Thermodynamic profiling of HIV RREIIB RNA-zinc finger interactions.
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ABSTRACT: The interactions between the HIV Rev-responsive element (RRE) RNA and the HIV regulatory protein Rev, are crucial for the HIV life-cycle. Earlier, we showed that single C(2)H(2) zinc fingers (znfs) have the same binding site as the Rev peptide and exhibit nanomolar affinity. In this study, the specific role of amino acid side chains and molecular processes involved with complex formation were investigated by perturbation of the binding energetics via changes in temperature, pH, buffers, and salt concentrations, as well as znf and RNA mutations, by isothermal titration calorimetry. Interestingly, despite the large cationic charge on the znfs, the number of interactions with the RNA phosphate backbone was lower than intuitively expected. The presence of binding induced protonation was established by ITC and localized by NMR to a histidine on the znf beta-sheet. The DeltaC(p) of znf-RNA binding was observed to be substantially negative and could not be accounted for by conventional solvent-accessible surface area models. An alternative model, based on the extent of hydrogen bond changes as a result of differences in ligand-induced water displacement at the binding site, provided reasonable explanation of the trends in DeltaC(p), as well as DeltaH and DeltaS. Our studies show that incorporation of favorable interactions at the solvent-excluded binding interface can be used to alleviate the unfavorable enthalpic penalties of displacing water molecules from the hydrated RNA surface.
Project description:Messenger RNA transcripts are coated from cap to tail with a dynamic combination of RNA binding proteins that process, package, and ultimately regulate the fate of mature transcripts. One class of RNA binding proteins essential for multiple aspects of mRNA metabolism consists of the poly(A) binding proteins. Previous studies have concentrated on the canonical RNA recognition motif-containing poly(A) binding proteins as the sole family of poly(A)-specific RNA binding proteins. In this study, we present evidence for a previously uncharacterized poly(A) recognition motif consisting of tandem CCCH zinc fingers. We have probed the nucleic acid binding properties of a yeast protein, Nab2, that contains this zinc finger motif. Results of this study reveal that the seven tandem CCCH zinc fingers of Nab2 specifically bind to polyadenosine RNA with high affinity. Furthermore, we demonstrate that a human protein, ZC3H14, which contains CCCH zinc fingers homologous to those found in Nab2, also specifically binds polyadenosine RNA. Thus, we propose that these proteins are members of an evolutionarily conserved family of poly(A) RNA binding proteins that recognize poly(A) RNA through a fundamentally different mechanism than previously characterized RNA recognition motif-containing poly(A) binding proteins.
Project description:The interactions of the human double-stranded RNA-binding zinc finger protein JAZ with RNA or DNA were investigated using electrophoretic mobility-shift assays, isothermal calorimetry, and nuclear magnetic resonance spectroscopy. Consistent with previous reports, JAZ has very low affinity for duplex DNA or single-stranded RNA, but it binds preferentially to double-stranded RNA (dsRNA) with no detectable sequence specificity. The affinity of JAZ for dsRNA is unaffected by local structural features such as loops, overhangs, and bulges, provided a sufficient length of reasonably well-structured A-form RNA (about 18 bp for a single zinc finger) is present. Full-length JAZ contains four Cys2His2 zinc fingers (ZF1-4) and has the highest apparent affinity for dsRNA; two-finger constructs ZF12 and ZF23 have lower affinity, and ZF34 binds even more weakly. The fourth zinc finger, ZF4, has no measurable RNA-binding affinity. Single zinc finger constructs ZF1, ZF2, and ZF3 show evidence for multiple-site binding on the minimal RNA. Fitting of quantitative NMR titration and isothermal calorimetry data to a two-site binding model gave Kd1 ∼ 10 μM and Kd2 ∼ 100 μM. Models of JAZ-RNA complexes were generated using the high-ambiguity-driven biomolecular docking (HADDOCK) program. Single zinc fingers bind to the RNA backbone without sequence specificity, forming complexes with contacts between the RNA minor groove and residues in the N-terminal β strands and between the major groove and residues in the helix-kink-helix motif. We propose that the non-sequence-specific interaction between the zinc fingers of JAZ with dsRNA is dependent only on the overall shape of the A-form RNA.
Project description:Genome editing using zinc finger nucleases (ZFNs) has been successfully applied to disrupt CCR5 or CXCR4 host factors and inhibit viral entry and infection. Gene therapy using ZFNs to modify the PSIP1 gene, which encodes the lens epithelium-derived growth factor (LEDGF) protein, might restrain an early step of the viral replication cycle at the integration level. ZFNs targeting the PSIP1 gene (ZFNLEDGF) were designed to specifically recognize the sequence after the integrase binding domain (IBD) of the LEDGF/p75 protein. ZFNLEDGF successfully recognized the target region of the PSIP1 gene in TZM-bl cells by heteroduplex formation and DNA sequence analysis. Gene editing induced a frameshift of the coding region and resulted in the abolishment of LEDGF expression at the mRNA and protein levels. Functional assays revealed that infection with the HIV-1 R5 BaL or X4 NL4-3 viral strains was impaired in LEDGF/p75 knockout cells regardless of entry tropism due to a blockade in HIV-1 proviral integration into the host genome. However, residual infection was detected in the LEDGF knockout cells. Indeed, LEDGF knockout restriction was overcome at a high multiplicity of infection, suggesting alternative mechanisms for HIV-1 genome integration rather than through LEDGF/p75. However, the observed residual integration was sensitive to the integrase inhibitor raltegravir. These results demonstrate that the described ZFNLEDGF effectively targets the PSIP1 gene, which is involved in the early steps of the viral replication cycle; thus, ZFNLEDGF may become a potential antiviral agent for restricting HIV-1 integration. Moreover, LEDGF knockout cells represent a potent tool for elucidating the role of HIV integration cofactors in virus replication.
Project description:Human Immunodeficiency Virus type 1 (HIV-1) is a lentivirus that causes a persistent viral infection and results in the demise of immune regulatory cells. Clearance of HIV-1 infection by the immune system is inefficient, and integration of proviral DNA into the genome of host cells provides a means for evasion and long-term persistence. A therapeutic compound that specifically targets and sustainably activates a latent HIV provirus could be transformative and is an overarching goal for the “shock and kill” approach to a functional cure for HIV. Substantial progress has been made towards the development of recombinant proteins that can target specific genomic loci for gene activation, repression or inactivation by directed mutations. However, most of these modalities are too large, or too complex, for efficient therapeutic application. We describe here the development and testing of a novel recombinant zinc finger protein transactivator, ZFPb-362-VPR, which specifically and potently enhances proviral HIV transcription both in established latency models and across different viral clades. Additionally, ZFP-362-VPR activated HIV reporter gene expression in a well-established primary human CD4+ T-cell latency model and was specific in targeting the HIV LTR as determined from off-target transcriptome analyses. This study provides clear proof of concept for the application of a novel, and therapeutically relevant, protein transactivator to purge cellular reservoirs of HIV-1.
Project description:Zinc finger antiviral protein (ZAP) is a host antiviral factor that selectively inhibits the replication of a variety of viruses. ZAP recognizes the CG-enriched RNA sequences and activates the viral RNA degradation machinery. In this work, we investigated the dynamics of a ZAP/RNA complex and computed the energetics of mutations in ZAP that affect its binding to the viral RNA. The crystal structure of a mouse-ZAP/RNA complex showed that RNA interacts with the zinc finger 2 (ZF2) and ZF3 domains. However, we found that due to the dynamic behavior of the single-stranded RNA, the terminal nucleotides C1 and G2 of RNA change their positions from the ZF3 to the ZF1 domain. Moreover, the electrostatic interactions between the zinc ions and the viral RNA provide further stability to the ZAP/RNA complex. We also provide structural and thermodynamic evidence for seven residue pairs (C1-Arg74, C1-Arg179, G2-Arg74, U3-Lys76, C4-Lys76, G5-Arg95, and U6-Glu204) that show favorable ZAP/RNA interactions, although these interactions were not observed in the ZAP/RNA crystal structure. Consistent with the observations from the mouse-ZAP/RNA crystal structure, we found that four residue pairs (C4-Lys89, C4-Leu90, C4-Tyr108, and G5-Lys107) maintained stable interactions in MD simulations. Based on experimental mutagenesis studies and our residue-level interaction analysis, we chose seven residues (Arg74, Lys76, Lys89, Arg95, Lys107, Tyr108, and Arg179) for individual alanine mutations. In addition, we studied mutations in those residues that are only observed in the crystal structures as interacting with RNA (Tyr98, Glu148, and Arg170). Out of these 10 mutations, we found that the Ala mutation in each of the five residues Arg74, Lys76, Lys89, Lys107, and Glu148 significantly reduced the binding affinity of ZAP to RNA.
Project description:BACKGROUND: Glucose inhibition of gluconeogenic growth suppressor 2 protein (Gis2p) and zinc-finger protein 9 (ZNF9) are conserved yeast and human zinc-finger proteins. The function of yeast Gis2p is unknown, but human ZNF9 has been reported to bind nucleic acids, and mutations in the ZNF9 gene cause the neuromuscular disease myotonic dystrophy type 2. To explore the impact of these proteins on RNA regulation, we undertook a systematic analysis of the RNA targets and of the global implications for gene expression. RESULTS: Hundreds of mRNAs were associated with Gis2p, mainly coding for RNA processing factors, chromatin modifiers and GTPases. Target mRNAs contained stretches of G(A/U)(A/U) trinucleotide repeats located in coding sequences, which are sufficient for binding to both Gis2p and ZNF9, thus implying strong structural conservation. Predicted ZNF9 targets belong to the same functional categories as seen in yeast, indicating functional conservation, which is further supported by complementation of the large cell-size phenotype of gis2 mutants with ZNF9. We further applied a matched-sample proteome-transcriptome analysis suggesting that Gis2p differentially coordinates expression of RNA regulons, primarily by reducing mRNA and protein levels of genes required for ribosome assembly and by selectively up-regulating protein levels of myosins. CONCLUSIONS: This integrated systematic exploration of RNA targets for homologous RNA-binding proteins indicates an unexpectedly high conservation of the RNA-binding properties and of potential targets, thus predicting conserved RNA regulons. We also predict regulation of muscle-specific genes by ZNF9, adding a potential link to the myotonic dystrophy related phenotypes seen in ZNF9 mouse models.
Project description:Integration of environmental signals and interactions among photoreceptors and transcriptional regulators is key in shaping plant development. TANDEM ZINC-FINGER PLUS3 (TZP) is an integrator of light and photoperiodic signaling that promotes flowering in Arabidopsis thaliana Here we elucidate the molecular role of TZP as a positive regulator of hypocotyl elongation. We identify an interacting partner for TZP, the transcription factor ZINC-FINGER HOMEODOMAIN 10 (ZFHD10), and characterize its function in coregulating the expression of blue-light-dependent transcriptional regulators and growth-promoting genes. By employing a genome-wide approach, we reveal that ZFHD10 and TZP coassociate with promoter targets enriched in light-regulated elements. Furthermore, using a targeted approach, we show that ZFHD10 recruits TZP to the promoters of key coregulated genes. Our findings not only unveil the mechanism of TZP action in promoting hypocotyl elongation at the transcriptional level but also assign a function to an uncharacterized member of the ZFHD transcription factor family in promoting plant growth.
Project description:PRDM9 is a trans-acting factor directing meiotic recombination to specific DNA-binding sites by its zinc finger (ZnF) array. It was suggested that PRDM9 is a multimer; however, we do not know the stoichiometry or the components inducing PRDM9 multimerization. In this work, we used in vitro binding studies and characterized with electrophoretic mobility shift assays, mass spectrometry, and fluorescence correlation spectroscopy the stoichiometry of the PRDM9 multimer of two different murine PRDM9 alleles carrying different tags and domains produced with different expression systems. Based on the migration distance of the PRDM9-DNA complex, we show that PRDM9 forms a trimer. Moreover, this stoichiometry is adapted already by the free, soluble protein with little exchange between protein monomers. The variable ZnF array of PRDM9 is sufficient for multimerization, and at least five ZnFs form already a functional trimer. Finally, we also show that only one ZnF array within the PRDM9 oligomer binds to the DNA, whereas the remaining two ZnF arrays likely maintain the trimer by ZnF-ZnF interactions.
Project description:Adenosine-to-inosine RNA editing, catalyzed by adenosine deaminase acting on RNA (ADAR) enzymes, alters RNA sequences from those encoded by DNA. These editing events are dynamically regulated, but few trans regulators of ADARs are known in vivo. Here, we screen RNA-binding proteins for roles in editing regulation with knockdown experiments in the Drosophila brain. We identify zinc-finger protein at 72D (Zn72D) as a regulator of editing levels at a majority of editing sites in the brain. Zn72D both regulates ADAR protein levels and interacts with ADAR in an RNA-dependent fashion, and similar to ADAR, Zn72D is necessary to maintain proper neuromuscular junction architecture and fly mobility. Furthermore, Zn72D's regulatory role in RNA editing is conserved because the mammalian homolog of Zn72D, Zfr, regulates editing in mouse primary neurons. The broad and conserved regulation of ADAR editing by Zn72D in neurons sustains critically important editing events.
Project description:Human immunodeficiency virus type 1 (HIV-1) causes a persistent viral infection resulting in the demise of immune regulatory cells. Clearance of HIV-1 infection results in integration of proviral DNA into the genome of host cells, which provides a means for evasion and long-term persistence. A therapeutic compound that specifically targets and sustainably activates a latent HIV-1 provirus could be transformative and is the goal for the "shock-and-kill" approach to a functional cure for HIV-1. Substantial progress has been made toward the development of recombinant proteins that target specific genomic loci for gene activation, repression, or inactivation by directed mutations. However, most of these modalities are too large or too complex for efficient therapeutic application. We describe here the development and testing of a novel recombinant zinc finger protein transactivator, ZFP-362-VPR, which specifically and potently enhances proviral HIV-1 transcription both in established latency models and activity across different viral clades. Additionally, ZFP-362-VPR-activated HIV-1 reporter gene expression in a well-established primary human CD4+ T cell latency model and off-target pathways were determined by transcriptome analyses. This study provides clear proof of concept for the application of a novel, therapeutically relevant, protein transactivator to purge cellular reservoirs of HIV-1.