Deborah L. Johnson

p53 represses RNA polymerase III transcription by targeting TBP and inhibiting promoter occupancy by TFIIIB

The tumor suppressor p53 is a transcription factor that controls cellular growth and proliferation. p53 targets include RNA polymerase (pol) III‐dependent genes encoding untranslated RNAs such as tRNA and 5S rRNA. These genes are repressed through interaction of p53 with TFIIIB, a TATA‐binding protein (TBP)‐containing factor. Although many studies have shown that p53 binds to TBP, the significance of this interaction has remained elusive. Here we demonstrate that the TBP–p53 interaction is of functional importance for regulating RNA pol III‐transcribed genes. Unlike RNA pol II‐dependent promoter repression, overexpressing TBP can reverse inhibition of tRNA gene transcription by p53. p53 does not disrupt the direct interaction between the TFIIIB subunits TBP and Brf1, but prevents the association of Brf1 complexes with TFIIIC2 and RNA pol III. Using chromatin immunoprecipitation assays, we found that TFIIIB occupancy on tRNA genes markedly decreases following p53 induction, whereas binding of TFIIIC2 to these genes is unaffected. Together our results support the idea that p53 represses RNA pol III transcription through direct interactions with TBP, preventing promoter occupancy by TFIIIB.

RNA-Dependent Replication and Transcription of Hepatitis Delta Virus RNA Involve Distinct Cellular RNA Polymerases

ABSTRACT Cellular DNA-dependent RNA polymerase II (pol II) has been postulated to carry out RNA-dependent RNA replication and transcription of hepatitis delta virus (HDV) RNA, generating a full-length (1.7-kb) RNA genome and a subgenomic-length (0.8-kb) mRNA. However, the supporting evidence for this hypothesis was ambiguous because the previous experiments relied on DNA-templated transcription to initiate HDV RNA synthesis. Furthermore, there is no evidence that the same cellular enzyme is involved in the synthesis of both RNA species. In this study, we used a novel HDV RNA-based transfection approach, devoid of any artificial HDV cDNA intermediates, to determine the enzymatic and metabolic requirements for the synthesis of these two RNA species. We showed that HDV subgenomic mRNA transcription was inhibited by a low concentration of α-amanitin (<3 μg/ml) and could be partially restored by an α-amanitin-resistant mutant pol II; however, surprisingly, the synthesis of the full-length (1.7-kb) antigenomic RNA was not affected by α-amanitin to a concentration higher than 25 μg/ml. By several other criteria, such as the differing requirement for the de novo-synthesized hepatitis delta antigen and temperature dependence, we further showed that the metabolic requirements of subgenomic HDV mRNA synthesis are different from those for the synthesis of genomic-length HDV RNA and cellular pol II transcripts. The synthesis of the two HDV RNA species could also be uncoupled under several different conditions. These findings provide strong evidence that pol II, or proteins derived from pol II transcripts, is involved in mRNA transcription from the HDV RNA template. In contrast, the synthesis of the 1.7-kb HDV antigenomic RNA appears not to be dependent on pol II. These results reveal that there are distinct molecular mechanisms for the synthesis of these two RNA species.

Enhanced RNA Polymerase III-dependent Transcription Is Required for Oncogenic Transformation

RNA polymerase (pol) III transcription, responsible for the synthesis of various stable RNAs, including 5 S rRNAs and tRNAs, is regulated by oncogenic proteins and tumor suppressors. Although it is well established that RNA pol III-dependent transcription is deregulated in transformed cells and malignant tumors, it has not been determined whether this represents a cause or consequence of these processes. We show that Rat1a fibroblasts undergoing oncogenic transformation by the TATA-binding protein or c-Myc display enhanced RNA pol III transcription. Decreased expression of the RNA pol III-specific transcription factor Brf1 prevented this increase in RNA pol III transcription. Although the overall proliferation rates of these cells remained unchanged, the ability of cells to grow in an anchorage-independent manner and form tumors in mice was markedly reduced. Although overexpression of Brf1 modestly stimulated RNA pol III transcription, expression of a phosphomimic, Brf1-T145D, more significantly induced transcription. However, these increases in transcription were not sufficient to promote cellular transformation. Together, these results demonstrate that enhanced RNA pol III transcription is essential for anchorage-independent growth and tumorigenesis and that these events can be uncoupled from effects on anchorage-dependent proliferation.

Hepatitis B Virus X Protein Induces RNA Polymerase III- Dependent Gene Transcription and Increases Cellular TATA- Binding Protein by Activating the Ras Signaling Pathway

Our previous studies have shown that the hepatitis B virus protein, X, activates all three classes of RNA polymerase III (pol III)-dependent promoters by increasing the cellular level of TATA-binding protein (TBP) (H.-D. Wang et al., Mol. Cell. Biol. 15:6720-6728, 1995), a limiting transcription component (A. Trivedi et al., Mol. Cell. Biol. 16:6909-6916, 1996). We have investigated whether these X-mediated events are dependent on the activation of the Ras/Raf-1 signaling pathway. Transient expression of a dominant-negative mutant Ras gene (Ras-ala15) in a Drosophila S-2 stable cell line expressing X (X-S2), or incubation of the cells with a Ras farnesylation inhibitor, specifically blocked both the X-dependent activation of a cotransfected tRNA gene and the increase in cellular TBP levels. Transient expression of a constitutively activated form of Ras (Ras-val12) in control S2 cells produced both an increase in tRNA gene transcription and an increase in cellular TBP levels. These events are not cell type specific since X-mediated gene induction was also shown to be dependent on Ras activation in a stable rat 1A cell line expressing X. Furthermore, increases in RNA pol III-dependent gene activity and TBP levels could be restored in X-S2 cells expressing Ras-ala15 by coexpressing a constitutively activated form of Raf-1. These events are serum dependent, and when the cells are serum deprived, the X-mediated effects are augmented. Together, these results demonstrate that the X-mediated induction of RNA pol III-dependent genes and increase in TBP are both dependent on the activation of the Ras/Raf-1 signaling cascade. In addition, these studies define two new and important consequences mediated by the activation of the Ras signal transduction pathway: an increase in the central transcription factor, TBP, and the induction of RNA pol III-dependent gene activity.

Regulation of RNA Polymerase I-Dependent Promoters by the Hepatitis B Virus X Protein via Activated Ras and TATA-Binding Protein

ABSTRACT 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 anX 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.

PTEN represses RNA Polymerase I transcription by disrupting the SL1 complex.

ABSTRACT PTEN is a tumor suppressor whose function is frequently lost in human cancer. It possesses a lipid phosphatase activity that represses the activation of PI3 kinase/Akt signaling, leading to decreased cell growth, proliferation, and survival. The potential for PTEN to regulate transcription of the large rRNAs by RNA polymerase I (RNA Pol I) was investigated. As increased synthesis of rRNAs is a hallmark of neoplastic transformation, the ability of PTEN to control the transcription of rRNAs might be crucial for its tumor suppressor function. The expression of PTEN in PTEN-deficient cells represses RNA Pol I transcription, while decreasing PTEN expression enhances transcription. PTEN-mediated repression requires its lipid phosphatase activity and is independent of the p53 status of the cell. This event can be uncoupled from PTEN′s ability to regulate the cell cycle. RNA Pol I is regulated through PI3 kinase/Akt/mammalian target of rapamycin/S6 kinase, and the expression of constitutively activated S6 kinase is able to abrogate transcription repression by PTEN. No change in the expression of the RNA Pol I transcription components, upstream binding factor or SL1, was observed upon PTEN expression. However, chromatin immunoprecipitation assays demonstrate that PTEN differentially reduces the occupancy of the SL1 subunits on the rRNA gene promoter. Furthermore, PTEN induces dissociation of the SL1 subunits. Together, these results demonstrate that PTEN represses RNA Pol I transcription through a novel mechanism that involves disruption of the SL1 complex.

PTEN Represses RNA Polymerase III-Dependent Transcription by Targeting the TFIIIB Complex

ABSTRACT PTEN, a tumor suppressor whose function is frequently lost in human cancers, possesses a lipid phosphatase activity that represses phosphatidylinositol 3-kinase (PI3K) signaling, controlling cell growth, proliferation, and survival. The potential for PTEN to regulate the synthesis of RNA polymerase (Pol) III transcription products, including tRNAs and 5S rRNAs, was evaluated. The expression of PTEN in PTEN-deficient cells repressed RNA Pol III transcription, whereas decreased PTEN expression enhanced transcription. Transcription repression by PTEN was uncoupled from PTEN-mediated effects on the cell cycle and was independent of p53. PTEN acts through its lipid phosphatase activity, inhibiting the PI3K/Akt/mTOR/S6K pathway to decrease transcription. PTEN, through the inactivation of mTOR, targets the TFIIIB complex, disrupting the association between TATA-binding protein and Brf1. Kinetic analysis revealed that PTEN initially induces a decrease in the serine phosphorylation of Brf1, leading to a selective reduction in the occupancy of all TFIIIB subunits on tRNALeu genes, whereas prolonged PTEN expression results in the enhanced serine phosphorylation of Bdp1. Together, these results demonstrate a new class of genes regulated by PTEN through its ability to repress the activation of PI3K/Akt/mTOR/S6K signaling.

The hepatitis B virus X protein increases the cellular level of TATA-binding protein, which mediates transactivation of RNA polymerase III genes.

The hepatitis B virus X gene product transactivates a variety of cellular and viral genes. The mechanism for X induction of RNA polymerase (pol) III genes was investigated. By using Drosophila S-2 cells stably transformed with the X gene, the transient expression of a tRNA gene is enhanced. Comparing the transcriptional activities of extracts derived from these cells, all three types of RNA pol III promoters are stimulated by X. Interestingly, both S-2 and rat 1A cells stably transformed with the X gene produce increased cellular levels of the TATA-binding protein (TBP). By using various kinase inhibitors, it was found that the X-mediated increases in both transcription and TBP are dependent upon protein kinase C activation. Since TBP is a subunit of TFIIIB, the activity of this component fractionated from extracts derived from control and X-transformed cells was analyzed. These studies reveal that TFIIIB activity is substantially more limiting in control cells and that TFIIIB isolated from X-transformed cells has increased activity in reconstitution assays compared with TFIIIB isolated from control cells. Conversely, comparison of TFIIIC from control and X-transformed cell extracts revealed that there is relatively little change in its ability either to reconstitute transcription or to bind to DNA and that there is no change in the catalytic activity of RNA pol III. Studies were performed to determine whether directly increasing cellular TBP alone could enhance RNA pol III gene transcription. Transient expression of a TBP cDNA in rat 1A cells was capable of stimulating transcription activity from the resultant extracts in vitro. Together, these results demonstrate that one mechanism by which X mediates transactivation of RNA poll III genes is by increasing limiting TBP via the activation of cellular signaling pathways. The discovery that X increases cellular TBP, the universal transcription factor, provides a novel mechanism for the function of a viral transactivator protein and may explain the ability of X to produce such large and diverse effects on cellular gene expression.

Increased Expression of TATA-Binding Protein, the Central Transcription Factor, Can Contribute to Oncogenesis

ABSTRACT Despite the central role of TATA-binding protein (TBP) in transcription, changes in cellular TBP concentration produce selective effects on gene expression. Moreover, TBP is up-regulated by oncogenic signaling pathways. These findings suggest that TBP could be a nexus in pathways that regulate cell proliferation and that genetic lesions that result in cellular transformation may produce their effects at least in part through TBP. We provide evidence consistent with this hypothesis, demonstrating that increases in TBP expression contribute to cellular transformation. A Ras-mediated increase in TBP expression is required for full Ras transforming activity. TBP overexpression induces cells to grow in an anchorage-independent manner and to form tumors in athymic mice. These effects on cellular transformation require changes in RNA polymerase II-dependent transcription and on the selective recruitment of TBP to promoters via its DNA binding activity. TBP expression is elevated in human colon carcinomas relative to normal colon epithelium. Both Ras-dependent and Ras-independent mechanisms mediate increases in TBP expression in colon carcinoma cell lines. We conclude that TBP may be a critical component in dysregulated signaling that occurs downstream of genetic lesions that cause tumors.

High molecular mass amino acyl-tRNA synthetase complexes in eukaryotes

Aminoacyl-t RNA synthetases (AARS) are enzymes which play an indispensable role in protein biosynthe- sis by catalyzing the formation of aminoacyl-tRNA from amino acid, the cognate tRNA, and ATP by highly selective intermolecular interactions [57]. Joachimiak and Barciszewski [41] have provided an extensive compilation of the properties of the amino- acyl-tRNA synthetases; however, information on the eukaryotic highM r (HMr) complexes of aminoacyl-tRNA synthetases was lacking. Here, we intend to fill this void by providing a summary of the properties of the eukaryotic aminoacyl-tRNA synthetase complexes. Eukaryotic aminoacyl-tRNA synthetases may occur as complexes with Mr-values of >106 in contrast to the prokaryotic counterparts which have Mr-values of <250 000. These eukaryotic HMr--AARS complexes appear ubiquitous in a wide spectrum of cell types from yeast to human placenta as shown in table 1. Although not all 20 aminoacyl-tRNA synthetases were examined in each case shown in table 1, it appears that the AARS commonly associated with M r complexes are those specific for Arg, Gin, Glu, fie, Leu, Lys and Met. The properties of these HMr-AARS complexes are most consistent with multienzyme complexes of aminoacyl-tRNA synthetases [ 19,20,43, 46]. The physicochemical properties, composition, and stoichiometry of the more rigorously character- ized complexes are shown in table 2. The mechanism(s) of intermolecular interaction between the aminoacyl-tRNA synthetases is not known, but the putative interactions of aminoacyl- tRNA synthetases with a variety of biomolecules have been suggested to play a role in complex formation as shown in table 3. Our present knowledge of the func- * To whom correspondences should be addressed tional significance of HMr--AARS is profoundly lack- ing; however, interactions of the aminoacyl-tRNA synthetases with other components of the protein biosynthetic machinery and other enzymes suggest the intriguing possibility of higher organization of eukaryotic protein biosynthesis. Table 4 is a summary of the possible interactions of the aminoacyl-tRNA synthetases with subcellular components and other enzymes. This presentation is a brief summary of the prop- erties of the high molecular weight eukaryotic amino- acyl-tRNA synthetase complexes. We hope that this compilation will complement that presented in [41 ] and will provide useful information for workers in this and other related fields.