Cytosolic RGG RNA-binding proteins are temperature sensitive flowering time regulators in Arabidopsis.

mRNA translation is tightly regulated by various classes of RNA-binding proteins (RBPs) during development and in response to changing environmental conditions. In this study, we characterize the arginine-glycine-glycine (RGG) motif containing RBP family of Arabidopsis thaliana representing homologues of the multifunctional translation regulators and ribosomal preservation factors Stm1 from yeast (ScStm1) and human SERBP1 (HsSERBP1). The Arabidopsis genome encodes three RGG proteins named AtRGGA, AtRGGB and AtRGGC. While AtRGGA is ubiquitously expressed, AtRGGB and AtRGGC are enriched in dividing cells. All AtRGGs localize almost exclusively to the cytoplasm and bind with high affinity to ssRNA, while being capable to interact with most nucleic acids, except dsRNA. A protein-interactome study shows that AtRGGs interact with ribosomal proteins and proteins involved in RNA processing and transport. In contrast to ScStm1, AtRGGs are enriched in ribosome-free fractions in polysome profiles, suggesting additional plant-specific functions. Mutant studies show that AtRGG proteins differentially regulate flowering time, with a distinct and complex temperature dependency for each AtRGG protein. In conclusion, we suggest that AtRGGs function in fine-tuning translation efficiency to control flowering time and potentially other developmental processes in response to environmental changes.

Structural basis of an essential interaction between influenza polymerase and Pol II CTD.

The heterotrimeric influenza polymerase (FluPol), comprising subunits PA, PB1 and PB2, binds to the conserved 5' and 3' termini (the 'promoter') of each of the eight single-stranded viral RNA (vRNA) genome segments and performs both transcription and replication of vRNA in the infected cell nucleus. To transcribe viral mRNAs, FluPol associates with cellular RNA polymerase II (Pol II), which enables it to take 5'-capped primers from nascent Pol II transcripts. Here we present a co-crystal structure of bat influenza A polymerase bound to a Pol II C-terminal domain (CTD) peptide mimic, which shows two distinct phosphoserine-5 (SeP5)-binding sites in the polymerase PA subunit, accommodating four CTD heptad repeats overall. Mutagenesis of the SeP5-contacting basic residues (PA K289, R454, K635 and R638) weakens CTD repeat binding in vitro without affecting the intrinsic cap-primed (transcription) or unprimed (replication) RNA synthesis activity of recombinant polymerase, whereas in cell-based minigenome assays the same mutations substantially reduce overall polymerase activity. Only recombinant viruses with a single mutation in one of the SeP5-binding sites can be rescued, but these viruses are severely attenuated and genetically unstable. Several previously described mutants that modulate virulence can be rationalized by our results, including a second site mutation (PA(C453R)) that enables the highly attenuated mutant virus (PA(R638A)) to revert to near wild-type infectivity. We conclude that direct binding of FluPol to the SeP5 Pol II CTD is fine-tuned to allow efficient viral transcription and propose that the CTD-binding site on FluPol could be targeted for antiviral drug development.

Shack-Hartmann wavefront sensors based on 2D refractive lens arrays and super-resolution multi-contrast X-ray imaging.

Different approaches of 2D lens arrays as Shack-Hartmann sensors for hard X-rays are compared. For the first time, a combination of Shack-Hartmann sensors for hard X-rays (SHSX) with a super-resolution imaging approach to perform multi-contrast imaging is demonstrated. A diamond lens is employed as a well known test object. The interleaving approach has great potential to overcome the 2D lens array limitation given by the two-photon polymerization lithography. Finally, the radiation damage induced by continuous exposure of an SHSX prototype with a white beam was studied showing a good performance of several hours. The shape modification and influence in the final image quality are presented.

The zinc finger domains in U2AF26 and U2AF35 have diverse functionalities including a role in controlling translation.

Recent work has associated point mutations in both zinc fingers (ZnF) of the spliceosome component U2AF35 with malignant transformation. However, surprisingly little is known about the functionality of the U2AF35 ZnF domains in general. Here we have analysed key functionalities of the ZnF domains of mammalian U2AF35 and its paralog U2AF26. Both ZnFs are required for splicing regulation, whereas only ZnF2 controls protein stability and contributes to the interaction with U2AF65. These features are confirmed in a naturally occurring splice variant of U2AF26 lacking ZnF2, that is strongly induced upon activation of primary mouse T cells and localized in the cytoplasm. Using Ribo-Seq in a model T cell line we provide evidence for a role of U2AF26 in activating cytoplasmic steps in gene expression, notably translation. Consistently, an MS2 tethering assay shows that cytoplasmic U2AF26/35 increase translation when localized to the 5'UTR of a model mRNA. This regulation is partially dependent on ZnF1 thus providing a connection between a core splicing factor, the ZnF domains and the regulation of translation. Altogether, our work reveals unexpected functions of U2AF26/35 and their ZnF domains, thereby contributing to a better understanding of their role and regulation in mammalian cells.

In situ structural kinetics of picosecond laser-induced heating and fragmentation of colloidal gold spheres.

Fragmentation of colloidal 54 nm gold nanoparticles by picosecond laser pulses is recorded by time-resolved X-ray scattering, giving access to structural dynamics down to a 80 ps resolution. Lattice temperature and energy dissipation have been quantified to verify that the maximum applied fluence of 1800 J m-2 heats up the particles close to boiling. Already within 30 ns, particles with significantly lower particle sizes of 2 to 3 nm are detected, which hints towards an ultrafast process either by a thermal phase explosion or Coulomb instability. An arrested growth is observed on a microsecond time scale resulting in a final particle size of 3-4 nm with high yield. In this context, the fragmentation in a NaCl/NaOH solution seems to limit growth by electrostatic stabilization of fragments, whereas it does not modify the initial product sizes. The laser-induced fragmentation process is identified as a single-step, instantaneous reaction.

Structure of influenza A polymerase bound to the viral RNA promoter.

The influenza virus polymerase transcribes or replicates the segmented RNA genome (viral RNA) into viral messenger RNA or full-length copies. To initiate RNA synthesis, the polymerase binds to the conserved 3' and 5' extremities of the viral RNA. Here we present the crystal structure of the heterotrimeric bat influenza A polymerase, comprising subunits PA, PB1 and PB2, bound to its viral RNA promoter. PB1 contains a canonical RNA polymerase fold that is stabilized by large interfaces with PA and PB2. The PA endonuclease and the PB2 cap-binding domain, involved in transcription by cap-snatching, form protrusions facing each other across a solvent channel. The 5' extremity of the promoter folds into a compact hook that is bound in a pocket formed by PB1 and PA close to the polymerase active site. This structure lays the basis for an atomic-level mechanistic understanding of the many functions of influenza polymerase, and opens new opportunities for anti-influenza drug design.

Structural insight into cap-snatching and RNA synthesis by influenza polymerase.

Influenza virus polymerase uses a capped primer, derived by 'cap-snatching' from host pre-messenger RNA, to transcribe its RNA genome into mRNA and a stuttering mechanism to generate the poly(A) tail. By contrast, genome replication is unprimed and generates exact full-length copies of the template. Here we use crystal structures of bat influenza A and human influenza B polymerases (FluA and FluB), bound to the viral RNA promoter, to give mechanistic insight into these distinct processes. In the FluA structure, a loop analogous to the priming loop of flavivirus polymerases suggests that influenza could initiate unprimed template replication by a similar mechanism. Comparing the FluA and FluB structures suggests that cap-snatching involves in situ rotation of the PB2 cap-binding domain to direct the capped primer first towards the endonuclease and then into the polymerase active site. The polymerase probably undergoes considerable conformational changes to convert the observed pre-initiation state into the active initiation and elongation states.

Capped RNA primer binding to influenza polymerase and implications for the mechanism of cap-binding inhibitors.

Influenza polymerase uses short capped primers snatched from nascent Pol II transcripts to initiate transcription of viral mRNAs. Here we describe crystal structures of influenza A and B polymerase bound to a capped primer in a configuration consistent with transcription initiation ('priming state') and show by functional assays that conserved residues from both the PB2 midlink and cap-binding domains are important for positioning the capped RNA. In particular, mutation of PB2 Arg264, which interacts with the triphosphate linkage in the cap, significantly and specifically decreases cap-dependent transcription. We also compare the configuration of the midlink and cap-binding domains in the priming state with their very different relative arrangement (called the 'apo' state) in structures where the potent cap-binding inhibitor VX-787, or a close analogue, is bound. In the 'apo' state the inhibitor makes additional interactions to the midlink domain that increases its affinity beyond that to the cap-binding domain alone. The comparison suggests that the mechanism of resistance of certain mutations that allow virus to escape from VX-787, notably PB2 N510T, can only be rationalized if VX-787 has a dual mode of action, direct inhibition of capped RNA binding as well as stabilization of the transcriptionally inactive 'apo' state.

Inverted Hartmann mask for single-shot phase-contrast x-ray imaging of dynamic processes.

In this Letter, we present the application of the inverted Hartmann mask for time-resolved single-shot phase-contrast x-ray imaging. The inverted Hartmann mask is a periodic array of free-standing gold pillars. The array is manufactured by UV lithography and electroplating. Time-resolved measurements are performed for imaging of pulsed laser ablation in liquids using white-beam synchrotron radiation. The inverted Hartmann mask in combination with a single-shot imaging technique provides sufficient differential phase contrast even at very short exposure times. It can be effectively used for phase-contrast x-ray imaging of fast dynamic processes with temporal resolution on the millisecond scale.

Incubation Effect of Pre-Irradiation on Bubble Formation and Ablation in Laser Ablation in Liquids.

Pulsed laser ablation in liquids (PLAL) is a multi-scale process, which is widely studied either in batch ablation with prolonged target irradiation as well as mechanistic investigations, in a defined (single-shot) process. However, fundamental studies on defined pulse series are rare. We have investigated the effect of a developing rough morphology of the target surface on the PLAL process with nanosecond pulses and, partially, picosecond pulses. At low fluence the cavitation bubble growth as well as the ablation yield depend on the irradiation history of the target. The bubble size increases with repeated irradiation on one spot for the first 2-30 pulses as well as with the applied dose. This is discussed within the framework of incubation effects. Incubation is found to be important, resulting in a bubble volume increase by a factor of six or more between pristine and corrugated targets. The target surface, changing from smooth to corrugated, induces a more efficient localization of laser energy at the solid-liquid interface. This is accompanied by a suppressed reflectivity and more efficient coupling of energy into the laser-induced plasma. Thus, the cavitation bubble size increases as well as ablation being enhanced. At high fluence, such incubation is masked by the rapid development of surface damage within the first shots, which eventually would lead to a reduction of bubble sizes.

Time and Mechanism of Nanoparticle Functionalization by Macromolecular Ligands during Pulsed Laser Ablation in Liquids.

Laser ablation of gold in liquids with nanosecond laser pulses in aqueous solutions of inorganic electrolytes and macromolecular ligands for gold nanoparticle size quenching is probed inside the laser-induced cavitation bubble by in situ X-ray multicontrast imaging with a Hartmann mask (XHI). It is found that (i) the in situ size quenching power of sodium chloride (NaCl) in comparison to the ablation in pure water can be observed by the scattering contrast from XHI already inside the cavitation bubble, while (ii) for polyvinylpyrrolidone (PVP) as a macromolecular model ligand an in situ size quenching cannot be observed. Complementary ex situ characterization confirms the overall size quenching ability of both additive types NaCl and PVP. The macromolecular ligand as well as its monomer N-vinylpyrrolidone (NVP) are mainly effective for growth quenching of larger nanoparticles on later time scales, leading to the conclusion of an alternative interaction mechanism with ablated nanoparticles compared to the electrolyte NaCl, probably outside of the cavitation bubble, in the surrounding liquid phase. While monomer and polymer have similar effects on the particle properties, with the polymer being slightly more efficient, only the polymer is effective against hydrodynamic aggregation.

An in vitro fluorescence based study of initiation of RNA synthesis by influenza B polymerase.

Influenza polymerase replicates, via a complementary RNA intermediate (cRNA), and transcribes the eight viral RNA (vRNA) genome segments. To initiate RNA synthesis it is bound to the conserved 5΄ and 3΄ extremities of the vRNA or cRNA (the 'promoter'). 5΄-3΄ base-pairing in the distal promoter region is essential to position the template RNA at the polymerase active site, as shown by a new crystal structure with the 3΄ end threading through the template entry tunnel. We develop fluorescence polarization assays to quantify initiation of cap-primed (transcription) or unprimed (replication) RNA synthesis by recombinant influenza B polymerase bound to the vRNA or cRNA promoter. The rate-limiting step is formation of a primed initiation complex with minimally ApG required to stabilize the 3΄ end of the template within the active-site. Polymerase bound to the vRNA promoter initiates RNA synthesis terminally, while the cRNA promoter directs internal initiation at a significantly lower rate. Progression to elongation requires breaking the promoter 5΄-3΄ base-pairing region and favourable compensation by the emerging template-product base-pairs. The RNA synthesis assay is adaptable to high-throughput screening for polymerase inhibitors. In a pilot study, we find that initiation at the cRNA promoter is unusually susceptible to inhibition by 2΄F-2΄dNTPs.

Domain Organization of Vaccinia Virus Helicase-Primase D5.

UNLABELLED: Poxviridae are viruses with a large linear double-stranded DNA genome coding for up to 250 open reading frames and a fully cytoplasmic replication. The double-stranded DNA genome is covalently circularized at both ends. Similar structures of covalently linked extremities of the linear DNA genome are found in the African swine fever virus (asfarvirus) and in the Phycodnaviridae We are studying the machinery which replicates this peculiar genome structure. From our work with vaccinia virus, we give first insights into the overall structure and function of the essential poxvirus virus helicase-primase D5 and show that the active helicase domain of D5 builds a hexameric ring structure. This hexamer has ATPase and, more generally, nucleoside triphosphatase activities that are indistinguishable from the activities of full-length D5 and that are independent of the nature of the base. In addition, hexameric helicase domains bind tightly to single- and double-stranded DNA. Still, the monomeric D5 helicase construct truncated within the D5N domain leads to a well-defined structure, but it does not have ATPase or DNA-binding activity. This shows that the full D5N domain has to be present for hexamerization. This allowed us to assign a function to the D5N domain which is present not only in D5 but also in other viruses of the nucleocytoplasmic large DNA virus (NCLDV) clade. The primase domain and the helicase domain were structurally analyzed via a combination of small-angle X-ray scattering and, when appropriate, electron microscopy, leading to consistent low-resolution models of the different proteins. IMPORTANCE: Since the beginning of the 1980s, research on the vaccinia virus replication mechanism has basically stalled due to the absence of structural information. As a result, this important class of pathogens is less well understood than most other viruses. This lack of information concerns in general viruses of the NCLDV clade, which use a superfamily 3 helicase for replication, as do poxviruses. Here we provide for the first time information about the domain structure and DNA-binding activity of D5, the poxvirus helicase-primase. This result not only refines the current model of the poxvirus replication fork but also will lead in the long run to a structural basis for antiviral drug design.

Fluence Threshold Behaviour on Ablation and Bubble Formation in Pulsed Laser Ablation in Liquids.

The ablation yield and bubble-formation process during nanosecond pulsed-laser ablation of silver in water are analysed by stroboscopic videography, time-resolved X-ray radiography and in situ UV/Vis spectroscopy. This process is studied as function of lens-target distance and laser fluence. Both the ablation yield and the bubble-cavitation process exhibit threshold behaviour as a function of fluence, which is linked to the efficiency of coupling of energy at the water/target interface. Although ablation happens below this threshold, quantitative material emission is linked to bubble formation. Above the threshold, both bubble size and ablation show linear behaviour.

Initiation of RNA synthesis by the hepatitis C virus RNA-dependent RNA polymerase is affected by the structure of the RNA template.

The hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B is a central enzyme of the intracellular replication of the viral (+)RNA genome. Here, we studied the individual steps of NS5B-catalyzed RNA synthesis by a combination of biophysical methods, including real-time 1D (1)H NMR spectroscopy. NS5B was found to bind to a nonstructured and a structured RNA template in different modes. Following NTP binding and conversion to the catalysis-competent ternary complex, the polymerase revealed an improved affinity for the template. By monitoring the folding/unfolding of 3'(-)SL by (1)H NMR, the base pair at the stem's edge was identified as the most stable component of the structure. (1)H NMR real-time analysis of NS5B-catalyzed RNA synthesis on 3'(-)SL showed that a pronounced lag phase preceded the processive polymerization reaction. The presence of the double-stranded stem with the edge base pair acting as the main energy barrier impaired RNA synthesis catalyzed by NS5B. Our observations suggest a crucial role of RNA-modulating factors in the HCV replication process.

NeuroBus - Architecture for an Ultra-Flexible Neural Interface.

This article presents the system architecture for an implant concept called NeuroBus. Tiny distributed direct digitizing neural recorder ASICs on an ultra-flexible polyimide substrate are connected in a bus-like structure, allowing short connections between electrode and recording front-end with low wiring effort and high customizability. The small size (344 µm × 294 µm) of the ASICs and the ultraflexible substrate allow a low bending stiffness, enabling the implant to adapt to the curvature of the brain and achieving high structural biocompatibility. We introduce the architecture, the integrated building blocks, and the post-CMOS processes required to realize a NeuroBus, and we characterize the prototyped direct digitizing neural recorder front-end as well as polyimide-based ECoG brain interface. A rodent animal model is further used to validate the joint capability of the recording front-end and thin-film electrode array.

Change of dominant material properties in laser perforation process with high-energy lasers up to 120 kilowatt.

In laser materials processing, energy losses due to reflection, heat conduction and thermal radiation play an important role. In this publication, we show that with increasing laser intensity, the energy lost within the sample becomes less important for metal perforation processes. We compare the laser-matter interaction of aluminum and steel plates. Material parameters such as density, melting point and especially thermal conductivity differ strongly, leading to much longer perforation times for aluminum in comparison to steel at laser powers of 20 kW. However, this behavior changes at laser powers of more than 80 kW where the perforation times of aluminum become shorter than the corresponding times for steel. By comparing experimental data and simulations, we conclude that thermal conduction is the dominant factor of energy loss at low powers, but is reduced at high laser powers.

A Direct Digitizing Chopped Neural Recorder Using a Body-Induced Offset Based DC Servo Loop.

This article presents a direct digitizing neural recorder that uses a body-induced offset based DC servo loop to cancel electrode offset (EDO) on-chip. The bulk of the input pair is used to create an offset, counteracting the EDO. The architecture does not require AC coupling capacitors which enables the use of chopping without impedance boosting while maintaining a large input impedance of 238 M Ω over the whole 10 kHz bandwidth. Implemented in a 180 nm HV-CMOS process, the prototype occupies a silicon area of only 0.02 mm2 while consuming 12.8 µW and achieving 1.82 µV[Formula: see text] of input-referred noise in the local field potential (LFP) band and a NEF of 5.75.

2D and 3D Triangulation Are Suitable In Situ Measurement Tools for High-Power Large Spot Laser Penetration Processes to Visualize Depressions and Protrusions before Perforating.

During laser penetration, the irradiated samples form a melt pool before perforation. Knowledge of the dynamics of this melt pool is of interest for the correct physical description of the process and leads to improved simulations. However, a direct investigation, especially at the location of high-power laser interaction with large spot diameters in the centimeter range is missing until now. Here, the applicability of 2D triangulation for surface topology observations is demonstrated. With the designed bidirectional 2D triangulation setup, the material cross-section is measured by profile detection at the front and back side. This allows a comprehensive description of the penetration process to be established, which is important for a detailed explanation of the process. Specific steps such as surface melting, indentations, protrusions during melt pool development and their dynamics, and the perforation are visualized, which were unknown until now. Furthermore, a scanning 3D triangulation setup is developed to obtain more information about the entire melt pool at the front side, and not just a single intersection line. The measurements exhibit a mirror-symmetric melt pool and the possibility to extrapolate from the central profile to the outer regions in most cases.

Mechanisms of activity and inhibition of the hepatitis C virus RNA-dependent RNA polymerase.

The RNA-dependent RNA polymerase NS5B is a key enzyme of the replication of hepatitis C virus (HCV) and a major therapeutic target. Applying a novel continuous assay with highly purified protein and a fluorescent RNA-template we provide for the first time a comprehensive mechanistic description of the enzymatic reaction. Using fluorescence spectroscopy, the kinetics of NS5B was confirmed to consist of two half-reactions, namely substrate binding and turnover. Determining the binding constants of the substrates and the rate constants of individual reaction steps, NS5B was shown to bind the template single-stranded RNA with high affinity (nanomolar range) and in a stepwise process that reflects the substrate positioning. As demonstrated by CD, NTP(s) binding caused a tertiary structural change of the enzyme into an active conformation. The second half-reaction was dissected into a sequential polymerization and a subsequent, rate-limiting product release reaction. Taking advantage of these tools, we analyzed the mechanism of action of the NS5B inhibitor HCV-796, which was shown to interfere with the formation of double-stranded RNA by blocking the second half-reaction.