Crystal structure of the two-RRM domain of hnRNP A1 (UP1) complexed with single-stranded telomeric DNA

Human hnRNP A1 is a versatile single-stranded nucleic acid-binding protein that functions in various aspects of mRNA maturation and in telomere length regulation. The crystal structure of UP1, the amino-terminal domain of human hnRNP A1 containing two RNA-recognition motifs (RRMs), bound to a 12-nucleotide single-stranded telomeric DNA has been determined at 2.1 Angstrom resolution. The structure of the complex reveals the basis for sequence-specific recognition of the single-stranded overhangs of human telomeres by hnRNP A1. It also provides insights into the basis for high-affinity binding of hnRNP A1 to certain RNA sequences, and for nucleic acid binding and functional synergy between the RRMs. In the crystal structure, a UP1 dimer binds to two strands of DNA, and each strand contacts RRM1 of one monomer and RRM2 of the other. The two DNA strands are antiparallel, and regions of the protein flanking each RRM make important contacts with DNA. The extensive protein-protein interface seen in the crystal structure of the protein-DNA complex and the evolutionary conservation of the interface residues suggest the importance of specific protein-protein interactions for the sequence-specific recognition of single-stranded nucleic acids. Models for regular packaging of telomere 3' overhangs and for juxtaposition of alternative 5' splice sites are proposed

INPOP08, a 4-D planetary ephemeris: From asteroid and time-scale computations to ESA Mars Express and Venus Express contributions

The latest version of the planetary ephemerides developed at the Paris Observatory and at the Besancon Observatory is presented here. INPOP08 is a 4-dimension ephemeris since it provides to users positions and velocities of planets and the relation between TT and TDB. Investigations leading to improve the modeling of asteroids are described as well as the new sets of observations used for the fit of INPOP08. New observations provided by the European Space Agency (ESA) deduced from the tracking of the Mars Express (MEX) and Venus Express (VEX) missions are presented as well as the normal point deduced from the Cassini mission. We show the huge impact brought by these observations in the fit of INPOP08, especially in terms of Venus, Saturn and Earth-Moon barycenter orbits.Comment: 14 pages. submitted to A&A. accepted in A&

Analysis of adenovirus VA RNAI structure and stability using compensatory base pair modifications

Adenovirus VA RNAs are short non-coding transcripts that assist in maintaining viral protein expression in infected cells. Six sets of mismatch and compensatory base pair mutants of VA RNAI were examined by gel mobility and RNA UV melting to assess the contribution of each structural domain to its overall structure and stability. Each domain of VA RNAI was first assigned to one of two apparent unfolding transitions in the wild-type melting profile. The Terminal Stem and Central Domain unfold in a single cooperative apparent transition with an apparent Tm of ∼60°C. In contrast, the Apical Stem unfolds independently and with much higher apparent Tm of ∼83°C. Remarkably, this domain appears to behave as an almost entirely autonomous unit within the RNA, mirroring the functional division within the RNA between PKR binding and inhibition. The effects of mismatch and compensatory mutations at five of the six sites on the RNA melting profile are consistent with proposed base pairing and provide further validation of the current secondary structure model. Mutations in the Central Domain were tested in PKR inhibition assays and a component of the VA RNAI Central Domain structure essential for PKR inhibitory activity was identified

Orbital effects of a monochromatic plane gravitational wave with ultra-low frequency incident on a gravitationally bound two-body system

We analytically compute the long-term orbital variations of a test particle orbiting a central body acted upon by an incident monochromatic plane gravitational wave. We assume that the characteristic size of the perturbed two-body system is much smaller than the wavelength of the wave. Moreover, we also suppose that the wave's frequency is much smaller than the particle's orbital one. We make neither a priori assumptions about the direction of the wavevector nor on the orbital geometry of the planet. We find that, while the semi-major axis is left unaffected, the eccentricity, the inclination, the longitude of the ascending node, the longitude of pericenter and the mean anomaly undergo non-vanishing long-term changes. They are not secular trends because of the slow modulation introduced by the tidal matrix coefficients and by the orbital elements themselves. They could be useful to indepenedently constrain the ultra-low frequency waves which may have been indirectly detected in the BICEP2 experiment. Our calculation holds, in general, for any gravitationally bound two-body system whose characteristic frequency is much larger than the frequency of the external wave. It is also valid for a generic perturbation of tidal type with constant coefficients over timescales of the order of the orbital period of the perturbed particle.Comment: LaTex2e, 24 pages, no figures, no tables. Changes suggested by the referees include

The PKR-binding domain of adenovirus VA RNAI exists as a mixture of two functionally non-equivalent structures

VA RNAI is a non-coding adenoviral transcript that counteracts the host cell anti-viral defenses such as immune responses mediated via PKR. We investigated potential alternate secondary structure conformations within the PKR-binding domain of VA RNAI using site-directed mutagenesis, RNA UV-melting analysis and enzymatic RNA secondary structure probing. The latter data clearly indicated that the wild-type VA RNAI apical stem can adopt two different conformations and that it exists as a mixed population of these two structures. In contrast, in two sequence variants we designed to eliminate one of the possible structures, while leaving the other intact, each formed a unique secondary structure. This clarification of the apical stem pairing also suggests a small alteration to the apical stem–loop secondary structure. The relative ability of the two apical stem conformations to bind PKR and inhibit kinase activity was measured by isothermal titration calorimetry and PKR autophosphorylation inhibition assay. We found that the two sequence variants displayed markedly different activities, with one being a significantly poorer binder and inhibitor of PKR. Whether the presence of the VA RNAI conformation with reduced PKR inhibitory activity is directly beneficial to the virus in the cell for some other function requires further investigation

Observational constraints on spatial anisotropy of G from orbital motions

A phenomenological anisotropic variation \Delta G/G of the Newtonian gravitational coupling parameter G, if real, would affect the orbital dynamics of a two-body gravitationally bound system in a specific way. We analytically work out the long-term effects that such a putative modification of the usual Newtonian inverse-square law would induce on the trajectory of a test particle orbiting a central mass. Without making any a-priori simplifying assumptions concerning the orbital configuration of the test particle, it turns out that its osculating semi-major axis a, eccentricity e, pericenter \varpi and mean anomaly M undergo long-term temporal variations, while the inclination I and the node \Omega are left unaffected. Moreover, the radial and the transverse components of the position and the velocity vectors r and v of the test particle experience non-vanishing changes per orbit, contrary to the out-of-plane ones. Then, we compute our theoretical predictions for some of the major bodies of the solar system by orienting the gradient of G(r) towards the Galactic Center and keeping it fixed over the characteristic timescales involved. By comparing our calculation to the latest observational determinations for the same bodies, we infer \Delta G/G <= 10^-17 over about 1 au. Finally, we consider also the Supermassive Black Hole hosted by the Galactic Center in Sgr A^* and the main sequence star S2 orbiting it in about 16 yr, obtaining just \Delta G/G <= 10^-2 over 1 kau.Comment: LaTex2e, 18 pages, no figures, 4 tables. Accepted by Classical and Quantum Gravity (CQG). Typo fixed. Reference update

Hepatitis C Virus Controls Interferon Production through PKR Activation

Hepatitis C virus is a poor inducer of interferon (IFN), although its structured viral RNA can bind the RNA helicase RIG-I, and activate the IFN-induction pathway. Low IFN induction has been attributed to HCV NS3/4A protease-mediated cleavage of the mitochondria-adapter MAVS. Here, we have investigated the early events of IFN induction upon HCV infection, using the cell-cultured HCV JFH1 strain and the new HCV-permissive hepatoma-derived Huh7.25.CD81 cell subclone. These cells depend on ectopic expression of the RIG-I ubiquitinating enzyme TRIM25 to induce IFN through the RIG-I/MAVS pathway. We observed induction of IFN during the first 12 hrs of HCV infection, after which a decline occurred which was more abrupt at the protein than at the RNA level, revealing a novel HCV-mediated control of IFN induction at the level of translation. The cellular protein kinase PKR is an important regulator of translation, through the phosphorylation of its substrate the eIF2α initiation factor. A comparison of the expression of luciferase placed under the control of an eIF2α-dependent (IRESEMCV) or independent (IRESHCV) RNA showed a specific HCV-mediated inhibition of eIF2α-dependent translation. We demonstrated that HCV infection triggers the phosphorylation of both PKR and eIF2α at 12 and 15 hrs post-infection. PKR silencing, as well as treatment with PKR pharmacological inhibitors, restored IFN induction in JFH1-infected cells, at least until 18 hrs post-infection, at which time a decrease in IFN expression could be attributed to NS3/4A-mediated MAVS cleavage. Importantly, both PKR silencing and PKR inhibitors led to inhibition of HCV yields in cells that express functional RIG-I/MAVS. In conclusion, here we provide the first evidence that HCV uses PKR to restrain its ability to induce IFN through the RIG-I/MAVS pathway. This opens up new possibilities to assay PKR chemical inhibitors for their potential to boost innate immunity in HCV infection

The Efficacy of Generating Three Independent Anti-HIV-1 siRNAs from a Single U6 RNA Pol III-Expressed Long Hairpin RNA

RNA Interference (RNAi) effectors have been used to inhibit rogue RNAs in mammalian cells. However, rapidly evolving sequences such as the human immunodeficiency virus type 1 (HIV-1) require multiple targeting approaches to prevent the emergence of escape variants. Expressed long hairpin RNAs (lhRNAs) have recently been used as a strategy to produce multiple short interfering RNAs (siRNAs) targeted to highly variant sequences. We aimed to characterize the ability of expressed lhRNAs to generate independent siRNAs that silence three non-contiguous HIV-1 sites by designing lhRNAs comprising different combinations of siRNA-encoding sequences. All lhRNAs were capable of silencing individual target sequences. However, silencing efficiency together with concentrations of individual lhRNA-derived siRNAs diminished from the stem base (first position) towards the loop side of the hairpin. Silencing efficacy against HIV-1 was primarily mediated by siRNA sequences located at the base of the stem. Improvements could be made to first and second position siRNAs by adjusting spacing arrangements at their junction, but silencing of third position siRNAs remained largely ineffective. Although lhRNAs offer advantages for combinatorial RNAi, we show that good silencing efficacy across the span of the lhRNA duplex is difficult to achieve with sequences that encode more than two adjacent independent siRNAs