CapZyme-Seq Comprehensively Defines Promoter-Sequence Determinants for RNA 5' Capping with NAD.

Nucleoside-containing metabolites such as NAD+ can be incorporated as 5' caps on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report CapZyme-seq, a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-seq with multiplexed transcriptomics, we determine efficiencies of NAD+ capping by Escherichia coli RNAP for ∼16,000 promoter sequences. The results define preferred transcription start site (TSS) positions for NAD+ capping and define a consensus promoter sequence for NAD+ capping: HRRASWW (TSS underlined). By applying CapZyme-seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD+-capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs (sRNAs). Our findings define the promoter-sequence determinants for NCIN capping with NAD+ and provide a general method for analysis of NCIN capping in vitro and in vivo.

Human Transient Receptor Potential Ankyrin 1 Channel: Structure, Function, and Physiology.

The transient receptor potential ion channel TRPA1 is a Ca2+-permeable nonselective cation channel widely expressed in sensory neurons, but also in many nonneuronal tissues typically possessing barrier functions, such as the skin, joint synoviocytes, cornea, and the respiratory and intestinal tracts. Here, the primary role of TRPA1 is to detect potential danger stimuli that may threaten the tissue homeostasis and the health of the organism. The ability to directly recognize signals of different modalities, including chemical irritants, extreme temperatures, or osmotic changes resides in the characteristic properties of the ion channel protein complex. Recent advances in cryo-electron microscopy have provided an important framework for understanding the molecular basis of TRPA1 function and have suggested novel directions in the search for its pharmacological regulation. This chapter summarizes the current knowledge of human TRPA1 from a structural and functional perspective and discusses the complex allosteric mechanisms of activation and modulation that play important roles under physiological or pathophysiological conditions. In this context, major challenges for future research on TRPA1 are outlined.

The torpedo effect in Bacillus subtilis: RNase J1 resolves stalled transcription complexes.

RNase J1 is the major 5'-to-3' bacterial exoribonuclease. We demonstrate that in its absence, RNA polymerases (RNAPs) are redistributed on DNA, with increased RNAP occupancy on some genes without a parallel increase in transcriptional output. This suggests that some of these RNAPs represent stalled, non-transcribing complexes. We show that RNase J1 is able to resolve these stalled RNAP complexes by a "torpedo" mechanism, whereby RNase J1 degrades the nascent RNA and causes the transcription complex to disassemble upon collision with RNAP. A heterologous enzyme, yeast Xrn1 (5'-to-3' exonuclease), is less efficient than RNase J1 in resolving stalled Bacillus subtilis RNAP, suggesting that the effect is RNase-specific. Our results thus reveal a novel general principle, whereby an RNase can participate in genome-wide surveillance of stalled RNAP complexes, preventing potentially deleterious transcription-replication collisions.

The First In Vivo Study Shows That Gyrophoric Acid Changes Behavior of Healthy Laboratory Rats.

Gyrophoric acid (GA), a lichen secondary metabolite, has attracted more attention during the last years because of its potential biological effects. Until now, its effect in vivo has not yet been demonstrated. The aim of our study was to evaluate the basic physicochemical and pharmacokinetic properties of GA, which are directly associated with its biological activities. The stability of the GA in various pH was assessed by conducting repeated UV-VIS spectral measurements. Microsomal stability in rat liver microsomes was performed using Ultra-Performance LC/MS. Binding to human serum albumin (HSA) was assessed using synchronous fluorescence spectra, and molecular docking analysis was used to reveal the binding site of GA to HSA. In the in vivo experiment, 24 Sprague-Dawley rats (Velaz, Únetice, Czech Republic) were used. The animals were divided as follows. The first group (n = 6) included healthy males as control intact rats (♂INT), and the second group (n = 6) included healthy females as controls (♀INT). Groups three and four (♂GA/n = 6 and ♀GA/n = 6) consisted of animals with daily administered GA (10 mg/kg body weight) in an ethanol-water solution per os for a one-month period. We found that GA remained stable under various pH and temperature conditions. It bonded to human serum albumin with the binding constant 1.788 × 106 dm3mol-1 to reach the target tissue via this mechanism. In vivo, GA did not influence body mass gain, food, or fluid intake during the experiment. No liver toxicity was observed. However, GA increased the rearing frequency in behavioral tests (p < 0.01) and center crossings in the elevated plus-maze (p < 0.01 and p < 0.001, respectively). In addition, the time spent in the open arm was prolonged (p < 0.01 and p < 0.001, respectively). Notably, GA was able to pass through the blood-brain barrier, indicating its ability to permeate into the brain and to stimulate neurogenesis in the hilus and subgranular zone of the hippocampus. These observations highlight the potential role of GA in influencing brain function and neurogenesis.

Spatial arrangements of cyclodextrin host-guest complexes in solution studied by 13C NMR and molecular modelling.

13C NMR spectroscopic analyses of Cs symmetric guest molecules in the cyclodextrin host cavity, combined with molecular modelling and solid-state X-ray analysis, provides a detailed description of the spatial arrangement of cyclodextrin host-guest complexes in solution. The chiral cavity of the cyclodextrin molecule creates an anisotropic environment for the guest molecule resulting in a splitting of its prochiral carbon signals in 13C NMR spectra. This signal split can be correlated to the distance of the guest atoms from the wall of the host cavity and to the spatial separation of binding sites preferred by pairs of prochiral carbon atoms. These measurements complement traditional solid-state analyses, which rely on the crystallization of host-guest complexes and their crystallographic analysis.

Synthesis and cholinesterase inhibitory activity study of Amaryllidaceae alkaloid analogues with N-methyl substitution.

Polycyclic compounds with N-methyl substitution, structurally related to Amaryllidaceae alkaloids, have been synthesised, together with their analogues bearing a quaternary nitrogen atom. To prevent the lone electron pair of the nitrogen from interfering with the reaction sequence, two approaches to the synthesis were investigated: N-oxidation and Boc protection of the nitrogen. The second method was more successful due to the limited stability of N-oxides in the halocyclisation step. An asymmetric version of the synthesis was also developed for this type of compounds. The prepared products were tested in vitro for their cholinesterase inhibitory activity and the results were rationalised by molecular docking studies with human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBuChE). In general, our products were more active against BuChE than against AChE, and it was noted that larger ligands should be prepared for future studies, since in some cases acetylcholine can still fit into the active site along with the bound ligand.

Design and NMR characterization of reversible head-to-tail boronate-linked macrocyclic nucleic acids.

Inspired by the ability of boronic acids to bind with compounds containing diol moieties, we envisioned the formation in solution of boronate ester-based macrocycles by the head-to-tail assembly of a nucleosidic precursor that contains both a boronic acid and the natural 2',3'-diol of ribose. DOSY NMR spectroscopy experiments in water and anhydrous DMF revealed the dynamic assembly of this precursor into dimeric and trimeric macrocycles in a concentration-dependent fashion as well as the reversibility of the self-assembly process. NMR experimental values and quantum mechanics calculations provided further insight into the sugar pucker conformation profile of these macrocycles.

The mechanism of RNA 5' capping with NAD+, NADH and desphospho-CoA.

The chemical nature of the 5' end of RNA is a key determinant of RNA stability, processing, localization and translation efficiency, and has been proposed to provide a layer of 'epitranscriptomic' gene regulation. Recently it has been shown that some bacterial RNA species carry a 5'-end structure reminiscent of the 5' 7-methylguanylate 'cap' in eukaryotic RNA. In particular, RNA species containing a 5'-end nicotinamide adenine dinucleotide (NAD+) or 3'-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria. It has been proposed that NAD+, reduced NAD+ (NADH) and dpCoA caps are added to RNA after transcription initiation, in a manner analogous to the addition of 7-methylguanylate caps. Here we show instead that NAD+, NADH and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP). We further show that both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps, that promoter DNA sequences at and upstream of the transcription start site determine the efficiency of NCIN capping, that NCIN capping occurs in vivo, and that NCIN capping has functional consequences. We report crystal structures of transcription initiation complexes containing NCIN-capped RNA products. Our results define the mechanism and structural basis of NCIN capping, and suggest that NCIN-mediated 'ab initio capping' may occur in all organisms.

Phospho-Mimetic Mutation at Ser602 Inactivates Human TRPA1 Channel.

The Transient Receptor Potential Ankyrin 1 (TRPA1) channel is an integrative molecular sensor for detecting environmental irritant compounds, endogenous proalgesic and inflammatory agents, pressure, and temperature. Different post-translational modifications participate in the discrimination of the essential functions of TRPA1 in its physiological environment, but the underlying structural bases are poorly understood. Here, we explored the role of the cytosolic N-terminal residue Ser602 located near a functionally important allosteric coupling domain as a potential target of phosphorylation. The phosphomimetic mutation S602D completely abrogated channel activation, whereas the phosphonull mutations S602G and S602N produced a fully functional channel. Using mutagenesis, electrophysiology, and molecular simulations, we investigated the possible structural impact of a modification (mutation or phosphorylation) of Ser602 and found that this residue represents an important regulatory site through which the intracellular signaling cascades may act to reversibly restrict or "dampen" the conformational space of the TRPA1 channel and promote its transitions to the closed state.

Mapping of CaM, S100A1 and PIP2-Binding Epitopes in the Intracellular N- and C-Termini of TRPM4.

Molecular determinants of the binding of various endogenous modulators to transient receptor potential (TRP) channels are crucial for the understanding of necessary cellular pathways, as well as new paths for rational drug designs. The aim of this study was to characterise interactions between the TRP cation channel subfamily melastatin member 4 (TRPM4) and endogenous intracellular modulators-calcium-binding proteins (calmodulin (CaM) and S100A1) and phosphatidylinositol 4, 5-bisphosphate (PIP2). We have found binding epitopes at the N- and C-termini of TRPM4 shared by CaM, S100A1 and PIP2. The binding affinities of short peptides representing the binding epitopes of N- and C-termini were measured by means of fluorescence anisotropy (FA). The importance of representative basic amino acids and their combinations from both peptides for the binding of endogenous TRPM4 modulators was proved using point alanine-scanning mutagenesis. In silico protein-protein docking of both peptides to CaM and S100A1 and extensive molecular dynamics (MD) simulations enabled the description of key stabilising interactions at the atomic level. Recently solved cryo-Electron Microscopy (EM) structures made it possible to put our findings into the context of the entire TRPM4 channel and to deduce how the binding of these endogenous modulators could allosterically affect the gating of TRPM4. Moreover, both identified binding epitopes seem to be ideally positioned to mediate the involvement of TRPM4 in higher-order hetero-multimeric complexes with important physiological functions.

The Core and Holoenzyme Forms of RNA Polymerase from Mycobacterium smegmatis.

Bacterial RNA polymerase (RNAP) is essential for gene expression and as such is a valid drug target. Hence, it is imperative to know its structure and dynamics. Here, we present two as-yet-unreported forms of Mycobacterium smegmatis RNAP: core and holoenzyme containing σA but no other factors. Each form was detected by cryo-electron microscopy in two major conformations. Comparisons of these structures with known structures of other RNAPs reveal a high degree of conformational flexibility of the mycobacterial enzyme and confirm that region 1.1 of σA is directed into the primary channel of RNAP. Taken together, we describe the conformational changes of unrestrained mycobacterial RNAP.IMPORTANCE We describe here three-dimensional structures of core and holoenzyme forms of mycobacterial RNA polymerase (RNAP) solved by cryo-electron microscopy. These structures fill the thus-far-empty spots in the gallery of the pivotal forms of mycobacterial RNAP and illuminate the extent of conformational dynamics of this enzyme. The presented findings may facilitate future designs of antimycobacterial drugs targeting RNAP.

Cytoplasmic Inter-Subunit Interface Controls Use-Dependence of Thermal Activation of TRPV3 Channel.

The vanilloid transient receptor potential channel TRPV3 is a putative molecular thermosensor widely considered to be involved in cutaneous sensation, skin homeostasis, nociception, and pruritus. Repeated stimulation of TRPV3 by high temperatures above 50 °C progressively increases its responses and shifts the activation threshold to physiological temperatures. This use-dependence does not occur in the related heat-sensitive TRPV1 channel in which responses decrease, and the activation threshold is retained above 40 °C during activations. By combining structure-based mutagenesis, electrophysiology, and molecular modeling, we showed that chimeric replacement of the residues from the TRPV3 cytoplasmic inter-subunit interface (N251-E257) with the homologous residues of TRPV1 resulted in channels that, similarly to TRPV1, exhibited a lowered thermal threshold, were sensitized, and failed to close completely after intense stimulation. Crosslinking of this interface by the engineered disulfide bridge between substituted cysteines F259C and V385C (or, to a lesser extent, Y382C) locked the channel in an open state. On the other hand, mutation of a single residue within this region (E736) resulted in heat resistant channels. We propose that alterations in the cytoplasmic inter-subunit interface produce shifts in the channel gating equilibrium and that this domain is critical for the use-dependence of the heat sensitivity of TRPV3.

Solution structure of domain 1.1 of the σA factor from Bacillus subtilis is preformed for binding to the RNA polymerase core.

Bacterial RNA polymerase (RNAP) requires σ factors to recognize promoter sequences. Domain 1.1 of primary σ factors (σ1.1) prevents their binding to promoter DNA in the absence of RNAP, and when in complex with RNAP, it occupies the DNA-binding channel of RNAP. Currently, two 3D structures of σ1.1 are available: from Escherichia coli in complex with RNAP and from T. maritima solved free in solution. However, these two structures significantly differ, and it is unclear whether this difference is due to an altered conformation upon RNAP binding or to differences in intrinsic properties between the proteins from these two distantly related species. Here, we report the solution structure of σ1.1 from the Gram-positive bacterium Bacillus subtilis We found that B. subtilis σ1.1 is highly compact because of additional stabilization not present in σ1.1 from the other two species and that it is more similar to E. coli σ1.1. Moreover, modeling studies suggested that B. subtilis σ1.1 requires minimal conformational changes for accommodating RNAP in the DNA channel, whereas T. maritima σ1.1 must be rearranged to fit therein. Thus, the mesophilic species B. subtilis and E. coli share the same σ1.1 fold, whereas the fold of σ1.1 from the thermophile T. maritima is distinctly different. Finally, we describe an intriguing similarity between σ1.1 and δ, an RNAP-associated protein in B. subtilis, bearing implications for the so-far unknown binding site of δ on RNAP. In conclusion, our results shed light on the conformational changes of σ1.1 required for its accommodation within bacterial RNAP.

RNA-based boronate internucleosidic linkages: an entry into reversible templated ligation and loop formation.

The use of templates able to control the assembly and disassembly of supramolecular biopolymers is an attractive approach with applications ranging from engineering new biopolymers to the modulation of complex biological systems. Self-assembled nucleic acid-based systems hold thus substantive potential for the construction of well-defined and stimuli-responsive molecular architectures. We report here for the first time the synthesis of a 5'-boronoribonucleotidic phosphoramidite building block, its incorporation at the 5' extremities of RNA sequences, and its ability to generate boronate internucleosidic linkages by RNA- and DNA-templated ligation. Moreover, melting denaturation studies also revealed that 5'-boronic acid ended RNA sequences are able to promote the formation of RNA loops in the presence of RNA templating partners. Molecular-dynamics (MD) simulations were used to model the structural parameters governing these processes.

Influence of major-groove chemical modifications of DNA on transcription by bacterial RNA polymerases.

DNA templates containing a set of base modifications in the major groove (5-substituted pyrimidines or 7-substituted 7-deazapurines bearing H, methyl, vinyl, ethynyl or phenyl groups) were prepared by PCR using the corresponding base-modified 2'-deoxyribonucleoside triphosphates (dNTPs). The modified templates were used in an in vitro transcription assay using RNA polymerase from Bacillus subtilis and Escherichia coli Some modified nucleobases bearing smaller modifications (H, Me in 7-deazapurines) were perfectly tolerated by both enzymes, whereas bulky modifications (Ph at any nucleobase) and, surprisingly, uracil blocked transcription. Some middle-sized modifications (vinyl or ethynyl) were partly tolerated mostly by the E. colienzyme. In all cases where the transcription proceeded, full length RNA product with correct sequence was obtained indicating that the modifications of the template are not mutagenic and the inhibition is probably at the stage of initiation. The results are promising for the development of bioorthogonal reactions for artificial chemical switching of the transcription.

The plausible association of MTHFR and ADORA2A polymorphisms with nodules in rheumatoid arthritis patients treated with methotrexate.

OBJECTIVE: The treatment of rheumatoid arthritis (RA) patients with methotrexate (MTX) is linked to the development or progression of rheumatoid nodules. The aim of this study was to determine whether folate and adenosine pathways-related single nucleotide polymorphisms might be predictive of increased nodule formation in RA patients treated with oral MTX. METHODS: A total of 185 Caucasian RA patients were enrolled in this cross-sectional study, all of whom fulfilled the 1987 RA criteria of the American College of Rheumatology; each patient had a history of MTX treatment. RESULTS: A higher frequency of the MTHFR 1298AA genotype was found in 17 (70.8%) of 24 patients with general nodules [odds ratio (OR)=3.08, 95% confidence interval (CI): 1.20-7.69] and in 14 (73.7%) of 19 patients who developed nodules during MTX treatment (OR=3.55, 95% CI: 1.22-10.32). In contrast, a negative association with nodules during MTX treatment (OR=0.29, 95% CI: 0.08-1.10) was found for 19 (79.2%) patients with the TT genotype (rs2298383) in the adenosine A2a receptor gene (ADORA2A). However, the significance did not remain upon correction for multiple testing. The combination of MTHFR 1298AA along with ADORA2A rs2298383 CC or CT genotypes occurring in one-third of RA patients showed a higher frequency of general nodules 15/59 (25.4%) as well as developing nodules during MTX treatment 13/59 (22.0%) in comparison with the overall studied group: 24/185 (13.0%) and 19/185 (10.3%), respectively. CONCLUSION: This exploratory study indicates for the first time a plausible association of adenosine and folate pathways single nucleotide polymorphisms in nodules' etiopathogenesis.

RNA-directed off/on switch of RNase H activity using boronic ester formation.

RNase H is a non-specific endonuclease which degrades selectively the RNA strand in DNA/RNA duplexes. We demonstrate in the present study that 5'-boronic acid modified oligonucleotides hybridized to a RNA target sequence converts RNase H to an inactivated enzyme complex. The dynamic formation of a boronate ester upon addition of a diol moiety disrupts the enzyme-inhibitor complex and reactivates RNase H. Moreover, we show that reactivation of RNase H function can also be engineered through short RNA trimers inputs that fashion RNase H from a non-specific DNA-guided enzyme into an informational and programmable RNA-guided one. Examples of programmable RNA recognition and cleavage illustrate the potential of this new stimuli-responsive system.

Tuning the hybridization properties of modified oligonucleotides: from flexible to conformationally constrained phosphonate internucleotide linkages.

The concept of conformational restriction leading to the preorganization of modified strands has proven to be successful and has afforded nucleic acid analogues with many interesting properties suitable for various biochemical applications. We utilized this concept to prepare a set of constrained oligonucleotides derived from 1,4-dioxane and 1,3-dioxolane-locked nucleoside phosphonates and evaluated their hybridization affinities towards their complementary RNA strands. With an increase of ΔTm per modification up to +5.2 °C, the hybridization experiments revealed the (S)-2',3'-O-phosphonomethylidene internucleotide linkage as one of the most Tm-increasing modifications reported to date. Moreover, we introduced a novel prediction tool for the pre-selection of potentially interesting chemical modifications of oligonucleotides.

5'-O-Methylphosphonate nucleic acids--new modified DNAs that increase the Escherichia coli RNase H cleavage rate of hybrid duplexes.

Several oligothymidylates containing various ratios of phosphodiester and isopolar 5'-hydroxyphosphonate, 5'-O-methylphosphonate and 3'-O-methylphosphonate internucleotide linkages were examined with respect to their hybridization properties with oligoriboadenylates and their ability to induce RNA cleavage by ribonuclease H (RNase H). The results demonstrated that the increasing number of 5'-hydroxyphosphonate or 5'-O-methylphosphonate units in antisense oligonucleotides (AOs) significantly stabilizes the heteroduplexes, whereas 3'-O-methylphosphonate AOs cause strong destabilization of the heteroduplexes. Only the heteroduplexes with 5'-O-methylphosphonate units in the antisense strand exhibited a significant increase in Escherichia coli RNase H cleavage activity by up to 3-fold (depending on the ratio of phosphodiester and phosphonate linkages) in comparison with the natural heteroduplex. A similar increase in RNase H cleavage activity was also observed for heteroduplexes composed of miRNA191 and complementary AOs containing 5'-O-methylphosphonate units. We propose for this type of AOs, working via the RNase H mechanism, the abbreviation MEPNA (MEthylPhosphonate Nucleic Acid).

Synthesis and structural characterization of monomeric and dimeric peptide nucleic acids prepared by using microwave-promoted multicomponent reactions.

A solution phase synthesis of peptide nucleic acid monomers and dimers was developed by using microwave-promoted Ugi multicomponent reactions. A mixture of a functionalized amine, a carboxymethyl nucleobase, paraformaldehyde and an isocyanide as building blocks generates PNA monomers which are then partially deprotected and used in a second Ugi 4CC reaction, leading to PNA dimers. Conformational rotamers were identified by using NMR and MD simulations.