Biology subjects

Alcalde, J.

Publications and source records attributed to Alcalde, J..

4 recordsLinked to original sources

bioRxiv2026

RiboCollSensor: a sensitive real-time detector of ribosome collisions in mammalian cells based on split-NanoLuc complementation

Disruption of ribosome flux on translating mRNAs can result in ribosome collisions that activate key cellular responses. Despite growing interest in the field, current methods to detect ribosome collisions have limited sensitivity and are not suitable for use in living cells. Here, we describe a novel, reliable, and highly sensitive method based on split-nanoluciferase complementation to detect ribosome collisions in living cells. RiboCollSensor relies on the specific recruitment of EDF1-LgBiT to collided ribosomes near uS4-SmBiT, which generates luminescence due to the proximity of the partners in the ribosome. This biosensor showed unprecedented sensitivity, allowing detection of basal ribosome collisions in unstressed cells or under very low stress levels, enabling real-time analysis of collision kinetics. Thus, an increase in collisions could be detected within the first minute after translation disturbance, confirming the role of ribosomal flux as a rapid sensor of cell stress. Ribosome collisions rapidly disappeared after stress withdrawal, whereas under persistent stress, recovery was slower, taking up to two hours depending on the cell type. The ease and flexibility of this method, which requires only transient co-expression of sensor partners in target cells, make it applicable across many cell types to monitor the impact of internal and external cues on ribosome dynamics in real time.

molecular biology↗

bioRxiv2026

C9ORF72-derived polyGR polypeptides disrupt passive nucleocytoplasmic transport by tuning protein affinity for the nuclear pore barrier

Nucleocytoplasmic partitioning is an essential determinant of eukaryotic cellular function, governed by the nuclear pore complex, a molecular portal filled by a disordered phenylalanine-glycine (FG)-rich phase that governs selective entry and exit. Disruption of nucleocytoplasmic partitioning is seen across many different diseases, including viral infection, cancer, and neurodegeneration. However, what determines whether a given protein mislocalises when nucleocytoplasmic transport is disrupted remains unknown. This question is central to amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), where cytosolic mislocalisation of the nuclear RNA-binding protein TDP-43 is a defining pathological feature. The most common genetic cause of these diseases is a G4C2 repeat expansion in the gene C9ORF72, which produces aberrant neurotoxic polypeptides that induce nucleocytoplasmic transport defects. Here, we show how the highly toxic poly(glycine-arginine/GR) polypeptide engages the nuclear pore FG-rich selectivity barrier and retunes passive nucleocytoplasmic transport according to client surface chemistry. Using coarse-grained simulations, in vitro FG-phase reconstitution and human cell lines and neurons, we find that polyGR produces a non-linear, biphasic modulation of nuclear pore passage. Proteins with low affinity for the FG phase are unaffected, whereas proteins with higher affinity due to solvent-exposed hydrophobic residues exhibit enhanced transport up to a critical threshold, beyond which highly hydrophobic proteins experience transport suppression, cytoplasmic accumulation and aggregation. Together, these findings establish how disease-associated polypeptides retune the physicochemical rules governing passive nuclear pore transport, leading to biphasic outcomes determined by protein surface chemistry that alter protein compartmentalisation and aggregation. This provides a biophysical mechanism by which polyGR drives selective protein vulnerability to nuclear pore dysfunction in C9ORF72-associated ALS/FTD.

cell biology↗

bioRxiv2026

Breaking β-sheets in FUS prion-like domain preserves phase separation and function but prevents aggregation and toxicity

The RNA-binding protein Fused in Sarcoma (FUS) undergoes phase separation associated with RNA processing. However, the prion-like low complexity (LC) domain of FUS forms solid-like aggregates in neurodegenerative diseases. Whether the formation of {beta}-sheet structure associated with pathology is also physiologically/functionally relevant is debated. Similarly, if mislocalization alone or concomitant aggregation is responsible for FUS gain-of-function toxicity remains to be probed. Here, we introduce {beta}-sheet breaking proline residues into FUS LC with the goal of preventing cross-{beta}-driven aggregation without disrupting essential functions and phase separation. {beta}-sheet-deficient FUS variants maintain native-like global motions, disorder, and phase separation, but no longer show a liquid-to-solid transition (LST). Biochemical partitioning, cellular localization, and auto- and cross-regulatory functions of FUS all remain essentially unchanged. Conversely, FUS-induced neurodegeneration in several Drosophila models is drastically reduced. These findings suggest a strategy for mitigating disease-related toxicity through backbone structure modulation to prevent prion-like domain protein aggregation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/706410v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@d15f63org.highwire.dtl.DTLVardef@1cd6221org.highwire.dtl.DTLVardef@e58126org.highwire.dtl.DTLVardef@181ec67_HPS_FORMAT_FIGEXP M_FIG C_FIG SUMMARYThe RNA-binding protein Fused in Sarcoma (FUS) undergoes phase separation as part of its physiological function but can aberrantly aggregate into solid-like assemblies in amyotrophic lateral sclerosis and frontotemporal dementia. To dissect the role of {beta}-sheets in both function and pathological transition, we engineered {beta}-sheet-preventing FUS variants via targeted proline residue insertions in the prion-like disordered region. These variants retained native structure, motions, and phase behavior yet showed dramatically reduced aggregation, both as an isolated prion-like domain and in full-length FUS. Crucially, these variants maintained a panel of FUS cellular functions that depend on FUS condensation but prevented FUS toxicity in fly models of neurodegeneration. Our findings implicate {beta}-sheets as key drivers of FUS condensate maturation and neuronal toxicity, highlighting {beta}-sheet modulation as a therapeutic strategy against FUS-related neurodegeneration. HIGHLIGHTSO_LITargeted proline additions disrupt {beta}-sheet formation in FUS without altering native conformations, dynamics, or phase separation behavior C_LIO_LI{beta}-sheet-deficient FUS variants prevent aggregation and liquid-to-solid transitions while retaining key biological functions C_LIO_LIIn vivo models reveal attenuated toxicity of {beta}-sheet-deficient FUS in Drosophila C_LIO_LI{beta}-sheets are identified as central drivers of condensate maturation and neuronal death, offering a therapeutic entry point for modulating prion-like domain pathology C_LI

biochemistry↗

bioRxiv2026

The Structured RNA-binding Domains and Condensation Capacity of FUS Shape its RNA-binding Landscape and Function.

RNA-binding proteins (RBPs) are key regulators of gene expression and often contain intrinsically disordered regions that drive biomolecular condensation. Yet, how condensation affects RBP specificity and function remains poorly defined. Here, we use strategically designed point mutations to selectively impair canonical RNA-binding and condensation of the amyotrophic lateral sclerosis (ALS)-linked RBP Fused in Sarcoma (FUS). Using automated high-content imaging, we show that both properties shape nuclear ribonucleoprotein condensates and govern distinct aspects of FUS function in the DNA damage response. Transcriptome-wide mapping of FUS-RNA interactions reveals that the canonical RNA-binding domains recognise G-rich and C-rich motifs, whereas condensation selectively enhances binding to G-rich and structured sequences, often acting in concert with canonical RNA-binding to control transcriptional programs and splicing decisions. These findings provide a mechanistic framework for how FUS integrates condensation with sequence-specific RNA recognition to orchestrate nuclear organisation, genome stability and RNA metabolism. Furthermore, our rigorously validated mutants offer a platform for future mechanistic dissection of ALS pathogenesis and the development of targeted therapeutic strategies. HighlightsO_LIFUS condensation and RNA-binding can be functionally uncoupled with targeted point mutations C_LIO_LICondensation drives FUS recruitment and assembly of nuclear RNP condensates C_LIO_LIFUS condensation and RNA-binding play distinct roles in the DNA damage response C_LIO_LIThe structured RNA-binding domains confer specificity to G-rich and C-rich motifs C_LIO_LICondensation enhances FUS binding to G-rich, structured RNA elements C_LIO_LIFUS condensation and canonical RNA-binding synergistically regulate gene expression C_LI

molecular biology↗