Higher-order RNA assemblies have emerged as an intrinsic property of RNA under Mg2+ conditions, raising the question of whether conserved RNA architecture contributes to their organization and functional competence. Here, we investigated the contribution of conserved RNA architecture to higher-order RNA assembly using conserved PTC-derived RNA fragments representing the A- and P-site regions of the large ribosomal subunit. These RNA fragments exhibited distinct Mg2+-dependent condensation behavior and cooperatively formed dynamic higher-order RNA assemblies. Replacing one conserved PTC-derived fragment with an architectural-control RNA altered RNA assembly behavior and failed to support functional competence in modified ribosomal fragment assays. Primitive peptidyl transfer chemistry serves as a functional readout of higher-order RNA organization. Accordingly, our findings suggest that conserved RNA architecture organizes higher-order RNA assemblies into functionally competent structural states.

Fibroblast growth factor (FGF) signaling plays a pivotal role in the development, maintenance, and regeneration of musculoskeletal tissues. While its transcriptional regulation has been extensively characterized, accumulating evidence indicates that FGF activity is also modulated by a diverse array of post-transcriptional mechanisms. In this review, microRNAs, long non-coding RNAs, alternative splicing, and RNA modifications are examined as key regulators of FGF ligands and receptors across bone, cartilage, muscle, and tooth. RNA-binding proteins are also discussed as regulators of FGF transcript stability and translation. The effects of these RNA-based mechanisms on FGF ligand and receptor abundance, isoform production, and signaling responsiveness are compared, and a pathway-specific RNA regulome is proposed as an organizing framework to integrate these mechanisms across musculoskeletal tissues. This approach also distinguishes directly demonstrated RNA-FGF interactions from indirect associations with connected signaling pathways. The therapeutic implications of targeting post-transcriptional nodes, particularly through non-coding RNAs and epitranscriptomic marks, are discussed in the context of musculoskeletal disease and regeneration, highlighting promising avenues for future musculoskeletal interventions.

While cancer-associated SF3B1 K700E mutation altered production of circRNAs, including circATP9B, the underlying molecular mechanism remained unexplored. Here, we found that SF3B1 mutation enhanced interaction between SF3B1 and SFPQ, an RNA-binding protein involved in the formation of circRNA, and SFPQ bound to flanking introns of the back-splicing site of circATP9B. Deletion of the potential binding sites of SFPQ in the flanking introns decreased the production of circATP9B. Furthermore, enrichment analysis demonstrated that SFPQ binding sites were enriched in the flanking introns of the back-splicing sites of other dysregulated circRNAs associated with SF3B1 mutation. In addition, circATP9B bound to NPM1, resulting in the retention of NPM1 in cytoplasm, which suppressed p53-dependent apoptosis. In summary, we revealed the mechanism by which SF3B1 mutation promotes circATP9B biogenesis, and demonstrated that circATP9B mediates apoptosis through NPM1.

Amino acid transporters are essential for maintaining cellular amino acid homeostasis. The broad-specificity neutral amino acid transporter B[0]AT1 is predominantly expressed in the intestine and kidney, where it mediates the uptake and reuptake of neutral amino acids with varying affinities. Proper plasma-membrane localization of B[0]AT1 requires dimerization with angiotensin-converting enzyme 2 (ACE2) in the intestine or collectrin in the kidney. Structural information is available for apo and substrate-bound B[0]AT1-ACE2 complexes, but how B[0]AT1 interacts with different accessory proteins and recognizes smaller substrates remains enigmatic. Here, we compare cryo-EM structures of B[0]AT1 in complex with ACE2 or collectrin in substrate-free states, and bound to alanine, leucine or tryptophan. The complexes display only minor differences in accessory-protein binding through a conserved interaction surface. Substrate recognition follows a canonical architecture observed for other SLC6 family transporters, with subtle adaptations that potentially account for the different affinities of the three amino acids. Notably, we resolve several cholesterol molecules surrounding the transporter, highlighting a potential regulatory role of the membrane environment that we address by molecular dynamics simulations.

Protein kinase data originate from sources with different identifiers, definitions, and evidence models. KinomeX (https://kinomex.dokhlab.org) is an online platform that aligns the human protein kinase list from KinHub with reviewed entries from UniProtKB/Swiss-Prot and links each record to source-specific evidence. The catalog includes 678 protein entries: 522 KinHub core entries with 536 kinase-domain rows, and 156 reviewed UniProt entries labeled with the Kinase keyword (KW-0418), mostly phosphorylating lipids, nucleotides, sugars, or small molecules. Kinase profiles integrate reviewed annotations, 10,043 experimental structures, AlphaFold models, 1,021,421 ligand bioactivity records, 36,396 tissue-expression data from GTEx, 63,649 ClinVar variants, and live STRING connections. Missing records are marked as unavailable. We developed the Pharmaceutical Development Interest Score (PDIS), which combines citation, clinical-trial, structural, and chemical data with adjustable component weights. These rankings are consistent across numerous weightings (median Spearman ρ = 0.955) and better reflect Pharos target development levels than citation counts alone. Examples highlight 93 under-cited but structurally and chemically accessible kinases and 47 CNS-specific kinases, with evidence on structure, chemistry, and variants. KinomeX enables evidence review and hypothesis formation, using PDIS to indicate documented development efforts rather than biological significance.

We present a closed-form joint probability distribution of disulfide-bonded cysteine-cysteine conformations in proteins. The model is inferred under a Bayesian framework and extends the dihedral conformation verification capabilities of PhiSiCal-Checkup [1], previously restricted to individual amino acid conformations, to now include disulfide-bonded cysteine pairs. It further permits rapid, on-demand analytical derivation of marginal and conditional distributions over any subset of dihedral conformational angles, enabling efficient probability evaluation and random sampling of disulfide conformations. These capabilities provide a unified probabilistic framework for downstream analyses, including modelling, refinement, simulation, and design. Software to analyze disulfide bonded cysteine conformations is accessible fromhttps://lcb.infotech.monash.edu/phisical/disulfide/.

Off-target polypharmacology across the conserved kinase ATP pocket can limit the safety and efficacy of kinase-directed compounds. HuKSA is a transparent ligand-based framework that predicts kinase targets and estimates measured-kinome selectivity from a SMILES string while exposing the reference activities behind each estimate. The method compares ECFP4 fingerprints by Tanimoto similarity and applies five-neighbour, similarity-weighted read-across to an atlas of 794 compounds with measurements spanning 464 kinases. Selectivity is summarized by the S-score, the fraction of measured kinases with pActivity above 6. In this sparse matrix, a Gini coefficient calculated after padding unmeasured kinases closely tracked assay coverage, whereas the measured-panel S-score showed weak assay-count dependence. Nevertheless, S-scores remain conditional on which kinases were assayed and on the mixture of IC50, Ki and Kd measurements. In 125 atlas-excluded KLIFS and PKIDB inhibitors, an annotated target ranked first for 19.2% of compounds and within the top 10 for 55.2%. Top-10 recovery was 83.3% among compounds in the high structural-support band, but these bands are empirical similarity descriptors, not calibrated probabilities. Internal leave-one-compound-out validation gave a pooled target-activity ROC-AUC of 0.83 and S-score prediction RMSE of 0.123. Scaffold and similarity-exclusion analyses defined the applicability domain, indicating strongest performance for queries supported by close measured analogues. By displaying measured neighbours, target-specific support counts, and prediction coverage, HuKSA enables users to inspect target and selectivity hypotheses before experimental testing and follow-up biochemical validation. The publicly accessible online Streamlit web server is available at https://huksalab-tool.hf.space.

Piwi-interacting RNAs (piRNAs) are small non-coding RNAs (ncRNAs) that guide Piwi proteins to complementary RNA targets and trigger gene silencing through secondary 22G siRNA amplification. Accurate identification of piRNA target sites is therefore critical for understanding cellular gene silencing. Previously, we developed piRTarBase to catalog canonical piRNA-mRNA interactions using pirScan, but it was limited to mRNA targets and canonical binding rules. To address these limitations, we developed piRTarBase2, an updated database integrating CLASH-derived interactions processed by ChiRA. piRTarBase2 combined pirScan and RNAup to characterize both canonical and non-canonical binding sites across mRNAs and ncRNAs. It further integrates multiple lines of supporting evidence, including expression fold changes in prg-1 mutants, 22G-RNA signals, and cross-linking-induced mutation sites, for confidence evaluation of the piRNA binding sites. piRTarBase2 provides a comprehensive resource for exploring experimentally supported piRNA-target interactions and is available at https://cosbi7.ee.ncku.edu.tw/pirtarbase_v2/.

The identification of Pipistrellus bat coronavirus HKU5 lineage 2 (HKU5-CoV-2) as a human ACE2-adapted virus highlights the need for antiviral strategies to control emerging HKU5-related merbecoviruses. With no specific antivirals approved for merbecoviruses, repurposing SARS-CoV-2 M[pro] inhibitors is a promising strategy. However, their inhibitory activity and binding mechanisms against HKU5-CoV M[pro] remain poorly understood. Here, we obtained high-resolution crystal structures of HKU5-CoV-1 M[pro] in its apo state (1.75 Å) and in complex with the approved covalent inhibitor nirmatrelvir (1.91 Å) and the non-covalent inhibitor ensitrelvir (1.55 Å). These structures reveal an induced-fit mechanism, in which the S2 loop containing Leu49 adopts an open conformation in the apo state and undergoes inhibitor-specific conformational changes upon inhibitor binding. These structures provided a foundation for characterizing HKU5-CoV-2 M[pro] via modeling and molecular dynamics simulations, demonstrating conserved inhibitor-binding modes across both lineages. Consistent with this, biochemical assays revealed that HKU5-CoV-1 and HKU5-CoV-2 M[pro] exhibited nearly identical enzyme kinetic profiles, with turnover rates approximately two-fold higher than SARS-CoV-2 M[pro]. Structural comparison of the HKU5-CoV-1 and SARS-CoV-2 M[pro] revealed a conserved S1 subsite, alongside key variations: Met25 lining S1', a flexible Leu49-containing S2 loop, and a Ser171-mediated expanded S4 cavity. Despite these variations, nirmatrelvir and ensitrelvir showed potent inhibitory activity, with comparable double-digit nanomolar IC50 values across all three M[pro] proteins. Computational analyses indicated that HKU5-CoV-2 M[pro] engages nirmatrelvir and ensitrelvir in binding modes comparable to HKU5-CoV-1, supporting a conserved mechanism of inhibitor recognition and providing a structural basis for developing pan-coronavirus antivirals targeting emerging merbecoviruses.

UvrD-family Superfamily 1A helicases are processive ATP-dependent motor proteins that function during DNA replication, recombination, repair, and transcription. UvrD-family monomers translocate along single stranded (ss) DNA with 3'-to-5' directionality but must be activated by dimerization to become helicases in the absence of force or accessory factors. Mycobacterium tuberculosis (Mtb) UvrD1 helicase forms dimers via a disulfide bond between native cysteines in the 2B sub-domains of each monomer. E. coli UvrD forms non-covalent dimers using the same 2B domain interface as in Mtb UvrD1. Using both ensemble and single DNA molecule approaches we examined an E. coli UvrD variant (R421C), which forms covalent dimers with constitutive helicase activity. For the first time this has enabled us to compare the ssDNA translocation and helicase activities of covalent dimers and monomers. Crosslinked UvrD dimers exhibit much higher ssDNA translocation processivities than monomers, although with similar translocation rates. Crosslinked UvrD dimers also show highly processive DNA unwinding of thousands of base pairs, much higher than non-crosslinked UvrD dimers, while monomers show no DNA unwinding activity. DNA unwinding rates of crosslinked UvrD dimers are only ∼20% slower than ssDNA translocation rates, indicating they are "active" helicases that directly facilitate duplex destabilization.

The growing availability of 3D protein structures enables large-scale assessment of missense variants and their phenotypic impact. Missense3D, first developed in 2019, predicts the structural consequences of amino acid substitutions and provides mechanistic explanations by identifying features such as cavities, steric clashes and disrupted non-covalent bonds that may cause protein misfolding. The new Missense3D algorithm presented here accepts genomics, protein, and structural coordinates; models variants on both single proteins and protein-protein complexes using experimental and predicted structures, including AlphaFold models; performs automated sequence-structure mapping; and evaluates structural model quality before analysis. The Missense3D web server is freely available to all users, including commercial ones, at https://missense3d.bc.ic.ac.uk/∼missense3d2/.

Understanding and manipulating the 3D structure and dynamics of molecular systems is central to all molecular sciences including structural biology. MolecularWebXR's new HandMol module provides a browser-based, multiuser environment for immersive molecular visualization and interactive modeling across all modern devices. Leveraging the WebXR standard, HandMol requires no installation, allowing users to seamlessly access shared virtual rooms from VR/AR headsets, mobile devices, or computers. HandMol enables natural interaction through hand tracking, allowing users to query atom information by pointing or to manipulate structures using bare-hand gestures. Real-time physics feedback is provided via integrated engines, including ANI-2x for small molecules, AMBER14 for atomistic biomolecular systems, and CALVADOS coarse-grained models. We demonstrate HandMol's utility through 3D-difficult tasks like ligand (un)docking, driving conformational changes, and preparing starting poses for simulations. This globally accessible platform facilitates scientific discussion, hypothesis testing, and immersive education by bridging high-performance molecular dynamics with intuitive, natural control in a collaborative virtual space. Resource link:https://molecularwebxr.org/. Direct link to HandMol:https://molecularwebxr.org/create.

Type II topoisomerases are found in all kingdoms of life and perform key biological functions during replication, transcription, and other DNA metabolic events. These enzymes are also the targets of important classes of anti-bacterial and anti-cancer drugs. Clinically active drugs targeting topoisomerase II primarily act as poisons rather than as enzyme inhibitors. During the normal enzymatic reaction, topoisomerase poisons stabilize the normally reversible covalent enzyme/DNA adduct leading to DNA damage. More recently, Top2 mutants have been identified that exert the same effect as these small molecules, and expression of these mutants in cells leads to genome instability. Mutations that increase the potency of topoisomerase targeting drugs have the potential to mimic drug action and expand insight into the drug mechanism. Here we carried out an extensive screen of mutations in human Top2α to identify mutations that result in elevated levels of sensitivity to the anti-cancer drug etoposide. This screen was conducted by expressing a library of mutagenized human Top2α that complemented a yeast temperature sensitive Top2 allele in rad52- yeast strains. Etoposide hypersensitive mutations were identified throughout the catalytic domains of Top2α. Mutants were typically hypersensitive to other Top2 inhibitors, although some mutants had hypersensitivity that was specific for etoposide. Several examples of mutant proteins identified in the screen were purified and characterized biochemically. These proteins exhibited a broad range of alterations in enzyme activity including altered requirements for divalent cations, altered relaxation and decatenation activity, altered utilization of ATP, and enhanced DNA cleavage in the absence of inhibitors. Our results suggest that diverse mechanisms influence the ability of small molecules to convert type II topoisomerases to DNA damaging agents, and the etoposide hypersensitivity mutations found provide landmarks for the development of small molecules with improved potency.

MAGI1 is a multidomain scaffold protein that coordinates signaling complexes involved in cell adhesion, polarity, and signal transduction. While PDZ domains are generally regarded as independent interaction modules, the role of interdomain communication in regulating partner recognition remains poorly understood. Here, we investigated the molecular basis of partner recognition by its sixth PDZ domain (PDZ6) and the contribution of neighboring domains to this process. Using stopped-flow kinetics, we characterized the interactions of PDZ6 with two physiological partners, β-catenin and TRIP6. The two proteins displayed distinct kinetic and thermodynamic behaviors, indicating different recognition mechanisms despite targeting the same domain. Analysis of ionic strength and pH dependence revealed distinct molecular determinants underlying the two interactions. While β-catenin binding was largely insensitive to the multidomain context, TRIP6 recognition was selectively influenced by adjacent domains. Remarkably, binding of DLL1 to the neighboring PDZ5 domain strongly enhanced TRIP6 affinity while exerting minimal effects on β-catenin, and this effect was further modulated by pH. Together, these findings support a model in which MAGI1 behaves as a dynamically coupled scaffold whose binding properties are shaped by interdomain communication, highlighting the importance of studying PDZ-mediated interactions within their multidomain context.