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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.07.023 Byline: Patrick Allard (a)(b), Qin Yang (b), William F. Marzluff (c), Hugh J. Clarke (a)(b) Keywords: Oocyte; Histones; Translational control; SLBP; Meiotic maturation Abstract: Although messenger RNAs encoding the histone proteins are among the most abundant in mammalian oocytes, the mechanism regulating their translation has not been identified. The stem-loop binding protein (SLBP) binds to a highly conserved sequence in the 3'-untranslated region (utr) of the non-polyadenylated histone mRNAs in somatic cells and mediates their stabilization and translation. We previously showed that SLBP, which is expressed only during S-phase of proliferating cells, is expressed in growing oocytes at G2 of the cell cycle and accumulates substantially during meiotic maturation. We report here that elevating the amount of SLBP in immature (G2) oocytes is sufficient to increase translation of a reporter mRNA bearing the histone 3'-utr and endogenous histone synthesis and that this effect is not mediated through increased stability of the encoding mRNAs. We further report that translation of the reporter mRNA increases dramatically during meiotic maturation coincident with the accumulation of SLBP. Conversely, when SLBP accumulation during maturation is prevented using RNA interference, both translation of the reporter mRNA and synthesis of endogenous histones are significantly reduced. This effect is not mediated by a loss of the encoding mRNAs. Moreover, following fertilization, SLBP-depleted oocytes also show a significant decrease in pronuclear size and in the amount of acetylated histone detectable on the chromatin. These results demonstrate that histone synthesis in immature and maturing oocytes is governed by a translational control mechanism that is directly regulated by changes in the amount of SLBP. Author Affiliation: (a) Department of Biology, McGill University, Montreal, QC, Canada (b) Department of Obstetrics and Gynecology, McGill University, Montreal, QC, Canada (c) Program in Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA Article History: Received 21 April 2005; Revised 22 June 2005; Accepted 19 July 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2005.08.012 Byline: Rashu B. Seth, Lijun Sun, Chee-Kwee Ea, Zhijian J. Chen Abstract: Viral infection triggers host innate immune responses through activation of the transcription factors NF-[kappa]B and IRF3, which coordinately regulate the expression of type-I interferons such as interferon-[beta] (IFN-[beta]). Herein, we report the identification of a novel protein termed MAVS (mitochondrial antiviral signaling), which mediates the activation of NF-[kappa]B and IRF3 in response to viral infection. Silencing of MAVS expression through RNA interference abolishes the activation of NF-[kappa]B and IRF3 by viruses, thereby permitting viral replication. Conversely, overexpression of MAVS induces the expression of IFN-[beta] through activation of NF-[kappa]B and IRF3, thus boosting antiviral immunity. Epistasis experiments show that MAVS is required for the phosphorylation of IRF3 and I[kappa]B and functions downstream of RIG-I, an intracellular receptor for viral RNA. MAVS contains an N-terminal CARD-like domain and a C-terminal transmembrane domain, both of which are essential for MAVS signaling. The transmembrane domain targets MAVS to the mitochondria, implicating a new role of mitochondria in innate immunity. Author Affiliation: Howard Hughes Medical Institute, Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390 Article History: Received 8 June 2005; Revised 8 August 2005; Accepted 11 August 2005 Article Note: (miscellaneous) Published online: August 25, 2005

A series of oligonucleotides containing modified purines and pyrimidines at the recognition site were prepared to probe their mechanism of selective interaction to bind to the methyl-binding domain (MBD) of the methyl-CpG-binding protein 2 (MeCP2). DNA-protein interface mediated by structured water as opposed to hydrophobic interactions elucidated the improved binding of the MBD to oligonucleotides containing several cytosine analogues

The self-assembly properties of highly purified, recombinant L4-22K protein is characterized to understand the thermodynamics of assembly of two human adenoviral (Ad) proteins, IVa2 and L4-22K. L4-22K which reversibly assembles into higher-order structures according to an indefinite, isodesmic assembly scheme could provide significant insight into the thermodynamics of assembly of L4-22K and IVa2 on the PS.

A pair of furan-based cyclic homooligopeptides is prepared to specifically target G-quadruplexes and UV melting has shown that these ligands have effectively induced G-quadruplex structures in the G-rich 22-mer c-MYC DNA sequence and have stabilized the structure. The studies have shown that the present molecules are effective in specifically binding and stabilizing quadruplexes and have provided a suitable scaffold to develop into a quadruplex-targeting therapeutic agent.

The equilibrium binding Deinococcus radiodurans single-stranded (ss) DNA binding protein (DrSSB) to ssDNA is examined for comparison with that of Escherichia coli SSB (EcoSSB). The binding enthalpy has shown a large sensitivity to NaCl concentration, similar to that seen for EcoSSB and these common features for SSB proteins having multiple DNA binding domains enable versatility of SSB function in vivo.

Two X-ray structures of chicken smooth muscle tropomyosin overlap complex are described. The interactions between the N- and C-terminal coiled coils of smooth muscle tropomyosin have displayed curvature, which has differed between the two structures and has implied flexibility in the overlap complex, at least in solution.

The properties of six individually expressed human methylated DNA binding protein 2 (MeCP2) domains spanning the entire protein were analyzed in detail to better understand their interactions with each other, with DNA, and with nucleosomal arrays. The results offer new mechanistic and biochemical insights related to the conformational modulations of intrinsically disordered MeCP2 protein, and its context-dependent in vivo roles.

Isothermal titration calorimetry was used to probe the specificity and examine the thermodynamics of single-stranded DNA binding protein (SSB) binding to two Escherichia coli proteins important for DNA replication, the x subunit of DNA polymerase III holoenzyme and the PriA helicase. The results suggested that DNA binding proteins could use the interaction of SSB with both x and PriA to gain access to ssDNA by first interacting with the SSB C-termini.

NMR is used for describing the structure-function relationship on desamido-bacitracin (Bc) of low antibiotic activity, wherein D-Glu-4 is shown to be vital for the bioactivity of Bc. The interaction of the phosphor-moiety with Bc is reflected by DNA binding, wherein metal-free Bc does not bind DNA, whereas various metal complexes of Bc do and Cu(II)_Bc is shown to bind and oxidatively cleave DNA under reduction conditions in the air.

Coiled-coil arrays were used to study the interaction specificity of four viral basic-region leucine-zipper transcription factors (bZIPs) and assess their self-associations as well as heterointeractions with 33 representative human bZIPs. The new interactions detected for HBZ with MAFB, MAFG, ATF2, CEBPG, and CREBZF and MEQ with NFIL3 were confirmed in solution by circular dichroism.

A genetically encoded fluorescent copper(I) sensor based on the copper(I)-binding-induced conformational change of a copper-responsive transcriptional regulator is described. The reporter Amt1-FRET has operated in the dynamic range of the cellular copper buffer in mammalian cells, reported dynamic fluctuations of the cellular copper availability within minutes of perturbation and has visualized the tightly controlled copper availability in mammalian cells.

The use of silicon chips with 16 DNA-modified electrodes (DME chips) utilizing DNA-mediated charge transport for multiplexed detection of DNA and DNA-binding protein targets is described. The results have shown how DME chips have facilitated sensitive and selective detection of DNA and DNA-binding protein targets in a robust and internally standardized multiplexed format.

The fluorescent single-stranded DNA binding protein (SSB) is used to develop the kinetic mechanism by which the protein and ssDNA associate and dissociate and understand the interaction between single-stranded DNA (ssDNA) and SSB. The complex dissociation kinetics of the fluorescent SSB*ssDNA complex is found to be greatly accelerated by the presence of free wild-type SSB.

The conformational and physical properties of the disordered C-terminal domain of TAR DNA-binding protein 43 (TDP-43) are clarified by preparing many peptide fragments and showing that only D1 within D1-4 has formed twisted fibrils with a cross section under the incubation of phosphate buffer. The studies have shown that all the three peptides formed an amyloid structure, which has provided insights into the nature of its aggregation with regard to the other fragments in the C-terminus of TDP-43.

A high-resolution NMR solution structure of recombinant ribosomal protein L14e, a component of the large ribosomal subunit in both archaea and eukaryotes is characterized to understand the classic SH3 fold structure adopted by the N-terminal 57 residues. Great similarities observed between the N-terminal sequence of L14e of any Sulfolobus protein and N-terminal sequence of Sac8b, a DNA-binding protein indicated the potential use of structure of L14e to serve as model to study other members of the L14e family, including COG2163 group, L6e and L27e.

Max-47 is a designed hybrid protein that comprises of the Max DNA-binding basic region and E47 HLH dimerization subdomain. Modified yeast one-hybrid system (Y1H) is used for showing that Max-E47 has outcompeted the native MaxbHLHZ for the E-box and these hybrids have provided a minimalist scaffold for exploring the relationship between protein structure and DNA-binding function.

Cyclic 8-ring Py-Im polyamides have targeted the DNA-sequence 5'-WGWWCW-3', which has corresponded to the androgen response element (ARE) bound by the androgen receptor transcription factor to modulate gene expression. The studies have shown that cyclic Py-Im polyamides bind DNA with high affinities and regulate the expression of AR target genes in cell culture studies, which shows that the cycle is cell permeable.

The eukaryotic ssDNA binding protein, Replication Protein A (RPA) is used to demonstrate the application of NMR spectroscopy as a powerfull tool to characterize the remodeling of architecture in different functional states of modular proteins with multiple domains. The first direct evidence obtained for the remodeling of RPA upon binding ssDNA, including an alteration in the availability of the RPA32N domain might help explain damage-dependent phosphorylation in modular proteins.

Nuclear magnetic resonance spectroscopy and proton exchange were used to characterize two RNA-DNA hybrids from the tR2 intrinsic transcription terminator site of phage [lambda]. The results suggested for the first time that the RNA-DNA hybrid structures with the same base sequence, the opening dynamics and the stability of individual base pairs are strongly influenced by the chemical nature of each strand.

The functional significance of three conserved aromatic amino acids (Trp37, Tyr60, and Phe104) for heat shock transcription factor 1 (HSF1) trimerization is demonstrated. An intramolecular interaction between Tyr60 and [alpha]-helix 1 in the DNA-binding domain and intermolecular aromatic-aromatic interaction between the Trp37 and Phe104 facilitated the formation of intermolecular disulfide bonds, leading to the heat-induced HSF1 trimerization.

A new method is used for measuring forces between small protein domains based on double electron-electron resonance (DEER) spectroscopy by using a model peptide derived from the [alpha]-helical coiled-coil leucine zipper of yeast transcriptional activator GCN4. The equilibrium distribution of distances between two nitroxide spin labels rigidly attached to the helices of the dimer is determined by using DEER and has produced a closing force of 100[plusmn]10 pH between monomers.

Extensive replica exchange (REX) simulations are carried out in an optimized generalized Born (GB) force field to characterize the disordered states of a regulatory intrinsically disordered protein (IDP), KID domain of transcription factor CREB and its phosphorylated form, pKID. The results have shown that an implicit solvent-based modeling framework, despite various existing limitations, is feasible for accurate atomistic simulation of small IDPs.

The studies have shown that derivatives of wrenchnolol, a synthetic molecule that interacts with Sur-2 coactivator, serve as activation modules and stimulate gene transcription in vitro and in cells when tethered to a DNA-binding molecule. Thirteen derivatives of wrenchnolol are chemically prepared, optimized and tested for their ability to activate transcription in vitro and in cells.

The first biochemical characterization of XPB, the largest subunit of the eukaryotic transcription factor TFIIH, which is essential for both initiation of transcription by RNA polymerase II and nucleotide excision repair (NER) from bacteria, Mycobacterium tuberculosis and Kineococcus radiotolerans is reported. The ATP hydrolytic acttivity by the bacterial XPBs is strongly dependent on both the nature of a nucleotide triphosphate and that of a divalent metal.

Several properties of Helicobacter pylori NikR (HpNikR), a nickel-responsive transcription factor were explored to understand its role in the direct or indirect internal pH-regulation in modulation of the DNA-binding activity upon exposure to acidic shock. The results obtained from the site-directed mutagenesis of HpNikR revealed that either Asp7 or Asp8 in the N-terminus of HpNikR significant for the activation of DNA binding and the viability of H. pylori.

The isothermal titration calorimetry and NMR spectroscopy were utilized to map the interaction between the transactivation domain (TAD) of p53 and the transcriptional coactivators CBP KIX domain. The KIX-binding affinity of p53 TAD was observed to be enhanced by simultaneous phosphorylation of Thr18 and Ser20.

The screening of a compound library of synthetic DNA binding molecules for selective binding to the consensus sequence of the transcription factor LEF-1 followed by the assessment of the candidate compounds in a series of assays has provided two lead compounds, lefmycin-2 and lefmycin-2. The sequence of screens defining the method has assured that activity in the final functional assay is directly related to the inhibition of gene transcription and DNA binding properties of the molecules.

The stereoselective synthesis of subglutinols A and B and the potential of subglutinol A as an immunosuppressive drug with dose-dependent osteogenic activity are described. Activating protein 1 (AP-1) family transcription factors are one of the key regulators for the anabolic activity of subglutinol A.

The isothermal titration calorimetry approach is employed to explain the thermodynamics of the Jun-Fos heterodimeric transcription factor to the synthetic dsDNA oligos containing TGACTCA element. The TGACTCA cis motif is shown to play an extremely significant role in the modulation of energetics and orientation of the binding process.

The three-step mechanism that is involved in the binding and bending of the AdMLP TATA sequence (TATAAAAG) by a protein core domain is discussed. The structures and the properties of DNA bound in solution and the crystal are shown to be completely different.

A nuclear magnetic resonance (NMR) is employed to map the interaction between Bdp1 and Brf1, two of the transcription factors of TFIIIB at single-amino acid resolution, using minimal functional segments of the two proteins. The structural model derived from NMR revealed that the most important anchorage site of Brf1 is located on a convex surface of Bdp1 that encompasses helix 1 and helix 3 of its conserved SANT domain.

A biochemical study is carried out to examine the proteins containing both an A/T-rich interaction domain (ARID) and a high mobility group (HMG)-box domain from Arabidopsis thaliana. The results showed that both the ARID and the HMG-box domain contribute to the DNA interactions of ARID-HMG1 to give the protein characteristics of ARID and HMG-box proteins.