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PubMed2004

Evolution and distribution of RNA polymerase II regulatory sites from RNA polymerase III dependant mobile Alu elements.

BACKGROUND: The primate-specific Alu elements, which originated 65 million years ago, exist in over a million copies in the human genome. These elements have been involved in genome shuffling and various diseases not only through retrotransposition but also through large scale Alu-Alu mediated recombination. Only a few subfamilies of Alus are currently retropositionally active and show insertion/deletion polymorphisms with associated phenotypes. Retroposition occurs by means of RNA intermediates synthesised by a RNA polymerase III promoter residing in the A-Box and B-Box in these elements. Alus have also been shown to harbour a number of transcription factor binding sites, as well as hormone responsive elements. The distribution of Alus has been shown to be non-random in the human genome and these elements are increasingly being implicated in diverse functions such as transcription, translation, response to stress, nucleosome positioning and imprinting. RESULTS: We conducted a retrospective analysis of putative functional sites, such as the RNA pol III promoter elements, pol II regulatory elements like hormone responsive elements and ligand-activated receptor binding sites, in Alus of various evolutionary ages. We observe a progressive loss of the RNA pol III transcriptional potential with concomitant accumulation of RNA pol II regulatory sites. We also observe a significant over-representation of Alus harboring these sites in promoter regions of signaling and metabolism genes of chromosome 22, when compared to genes of information pathway components, structural and transport proteins. This difference is not so significant between functional categories in the intronic regions of the same genes. CONCLUSIONS: Our study clearly suggests that Alu elements, through retrotransposition, could distribute functional and regulatable promoter elements, which in the course of subsequent selection might be stabilized in the genome. Exaptation of regulatory elements in the preexisting genes through Alus could thus have contributed to evolution of novel regulatory networks in the primate genomes. With such a wide spectrum of regulatory sites present in Alus, it also becomes imperative to screen for variations in these sites in candidate genes, which are otherwise repeat-masked in studies pertaining to identification of predisposition markers.

Alu Elements↗
PubMed1999

Functional analysis of the Ume3p/ Srb11p-RNA polymerase II holoenzyme interaction.

The yeast C-type cyclin Ume3p/Srb11p and its cyclin-dependent kinase (Cdk) Ume5p are required for the full repression of genes involved in the stress response or meiosis. This cyclin-Cdk kinase copurifies with the RNA polymerase II holoenzyme complex, suggesting it functions through modification of the transcriptional machinery. This report describes two domains required for Ume3p-RNA Pol II holoenzyme association. One domain contains the highly conserved cyclin box that directs cyclin-Cdk interaction and requires Ume5p for holoenzyme binding. The second domain, termed HAD for holoenzyme associating domain, is located within the amino-terminal region of the cyclin and is sufficient for holoenzyme binding independent of Ume5p or the cyclin box. In addition to its role in RNA Pol II holoenzyme association, the HAD is also required for Ume3p-dependent repression in vivo. Finally, HAD mutations do not affect the ability of the Ume3p-Ume5p kinase to phosphorylate in vitro the carboxy-terminal domain (CTD) of RNA polymerase II, a reported target of cyclin C-Cdk activity. In conclusion, this study demonstrates that the association of the Ume3p to the holoenzyme is complex, involving two independent domains, both of which are required for full Ume3p-dependent repression in vivo. Furthermore, HAD-dependent repression does not appear to involve CTD phosphorylation, suggesting a different role for this domain in directing Ume3p-Ume5p activity.

Adenosine Triphosphatases↗
PubMed2003

RNA polymerase I transcribes procyclin genes and variant surface glycoprotein gene expression sites in Trypanosoma brucei.

In eukaryotes, RNA polymerase (pol) I exclusively transcribes the large rRNA gene unit (rDNA) and mRNA is synthesized by RNA pol II. The African trypanosome, Trypanosoma brucei, represents an exception to this rule. In this organism, transcription of genes encoding the variant surface glycoprotein (VSG) and the procyclins is resistant to alpha-amanitin, indicating that it is mediated by RNA pol I, while other protein-coding genes are transcribed by RNA pol II. To obtain firm proof for this concept, we generated a T. brucei cell line which exclusively expresses protein C epitope-tagged RNA pol I. Using an anti-protein C immunoaffinity matrix, we specifically depleted RNA pol I from transcriptionally active cell extracts. The depletion of RNA pol I impaired in vitro transcription initiated at the rDNA promoter, the GPEET procyclin gene promoter, and a VSG gene expression site promoter but did not affect transcription from the spliced leader (SL) RNA gene promoter. Fittingly, induction of RNA interference against the RNA pol I largest subunit in insect-form trypanosomes significantly reduced the relative transcriptional efficiency of rDNA, procyclin genes, and VSG expression sites in vivo whereas that of SL RNA, alphabeta-tubulin, and heat shock protein 70 genes was not affected. Our studies unequivocally show that T. brucei harbors a multifunctional RNA pol I which, in addition to transcribing rDNA, transcribes procyclin genes and VSG gene expression sites.

Animals↗
PubMed1998

Protein kinase C inhibits transcription from the RNA polymerase III promoter of the human c-myc gene.

The c-myc promoter has a unique characteristic showing both RNA polymerase II (pol II) and RNA polymerase III (pol III) activities. Previous studies demonstrated that activating PKC results in upregulation of c-myc expression from its pol II promoter. However, how PKC activation affects expression from the pol III promoter of the c-myc gene is not well understood. This study examines the effect of PKC on the pol III transcription from the c-myc gene by using an in vitro system. We report the inhibition of the c-myc pol III transcript by activating PKC. Further, either a phosphocellulose fraction of HeLa whole cell extract (WCE) enriched for transcription factor TF IIIB, or recombinant TATA-box binding protein could restore the inhibited c-myc pol III transcription under conditions that activate PKC. A role has been proposed for the c-myc pol III transcript in the regulation of c-myc gene expression. Therefore, this report discusses the significance of the downregulation of c-myc expression from its pol III promoter and the possible interplay between the pol II and pol III promoters of this gene.

Cell Fractionation↗
PubMed2006

Cockayne syndrome B protein regulates the transcriptional program after UV irradiation.

The phenotype of the human genetic disorder Cockayne syndrome (CS) is not only due to DNA repair defect but also (and perhaps essentially) to a severe transcription initiation defect. After UV irradiation, even undamaged genes are not transcribed in CSB cells. Indeed, neither RNA pol II nor the associated basal transcription factors are recruited to the promoters of the housekeeping genes, around of which histone H4 acetylation is also deficient. Transfection of CSB restores the recruitment process of RNA pol II. On the contrary, the p53-responsive genes do not require CSB and are transcribed in both wild-type and CSB cells upon DNA damage. Altogether, our data highlight the pivotal role of CSB in initiating the transcriptional program of certain genes after UV irradiation, and also may explain some of the complex traits of CS patients.

Cockayne Syndrome↗
PubMed2005

Structure of an RNA polymerase II-RNA inhibitor complex elucidates transcription regulation by noncoding RNAs.

The noncoding RNA B2 and the RNA aptamer FC bind RNA polymerase (Pol) II and inhibit messenger RNA transcription initiation, but not elongation. We report the crystal structure of FC(*), the central part of FC RNA, bound to Pol II. FC(*) RNA forms a double stem-loop structure in the Pol II active center cleft. B2 RNA may bind similarly, as it competes with FC(*) RNA for Pol II interaction. Both RNA inhibitors apparently prevent the downstream DNA duplex and the template single strand from entering the cleft after DNA melting and thus interfere with open-complex formation. Elongation is not inhibited, as nucleic acids prebound in the cleft would exclude the RNA inhibitors. The structure also indicates that A-form RNA could interact with Pol II similarly to a B-form DNA promoter, as suggested for the bacterial transcription inhibitor 6S RNA.

Base Sequence↗
PubMed2002

The C-terminal domain of the largest subunit of RNA polymerase II is required for stationary phase entry and functionally interacts with the Ras/PKA signaling pathway.

The Saccharomyces cerevisiae Ras proteins control cell growth by regulating the activity of the cAMP-dependent protein kinase (PKA). In this study, a genetic approach was used to identify cellular processes that were regulated by Ras/PKA signaling activity. Interestingly, we found that mutations affecting the C-terminal domain (CTD), of Rpb1p, the largest subunit of RNA polymerase II, were very sensitive to changes in Ras signaling activity. The Rpb1p CTD is a highly conserved, repetitive structure that is a key site of control during the production of a mature mRNA molecule. We found that mutations compromising the CTD were synthetically lethal with alterations that led to elevated levels of Ras/PKA signaling. Altogether, the data suggested that Ras/PKA activity was negatively regulating a protein that functioned in concert with the CTD during RNA pol II transcription. Consistent with this prediction, we found that elevated levels of Ras signaling caused growth and transcription defects that were very similar to those observed in mutants encoding an Rpb1p with a truncated CTD. In all, these data suggested that S. cerevisiae growth control and RNA pol II transcription might be coupled by using the Ras pathway to regulate CTD function.

Cyclic AMP-Dependent Protein Kinases↗
PubMed1998

Localization and characterization of the RNA binding protein TLS in skin and stratified mucosa.

Translocated in liposarcoma (TLS), a member of the Ewing's sarcoma family of RNA binding proteins, is targeted to the product of RNA POL II and functions in nuclear events as well as in nuclear-cytoplasmic transport of mRNA. It has been most extensively studied in cell lines, but was identified in several rat tissues by northern blot analysis, with adipose tissue showing the highest expression followed by whole skin. This paper describes a protein with amino acid sequence homology to TLS that was isolated from bovine tongue epithelium using an affinity column made with an antibody to the cornified envelope precursor sciellin. Using reverse transcriptase polymerase chain reaction technology and total RNA isolated from bovine tongue epithelium, a cDNA was obtained whose nucleotide sequence coded for a protein homologous to human TLS. Nuclear staining in all layers of human epidermis and bovine stratified epithelium was observed with an antibody to TLS, whereas peripheral staining of the upper layers of these tissues was observed with the antibody to sciellin. Cultured cells gave similar results; however, adult tissue required boiling in citrate buffer to unmask antigenic sites before reacting with the TLS antibody. Western blots of extracts of human and bovine keratinocytes using TLS and sciellin antibodies showed that the two proteins shared at least one epitope, but that they were different. TLS was lost from the nucleus following inhibition of RNA POL II activity and the protein was identified in CNBr extracts of purified keratinocytes cornified envelopes by western blot. These results clearly indicate that TLS functions as an RNA binding protein in keratinocytes in vivo and in vitro. Furthermore the sequestration of TLS to the cell envelope may play a role in regulating its nuclear-cytoplasmic transport and effect its role in transcription.

Amino Acid Sequence↗
PubMed2000

Specific HDV RNA-templated transcription by pol II in vitro.

RNA polymerase II is implicated in the RNA-templated RNA synthesis during replication of viroids and Hepatitis Delta Virus (HDV); however, neither the RNA template nor protein factor requirements for this process are well defined. We have developed an in vitro transcription system based on HeLa cell nuclear extract (NE), in which a segment of antigenomic RNA corresponding to the left-hand tip region of the HDV rod-like structure serves as a template for efficient and highly specific RNA synthesis. Accumulation of the unique RNA product is highly sensitive to alpha-amanitin in HeLa NE and only partially sensitive to this drug in NE from PMG cells that contain an allele of the alpha-amanitin-resistant subunit of pol II, strongly suggesting pol II involvement in this reaction. Detailed analysis of the RNA product revealed that it represents a chimeric molecule composed of a newly synthesized transcript covalently attached to the 5' half of the RNA template. Selection of the start site for transcription is remarkably specific and depends on the secondary structure of the RNA template, rather than on its primary sequence. Some features of this reaction resemble the RNA cleavage-extension process observed for pol II-arrested complexes in vitro. A possible involvement of the described reaction in HDV replication is discussed.

Amanitins↗
PubMed2005

RNA-interference-directed chromatin modification coupled to RNA polymerase II transcription.

RNA interference (RNAi) acts on long double-stranded RNAs (dsRNAs) in a variety of eukaryotes to generate small interfering RNAs that target homologous messenger RNA, resulting in their destruction. This process is widely used to 'knock-down' the expression of genes of interest to explore phenotypes. In plants, fission yeast, ciliates, flies and mammalian cells, short interfering RNAs (siRNAs) also induce DNA or chromatin modifications at the homologous genomic locus, which can result in transcriptional silencing or sequence elimination. siRNAs may direct DNA or chromatin modification by siRNA-DNA interactions at the homologous locus. Alternatively, they may act by interactions between siRNA and nascent transcript. Here we show that in fission yeast (Schizosaccharomyces pombe), chromatin modifications are only directed by RNAi if the homologous DNA sequences are transcribed. Furthermore, transcription by exogenous T7 polymerase is not sufficient. Ago1, a component of the RNAi effector RISC/RITS complex, associates with target transcripts and RNA polymerase II. Truncation of the regulatory carboxy-terminal domain (CTD) of RNA pol II disrupts transcriptional silencing, indicating that, like other RNA processing events, RNAi-directed chromatin modification is coupled to transcription.

Argonaute Proteins↗
PubMed1998

Regulation of RNA polymerase I-dependent promoters by the hepatitis B virus X protein via activated Ras and TATA-binding protein.

The hepatitis B virus (HBV) X protein is essential for viral infectivity, and evidence indicates that it is a strong contributor to HBV-mediated oncogenesis. X has been shown to transactivate a wide variety of RNA polymerase (Pol) II-dependent, as well as RNA Pol III-dependent, promoters. In this study, we have investigated the possibility that X modulates RNA Pol I-dependent rRNA transcription. In both human hepatoma Huh7 and Drosophila Schneider S2 cell lines, X expression stimulated rRNA promoter activity. Extracts prepared from X-expressing cells stably transfected with an X gene also exhibited an increased ability to transcribe the rRNA promoter. The mechanism for X transactivation was examined by determining whether this regulatory event was dependent on Ras activation and increased TATA-binding protein (TBP) levels. Our previous studies have demonstrated that X, and the activation of Ras, produces an increase in the cellular levels of TBP (H.-D. Wang, A. Trivedi, and D. L. Johnson, Mol. Cell. Biol. 17:6838-6846, 1997). Expression of a dominant negative form of Ras blocked the X-mediated induction of the rRNA promoters, whereas expression of a constitutively activated form of Ras mimicked the enhancing effect of X on rRNA promoter activity. When TBP was overexpressed in either Huh7 or S2 cells, a dose-dependent increase in rRNA promoter activity was observed. To analyze whether the increase in TBP was modulating rRNA promoter activity indirectly, by increasing activity of RNA Pol II-dependent promoters, a Drosophila TBP cDNA was constructed with a mutation that eliminated its ability to stimulate RNA Pol II-dependent promoters. Transient expression of wild-type TBP in S2 cells increased the activities of specific RNA Pol I- and Pol II-dependent promoters. Expression of the mutant TBP protein failed to enhance the activity of the RNA Pol II-dependent promoters, yet the protein completely retained its ability to stimulate the rRNA promoter. Furthermore, the addition of recombinant TBP to S2 extracts stimulated rRNA promoter activity in vitro. Together, these results demonstrate that the HBV X protein up-regulates RNA Pol I-dependent promoters via a Ras-activated pathway in two distinct cell lines. The enhanced promoter activity can, at least in part, be attributed to the X- and Ras-mediated increase in cellular TBP, a limiting transcription component.

Animals↗
PubMed2004

Silencing of antiapoptotic survivin gene by multiple approaches of RNA interference technology.

Silencing of mammalian gene expression by RNA interference (RNAi) technology can be achieved using small interfering RNA (siRNA) or short hairpin RNA (shRNA). However, the relative effectiveness of these two approaches is not known. It is also not clear whether gene-specific shRNA transcribed from an RNA polymerase II (Pol II)-directed promoter in a fusion form can disrupt the targeted gene expression. Here, we report that using both luciferase and antiapoptotic survivin genes as targets, both siRNA and shRNA approaches significantly silenced the targeted gene expression in cancer cells. We further demonstrated that shRNAs transcribed from an RNA Pol II-mediated promoter in a green fluorescent protein (GFP) fusion form at the 3'-untranslated region silenced luciferase and survivin expression as well, suggesting that the extra RNA sequence outside of the shRNA hairpin does not disrupt shRNA function. We also showed that silencing of survivin expression selectively induces apoptosis in transfected cells. Together, we have validated multiple approaches of RNAi technology using both survivin and luciferase genes as targets and demonstrated for the first time that GFP-shRNAs transcribed from an RNA Pol II-mediated promoter could mediate gene silencing, which may lead to new directions for the application of RNAi technology.

Apoptosis↗
PubMed2006

Linking splicing to Pol II transcription stabilizes pre-mRNAs and influences splicing patterns.

RNA processing is carried out in close proximity to the site of transcription, suggesting a regulatory link between transcription and pre-mRNA splicing. Using an in vitro transcription/splicing assay, we demonstrate that an association of RNA polymerase II (Pol II) transcription and pre-mRNA splicing is required for efficient gene expression. Pol II-synthesized RNAs containing functional splice sites are protected from nuclear degradation, presumably because the local concentration of the splicing machinery is sufficiently high to ensure its association over interactions with nucleases. Furthermore, the process of transcription influences alternative splicing of newly synthesized pre-mRNAs. Because other RNA polymerases do not provide similar protection from nucleases, and their RNA products display altered splicing patterns, the link between transcription and RNA processing is RNA Pol II-specific. We propose that the connection between transcription by Pol II and pre-mRNA splicing guarantees an extended half-life and proper processing of nascent pre-mRNAs.

DNA-Directed RNA Polymerases↗
PubMed2003

Architecture of initiation-competent 12-subunit RNA polymerase II.

RNA polymerase (Pol) II consists of a 10-polypeptide catalytic core and the two-subunit Rpb4/7 complex that is required for transcription initiation. Previous structures of the Pol II core revealed a "clamp," which binds the DNA template strand via three "switch regions," and a flexible "linker" to the C-terminal repeat domain (CTD). Here we derived a model of the complete Pol II by fitting structures of the core and Rpb4/7 to a 4.2-A crystallographic electron density map. Rpb4/7 protrudes from the polymerase "upstream face," on which initiation factors assemble for promoter DNA loading. Rpb7 forms a wedge between the clamp and the linker, restricting the clamp to a closed position. The wedge allosterically prevents entry of the promoter DNA duplex into the active center cleft and induces in two switch regions a conformation poised for template-strand binding. Interaction of Rpb4/7 with the linker explains Rpb4-mediated recruitment of the CTD phosphatase to the CTD during Pol II recycling. The core-Rpb7 interaction and some functions of Rpb4/7 are apparently conserved in all eukaryotic and archaeal RNA polymerases but not in the bacterial enzyme.

Amino Acid Sequence↗
PubMed2000

Repression of RNA polymerase I transcription by the tumor suppressor p53.

The tumor suppressor protein p53 is frequently inactivated in tumors. It functions as a transcriptional activator as well as a repressor for a number of viral and cellular promoters transcribed by RNA polymerase II (Pol II) and by RNA Pol III. Moreover, it appears that p53 also suppresses RNA Pol I transcription. In this study, we examined the molecular mechanism of Pol I transcriptional inhibition by p53. We show that wild-type, but not mutant, p53 can repress Pol I transcription from a human rRNA gene promoter in cotransfection assays. Furthermore, we show that recombinant p53 inhibits rRNA transcription in a cell-free transcription system. In agreement with these results, p53-null epithelial cells display an increased Pol I transcriptional activity compared to that of epithelial cells that express p53. However, both cell lines display comparable Pol I factor protein levels. Our biochemical analysis shows that p53 prevents the interaction between SL1 and UBF. Protein-protein interaction assays indicate that p53 binds to SL1, and this interaction is mostly mediated by direct contacts with TATA-binding protein and TAF(I)110. Moreover, template commitment assays show that while the formation of a UBF-SL1 complex can partially relieve the inhibition of transcription, only the assembly of a UBF-SL1-Pol I initiation complex on the rDNA promoter confers substantial protection against p53 inhibition. In summary, our results suggest that p53 represses RNA Pol I transcription by directly interfering with the assembly of a productive transcriptional machinery on the rRNA promoter.

Genes, Tumor Suppressor↗
PubMed1999

CpG island hypermethylation in human colorectal tumors is not associated with DNA methyltransferase overexpression.

The molecular basis of aberrant hypermethylation of CpG islands observed in a subset of human colorectal tumors is unknown. One potential mechanism is the up-regulation of DNA (cytosine-5)-methyltransferases. Recently, two new mammalian DNA methyltransferase genes have been identified, which are referred to as DNMT3A and DNMT3B. The encoded proteins differ from the predominant mammalian DNA methyltransferase DNMT1 in that they have a substantially higher ratio of de novo to maintenance methyltransferase activity. We have used a highly quantitative 5' nuclease fluorogenic reverse transcription-PCR method (TaqMan) to analyze the expression of all three DNA methyltransferase genes in 25 individual colorectal adenocarcinoma specimens and matched normal mucosa samples. In addition, we examined the methylation patterns of four CpG islands [APC, ESR1 (estrogen receptor), CDKN2A (p16), and MLH1] to determine whether individual tumors show a positive correlation between the level of DNA methyltransferase expression and the frequency of CpG island hypermethylation. All three methyltransferases appear to be up-regulated in tumors when RNA levels are normalized using either ACTB (beta-actin) or POLR2A (RNA pol II large subunit), but not when RNA levels are normalized with proliferation-associated genes, such as H4F2 (histone H4) or PCNA. The frequency or extent of CpG island hypermethylation in individual tumors did not correlate with the expression of any of the three DNA methyltransferases. Our results suggest that deregulation of DNA methyltransferase gene expression does not play a role in establishing tumor-specific abnormal DNA methylation patterns in human colorectal cancer.

Adenocarcinoma↗
PubMed2026

Emergent 3D genome reorganization and graded gene control from the stepwise assembly of transcriptional condensates.

Transcriptional condensates are clusters of transcription factors, coactivators, and RNA Pol II associated with gene activation, yet how they assemble and function within the cell remains unclear. Here, we show that transcriptional condensates form in a stepwise manner to enable both graded and three-dimensional (3D) gene control in the yeast heat shock response (HSR). First, the transcription factor Hsf1 (heat shock factor 1) clusters upon partial dissociation from the chaperone Hsp70. Next, the coactivator Mediator partitions following further Hsp70 dissociation and Hsf1 phosphorylation. Finally, Pol II condenses, driving emergent coalescence of HSR genes. Separation-of-function Hsf1 mutants revealed graded (non-switch-like) control of transcription and a decoupling of condensate formation and gene activation. Fully assembled HSR condensates promoted adaptive 3D genome reconfiguration, suggesting a role beyond transcription. In the HSR, differential condensation of the transcriptional machinery quantitatively tunes gene expression and qualitatively remodels the 3D genome.

3D genome↗
PubMed2002

Distinct parts of minichromosome maintenance protein 2 associate with histone H3/H4 and RNA polymerase II holoenzyme.

Minichromosome maintenance (MCM) proteins are part of the replication licensing factor (RLF-M), which limits the initiation of DNA replication to once per cell cycle. We have previously reported that higher order complexes of mammalian pol II and general pol II transcription factors, referred to as pol II holoenzyme, also contain MCM proteins. In the present study we have analyzed in detail the interaction between MCM2 and pol II holoenzyme. N- and C- terminal deletions were introduced into epitope-tagged MCM2 and the truncated proteins were transiently expressed in 293 cells. Affinity chromatography was used to purify RNA pol II holoenzyme and histone binding MCM complexes. We found that amino acids 168-230 of MCM2 are required for its binding to pol II holoenzyme in vivo. We also showed that bacterially expressed amino acids 169-212 of MCM2 associate with pol II and several general transcription factors in vitro. Point mutations within the 169-212 domain of MCM2 disrupted its interaction with pol II holoenzyme both in vitro and in vivo. This region is distinct from the previously characterized histone H3 binding domain of MCM2.

Acetyltransferases↗