Conformational plasticity of RNAs plays important roles in recognizing RNA-binding proteins, and is often modulated by their binding partners. Here, we investigate RNA conformational preferences in a non-redundant dataset of 263 protein-RNA complexes to characterize the structural landscape associated with protein recognition. RNA dinucleotide segments are analyzed using seven backbone torsion angles ({delta}1, {varepsilon}1, {zeta}1, 2, {beta}2, {gamma}2, and {delta}2), two glycosidic torsion angles ({chi}1 and {chi}2) and the pseudo-torsion angle . Focusing on dinucleotide steps present in both interface and non-interface regions, we performed density-based clustering using selected backbone torsion angles to identify recurrent conformational states. We identify 28 distinct RNA dinucleotide conformers containing at least ten members each. Among these, eight conformers represent previously unreported nucleotide conformers (NtCs), including the transitional and the non-canonical states AB06, AB07, BB21, BB22, OP32, OP33, IC08 and IC09. Several of these conformers are preferentially enriched at protein-binding interfaces, suggesting their involvement in local conformational adaptation during protein-RNA recognition. The newly identified conformers span transitional A-B geometries, distorted B-like states, open conformations and compact intercalated structures, highlighting the remarkable structural plasticity of RNA in ribonucleoprotein complexes. Overall, this study expands the current understanding of RNA conformational space and provides a refined RNA dinucleotide conformer library for protein-RNA complexes. These findings will facilitate the identification of novel RNA structural motifs and improved RNA structural modeling, docking protein-RNA complexes and deep learning-based prediction frameworks for describing RNA tertiary structures.
Science Journals
Protein labelling by covalent attachment of a specific substrate to a self-labelling protein tag has become a regular in the life sciences. Herein, we report the design of a two-component labelling system, comprised of a non-fluorescent difluorinated xanthene, called F2X, and a HaloTag mutant engineered for targeted reactivity towards F2X. Upon primary covalent locking of the ligand at the canonical aspartate residue, two proximal lysine residues located at the protein surface can undergo nucleophilic aromatic substitution with the F2X core, building a fluorescent rhodamine via triple-covalent fusion. We used a generalizable in silico pipeline for heuristic conformational sampling of covalent protein-ligand complexes to find suitable mutation sites, culminating in the curation of 7 double-lysine HaloTag mutants for targeted in vitro testing. Reaction with the best-performing mutant, HTPL161K_Q165K, is characterized by full protein mass spectrometry, fluorescence polarization fluorescence lifetime, and fluorescence anisotropy and rationalized by computational modelling. We showcase the system in single molecule microscopy, where obviation of post-labelling purification is a prime advantage when targeting recombinant proteins that may not be expressed in larger quantities, and employ F2X in living cells with reduced photobleaching. Lastly, a cell-impermeable version was obtained by means of sulfonation, exclusively targeting extracellularly exposed HTPKK fused to the neuromodulatory G protein-coupled receptor metabotropic glutamate receptor 2.
Humans comprehend language incrementally, updating the representation of sentence meaning with each incoming word. These updates are guided by the distance between each perceived word and prior expectations--the prediction error. The alignment between large language models (LLMs) and cortical activity inspires the hypothesis that the cortical computation of prediction error is Surface-based, driven by statistical patterns of word form co-occurrence. In contrast, psycholinguistic models propose that prediction error computation is Meaning-based, driven by word semantics. We used polysemic words with ambiguous semantics to distinguish these models: ambiguity would introduce uncertainty into meaning representations and hence the prediction error, if Meaning-based, but would not affect the prediction error, if Surface-based. We examined how ambiguity influenced prediction error signatures in self-paced reading times and magnetoencephalographic (MEG) neural responses during sentence processing. While an LLM-based proxy of prediction error robustly predicted reading times and neural responses to unambiguous words, it failed to predict either under ambiguity. That is, prediction error computation was altered by uncertainty in word meaning, which supports the Meaning-based model and corroborates the essential role of word meaning in predictive language processing. Our findings highlight an important limitation of LLMs as in silico models of the human language faculty.
Course-based undergraduate research experiences (CUREs) can expand undergraduates' access to research and motivate students to stay in science. Yet, little research has examined how CURE instruction shapes student motivation. We leveraged a motivation-related characterization of non-content talk of 48 CURE and non-CURE instructors to predict the motivation-related outcomes of 462 students. We fit a series of multi-level models (MLM) in which we regressed students' post-course scientific self-efficacy, task values, scientific identity, and science-related intentions onto instructors' self-efficacy and task values-related talk, controlling for students' pre-course levels. We also fit an MLM to explore whether instructors' relationship-building talk (immediacy talk) was associated with students' rapport with their instructor. Instructors' self-efficacy talk did not affect students' self-efficacy, and instructors' immediacy talk had a marginally positive but non-significant association with students' rapport ratings. Instructors' task values talk positively influenced students' scientific identity and some but not all of their task values. Instructors' task values talk also positively influenced students' intentions to pursue a science career, but not graduate education or research careers. Collectively, these results suggest that instructors' task values talk may underpin some of the motivational effects of CURE instruction, but that task values talk need not be limited to CUREs.
Conventional diagonal stride skiing traditionally includes a glide phase, characterised by a period of relatively passive gliding on one ski. While the glide phase may take advantage of low ski-snow friction, it does not exhibit the same whole-cycle mechanical energy fluctuations seen in running or walking on foot. A new sub-technique, known as running style, substantially reduces the glide phase and may alter the role of elastic tissues, making the movement pattern more similar to uphill running on foot in its temporal organisation. We examined knee extensor and plantar flexor muscle-tendon behaviour in eight competitive skiers performing conventional diagonal and running techniques on a treadmill inclined at 10 deg. Using synchronised ultrasonography, 3D kinematics, ski forces and EMG, we quantified gastrocnemius medialis and vastus lateralis fascicle and muscle-tendon unit (MTU) dynamics in both the running (RUN) and conventional (CON) styles. Shorter glide and total cycle durations during RUN shifted MTU peak length and velocity earlier during the kick phase. Fascicles in both muscles operated at similar velocities across techniques, showing MTU-fascicle decoupling. Vastus lateralis fascicles shortened at higher absolute peak velocities than gastrocnemius in both conditions, while normalised velocities were similar. RUN increased preactivation and advanced EMG timing, while integrated EMG during the kick was lower compared to CON. These findings suggest that, despite large shifts in external mechanics between glide-based and more running-like skiing, elastic tissues may help stabilise fascicle behaviour and preserve a similar contractile strategy across muscles and techniques.
Staphylococcus aureus encounters diverse environmental conditions during colonization and infection, including fluctuations in nutrient availability, oxidative stress, and oxygen limitation. Adaptation to these environments requires regulatory systems that coordinate stress responses with metabolic remodeling. The extracytoplasmic function sigma factor SigS contributes to stress adaptation and virulence in S. aureus and directly activates expression of the sroAB operon, which encodes the small proteins SroA and SroB. While previous work demonstrated that SroA participates in feedback regulation of sigS expression, the broader physiological role of SroA has remained unclear. To define the regulatory functions of SroA, we performed RNA sequencing following inducible overexpression of sroA in S. aureus. Transcriptome analysis revealed extensive remodeling of gene expression, with approximately 200 transcripts significantly altered. Transcriptome analysis revealed coordinated repression of metabolic pathways (including nitrate respiration and nucleotide biosynthesis) alongside activation of stress-response and nutrient acquisition genes. Northern blot and quantitative RT-PCR analysis confirmed repression of narG and narJ transcripts following SroA overexpression. Consistent with these transcriptional changes, nitrate reduction assays demonstrated that SroA overexpression reduces nitrate respiration activity. In addition to repression of nitrate respiration genes, SroA overexpression broadly suppressed genes involved in de novo purine and pyrimidine biosynthesis. In contrast, transcripts associated with stress responses and nutrient acquisition, including the SOS-associated gene sosA and the phosphate transport gene pstS, were upregulated. Together, these findings identify SroA as a regulator that links stress-responsive signaling to metabolic remodeling in S. aureus, particularly through modulation of nitrate respiration pathways.
WD40 domains are major protein-protein interaction (PPI) scaffolds, yet their contributions to fungal pathogenicity remain poorly defined. We systematically analysed 94 canonical WD40 proteins in Cryptococcus neoformans. Conditional knockdown and sporulation identified 36 essential WD40 proteins, while in vitro and in vivo profiling of 103 signature -tagged deletion strains spanning 52 genes uncovered 31 pathogenicity-related WD40 proteins, including epigenetic and post -transcriptional regulators. We identified Wcp1, a dual-domain protein whose WD40-repeat and cyclophilin domains are required for growth at 37{degrees}C under 5% CO2. Its WD40 scaffold and PPIase domain supported CO2/heat tolerance and virulence. Notably, Wcp1 couples these functions to acidic pH adaptation: wcp1{Delta} failed to grow under elevated temperature and CO2 at acidic pH, exhibited enhanced intracellular acidification, reduced macrophage survival and attenuated virulence in Drosophila and mice. Integrated transcriptomic and proteomic analyses place Wcp1 at the centre of intracellular pH homeostasis, coordinating proton transport, metabolic adaptation and stress-buffering networks.
Quantifying the lipid biosynthesis rate of archaea in hot spring sediments is necessary to interpret the abundance, isotopic patterns, and environmental significance of archaeal lipid biosignatures, with implications for modern biogeochemical cycling and astrobiology. Here, we performed lipid hydrogen stable isotope probing (LH-SIP) experiments on whole sediments collected from two high-temperature, suboxic, circumneutral hot springs in Yellowstone National Park (USA) and El Tatio Geyserfield (Chile). We determined the incorporation of 2H2O into intact polar lipids (IPLs) which provides a taxon- and metabolism-agnostic quantification of biosynthesis under near-natural conditions. We targeted isoprenoid glycerol dialkyl glycerol tetraether lipids (IPL iGDGTs) and recovered structures with 0 to 7 cyclopentyl rings from both springs. We observed minor 2H-uptake into archaeal IPLs in spring sediments in Yellowstone, corresponding to decadal-scale apparent generation times (16 {+/-} 7 years), and no 2H-uptake in El Tatio sediments (consistent with minimum generation times of 35 {+/-} 5 years). We infer that net production of sedimentary IPL-iGDGTs is very slow, consistent with a combination of slow archaeal growth, persistence of older IPLs, lipid recycling, and/or contributions from recently sedimented planktonic biomass. These are the first direct, ex situ estimates of archaeal lipid production rates in terrestrial hydrothermal systems using LH-SIP incubations and provide critical constraints for interpreting archaeal lipids in ancient hot spring deposits. This research establishes a framework for assessing activity by slow-growing extremophilic archaea in hydrothermal environments and provides support for targeting hydrothermal deposits on Mars for biosignature detection efforts.
Advancing the utility of plant synthetic biology requires the continued development of protein engineering tools. Self-assembling protein compartments, such as virus-like particles (VLPs), provide versatile scaffolds for synthetic biology. However, few plant-expressed VLPs have demonstrated broad amenability to protein engineering, restricting their applications to specific contexts. Here, the Salmonella typhimurium bacteriophage P22 VLP is explored as a novel protein scaffold for plant synthetic biology, demonstrating its application in a eukaryote for the first time. Through transient expression in the biofactory plant Nicotiana benthamiana, the capacity for P22 VLPs to correctly assemble and selectively encapsulate recombinant protein cargo is demonstrated. The durability of this protein scaffold is explored, through co-encapsulation of multiple cargo protein species and by encapsulation through direct fusion to the P22 coat protein. Finally, the ability to simultaneously program cargo encapsulation and external protein display on P22 VLPs in vivo is demonstrated through SpyTag/SpyCatcher-mediated protein conjugation. This work demonstrates the broad utility of P22 VLPs as nanoscale protein scaffolds for plant synthetic biology. Keywords: protein scaffolds, cargo encapsulation, protein display, SpyTag/SpyCatcher, transient expression, Nicotiana benthamiana.
How do humans store sequences that far exceed working memory capacity? Using visuo-spatial and binary auditory sequences, we previously showed that a Language of Thought (LoT) architecture, in which simple primitives are recursively combined into hierarchical programs, enables efficient storage of structured sequences. Here we ask whether this principle extends to purely ordinal structure: sequences defined by how items repeat and in what order, as in AABBCCAABBCC, independently of their spatial content. Across three experiments, participants reproduced 12-item sequences of spatial locations with various ordinal structures. The minimal description length derived from the LoT model predicted recall accuracy with remarkable precision (r = .96), substantially outperforming Shannon entropy, Lempel-Ziv complexity, chunking models and subjective complexity ratings. Critically, fine-grained analyses of participants' inter-click intervals during reproduction revealed systematic slowdowns at the hierarchical boundaries predicted by the LoT programs, providing a behavioral signature of the underlying mental syntax. These results identify a compact vocabulary of mental primitives, repetition, mirroring, and interleaving, whose composition accounts for the symbolic compression of ordinal structures. For ordinal regularities, human sequence memory operates as a form of program induction, leveraging a domain-general capacity for hierarchical compression to encode complex structured information.
Coxiella burnetii is the only member of the order Legionellales known to primarily infect vertebrates. The Q fever pathogen is also unusual in that it replicates within an acidified phagolysosome-like vacuole. The evolutionary origins of the virulence determinants underlying this lifestyle remain unclear. More broadly, little is known about how virulence-related traits arise in specialized intracellular lineages, where access to foreign-origin DNA may be more episodic. To address this question, we used Legionellales-wide comparative phylogenomics to reconstruct the gain and loss of traits affecting host interaction, immune evasion, intracellular survival, and metabolism. We found that many virulence-associated traits in C. burnetii predate the modern pathogen and were assembled stepwise in ancestors that likely occupied niches distinct from the acidified vacuolar niche of modern C. burnetii. The common ancestor shared with soft-tick Coxiella endosymbionts likely encoded most C. burnetii type IVB secretion system effectors, indicating that much of the host-manipulation repertoire in C. burnetii was already present before the emergence of the modern pathogen. Distinctive lipopolysaccharide features associated with immune evasion also appear to have accumulated progressively within the Coxiella lineage, including genes implicated in synthesis of virenose, a unique O-antigen sugar critical for C. burnetii virulence. Traits likely to support replication in the acidic Coxiella-containing vacuole likewise accumulated gradually, with generalized stress-tolerance functions predating acquisition of an Mrp cation/proton antiporter that may have further supported pH homeostasis. Additional changes in sugar transport and catabolism, glycolytic control, and respiratory metabolism may have enhanced metabolic flexibility and access to diverse substrates in this nutrient-rich niche. Together, these findings support a model in which vertebrate pathogenicity in C. burnetii emerged …