ABSTRACT
The heterodimeric nuclear factor (NF) 90/NF45 complex (NF90/NF45) binds nucleic acids and is a multifunctional regulator of gene expression. Here we report that depletion of NF90/NF45 restores the expression of the p53 and p21 proteins in cervical carcinoma cells infected with high-risk human papillomaviruses (HPVs). Knockdown of either NF90 or NF45 by RNA interference led to greatly elevated levels of p53 and p21 proteins in HPV-derived HeLa and SiHa cells but not in other cancerous or normal cell lines. In HeLa cells, p21 messenger-RNA (mRNA) increased concomitantly but the level of p53 mRNA was unaffected. RNA interference directed against p53 prevented the induction of both proteins. These results indicated that the upregulation of p21 is due to p53-dependent transcription, whereas p53 is regulated post-transcriptionally. Proteasome-mediated turnover of p53 is accelerated by the HPV E6 and cellular E6AP proteins. We therefore examined the hypothesis that this pathway is regulated by NF90/NF45. Indeed, depletion of NF90 attenuated the expression of E6 RNA and inhibited transcription from the HPV early promoter, revealing a new role for NF90/NF45 in HPV gene expression. The transcription inhibition was largely independent of the reduction of P-TEFb (positive transcription elongation factor b) levels caused by NF90 depletion. Consistent with p53 derepression, NF90/NF45-depleted HeLa cells displayed elevated poly ADP-ribose polymerase (PARP) cleavage and susceptibility to camptothecin-induced apoptosis. We conclude that high-risk strains of HPV utilize the cellular NF90/NF45 complex for viral E6 expression in infected cervical carcinoma cell lines. Interference with NF90/NF45 function could assist in controlling cervical carcinoma.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p21/metabolism , Nuclear Factor 45 Protein/genetics , Nuclear Factor 90 Proteins/genetics , Tumor Suppressor Protein p53/metabolism , Uterine Cervical Neoplasms/genetics , Apoptosis/genetics , Carcinoma/genetics , Carcinoma/pathology , Carcinoma/virology , Cell Transformation, Neoplastic , Cyclin-Dependent Kinase Inhibitor p21/genetics , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , Female , Gene Expression Regulation, Neoplastic/genetics , HeLa Cells , Human papillomavirus 18/genetics , Humans , Multiprotein Complexes/genetics , Multiprotein Complexes/metabolism , Nuclear Factor 45 Protein/metabolism , Nuclear Factor 90 Proteins/metabolism , Oncogene Proteins, Viral/biosynthesis , Oncogene Proteins, Viral/genetics , Papillomaviridae/genetics , Papillomaviridae/metabolism , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/metabolism , Signal Transduction , Tumor Suppressor Protein p53/genetics , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/virologyABSTRACT
Expression of the cellular inhibitor of apoptosis protein 1 (cIAP1) is unexpectedly repressed at the level of translation under normal physiological conditions in many cell lines. We have previously shown that the 5' untranslated region of cIAP1 mRNA contains a stress-inducible internal ribosome entry site (IRES) that governs expression of cIAP1 protein. Although inactive in unstressed cells, the IRES supports cap-independent translation of cIAP1 in response to endoplasmic reticulum stress. To gain an insight into the mechanism of cIAP1 IRES function, we empirically derived the minimal free energy secondary structure of the cIAP1 IRES using enzymatic cleavage mapping. We subsequently used RNA affinity chromatography to identify several cellular proteins, including nuclear factor 45 (NF45) as cIAP1 IRES binding proteins. In this report we show that NF45 is a novel RNA binding protein that enhances IRES-dependent translation of endogenous cIAP1. Further, we show that NF45 is required for IRES-mediated induction of cIAP1 protein during the unfolded protein response. The data presented are consistent with a model in which translation of cIAP1 is governed, at least in part, by NF45, a novel cellular IRES trans-acting factor.
Subject(s)
Apoptosis/physiology , Inhibitor of Apoptosis Proteins/metabolism , Nuclear Factor 45 Protein/metabolism , Protein Biosynthesis/physiology , Transcription Factors/metabolism , Unfolded Protein Response/physiology , Cell Line , Cell Line, Tumor , Chromatography, Affinity , Endoplasmic Reticulum/genetics , Endoplasmic Reticulum/metabolism , Enzyme Activation/physiology , HeLa Cells , Humans , Inhibitor of Apoptosis Proteins/chemistry , Inhibitor of Apoptosis Proteins/genetics , Nuclear Factor 45 Protein/genetics , Protein Structure, Tertiary/physiology , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Stress, Physiological/physiology , Transcription Factors/geneticsABSTRACT
We identified a novel family of human noncoding RNAs by in vivo cross-linking to the nuclear factor 90 (NF90) protein. These small NF90-associated RNAs (snaRs) are transcribed by RNA polymerase III and display restricted tissue distribution, with high expression in testis and discrete areas of the brain. The most abundant human transcript, snaR-A, interacts with the cell's transcription and translation systems. snaR genes have evolved in African Great Apes (human, chimpanzee, and gorilla) and some are unique to humans. We traced their ancestry to the Alu SINE (short interspersed nucleotide element) family, via two hitherto unreported sets of short genetic elements termed ASR (Alu/snaR-related) and CAS (Catarrhine ancestor of snaR). This derivation entails a series of internal deletions followed by expansions. The evolution of these genes coincides with major primate speciation events: ASR elements are found in all monkeys and apes, whereas CAS elements are limited to Old World monkeys and apes. In contrast to ASR and CAS elements, which are retrotransposons, human snaR genes are predominantly located in three clusters on chromosome 19 and have been duplicated as part of a larger genetic element. Insertion of the element containing snaR-G into a gene encoding a chorionic gonadotropin beta subunit generated new hormone genes in African Great Apes.
Subject(s)
Chorionic Gonadotropin/genetics , Evolution, Molecular , Alu Elements , Animals , Base Sequence , Genetic Speciation , Humans , Molecular Sequence Data , Multigene Family , Nuclear Factor 90 Proteins/genetics , Nuclear Factor 90 Proteins/metabolism , Nucleic Acid Conformation , Phylogeny , Primates/genetics , RNA, Untranslated/chemistry , RNA, Untranslated/genetics , RNA, Untranslated/metabolism , Segmental Duplications, Genomic , Sequence Homology, Nucleic Acid , Short Interspersed Nucleotide ElementsABSTRACT
The Cold Spring Harbor Symposium on The Ribosome was held on 31 May - 5 June in Cold Spring Harbor, NY. USA.
Subject(s)
Genome , Ribosomes/genetics , Ribosomes/metabolism , Cryoelectron Microscopy , Crystallography, X-Ray , Models, Molecular , RNA/genetics , RNA/metabolism , RNA, Ribosomal/chemistry , Ribosomal Proteins/chemistry , Ribosomes/chemistry , Ribosomes/ultrastructureSubject(s)
Eukaryotic Cells/metabolism , Protein Biosynthesis/physiology , Animals , Artifacts , Binding Sites/physiology , Genes , Genetic Vectors , Humans , Peptide Initiation Factors/metabolism , RNA Processing, Post-Transcriptional/physiology , RNA, Messenger/metabolism , RNA, Transfer/metabolism , RNA, Viral/metabolism , Reproducibility of Results , Research Design , Ribosomes/metabolismABSTRACT
Nuclear factor 90 (NF90) is a member of an expanding family of double-stranded (ds) RNA-binding proteins thought to be involved in gene expression. Originally identified in complex with nuclear factor 45 (NF45) as a sequence-specific DNA-binding protein, NF90 contains two double stranded RNA-binding motifs (dsRBMs) and interacts with highly structured RNAs as well as the dsRNA-activated protein kinase, PKR. In this report, we characterize the biochemical interactions between these two dsRBM containing proteins. NF90 binds to PKR through two independent mechanisms: an RNA-independent interaction occurs between the N terminus of NF90 and the C-terminal region of PKR, and an RNA-dependent interaction is mediated by the dsRBMs of the two proteins. Co-immunoprecipitation analysis demonstrates that NF90, NF45, and PKR form a complex in both nuclear and cytosolic extracts, and both proteins serve as substrates for PKR in vitro. NF90 is phosphorylated by PKR in its RNA-binding domain, and this reaction is partially blocked by the NF90 N-terminal region. The C-terminal region also inhibits PKR function, probably through competitive binding to dsRNA. A model for NF90-PKR interactions is proposed.
Subject(s)
DNA-Binding Proteins/metabolism , Transcription Factors/metabolism , eIF-2 Kinase/metabolism , Animals , Binding Sites , Cell Nucleus/metabolism , Cytosol/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Kinetics , Models, Molecular , NFATC Transcription Factors , Nuclear Factor 45 Protein , Nuclear Factor 90 Proteins , Nuclear Proteins/metabolism , Nucleic Acid Conformation , Protein Conformation , RNA/chemistry , RNA/metabolism , RNA, Double-Stranded/chemistry , RNA, Double-Stranded/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Substrate Specificity , Transcription Factors/chemistry , Transcription Factors/genetics , eIF-2 Kinase/chemistryABSTRACT
Protein kinase PKR is activated by double-stranded RNA (dsRNA) and phosphorylates translation initiation factor 2alpha to inhibit protein synthesis in virus-infected mammalian cells. PKR contains two dsRNA binding motifs (DRBMs I and II) required for activation by dsRNA. There is strong evidence that PKR activation requires dimerization, but the role of dsRNA in dimer formation is controversial. By making alanine substitutions predicted to remove increasing numbers of side chain contacts between the DRBMs and dsRNA, we found that dimerization of full-length PKR in yeast was impaired by the minimal combinations of mutations required to impair dsRNA binding in vitro. Mutation of Ala-67 to Glu in DRBM-I, reported to abolish dimerization without affecting dsRNA binding, destroyed both activities in our assays. By contrast, deletion of a second dimerization region that overlaps the kinase domain had no effect on PKR dimerization in yeast. Human PKR contains at least 15 autophosphorylation sites, but only Thr-446 and Thr-451 in the activation loop were found here to be critical for kinase activity in yeast. Using an antibody specific for phosphorylated Thr-451, we showed that Thr-451 phosphorylation is stimulated by dsRNA binding. Our results provide strong evidence that dsRNA binding is required for dimerization of full-length PKR molecules in vivo, leading to autophosphorylation in the activation loop and stimulation of the eIF2alpha kinase function of PKR.
Subject(s)
RNA, Double-Stranded/metabolism , eIF-2 Kinase/metabolism , Amino Acid Sequence , Dimerization , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Models, Molecular , Molecular Sequence Data , Mutation , Phosphorylation , Plasmids , Poly I-C/metabolism , Threonine/metabolism , Yeasts/enzymology , eIF-2 Kinase/geneticsABSTRACT
CDK7, CDK8, and CDK9 are cyclin-dependent kinases (CDKs) that phosphorylate the C-terminal domain (CTD) of RNA polymerase II. They have distinct functions in transcription. Because the three CDKs target only serine 5 in the heptad repeat of model CTD substrates containing various numbers of repeats, we tested the hypothesis that the kinases differ in their ability to phosphorylate CTD heptad arrays. Our data show that the kinases display different preferences for phosphorylating individual heptads in a synthetic CTD substrate containing three heptamer repeats and specific regions of the CTD in glutathione S-transferase fusion proteins. They also exhibit differences in their ability to phosphorylate a synthetic CTD peptide that contains Ser-2-PO(4). This phosphorylated peptide is a poor substrate for CDK9 complexes. CDK8 and CDK9 complexes, bound to viral activators E1A and Tat, respectively, target only serine 5 for phosphorylation in the CTD peptides, and binding to the viral activators does not change the substrate preference of these kinases. These results imply that the display of different CTD heptads during transcription, as well as their phosphorylation state, can affect their phosphorylation by the different transcription-associated CDKs.
Subject(s)
Cyclin-Dependent Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Amino Acid Sequence , Cyclin-Dependent Kinase 8 , Cyclin-Dependent Kinase 9 , Cyclin-Dependent Kinases/genetics , HeLa Cells , Humans , Molecular Sequence Data , Phosphorylation , Protein Serine-Threonine Kinases/genetics , RNA Polymerase II/metabolism , Substrate Specificity , Transcription, Genetic , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
Double-stranded-RNA (dsRNA)-dependent protein kinase PKR is induced by interferon and activated upon autophosphorylation. We previously identified four autophosphorylated amino acids and elucidated their participation in PKR activation. Three of these sites are in the central region of the protein, and one is in the kinase domain. Here we describe the identification of four additional autophosphorylated amino acids in the spacer region that separates the two dsRNA-binding motifs in the RNA-binding domain. Eight amino acids, including these autophosphorylation sites, are duplicated in hepatitis C virus (HCV) envelope protein E2. This region of E2 is required for its inhibition of PKR although the mechanism of inhibition is not known. Replacement of all four of these residues in PKR with alanines did not dramatically affect kinase activity in vitro or in yeast Saccharomyces cerevisiae. However, when coupled with mutations of serine 242 and threonines 255 and 258 in the central region, these mutations increased PKR protein expression in mammalian cells, consistent with diminished kinase activity. A synthetic peptide corresponding to this region of PKR was phosphorylated in vitro by PKR, but phosphorylation was strongly inhibited after PKR was preincubated with HCV E2. Another synthetic peptide, corresponding to the central region of PKR and containing serine 242, was also phosphorylated by active PKR, but E2 did not inhibit this peptide as efficiently. Neither of the PKR peptides was able to disrupt the HCV E2-PKR interaction. Taken together, these results show that PKR is autophosphorylated on serine 83 and threonines 88, 89, and 90, that this autophosphorylation may enhance kinase activation, and that the inhibition of PKR by HCV E2 is not solely due to duplication of and competition with these autophosphorylation sites.
Subject(s)
RNA, Viral/metabolism , Viral Envelope Proteins/physiology , eIF-2 Kinase/antagonists & inhibitors , Amino Acid Sequence , Animals , Binding Sites , COS Cells , Molecular Sequence Data , Molecular Weight , Phosphorylation , eIF-2 Kinase/chemistry , eIF-2 Kinase/metabolismABSTRACT
The interferon-inducible double-stranded RNA (dsRNA)-activated protein kinase PKR is regulated by dsRNAs that interact with the two dsRNA-binding motifs (dsRBMs) in its N terminus. The dsRBM is a conserved protein motif found in many proteins from most organisms. In this study, we investigated the biochemical functions and cytological activities of the two PKR dsRBMs (dsRBM1 and dsRBM2) and the cooperation between them. We found that dsRBM1 has a higher affinity for binding to dsRNA than dsRBM2. In addition, dsRBM1 has RNA-annealing activity that is not displayed by dsRBM2. Both dsRBMs have an intrinsic ability to dimerize (dsRBM2) or multimerize (dsRBM1). Binding to dsRNA inhibits oligomerization of dsRBM1 but not dsRBM2 and strongly inhibits the dimerization of the intact PKR N terminus (p20) containing both dsRBMs. dsRBM1, like p20, activates reporter gene expression in transfection assays, and it plays a determinative role in localizing PKR to the nucleolus and cytoplasm of the cell. Thus, dsRBM2 has weak or no activity in dsRNA binding, stimulation of gene expression, and PKR localization, but it strongly enhances these functions of dsRBM1 when contained in p20. However, dsRBM2 does not enhance the annealing activity of dsRBM1. This study shows that the dsRBMs of PKR possess distinct properties and that some, but not all, of the functions of the enzyme depend on cooperation between the two motifs.
Subject(s)
RNA, Double-Stranded/metabolism , eIF-2 Kinase/chemistry , eIF-2 Kinase/metabolism , Amino Acid Motifs , Amino Acid Sequence , Cell Line , Cross-Linking Reagents/pharmacology , Dimerization , Electrophoresis, Polyacrylamide Gel , Enzyme Activation , Escherichia coli/metabolism , Genes, Reporter , Glutathione Transferase/metabolism , Green Fluorescent Proteins , HeLa Cells , Humans , Kinetics , Luciferases/metabolism , Luminescent Proteins/metabolism , Models, Biological , Models, Genetic , Molecular Sequence Data , Plasmids/metabolism , Protein Binding , Protein Structure, Tertiary , RNA/metabolism , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Transcriptional Activation , TransfectionSubject(s)
Cell Division/genetics , Protein Biosynthesis , RNA-Binding Proteins/genetics , Animals , DNA-Binding Proteins/genetics , Homeostasis , Humans , Kinetics , Models, Genetic , NFATC Transcription Factors , Nuclear Proteins/genetics , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Transcription Factors/geneticsABSTRACT
PKR is a double-stranded (ds) RNA activated protein kinase whose expression is induced by interferon. Activated PKR phosphorylates its cellular substrate, eIF2, an essential initiation factor of translation. Prior evidence from a murine model system suggested that PKR may act as a tumor suppressor, but the evidence from human tumors is equivocal. To study PKR function in human breast cancer, PKR activity was measured in mammary carcinoma cell lines and nontransformed mammary epithelial cell lines. If PKR functioned as a tumor suppressor in this system, its activity would be higher in nontransformed cells than in carcinoma cells. On the contrary, PKR autophosphorylation and the phosphorylation of its substrate, the alpha-subunit of eIF2, is 7 - 40-fold higher in lysates prepared from breast carcinoma cell lines than in those from nontransformed epithelial cell lines. Correspondingly, a larger proportion of eIF2alpha is present in a phosphorylated state in carcinoma cell lines than in nontransformed cell lines. Protein synthesis is not inhibited by the high eIF2alpha phosphorylation in carcinoma cells, probably because they contain higher levels of eIF2B, the initiation factor that is inhibited by eIF2alpha phosphorylation. The dramatically lower PKR activity in nontransformed cell lines is partially due to lower PKR protein levels (2 - 4-fold) as well as to the presence of a PKR inhibitor. The nontransformed cells contain P58, a known cellular inhibitor of PKR that physically interacts with PKR and may be responsible for the low PKR activity in these cells. Taken together, these observations call into question the role of PKR as a tumor suppressor and suggest a positive regulatory role of PKR in growth control of breast cancer cells.
Subject(s)
Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/metabolism , Neoplasm Proteins/metabolism , eIF-2 Kinase/metabolism , Blotting, Western , Breast/metabolism , Cell Line, Transformed , Female , Fibrocystic Breast Disease/metabolism , Humans , Isoelectric Focusing , Phosphorylation , Receptors, Cytoplasmic and Nuclear/metabolism , Serine/metabolism , Tumor Cells, Cultured , Lamin B ReceptorABSTRACT
Highly structured and double-stranded (ds) RNAs are adaptable and potent biochemical entities. They interact with dsRNA-binding proteins (RBPs), the great majority of which contain a sequence called the dsRNA-binding motif (dsRBM). This approximately 70-amino-acid sequence motif forms a tertiary structure that interacts with dsRNA, with partially duplexed RNA and, in some cases, with RNA-DNA hybrids, generally without obvious RNA sequence specificity. At least nine families of functionally diverse proteins contain one or more dsRBMs. The motif also participates in complex formation through protein-protein interactions.
Subject(s)
Amino Acid Motifs , RNA-Binding Proteins/metabolism , Amino Acid Sequence , Molecular Sequence Data , Protein Conformation , RNA-Binding Proteins/chemistry , Sequence Homology, Amino AcidABSTRACT
Myotonic dystrophy is caused by an expanded CTG repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. The expanded repeat triggers the nuclear retention of mutant DMPK transcripts, but the resulting underexpression of DMPK probably does not fully account for the severe phenotype. One proposed disease mechanism is that nuclear accumulation of expanded CUG repeats may interfere with nuclear function. Here we show by thermal melting and nuclease digestion studies that CUG repeats form highly stable hairpins. Furthermore, CUG repeats bind to the dsRNA-binding domain of PKR, the dsRNA-activated protein kinase. The threshold for binding to PKR is approximately 15 CUG repeats, and the affinity increases with longer repeat lengths. Finally, CUG repeats that are pathologically expanded can activate PKR in vitro. These results raise the possibility that the disease mechanism could be, in part, a gain of function by mutant DMPK transcripts that involves sequestration or activation of dsRNA binding proteins.
Subject(s)
RNA, Double-Stranded/metabolism , Trinucleotide Repeat Expansion , eIF-2 Kinase/metabolism , Base Pairing , Endoribonucleases/metabolism , Enzyme Activation , Nucleic Acid Conformation , Protein Binding , RNA, Double-Stranded/analysis , Ribonuclease IIIABSTRACT
Sera of some patients afflicted with the inflammatory disease myositis contain antibodies of the anti-PL-12 type. A fraction of these polyclonal autoantibodies specifically precipitates the fully matured human tRNA(Ala) bearing the anticodon IGC (PL-12 antigen). Earlier work (Bunn & Mathews, 1987, Science 238:116-119) had shown that the epitopes are located entirely within the anticodon stem-loop of the tRNA(Ala). Here we demonstrate that human anti-tRNA(Ala) autoantibodies immunoprecipitate a synthetic polyribonucleotide containing inosine (I) and N1-methylinosine (m1I) separated by 2 nt as in the anticodon stem-loop of human tRNA(Ala). The shortest polyribonucleotide that can be immunoprecipitated corresponds to the pentanucleotide IpGpCpm1IpUp, which corresponds to part of the anticodon loop of human tRNA(Ala) and lacks the stem-loop structure. The efficiency of immunoprecipitation was about four times greater with longer polyribonucleotides capable of forming a stem-loop structure, and was abolished by altering the relative positions of I and m1I within the synthetic polynucleotide. Synthetic oligodeoxyribonucleotide analogs of the tRNA(Ala) stem-loop, containing the sequence dIpdGdCdm1Ip, are not antigenic. Our results show that human anti-tRNA(Ala) autoantibodies selectively recognize chemical details of modified nucleotides (the 6-keto group of inosine-34 and the 6-keto group and the N1-methyl groups of N1-methylinosine-37) within an anticodon loop structure of a tRNA molecule. We also describe the chemical synthesis of the phosphoramidite derivatives corresponding to N1-methylinosine and N1-methyl-2'-deoxyinosine for use in the automatic chemical synthesis of oligonucleotides containing N1-methylinosine and N1-methyl-2'-deoxyinosine.
Subject(s)
Antibodies, Antiphospholipid/chemistry , Anticodon , Epitopes/chemistry , Inosine/analogs & derivatives , Inosine/chemistry , Myositis/immunology , RNA, Transfer, Ala/chemistry , Base Sequence , Chromatography, Thin Layer/methods , DNA/chemistry , Humans , Molecular Mimicry , Nucleic Acid Conformation , Nucleic Acid Hybridization , Precipitin TestsABSTRACT
The human immunodeficiency virus type 1 transcriptional regulator Tat increases the efficiency of elongation, and complexes containing the cellular kinase CDK9 have been implicated in this process. CDK9 is part of the Tat-associated kinase TAK and of the elongation factor P-TEFb (positive transcription elongation factor-b), which consists minimally of CDK9 and cyclin T. TAK and P-TEFb are both able to phosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II, but their relationships to one another and to the stimulation of elongation by Tat are not well characterized. Here we demonstrate that human cyclin T1 (but not cyclin T2) interacts with the activation domain of Tat and is a component of TAK as well as of P-TEFb. Rodent (mouse and Chinese hamster) cyclin T1 is defective in Tat binding and transactivation, but hamster CDK9 interacts with human cyclin T1 to give active TAK in hybrid cells containing human chromosome 12. Although TAK is phosphorylated on both serine and threonine residues, it specifically phosphorylates serine 5 in the CTD heptamer. TAK is found in the nuclear and cytoplasmic fractions of human cells as a large complex (approximately 950 kDa). Magnesium or zinc ions are required for the association of Tat with the kinase. We suggest a model in which Tat first interacts with P-TEFb to form the TAK complex that engages with TAR RNA and the elongating transcription complex, resulting in hyperphosphorylation of the CTD on serine 5 residues.
Subject(s)
Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , Gene Products, tat/metabolism , HIV-1/metabolism , Protein Serine-Threonine Kinases/metabolism , RNA Polymerase II/metabolism , Amino Acid Sequence , Animals , CHO Cells , Cations , Cell Line , Cricetinae , Cyclin T , Cyclin-Dependent Kinase 9 , Gene Products, tat/genetics , Humans , Magnesium , Mice , Molecular Sequence Data , Phosphorylation , Positive Transcriptional Elongation Factor B , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Rodentia , Serine/metabolism , Spodoptera/cytology , Substrate Specificity , Threonine/metabolism , tat Gene Products, Human Immunodeficiency VirusABSTRACT
The proliferating cell nuclear antigen (PCNA) is an essential eukaryotic DNA replication factor that is transcriptionally regulated by the adenovirus oncoprotein E1A 243R. Inducibility of the human PCNA promoter by E1A 243R is conferred by the cis-acting PCNA E1A-responsive element (PERE), which associates with the ATF-1, cAMP response element-binding protein (CREB), and RFX1 transcription factors and is modulated by cellular proteins such as the coactivator CREB-binding protein (CBP) and tumor suppressor p107 (Labrie, C., Lee, B. H., and Mathews, M. B. (1995) Nucleic Acids Res. 23, 3732-3741; Lee, B. H., and Mathews, M. B. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4481-4486; Lee, B. H., Liu, M., and Mathews, M. B. (1998) J. Virol. 72, 1138-1145). RFX1 also forms a complex with sequences in the PCNA promoter of mouse and rat that share homology with the RFX1 consensus site. To explore the role of RFX1 in regulating the PCNA promoter, we examined the effects of mutations in the human PERE on RFX1 binding and gene expression. Mutations within the RFX1 consensus binding site reduced RFX1 binding, whereas mutations upstream of the site, or on its border, increased RFX1 binding. These mutations also affected the transcriptional activity of PCNA-chloramphenicol acetyltransferase reporter constructs in transient expression assays. The relative transcriptional activity of mutant PCNA promoters, both in the presence and absence of E1A 243R, was inversely related to their ability to complex with RFX1. These findings suggest that the binding of RFX1 is influenced by sequences outside its consensus binding site and that this transcription factor plays an inhibitory role in the regulation of PCNA gene expression.
Subject(s)
DNA-Binding Proteins/metabolism , Proliferating Cell Nuclear Antigen/genetics , Transcription Factors/metabolism , Adenovirus E1A Proteins/genetics , Binding Sites , DNA Probes , Gene Expression Regulation , Genes, Reporter , HeLa Cells , Humans , Mutation , Nuclear Proteins/analysis , Promoter Regions, Genetic , Regulatory Factor X Transcription Factors , Regulatory Factor X1 , Regulatory Sequences, Nucleic Acid , Sequence Homology, Nucleic AcidABSTRACT
RNA polymerase III (pol III) transcription generally terminates at a run of four or more thymidine (T) residues but some pol III genes contain runs of T residues that are not recognized as termination signals. Here, we investigate the terminal signal requirements that are operative in adenovirus virus-associated (VA) RNA genes. In the Xenopus 5 S RNA gene, efficient termination requires the T residues to be in a G+C-rich sequence context, but a run of five T residues in a G+C-rich context does not cause pol III termination when placed 30 nt downstream of the adenovirus-2 VA RNAI promoter in a VA-Tat chimeric gene. The failure of pol III to recognize this putative termination signal is not due to the chimeric nature of the gene or to the proximity of the signal to the promoter, but to its sequence context. Termination at the VA RNA gene site requires a T-rich sequence and is inhibited by the proximity of G residues, but is insensitive to the presence of A residues. The T-rich sequence need not be uninterrupted, however. In the VA RNA gene of the avian adenovirus, CELO, the first of two tandem termination signals contains an interrupted run of T residues, TTATT, which functions as a terminator with high (although not complete) efficiency. These findings, together with a survey of sequences neighboring the terminal site of other pol III genes, lead to the conclusion that pol III termination signals are more complex than hitherto recognized, and that sequence context requirements differ between members of the class 1 and class 2 families of pol III genes.
Subject(s)
RNA Polymerase III/genetics , RNA, Ribosomal, 5S/genetics , Transcription, Genetic/genetics , Animals , Aviadenovirus/genetics , Conserved Sequence/genetics , DNA Mutational Analysis , Dependovirus/genetics , Genes, Viral/genetics , Kinetics , Promoter Regions, Genetic/genetics , RNA Polymerase III/classification , RNA, Viral/genetics , Xenopus/geneticsABSTRACT
The promoter of the human proliferating cell nuclear antigen (PCNA) gene is activated by the adenovirus oncoprotein E1A 243R in HeLa cells. To understand the effect of this oncoprotein on PCNA expression in cells that are sensitive to oncogenic transformation by adenovirus, we studied the effect of E1A 243R on PCNA promoter-directed reporter gene expression in cloned rat embryo fibroblast (CREF) and primary baby rat kidney cells. In contrast to the results obtained in HeLa cells, E1A repressed the PCNA promoter in both cell-types. Promoter analysis identified a p53-responsive element that mediates E1A-induced repression. Repression required the intact N-terminus of E1A 243R, as shown by the ability of mutant E1A proteins to repress the promoter, and correlated with the p300-binding region of E1A. The adenovirus E1B 19K protein relieved repression by E1A 243R. These results reveal dual pathways for induction of this essential DNA replication factor and suggest a mechanism for oncogenic cooperativity between the E1A and E1B oncoproteins.