Biology subjects

Simon, B.

Publications and source records attributed to Simon, B..

At least 19 recordsLinked to original sources

bioRxiv2026

Transient interdomain interactions shape the conformational ensemble governing RNA recognition by the tandem RRMs of Sex-lethal

RNA recognition motif (RRM) proteins frequently contain multiple RNA-binding domains connected by flexible linkers, yet the contribution of transient interdomain interactions to RNA recognition remains incompletely understood. Here, we investigated the structural organization of the tandem RRMs of the Drosophila melanogaster splicing regulator Sex-lethal (Sxl) using solution NMR spectroscopy in combination with rational protein engineering, restrained docking and RNA-binding studies. Progressive extension of the native interdomain linker resulted in a gradual decrease in rotational coupling between the two RRMs and continuous chemical shift changes, demonstrating that the RNA-free protein samples a dynamic conformational ensemble rather than behaving as two independently tumbling domains. NMR-guided docking identified a compact arrangement compatible with the experimental data and suggested a transient interface partially overlapping the RNA-binding surfaces. Surprisingly, a mutant designed to weaken this interface produced the opposite effect: instead of increasing interdomain mobility, it exhibited enhanced rotational coupling while remaining natively folded, indicating a redistribution of the conformational ensemble rather than disruption of the domain architecture. Both linker extension and the mutant reduced RNA-binding affinity, and the mutant additionally diminished sequence discrimination, demonstrating that perturbations shifting the conformational equilibrium in either direction compromise RNA recognition. Together, our results demonstrate that RNA recognition by Sxl is governed not by a single apo structure but by a finely balanced conformational ensemble, and that perturbing this equilibrium in either direction compromises high-affinity and sequence-selective RNA binding.

biophysics↗

bioRxiv2026

Routine FFPE sections support clinically compatible single-nucleus transcriptomics across six human cancer types

Tumor cellular composition--including malignant cell states, immune populations, and stromal populations--is increasingly recognized as a determinant of therapeutic response and resistance to anti-cancer agents, yet comprehensive cellular profiling remains largely confined to research settings. Here, we present a clinically compatible sample-to-report workflow for tumor composition profiling from routine formalin-fixed paraffin-embedded (FFPE) clinical specimens. By combining low-input single-nucleus RNA sequencing with foundation model- based automated cell annotation, this workflow enables prospective sample-by-sample analysis without dedicated research material or cohort-based processing. Across 116 clinical specimens representing six cancer types, we generated reproducible measurements of cellular composition and cell-type-specific gene expression, demonstrated high technical reproducibility, and showed concordance with pathological assessment of immune infiltration. The workflow was similarly applicable to archival FFPE material and ultra-low-input biopsy specimens. Together, these findings establish a practical framework for routine single-cell profiling from standard pathology specimens and open the perspective of prospective evaluation of cellular composition as a clinical biomarker in precision oncology.

cancer biology↗

bioRxiv2025

A single-nucleus multimodal framework reveals epigenomic priming of chemoresistant states in ovarian cancer

Non-genetic intratumor heterogeneity (ITH) drives therapeutic failure in cancer, yet its clinical monitoring remains challenging. We develop a multimodal single-nucleus framework that simultaneously profiles transcriptional and histone modification landscapes from frozen biopsies. Applied to longitudinal samples from 16 patients with high-grade serous ovarian cancer (HGSOC), this approach reveals reproducible tumor evolution under chemotherapy: proliferative and interferon-responsive states are lost, while those associated with TNF- and epithelial-mesenchymal transition (EMT) expand. Chromatin profiling shows that these chemoresistant programs are epigenetically primed through H3K4me1 marks before treatment and nominates transcription-factor drivers, including ZBTB7A. Non-genetic baseline tumor composition predicts survival, and fibroblast remodeling parallels malignant adaptation. These findings establish a clinically scalable strategy for mapping functional ITH and identify epigenomic priming as a determinant of therapeutic failure. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=181 HEIGHT=200 SRC="FIGDIR/small/692102v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@432524org.highwire.dtl.DTLVardef@3b7843org.highwire.dtl.DTLVardef@54a2eforg.highwire.dtl.DTLVardef@95273d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Molecular basis of ParA ATPase activation by the CTPase ParB during bacterial chromosome segregation

DNA segregation by bacterial ParABS systems is mediated by transient tethering interactions between nucleoid-bound dimers of the ATPase ParA and centromere (parS)-associated complexes of the clamp-forming CTPase ParB. The lifetime of these interactions is limited by the ParB-dependent activation of ParA ATPase activity. Here, we elucidate the functional interplay between ParA and ParB in the model bacterium Myxococcus xanthus. We demonstrate that the N-terminal ParA-binding motif of ParB associates with a conserved bipartite binding pocket at the ParA dimer interface, in a manner dependent on ParB clamp closure. Moreover, we show that ParB and non-specific DNA interact cooperatively with ParA and synergistically induce structural changes at its Walker A and Walker B motifs that correlate with the activation of ParA ATPase activity. These results advance our understanding of the mechanism underlying DNA transport by the ParABS system and may help to unravel the mode of action of related cargo-positioning systems.

microbiology↗

bioRxiv2025

CombiCTx: Screening diffusion gradients of anti-cancer drug combinations

The reduced effectiveness of chemotherapy in many patients highlights the need for novel drug combinations and optimal ratios that target multiple survival mechanisms, which tumors may engage to confer drug resistance. Dynamic conditions within the tumor microenvironment shape cell behavior and influence the response to anti-cancer drugs, varying by cell type and local context. Accordingly, assays that identify effective concentrations and drug interactions (additive, synergistic, or antagonistic) in a relevant tumor tissue model are required to discover new combination treatments. To address this need for combinatorial chemotherapeutic (CTx) screening assays, we reconfigured CombiANT, a device for testing antibiotic interactions, and present a new assay called CombiCTx. The assay uses a device with three reservoirs containing gels loaded with chemotherapeutics or other anti-cancer drugs. The drug-loaded device is inverted and placed in a standard culture dish containing cancer cells, and both are then enclosed in gel. As drugs diffuse from the reservoirs, cancer cells are exposed to overlapping dynamic gradients of anti-cancer drugs, which can interact in various ways. We imaged doxorubicin diffusion in the assay using timelapse microscopy and employed the apoptotic agent staurosporine as a model drug, and established an imaging protocol for quantifying MDA-MB-231 breast cancer cell apoptosis along drug diffusion gradients. Finally, we tested navitoclax and gemcitabine to demonstrate the capacity of CombiCTx to evaluate combined cytotoxic effects while accounting for drug diffusion. Evaluating drug combinations with an assay that accounts for drug properties that influence diffusion in a complex tumor matrix may provide clinically relevant information.

cancer biology↗

bioRxiv2024

A direct interaction between the RNA-binding proteins Staufen and Tm1-I/C regulates oskar mRNP composition and transport

In the Drosophila female germline, oskar messenger RNA is transported on microtubules from the nurse cells to the posterior pole of the oocyte, where it is translated. Transport of oskar transcripts from the nurse cells into the oocyte requires dynein, while localization of the mRNAs within the oocyte to the posterior pole is dependent upon kinesin-1. Staufen, a dsRNA-binding protein, has been shown to bind the oskar mRNA transport complex in the oocyte and inactivate dynein; however, it remains unclear how kinesin is activated. Here, using surface plasmon resonance, nuclear magnetic resonance spectroscopy and RNA imaging within egg chambers, we demonstrate that Staufen directly interacts with Tm1, a non-canonical kinesin adaptor. This work provides a molecular explanation for the previously unclear role of Staufen in oskar mRNA localization.

molecular biology↗

bioRxiv2024

Evolutionary branching in multi-level selection models

We study a model of group-structured populations featuring individual-level birth and death events, and group-level fission and extinction events. Individuals play games within their groups, while groups play games against other groups. Pay-offs from individual-level games affect birth rates of individuals, and payoffs from group-level games affect group extinction rates. We focus on the the evolutionary dynamics of continuous traits with particular emphasis on the phenomenon of evolutionary diversification. Specifically, we consider two-level processes in which individuals and groups play continuous snowdrift or prisoners dilemma games. Individual game strategies evolve due to selection pressure from both the individual and group level interactions. The resulting evolutionary dynamics turns out to be very complex, including branching and type-diversification at one level or the other. We observe that a weaker selection pressure at the individual level results in more adaptable groups and sometimes group-level branching. Stronger individual-level selection leads to more effective adaptation within each group while preventing the groups from optimizing their strategies for group-level games.

evolutionary biology↗

bioRxiv2024

The Drosophila RNA binding protein Hrp48 binds a specificRNA sequence of the msl-2 mRNA 3' UTR to regulatetranslation

Repression of msl-2 mRNA translation is essential for viability of Drosophila melanogaster females to prevent hypertranscription of both X chromosomes. This translational control event is coordinated by the female-specific protein Sex-lethal (Sxl) which recruits the RNA binding proteins Unr and Hrp48 to the 3 untranslated region (UTR) of the msl-2 transcript and represses translation initiation. The mechanism exerted by Hrp48 during translation repression and its interaction with msl-2 are not well understood. Here we investigate the RNA binding specificity and affinity of the tandem RNA recognition motifs of Hrp48. Using NMR spectroscopy, molecular dynamics simulations and isothermal titration calorimetry, we identified the exact region of msl-2 3 UTR recognized by Hrp48. Additional biophysical experiments and translation assays give further insights into complex formation of Hrp48, Unr, Sxl and RNA. Our results show that Hrp48 binds independent of Sxl and Unr downstream of the E and F binding sites of Sxl and Unr to msl-2.

biophysics↗

bioRxiv2023

The Listeria monocytogenes persistence factor ClpL is a potent stand-alone disaggregase

Heat stress can cause cell death by triggering the aggregation of essential proteins. In bacteria, aggregated proteins are rescued by the canonical Hsp70/AAA+ (ClpB) bi-chaperone disaggregase. Man-made, severe stress conditions applied during e.g. food-processing represent a novel threat for bacteria by exceeding the capacity of the Hsp70/ClpB system. Here, we report on the potent autonomous AAA+ disaggregase ClpL from Listeria monocytogenes that provides enhanced heat resistance to the food-borne pathogen enabling persistence in adverse environments. ClpL shows increased thermal stability and enhanced disaggregation power compared to Hsp70/ClpB, enabling it to withstand severe heat stress and to solubilize tight aggregates. ClpL binds to protein aggregates via aromatic residues present in its N-terminal domain (NTD) that adopts a partially folded and dynamic conformation. Target specificity is achieved by simultaneous interactions of multiple NTDs with the aggregate surface. ClpL shows remarkable structural plasticity by forming diverse higher assembly states through interacting ClpL rings. NTDs become largely sequestered upon ClpL ring interactions. Stabilizing ring assemblies by engineered disulfide bonds strongly reduces disaggregation activity, suggesting that they represent storage states.

biochemistry↗

bioRxiv2023

Structural basis of aggregate binding by the AAA+ disaggregase ClpG

Severe heat stress causes massive loss of essential proteins by aggregation necessitating a cellular activity that rescues aggregated proteins. This activity is executed by ATP-dependent, ring-forming, hexameric AAA+ disaggregases. Little is known about the recognition principles of stress-induced protein aggregates. How can disaggregases specifically target aggregated proteins while avoiding binding to soluble non-native proteins? Here, we determined by NMR spectroscopy the core structure of the aggregate targeting N1 domain of the bacterial AAA+ disaggregase ClpG, which confers extreme heat resistance to bacteria. N1 harbors a Zn2+-coordination site that is crucial for structural integrity and disaggregase functionality. Conserved hydrophobic N1 residues located on a {beta}-strand were found crucial for aggregate targeting and disaggregation activity. Mixing experiments with N1-truncated AAA+ hexamers revealed that a minimal number of four N1 domains must be present in a AAA+ ring for high disaggregation activity. We suggest that multiple N1 domains increase substrate affinity through avidity effects. These findings define the recognition principle of a protein aggregate by a disaggregase, involving simultaneous contacts with multiple hydrophobic substrate patches located in close vicinity on an aggregate surface. This binding mode ensures selectivity for aggregated proteins while sparing soluble, non-native protein structures from disaggregase activity.

biochemistry↗

bioRxiv2022

Structural basis for specific RNA recognition by the alternative splicing factor RBM5

The RNA-binding motif protein RBM5 belongs to a family of multi-domain RNA binding proteins that are implicated in cancer and regulate alternative splicing of genes important for apoptosis and cell proliferation and have been implicated in cancer. RBM5 harbors structural modules for RNA recognition, such as RRM domains and a Zn finger, and protein-protein interactions such as an OCRE domain. Here, we characterize binding of the RBM5 RRM1-ZnF1-RRM2 domains to cis-regulatory RNA elements. A structure of the RRM1-ZnF1 region in complex with RNA shows how the tandem domains cooperate to sandwich target RNA and specifically recognize a GG dinucleotide in a non-canonical fashion. While the RRM1-ZnF1 domains act as a single structural module, RRM2 is connected by a flexible linker and tumbles independently. However, all three domains participate in RNA binding and adopt a closed architecture upon RNA binding. Our data highlight how cooperativity and conformational modularity of multiple RNA binding domains enable the recognition of distinct RNA motifs, thereby contributing to the regulation of alternative splicing. Remarkably, we observe surprising differences in coupling of the RNA binding domains between the closely related homologs RBM5 and RBM10. HighlightsO_LIMultiple RNA binding domains enable differential recognition of distinct RNA motifs to regulate alternative splicing C_LIO_LIThe RRM1-ZnF1 domains of RBM5 mediate specific recognition of cis regulatory RNA motifs to modulate alternative splicing C_LIO_LIRRM1-ZnF1 sandwich the target RNA for non-canonical recognition of a GG dinucleotide C_LIO_LIRRM1-ZnF1-RRM2 bind cooperatively to contiguous cis-regulatory motifs in caspase-2 and NUMB pre-mRNAs C_LI

biochemistry↗

bioRxiv2022

Upstream of N-Ras C-terminal cold shock domains mediate poly(A) specificity in a novel RNA recognition mode and bind poly(A) binding protein during translation regulation

RNA binding proteins (RBPs) often engage multiple RNA binding domains (RBDs) to increase target specificity and affinity. However, the complexity of target recognition of multiple RBDs remains largely unexplored. Here we use Upstream of N-Ras (Unr), a multidomain RBP, to demonstrate how multiple RBDs orchestrate target specificity. A crystal structure of the three C-terminal RNA binding cold-shock domains (CSD) of Unr bound to a poly(A) sequence exemplifies how recognition goes beyond the classical {pi}-{pi}-stacking in CSDs. Further structural studies reveal several interaction surfaces between the N-terminal and C-terminal part of Unr with the poly(A)-binding protein (pAbp). This provides first atomistic details towards understanding regulation of translation initiation that is mediated by the interplay of these two proteins with each other and RNA.

molecular biology↗

bioRxiv2022

Tropomyosin 1-I/C co-ordinates kinesin-1 and dynein motors during oskar mRNA transport

Dynein and kinesin motors mediate long-range intracellular transport, translocating towards microtubule minus and plus ends, respectively. Cargoes often undergo bidirectional transport by binding to both motors simultaneously. However, it is not known how motor activities are coordinated in such circumstances. In Drosophila, sequential activities of the dynein-dynactin-BicD-Egalitarian (DDBE) complex and of kinesin-1 deliver oskar mRNA from nurse cells to the oocyte, and within the oocyte to the posterior pole. Here, through in vitro reconstitution, we show that Tm1-I/C, a Tropomyosin-1 isoform, links kinesin-1 in an inactive state to DDBE-associated oskar mRNA. NMR spectroscopy, small-angle X-ray scattering and structural modeling indicate that Tm1-I/C suppresses kinesin-1 activity by stabilizing its autoinhibited conformation, thus preventing a tug-of-war between the opposite polarity motors until kinesin-1 is activated in the oocyte. Our work reveals a novel strategy ensuring sequential activity of microtubule motors.

biochemistry↗

bioRxiv2022

In vitro floral development in poplar: Insights into seed trichome and trimonoecy regulation

Woody perennials including Populus spp. (poplars) have a juvenile phase that ranges from several years to decades in length. This and the year-long floral development process are major impediments to breeding and to fundamental research of reproductive traits. Here we report a CRISPR-empowered in vitro flowering system and demonstrate its application using three reproductive traits: sex, seed trichomes, and a previously undescribed potential for trimonoecy in poplar.

plant biology↗

bioRxiv2022

Mechanistic insights into RNA surveillance by the canonical poly(A) polymerase Pla1 of the MTREC complex.

The S. pombe orthologue of the human PAXT complex, Mtl1-Red1 Core (MTREC), is an eleven-subunit complex which targets cryptic unstable transcripts (CUTs) to the nuclear RNA exosome for degradation. It encompasses the canonical poly(A) polymerase Pla1, responsible for polyadenylation of nascent RNA transcripts as part of the cleavage and polyadenylation factor (CPF/CPSF). In this study we identified and characterised the interaction between Pla1 and the MTREC complex core component Red1 and analysed the functional relevance of this interaction in vivo. Our crystal structure of the Pla1-Red1 complex showed that a 58-residue fragment in Red1 binds to the RNA recognition motif domain of Pla1 and tethers it to the MTREC complex. Structure-based Pla1-Red1 interaction mutations showed that Pla1, as part of MTREC complex, hyper-adenylates CUTs for their efficient degradation. Interestingly, the Red1-Pla1 interaction was also required for the efficient assembly of the fission yeast facultative heterochromatic islands. Together, our data suggest a complex interplay between the RNA surveillance and 3-end processing machineries.

molecular biology↗

bioRxiv2021

Genetic diversity of local and introduced cassava germplasm in Burundi using DArTseq molecular analyses

In Burundi, most of small-scale farmers still grow traditional cassava landraces that are adapted to local conditions and have been selected for consumer preferred attributes. They tend to be susceptible, in varying degrees, to devastating cassava viral diseases such as Cassava Brown Steak Disease (CBSD) and Cassava Mosaic Disease (CMD) with production annual losses of US$1 billion annually. For long term resistance to the disease, several breeding strategies have been proposed. A sound basis for a breeding program is to understand the genetic diversity of both landraces and elite introduced breeding cultivars. This will also assist in efforts to conserve landraces ahead of the broad distribution of improved varieties which have the possibility of replacing landraces. Our study aimed at determining the genetic diversity and relationships of local landraces and introduced elite germplasm using morphological and simple nucleotide polymorphism (SNP) markers as well as identifying a core set of germplasm from the local varieties to be used in the cassava breeding program. A total of 118 cultivars were characterized for morphological trait variation based on leaf, stem and root traits, and genetic variation using SNP markers. Results of morphological characterization based on Wards Method revealed three main clusters and five accessions sharing similar characteristics. Molecular characterization identified over 18,000 SNPs and six main clusters and three pairs of duplicates which should be pooled together as one cultivar to avoid redundancy. Results of population genetic analysis showed low genetic distance between populations and between local landraces and elite germplasm. Accessions that shared similar morphological traits were divergent at the molecular level indicating that clustering using morphological traits was inconsistent. Despite the variabilities found within the collection, it was observed that cassava germplasm in Burundi have a narrow genetic base.

genetics↗

bioRxiv2020

Structure and dynamics of the quaternary hunchback mRNA translation repression complex

A key regulatory process during Drosophila development is the localized suppression of the hunchback mRNA translation at the posterior, which gives rise to a hunchback gradient governing the formation of the anterior-posterior body axis. The suppression of the RNA is achieved by a concerted action of Brain Tumour (Brat), Pumilio (Pum) and Nanos. Each protein is necessary for proper Drosophila development. The RNA contacts have been elucidated for the proteins individually in several atomic-resolution structures. However, the interplay of all three proteins in the RNA suppression remains a long-standing open question. We characterize the quaternary complex of the RNA-binding domains of Brat, Pum and Nanos with hunchback mRNA by combining NMR spectroscopy, SANS/SAXS, XL/MS with MD simulations and ITC assays. The quaternary hunchback mRNA suppression complex is flexible with the unoccupied nucleotides of the RNA functioning as a flexible linker between the Brat and Pum-Nanos moieties of the complex. Moreover, Brat and Pum with Nanos bind the RNA completely independently. In accordance with previous studies, showing that Brat can suppress hunchback mRNA independently and is distributed uniformly throughout the embryo, this suggests that hunchback mRNA suppression by Brat is functionally separate from the suppression by Pumilio and Nanos.

molecular biology↗

bioRxiv2020

RNA binding regulates TRIM25-mediated RIG-I ubiquitylation

TRIM25 is a ubiquitin E3 ligase active in innate immunity and cell fate decisions. Mounting evidence suggests that TRIM25's E3 ligase activity is regulated by RNAs. However, while mutations affecting RNA binding have been described, neither the precise RNA binding site has been identified nor which domains are involved. Here, we present biophysical evidence for the presence of RNA binding sites on both TRIM25 PRY/SPRY and coiled-coil domains, and map the binding site on the PRY/SPRY with residue resolution. Cooperative RNA-binding of both domains enhances their otherwise transient interaction in solution and increases the E3 ligase activity of TRIM25. We also show that TRIM25 not only binds RNA in mammalian cells but that interfering with RNA binding has an effect on cellular RIG-I ubiquitination.

molecular biology↗