Reduction In RNA Synthesis Research Articles

Structural basis of transcription reduction by a promoter-proximal +1 nucleosome

At active human genes, the+1 nucleosome is located downstream of the RNA polymerase II (RNA Pol II) pre-initiationcomplex (PIC). However, at inactive genes, the+1 nucleosome is found further upstream, at a promoter-proximal location. Here, we establish a model system to show that a promoter-proximal+1 nucleosome can reduce RNA synthesis invivo and invitro, and we analyze its structural basis. We find that the PIC assembles normally when the edge of the+1 nucleosome is located 18 base pairs (bp) downstream of the transcription start site (TSS). However, when the nucleosome edge is located further upstream, only 10bp downstream of the TSS, the PIC adopts an inhibited state. The transcription factor IIH (TFIIH) shows a closed conformation and its subunit XPB contacts DNA with only one of its two ATPase lobes, inconsistent with DNA opening. These results provide a mechanism for nucleosome-dependent regulation of transcription initiation.

Specific Residues in the C-Terminal Domain of the Human Metapneumovirus Phosphoprotein Are Indispensable for Formation of Viral Replication Centers and Regulation of the Function of the Viral Polymerase Complex.

Human metapneumovirus (HMPV) is a negative-strand RNA virus that frequently causes respiratory tract infections in infants, the elderly, and the immunocompromised. A hallmark of HMPV infection is the formation of membraneless, liquid-like replication and transcription centers in the cytosol termed inclusion bodies (IBs). The HMPV phosphoprotein (P) and nucleoprotein (N) are the minimal viral proteins necessary to form IB-like structures, and both proteins are required for the viral polymerase to synthesize RNA during infection. HMPV P is a homotetramer with regions of intrinsic disorder and has several known and predicted phosphorylation sites of unknown function. In this study, we found that the P C-terminal intrinsically disordered domain (CTD) must be present to facilitate IB formation with HMPV N, while either the N-terminal intrinsically disordered domain or the central oligomerization domain was dispensable. Alanine substitution at a single tyrosine residue within the CTD abrogated IB formation and reduced coimmunoprecipitation with HMPV N. Mutations to C-terminal phosphorylation sites revealed a potential role for phosphorylation in regulating RNA synthesis and P binding partners within IBs. Phosphorylation mutations which reduced RNA synthesis in a reporter assay produced comparable results in a recombinant viral rescue system, measured as an inability to produce infectious viral particles with genomes containing these single P mutations. This work highlights the critical role HMPV P plays in facilitating a key step of the viral life cycle and reveals the potential role for phosphorylation in regulating the function of this significant viral protein. IMPORTANCE Human metapneumovirus (HMPV) infects global populations, with severe respiratory tract infections occurring in infants, the elderly, and the immunocompromised. There are currently no FDA-approved therapeutics available to prevent or treat HMPV infection. Therefore, understanding how HMPV replicates is vital for the identification of novel targets for therapeutic development. During HMPV infection, viral RNA synthesis proteins localize to membraneless structures called inclusion bodies (IBs), which are sites of genome replication and transcription. The HMPV phosphoprotein (P) is necessary for IBs to form and for the virus to synthesize RNA, but it is not known how this protein contributes to IB formation or if it is capable of regulating viral replication. We show that the C-terminal domain of P is the location of a molecular interaction driving IB formation and contains potential phosphorylation sites where amino acid charge regulates the function of the viral polymerase complex.

Escherichia coli protein synthesis is limited by mRNA availability rather than ribosomal capacity during phosphate starvation.

Protein synthesis is the most energetically costly process in the cell. Consequently, it is a tightly regulated process, and regulation of the resources allocated to the protein synthesis machinery is at the heart of bacterial growth optimization theory. However, the molecular mechanisms that result in dynamic downregulation of protein synthesis in response to nutrient starvation are not well described. Here, we first quantify the Escherichia coli response to phosphate starvation at the level of accumulation rates for protein, RNA and DNA. Escherichia coli maintains a low level of protein synthesis for hours after the removal of phosphate while the RNA contents decrease, primarily as a consequence of ribosomal RNA degradation combined with a reduced RNA synthesis rate. To understand the molecular basis for the low protein synthesis rate of phosphate-starved cells, template mRNA for translation was overproduced in the form of a highly induced long-lived mRNA. Remarkably, starved cells increased the rate of protein synthesis and reduced the rate of ribosomal RNA degradation upon mRNA induction. These observations suggest that protein synthesis in phosphate-starved cells is primarily limited by the availability of template, and does not operate at the maximum capacity of the ribosomes. We suggest that mRNA limitation is an adaptive response to phosphate starvation that prevents the deleterious consequences of overcommitting resources to protein synthesis. Moreover, our results support the model that degradation of ribosomal RNA occurs as a consequence of the availability of idle ribosomal subunits.

Distinct transcription kinetics of pluripotent cell states

Mouse embryonic stem cells (mESCs) can adopt naïve, ground, and paused pluripotent states that give rise to unique transcriptomes. Here, we use transient transcriptome sequencing (TT‐seq) to define both coding and non‐coding transcription units (TUs) in these three pluripotent states and combine TT‐seq with RNA polymerase II occupancy profiling to unravel the kinetics of RNA metabolism genome‐wide. Compared to the naïve state (serum), RNA synthesis and turnover rates are globally reduced in the ground state (2i) and the paused state (mTORi). The global reduction in RNA synthesis goes along with a genome‐wide decrease of polymerase elongation velocity, which is related to epigenomic features and alterations in the Pol II termination window. Our data suggest that transcription activity is the main determinant of steady state mRNA levels in the naïve state and that genome‐wide changes in transcription kinetics invoke ground and paused pluripotent states.

LEO1 is a partner for Cockayne syndrome protein B (CSB) in response to transcription-blocking DNA damage.

Cockayne syndrome (CS) is an autosomal recessive genetic disorder characterized by photosensitivity, developmental defects, neurological abnormalities, and premature aging. Mutations in CSA (ERCC8), CSB (ERCC6), XPB, XPD, XPG, XPF (ERCC4)and ERCC1 can give rise to clinical phenotypes resembling classic CS. Using a yeast two-hybrid (Y2H) screening approach, we identified LEO1 (Phe381-Ser568 region) as an interacting protein partner of full-length and C-terminal (Pro1010-Cys1493) CSB in two independent screens. LEO1 is a member of the RNA polymerase associated factor 1 complex (PAF1C) with roles in transcription elongation and chromatin modification. Supportive of the Y2H results, purified, recombinant LEO1 and CSB directly interact in vitro, and the two proteins exist in a common complex within human cells. In addition, fluorescently tagged LEO1 and CSB are both recruited to localized DNA damage sites in human cells. Cell fractionation experiments revealed a transcription-dependent, coordinated association of LEO1 and CSB to chromatin following either UVC irradiation or cisplatin treatment of HEK293T cells, whereas the response to menadione was distinct, suggesting that this collaboration occurs mainly in the context of bulky transcription-blocking lesions. Consistent with a coordinated interaction in DNA repair, LEO1 knockdown or knockout resulted in reduced CSB recruitment to chromatin, increased sensitivity to UVC light and cisplatin damage, and reduced RNA synthesis recovery and slower excision of cyclobutane pyrimidine dimers following UVC irradiation; the absence of CSB resulted in diminished LEO1 recruitment. Our data indicate a reciprocal communication between CSB and LEO1 in the context of transcription-associated DNA repair and RNA transcription recovery.

Thirdhand smoke exposure causes replication stress and impaired transcription in human lung cells.

Thirdhand cigarette smoke (THS) is a newly described toxin that lingers in the indoor environment long after cigarettes have been extinguished. Emerging results from both cellular and animal model studies suggest that THS is a potential human health hazard. DNA damage derived from THS exposure could have genotoxic consequences that would lead to the development of diseases. However, THS exposure-induced interference with fundamental DNA transactions such as replication and transcription, and the role of DNA repair in ameliorating such effects, remain unexplored. Here, we found that THS exposure increased the percentage of cells in S-phase, suggesting impaired S-phase progression. Key DNA damage response proteins including RPA, ATR, ATM, CHK1, and BRCA1 were activated in lung cells exposed to THS, consistent with replication stress. In addition, THS exposure caused increased 53BP1 foci, indicating DNA double-strand break induction. Consistent with these results, we observed increased micronuclei formation, a marker of genomic instability, in THS-exposed cells. Exposure to THS also caused a significant increase in phosphorylated RNA Polymerase II engaged in transcription elongation, suggesting an increase in transcription-blocking lesions. In agreement with this conclusion, ongoing RNA synthesis was very significantly reduced by THS exposure. Loss of nucleotide excision repair exacerbated the reduction in RNA synthesis, suggesting that bulky DNA adducts formed by THS are blocks to transcription. The adverse impact on both replication and transcription supports genotoxic stress as a result of THS exposure, with important implications for both cancer and other diseases.

Zinc Finger-Containing Cellular Transcription Corepressor ZBTB25 Promotes Influenza Virus RNA Transcription and Is a Target for Zinc Ejector Drugs.

Influenza A virus (IAV) replication relies on an intricate interaction between virus and host cells. How the cellular proteins are usurped for IAV replication remains largely obscure. The aim of this study was to search for novel and potential cellular factors that participate in IAV replication. ZBTB25, a transcription repressor of a variety of cellular genes, was identified by an RNA interference (RNAi) genomic library screen. Depletion of ZBTB25 significantly reduced IAV production. Conversely, overexpression of ZBTB25 enhanced it. ZBTB25 interacted with the viral RNA-dependent RNA polymerase (RdRp) protein and modulated its transcription activity. In addition, ZBTB25 also functioned as a viral RNA (vRNA)-binding protein, binding preferentially to the U-rich sequence within the 5' untranslated region (UTR) of vRNA. Both protein-protein and protein-RNA interactions involving ZBTB25 facilitated viral RNA transcription and replication. In addition, ZBTB25 suppressed interferon production, further enhancing viral replication. ZBTB25-associated functions required an intact zinc finger domain and posttranslational SUMO-1 modification of ZBTB25. Furthermore, treatment with disulfiram (a zinc ejector) of ZBTB25-overexpressing cells showed significantly reduced IAV production as a result of reduced RNA synthesis. Our findings indicate that IAV usurps ZBTB25 for IAV RNA synthesis and serves as a novel and potential therapeutic antiviral target.IMPORTANCE IAV-induced seasonal influenza causes severe illness and death in high-risk populations. However, IAV has developed resistance to current antiviral drugs due to its high mutation rate. Therefore, development of drugs targeting cellular factors required for IAV replication is an attractive alternative for IAV therapy. Here, we discovered a cellular protein, ZBTB25, that enhances viral RdRp activity by binding to both viral RdRp and viral RNA to stimulate viral RNA synthesis. A unique feature of ZBTB25 in the regulation of viral replication is its dual transcription functions, namely, promoting viral RNA transcription through binding to the U-rich region of vRNA and suppressing cellular interferon production. ZBTB25 contains a zinc finger domain that is required for RNA-inhibitory activity by chelating zinc ions. Disulfiram treatment disrupts the zinc finger functions, effectively repressing IAV replication. Based on our findings, we demonstrate that ZBTB25 regulates IAV RNA transcription and replication and serves as a promising antiviral target for IAV treatment.

Mutations conferring a noncytotoxic phenotype on chikungunya virus replicons compromise enzymatic properties of nonstructural protein 2.

Chikungunya virus (CHIKV) (genus Alphavirus) has a positive-sense RNA genome. CHIKV nonstructural protein 2 (nsP2) proteolytically processes the viral nonstructural polyprotein, possesses nucleoside triphosphatase (NTPase), RNA triphosphatase, and RNA helicase activities, and induces cytopathic effects in vertebrate cells. Although alphaviral nsP2 mutations can result in a noncytotoxic phenotype, the effects of such mutations on nsP2 enzymatic activities are not well understood. In this study, we introduced a P718G (PG) mutation and selected for additional mutations in CHIKV nsP2 that resulted in a CHIKV replicon with a noncytotoxic phenotype in BHK-21 cells. Combinations of PG and either an E117K (EK) substitution or a GEEGS sequence insertion after residue T647 (5A) markedly reduced RNA synthesis; however, neither PG nor 5A prevented nsP2 nuclear translocation. Introducing PG into recombinant nsP2 inhibited proteolytic cleavage of nsP1/nsP2 and nsP3/nsP4 sites, reduced GTPase and RNA helicase activities, and abolished RNA stimulation of GTPase activity. 5A and EK modulated the effects of PG. However, only the RNA helicase activity of nsP2 was reduced by both of these mutations, suggesting that defects in this activity may be linked to a noncytotoxic phenotype. These results increase our understanding of the molecular basis for the cytotoxicity that accompanies alphaviral replication. Furthermore, adaptation of the CHIKV replicon containing both 5A and PG allowed the selection of a CHIKV replicon with adaptive mutations in nsP1 and nsP3 that enable persistence in human cell line. Such cell lines represent valuable experimental systems for discovering host factors and for screening inhibitors of CHIKV replication at lower biosafety levels. CHIKV is a medically important pathogen that causes febrile illness and can cause chronic arthritis. No approved vaccines or antivirals are available for CHIKV. The attenuation of CHIKV is critical to the establishment of experimental systems that can be used to conduct virus replication studies at a lower biosafety level. We applied a functional selection approach to develop, for the first time, a noncytotoxic CHIKV replicon capable of persisting in human cell lines. We anticipate that this safe and efficient research tool will be valuable for screening CHIKV replication inhibitors and for identifying and analyzing host factors involved in viral replication. We also analyzed, from virological and protein biochemistry perspectives, the functional defects caused by mutations conferring noncytotoxic phenotypes; we found that all known enzymatic activities of CHIKV nsP2, as well as its RNA-binding capability, were compromised by these mutations, which led to a reduced capacity for replication.

Recent advances in gastric cancer.

Recent advances in gastric cancer.

Investigations on the Tobacco Necrosis Virus D p60 Replicase Protein

Tobacco Necrosis Virus D (TNV-D), in the genus Betanecrovirus (family Tombusviridae), possesses a single-stranded, positive-sense RNA genome containing six open reading frames (ORFs). Two 5'-proximal ORFs (1 and 2) encode overlapping polypeptides of 22 and 82 kDa (p22 and p82, respectively) which are both required for replication. The p22 auxiliary protein contains no replication motifs but the C-terminal region, downstream of a leaky stop codon, encodes a 60 kDa polypeptide (p60) which contains conserved RNA-dependent RNA polymerase (RdRP) motifs. Here we have expressed and purified recombinant p60 and show that in vitro it binds and efficiently synthesises both TNV-D RNA and Satellite tobacco necrosis virus C RNA. Alanine scanning mutagenesis of conserved amino acids in characteristic motifs in p60 revealed that some mutations significantly reduced RNA synthesis but mutating the second asparagine residue in the conserved GDD box was lethal. The effects of mutating identical amino acids in p60 on virus replication in vivo were examined in Nicotiana benthamiana plants following infection with RNA transcribed from wild type (wt) and mutant constructs. In inoculated leaves the behaviour of the mutants paralleled the in vitro data but systemic infection was precluded in all but one mutant which had reverted to wt. This study is the first to demonstrate the nucleic acid-binding and synthetic capabilities of a betanecrovirus polymerase.

ΔNp63α promotes cellular quiescence via induction and activation of Notch3

Genetic analysis of TP63 indicates that ΔNp63 isoforms are required for preservation of self-renewing capacity in the stem cell compartments of diverse epithelial structures; however, the underlying cellular and molecular mechanisms remain incompletely defined. Cellular quiescence is a common feature of adult stem cells that may account for their ability to retain long-term replicative capacity while simultaneously limiting cellular division. Similarly, quiescence within tumor stem cell populations may represent a mechanism by which these populations evade cytotoxic therapy and initiate tumor recurrence. Here, we present evidence that ΔNp63α, the predominant TP63 isoform in the regenerative compartment of diverse epithelial structuresm, promotes cellular quiescence via activation of Notch signaling. In HC11 cells, ectopic ΔNp63α mediates a proliferative arrest in the 2N state coincident with reduced RNA synthesis characteristic of cellular quiescence. Additionally, ΔNp63α and other quiescence-inducing stimuli enhanced expression of Notch3 in HC11s and breast cancer cell lines, and ectopic expression of the Notch3 intracellular domain (N3ICD) was sufficient to cause accumulation in G0/G1 and increased expression of two genes associated with quiescence, Hes1 and Mxi1. Pharmacologic inhibition of Notch signaling or shRNA-mediated suppression of Notch3 were sufficient to bypass quiescence induced by ΔNp63α and other quiescence-inducing stimuli. These studies identify a novel mechanism by which ΔNp63α preserves long-term replicative capacity by promoting cellular quiescence and identify the Notch signaling pathway as a mediator of multiple quiescence-inducing stimuli, including ΔNp63α expression.

Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1

The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser2. This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology.

Seven nucleotide changes characteristic of the hepatitis C virus genotype 3 5′ untranslated region: correlation with reduced in vitro replication

Computer analysis of 158 hepatitis C virus (HCV) 5' untranslated region (5' UTR) sequences from the six genotypes showed that the 5' UTR from genotype 3 displays seven specific non-contiguous nucleotide changes, at positions 8, 13, 14, 70, 97, 203 and 224. The purpose of this study was to investigate the impact of these changes on translation and replication activities. Indeed, these modifications could alter both the internal ribosome entry site (IRES) present in the 5' UTR of the plus-strand RNA and the 3' end of the minus strand involved in the initiation of plus-strand RNA synthesis. We found that the genotype 3-specific nucleotide changes do not modify the in vitro or ex vivo translation activity of the corresponding IRES, in comparison with that of genotype 1. In contrast, in vitro replication from the minus-strand RNA is eight times less efficient for genotype 3 than for genotype 1 RNA, suggesting the involvement of some nucleotide changes in the reduction of RNA synthesis. Nucleotides 13, 14 and 224 were found to be responsible for this effect. Moreover, a reduced replicative activity was confirmed ex vivo for genotype 3, but to a lesser extent than that observed in vitro, using an RNA minigenome.

Stimulation of picornavirus replication by the poly(A) tail in a cell-free extract is largely independent of the poly(A) binding protein (PABP)

Picornavirus infectivity is dependent on the RNA poly(A) tail, which binds the poly(A) binding protein (PABP). PABP was reported to stimulate viral translation and RNA synthesis. Here, we studied encephalomyocarditis virus (EMCV) and poliovirus (PV) genome expression in Krebs-2 and HeLa cell-free extracts that were drastically depleted of PABP (96%-99%). Although PABP depletion markedly diminished EMCV and PV internal ribosome entry site (IRES)-mediated translation of a polyadenylated luciferase mRNA, it displayed either no (EMCV) or slight (PV) deleterious effect on the translation of the full-length viral RNAs. Moreover, PABP-depleted extracts were fully competent in supporting EMCV and PV RNA replication and virus assembly. In contrast, removing the poly(A) tail from EMCV RNA dramatically reduced RNA synthesis and virus yields in cell-free reactions. The advantage conferred by the poly(A) tail to EMCV synthesis was more pronounced in untreated than in nuclease-treated extract, indicating that endogenous cellular mRNAs compete with the viral RNA for a component(s) of the RNA replication machinery. These results suggest that the poly(A) tail functions in picornavirus replication largely independent of PABP.

The BRO proteins of Bombyx mori nucleopolyhedrovirus are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway

The BRO proteins of Bombyx mori nucleopolyhedrovirus (BmNPV) display a biphasic pattern of intracellular localization during infection. At early times, they reside in the nucleus but then show both cytoplasmic and nuclear localization as the infection proceeds. Therefore, we examined the possibility of nuclear export. Using inhibitors, we reveal that BmNPV BRO proteins shuttle between the nucleus and cytoplasm. Mutations on the leucine-rich region of BRO proteins resulted in nuclear accumulation of transiently expressed proteins, suggesting that this region functions as a CRM1-dependent nuclear export signal (NES). On the contrary, mutant BRO-D with an altered NES did not show nuclear accumulation in infected cells, although protein production seemed to be reduced. RT-PCR analysis showed that the lower level of protein production was due to a reduction in RNA synthesis. Taken together, our results suggest that BRO proteins are nucleocytoplasmic shuttling proteins that utilize the CRM1-mediated nuclear export pathway.

Site-directed mutagenesis of the Nidovirus replicative endoribonuclease NendoU exerts pleiotropic effects on the arterivirus life cycle.

The highly conserved NendoU replicative domain of nidoviruses (arteriviruses, coronaviruses, and roniviruses) belongs to a small protein family whose cellular branch is prototyped by XendoU, a Xenopus laevis endoribonuclease involved in nucleolar RNA processing. Recently, sequence-specific in vitro endoribonuclease activity was demonstrated for the NendoU-containing nonstructural protein (nsp) 15 of several coronaviruses. To investigate the biological role of this novel enzymatic activity, we have characterized a comprehensive set of arterivirus NendoU mutants. Deleting parts of the NendoU domain from nsp11 of equine arteritis virus was lethal. Site-directed mutagenesis of conserved residues exerted pleiotropic effects. In a first-cycle analysis, replacement of two conserved Asp residues in the C-terminal part of NendoU rendered viral RNA synthesis and virus production undetectable. In contrast, mutagenesis of other conserved residues, including two putative catalytic His residues that are absolutely conserved in NendoU and cellular homologs, produced viable mutants displaying reduced plaque sizes (20 to 80% reduction) and reduced yields of infectious progeny of up to 5 log units. A more detailed analysis of these mutants revealed a moderate reduction in RNA synthesis, with subgenomic RNA synthesis consistently being more strongly affected than genome replication. Our data suggest that the arterivirus nsp11 is a multifunctional protein with a key role in viral RNA synthesis and additional functions in the viral life cycle that are as yet poorly defined.

Nuclear organization and dynamics of transcription sites in rat sensory ganglia neurons detected by incorporation of 5′-fluorouridine into nascent RNA

In this study we have used the transcription assay with 5′-fluorouridine incorporation into nascent RNA to analyze the nuclear organization and dynamics of transcription sites in rat trigeminal ganglia neurons. The 5′-FU administrated by i.p. injection was successfully incorporated into nuclear domains containing actively transcribing genes of trigeminal neurons. 5′-Fluorouridine RNA-labeling was detected with immunocytochemistry at light and electron microscopy levels. The 5′-fluorouridine incorporation sites were detected in the nucleolus, particularly on the dense fibrillar component, and in numerous transcription foci spread throughout the euchromatin regions, without preferential positioning at the nuclear periphery or in the nuclear interior. Double labeling experiments to combine 5′-fluorouridine incorporation with molecular markers of nuclear compartments showed the absence of transcription sites in Cajal bodies and nuclear speckles of splicing factors. Similarly, no 5′-fluorouridine labeling was detected in well-characterized chromatin silencing domain, the telomeric heterochromatin. The specificity and sensitivity of the run-on transcription assay in trigeminal ganglia neurons was verified by the i.p. administration of the transcription inhibitor actinomycin D. The dramatic reduction in RNA synthesis upon actinomycin D treatment was associated with two important cellular events, heterochromatin silencing and formation of DNA damage/repair nuclear foci, demonstrated by the expression of tri-methylated histone H4 and phosphorylated H2AX, respectively. 5′-Fluorouridine incorporation in animal models provides a useful tool to investigate the organization of gene expression in mammalian neurons in both normal physiology and experimental pathology systems.

Functional Analysis of RNA Binding by the Hepatitis C Virus RNA-dependent RNA Polymerase

Protein-RNA interaction plays a critical role in regulating RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp). RNAs of 7 nucleotides (nt) or longer had affinities 5-fold better than an RNA of 5 nt, suggesting a minimal length required for binding. To identify RNA contact sites on the HCV RdRp, a biotinylated 7-nt RNA capable of directing de novo initiation was used in a process that coupled reversible formaldehyde cross-linking, RNA affinity chromatography, and mass spectrometry. By this process, we identified 18 peptides cross-linked to the 7-nt RNA. When these identified peptides were overlaid on the three-dimensional structures of NS5B, most mapped to the fingers subdomain, connecting loops between fingers and thumb subdomains and in the putative RNA binding channel. Two of the identified peptides resided in the active site cavity of the RdRp. Recombinant HCV RdRp with single residue changes in likely RNA contact sites were generated and characterized for effects on HCV RdRp activity. Mutant proteins had significant effects on cross-linking to 7-nt RNA and reduced RNA synthesis in vitro by 2- to 20-fold compared with wild type protein. When the mutations were tested for the replication of HCV RNA in the context of the cells transfected with the HCV subgenomic replicon, all except one prevented colony formation, indicating a defect in HCV RNA replication. These biochemical and functional analyses identified a number of residues in the HCV RdRp that are important for HCV RNA synthesis.

Transcription inhibition by flavopiridol: mechanism of chronic lymphocytic leukemia cell death

AbstractFlavopiridol is active against chronic lymphocytic leukemia (CLL) cells in vitro and in the treatment of advanced stage disease, but the mechanisms of these actions remain unclear. Originally developed as a general cyclin-dependent kinase inhibitor, flavopiridol is a potent transcriptional suppressor through the inhibition of positive transcription elongation factor b (P-TEFb; CDK9/cyclin T). P-TEFb phosphorylates the C-terminal domain (CTD) of RNA polymerase II to promote transcriptional elongation. Because most CLL cells are not actively cycling, and their viability is dependent upon the continuous expression of antiapoptotic proteins, we hypothesized that flavopiridol induces apoptosis in CLL cells through the transcriptional down-regulation of such proteins. This study demonstrated that flavopiridol inhibited the phosphorylation of the CTD of RNA polymerase II in primary CLL cells and reduced RNA synthesis. This was associated with a decline of the transcripts and the levels of short-lived antiapoptotic proteins such as myeloid cell leukemia 1 (Mcl-1), and resulted in the induction of apoptosis. The B-cell lymphoma 2 (Bcl-2) protein level remained stable, although its mRNA was consistently reduced, suggesting that the outcome of transcriptional inhibition by flavopiridol is governed by the intrinsic stability of the individual transcripts and proteins. The dependence of CLL-cell survival on short-lived oncoproteins may provide the biochemical basis for the therapeutic index in response to flavopiridol. (Blood. 2005;106:2513-2519)

Functional Interaction of Heterogeneous Nuclear Ribonucleoprotein C with Poliovirus RNA Synthesis Initiation Complexes

We had previously demonstrated that a cellular protein specifically interacts with the 3' end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione S-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3D(pol), as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1DeltaC) diminished these protein-protein interactions. When GST/hnRNP C1DeltaC was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.