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RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein–RNA and protein–protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large ribonucleoprotein particles (RNPs) these proteins function in. Using UV-induced RNA–protein crosslinking of entire cells, protein complex purification and mass spectrometric analysis, we identified >100 in vivo RNA crosslinks in 16 nuclear mRNP components in Saccharomyces cerevisiae. For functional analysis, we chose Npl3, which displayed crosslinks in its two RNA recognition motifs (RRMs) and in the connecting flexible linker region. Both RRM domains and the linker uniquely contribute to RNA recognition as revealed by NMR and structural analyses. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains. Notably, an npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of other mRNP components into nuclear mRNPs, establishing a so far unknown function of Npl3 in nuclear mRNP assembly. Taken together, our integrative analysis uncovers a specific function of the RNA-binding activity of the nuclear mRNP component Npl3. This approach can be readily applied to RBPs in any RNA metabolic process.

RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large messenger ribonucleoprotein particle (mRNP) assemblies, these proteins often function in. Using UV-induced RNA-protein crosslinking and subsequent mass spectrometric analysis, we first identified more than 100 in vivo RNA crosslinks in 16 nuclear mRNP components in S. cerevisiae. For functional analysis, we chose Npl3, for which we determined crosslinks in its two RNA recognition motifs (RRM) and in the flexible linker region connecting the two. Using NMR and structural analyses, we show that both RRM domains and the linker uniquely contribute to RNA recognition. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains of Npl3. Notably, the npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of further mRNP components into nuclear mRNPs, establishing a function of Npl3 in nuclear mRNP assembly. Taken together, we determined the specific function of the RNA-binding activity of the nuclear mRNP component Npl3, an approach that can be applied to many RBPs in any RNA metabolic process.

The mechanisms by which viruses hijack their host’s genetic machinery are of enormous current interest. One mechanism is adenosine diphosphate (ADP) ribosylation, where ADP-ribosyltransferases (ARTs) transfer an ADP-ribose fragment from the ubiquitous coenzyme nicotinamide adenine dinucleotide (NAD) to acceptor proteins. When bacteriophage T4 infects Escherichia coli, three different ARTs reprogram the host’s transcriptional and translational apparatus. Recently, NAD was identified as a 5’-modification of cellular RNA molecules in bacteria and higher organisms. Here, we report that bacteriophage T4 ARTs accept not only NAD, but also NAD-RNA as substrate, thereby covalently linking entire RNA chains to acceptor proteins in an “RNAylation” reaction. One of these ARTs, ModB, efficiently RNAylates its host protein target, ribosomal protein S1, at arginine residues and strongly prefers NAD-RNA over NAD. Mutation of a single arginine at position 139 abolishes ADP-ribosylation and RNAylation. Overexpression of mammalian ADP-ribosylarginine hydrolase 1 (ARH1), which cleaves arginine-phosphoribose bonds, shows a decelerated lysis of E. coli when infected with T4. Our findings not only challenge the established views of the phage replication cycle, but also reveal a distinct biological role of NAD-RNA, namely activation of the RNA for enzymatic transfer. Our work exemplifies the first direct connection between RNA modification and post-translational protein modification. As ARTs play important roles in different viral infections, as well as in antiviral defence by the host, RNAylation may have far-reaching implications.

The mechanisms by which viruses hijack their host’s genetic machinery are of enormous current interest. One mechanism is adenosine diphosphate (ADP) ribosylation, where ADP-ribosyltransferases (ARTs) transfer an ADP-ribose fragment from the ubiquitous coenzyme nicotinamide adenine dinucleotide (NAD) to acceptor proteins 1. When bacteriophage T4 infects Escherichia coli, three different ARTs reprogram the host’s transcriptional and translational apparatus 2,3. Recently, NAD was identified as a 5′-modification of cellular RNA molecules in bacteria and higher organisms 4-6. Here, we report that bacteriophage T4 ARTs accept not only NAD, but also NAD-RNA as substrate, thereby covalently linking entire RNA chains to acceptor proteins in an “RNAylation” reaction. One of these ARTs, ModB, efficiently RNAylates its host protein target, ribosomal protein S1, at arginine residues and strongly prefers NAD-RNA over NAD. Mutation of a single arginine at position 139 abolishes ADP-ribosylation and RNAylation. Overexpression of mammalian ADP-ribosylarginine hydrolase 1 (ARH1), which cleaves arginine-phosphoribose bonds, shows a decelerated lysis of E. coli when infected with T4. Our findings not only challenge the established views of the phage replication cycle, but also reveal a distinct biological role of NAD-RNA, namely activation of the RNA for enzymatic transfer. Our work exemplifies the first direct connection between RNA modification and post-translational protein modification. As ARTs play important roles in different viral infections, as well as in antiviral defence by the host 7, RNAylation may have far-reaching implications.Competing Interest StatementThe authors have declared no competing interest.

Sleep is an essential state that allows for recuperation and survival processes. Disturbing sleep triggers stress responses that promote protective gene expression. Sleep and its deprivation grossly impact gene expression, but little is known about how normal or disturbed sleep control gene expression. Central to the induction of sleep are sleep-active neurons, which inhibit wakefulness and promote survival. Sleep and sleep-active neurons are highly conserved. In Caenorhabditis elegans, the sleep-active RIS neuron is crucial for sleep and survival. Here, we show that RIS depolarization promotes the protective gene expression response that occurs during developmental arrest. This response includes the activation of FOXO/DAF-16 and expression of DAF-16 target genes such as HSP-12.6, a small heat-shock protein that is required for starvation survival. Disturbing sleep by mechanical stimulation increases RIS depolarization. RIS activation in turn activates DAF-16 and other genes required for survival. Hence, during normal sleep, RIS depolarization promotes protective gene expression. When sleep is disturbed, protective gene expression gets further increased by raised RIS depolarization. We thus link sleep-active neuron depolarization to protective gene expression changes and suggest that the cellular stress response following sleep deprivation could be understood as a safeguarding process that is caused by the overactivation of sleep-active neurons.

Protein–DNA interactions are key to the functionality and stability of the genome. Identification and mapping of protein–DNA interaction interfaces and sites is crucial for understanding DNA-dependent processes. Here, we present a workflow that allows mass spectrometric (MS) identification of proteins in direct contact with DNA in reconstituted and native chromatin after cross-linking by ultraviolet (UV) light. Our approach enables the determination of contact interfaces at amino-acid level. With the example of chromatin-associated protein SCML2 we show that our technique allows differentiation of nucleosome-binding interfaces in distinct states. By UV cross-linking of isolated nuclei we determined the cross-linking sites of several factors including chromatin-modifying enzymes, demonstrating that our workflow is not restricted to reconstituted materials. As our approach can distinguish between protein–RNA and DNA interactions in one single experiment, we project that it will be possible to obtain insights into chromatin and its regulation in the future., Cross-linking mass spectrometry (XLMS) allows mapping of protein-protein and protein-RNA interactions, but the analysis of protein-DNA complexes remains challenging. Here, the authors develop a UV light-based XLMS workflow to determine protein-DNA interfaces in reconstituted chromatin and isolated nuclei.

Type IV CRISPR–Cas modules belong to class 1 prokaryotic adaptive immune systems, which are defined by the presence of multisubunit effector complexes. They usually lack the known Cas proteins involved in adaptation and target cleavage, and their function has not been experimentally addressed. To investigate RNA and protein components of this CRISPR–Cas type, we located a complete type IV cas gene locus and an adjacent CRISPR array on a megaplasmid of Aromatoleum aromaticum EbN1, which contains an additional type I-C system on its chromosome. RNA sequencing analyses verified CRISPR RNA (crRNA) production and maturation for both systems. Type IV crRNAs were shown to harbour unusually short 7 nucleotide 5′-repeat tags and stable 3′ hairpin structures. A unique Cas6 variant (Csf5) was identified that generates crRNAs that are specifically incorporated into type IV CRISPR–ribonucleoprotein (crRNP) complexes. Structures of RNA-bound Csf5 were obtained. Recombinant production and purification of the type IV Cas proteins, together with electron microscopy, revealed that Csf2 acts as a helical backbone for type IV crRNPs that include Csf5, Csf3 and a large subunit (Csf1). Mass spectrometry analyses identified protein–protein and protein–RNA contact sites. These results highlight evolutionary connections between type IV and type I CRISPR–Cas systems and demonstrate that type IV CRISPR–Cas systems employ crRNA-guided effector complexes. The characterization of the type IV CRISPR system from Aromatoleum aromaticum shows that Csf2 serves as a backbone to which Csf5, Csf3 and Csf1 bind to form CRISPR–ribonucleoprotein complexes. Csf5 (a Cas6 variant) generates CRISPR RNAs with short, 7 nucleotide 5′-repeat tags and stable 3′ hairpin structures.

Scientific reports 8(1), 8880 (2018). doi:10.1038/s41598-018-27118-5, Dnmt2 methylates cytosine at position 38 of tRNA$^{Asp}$ in a variety of eukaryotic organisms. A correlation between the presence of the hypermodified nucleoside queuosine (Q) at position 34 of tRNAAsp and the Dnmt2 dependent C38 methylation was recently found in vivo for S. pombe and D. discoideum. We demonstrate a direct effect of the Q-modification on the methyltransferase catalytic efficiency in vitro, as V$_{max}$/K$_{0.5}$ of purified S. pombe Dnmt2 shows an increase for in vitro transcribed tRNA$^{Asp}$ containing Q34 to 6.27 ∗ 10$^{–3}$ s$^b{−1} \mu M^{−1}$ compared to 1.51 ∗ 10$^{–3}$ s$^{−1} µ$M$^{−1}$ for the unmodified substrate. Q34tRNA$^{Asp}$ exhibits an only slightly increased affinity for Dnmt2 in comparison to unmodified G34tRNA. In order to get insight into the structural basis for the Q-dependency, the crystal structure of S. pombe Dnmt2 was determined at 1.7 Å resolution. It closely resembles the known structures of human and E. histolytica Dnmt2, and contains the entire active site loop. The interaction with tRNA was analyzed by means of mass-spectrometry using UV cross-linked Dnmt2-tRNA complex. These cross-link data and computational docking of Dnmt2 and tRNA$^{Asp}$ reveal Q34 positioned adjacent to the S-adenosylmethionine occupying the active site, suggesting that the observed increase of Dnmt2 catalytic efficiency by queuine originates from optimal positioning of the substrate molecules and residues relevant for methyl transfer., Published by Springer Nature, [London]

Using a set of IR spectroscopy methods the OH- and OD-stretch region of H2O, D2O and HDO were investigated. With a combination of attenuated total reflection infrared (ATR-IR) and transmission infrared Fourier Transform spectroscopy, temperature dependent experiments were carried out. HDO experiments were done with varying isotopic concentrations. We focused on contributions at higher wavenumbers within the OH/OD-stretch region to discuss coupling effects. We also compared results from ATR and transmission spectroscopy in order to check the comparability of these techniques. In addition we tried to investigate how the overtone of the bending vibration may be included into the analysis of the OH-stretch region of pure water.

We used a combination of theoretical and experimental methods to derive the spectroscopic properties of imidazolium-based ionic liquids. Vibrational frequencies, NMR chemical shifts, and quadrupole coupling constants react in comparable manner to changes in the chemical environment. This suggests that both the IR and the NMR spectroscopic properties reflect a similar type of electronic perturbation caused by hydrogen bonding. These relationships of the spectroscopic properties provide detailed information about structural complexes and may thus serve as good indicators of ion-pair formation. They also help to decide which spectroscopic tool is the most sensitive for investigating molecular interactions. The measurement of only one spectroscopic property allows the prediction of other properties that cannot be so easily measured. In some cases, this is the only way to obtain reliable coupling constants for deriving molecular correlation times from macroscopic NMR relaxation times, thus opening a new path for studying structure-dynamics relations in ionic liquids.

We have used 2H magnetic relaxation to study the single-particle reorientation of the 1-ethyl-3-methylimidazolium ([emim]+) ion in two prototypical low-viscosity ionic liquids comprising the bis(trifluoromethylsulfonyl)imide ([Tf2N]−) and dicyanimide ([(CN)2N]−) anions. Hydrodynamic analysis reveals extremely low (‘subslip’) coupling factors not captured by existing theories. Comparison with the microwave dielectric spectrum of [emim][Tf2N] shows that the dominant diffusive component of the dielectric response can be rationalized by single-particle reorientation of the cations.

The formation, stabilisation and reactivity of contact ion pairs of non-protic imidazolium ionic liquids (ILs) in solution are conceptualized in light of selected experimental evidence as well theoretical calculations reported mainly in the last ten years. Electric conductivity, NMR, ESI-MS and IR data as well as theoretical calculations support not only the formation of contact ion pairs in solution, but also the presence of larger ionic and neutral aggregates even when dissolved in solvents with relatively high dielectric constants, such as acetonitrile and DMSO. The presence of larger imidazolium supramolecular aggregates is favoured at higher salt concentrations in solvents of low dielectric constant for ILs that contain shorter N-alkyl side chains associated with anions of low coordination ability. The stability and reactivity of neutral contact species are also dependent on the nature of the anion, imidazolium substituents, and are more abundant in ILs containing strong coordinating anions, in particular those that can form charge transfer complexes with the imidazolium cation. Finally, some ILs display reactivities as contact ion pairs rather than solvent-separated ions.

With a combination of FTIR and ab initio calculations we studied the structure of water molecules confined in ionic liquids (ILs). Most of the single water molecules are H-bonded in double donor (DD) configurations either to one anion or to two anions in a 1 : 2 complex. We show that the vibrational stretching modes of such water molecules can be used as sensitive probes for the polarity of the ILs, expressed through dielectric constants. We apply our method to imidazolium salts of the third and fourth generation. We discuss the anion- and cation-dependence and compare our results with other polarity parameters.

Similarities and differences: Far-infrared spectra of protic ionic liquids could be assigned to intermolecular bending and stretching modes of hydrogen bonds. The characteristics of the low-frequency spectra resemble those of water. Both liquids form three-dimensional network structures, but only water is capable of building tetrahedral configurations. EAN: ethylammonium nitrate, PAN: propylammonium nitrate, DMAN: dimethylammonium nitrate.

Cohesion energies determine the phase behavior of materials. The understanding of interaction energies is in particular interesting for ionic liquids. Here we show experimentally that, in accord with theoretical work, the intermolecular cation-anion interactions in ionic liquids can be detected by far FTIR spectroscopy. The measured vibrational bands of aprotic and protic ionic liquids in the low-frequency range can be referred to the interaction strength between cations and anions in various combinations. It can be shown by DFT B3LYP calculations that these interactions are described by characteristic ratios between Coulomb forces and hydrogen bonds. These ratios can be tuned towards increasing hydrogen bond contributions which is reflected in important macroscopic properties of ionic liquids such as enthalpies of vaporization and viscosities. This opens a new path for tuning the desired properties of this new class of material.

We study the structure and dynamics of hydrogen-bonded complexes of H 2 O/D 2 O and dimethyl sulfoxide (DMSO) by infrared spectroscopy, NMR spectroscopy and ab initio calculations. We find that single water molecules occur in two configurations. For one half of the water monomers both OH/OD groups form strong hydrogen bonds to DMSO molecules, whereas for the other half only one of the two OH/OD groups is hydrogen-bonded to a solvent molecule. The H-bond strength between water and DMSO is in the order of that in bulk water. NMR deuteron relaxation rates and calculated deuteron quadrupole coupling constants yield rotational correlation times of water. The molecular reorientation of water monomers in DMSO is two-and-a-half times slower than in bulk water. This result can be explained by local structure behavior.

A model system for long semiflexible rods is introduced to investigate the nematic mesophase. The free energy is separated into two parts F = F″ + Δ, where F″ represents a fully aligned, hard system and Δ a correction which in our case is evaluated by the counting technique of DiMarzio extended to the case of nonrigid rods. F″ may be estimated by a variety of techniques. We find several interesting features: (1) for both rigid and flexible cases the anisotropic phase undergoes a second transition at still higher densities to what we take to be the solid phase; (2) for the flexible case sensible values of length‐to‐breadth ratio and flexibility give the isotropic–anisotropic transition at liquid densities and with Δρ / ρ ∼ 0(1%) in keeping with experiment and in marked contrast to all available results for strictly rigid systems; (3) for a given temperature there is a lower bound to the stiffness energy (associated with making a bend) required for the existence of an isotropic–anisotropic transition. In t...

The existence of an isotropic-anisotropic phase transition in a system of long rigid rods has been noted by a number of workers. Apart from its intrinsic interest, this result has been considered to have application to at least the nematic-isotropic phase transition observed for nematic mesophases, and perhaps more generally to other mesophases as well.