Several SARS-CoV-2 variants have evolved with clinical relevance due to their structural and functional impact on viral proteins and genomic RNA structures. A comprehensive structural and functional characterization of single-nucleotide variations in the 5 '-untranslated region (UTR) of SARS-CoV-2 has remained elusive. The co-evolution of 5 '-UTR stem-loop 1 (SL1) and non-structural protein (nsp) 1 mutants are of particular importance, as both are key in directing the translation of the viral genome. Here, we investigate the structure and function of the most frequently emerging mutations in SL1 and nsp1. Mutation C21U in the apical loop of SL1 shows changed loop dynamics and reduced escape from repression by nsp1 mutants. Mutation analyses of the pyrimidine loop and the apical helix of SL1 identify preferred sequence motifs for escape from nsp1 repression. Importantly, sequence preferences are governed by the structural features, with suboptimal pyrimidine sequences escaping repression when presented in the SL1 context. Compared to the nsp1 wild type (wt), the currently circulating nsp1 linker variant S135R is much more sensitive toward sequence and structure variations of the apical loop of SL1. Thus, our study provides new insights into the structure-function relationship and co-evolution between viral RNA structures and viral proteins.

Targeting the RNA genome of SARS-CoV-2 is a viable option for antiviral drug development. We explored three ligand binding sites of the core pseudoknot RNA of the SARS-CoV-2 frameshift element. We iteratively optimized ligands, based on improved affinities, targeting these binding sites and report on structural and dynamic properties of the three identified binding sites. Available experimental 3D structures of the pseudoknot element were compared to SAXS and NMR data to validate its dominant folding state in solution. In order to experimentally map in silico predicted binding sites, NMR assignments of the majority of nucleobases were achieved by segmental labeling of the pseudoknot RNA and isotope-filtered NMR experiments at 1.2 GHz, demonstrating the value of NMR spectroscopy to supplement modelling and docking data. Optimized ligands with enhanced affinity were shown to specifically inhibit frameshifting without affecting 0-frame translation in cell-free translation assays, establishing the frameshift element as target for drug-like ligands of low molecular weight.

HnRNPs are ubiquitously expressed RNA-binding proteins, tightly controlling posttranscriptional gene regulation. Consequently, hnRNP networks are essential for cellular homeostasis and their dysregulation is associated with cancer and other diseases. However, the physiological function of hnRNPs in non-cancerous cell systems are poorly understood. We analyzed the importance of HNRNPDL in endothelial cell functions. Knockdown of HNRNPDL led to impaired proliferation, migration and sprouting of spheroids. Transcriptome analysis identified cyclin D1 (CCND1) and tropomyosin 4 (TPM4) as targets of HNRNPDL, reflecting the phenotypic changes after knockdown. Our findings underline the importance of HNRNPDL for the homeostasis of physiological processes in endothelial cells.

AT-rich interacting domain (ARID)-containing proteins, Arids, are a heterogeneous DNA-binding protein family involved in transcription regulation and chromatin processing. For the member Arid5a, no exact DNA-binding preference has been experimentally defined so far. Additionally, the protein binds to mRNA motifs for transcript stabilization, supposedly through the DNA-binding ARID domain. To date, however, no unbiased RNA motif definition and clear dissection of nucleic acid-binding through the ARID domain have been undertaken. Using NMR-centered biochemistry, we here define the Arid5a DNA preference. Further, high-throughput in vitro binding reveals a consensus RNA-binding motif engaged by the core ARID domain. Finally, transcriptome-wide binding (iCLIP2) reveals that Arid5a has a weak preference for (A)U-rich regions in pre-mRNA transcripts of factors related to RNA processing. We fi nd that the intrinsically disordered regions fl anking the ARID domain modulate the specificity and affinity of DNA binding, while they appear crucial for RNA interactions. Ultimately, our data suggest that Arid5a uses its extended ARID domain for bifunctional gene regulation and that the involvement of IDR extensions is a more general feature of Arids in interacting with different nucleic acids at the chromatin- mRNA interface.

ABSTRACTZinc finger (ZnF) domains appear in a pool of structural contexts and despite their small size achieve varying target specificities, covering single-stranded and double-stranded DNA and RNA as well as proteins. Combined with other RNA-binding domains, ZnFs enhance affinity and specificity of RNA-binding proteins (RBPs). The ZnF-containing immunoregulatory RBP Roquin initiates mRNA decay, thereby controlling the adaptive immune system. Its unique ROQ domain shape-specifically recognizes stem-looped cis-elements in mRNA 3’-untranslated regions (UTR). The N-terminus of Roquin contains a RING domain for protein-protein interactions and a ZnF, which was suggested to play an essential role in RNA decay by Roquin. The ZnF domain boundaries, its RNA motif preference and its interplay with the ROQ domain have remained elusive, also driven by the lack of high-resolution data of the challenging protein. We provide the solution structure of the Roquin-1 ZnF and use an RBNS-NMR pipeline to show that the ZnF recognizes AU-rich elements (ARE). We systematically refine the contributions of adenines in a poly(U)-background to specific complex formation. With the simultaneous binding of ROQ and ZnF to a natural target transcript of Roquin, our study for the first time suggests how Roquin integrates RNA shape and sequence specificity through the ROQ-ZnF tandem.

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.

The cellular levels of mRNAs are controlled post-transcriptionally by cis-regulatory elements located in the 3'-untranslated region. These linear or structured elements are recognized by RNA-binding proteins (RBPs) to modulate mRNA stability. The Roquin-1 and -2 proteins specifically recognize RNA stem-loop motifs, the trinucleotide loop-containing constitutive decay elements (CDEs) and the hexanucleotide loop-containing alternative decay elements (ADEs), with their unique ROQ domain to initiate mRNA degradation. However, the RNA-binding capacity of Roquin towards different classes of stem-loops has not been rigorously characterized, leaving its exact binding preferences unclear. Here, we map the RNA-binding preference of the ROQ domain at nucleotide resolution introducing sRBNS (structured RNA Bind-n-Seq), a customized RBNS workflow with pre-structured RNA libraries. We found a clear preference of Roquin towards specific loop sizes and extended the consensus motifs for CDEs and ADEs. The newly identified motifs are recognized with nanomolar affinity through the canonical RNA-ROQ interface. Using these new stem-loop variants as blueprints, we predicted novel Roquin target mRNAs and verified the expanded target space in cells. The study demonstrates the power of high-throughput assays including RNA structure formation for the systematic investigation of (structural) RNA-binding preferences to comprehensively identify mRNA targets and elucidate the biological function of RBPs.

The outbreak of COVID-19 in December 2019 required the formation of international consortia for a coordinated scientific effort to understand and combat the virus. In this Viewpoint Article, we discuss how the NMR community has gathered to investigate the genome and proteome of SARS-CoV-2 and tested them for binding to low-molecular-weight binders. External factors including extended lockdowns due to the global pandemic character of the viral infection triggered the transition from locally focused collaborative research conducted within individual research groups to digital exchange formats for immediate discussion of unpublished results and data analysis, sample sharing, and coordinated research between more than 50 groups from 18 countries simultaneously. We discuss key lessons that might pertain after the end of the pandemic and challenges that we need to address.

Abstract 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.

Engineering in vitro selected RNA aptamers into in vivo functional riboswitches represents a long-standing challenge in molecular biology. The highly specific aptamer domain of the riboswitch undergoes a conformational adjustment in response to ligand sensing, which in turn exerts the regulatory function. Besides essential factors like structural complexity and ligand binding kinetics, the active role of magnesium ions in stabilizing RNA tertiary structures and assisting in ligand binding can be a vital criterion. We present spectroscopic studies on the magnesium ion-driven folding of the Tetracycline binding aptamer. Using fluorescent labels, the aptamer pre-folding and subsequent ligand binding is monitored by magnesium titration experiments and time-resolved stopped-flow measurements. A minimum concentration of 0.5 mM magnesium is required to fold into a magnesium ion-stabilized binding-competent state with a preformed binding pocket. Tetracycline binding causes a pronounced conformational change that results in the establishment of the triple helix core motif, and that further propagates towards the closing stem. By a dynamic acquisition of magnesium ions, a kink motif is formed at the intersection of the triple helix and closing stem regions. This ultimately entails a stabilization of the closing stem which is discussed as a key element in the regulatory function of the Tetracycline aptamer.

AbstractDer Ausbruch von COVID‐19 im Dezember 2019 erforderte eine Koordination wissenschaftlicher Beiträge zum Verständnis und zur Bekämpfung des Virus und führte so zur Bildung internationaler Forschungskonsortien. In diesem Viewpoint Article erläutern wir, wie sich die NMR‐Community versammelt hat, um sowohl das Genom als auch das Proteom von SARS‐CoV‐2 strukturell zu untersuchen und für beide die Bindung an niedermolekulare Verbindungen zu testen. Externe Faktoren, einschließlich längerfristiger Lockdowns aufgrund der Pandemie, begünstigten den Übergang von lokaler, auf einzelne Forschungsgruppen ausgerichteter Verbundforschung hin zu hochkoordinierter Forschung mit über 50 Gruppen aus 18 Ländern, die durch digitale Austauschformate unmittelbar gemeinsam Daten analysierten und unveröffentlichte Ergebnisse diskutierten und darüber hinaus Proben untereinander teilten. Wir diskutieren hier einige wichtige Lehren, die nach dem Ende der Pandemie gezogen werden sollten, und Herausforderungen, die es anzugehen gilt.

The stem-loop (SL1) is the 5'-terminal structural element within the single-stranded SARS-CoV-2 RNA genome. It is formed by nucleotides 7–33 and consists of two short helical segments interrupted by an asymmetric internal loop. This architecture is conserved among Betacoronaviruses. SL1 is present in genomic SARS-CoV-2 RNA as well as in all subgenomic mRNA species produced by the virus during replication, thus representing a ubiquitous cis-regulatory RNA with potential functions at all stages of the viral life cycle. We present here the 1H, 13C and 15N chemical shift assignment of the 29 nucleotides-RNA construct 5_SL1, which denotes the native 27mer SL1 stabilized by an additional terminal G-C base-pair.

SARS‐CoV‐2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti‐virals. Within the international Covid19‐NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR‐detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure‐based drug design against the SCoV2 proteome.

Proteome integrity is crucial for cellular homeostasis and adaptation to stress conditions such as hypoxia. One mechanism for rapid adaptation of the proteome in response to changing environmental signals is alternative splicing. In addition to generating different protein isoforms, alternative splicing is also capable of controlling total protein levels by the regulated synthesis of non-productive mRNA isoforms. The hypoxia-induced isoform E of the tumor suppressor MAX is produced by retention and translation of the last intron. This leads to an alternative C-terminus that harbors a potent C-degron, the isoE degron. Strikingly, the isoE degron represents a universal protein degradation signal that is not only functional in mammalian cells, but also in yeast and even in bacteria. Essential for efficient protein decay is a conserved (F/W)xxW motif. Degradation of isoE tagged proteins is mediated by the proteasome in eukaryotes and Lon protease in bacteria. Thus, the isoE degron is a broadly applicable and highly efficient tool in protein analyses.

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.

Posttranscriptional regulation at mRNA level is defined not only by sequence, but also by the structure of the mRNA. Structures in the untranslated regions control stability, translation and localization of an mRNA. Evolutionary conservation can be used to identify such regulatory active structures in mRNAs. For example, constitutive decay element (CDE) stem-loops are recognized by Roquin proteins in an exclusively shape-specific manner. Strikingly, some CDEs can serve a dual function in gene repression depending on their folding status.

The SARS-CoV-2 virus is the cause of the respiratory disease COVID-19. As of today, therapeutic interventions in severe COVID-19 cases are still not available as no effective therapeutics have been developed so far. Despite the ongoing development of a number of effective vaccines, therapeutics to fight the disease once it has been contracted will still be required. Promising targets for the development of antiviral agents against SARS-CoV-2 can be found in the viral RNA genome. The 5′- and 3′-genomic ends of the 30 kb SCoV-2 genome are highly conserved among Betacoronaviruses and contain structured RNA elements involved in the translation and replication of the viral genome. The 40 nucleotides (nt) long highly conserved stem-loop 4 (5_SL4) is located within the 5′-untranslated region (5′-UTR) important for viral replication. 5_SL4 features an extended stem structure disrupted by several pyrimidine mismatches and is capped by a pentaloop. Here, we report extensive 1H, 13C, 15N and 31P resonance assignments of 5_SL4 as the basis for in-depth structural and ligand screening studies by solution NMR spectroscopy.

SARS-CoV-2 contains a positive single-stranded RNA genome of approximately 30 000 nucleotides. Within this genome, 15 RNA elements were identified as conserved between SARS-CoV and SARS-CoV-2. By nuclear magnetic resonance (NMR) spectroscopy, we previously determined that these elements fold independently, in line with data from in vivo and ex-vivo structural probing experiments. These elements contain non-base-paired regions that potentially harbor ligand-binding pockets. Here, we performed an NMR-based screening of a poised fragment library of 768 compounds for binding to these RNAs, employing three different 1 H-based 1D NMR binding assays. The screening identified common as well as RNA-element specific hits. The results allow selection of the most promising of the 15 RNA elements as putative drug targets. Based on the identified hits, we derive key functional units and groups in ligands for effective targeting of the RNA of SARS-CoV-2.

The SARS-CoV-2 virus is the cause of the respiratory disease COVID-19. As of today, therapeutic interventions in severe COVID-19 cases are still not available as no effective therapeutics have been developed so far. Despite the ongoing development of a number of effective vaccines, therapeutics to fight the disease once it has been contracted will still be required. Promising targets for the development of antiviral agents against SARS-CoV-2 can be found in the viral RNA genome. The 5′- and 3′-genomic ends of the 30 kb SCoV-2 genome are highly conserved among Betacoronaviruses and contain structured RNA elements involved in the translation and replication of the viral genome. The 40 nucleotides (nt) long highly conserved stem-loop 4 (5_SL4) is located within the 5′-untranslated region (5′-UTR) important for viral replication. 5_SL4 features an extended stem structure disrupted by several pyrimidine mismatches and is capped by a pentaloop. Here, we report extensive 1 H, 13 C, 15 N and 31 P resonance assignments of 5_SL4 as the basis for in-depth structural and ligand screening studies by solution NMR spectroscopy.