Marshallsay ◽  
D Leader ◽  
J W Brown ◽  
W Filipowicz
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Gene Promoters ◽  
Small Nuclear Rna ◽  
Promoter Elements ◽  
Pol Ii ◽  
Snrna Gene ◽  
Nuclear Rna ◽  
Pol Iii

RNA polymerase (Pol) II- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes of dicotyledonous plants contain two essential upstream promoter elements, the USE and TATA. The USE is a highly conserved plant snRNA gene-specific element, and its distance from the -30 TATA box, corresponding to approximately three and four helical DNA turns in Pol III and Pol II genes, respectively, is crucial for determining RNA Pol specificity of transcription. Sequences upstream of the USE play no role in snRNA gene transcription in dicot plants. Here we show that for expression of snRNA genes in maize, a monocotyledonous plant, the USE and TATA elements are essential, but not sufficient, for transcription. Efficient expression of both Pol II- and Pol III-specific snRNA genes in transfected maize protoplasts requires an additional element(s) positioned upstream of the USE. This element, named MSP (for monocot-specific promoter; consensus, RGCCCR), is present in one to three copies in monocot snRNA genes and is interchangeable between Pol II- and Pol III-specific genes. The efficiency of snRNA gene expression in maize protoplast is determined primarily by the strength of the MSP element(s); this contrasts with the situation in protoplasts of a dicot plant, Nicotiana plumbaginifolia, where promoter strength is a function of the quality of the USE element. Interestingly, the organization of monocot Pol III-specific snRNA gene promoters closely resembles those of equivalent vertebrate promoters. The data are discussed in the context of the coevolution of Pol II- and Pol III-specific snRNA gene promoters within many eukaryotic organisms.

scholarly journals Structure-Function Analysis of the Human TFIIB-Related Factor II Protein Reveals an Essential Role for the C-Terminal Domain in RNA Polymerase III Transcription

2005 ◽  
Vol 25 (21) ◽  
pp. 9406-9418 ◽  
Author(s):  
Ashish Saxena ◽  
Beicong Ma ◽  
Laura Schramm ◽  
Nouria Hernandez
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Function Analysis ◽  
Rna Polymerase Iii ◽  
Related Factor ◽  
Pol Ii ◽  
Terminal Domain ◽  
U6 Promoter ◽  
Pol Iii ◽  
Type 3

ABSTRACT The transcription factors TFIIB, Brf1, and Brf2 share related N-terminal zinc ribbon and core domains. TFIIB bridges RNA polymerase II (Pol II) with the promoter-bound preinitiation complex, whereas Brf1 and Brf2 are involved, as part of activities also containing TBP and Bdp1 and referred to here as Brf1-TFIIIB and Brf2-TFIIIB, in the recruitment of Pol III. Brf1-TFIIIB recruits Pol III to type 1 and 2 promoters and Brf2-TFIIIB to type 3 promoters such as the human U6 promoter. Brf1 and Brf2 both have a C-terminal extension absent in TFIIB, but their C-terminal extensions are unrelated. In yeast Brf1, the C-terminal extension interacts with the TBP/TATA box complex and contributes to the recruitment of Bdp1. Here we have tested truncated Brf2, as well as Brf2/TFIIB chimeric proteins for U6 transcription and for assembly of U6 preinitiation complexes. Our results characterize functions of various human Brf2 domains and reveal that the C-terminal domain is required for efficient association of the protein with U6 promoter-bound TBP and SNAPc, a type 3 promoter-specific transcription factor, and for efficient recruitment of Bdp1. This in turn suggests that the C-terminal extensions in Brf1 and Brf2 are crucial to specific recruitment of Pol III over Pol II.

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