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.

Protocol to study the direct binding of proteins to RNA:DNA hybrids or RNA-DNA chimeras in living cells using cross-linking immunoprecipitation.

RNA-binding proteins (RBPs) are involved in many biological processes. The direct interaction between protein and RNA can be studied using cross-linking immunoprecipitation (CLIP) techniques in living cells. Here, we present a protocol to characterize the direct binding of proteins to RNA:DNA hybrids or RNA-DNA chimeras in living cells using CLIP. We describe steps for RNA-protein UV-C cross-linking in living cells, isolating RNA-protein complexes, RNA labeling, and extracting nucleic acid. We then detail procedures for nuclease treatment and nucleic acid migration.

A Study on the Binding Mechanism and the Impact of Key Residue Mutations between SND1 and MTDH Peptide through Molecular Dynamics Simulations.

Metastasis of breast cancer is the main cause of death for patients with breast cancer. The interaction between metadherin (MTDH) and staphylococcal nuclease domain 1 (SND1) plays a pivotal role in promoting breast cancer development. However, the binding details between MTDH and SND1 remain unclear. In this study, we employed all-atom molecular dynamics simulations (MDs) and conducted binding energy calculations to investigate the binding details and the impact of key residue mutations on binding. The mutations in key residues have not significantly affected the overall stability of the structure and the fluctuation of residues near the binding site; they have exerted a substantial impact on the binding of SND1 and MTDH peptide. The electrostatic interactions and van der Waals interactions play an important role in the binding of SND1 and the MTDH peptide. The mutations in the key residues have a significant impact on electrostatic and van der Waals interactions, resulting in weakened binding. The energy contributions of key residues mainly come from the electrostatic energy and van der Waals interactions of the side chain. In addition, the key residues form an intricate and stable network of hydrogen bonds and salt-bridge interactions with the MTDH peptide. The mutations in key residues have directly disrupt the interactions formed between SND1 and MTDH peptide, consequently leading to changes in the binding mode of the MTDH peptide. These analyses unveil the detailed atomic-level interaction mechanism between SND1 and the MTDH peptide, providing a molecular foundation for the development of antibreast cancer drugs.

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.

Xmas-2 Protein, the Core Protein of the TREX-2 mRNA Export Complex, Does not Determine the Specificity of ras2 mRNA Binding by the Complex.

The TREX-2 complex of eukaryotes is responsible for the export of a wide range of mRNAs from the nucleus to the cytoplasm. Previously, we showed that a subunit of the D. melanogaster TREX-2 complex, the PCID2 protein, has a domain that specifically interacts with RNA. However, it remains unknown whether other components of the complex are involved in interaction with and recognition of the target mRNA. In the present study, we determined the role of Xmas-2, the core structural subunit of the complex, in the specific recognition of ras2 mRNA fragments. In this work, we showed that Xmas-2 interacts with ras2 mRNA independently of other subunits of the complex. We showed that RNA-binding domains are located in both the N-terminal domain and the C-terminal domain of Xmas-2. However, the interaction of the protein with ras2 mRNA fragments is independent of RNA sequence and structure and is nonspecific. Thus, the Xmas-2 subunit is not involved in the recognition of specific RNA sequences by the complex.

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.

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.

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.

Rational design of base, sugar and backbone modifications improves ADAR-mediated RNA editing.

AIMers are short, chemically modified oligonucleotides that induce A-to-I RNA editing through interaction with endogenous adenosine deaminases acting on RNA (ADAR) enzymes. Here, we describe the development of new AIMer designs with base, sugar and backbone modifications that improve RNA editing efficiency over our previous design. AIMers incorporating a novel pattern of backbone and 2' sugar modifications support enhanced editing efficiency across multiple sequences. Further efficiency gains were achieved through incorporation of an N-3-uridine (N3U), in place of cytidine (C), in the 'orphan base' position opposite the edit site. Molecular modeling suggests that N3U might enhance ADAR catalytic activity by stabilizing the AIMer-ADAR interaction and potentially reducing the energy required to flip the target base into the active site. Supporting this hypothesis, AIMers containing N3U consistently enhanced RNA editing over those containing C across multiple target sequences and multiple nearest neighbor sequence combinations. AIMers combining N3U and the novel pattern of 2' sugar chemistry and backbone modifications improved RNA editing both in vitro and in vivo. We provide detailed N3U synthesis methods and, for the first time, explore the impact of N3U and its analogs on ADAR-mediated RNA editing efficiency and targetable sequence space.

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.

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.

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.

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.

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.

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.

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.

Phase Separation Modulates the Thermodynamics and Kinetics of RNA Hybridization.

Biomolecular condensates can influence cellular function in a number of ways, including by changing the structural dynamics and conformational equilibria of the molecules partitioned within them. Here we use methyl transverse relaxation optimized spectroscopy (methyl-TROSY) NMR in conjunction with 2'-O-methyl labeling of RNA to characterize the thermodynamics and kinetics of RNA-RNA base pairing in condensates formed by the C-terminal intrinsically disordered region of CAPRIN1, an RNA-binding protein involved in RNA transport, translation, and stability. CAPRIN1 condensates destabilize RNA-RNA base pairing, resulting from a ∼270-fold decrease and a concomitant ∼15-fold increase in the on- and off-rates for duplex formation, respectively. The ∼30-fold slower diffusion of RNA single strands within the condensed phase partially accounts for the reduced on-rate, but the further ∼9-fold reduction likely reflects shedding of CAPRIN1 chains that are interacting with the RNA prior to hybridization. Our study emphasizes the important role of protein solvation in modulating nucleic acid recognition processes inside condensates.

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.

Structural basis of tRNA recognition by the widespread OB fold.

The widespread oligonucleotide/oligosaccharide-binding (OB)-fold recognizes diverse substrates from sugars to nucleic acids and proteins, and plays key roles in genome maintenance, transcription, translation, and tRNA metabolism. OB-containing bacterial Trbp and yeast Arc1p proteins are thought to recognize the tRNA elbow or anticodon regions. Here we report a 2.6 Å co-crystal structure of Aquifex aeolicus Trbp111 bound to tRNAIle, which reveals that Trbp recognizes tRNAs solely by capturing their 3' ends. Structural, mutational, and biophysical analyses show that the Trbp/EMAPII-like OB fold precisely recognizes the single-stranded structure, 3' terminal location, and specific sequence of the 3' CA dinucleotide - a universal feature of mature tRNAs. Arc1p supplements its OB - tRNA 3' end interaction with additional contacts that involve an adjacent basic region and the tRNA body. This study uncovers a previously unrecognized mode of tRNA recognition by an ancient protein fold, and provides insights into protein-mediated tRNA aminoacylation, folding, localization, trafficking, and piracy.