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.

A transcriptionally active tRNA gene interferes with nucleosome positioning in vivo

1992 ◽  
Vol 12 (9) ◽  
pp. 4015-4025
Author(s):  
R H Morse ◽  
S Y Roth ◽  
R T Simpson
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
Nucleosome Positioning ◽  
Yeast Cells ◽  
Trna Genes ◽  
Start Site ◽  
Cis Acting

Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo [R. T. Simpson, Nature (London) 343:387-389, 1990]. Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells. We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected. A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element. These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids. Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes. Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted. This finding demonstrates that the transcriptional competence of the tRNA gene determined its ability to override a nucleosome positioning signal in vivo and establishes that a hierarchy exists between cis-acting elements and nucleosome positioning signals.

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 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).

Gene insulation. Part I: natural strategies in yeast and Drosophila

2010 ◽  
Vol 88 (6) ◽  
pp. 875-884 ◽  
Author(s):  
Michèle Amouyal
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Rna Polymerase Iii ◽  
Eukaryotic Cells ◽  
Trna Genes ◽  
Initiation Complex ◽  
Heterochromatin Formation ◽  
Transcription Initiation Complex

This review in two parts deals with the increasing number of processes known to be used by eukaryotic cells to protect gene expression from undesired genomic enhancer or chromatin effects, by means of the so-called insulators or barriers. The most advanced studies in this expanding field concern yeasts and Drosophila (this article) and the vertebrates (next article in this issue). Clearly, the cell makes use of every gene context to find the appropriate, economic, solution. Thus, besides the elements formerly identified and specifically dedicated to insulation, a number of unexpected elements are diverted from their usual function to structure the genome and enhancer action or to prevent heterochromatin spreading. They are, for instance, genes actively transcribed by RNA polymerase II or III, partial elements of these transcriptional machineries (stalled RNA polymerase II, normally required by genes that must respond quickly to stimuli, or TFIIIC bound at its B-box, normally required by RNA polymerase III for assembly of the transcription initiation complex at tRNA genes), or genomic sequences occupied by variants of standard histones, which, being rapidly and permanently replaced, impede heterochromatin formation.

scholarly journals A transcriptionally active tRNA gene interferes with nucleosome positioning in vivo.

1992 ◽  
Vol 12 (9) ◽  
pp. 4015-4025 ◽  
Author(s):  
R H Morse ◽  
S Y Roth ◽  
R T Simpson
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Trna Gene ◽  
Rna Polymerase Iii ◽  
Nucleosome Positioning ◽  
Yeast Cells ◽  
Trna Genes ◽  
Start Site ◽  
Cis Acting

Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo [R. T. Simpson, Nature (London) 343:387-389, 1990]. Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells. We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected. A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element. These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids. Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes. Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted. This finding demonstrates that the transcriptional competence of the tRNA gene determined its ability to override a nucleosome positioning signal in vivo and establishes that a hierarchy exists between cis-acting elements and nucleosome positioning signals.

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 RNA polymerase II pausing as a context-dependent reader of the genome

2016 ◽  
Vol 94 (1) ◽  
pp. 82-92 ◽  
Author(s):  
Adam Scheidegger ◽  
Sergei Nechaev
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cell Types ◽  
Gene Promoters ◽  
Cell Type ◽  
Pol Ii ◽  
Cell Type Specific ◽  
Pol Ii Pausing ◽  
Context Dependent ◽  
Specific Regulation

The RNA polymerase II (Pol II) transcribes all mRNA genes in eukaryotes and is among the most highly regulated enzymes in the cell. The classic model of mRNA gene regulation involves recruitment of the RNA polymerase to gene promoters in response to environmental signals. Higher eukaryotes have an additional ability to generate multiple cell types. This extra level of regulation enables each cell to interpret the same genome by committing to one of the many possible transcription programs and executing it in a precise and robust manner. Whereas multiple mechanisms are implicated in cell type-specific transcriptional regulation, how one genome can give rise to distinct transcriptional programs and what mechanisms activate and maintain the appropriate program in each cell remains unclear. This review focuses on the process of promoter-proximal Pol II pausing during early transcription elongation as a key step in context-dependent interpretation of the metazoan genome. We highlight aspects of promoter-proximal Pol II pausing, including its interplay with epigenetic mechanisms, that may enable cell type-specific regulation, and emphasize some of the pertinent questions that remain unanswered and open for investigation.

scholarly journals Lack of gene- and strand-specific DNA repair in RNA polymerase III-transcribed human tRNA genes.

1997 ◽  
Vol 17 (1) ◽  
pp. 219-229 ◽  
Author(s):  
R Dammann ◽  
G P Pfeifer
Keyword(s):  
Dna Repair ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Human Fibroblasts ◽  
Rna Polymerase Iii ◽  
Dna Lesions ◽  
Rrna Genes ◽  
Trna Genes ◽  
Polymerase Iii ◽  
Flanking Sequences

UV light induces DNA lesions which are removed by nucleotide excision repair. Genes transcribed by RNA polymerase II are repaired faster than the flanking chromatin, and the transcribed strand is repaired faster than the coding strand. Transcription-coupled repair is not seen in RNA polymerase I-transcribed human rRNA genes. Since repair of genes transcribed by RNA polymerase III has not been analyzed before, we investigated DNA repair of tRNA genes after irradiation of human fibroblasts with UVC. We studied the repair of UV-induced cyclobutane pyrimidine dimers at nucleotide resolution by ligation-mediated PCR. A single-copy gene encoding selenocysteine tRNA, a tRNA valine gene, and their flanking sequences were analyzed. Protein-DNA footprinting showed that both genes were occupied by regulatory factors in vivo, and Northern blotting and nuclear run-on analysis of the tRNA indicated that these genes were actively transcribed. We found that both genes were repaired slower than RNA polymerase II-transcribed genes. No major difference between repair of the transcribed and the coding DNA strands was detected. Transcribed sequences of the tRNA genes were not repaired faster than flanking sequences. Indeed, several sequence positions in the 5' flanking region of the tRNA(Val) gene were repaired more efficiently than the gene itself. These results indicate that unlike RNA polymerase II, RNA polymerase III has no stimulatory effect on DNA repair. Since tRNA genes are covered by the regulatory factor TFIIIC and RNA polymerase III, these proteins may actually inhibit the DNA's accessibility to repair enzymes.

scholarly journals Disruption of cardiac Med1 inhibits RNA polymerase II promoter occupancy and promotes chromatin remodeling

2019 ◽  
Vol 316 (2) ◽  
pp. H314-H325 ◽  
Author(s):  
Duane D. Hall ◽  
Kathryn M. Spitler ◽  
Chad E. Grueter
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Immunoprecipitation ◽  
Chromatin Accessibility ◽  
Gene Promoters ◽  
Specific Expression ◽  
Pol Ii ◽  
Transcriptional Start Sites

The Mediator coactivator complex directs gene-specific expression by binding distal enhancer-bound transcription factors through its Med1 subunit while bridging to RNA polymerase II (Pol II) at gene promoters. In addition, Mediator scaffolds epigenetic modifying enzymes that determine local DNA accessibility. Previously, we found that deletion of Med1 in cardiomyocytes deregulates more than 5,000 genes and promotes acute heart failure. Therefore, we hypothesized that Med1 deficiency disrupts enhancer-promoter coupling. Using chromatin immunoprecipitation-coupled deep sequencing (ChIP-seq; n = 3/ChIP assay), we found that the Pol II pausing index is increased in Med1 knockout versus floxed control mouse hearts primarily due to a decrease in Pol II occupancy at the majority of transcriptional start sites without a corresponding increase in elongating species. Parallel ChIP-seq assays reveal that Med1-dependent gene expression correlates strongly with histone H3 K27 acetylation, which is indicative of open and active chromatin at transcriptional start sites, whereas H3 K27 trimethylated levels, representing condensed and repressed DNA, are broadly increased and inversely correlate with absolute expression levels. Furthermore, Med1 deletion leads to dynamic changes in acetyl-K27 associated superenhancer regions and their enriched transcription factor-binding motifs that are consistent with altered gene expression. Our findings suggest that Med1 is important in establishing enhancer-promoter coupling in the heart and supports the proposed role of Mediator in establishing preinitiation complex formation. We also found that Med1 determines chromatin accessibility within genes and enhancer regions and propose that the composition of transcription factors associated with superenhancer changes to direct gene-specific expression. NEW & NOTEWORTHY Based on our previous findings that transcriptional homeostasis and cardiac function are disturbed by cardiomyocyte deletion of the Mediator coactivator Med1 subunit, we investigated potential underlying changes in RNA polymerase II localization and global chromatin accessibility. Using chromatin immunoprecipitation sequencing, we found that disrupted transcription arises from a deficit in RNA polymerase II recruitment to gene promoters. Furthermore, active versus repressive chromatin marks are redistributed within gene loci and at enhancer regions correlated with gene expression changes.

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.

Sign in / Sign up

Export Citation Format

Share Document