Induction of p53, p21 and apoptosis by silencing the NF90/NF45 complex in human papilloma virus-transformed cervical carcinoma cells.

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.

The machine that decodes the genome.

The Cold Spring Harbor Symposium on The Ribosome was held on 31 May - 5 June in Cold Spring Harbor, NY. USA.

Three RNA polymerase II carboxyl-terminal domain kinases display distinct substrate preferences.

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.

Human and rodent transcription elongation factor P-TEFb: interactions with human immunodeficiency virus type 1 tat and carboxy-terminal domain substrate.

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.

Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro.

P-TEFb is a key regulator of the process controlling the processivity of RNA polymerase II and possesses a kinase activity that can phosphorylate the carboxy-terminal domain of the largest subunit of RNA polymerase II. Here we report the cloning of the small subunit of Drosophila P-TEFb and the finding that it encodes a Cdc2-related protein kinase. Sequence comparison suggests that a protein with 72% identity, PITALRE, could be the human homolog of the Drosophila protein. Functional homology was suggested by transcriptional analysis of an RNA polymerase II promoter with HeLa nuclear extract depleted of PITALRE. Because the depleted extract lost the ability to produce long DRB-sensitive transcripts and this loss was reversed by the addition of purified Drosophila P-TEFb, we propose that PITALRE is a component of human P-TEFb. In addition, we found that PITALRE associated with the activation domain of HIV-1 Tat, indicating that P-TEFb is a Tat-associated kinase (TAK). An in vitro transcription assay demonstrates that the effect of Tat on transcription elongation requires P-TEFb and suggests that the enhancement of transcriptional processivity by Tat is attributable to enhanced function of P-TEFb on the HIV-1 LTR.

Synthesis and purification of single-stranded RNA for use in experiments with PKR and in cell-free translation systems.

The biosynthesis of RNA in vitro using bacteriophage RNA polymerases has opened up many avenues of research. Large amounts of specific RNA species can be readily produced but small amounts of contaminants that are simultaneously generated can interfere with biological assays, PKR, a ribosome-associated and double-stranded (ds) RNA-dependent protein kinase, is an important regulator of the initiation of protein synthesis. It can be activated by very low concentrations of dsRNA and inhibited by small structured RNAs or high concentrations of dsRNA. The best-studied inhibitor of PKR activation is adenovirus VA RNA1. Its gene was cloned into a plasmid under the control of the T7 RNA polymerase promoter, and the optimization of VA RNA transcription is described. A dsRNA by-product of the transcription reaction activates PKR in kinase autophosphorylation assays, and hence a purification protocol that allows the separation and removal of dsRNA contaminants was developed. A scheme to analyze the RNA product with specific nucleases is discussed. In a reticulocyte cell-free translation system the activation of PKR by dsRNA contaminating a synthetic mRNA preparation is likely to lead to shut-off of translation. An assay to directly visualize and measure the level of PKR phosphorylation in the lysate is detailed.

Structural features of adenovirus 2 virus-associated RNA required for binding to the protein kinase DAI.

The double-stranded RNA activated protein kinase DAI contains an RNA binding domain consisting of two copies of a double-stranded RNA binding motif. We have investigated the role of RNA structure in the interaction between DAI and the structured single-stranded RNA, adenovirus VA RNAI, which inhibits DAI activation. Mutations in the apical stem, terminal stem, and central domain of the RNA were tested to assess the contribution of these elements to DAI binding in vitro. The data demonstrate that over half a turn of intact apical stem is required for the interaction and that there is a correlation between the binding of apical stem mutants and their ability to function both in vivo and in vitro. There was also evidence of preference for GC-rich sequence in the proximal region of the apical stem. In the central domain the correlation between binding and function of mutant RNAs was poor, suggesting that at least some of this region plays no direct role in binding to DAI, despite its functional importance. Exceptionally, central domain mutations that encroached on the phylogenetically conserved stem 4 of VA RNA disrupted binding, and complementary mutations in this sequence partially restored binding. Measurement of the binding of wild-type VA RNAI to DAI and p20, a truncated form of the protein containing the RNA binding domains alone, under various ionic conditions imply that the major interactions are electrostatic and occur via the protein's RNA binding domain. However, differences between full-length DAI and p20 in their binding to mutants in the conserved stem suggest that regions outside the RNA binding domain also participate in the binding. The additional interactions are likely to be non-ionic, and may be important for preventing DAI activation during virus infection.

Mutational analysis of the central domain of adenovirus virus-associated RNA mandates a revision of the proposed secondary structure.

Protein synthesis in adenovirus-infected cells is regulated during the late phase of infection. The rate of initiation is maintained by a small viral RNA, virus-associated (VA) RNAI, which prevents the phosphorylation of eukaryotic initiation factor eIF-2 by a double-stranded RNA-activated protein kinase, DAI. On the basis of nuclease sensitivity analysis, a secondary-structure model was proposed for VA RNA. The model predicts a complex stem-loop structure in the central part of the molecule, the central domain, joining two duplexed stems. The central domain is required for the inhibition of DAI activation and participates in the binding of VA RNA to DAI. To assess the significance of the postulated stem-loop structure in the central domain, we generated compensating, deletion, and substitution mutations. A substitution mutation which disrupts the structure in the central domain abolishes VA RNA function in vitro and in vivo. Base-compensating mutations failed to restore the function or structure of the mutant, implying that the stem-loop structure may not exist. To confirm this observation, we tested mutants with alterations in the hypothetical loop and short stem that constitute the main features of the wild-type model structure. The upper part of the hypothetical loop could be deleted without abolishing the ability of the RNA to block DAI activation in vitro, whereas other loop mutations were deleterious for function and caused major rearrangements in the molecule. Base-compensating mutations in the stem did not restore the expected base pairing, even though the mutant RNAs were still functional in vitro. Surprisingly, a mutant with a noncompensating substitution mutation in the stem was more effective than wild-type VA RNAI in DAI inhibition assays but was ineffective in vivo. The structural and functional consequences of these mutations do not support the proposed model structure for the central domain, and we therefore suggest an alternative structure in which tertiary interactions may play a significant role in shaping the specificity of VA RNA function in the infected cell. Discrepancies between the functionality of mutant forms of VA RNA in vivo and in vitro are consistent with the existence of additional roles for VA RNA in the cell.

Role of the apical stem in maintaining the structure and function of adenovirus virus-associated RNA.

Adenovirus virus-associated (VA) RNAI maintains efficient protein synthesis during the late phase of infection by preventing the activation of the double-stranded-RNA-dependent protein kinase, DAI. A secondary structure model for VA RNAI predicts the existence of two stems joined by a complex stem-loop structure, the central domain. The structural consequences of mutations and compensating mutations introduced into the apical stem lend support to this model. In transient expression assays for VA RNA function, foreign sequences inserted into the apical stem were fully tolerated provided that the stem remained intact. Mutants in which the base of the apical stem was disrupted retained partial activity, but truncation of the apical stem abolished the ability of the RNA to block DAI activation in vitro, suggesting that the length and position of the stem are both important for VA RNA function. These results imply that VA RNAI activity depends on secondary structure at the top of the apical stem as well as in the central domain and are consistent with a two-step mechanism involving DAI interactions with both the apical stem and the central domain.

Removal of double-stranded contaminants from RNA transcripts: synthesis of adenovirus VA RNAI from a T7 vector.

Bacteriophage RNA polymerases are widely used to synthesize defined RNAs on a large scale in vitro. Unfortunately, the RNA product contains a small proportion of contaminating RNAs, including complementary species, which can lead to errors of interpretation. We cloned the gene encoding Ad2 VA RNAI into a vector containing a T7 RNA polymerase promoter in order to generate large quantities of VA RNA for the study of its interaction with the dsRNA-dependent protein kinase DAI. Exact copies of VA RNAI were synthesized efficiently, but were contaminated with small amounts of dsRNA which activated DAI and confounded interpretation of kinase assays. We therefore developed a method to remove the dsRNA contaminants, allowing VA RNAI and mutants to be tested for their ability to activate or inhibit DAI. This method appears to be generally applicable.