Biology subjects

Byun, D.

Publications and source records attributed to Byun, D..

8 recordsLinked to original sources

bioRxiv2026

Proteomic Analysis Reveals Widespread Regulation of Substrate Protein Abundance by O-fucosylation and O-GlcNAcylation

O-glycosylation of nucleocytosolic proteins by the Arabidopsis enzymes SPINDLY (SPY; O-fucosyltransferase) and SECRET AGENT (SEC; O-GlcNAc transferase) is essential for plant growth and development, yet the scope of their substrates and regulatory impact remains poorly defined. Here, we combined TurboID-based proximity labeling with quantitative proteomics to systematically map the SPY interactome and determine how SPY- and SEC-dependent modifications influence protein abundance. A functional SPY-TD enriched 221 proxiome proteins, including 80 known O-fucosylated substrates and 141 new interactors. The SPY-TD proxiome is enriched in nuclear pore components, chromatin regulators, transcription factors, and RNA-processing proteins. Integration with O-fucose and O-GlcNAc datasets yielded a comprehensive Arabidopsis SPY/SEC (At-S/S) protein list of 886 candidates. We quantified proteome-wide changes in spy single mutants and inducible spy sec double mutants. Loss of SPY alone caused selective stabilization or destabilization of targets, whereas combined SPY/SEC depletion triggered widespread, synergistic protein abundance changes, particularly affecting nucleoporins, transcriptional regulators, and RNA-binding proteins. Integration with ubiquitination datasets revealed extensive overlap, supporting potential crosstalk between O-fucosylation, O-GlcNAcylation, and ubiquitin-mediated protein turnover. Together, our study establishes proximity labeling as a powerful strategy to define plant O-glycosylation networks and reveals dual, context-dependent roles of SPY and SEC in controlling protein homeostasis and stress-responsive pathways.

systems biology↗

bioRxiv2025

Next-Generation Mapping of the ACINUS-Mediated Alternative Splicing Machinery and Its Regulation by O-glycosylation in Arabidopsis

Alternative splicing (AS) is a key mechanism of gene regulation, but the full repertoire of proteins involved and the regulatory mechanisms governing this process remain poorly understood. Using TurboID-based proximity labeling coupled with mass spectrometry (PL-MS), we comprehensively mapped the Arabidopsis AS machinery, focusing on the evolutionarily conserved splicing factor ACINUS, its paralog PININ, and the stable interactor SR45. We identified 298 high-confidence components, including both established and novel interactors, providing strong evidence that alternative splicing is coupled to transcription and that multiple RNA processing steps occur simultaneously in plants. Bioinformatic analysis reveals high redundancy, conserved mechanisms, and unique plant-specific features. Selected known and novel interactors were validated by AS readouts and phenotypic analysis, which also revealed a coordinated influence on splicing. Furthermore, a systematic evaluation of O-glycosylation double mutants revealed that SECRET AGENT (O-GlcNAc transferase) and SPINDLY (O-fucose transferase) modulate AS through both ACINUS-dependent and -independent pathways. Our results reveal the conserved as well as plant-specific AS regulatory network and highlight the global role of sugar modification in RNA processing.

plant biology↗

bioRxiv2024

COmapper: High-resolution mapping of meiotic crossovers by long-read sequencing in Arabidopsis

O_LIMeiotic crossovers rearrange existing genetic variation between homologous chromosomes, profoundly affecting genomic diversity. Crossovers are typically constrained to 1-3 events per chromosome pair, and their distribution is shaped by chromatin accessibility and DNA polymorphisms. Genome-wide crossover maps can be generated in plants by high-throughput short-read sequencing or linked-read sequencing. C_LIO_LIHere, we use long-read nanopore sequencing technology to develop a crossover mapping pipeline, COmapper, for high-resolution mapping of genome-wide crossovers from pooled DNA of F1 hybrid pollen and F2 recombinant seedlings derived from a cross between Arabidopsis thaliana accessions Col and Ler. We validate the high accuracy of COmapper by applying nanopore long-read sequencing to pooled DNA of Arabidopsis F2 individuals with crossovers mapped by short-read sequencing. C_LIO_LIUsing the COmapper, we constructed high-resolution genomic maps of crossovers using F1 hybrid pollen and F2 seedlings in wild type and crossover-elevated recq4a recq4b mutant, showing results comparable to short-read sequencing. Crossovers were enriched at gene-proximal promoters in wild type and increased but reshaped by high polymorphism density in recq4a recq4b. C_LIO_LIWe propose that COmapper will be widely applicable for exploring the effects of genetic, epigenetic and environmental changes on the crossover patterns across diverse plant species. C_LI

plant biology↗

bioRxiv2024

Impact of alternative splicing on Arabidopsis proteome

Limited proteomic evidence makes it unclear to what extent alternative splicing (AS) isoforms are translated and functionally relevant in eukaryotes. Here, we present a comprehensive proteomic analysis in plants using large-scale data mining, extensive fractionation of AspN-and trypsin-digested proteomes, and both label-free and TMT labeling. In total, we identified 471,196 peptides from 22,479 proteins by searching against Araport11, revealing 32,110 isoform-specific peptides. Using an integrated proteogenomic workflow coupled with SUPPA, we classified these peptides into 2,442 AS events, 879 of which involved intron retention (IR). Further analysis of unannotated events revealed 91 additional IRs that are translated, confirming that retained introns can give rise to peptides. AlphaFold modeling predicted the structural and functional impacts of these isoforms. Our dataset improved existing gene model annotations. By comparing wild-type plants with the AS mutant acinus pinin, we found that IR regulates transcript and protein abundance nonlinearly. Phenotypic assays confirmed the functional consequences, including reduced chlorophyll, impaired growth, and increased anthocyanin. Take together, our results provide large-scale proteomic evidence that AS isoforms are translated in plants, demonstrating how AS diversifies the proteome, regulates protein abundance, and affects growth and development.

plant biology↗

bioRxiv2024

Workflow enhancement of TurboID-mediated proximity labeling for SPY signaling network mapping

TurboID-based proximity labeling coupled to mass spectrometry (PL-MS) has emerged as a powerful tool for mapping protein-protein interactions in both plant and animal systems. Despite advances in sensitivity, PL-MS studies can still suffer from false negatives, especially when dealing with low abundance bait proteins and their transient interactors. Protein-level enrichment for biotinylated proteins is well developed and popular, but direct detection of biotinylated proteins by peptide-level enrichment and the difference in results between direct and indirect detection remain underexplored. To address this gap, we compared and improved enrichment and data analysis methods using TurboID fused to SPY, a low-abundance O-fucose transferase, using an AAL-enriched SPY target library for cross-referencing. Our results showed that MyOne and M280 streptavidin beads significantly outperformed antibody beads for peptide-level enrichment, with M280 performing best. In addition, while a biotin concentration [≤] 50 {micro}M is recommended for protein-level enrichment in plants, higher biotin concentrations can be used for peptide-level enrichment, allowing us to improve detection and data quality. FragPipes MSFragger protein identification and quantification software outperformed Maxquant and Protein Prospector for SPY interactome enrichment due to its superior detection of biotinylated peptides. Our improved washing protocols for protein-level enrichment mitigated bead collapse issues, improving data quality, and reducing experimental time. We found that the two enrichment methods provided complementary results and identified a total of 160 SPY-TurboID-enriched interactors, including 60 previously identified in the AAL-enriched SPY target list and 100 additional novel interactors. SILIA quantitative proteomics comparing WT and spy-4 mutants showed that SPY affects the protein levels of some of the identified interactors, such as nucleoporin proteins. We expect that our improvement will extend beyond TurboID to benefit other PL systems and hold promise for broader applications in biological research.

molecular biology↗

bioRxiv2021

Noninvasive Stimulation of Peripheral Nerves using Temporally-Interfering Electrical Fields

Electrical stimulation of peripheral nerves is a cornerstone of bioelectronic medicine. Effective ways to accomplish peripheral nerve stimulation noninvasively without surgically implanted devices is enabling for fundamental research and clinical translation. Here we demonstrate how relatively high frequency sine-wave carriers (3 kHz) emitted by two pairs of cutaneous electrodes can temporally interfere at deep peripheral nerve targets. The effective stimulation frequency is equal to the offset frequency (0.5 - 4 Hz) between the two carriers. We validate this principle of temporal interference nerve stimulation (TINS) in vivo using the murine sciatic nerve model. Effective actuation is delivered at significantly lower current amplitudes than standard transcutaneous electrical stimulation. Further, we demonstrate how flexible and conformable on-skin multielectrode arrays can facilitate precise alignment of TINS onto a nerve. Our method is simple, relying on repurposing of existing clinically-approved hardware. TINS opens the possibility of precise noninvasive stimulation with depth and efficiency previously impossible with transcutaneous techniques.

neuroscience↗

bioRxiv2021

HEAT SHOCK FACTOR BINDING PROTEIN limits meiotic crossovers by repressing HEI10 transcription

The number of meiotic crossovers is tightly controlled and most depend on pro-crossover ZMM proteins, such as the E3 ligase HEI10. Despite the importance of HEI10 dosage for crossover formation, how HEI10 transcription is controlled remains unexplored. In a forward genetic screen using a sensitive fluorescent seed crossover reporter in Arabidopsis thaliana we identify heat shock factor binding protein (HSBP) as a repressor of HEI10 transcription and crossover numbers. Using genome-wide crossover mapping and cytogenetics, we show that hsbp mutations or meiotic HSBP knockdowns increase ZMM-dependent crossovers towards the telomeres, mirroring the effects of HEI10 overexpression. Through RNA sequencing, DNA methylome and chromatin immunoprecipitation analysis, we reveal that HSBP directly represses HEI10 transcription by binding with heat shock factors (HSFs) at the HEI10 promoter and maintaining DNA methylation over the HEI10 5' untranslated region. Our findings provide insights into how the temperature response regulator HSBP restricts meiotic HEI10 transcription and crossover number by attenuating HSF activity.

plant biology↗

bioRxiv2019

DeepTetrad: high-throughput analysis of meiotic tetrads by deep learning in plants

Meiotic crossovers facilitate chromosome segregation and create new combinations of alleles in gametes. Crossover frequency varies along chromosomes and crossover interference limits the coincidence of closely spaced crossovers. Crossovers can be measured by observing the inheritance of linked transgenes expressing different colors of fluorescent protein in Arabidopsis pollen tetrads. Here we establish DeepTetrad, a deep learning-based image recognition package for pollen tetrad analysis that enables high-throughput measurements of crossover frequency and interference in individual plants. DeepTetrad will accelerate genetic dissection of mechanisms that control meiotic recombination.

plant biology↗