ECT2 peptide sequences outside the YTH domain regulate its m6A-RNA binding.

The m6A epitranscriptomic mark is the most abundant and widespread internal RNA chemical modification, which through the control of RNA acts as an important factor of eukaryote reproduction, growth, morphogenesis and stress response. The main m6A readers constitute a super family of proteins with hundreds of members that share a so-called YTH RNA binding domain. The majority of YTH proteins carry no obvious additional domain except for an Intrinsically Disordered Region (IDR). In Arabidopsis thaliana IDRs are important for the functional specialization among the different YTH proteins, known as Evolutionarily Conserved C-Terminal region, ECT 1 to 12. Here by studying the ECT2 protein and using an in vitro biochemical characterization, we show that full-length ECT2 and its YTH domain alone have a distinct ability to bind m6A, conversely to previously characterized YTH readers. We identify peptide regions outside of ECT2 YTH domain, in the N-terminal IDR, that regulate its binding to m6A-methylated RNA. Furthermore, we show that the selectivity of ECT2 binding for m6A is enhanced by a high uridine content within its neighbouring sequence, where ECT2 N-terminal IDR is believed to contact the target RNA in vivo. Finally, we also identify small structural elements, located next to ECT2 YTH domain and conserved in a large set of YTH proteins, that enhance its binding to m6A-methylated RNA. We propose from these findings that some of these regulatory regions are not limited to ECT2 or YTH readers of flowering plants but may be widespread among eukaryotic YTH readers.

Peptide nucleic acids can form hairpins and bind RNA-binding proteins.

RNA-binding proteins (RBPs) are a major class of proteins that interact with RNAs to change their fate or function. RBPs and the ribonucleoprotein complexes they constitute are involved in many essential cellular processes. In many cases, the molecular details of RBP:RNA interactions differ between viruses, prokaryotes and eukaryotes, making prokaryotic and viral RBPs good potential drug targets. However, targeting RBPs with small molecules has so far been met with limited success as RNA-binding sites tend to be extended, shallow and dynamic with a mixture of charged, polar and hydrophobic interactions. Here, we show that peptide nucleic acids (PNAs) with nucleic acid-like binding properties and a highly stable peptide-like backbone can be used to target some RBPs. We have designed PNAs to mimic the short RNA stem-loop sequence required for the initiation of prokaryotic signal recognition particle (SRP) assembly, a target for antibiotics development. Using a range of biophysical and biochemical assays, the designed PNAs were demonstrated to fold into a hairpin structure, bind the targeted protein and compete with the native RNA hairpin to inhibit SRP formation. To show the applicability of PNAs against other RBPs, a PNA was also shown to bind Nsp9 from SARS-CoV-2, a protein that exhibits non-sequence-specific RNA binding but preferentially binds hairpin structures. Taken together, our results support that PNAs can be a promising class of compounds for targeting RNA-binding activities in RBPs.

Molecular Simulations to Investigate the Impact of N6-Methylation in RNA Recognition: Improving Accuracy and Precision of Binding Free Energy Prediction.

N6-Methyladenosine (m6A) is a prevalent RNA post-transcriptional modification that plays crucial roles in RNA stability, structural dynamics, and interactions with proteins. The YT521-B (YTH) family of proteins, which are notable m6A readers, functions through its highly conserved YTH domain. Recent structural investigations and molecular dynamics (MD) simulations have shed light on the mechanism of recognition of m6A by the YTHDC1 protein. Despite advancements, using MD to predict the stabilization induced by m6A on the free energy of binding between RNA and YTH proteins remains challenging due to inaccuracy of the employed force field and limited sampling. For instance, simulations often fail to sufficiently capture the hydration dynamics of the binding pocket. This study addresses these challenges through an innovative methodology that integrates metadynamics, alchemical simulations, and force-field refinement. Importantly, our research identifies hydration of the binding pocket as giving only a minor contribution to the binding free energy and emphasizes the critical importance of precisely tuning force-field parameters to experimental data. By employing a fitting strategy built on alchemical calculations, we refine the m6A partial charge parameters, thereby enabling the simultaneous reproduction of N6 methylation on both the protein binding free energy and the thermodynamic stability of nine RNA duplexes. Our findings underscore the sensitivity of binding free energies to partial charges, highlighting the necessity for thorough parametrization and validation against experimental observations across a range of structural contexts.

IGF2BP1-Mediated Regulation of CCN1 Expression by Specific Binding to G-Quadruplex Structure in Its 3'UTR.

The intricate regulation of gene expression is fundamental to the biological complexity of higher organisms, and is primarily governed by transcriptional and post-transcriptional mechanisms. The 3'-untranslated region (3'UTR) of mRNA is rich in cis-regulatory elements like G-quadruplexes (G4s), and plays a crucial role in post-transcriptional regulation. G4s have emerged as significant gene regulators, impacting mRNA stability, translation, and localization. In this study, we investigate the role of a robust parallel G4 structure situated within the 3'UTR of CCN1 mRNA in post-transcriptional regulation. This G4 structure is proximal to the stop codon of human CCN1, and evolutionarily conserved. We elucidated its interaction with the insulin-like growth factor 2 binding protein 1 (IGF2BP1), a noncanonical RNA N6-methyladenosine (m6A) modification reader, revealing a novel interplay between RNA modifications and G-quadruplex structures. Knockdown experiments and mutagenesis studies demonstrate that IGF2BP1 binds specifically to the G4 structure, modulating CCN1 mRNA stability. Additionally, we unveil the role of IGF2BP1's RNA recognition motifs in G4 recognition, highlighting this enthalpically driven interaction. Our findings offer fresh perspectives on the complex mechanisms of post-transcriptional gene regulation mediated by G4 RNA secondary structures.

Human tumor suppressor protein Pdcd4 binds at the mRNA entry channel in the 40S small ribosomal subunit.

Translation is regulated mainly in the initiation step, and its dysregulation is implicated in many human diseases. Several proteins have been found to regulate translational initiation, including Pdcd4 (programmed cell death gene 4). Pdcd4 is a tumor suppressor protein that prevents cell growth, invasion, and metastasis. It is downregulated in most tumor cells, while global translation in the cell is upregulated. To understand the mechanisms underlying translational control by Pdcd4, we used single-particle cryo-electron microscopy to determine the structure of human Pdcd4 bound to 40S small ribosomal subunit, including Pdcd4-40S and Pdcd4-40S-eIF4A-eIF3-eIF1 complexes. The structures reveal the binding site of Pdcd4 at the mRNA entry site in the 40S, where the C-terminal domain (CTD) interacts with eIF4A at the mRNA entry site, while the N-terminal domain (NTD) is inserted into the mRNA channel and decoding site. The structures, together with quantitative binding and in vitro translation assays, shed light on the critical role of the NTD for the recruitment of Pdcd4 to the ribosomal complex and suggest a model whereby Pdcd4 blocks the eIF4F-independent role of eIF4A during recruitment and scanning of the 5' UTR of mRNA.

Cheminformatics-based analysis identified (Z)-2-(2,5-dimethoxy benzylidene)-6-(2-(4-methoxyphenyl)-2-oxoethoxy) benzofuran-3(2H)-one as an inhibitor of Marburg replication by interacting with NP.

The highly pathogenic Marburg virus (MARV) is a member of the Filoviridae family, a non-segmented negative-strand RNA virus. This article represents the computer-aided drug design (CADD) approach for identifying drug-like compounds that prevent the MARV virus disease by inhibiting nucleoprotein, which is responsible for their replication. This study used a wide range of in silico drug design techniques to identify potential drugs. Out of 368 natural compounds, 202 compounds passed ADMET, and molecular docking identified the top two molecules (CID: 1804018 and 5280520) with a high binding affinity of -6.77 and -6.672 kcal/mol, respectively. Both compounds showed interactions with the common amino acid residues SER_216, ARG_215, TYR_135, CYS_195, and ILE_108, which indicates that lead compounds and control ligands interact in the common active site/catalytic site of the protein. The negative binding free energies of CID: 1804018 and 5280520 were -66.01 and -31.29 kcal/mol, respectively. Two lead compounds were re-evaluated using MD modeling techniques, which confirmed CID: 1804018 as the most stable when complexed with the target protein. PC3 of the (Z)-2-(2,5-dimethoxybenzylidene)-6-(2-(4-methoxyphenyl)-2-oxoethoxy) benzofuran-3(2H)-one (CID: 1804018) was 8.74 %, whereas PC3 of the 2'-Hydroxydaidzein (CID: 5280520) was 11.25 %. In this study, (Z)-2-(2,5-dimethoxybenzylidene)-6-(2-(4-methoxyphenyl)-2-oxoethoxy) benzofuran-3(2H)-one (CID: 1804018) unveiled the significant stability of the proteins' binding site in ADMET, Molecular docking, MM-GBSA and MD simulation analysis studies, which also showed a high negative binding free energy value, confirming as the best drug candidate which is found in Angelica archangelica which may potentially inhibit the replication of MARV nucleoprotein.

Dynamic interactions drive early spliceosome assembly.

Splicing is a critical processing step during pre-mRNA maturation in eukaryotes. The correct selection of splice sites during the early steps of spliceosome assembly is highly important and crucial for the regulation of alternative splicing. Splice site recognition and alternative splicing depend on cis-regulatory sequence elements in the RNA and trans-acting splicing factors that recognize these elements and crosstalk with the canonical splicing machinery. Structural mechanisms involving early spliceosome complexes are governed by dynamic RNA structures, protein-RNA interactions and conformational flexibility of multidomain RNA binding proteins. Here, we highlight structural studies and integrative structural biology approaches, which provide complementary information from cryo-EM, NMR, small angle scattering, and X-ray crystallography to elucidate mechanisms in the regulation of early spliceosome assembly and quality control, highlighting the role of conformational dynamics.

Structures of influenza A and B replication complexes give insight into avian to human host adaptation and reveal a role of ANP32 as an electrostatic chaperone for the apo-polymerase.

Replication of influenza viral RNA depends on at least two viral polymerases, a parental replicase and an encapsidase, and cellular factor ANP32. ANP32 comprises an LRR domain and a long C-terminal low complexity acidic region (LCAR). Here we present evidence suggesting that ANP32 is recruited to the replication complex as an electrostatic chaperone that stabilises the encapsidase moiety within apo-polymerase symmetric dimers that are distinct for influenza A and B polymerases. The ANP32 bound encapsidase, then forms the asymmetric replication complex with the replicase, which is embedded in a parental ribonucleoprotein particle (RNP). Cryo-EM structures reveal the architecture of the influenza A and B replication complexes and the likely trajectory of the nascent RNA product into the encapsidase. The cryo-EM map of the FluB replication complex shows extra density attributable to the ANP32 LCAR wrapping around and stabilising the apo-encapsidase conformation. These structures give new insight into the various mutations that adapt avian strain polymerases to use the distinct ANP32 in mammalian cells.

Identification of whole-cell dsRNA-binding proteins by phase separation.

Interactions between double-stranded RNA (dsRNA) and proteins play an important role in cellular homeostasis by regulating the editing, stability, and splicing of intracellular RNA. The identification of dsRNA-binding proteins (dsRBPs) is key; however, it has long been challenging to purify dsRBPs from cells. In this study, we developed a novel method, dsRBPC (dsRNA-binding protein capture), to purify cellular dsRBPs based on classic phase separation purification procedures. A global dsRNA-binding proteome of LLC-PK1 cells was obtained, and we identified 1326 dsRBPs, including 1303 putative novel dsRBPs. Functional analyses suggested that these enriched dsRBPs are mainly associated with rRNA processing, RNA splicing, transcriptional regulation, and nucleocytoplasmic transport. We also found that the ARM (armadillo/beta-catenin-like repeats) motif is a previously unknown dsRNA-binding domain, as demonstrated by biochemical experiments. Collectively, this study provides a useful approach for dsRBP identification and the discovery of a global dsRNA-binding proteome to comprehensively map the dsRNA - protein interaction network.

Intrinsic disorder and salt-dependent conformational changes of the N-terminal region of TFIP11 splicing factor.

Tuftelin Interacting Protein 11 (TFIP11) was identified as a critical human spliceosome assembly regulator, interacting with multiple proteins and localising in membrane-less organelles. However, a lack of structural information on TFIP11 limits the rationalisation of its biological role. TFIP11 is predicted as an intrinsically disordered protein (IDP), and more specifically concerning its N-terminal (N-TER) region. IDPs lack a defined tertiary structure, existing as a dynamic conformational ensemble, favouring protein-protein and protein-RNA interactions. IDPs are involved in liquid-liquid phase separation (LLPS), driving the formation of subnuclear compartments. Combining disorder prediction, molecular dynamics, and spectroscopy methods, this contribution shows the first evidence TFIP11 N-TER is a polyampholytic IDP, exhibiting a structural duality with the coexistence of ordered and disordered assemblies, depending on the ionic strength. Increasing the salt concentration enhances the protein conformational flexibility, presenting a more globule-like shape, and a fuzzier unstructured arrangement that could favour LLPS and protein-RNA interaction. The most charged and hydrophilic regions are the most impacted, including the G-Patch domain essential to TFIP11 function. This study gives a better understanding of the salt-dependent conformational behaviour of the N-TER TFIP11, supporting the hypothesis of the formation of different types of protein assembly, in line with its multiple biological roles.

Aptamer-Loop DNA Nanoflower Recognition and Multicolor Fluorescent Carbon Quantum Dots Labeling System for Multitarget Living Cell Imaging.

Visualization of multiple targets in living cells is important for understanding complex biological processes, but it still faces difficulties, such as complex operation, difficulty in multiplexing, and expensive equipment. Here, we developed a nanoplatform integrating a nucleic acid aptamer and DNA nanotechnology for living cell imaging. Aptamer-based recognition probes (RPs) were synthesized through rolling circle amplification, which were further self-assembled into DNA nanoflowers encapsulated by an aptamer loop. The signal probes (SPs) were obtained by conjugation of multicolor emission carbon quantum dots with oligonucleotides complementary to RPs. Through base pairing, RPs and SPs were hybridized to generate aptamer sgc8-, AS1411-, and Apt-based imaging systems. They were used for individual/simultaneous imaging of cellular membrane protein PTK7, nucleolin, and adenosine triphosphate (ATP) molecules. Fluorescence imaging and intensity analysis showed that the living cell imaging system can not only specifically recognize and efficiently bind their respective targets but also provide a 5-10-fold signal amplification. Cell-cycle-dependent distribution of nucleolin and concentration-dependent fluorescence intensity of ATP demonstrated the utility of the system for tracking changes in cellular status. Overall, this system shows the potential to be a simple, low-cost, highly selective, and sensitive living cell imaging platform.

Differential conformational dynamics in two type-A RNA-binding domains drive the double-stranded RNA recognition and binding.

Trans-activation response (TAR) RNA-binding protein (TRBP) has emerged as a key player in the RNA interference pathway, wherein it binds to different pre-microRNAs (miRNAs) and small interfering RNAs (siRNAs), each varying in sequence and/or structure. We hypothesize that TRBP displays dynamic adaptability to accommodate heterogeneity in target RNA structures. Thus, it is crucial to ascertain the role of intrinsic and RNA-induced protein dynamics in RNA recognition and binding. We have previously elucidated the role of intrinsic and RNA-induced conformational exchange in the double-stranded RNA-binding domain 1 (dsRBD1) of TRBP in shape-dependent RNA recognition. The current study delves into the intrinsic and RNA-induced conformational dynamics of the TRBP-dsRBD2 and then compares it with the dsRBD1 study carried out previously. Remarkably, the two domains exhibit differential binding affinity to a 12-bp dsRNA owing to the presence of critical residues and structural plasticity. Furthermore, we report that dsRBD2 depicts constrained conformational plasticity when compared to dsRBD1. Although, in the presence of RNA, dsRBD2 undergoes induced conformational exchange within the designated RNA-binding regions and other residues, the amplitude of the motions remains modest when compared to those observed in dsRBD1. We propose a dynamics-driven model of the two tandem domains of TRBP, substantiating their contributions to the versatility of dsRNA recognition and binding.

Atomic resolution map of the solvent interactions driving SOD1 unfolding in CAPRIN1 condensates.

Biomolecules can be sequestered into membrane-less compartments, referred to as biomolecular condensates. Experimental and computational methods have helped define the physical-chemical properties of condensates. Less is known about how the high macromolecule concentrations in condensed phases contribute "solvent" interactions that can remodel the free-energy landscape of other condensate-resident proteins, altering thermally accessible conformations and, in turn, modulating function. Here, we use solution NMR spectroscopy to obtain atomic resolution insights into the interactions between the immature form of superoxide dismutase 1 (SOD1), which can mislocalize and aggregate in stress granules, and the RNA-binding protein CAPRIN1, a component of stress granules. NMR studies of CAPRIN1:SOD1 interactions, focused on both unfolded and folded SOD1 states in mixed phase and demixed CAPRIN1-based condensates, establish that CAPRIN1 shifts the SOD1 folding equilibrium toward the unfolded state through preferential interactions with the unfolded ensemble, with little change to the structure of the folded conformation. Key contacts between CAPRIN1 and the H80-H120 region of unfolded SOD1 are identified, as well as SOD1 interaction sites near both the arginine-rich and aromatic-rich regions of CAPRIN1. Unfolding of immature SOD1 in the CAPRIN1 condensed phase is shown to be coupled to aggregation, while a more stable zinc-bound, dimeric form of SOD1 is less susceptible to unfolding when solvated by CAPRIN1. Our work underscores the impact of the condensate solvent environment on the conformational states of resident proteins and supports the hypothesis that ALS mutations that decrease metal binding or dimerization function as drivers of aggregation in condensates.

Structural basis for activity switching in polymerases determining the fate of let-7 pre-miRNAs.

Tumor-suppressor let-7 pre-microRNAs (miRNAs) are regulated by terminal uridylyltransferases TUT7 and TUT4 that either promote let-7 maturation by adding a single uridine nucleotide to the pre-miRNA 3' end or mark them for degradation by the addition of multiple uridines. Oligo-uridylation is increased in cells by enhanced TUT7/4 expression and especially by the RNA-binding pluripotency factor LIN28A. Using cryogenic electron microscopy, we captured high-resolution structures of active forms of TUT7 alone, of TUT7 plus pre-miRNA and of both TUT7 and TUT4 bound with pre-miRNA and LIN28A. Our structures reveal that pre-miRNAs engage the enzymes in fundamentally different ways depending on the presence of LIN28A, which clamps them onto the TUTs to enable processive 3' oligo-uridylation. This study reveals the molecular basis for mono- versus oligo-uridylation by TUT7/4, as determined by the presence of LIN28A, and thus their mechanism of action in the regulation of cell fate and in cancer.

SART3 reads methylarginine-marked glycine- and arginine-rich motifs.

Glycine- and arginine-rich (GAR) motifs, commonly found in RNA-binding and -processing proteins, can be symmetrically (SDMA) or asymmetrically (ADMA) dimethylated at the arginine residue by protein arginine methyltransferases. Arginine-methylated protein motifs are usually read by Tudor domain-containing proteins. Here, using a GFP-Trap, we identify a non-Tudor domain protein, squamous cell carcinoma antigen recognized by T cells 3 (SART3), as a reader for SDMA-marked GAR motifs. Structural analysis and mutagenesis of SART3 show that aromatic residues lining a groove between two adjacent aromatic-rich half-a-tetratricopeptide (HAT) repeat domains are essential for SART3 to recognize and bind to SDMA-marked GAR motif peptides, as well as for the interaction between SART3 and the GAR-motif-containing proteins fibrillarin and coilin. Further, we show that the loss of this reader ability affects RNA splicing. Overall, our findings broaden the range of potential SDMA readers to include HAT domains.

Live Cell Protein Imaging of Tandem Complemented-GFP11-Tagged Coiled-Coil Domain-Containing Protein-124 Identifies this Factor in G3BP1-Induced Stress-Granules.

Coiled-coil domain-containing 124 protein is a multifunctional RNA-binding factor, and it was previously reported to interact with various biomolecular complexes localized at diverse subcellular locations, such as the ribosome, centrosome, midbody, and nucleoli. We aimed to better characterize the subcellular CCDC124 translocation by labelling this protein with a fluorescent tag, followed by laser scanning confocal microscopy methods. As traditional GFP-tagging of small proteins such as CCDC124 often faces limitations like potential structural perturbations of labeled proteins, and interference of the fluorescent-tag with their endogenous cellular functions, we aimed to label CCDC124 with the smallest possible split-GFP associated protein-tagging system (GFP11/GFP1-10) for better characterization of its subcellular localizations and its translocation dynamics. By recombinant DNA techniques we generated CCDC124-constructs labelled with either single of four tandem copies of GFP11 (GFP11 × 1::CCDC124, GFP11 × 4::CCDC124, or CCDC124::GFP11 × 4). We then cotransfected U2OS cells with these split-GFP constructs (GFP11 × 1(or X4)::CCDC124/GFP1-10) and analyzed subcellular localization of CCDC124 protein by laser scanning confocal microscopy. Tagging CCDC124 with four tandem copies of a 16-amino acid short GFP-derived peptide-tag (GFP11 × 4::CCDC124) allowed better characterization of the subcellular localization of CCDC124 protein in our model human bone osteosarcoma (U2OS) cells. Thus, by this novel methodology we successfully identified GFP11 × 4::CCDC124 molecules in G3BP1-overexpression induced stress-granules by live cell protein imaging for the first time. Our findings propose CCDC124 as a novel component of the stress granule which is a membraneless organelle involved in translational shut-down in response to cellular stress.

Phage anti-CRISPR control by an RNA- and DNA-binding helix-turn-helix protein.

In all organisms, regulation of gene expression must be adjusted to meet cellular requirements and frequently involves helix-turn-helix (HTH) domain proteins1. For instance, in the arms race between bacteria and bacteriophages, rapid expression of phage anti-CRISPR (acr) genes upon infection enables evasion from CRISPR-Cas defence; transcription is then repressed by an HTH-domain-containing anti-CRISPR-associated (Aca) protein, probably to reduce fitness costs from excessive expression2-5. However, how a single HTH regulator adjusts anti-CRISPR production to cope with increasing phage genome copies and accumulating acr mRNA is unknown. Here we show that the HTH domain of the regulator Aca2, in addition to repressing Acr synthesis transcriptionally through DNA binding, inhibits translation of mRNAs by binding conserved RNA stem-loops and blocking ribosome access. The cryo-electron microscopy structure of the approximately 40 kDa Aca2-RNA complex demonstrates how the versatile HTH domain specifically discriminates RNA from DNA binding sites. These combined regulatory modes are widespread in the Aca2 family and facilitate CRISPR-Cas inhibition in the face of rapid phage DNA replication without toxic acr overexpression. Given the ubiquity of HTH-domain-containing proteins, it is anticipated that many more of them elicit regulatory control by dual DNA and RNA binding.

Production of stable and pure ZC3H11A - An extensively disordered RNA binding protein.

Human ZC3H11A is an RNA-binding zinc finger protein involved in mRNA export and required for the efficient growth of human nuclear replicating viruses. Its biochemical properties are largely unknown so our goal has been to produce the protein in a pure and stable form suitable for its characterization. This has been challenging since the protein is large (810 amino acids) and with only the N-terminal zinc finger domain (amino acids 1-86) being well structured, the remainder is intrinsically disordered. Our production strategies have encompassed recombinant expression of full-length, truncated and mutated ZC3H11A variants with varying purification tags and fusion proteins in several expression systems, with or without co-expression of chaperones and putative interaction partners. A range of purification schemes have been explored. Initially, only truncated ZC3H11A encompassing the zinc finger domain could successfully be produced in a stable form. It required recombinant expression in insect cells since expression in E. coli gave a protein that aggregated. To reduce problematic nucleic acid contaminations, Cys8, located in one of the zinc fingers, was substituted by Ala and Ser. Interestingly, this did not affect nucleic acid binding, but the full-length protein was stabilised while the truncated version was insoluble. Ultimately, we discovered that when using alkaline buffers (pH 9) for purification, full-length ZC3H11A expressed in Sf9 insect cells was obtained in a stable and >90 % pure form, and as a mixture of monomers, dimers, tetramers and hexamers. Many of the challenges experienced are consistent with its predicted structure and unusual charge distribution.

Structure and RNA-binding of the helically extended Roquin CCCH-type zinc finger.

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.

Structural impacts of two disease-linked ADAR1 mutants: a molecular dynamics study.

Adenosine deaminases acting on RNA (ADARs) are pivotal RNA-editing enzymes responsible for converting adenosine to inosine within double-stranded RNA (dsRNA). Dysregulation of ADAR1 editing activity, often arising from genetic mutations, has been linked to elevated interferon levels and the onset of autoinflammatory diseases. However, understanding the molecular underpinnings of this dysregulation is impeded by the lack of an experimentally determined structure for the ADAR1 deaminase domain. In this computational study, we utilized homology modeling and the AlphaFold2 to construct structural models of the ADAR1 deaminase domain in wild-type and two pathogenic variants, R892H and Y1112F, to decipher the structural impact on the reduced deaminase activity. Our findings illuminate the critical role of structural complementarity between the ADAR1 deaminase domain and dsRNA in enzyme-substrate recognition. That is, the relative position of E1008 and K1120 must be maintained so that they can insert into the minor and major grooves of the substrate dsRNA, respectively, facilitating the flipping-out of adenosine to be accommodated within a cavity surrounding E912. Both amino acid replacements studied, R892H at the orthosteric site and Y1112F at the allosteric site, alter K1120 position and ultimately hinder substrate RNA binding.