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 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.

scholarly journals The Selenocysteine tRNA Gene in Leishmania major Is Transcribed by both RNA Polymerase II and RNA Polymerase III

2014 ◽  
Vol 14 (3) ◽  
pp. 216-227 ◽  
Author(s):  
Norma E. Padilla-Mejía ◽  
Luis E. Florencio-Martínez ◽  
Rodrigo Moreno-Campos ◽  
Juan C. Vizuet-de-Rueda ◽  
Ana M. Cevallos ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Leishmania Major ◽  
Single Gene ◽  
Rna Polymerase Iii ◽  
Transcriptional Analysis ◽  
Pol Ii ◽  
Content Type ◽  
Polymerase Iii ◽  
Pol Iii

ABSTRACT Eukaryotic tRNAs, transcribed by RNA polymerase III (Pol III), contain boxes A and B as internal promoter elements. One exception is the selenocysteine (Sec) tRNA (tRNA-Sec), whose transcription is directed by an internal box B and three extragenic sequences in vertebrates. Here we report on the transcriptional analysis of the tRNA-Sec gene in the protozoan parasite Leishmania major . This organism has unusual mechanisms of gene expression, including Pol II polycistronic transcription and maturation of mRNAs by trans splicing, a process that attaches a 39-nucleotide miniexon to the 5′ end of all the mRNAs. In L. major , tRNA-Sec is encoded by a single gene inserted into a Pol II polycistronic unit, in contrast to most tRNAs, which are clustered at the boundaries of polycistronic units. 5′ rapid amplification of cDNA ends and reverse transcription-PCR experiments showed that some tRNA-Sec transcripts contain the miniexon at the 5′ end and a poly(A) tail at the 3′ end, indicating that the tRNA-Sec gene is polycistronically transcribed by Pol II and processed by trans splicing and polyadenylation, as was recently reported for the tRNA-Sec genes in the related parasite Trypanosoma brucei . However, nuclear run-on assays with RNA polymerase inhibitors and with cells that were previously UV irradiated showed that the tRNA-Sec gene in L. major is also transcribed by Pol III. Thus, our results indicate that RNA polymerase specificity in Leishmania is not absolute in vivo , as has recently been found in other eukaryotes.

scholarly journals Diversity in the signals required for nuclear accumulation of U snRNPs and variety in the pathways of nuclear transport.

1991 ◽  
Vol 113 (4) ◽  
pp. 705-714 ◽  
Author(s):  
U Fischer ◽  
E Darzynkiewicz ◽  
S M Tahara ◽  
N A Dathan ◽  
R Lührmann ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Nuclear Accumulation ◽  
Nuclear Targeting ◽  
Stringent Requirement ◽  
Pol Ii ◽  
Nuclear Localization Signals ◽  
U6 Snrna

The requirements for nuclear targeting of a number of U snRNAs have been studied by analyzing the behavior of in vitro-generated transcripts after microinjection into the cytoplasm of Xenopus oocytes. Like the previously studied U1 snRNA, U2 snRNA is excluded from the nucleus when it does not have the 2,2,7mGpppN cap structure typical of the RNA polymerase II (pol II)-transcribed U snRNAs. Surprisingly, two other pol II-transcribed U snRNAs, U4 and U5, have a much less stringent requirement for the trimethyl cap structure. The gamma-monomethyl triphosphate cap structure of the RNA polymerase III-transcribed U6 snRNA, on the other hand, is shown not to play a role in nuclear targeting. Wheat germ agglutinin, which is known to prevent the import of many proteins into the nucleus, inhibits nuclear uptake of U6, but not of U1 or U5 snRNAs. Conversely, a 2,2,7mGpppG dinucleotide analogue of the trimethyl cap structure inhibits transport of the pol II U snRNAs, but does not detectably affect the transport of either U6 snRNA or a karyophilic protein. From these results it can be deduced that U6 enters the nucleus by a pathway similar or identical to that used by karyophilic proteins. The composite nuclear localization signals of the trimethyl cap-containing U snRNPs, however, do not function in the same way as previously defined nuclear targeting signals.

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 Induced RPB1 depletion reveals a direct gene-specific control of RNA Polymerase III function by RNA Polymerase II

2019 ◽  
Author(s):  
Alan Gerber ◽  
Keiichi Ito ◽  
Chi-Shuen Chu ◽  
Robert G. Roeder
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Polymerase Iii ◽  
Serum Starvation ◽  
Trna Genes ◽  
Large Set ◽  
Specific Expression ◽  
Pol Ii ◽  
Direct Gene ◽  
Specific Regulation

SummaryIncreasing evidence suggests that tRNA levels are dynamically and specifically regulated in response to internal and external cues to modulate the cellular translational program. However, the molecular players and the mechanisms regulating the gene-specific expression of tRNAs are still unknown. Using an inducible auxin-degron system to rapidly deplete RPB1 (the largest subunit of RNA Pol II) in living cells, we identified Pol II as a direct gene-specific regulator of tRNA transcription. Our data suggest that Pol II transcription robustly interferes with Pol III function at specific tRNA genes. This activity was further found to be essential for MAF1-mediated repression of a large set of tRNA genes during serum starvation, indicating that repression of tRNA genes by Pol II is dynamically regulated. Hence, Pol II plays a direct and central role in the gene-specific regulation of tRNA expression.

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 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 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 of the TFIIIC subcomplex τA provides insights into RNA polymerase III pre-initiation complex formation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Matthias K. Vorländer ◽  
Anna Jungblut ◽  
Kai Karius ◽  
Florence Baudin ◽  
Helga Grötsch ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Dual Function ◽  
Start Site ◽  
Transcription Start ◽  
Initiation Complex ◽  
Pol Ii ◽  
Negative Stain ◽  
Pol Iii

Abstract Transcription factor (TF) IIIC is a conserved eukaryotic six-subunit protein complex with dual function. It serves as a general TF for most RNA polymerase (Pol) III genes by recruiting TFIIIB, but it is also involved in chromatin organization and regulation of Pol II genes through interaction with CTCF and condensin II. Here, we report the structure of the S. cerevisiae TFIIIC subcomplex τA, which contains the most conserved subunits of TFIIIC and is responsible for recruitment of TFIIIB and transcription start site (TSS) selection at Pol III genes. We show that τA binding to its promoter is auto-inhibited by a disordered acidic tail of subunit τ95. We further provide a negative-stain reconstruction of τA bound to the TFIIIB subunits Brf1 and TBP. This shows that a ruler element in τA achieves positioning of TFIIIB upstream of the TSS, and suggests remodeling of the complex during assembly of TFIIIB by TFIIIC.

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