NMR characterization and ligand binding site of the stem-loop 2 motif from the Delta variant of SARS-CoV-2.

The stem-loop 2 motif (s2m) in SARS-CoV-2 (SCoV-2) is located in the 3'-UTR. Although s2m has been reported to display characteristics of a mobile genomic element that might lead to an evolutionary advantage, its function has remained unknown. The secondary structure of the original SCoV-2 RNA sequence (Wuhan-Hu-1) was determined by NMR in late 2020, delineating the base-pairing pattern and revealing substantial differences in secondary structure compared to SARS-CoV-1 (SCoV-1). The existence of a single G29742-A29756 mismatch in the upper stem of s2m leads to its destabilization and impedes a complete NMR analysis. With Delta, a variant of concern has evolved with one mutation compared to the original sequence that replaces G29742 by U29742. We show here that this mutation results in a more defined structure at ambient temperature accompanied by a rise in melting temperature. Consequently, we were able to identify >90% of the relevant NMR resonances using a combination of selective RNA labeling and filtered 2D NOESY as well as 4D NMR experiments. We present a comprehensive NMR analysis of the secondary structure, (sub)nanosecond dynamics, and ribose conformation of s2m Delta based on heteronuclear 13C NOE and T 1 measurements and ribose carbon chemical shift-derived canonical coordinates. We further show that the G29742U mutation in Delta has no influence on the druggability of s2m compared to the Wuhan-Hu-1 sequence. With the assignment at hand, we identify the flexible regions of s2m as the primary site for small molecule binding.

RNA G-quadruplex folding is a multi-pathway process driven by conformational entropy.

The kinetics of folding is crucial for the function of many regulatory RNAs including RNA G-quadruplexes (rG4s). Here, we characterize the folding pathways of a G-quadruplex from the telomeric repeat-containing RNA by combining all-atom molecular dynamics and coarse-grained simulations with circular dichroism experiments. The quadruplex fold is stabilized by cations and thus, the ion atmosphere forming a double layer surrounding the highly charged quadruplex guides the folding process. To capture the ionic double layer in implicit solvent coarse-grained simulations correctly, we develop a matching procedure based on all-atom simulations in explicit water. The procedure yields quantitative agreement between simulations and experiments as judged by the populations of folded and unfolded states at different salt concentrations and temperatures. Subsequently, we show that coarse-grained simulations with a resolution of three interaction sites per nucleotide are well suited to resolve the folding pathways and their intermediate states. The results reveal that the folding progresses from unpaired chain via hairpin, triplex and double-hairpin constellations to the final folded structure. The two- and three-strand intermediates are stabilized by transient Hoogsteen interactions. Each pathway passes through two on-pathway intermediates. We hypothesize that conformational entropy is a hallmark of rG4 folding. Conformational entropy leads to the observed branched multi-pathway folding process for TERRA25. We corroborate this hypothesis by presenting the free energy landscapes and folding pathways of four rG4 systems with varying loop length.

The 5'-terminal stem-loop RNA element of SARS-CoV-2 features highly dynamic structural elements that are sensitive to differences in cellular pH.

We present the nuclear magnetic resonance spectroscopy (NMR) solution structure of the 5'-terminal stem loop 5_SL1 (SL1) of the SARS-CoV-2 genome. SL1 contains two A-form helical elements and two regions with non-canonical structure, namely an apical pyrimidine-rich loop and an asymmetric internal loop with one and two nucleotides at the 5'- and 3'-terminal part of the sequence, respectively. The conformational ensemble representing the averaged solution structure of SL1 was validated using NMR residual dipolar coupling (RDC) and small-angle X-ray scattering (SAXS) data. We show that the internal loop is the major binding site for fragments of low molecular weight. This internal loop of SL1 can be stabilized by an A12-C28 interaction that promotes the transient formation of an A+•C base pair. As a consequence, the pKa of the internal loop adenosine A12 is shifted to 5.8, compared to a pKa of 3.63 of free adenosine. Furthermore, applying a recently developed pH-differential mutational profiling (PD-MaP) approach, we not only recapitulated our NMR findings of SL1 but also unveiled multiple sites potentially sensitive to pH across the 5'-UTR of SARS-CoV-2.

Structure of an internal loop motif with three consecutive U•U mismatches from stem-loop 1 in the 3'-UTR of the SARS-CoV-2 genomic RNA.

The single-stranded RNA genome of SARS-CoV-2 is highly structured. Numerous helical stem-loop structures interrupted by mismatch motifs are present in the functionally important 5'- and 3'-UTRs. These mismatches modulate local helical geometries and feature unusual arrays of hydrogen bonding donor and acceptor groups. However, their conformational and dynamical properties cannot be directly inferred from chemical probing and are difficult to predict theoretically. A mismatch motif (SL1-motif) consisting of three consecutive U•U base pairs is located in stem-loop 1 of the 3'-UTR. We combined NMR-spectroscopy and MD-simulations to investigate its structure and dynamics. All three U•U base pairs feature two direct hydrogen bonds and are as stable as Watson-Crick A:U base pairs. Plasmodium falciparum 25S rRNA contains a triple U•U mismatch motif (Pf-motif) differing from SL1-motif only with respect to the orientation of the two closing base pairs. Interestingly, while the geometry of the outer two U•U mismatches was identical in both motifs the preferred orientation of the central U•U mismatch was different. MD simulations and potassium ion titrations revealed that the potassium ion-binding mode to the major groove is connected to the different preferred geometries of the central base pair in the two motifs.

Cell-free transcription-translation system: a dual read-out assay to characterize riboswitch function.

Cell-free protein synthesis assays have become a valuable tool to understand transcriptional and translational processes. Here, we established a fluorescence-based coupled in vitro transcription-translation assay as a read-out system to simultaneously quantify mRNA and protein levels. We utilized the well-established quantification of the expression of shifted green fluorescent protein (sGFP) as a read-out of protein levels. In addition, we determined mRNA quantities using a fluorogenic Mango-(IV) RNA aptamer that becomes fluorescent upon binding to the fluorophore thiazole orange (TO). We utilized a Mango-(IV) RNA aptamer system comprising four subsequent Mango-(IV) RNA aptamer elements with improved sensitivity by building Mango arrays. The design of this reporter assay resulted in a sensitive read-out with a high signal-to-noise ratio, allowing us to monitor transcription and translation time courses in cell-free assays with continuous monitoring of fluorescence changes as well as snapshots of the reaction. Furthermore, we applied this dual read-out assay to investigate the function of thiamine-sensing riboswitches thiM and thiC from Escherichia coli and the adenine-sensing riboswitch ASW from Vibrio vulnificus and pbuE from Bacillus subtilis, which represent transcriptional and translational on- and off-riboswitches, respectively. This approach enabled a microplate-based application, a valuable addition to the toolbox for high-throughput screening of riboswitch function.

Fendrr synergizes with Wnt signalling to regulate fibrosis related genes during lung development via its RNA:dsDNA triplex element.

Long non-coding RNAs are a very versatile class of molecules that can have important roles in regulating a cells function, including regulating other genes on the transcriptional level. One of these mechanisms is that RNA can directly interact with DNA thereby recruiting additional components such as proteins to these sites via an RNA:dsDNA triplex formation. We genetically deleted the triplex forming sequence (FendrrBox) from the lncRNA Fendrr in mice and found that this FendrrBox is partially required for Fendrr function in vivo. We found that the loss of the triplex forming site in developing lungs causes a dysregulation of gene programs associated with lung fibrosis. A set of these genes contain a triplex site directly at their promoter and are expressed in lung fibroblasts. We biophysically confirmed the formation of an RNA:dsDNA triplex with target promoters in vitro. We found that Fendrr with the Wnt signalling pathway regulates these genes, implicating that Fendrr synergizes with Wnt signalling in lung fibrosis.

High-resolution structure of stem-loop 4 from the 5'-UTR of SARS-CoV-2 solved by solution state NMR.

We present the high-resolution structure of stem-loop 4 of the 5'-untranslated region (5_SL4) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) genome solved by solution state nuclear magnetic resonance spectroscopy. 5_SL4 adopts an extended rod-like structure with a single flexible looped-out nucleotide and two mixed tandem mismatches, each composed of a G•U wobble base pair and a pyrimidine•pyrimidine mismatch, which are incorporated into the stem-loop structure. Both the tandem mismatches and the looped-out residue destabilize the stem-loop structure locally. Their distribution along the 5_SL4 stem-loop suggests a role of these non-canonical elements in retaining functionally important structural plasticity in particular with regard to the accessibility of the start codon of an upstream open reading frame located in the RNA's apical loop. The apical loop-although mostly flexible-harbors residual structural features suggesting an additional role in molecular recognition processes. 5_SL4 is highly conserved among the different variants of SARS-CoV-2 and can be targeted by small molecule ligands, which it binds with intermediate affinity in the vicinity of the non-canonical elements within the stem-loop structure.

Biophysical Investigation of RNA ⋠DNA : DNA Triple Helix and RNA : DNA Heteroduplex Formation by the lncRNAs MEG3 and Fendrr.

Long non-coding RNAs (lncRNAs) are important regulators of gene expression and can associate with DNA as RNA : DNA heteroduplexes or RNA ⋅ DNA : DNA triple helix structures. Here, we review in vitro biochemical and biophysical experiments including electromobility shift assays (EMSA), circular dichroism (CD) spectroscopy, thermal melting analysis, microscale thermophoresis (MST), single-molecule Förster resonance energy transfer (smFRET) and nuclear magnetic resonance (NMR) spectroscopy to investigate RNA ⋅ DNA : DNA triple helix and RNA : DNA heteroduplex formation. We present the investigations of the antiparallel triplex-forming lncRNA MEG3 targeting the gene TGFB2 and the parallel triplex-forming lncRNA Fendrr with its target gene Emp2. The thermodynamic properties of these oligonucleotides lead to concentration-dependent heterogeneous mixtures, where a DNA duplex, an RNA : DNA heteroduplex and an RNA ⋅ DNA : DNA triplex coexist and their relative populations are modulated in a temperature-dependent manner. The in vitro data provide a reliable readout of triplex structures, as RNA ⋅ DNA : DNA triplexes show distinct features compared to DNA duplexes and RNA : DNA heteroduplexes. Our experimental results can be used to validate computationally predicted triple helix formation between novel disease-relevant lncRNAs and their DNA target genes.

A universal model of RNA.DNA:DNA triplex formation accurately predicts genome-wide RNA-DNA interactions.

RNA.DNA:DNA triple helix (triplex) formation is a form of RNA-DNA interaction which regulates gene expression but is difficult to study experimentally in vivo. This makes accurate computational prediction of such interactions highly important in the field of RNA research. Current predictive methods use canonical Hoogsteen base pairing rules, which whilst biophysically valid, may not reflect the plastic nature of cell biology. Here, we present the first optimization approach to learn a probabilistic model describing RNA-DNA interactions directly from motifs derived from triplex sequencing data. We find that there are several stable interaction codes, including Hoogsteen base pairing and novel RNA-DNA base pairings, which agree with in vitro measurements. We implemented these findings in TriplexAligner, a program that uses the determined interaction codes to predict triplex binding. TriplexAligner predicts RNA-DNA interactions identified in all-to-all sequencing data more accurately than all previously published tools in human and mouse and also predicts previously studied triplex interactions with known regulatory functions. We further validated a novel triplex interaction using biophysical experiments. Our work is an important step towards better understanding of triplex formation and allows genome-wide analyses of RNA-DNA interactions.

Dietary methionine starvation impairs acute myeloid leukemia progression.

Targeting altered tumor cell metabolism might provide an attractive opportunity for patients with acute myeloid leukemia (AML). An amino acid dropout screen on primary leukemic stem cells and progenitor populations revealed a number of amino acid dependencies, of which methionine was one of the strongest. By using various metabolite rescue experiments, nuclear magnetic resonance-based metabolite quantifications and 13C-tracing, polysomal profiling, and chromatin immunoprecipitation sequencing, we identified that methionine is used predominantly for protein translation and to provide methyl groups to histones via S-adenosylmethionine for epigenetic marking. H3K36me3 was consistently the most heavily impacted mark following loss of methionine. Methionine depletion also reduced total RNA levels, enhanced apoptosis, and induced a cell cycle block. Reactive oxygen species levels were not increased following methionine depletion, and replacement of methionine with glutathione or N-acetylcysteine could not rescue phenotypes, excluding a role for methionine in controlling redox balance control in AML. Although considered to be an essential amino acid, methionine can be recycled from homocysteine. We uncovered that this is primarily performed by the enzyme methionine synthase and only when methionine availability becomes limiting. In vivo, dietary methionine starvation was not only tolerated by mice, but also significantly delayed both cell line and patient-derived AML progression. Finally, we show that inhibition of the H3K36-specific methyltransferase SETD2 phenocopies much of the cytotoxic effects of methionine depletion, providing a more targeted therapeutic approach. In conclusion, we show that methionine depletion is a vulnerability in AML that can be exploited therapeutically, and we provide mechanistic insight into how cells metabolize and recycle methionine.

m6A Methylation of Transcription Leader Sequence of SARS-CoV-2 Impacts Discontinuous Transcription of Subgenomic mRNAs.

The SARS-CoV-2 genome has been shown to be m6A methylated at several positions in vivo. Strikingly, a DRACH motif, the recognition motif for adenosine methylation, resides in the core of the transcriptional regulatory leader sequence (TRS-L) at position A74, which is highly conserved and essential for viral discontinuous transcription. Methylation at position A74 correlates with viral pathogenicity. Discontinuous transcription produces a set of subgenomic mRNAs that function as templates for translation of all structural and accessory proteins. A74 is base-paired in the short stem-loop structure 5'SL3 that opens during discontinuous transcription to form long-range RNA-RNA interactions with nascent (-)-strand transcripts at complementary TRS-body sequences. A74 can be methylated by the human METTL3/METTL14 complex in vitro. Here, we investigate its impact on the structural stability of 5'SL3 and the long-range TRS-leader:TRS-body duplex formation necessary for synthesis of subgenomic mRNAs of all four viral structural proteins. Methylation uniformly destabilizes 5'SL3 and long-range duplexes and alters their relative equilibrium populations, suggesting that the m6A74 modification acts as a regulator for the abundance of viral structural proteins due to this destabilization.

Synonymous Codons Direct Cotranslational Folding toward Different Protein Conformations.

In all genomes, most amino acids are encoded by more than one codon. Synonymous codons can modulate protein production and folding, but the mechanism connecting codon usage to protein homeostasis is not known. Here we show that synonymous codon variants in the gene encoding gamma-B crystallin, a mammalian eye-lens protein, modulate the rates of translation and cotranslational folding of protein domains monitored in real time by Förster resonance energy transfer and fluorescence-intensity changes. Gamma-B crystallins produced from mRNAs with changed codon bias have the same amino acid sequence but attain different conformations, as indicated by altered in vivo stability and in vitro protease resistance. 2D NMR spectroscopic data suggest that structural differences are associated with different cysteine oxidation states of the purified proteins, providing a link between translation, folding, and the structures of isolated proteins. Thus, synonymous codons provide a secondary code for protein folding in the cell.

The cotranscriptional folding landscape for two cyclic di-nucleotide-sensing riboswitches with highly homologous aptamer domains acting either as ON- or OFF-switches.

Riboswitches are gene regulatory elements located in untranslated mRNA regions. They bind inducer molecules with high affinity and specificity. Cyclic-di-nucleotide-sensing riboswitches are major regulators of genes for the environment, membranes and motility (GEMM) of bacteria. Up to now, structural probing assays or crystal structures have provided insight into the interaction between cyclic-di-nucleotides and their corresponding riboswitches. ITC analysis, NMR analysis and computational modeling allowed us to gain a detailed understanding of the gene regulation mechanisms for the Cd1 (Clostridium difficile) and for the pilM (Geobacter metallireducens) riboswitches and their respective di-nucleotides c-di-GMP and c-GAMP. Binding capability showed a 25 nucleotide (nt) long window for pilM and a 61 nt window for Cd1. Within this window, binding affinities ranged from 35 µM to 0.25 µM spanning two orders of magnitude for Cd1 and pilM showing a strong dependence on competing riboswitch folds. Experimental results were incorporated into a Markov simulation to further our understanding of the transcriptional folding pathways of riboswitches. Our model showed the ability to predict riboswitch gene regulation and its dependence on transcription speed, pausing and ligand concentration.

Mechanistic and structural diversity between cytochrome bd isoforms of Escherichia coli.

The treatment of infectious diseases caused by multidrug-resistant pathogens is a major clinical challenge of the 21st century. The membrane-embedded respiratory cytochrome bd-type oxygen reductase is a critical survival factor utilized by pathogenic bacteria during infection, proliferation and the transition from acute to chronic states. Escherichia coli encodes for two cytochrome bd isoforms that are both involved in respiration under oxygen limited conditions. Mechanistic and structural differences between cydABX (Ecbd-I) and appCBX (Ecbd-II) operon encoded cytochrome bd variants have remained elusive in the past. Here, we demonstrate that cytochrome bd-II catalyzes oxidation of benzoquinols while possessing additional specificity for naphthoquinones. Our data show that although menaquinol-1 (MK1) is not able to directly transfer electrons onto cytochrome bd-II from E. coli, it has a stimulatory effect on its oxygen reduction rate in the presence of ubiquinol-1. We further determined cryo-EM structures of cytochrome bd-II to high resolution of 2.1 Å. Our structural insights confirm that the general architecture and substrate accessible pathways are conserved between the two bd oxidase isoforms, but two notable differences are apparent upon inspection: (i) Ecbd-II does not contain a CydH-like subunit, thereby exposing heme b595 to the membrane environment and (ii) the AppB subunit harbors a structural demethylmenaquinone-8 molecule instead of ubiquinone-8 as found in CydB of Ecbd-I Our work completes the structural landscape of terminal respiratory oxygen reductases of E. coli and suggests that structural and functional properties of the respective oxidases are linked to quinol-pool dependent metabolic adaptations in E. coli.

Identifying and Overcoming Artifacts in 1 H-Based Saturation Transfer NOE NMR Experiments.

Magnetization transfer experiments are versatile nuclear magnetic resonance (NMR) tools providing site-specific information. We have recently discussed how saturation magnetization transfer (SMT) experiments could leverage repeated repolarizations arising from exchanges between labile and water protons to enhance connectivities revealed via the nuclear Overhauser effect (NOE). Repeated experience with SMT has shown that a number of artifacts may arise in these experiments, which may confound the information being sought - particularly when seeking small NOEs among closely spaced resonances. One of these pertains to what we refer to as "spill-over" effects, originating from the use of long saturation pulses leading to changes in the signals of proximate peaks. A second, related but in fact different effect, derives from what we describe as NOE "oversaturation", a phenomenon whereby the use of overtly intense RF fields overwhelms the cross-relaxation signature. The origin and ways to avoid these two effects are described. A final source of potential artifact arises in applications where the labile 1Hs of interest are bound to 15N-labeled heteronuclei. SMT's long 1H saturation times will then be usually implemented while under 15N decoupling based on cyclic schemes leading to decoupling sidebands. Although these sidebands usually remain invisible in NMR, they may lead to a very efficient saturation of the main resonance when touched by SMT frequencies. All of these phenomena are herein experimentally demonstrated, and solutions to overcome them are proposed.

Integrated NMR/Molecular Dynamics Determination of the Ensemble Conformation of a Thermodynamically Stable CUUG RNA Tetraloop.

Both experimental and theoretical structure determinations of RNAs have remained challenging due to the intrinsic dynamics of RNAs. We report here an integrated nuclear magnetic resonance/molecular dynamics (NMR/MD) structure determination approach to describe the dynamic structure of the CUUG tetraloop. We show that the tetraloop undergoes substantial dynamics, leading to averaging of the experimental data. These dynamics are particularly linked to the temperature-dependent presence of a hydrogen bond within the tetraloop. Interpreting the NMR data by a single structure represents the low-temperature structure well but fails to capture all conformational states occurring at a higher temperature. We integrate MD simulations, starting from structures of CUUG tetraloops within the Protein Data Bank, with an extensive set of NMR data, and provide a structural ensemble that describes the dynamic nature of the tetraloop and the experimental NMR data well. We thus show that one of the most stable and frequently found RNA tetraloops displays substantial dynamics, warranting such an integrated structural approach.

Modulation of Aß42 Aggregation Kinetics and Pathway by Low-Molecular-Weight Inhibitors.

The aggregation of amyloid-ß 42 (Aß42) is directly related to the pathogenesis of Alzheimer's disease. Here, we have investigated the early stages of the aggregation process, during which most of the cytotoxic species are formed. Aß42 aggregation kinetics, characterized by the quantification of Aß42 monomer consumption, were tracked by real-time solution NMR spectroscopy (RT-NMR) allowing the impact that low-molecular-weight (LMW) inhibitors and modulators exert on the aggregation process to be analysed. Distinct differences in the Aß42 kinetic profiles were apparent and were further investigated kinetically and structurally by using thioflavin T (ThT) and transmission electron microscopy (TEM), respectively. LMW inhibitors were shown to have a differential impact on early-state aggregation. Insight provided here could direct future therapeutic design based on kinetic profiling of the process of fibril formation.

Site-Specific Labeling of RNAs with Modified and 19 F-Labeled Nucleotides by Chemo-Enzymatic Synthesis.

More than 170 post-transcriptional modifications of RNAs have currently been identified. Detailed biophysical investigations of these modifications have been limited since large RNAs containing these post-transcriptional modifications are difficult to produce. Further, adequate readout of spectroscopic fingerprints are important, necessitating additional labeling procedures beyond the naturally occurring RNA modifications. Here, we report the chemo-enzymatic synthesis of RNA modifications and several structurally similar fluorine-modified analogs further optimizing a recently developed methodology.[1] This chemo-enzymatic method allows synthesis of also large RNAs. We were able to incorporate 16 modified nucleotides and 6 19 F-labeled nucleotides. To showcase the applicability of such modified large RNAs, we incorporated a 19 F-labeled cytidine into the aptamer domain of the 2'dG sensing riboswitch (2'dG-sw) from Mesoplasma florum, enabling characterizing RNA fold, ligand binding and kinetics. Thanks to the large chemical shift dispersion of 19 F, we can detect conformational heterogeneity in the apo state of the riboswitch.

Optimization of the Lead Compound NVP-BHG712 as a Colorectal Cancer Inhibitor.

The ephrin type-A receptor 2 (EPHA2) kinase belongs to the largest family of receptor tyrosine kinases. There are several indications of an involvement of EPHA2 in the development of infectious diseases and cancer. Despite pharmacological potential, EPHA2 is an under-examined target protein. In this study, we synthesized a series of derivatives of the inhibitor NVP-BHG712 and triazine-based compounds. These compounds were evaluated to determine their potential as kinase inhibitors of EPHA2, including elucidation of their binding mode (X-ray crystallography), affinity (microscale thermophoresis), and selectivity (Kinobeads assay). Eight inhibitors showed affinities in the low-nanomolar regime (KD <10 nM). Testing in up to seven colon cancer cell lines that express EPHA2 reveals that several derivatives feature promising effects for the control of human colon carcinoma. Thus, we have developed a set of powerful tool compounds for fundamental new research on the interplay of EPH receptors in a cellular context.

Parallel reaction pathways accelerate folding of a guanine quadruplex.

G-quadruplexes (G4s) are four-stranded, guanine-rich nucleic acid structures that can influence a variety of biological processes such as the transcription and translation of genes and DNA replication. In many cases, a single G4-forming nucleic acid sequence can adopt multiple different folded conformations that interconvert on biologically relevant timescales, entropically stabilizing the folded state. The coexistence of different folded conformations also suggests that there are multiple pathways leading from the unfolded to the folded state ensembles, potentially modulating the folding rate and biological activity. We have developed an experimental method for quantifying the contributions of individual pathways to the folding of conformationally heterogeneous G4s that is based on mutagenesis, thermal hysteresis kinetic experiments and global analysis, and validated our results using photocaged kinetic NMR experiments. We studied the regulatory Pu22 G4 from the c-myc oncogene promoter, which adopts at least four distinct folded isomers. We found that the presence of four parallel pathways leads to a 2.5-fold acceleration in folding; that is, the effective folding rate from the unfolded to folded ensembles is 2.5 times as large as the rate constant for the fastest individual pathway. Since many G4 sequences can adopt many more than four isomers, folding accelerations of more than an order of magnitude are possible via this mechanism.