Biology subjects

Flatley, A.

Publications and source records attributed to Flatley, A..

4 recordsLinked to original sources

bioRxiv2026

RTTN moonlights beyond the centrosome to control ribosome biogenesis and tRNA modification in human brain organoids

RTTN (rotatin) is a centrosomal protein mutated in severe malformations of cortical development, yet how its dysfunction disrupts human corticogenesis has remained unclear. Here, we show that RTTN has an unrecognized function at the core of the translation machinery. Using human telencephalic and hippocampal organoids carrying distinct RTTN alleles, together with single-cell and bulk transcriptomics, polysome profiling, and tRNA pseudouridine sequencing, we find that RTTN is enriched in cycling first-trimester neural progenitors and physically associates with ribosome-biogenesis and RNA-processing factors. RTTN mutations impair rRNA biogenesis and polysome assembly, reduce cytoplasmic ribosome density and nascent protein synthesis, and remodel the tRNA pseudouridylation landscape through both a PUS7L-dependent variable-arm signature and a broader RTTN-specific defect. These translational deficits are accompanied by prolonged mitosis, reduced entry into S-phase, and impaired interkinetic nuclear migration in mutant progenitors. Our findings redefine RTTN as a regulator of ribosome homeostasis and mRNA translation and implicate defective translational capacity as a driver of RTTN-associated microcephaly. Graphical AbstractRTTN sustains ribosome and tRNA homeostasis in human neural progenitors; its mutation disrupts mRNA translation, stalling progenitor proliferation and interkinetic nuclear migration, and driving cortical malformation and growth failure. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/744412v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@16124aaorg.highwire.dtl.DTLVardef@ae23f7org.highwire.dtl.DTLVardef@bb588forg.highwire.dtl.DTLVardef@1b35028_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Development of Motif-Specific Monoclonal Antibodies for Global Protein Citrullination Detection with Minimal Cross-Reactivity to Homocitrullination

Protein citrullination, a post-translational modification (PTM) catalyzed by peptidylarginine deiminases (PADs), plays critical roles in biological processes such as immunity, gene regulation, and inflammation. Dysregulated citrullination is implicated in diseases including rheumatoid arthritis, multiple sclerosis, and cancer, making it a potential biomarker and therapeutic target. Immunodetection is the most commonly used technique to study citrullination. However, most commercially available antibodies against citrullination are either protein-specific or lack the sensitivity and specificity of the broad variety of modified proteins. In addition, existing anti-pan citrullination antibodies often fail to distinguish citrullination from homocitrullination, a chemically similar modification on lysine. This cross-reactivity limits their utility in deciphering the distinct biological roles of these PTMs. To address these challenges, we employed a motif-based strategy to generate monoclonal antibodies against citrullination. This approach leverages PAD enzyme sequence preferences from human proteome, enhancing specificity for citrullination while minimizing cross-reactivity with homocitrullination. We immunized rats with a pool of over 490,000 citrullinated peptides, designed to represent common citrullination motifs in human tissue proteomes. Two monoclonal antibody clones were established and validated for sensitivity and specificity using ELISA and western blot against in vitro citrullinated and homocitrullinated proteomes, as well as ionomycin-activated human neutrophils. Both clones demonstrated great sensitivity to diverse citrullinated proteins, robust discrimination against homocitrullination, and quantitative readout in biological samples. These novel antibodies provide powerful tools for studying global citrullination dynamics and hold promise for biomarker discovery and diagnostic applications in diseases involving PAD dysregulation.

molecular biology↗

bioRxiv2023

Structures of Arabidopsis thaliana MDL Proteins and Synergistic Effects with the Cytokine MIF on Human Receptors

Vertebrates have developed effective immune mechanisms to fight microbial attacks, relying on a sophisticated network of innate and adaptive responses, a circulatory system, and numerous orchestrating soluble mediators such as cytokines. Mammalian macrophage migration inhibitory factor (MIF) and its paralog D-dopachrome tautomerase (D-DT/MIF-2) are multifunctional inflammatory cytokines with chemokine-like properties that modulate immunity. Plants possess orthologous MIF/D-DT-like (MDL) proteins, whose function is largely unexplored. Driven by the previous discovery of cross-kingdom mimicry of plant (Arabidopsis thaliana) MDL proteins and human MIF receptor signaling, we here characterized the structures of the three A. thaliana MDLs by X-ray crystallography and explored the mechanism underlying the interplay between plant MDLs, human MIF, and its receptors. We obtained high-resolution structures at 1.56 [A], 1.40 [A], and 2.00 [A] resolution for MDL1, MDL2, and MDL3, respectively, revealing a typical trimeric assembly and a high three-dimensional similarity to human MIF. Although residues at the catalytic site of the three MDLs show high identity to human MIF, the proteins showed low tautomerase activity for the substrate 4-hydroxyphenylpyruvate (HPP). Structural differences likely explain the enzymatic inactivity of plant MDLs for HPP. Strikingly, employing in vitro, in vivo, and in planta test systems, we found that MIF and MDL proteins interact with each other and have the capacity to form hetero-oligomeric complexes. The functional consequences of this interaction were demonstrated applying a yeast-based reporter system specific for the MIF chemokine receptors CXCR2 and CXCR4. MDLs not only triggered receptor signaling on their own, but exhibited pronounced synergism regarding the activation of the CXCR2- and CXCR4-dependent signaling pathways, when co-applied with MIF. These findings were substantiated by the co-administration of pharmacological inhibitors that either disrupt MIF receptor binding or block the catalytic cavity. Moreover, biochemical and biophysical experiments using an allosteric oligomer-specific MIF inhibitor established hexa-oligomer formation between MIF and MDLs as the putative basis for the synergistic effect. Our results are the starting point for a mechanistic understanding of the immunomodulatory activity of a family of highly conserved plant proteins. One Sentence SummaryA. thaliana MDLs and human macrophage inhibitory factor interact with each other and induce synergistic effects in activating CXCR2 and CXCR4.

immunology↗

bioRxiv2018

Intricate structure of the interphase chromocenter revealed by the analysis of a factor involved in species formation

In higher eukaryotes centromeres often coalesce into a large intranuclear domain called the chromocenter. Chromocenters are important for the organization of pericentric heterochromatin and a disturbance of their formation results in an upregulation of repetitive elements and causes defects in chromosome segregation. Mutations in the gene encoding for the centromere associated Drosophila speciation factor HMR show very similar phenotypes suggesting a role of HMR in chromocenter architecture and function. We performed confocal and super resolution microscopy as well as proximity based biotinylation experiments of HMR and its associated protein HP1a to generate a molecular map of HMR and HP1a bound chromatin. Our work reveals an intricate internal structure of the centromeric chromatin region, which suggests a role of HMR in separating heterochromatin from centromeric chromatin.

biochemistry↗