Biology subjects

Allain, F. H.- T.

Publications and source records attributed to Allain, F. H.- T..

14 recordsLinked to original sources

bioRxiv2026

A qualitative and quantitative framework for the characterization of protein-RNA interactions by cross-linking mass spectrometry (XL-MS)

Protein-RNA interactions regulate nearly all levels of gene expression. Their characterization at the molecular basis is critical for understanding the function of protein-RNA complexes, both in cell homeostasis and pathology. Yet, molecular interactions underlying the formation of these complexes remain understudied. UV cross-linking mass spectrometry (XL-MS) is one of the leading approaches for in vitro identification of protein-RNA contact sites, and for providing structural information in the form of distance constraints crucial for the refinement of structural models. Our previously published CLIR-MS method enables qualitative characterization of protein-RNA interactions at residue-specific resolution but was not designed for highly accurate quantitative interpretation. Here, we present a XL-MS workflow based on irradiation with UV light that combines data-dependent acquisition (DDA) and parallel reaction monitoring (PRM) for simultaneous detection and characterization of protein-RNA interaction sites at residue-specific resolution, followed by label-free quantification of cross-linked products. We successfully apply our combined workflow to in vitro reconstituted protein-RNA complexes formed by the proteins TDP-43 and FOX1. We report on novel RNA interaction sites located in the oligomerization interface of the N-terminal domain (NTD) of TDP-43, and characterize quantitatively the process of RNA-induced monomerization of the NTD in an RNA titration experiment. We also investigate temperature-dependent differences in the efficiency of cross-link formation in the FOX1 complex. Our framework offers a unique and novel approach to comparative quantification of protein-RNA cross-links, overcoming the limitations imposed by conventional UV XL-MS methods.

biophysics↗

bioRxiv2026

Monomeric TDP-43 binds GU-rich RNA via its NTD but only its oligomers block cryptic exon inclusion

TDP-43 aggregation is a pathological feature of several neurodegenerative diseases, resulting in nuclear loss of function and RNA dysregulation, including cryptic exon derepression. TDP-43 comprises two RNA recognition motifs required for canonical RNA binding, a low-complexity domain implicated in phase separation, and an N-terminal domain (NTD) that mediates oligomerization. While NTD-driven oligomerization is critical for TDP-43 splicing function, some studies suggest a direct interaction of NTD with RNA. Here, we investigated RNA binding by the NTD and its functional interplay with oligomerization. We show that the NTD preferentially binds UG-rich RNA via residues located at the oligomerization interface. The NTD engaged RNA only on monomeric TDP-43, as RNA binding and oligomerization compete for the same interface and rely on similar interaction modes. By disentangling these competing activities, we demonstrated that oligomerization, but not NTD-RNA interaction, is essential for TDP-43-dependent alternative splicing and repression of cryptic exons. Our findings uncover a previously unappreciated RNA-binding activity within the TDP-43 NTD that directly competes with oligomerization and establish oligomerization as a key determinant of TDP-43 cryptic exon repression.

molecular biology↗

bioRxiv2026

Phosphorylation tunes electrostatically driven protein-RNA interactions

Phosphorylation of intrinsically disordered proteins (IDPs) is essential for regulating biomolecular interactions in many cellular processes. However, a quantitative understanding of how phosphorylation tunes the affinity between highly charged IDPs and nucleic acids is lacking. Here, we show that multi-site phosphorylation of the disordered arginine/serine-rich (RS) domain of the splicing factor SRSF1 acts as an electrostatic rheostat that governs RNA binding. By combining enzymatic phosphorylation, phosphomimetic variants, and chemically synthesised phosphopeptides with single-molecule Forster resonance energy transfer measurements, we reveal the RS domain to be a potent driver of protein-RNA association. Increasing phosphorylation progressively reduces this interaction, and extensive phosphorylation eliminates detectable RNA binding. Remarkably, the binding free energy depends linearly on RS-domain net charge, regardless of whether the charge arises from phosphorylation or acidic residues introduced as phosphomimetics. Together, our findings uncover a quantitative framework for how phosphorylation tunes the interactions of charged IDPs and rationalize why two acidic residues are required to mimic a single phosphorylation event.

biophysics↗

bioRxiv2025

Multiplexed analysis of multicomponent biomolecular condensates without any tag

Biomolecular condensates, formed in the process of liquid-liquid phase separation (LLPS), play key roles in RNA metabolism and cellular organization. These dynamic assemblies often contain several components such as proteins and RNAs. Experimental methods to study biological condensates commonly involve fluorophore-labelling of the biomolecules, which may affect phase separation behavior and in addition cannot access dilute-phase components due to low dye concentrations. Here we introduce a non-invasive, multiplexed NMR approach that enables selective observation of multiple components in hydrogel-stabilized biphasic condensates without using tags. The introduced multiplexing filter combines enhanced spin-spin cross-relaxation in the condensed phase with diffusion and isotope filters to resolve signals from distinct protein pools from both dilute and condensed phases. We demonstrate the robust performance of this 1D 1H NMR experiment using biphasic samples containing condensates of FUS N-terminal domain, FUS full-length, and the multicomponent condensate formed by two intrinsically disordered regions of hnRNPC1 protein with highly overlapping resonances. Integrating the multiplexing filter into multidimensional experiments enables site-specific structural and dynamic insights from diverse protein populations, extending NMR applications in LLPS.

biophysics↗

bioRxiv2024

LLPS REDIFINE allows the biophysical characterization of multicomponent condensates without tags or labels

Liquid-liquid phase separation (LLPS) phenomenon plays a vital role in multiple cell biology processes, providing a mechanism to concentrate biomolecules and promote cellular reactions locally. Despite its significance in biology, there is a lack of conventional techniques suitable for studying biphasic samples in their biologically relevant form. Here, we present a label-free and non-invasive approach to characterize protein, RNA and water in biomolecular condensates termed LLPS REstricted DIFusion of INvisible speciEs (REDIFINE). Relying on diffusion NMR measurements, REDIFINE exploits the exchange dynamics between the condensed and dispersed phases to allow the determination of not only diffusion constants in both phases but also the fractions of the species, the average radius of the condensed droplets and the exchange rate between the phases. We can also access the concentration of proteins in both phases. Observing proteins, RNAs, water, and even small molecules, REDIFINE analysis allows a rapid biophysical characterization of multicomponent condensates which is important to understand their functional roles. In comparing multiple systems, REDIFINE reveals that folded RNA-binding proteins form smaller and more dynamic droplets compared to the disordered ones. In addition, REDIFINE proved to be valuable beyond LLPS for the determination of binding constants in soluble protein-RNA without the need for titration.

biophysics↗

bioRxiv2023

A solid beta-sheet structure is formed at the surface of FUS liquid droplets during aging

Insights into liquid droplet formation via liquid-liquid phase separation and the subsequent liquid-to-solid phase transition are important for understanding cell dynamics, as well as a number of neurodegenerative disorders. We report here, using the example of the FUsed in Sarcoma (FUS) protein, an investigation of the liquid droplet maturation process combining solution- and solid-state NMR spectroscopy, Raman spectroscopy, and light and electron microscopies. Our study reveals that the surface of the droplets plays a critical role in this process. Indeed, when comparing a biphasic sample, in which liquid droplets are stabilized in an agarose matrix, with a pure monophasic condensed phase sample, we find that the liquid-droplet maturation kinetics is faster in the biphasic FUS sample, owing to the larger surface-to-volume ratio. In addition, using Raman spectroscopy, we observe structural differences upon liquid-droplet maturation between the inside and the surface of liquid droplets, which is of {beta}-sheet content as revealed by solid-state NMR. This is detected very early on and increases over time. In agreement with these observations, a solid crust-like shell is visually seen by microaspiration experiments. After several months, electron microscopy reveals that the matured FUS droplets have converted into solid linear fibrils distinct from the fibril core of seeded fibrils reported previously, as arginine side-chains from the arginine-and-glycine-rich domain (RGG) motif are partially rigidified, highlighting the participation of this motif in the liquid-to-solid transition. In presence of RNA, this aging process is not taking place.

biochemistry↗

bioRxiv2023

The diversity of splicing modifiers acting on A-1 bulged 5'-splice sites reveals rules to guide rational design

Non-physiological alternative splicing patterns are associated with numerous human diseases. Among the strategies developed to treat these diseases, small molecule splicing modifiers are emerging as a new class of RNA therapeutics. The SMN2 splicing modifier SMN-C5 was used as a prototype to understand their mode of action and discover the concept of 5-splice site bulge repair. However, different small molecules harbouring a similar activity were also identified. In this study, we combined NMR spectroscopy and computational approaches to determine the binding modes of other SMN2 and HTT splicing modifiers at the interface between U1 snRNP and an A-1 bulged 5-splice site. Our results show that the other splicing modifiers interact with the intermolecular RNA helix epitope containing an unpaired adenine within a G-2A-1G+1U+2 motif, which is essential for their biological activity. We also determined structural models of risdiplam, SMN-CX, and branaplam bound to RNA, and solved the solution structure of the most divergent SMN2 splicing modifier, SMN-CY, in complex with the RNA helix. These findings not only deepen our understanding of the chemical diversity of splicing modifiers that target A-1 bulged 5-splice sites, but also identify common pharmacophores required for modulating 5-splice site selection with small molecules.

molecular biology↗

bioRxiv2022

A hybrid structure determination approach to investigate the druggability of the nucleocapsid protein of SARS-CoV-2

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/507991v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@183d480org.highwire.dtl.DTLVardef@1f4235aorg.highwire.dtl.DTLVardef@13d1aedorg.highwire.dtl.DTLVardef@b2c87f_HPS_FORMAT_FIGEXP M_FIG C_FIG The ongoing pandemic caused by SARS-CoV-2 has called for concerted efforts to generate new insights into the biology of betacoronaviruses to inform drug screening and development. Here, we establish a workflow to determine the RNA recognition and druggability of the nucleocapsid N-protein of SARS-CoV-2, a highly abundant protein crucial for the viral life cycle. We use a synergistic method that combines NMR spectroscopy and protein-RNA cross-linking coupled to mass spectrometry to quickly determine the RNA binding of two RNA recognition domains of the N-protein. Finally, we explore the druggability of these domains by performing an NMR fragment screening. This workflow identified small molecule chemotypes that bind to RNA binding interfaces and that have promising properties for further drug development.

biophysics↗

bioRxiv2022

The cancer-associated RBM39 bridges the pre-mRNA, U1 and U2 snRNPs to regulate alternative splicing

Pharmacologic depletion of RNA-binding motif 39 (RBM39) using aryl sulfonamides represents a promising anti-cancer therapy. However, its efficiency correlates with the expression level of DCAF15 which acts at the interface between RBM39, the drug and the E3-ubiquitin ligase. Consequently, the identification of alternative approaches to deplete RBM39 independently of DCAF15 is required. Here, we combined transcriptomic analysis, functional assays, and structural biology to elucidate the molecular mechanisms governing RBM39 homeostasis. Our data revealed that RBM39 autoregulates the splicing of its own pre-mRNA by triggering the inclusion of a poison exon. During this process, RBM39 selects the 3-splice site of the toxic exon, helps the recruitment of U1 snRNP on its weak 5-splice site and bridges the 3-splice site recognition machinery. The elucidation of the molecular mechanisms controlling RBM39 homeostasis provides unprecedented insights into alternative 3-splice site selection and a solid frame to design alternative anti-cancer therapies.

molecular biology↗

bioRxiv2022

Integrative solution structure of a PTBP1-viral IRES complex reveals strong compaction and ordering with residual conformational flexibility

RNA-binding proteins (RBPs) are crucial regulators of gene expression and often comprise well-defined domains interspersed by flexible, intrinsically disordered regions. The structure determination of ribonucleoprotein complexes involving such RBPs is not common practice and requires integrative structural modeling approaches due to the fact that they often do not form a single stable globular state. Here, we integrate data from magnetic resonance, mass spectrometry, and small angle scattering to determine the solution structure of the polypyrimidine-tract binding protein 1 (PTBP1 also called hnRNP I) bound to an RNA which is part of the internal ribosome entry site (IRES) of the encephalomyocarditis virus (EMCV). PTBP1 binding to this IRES element enhances translation of the viral RNA. The determined structural ensemble reveals that both RNA and protein experience a strong compaction upon complex formation, get ordered but still maintain a substantial conformational flexibility. The C-terminal RNA recognition motif (RRM4) of PTBP1 rigidifies the complex by binding a single-strand RNA linker and, in turn, is essential for IRES-mediated translation. PTBP1 acts as an RNA chaperone for the IRES, by ordering the RNA into a few discrete conformations that expose the RNA stems to the outer surface of the RNP complex for subsequent interactions with the translation machinery. The conformational diversity within this structural ensemble is likely common among RNP complexes and important for their functionality. The presented approach opens the possibility to characterize heterogeneous RNP structures at atomic level.

biochemistry↗

bioRxiv2022

TDP-43 oligomerization and RNA binding are codependent but their loss elicits distinct pathologies

Aggregation of the RNA-binding protein TDP-43 is the main common neuropathological feature of TDP-43 proteinopathies. In physiological conditions, TDP-43 is predominantly nuclear and contained in biomolecular condensates formed via liquid-liquid phase separation (LLPS). However, in disease, TDP-43 is depleted from these compartments and forms cytoplasmic or, sometimes, intranuclear inclusions. How TDP-43 transitions from physiological to pathological states remains poorly understood. Here, we show that self-oligomerization and RNA binding cooperatively govern TDP-43 stability, functionality, LLPS and cellular localization. Importantly, our data reveal that TDP-43 oligomerization is connected to, and conformationally modulated by, RNA binding. Mimicking the impaired proteasomal activity observed in patients, we found that TDP-43 forms nuclear aggregates via LLPS and cytoplasmic aggregates via aggresome formation. The favored aggregation pathway depended on the TDP-43 state -monomeric/oligomeric, RNA-bound/-unbound- and the subcellular environment -nucleus/cytoplasm. Our work unravels the origins of heterogeneous pathological species occurring in TDP-43 proteinopathies.

cell biology↗

bioRxiv2022

The structure of yeast Npl3 bound to RNA reveals a cooperative sequence-specific recognition and an RNA chaperone role in splicing

The conserved SR-like protein Npl3 promotes splicing of diverse pre-mRNAs. However, the RNA sequence(s) recognized by the RNA Recognition Motifs (RRM1 & RRM2) of Npl3 during the splicing reaction remain elusive. Here, we developed a split-iCRAC approach in yeast to uncover the consensus sequence bound to each RRM. High-resolution NMR structures show that RRM2 recognizes a 5-GNGG-3 motif leading to an unusual mille-feuille topology. These structures also reveal how RRM1 preferentially interacts with a CC-dinucleotide upstream of this motif, and how the inter-RRM linker and the region C-terminal to RRM2 contributes to cooperative RNA-binding. Structure-guided functional studies show that Npl3 genetically interacts with U2 snRNP specific factors and we provide evidence that Npl3 melts U2 snRNA stem-loop I, a prerequisite for U2/U6 duplex formation within the catalytic center of the Bact spliceosomal complex. Thus, our findings provide insights into an unanticipated RNA chaperoning role for Npl3 during spliceosome active site formation.

biophysics↗

bioRxiv2022

Sensitive detection and structural characterisation of UV-induced cross-links in protein-RNA complexes using CLIR-MS

Protein-RNA interactions underpin many critical biological processes, demanding the development of technologies to precisely characterize their nature and functions. Many such technologies depend upon cross-linking under mild irradiation conditions to stabilize contacts between amino acids and nucleobases; for example, the cross-linking of stable isotope labelled RNA coupled to mass spectrometry (CLIR-MS) method. A deeper understanding of the CLIR-MS workflow is required to maximize its impact for structural biology, particularly addressing the low abundance of cross-linking products and the information content of spatial/geometric restraints reflected by a cross-link. Here, we present a vastly improved CLIR-MS pipeline that features enhanced sample preparation, data acquisition and interpretation. These advances significantly increase the number of detected cross-link products per sample. We demonstrate that the procedure is robust against variation of key experimental parameters, including irradiation energy and temperature. Using this improved protocol on four protein-RNA complexes representing canonical and non-canonical RNA-binding domains, we propose for the first time the distances encoded by protein-RNA cross-links, enabling their use as structural restraints. We also compared the cross-linking of canonical RNA with 4-thiouracil-labeled counterparts, showing slight, but noticeable differences. The improved understanding of protein-RNA cross-links refines the structural interpretation of complexes and facilitates the adoption of the method in integrative/hybrid structural biology.

biochemistry↗

bioRxiv2019

The solution structure of Dead End bound to AU-rich RNA reveals an unprecedented mode of tandem RRM-RNA recognition required for mRNA repression

Dead End (DND1) is an RNA-binding protein essential for germline development through its role in post-transcriptional gene regulation. The molecular mechanisms behind selection and regulation of its targets are unknown. Here, we present the solution structure of DND1s tandem RNA Recognition Motifs (RRMs) bound to AU-rich RNA. The structure reveals how an NYAYUNN element is specifically recognized, reconciling seemingly contradictory sequence motifs discovered in recent genome-wide studies. RRM1 acts as a main binding platform, including atypical extensions to the canonical RRM fold. RRM2 acts cooperatively with RRM1, capping the RNA using an unusual binding pocket, leading to an unprecedented mode of tandem RRM-RNA recognition. We show that the consensus motif is sufficient to mediate upregulation of a reporter gene in human cells and that this process depends not only on RNA binding by the RRMs, but also on DND1s double-stranded RNA binding domain (dsRBD), which is dispensable for target binding in cellulo. Our results point to a model where DND1 target selection is mediated by a non-canonical mode of AU-rich RNA recognition by the tandem RRMs and a role for the dsRBD in the recruitment of effector complexes responsible for target regulation.

molecular biology↗