Julia E. Weigand

The 5’-untranslated region (5’-UTR) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly structured and comprises five conserved stem-loops that serve as cis -acting regulatory elements. During infection, host proteins are recruited to these elements and regulate multiple steps during the viral life cycle. Stem-loop 5 (SL5) is highly conserved among different alpha- and betacoronaviruses and includes the start codon of non-structural protein 1 (nsp1). However, the function of SL5 in viral replication and its interacting proteins remain to be determined. Here, we identified heterogeneous nuclear ribonucleoprotein M (HNRNPM) as a host protein functionally interacting with SL5. HNRNPM translocates into the cytoplasm during SARS-CoV-2 infection and promotes protein synthesis from the viral 5’-UTR. Mutations in the lower stem of SL5 impair HNRNPM binding and reduce protein synthesis. Notably, the interaction between HNRNPM and SL5 is conserved among different alpha- and betacoronaviruses. Our findings indicate that HNRNPM is an important host factor that regulates viral translation to promote SARS-CoV-2 replication. IMPORTANCE: SARS-CoV-2 causes the coronavirus disease 2019 (COVID-19), which has demonstrated the pandemic potential of coronavirus infection and the need for effective antiviral treatment. The 5’-UTR of SARS-CoV-2 contains essential structured RNA elements, yet the function of many of these elements, including SL5, remain poorly understood. Our findings provide the first indication that SL5 can serve as a platform for modulation of viral protein synthesis by host factors and offer the first mechanistic understanding into the function of the proviral factor HNRNPM in SARS-CoV-2 infection. This regulation provides new insight into how SARS-CoV-2 employs host factors and could provide a strategy to limit viral replication.

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.

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.

Die Konzentration von mRNAs in der Zelle wird posttranskriptionell durch cis‐regulierende Elemente in der 3’‐untranslatierten Region gesteuert. Diese linearen oder strukturierten Elemente werden von RNA‐bindenden Proteinen (RBPs) erkannt, um die mRNA‐Stabilität zu kontrollieren. Die Proteine Roquin‐1 und −2 erkennen spezifisch RNA‐Stammschleifenmotive. Mit ihrer einzigartigen ROQ‐Domäne erkennen sie Trinukleotid‐Schleifen enthaltende konstitutive Degradationselemente (engl. constitutive decay elements, CDEs) und Hexanukleotid‐Schleifen enthaltende alternative Degradationselemente (engl. alternative decay elements, ADEs), um den mRNA‐Abbau zu initiieren. Die RNA‐Bindekapazität von Roquin gegenüber verschiedenen Klassen von Stammschleifen ist bis jetzt nicht genau charakterisiert, so dass die genauen Bindepräferenzen unklar sind. Hier katalogisieren wir die RNA‐Bindepräferenz der ROQ‐Domäne auf Nukleotidebene mit sRBNS (engl. structured RNA Bind‐n‐Seq), einem neuartigen und angepassten RBNS Protokoll mit strukturierten RNA‐Bibliotheken. Dadurch konnte eine klare Präferenz von Roquin für bestimmte Schleifengrößen festgestellt werden und die bekannten Konsensusmotive für CDEs und ADEs erweitert werden. Die neu identifizierten Motive werden mit nanomolarer Affinität über die bekannte RNA‐ROQ‐Bindung erkannt. Unter Verwendung dieser neuen Stammschleifenvarianten als Blaupausen haben wir neue Ziel‐mRNAs von Roquin vorhergesagt und den erweiterten Targetraum in Zellen verifiziert. Diese Studie demonstriert die Leistungsfähigkeit von Hochdurchsatzverfahren mit RNA‐Strukturen für die systematische Untersuchung von (strukturellen) RNA‐Bindepräferenzen zur detaillierten Identifizierung von mRNA‐Targets und zur Aufklärung der biologischen Funktion von RBPs.

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.

Zinc 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 RNAs. 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 features 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.

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 (RBNS) 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 find that the intrinsically disordered regions (IDR) flanking 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 bi-functional 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.