Biology subjects

Lee, H.-C.

Publications and source records attributed to Lee, H.-C..

At least 19 recordsLinked to original sources

bioRxiv2026

Germ granules function as stress-responsive hubs to protect germ cell resilience

Biomolecular condensates are dynamic, membrane-less compartments that perform critical cellular functions in the adaptation to environmental stressors. Within germ cells, germ granules are specialized condensates essential for germ cell viability, genome defense, and epigenetic inheritance. Notably, environmental stressors such as heat and starvation can disrupt genome defense and epigenetic inheritance, yet germ cell viability is often preserved. How germ cells adapt to environmental stress and recover viability remains poorly understood. Here, we investigate the impact of heat stress on the biomolecular condensates of adult germ cells in C. elegans. Upon heat stress, we observe extensive remodeling of the germ granule compartments, including reduced enrichment of key genome defense and epigenetic inheritance factors. Adjacent to remodeled germ granules, stress granules and P-bodies robustly assemble, suggesting these distinct condensates may functionally interact during the stress response. Indeed, we find stress granule and P-body formation within germ cells largely requires germ granule assembly factors GLH-1/VASA and MIP-1. Furthermore, we show these germ granule assembly factors and stress granule factors GTBP-1/G3BP1 and TIAR-1/TIA1, are essential for recovery of germ cell viability after heat stress. Intriguingly, stress granule factor TIAR-1/ TIA1 localizes to germ granules with and without stress, indicating germ granules are poised for the stress response. Together, our results support a model in which germ granules function as stress-responsive hubs that reorganize RNA granule networks to promote germline resilience.

developmental biology↗

bioRxiv2026

Distinct subcellular localizations of DUF1218 proteins in Marchantia polymorpha and Nicotiana benthamiana reveal two different plasmodesmata-targeting mechanisms

Plasmodesmata are membrane-lined channels connecting plant cells to facilitate intercellular transport of molecules. Although many plasmodesmata-localized proteins have evolved throughout plant evolution, whether they use a conserved targeting system remains unclear. In the bryophyte Marchantia polymorpha, we identified two DUF1218-domain proteins homologous to the Arabidopsis plasmodesmata-localized AtTVA. When ectopically expressed, MpDUF1218-1 localized to plasmodesmata in both Nicotiana benthamiana and M. polymorpha, whereas MpDUF1218-2 formed cytoplasmic puncta in both species. Unexpectedly, AtTVA formed cytoplasmic puncta rather than localizing to plasmodesmata in M. polymorpha. Domain-swap analyses revealed that the first helix of MpDUF1218-1 is crucial for plasmodesmata localization in N. benthamiana, while the first two helices are required in M. polymorpha. In contrast, the second and third helices of AtTVA contribute to its plasmodesmata localization in N. benthamiana. Further domain dissection indicated that other regions of MpDUF1218-1 also contribute to accurate targeting by regulating its distribution among the ER, cytoplasmic puncta, and plasma membrane. Together, our findings suggest that MpDUF1218-1 is targeted by a mechanism shared between the two species, whereas AtTVA relies on a distinct mechanism present in N. benthamiana but absent in M. polymorpha, suggesting the emergence of alternative plasmodesmata-targeting pathways during land plant evolution.

plant biology↗

bioRxiv2026

If the shoe fits? -- technical nuances of plasma proteomic workflows in clinical and preclinical contexts

The push for new clinical biomarkers has seen rapid innovation in biofluid analysis, particularly for plasma. For mass-spectrometry (MS)-based analysis, achieving depth and quantitative accuracy whilst ensuring throughput continues to shape plasma methods development. Numerous workflows have emerged that mitigate high-abundance suppression and expand dynamic range, especially when paired with next-generation MS instrumentation. Yet systematic evaluations that also consider biological variables (e.g., biofluid type, species) and technical parameters (e.g., MS methods) are limited. Here, we benchmarked eight sample-preparation workflows spanning neat approaches (SP3, STrap), depletion (perchloric acid, PerCA), and corona-enrichment strategies (MagNet HILIC/SAX, Enrich-iST, ProteoNano). We compared their performance across human plasma, human serum, and rat plasma, analysing all samples on an Orbitrap Astral (Thermo) using two plasma-optimised data-independent acquisition (DIA) methods: one discovery-maximised and one throughput-maximised. We identified 2,726 human and 3,767 rat proteins across workflows and methods, including [~]1,000 from neat plasma. Increasing throughput incurred a [~]20-30% reduction in depth, depending on workflow and species. EV-enrichment produced the deepest proteomes but with distinct compositions relative to neat, depleted, and secreted-protein-enriched samples, revealing a unique sub-proteome niche. Several workflows also performed markedly better in rat plasma, supporting improved sensitivity for preclinical analyses. Enrichment or depletion dramatically reshaped the balance of tissue-and cell-specific proteins detectable in plasma, suggesting that workflow choice should be guided by the organs, immune targets, or inflammatory signals most relevant to the study. In this vein, statistical analysis of differentially abundant proteins showed that >90% of detected proteins were significantly altered between workflows, with the largest numbers arising from the corona-enrichment strategies, underscoring how strongly workflow choice shapes the downstream proteome. Taken together, these findings emphasise a rapidly expanding plasma methodological landscape, where the most effective workflow is the one most precisely tailored to a cohorts biology.

systems biology↗

bioRxiv2026

Functional Evaluation of TRPC6 Missense Variants in Cancer Patients via Molecular Docking Analysis Compared with Patch Clamp Electrophysiology

Gain-of-function mutations in the transient receptor potential 6 (TRPC6) channel have recently been recognized as risk factors for both doxorubicin (DOX)-induced cardiomyopathy. Functional evaluation of TRPC6 missense variants is therefore important for cancer patients undergoing anthracycline treatment. However, traditional electrophysiological methods are labor-intensive and time-consuming. In this study, we compared the functional responses of TRPC6 missense variants to 1-oleoyl-2-acetyl-sn-glycerol (OAG), a TRPC6 agonist, using molecular docking and patch clamp recording techniques. For the wild-type (WT) TRPC6 structure (PDB ID: 6UZ8), OAG exhibited a binding energy of -4.49 kcal/mol and a dissociation constant (Kd) of 0.511 mM. Twenty TRPC6 missense variants were identified from cancer patients in the Mayo Clinic database. Of these, fifteen variants had resolvable structures, nine of which displayed increased Kd values and six decreased Kd values compared to WT in molecular docking analysis. Patch clamp recordings revealed that TRPC6 WT and mutant channels were inactive at baseline but were activated upon 50 M OAG stimulation, except two loss-of-function variants. Moreover, a 24-h treatment with 0.5 M DOX significantly enhanced OAG-induced channel activation. All three variants identified in patients with heart failure demonstrated gain-of-function properties in both electrophysiological measurements and in-silico predictions. Importantly, the results obtained from molecular docking and patch clamp recordings were strongly correlated, showing an 82% concordance, higher than the predictions from AlphaMissense. These findings indicate that our computational analysis provides a rapid and reliable method for predicting the functional impact of TRPC6 missense variants, which may aid clinical decision-making in cancer patients receiving chemotherapy.

physiology↗

bioRxiv2025

RNA Helicase A promotes small RNA biogenesis and sorting in germ cells

In germ cells, small RNAs function as a defense system to silence invading RNAs like viruses and transposons to protect genome integrity. The ability of small RNAs to robustly silence diverse RNA sequences prompts the question of how endogenous mRNAs avoid this silencing. In C. elegans, small RNAs bound by the Argonaute CSR-1 protect endogenous mRNAs from silencing, while also fine-tuning a subset of these mRNAs. Here, we identify RNA Helicase A (RHA-1) as a key regulator of CSR-1 small RNA biogenesis and function in mRNA fine-tuning. RHA-1 localizes to germ granules dependent on EGO-1, which synthesizes CSR-1 small RNAs. We find RHA-1 promotes small RNA production from the 5 regions of mRNAs and small RNA sorting to CSR-1. Loss of RHA-1 leads to elevated CSR-1 target mRNA levels and compromised fertility. Our study highlights the importance of small RNA regulation, mediated by RHA-1, to protect endogenous gene expression programs and germ cell function.

genetics↗

bioRxiv2025

Analysis of crosslinking sites suggests C. elegnas PIWI Argonaute exhibits flexible conformations for target recognition

Small RNAs play critical roles in gene regulation in diverse processes across organisms. Crosslinking, ligation, and analyses of sequence hybrid (CLASH) experiments have shown PIWI and Argonaute proteins bind to diverse mRNA targets, raising questions about their functional relevance and the degree of flexibility in target recognition. As crosslinking-induced mutations (CIMs) provides nucleotide-resolution of RNA binding sites, we developed MUTACLASH to systematically analyze CIMs in piRNA and miRNA CLASH data in C. elegans. We found CIMs are enriched at the nucleotide positions of mRNA corresponding to the center of targeting piRNAs and miRNAs. Notably, CIMs are also enriched at nucleotides with local pairing mismatches to piRNA. In addition, distinct patterns of CIMs are observed between canonical and non-canonical base pairing interactions, suggesting that the worm PIWI Argonaute PRG-1 adopts distinct conformations for canonical vs. non-canonical interactions. Critically, non-canonical miRNA or piRNA binding sites with CIMs exhibit more regulatory effects than those without CIMs, demonstrating CIM analysis as a valuable approach in assessing functional significance of small RNA targeting sites in CLASH data. Together, our analyses reveal the landscapes of Argonaute crosslinking sites on mRNAs and highlight MUTACLASH as an advanced tool in analyzing CLASH data.

molecular biology↗

bioRxiv2025

Nucleoporins shape germ granule architecture and balance small RNA silencing pathways

Animals have evolved distinct small RNA pathways, including piRNA and siRNA, to silence invasive and selfish nucleic acids. piRNA pathway factors are concentrated in perinuclear germ granules that frequently associate with nuclear pore complexes (NPCs). However, the factors mediating germ granule-NPC association and the functional relevance of such association remain unknown. Here we show that the conserved nucleoporins NPP-14 (NUP-214) and NPP-24 (NUP-88), components of the cytoplasmic filaments of NPC, play critical roles in anchoring germ granule to NPC and in attenuating piRNA silencing In C. elegans. Proximity labeling experiments further identified EPS-1 (enhanced piRNA silencing) as a key germ granule factor contributing to germ granule-NPC interaction. In npp-14, npp-24, or eps-1 mutant animals, we observed fewer but enlarged, unorganized germ granules, accompanied by the over-amplification of secondary small RNAs at piRNA targeting sites. Nonetheless, we found this enhancement of piRNA silencing comes at the cost of dampened RNAi efficiency and RNAi inheritance. Together, our studies uncovered factors contributing to germ granule-NPC association and underscored the importance of spatial organization of germ granules in balancing small RNA silencing pathways.

genetics↗

bioRxiv2024

Adherent-suspension plasticity promotes the dissemination and colonization of circulating tumor cells

Cell competition within the primary tumor drives tumor growth by promoting the uncontrolled proliferation of winner cells and eliminating loser cells by sensing cell fitness. However, the mechanism of how cell competition confers the loser cells with the metastatic potential of circulating tumor cells (CTCs) to transit between dissemination and colonization remains elusive. Here we found cell competition gives rise to less fit but viable cells that transformed into CTCs via adherent-to-suspension transition (AST) mechanisms. The induction of hematopoietic transcription factors hijacked by solid tumor cells rendered CTCs competent to reprogram their anchorage dependency and disseminate into the bloodstream, while subsequent suppression of these factors was critical to regain adhesion and colonize metastatic lesions. Disrupting the oscillatory dynamics of AST factors blocked the adherent-suspension plasticity (ASP) of breast cancer CTCs and suppressed lung metastasis. Furthermore, multiregional single-cell transcriptomic analyses of matched primary tumors, CTCs, and metastatic lesions from de novo metastatic breast cancer patients demonstrate the critical role of ASP in metastasis. SignificanceWe demonstrate cell competition-mediated displacement of loser cells manifests dynamic oscillation of AST factors that confer anchorage plasticity to circulating tumor cells critical for their dissemination and colonization in metastasis. These findings highlight the potential of targeting AST factors to develop effective anti-metastatic therapies.

cancer biology↗

bioRxiv2023

Hypoxic injury triggers maladaptive repair in human kidney organoids

Acute kidney injury (AKI) is a common clinical disorder linked to high rates of illness and death. Ischemia is a leading cause of AKI, which can result in chronic kidney disease (CKD) through maladaptive repair marked by impaired epithelial regeneration, inflammation, and metabolic dysregulation. There are no targeted therapies for AKI or to prevent progression to CKD and insight into human disease mechanisms remains limited. Here we show that human kidney organoids recapitulate key molecular and metabolic signatures of AKI and maladaptive repair in response to hypoxic injury. Transcriptional, proteomic, and metabolomic profiling revealed tubular injury, cell death, cell cycle arrest and metabolic reprogramming in organoids exposed to hypoxia. Following return to normoxic conditions, injured organoids had increased signatures of TNF and NF-{kappa}B signalling pathways and S100A8/9, associated with maladaptive repair. Single cell RNA sequencing localized AKI and maladaptive repair markers including GDF15, MMP7, ICAM1, IL32, SPP1, C3 and CCN1 to injured tubules. Metabolic phenotypes linked to CKD were also evident, including dysregulated gluconeogenesis, altered amino acid metabolism and lipid peroxidation. iPSC-derived macrophages incorporated into organoids displayed a robust activation and inflammatory response to hypoxia. Spatial transcriptomics revealed a shift from a tissue resident-like to inflammatory macrophage states and localized effects on tubular injury and inflammation. This multi-omic analysis defines conserved mechanisms of human ischemic AKI and maladaptive repair, highlighting new opportunities to test therapeutics and model immune-mediated interactions.

systems biology↗

bioRxiv2023

The HOMEODOMAIN-Like protein HDL mediates chromatin organization and rewires leaf epidermal patterning

The Arabidopsis leaf epidermis is comprised of trichomes, pavement cells, and stomata, originating from versatile precursor cells capable of dividing or differentiating to create unique epidermal patterns. The mechanism governing these transitions and the maintenance of cell heterogeneity remains unclear. In this study, we identified a novel homeodomain-like superfamily protein, HDL, localized in chromocenters and playing a role in chromatin organization. HDL interacts with histone deacetylase 6 (HDA6) and methyltransferases, indicating its function in modulating chromatin accessibility. In hdl mutants, we observed moderately increased chromatin accessibility in the promoter region of protein-coding genes, along with reduced stomatal density and elevated trichome numbers on leaf surfaces. Corresponding to these phenotypes, stomatal-related gene expression decreased, while a transcriptional reporter for GLABRA2, a trichome initiation gene, exhibited higher and more variable expression levels. These findings highlight how HDL-mediated chromatin organization influences epidermal cell fate by modulating gene expression and enhancing cell heterogeneity within the leaf epidermis. One Sentence SummaryA homeodomain-like protein functions with histone modifiers to regulate chromatin and influence cell fate decisions.

plant biology↗

bioRxiv2023

An obligate aerobe adapts to hypoxia by hybridising fermentation with carbon storage

In soil ecosystems, obligately aerobic bacteria survive oxygen deprivation (hypoxia) by entering non-replicative persistent states. Little is known about how these bacteria rewire their metabolism to stay viable in these states. The model obligate aerobe Mycobacterium smegmatis maintains redox homeostasis during hypoxia by mediating fermentative hydrogen production. However, the fate of organic carbon during fermentation, and the associated remodeling of carbon metabolism, is unresolved. Here we systematically profiled the metabolism of M. smegmatis during aerobic growth, hypoxic persistence, and the transition between these states. Using differential isotope labelling, and paired metabolomics and proteomics, we observed rerouting of central carbon metabolism through the pentose phosphate pathway and Entner-Doudoroff pathway during hypoxia. We show that M. smegmatis excretes high levels of hydrogen concurrently with upregulating triacylglyceride synthases and accumulating glycerides as carbon stores. Using electron cryotomography (cryo-ET), we observed the presence of large spheroid structures consistent with the appearance of lipid droplets. Thus, in contrast to obligately and facultative anaerobic fermentative bacteria, M. smegmatis stores rather than excretes organic carbon during hypoxia. This novel hybrid metabolism likely provides a competitive advantage in resource-variable environments by allowing M. smegmatis to simultaneously dispose excess reductant during hypoxia and maintain carbon stores to rapidly resume growth upon reoxygenation.

microbiology↗

bioRxiv2023

Sensitized piRNA reporter identifies multiple RNA processing factors involved in piRNA-mediated gene silencing

Metazoans guard their germlines against transposons and other foreign transcripts with PIWI-interacting RNAs (piRNAs). Due to the robust heritability of the silencing initiated by piRNAs in C.elegans, previous screens using Caenorhabditis elegans were strongly biased to uncover members of this pathway in the maintenance process but not in the initiation process. To identify novel piRNA pathway members, we have utilized a sensitized reporter strain which detects defects in initiation, amplification, or regulation of piRNA silencing. Using our reporter, we have identified Integrator complex subunits, nuclear pore components, protein import components, and pre-mRNA splicing factors as essential for piRNA-mediated gene silencing. We found the snRNA processing cellular machine termed the Integrator complex is required for both type I and type II piRNA production. Notably, we identified a role for nuclear pore and nucleolar components in promoting the perinuclear localization of anti-silencing CSR-1 Argonaute, as well as a role for Importin factor IMA-3 in nuclear localization of silencing Argonaute HRDE-1. Together, we have shown that piRNA silencing is dependent on evolutionarily ancient RNA processing machinery that has been co-opted to function in the piRNA mediated genome surveillance pathway.

genetics↗

bioRxiv2022

P bodies coat germ granules to promote transgenerational gene silencing in C. elegans

The formation of biomolecular condensates has emerged as a critical mechanism for compartmentation in living cells. Despite interactions between distinct condensates having been reported, the biological relevance of these interactions remains elusive. In germ cells, small RNA silencing factors are enriched in germ granule condensates, where distinct factors are organized into sub-compartments with specific functions linked to genome surveillance or transgenerational gene silencing. Here we showed that perinuclear germ granules are coated by P body condensates, which are known for housing translationally-inactive mRNAs and mRNA degradation factors. Disruption of P body factors, including CGH-1/DDX6 and CAR-1/LSM14, lead to dispersal of small RNA factors from perinuclear germ granules and disorganization of sub-compartments within germ granules. We further found that CAR-1 promotes the interaction between CGH-1 and germ granule factors, and these interactions are critical for the ability of CGH-1 to promote piRNA-mediated gene silencing. Importantly, we observed that cgh-1 mutants are competent in triggering gene silencing but exhibit defects in maintaining gene silencing in subsequent generations. Small RNA sequencing further showed that cgh-1 mutants exhibit defects in amplifying secondary small RNAs, known carriers of gene silencing memories. Together, our results uncover the function of P body factors in small RNA-mediated transgenerational gene silencing and highlight how the formation and function of one condensate can be regulated by an adjacent, interacting condensate in cells.

cell biology↗

bioRxiv2022

Transcriptome-wide analysis suggests piRNAs preferentially recognize the coding region of mRNAs in C. elegans

BackgroundPIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposon mRNAs and some endogenous mRNAs in various animals. However, C. elegans piRNAs fail to trigger gene silencing at many sequence-based predicted targeting sites. ResultsTo gain insights into the mechanisms that control piRNA silencing capability, we compared the transcriptome-wide predicted piRNA targeting sites to the in vivo piRNA binding sites. Surprisingly, while predicted piRNA targeting sites are enriched in 3 UTRs, we found that C. elegans piRNAs preferentially bind to coding regions (CDS) of target mRNAs, leading to preferential production of secondary silencing small RNAs in the CDS. Furthermore, our analyses suggest that Argonaute protein CSR-1 protects mRNAs from piRNA silencing through two distinct mechanisms - by inhibiting piRNA binding across the entire CSR-1 targeted transcript, and by inhibiting secondary silencing small RNA production locally at CSR-1 bound sites. However, CSR-1 is not responsible for the piRNA binding preference for the CDS. ConclusionsOur work identifies the CDS as the critical region that is uniquely competent for piRNA silencing in C. elegans. We speculate that the preference for CDS recognition by piRNAs may represent a mechanism to counteract the evolution of foreign protein-coding RNAs that evade piRNA surveillance.

genomics↗

bioRxiv2022

GLH/VASA helicases promote germ granule formation to ensure the fidelity of piRNA-mediated transcriptome surveillance

The ability to distinguish non-self from self is the key characteristic for any defense system. piRNAs function as guardians of the genome by silencing non- self nucleic acids and transposable elements in animals. Many piRNA factors are enriched in perinuclear germ granules, but whether their localization is required for piRNA biogenesis or function is not known. Here we show that GLH/VASA helicase mutants exhibit defects in forming perinuclear condensates containing PIWI and other small RNA cofactors. These mutant animals produce largely normal levels of piRNA but are defective in triggering piRNA silencing. Strikingly, while many piRNA targets are activated in GLH mutants, we observed that hundreds of endogenous genes are aberrantly silenced by piRNAs. This defect in self versus non-self recognition was also observed in other mutants where perinuclear P granules are disrupted. Together, our results argue that perinuclear germ granules function critically to promote the fidelity of piRNA- based transcriptome surveillance in C. elegans and preserve self versus non-self distinction.

cell biology↗

bioRxiv2021

Maize AFP1 confers antifungal activity by inhibiting chitin deacetylases from a broad range of fungi

Adapted plant pathogenic fungi deacetylate chitin to chitosan to avoid host perception and disarm the chitin-triggered plant immunity. Whether plants have evolved factors to counteract this fungal evasion mechanism in the plant-pathogen interface remains obscure. Here, we decipher the underlying mechanism of maize cysteine-rich receptor-like secreted proteins (CRRSPs)-AFP1, which exhibits mannose-binding dependent antifungal activity. AFP1 initials the action by binding to specific sites on the surface of yeast-like cells, filaments, and germinated spores of the biotrophic fungi Ustilago maydis. This could result in fungal cell growth and cell budding inhibition, delaying spore germination and subsequently reducing fungal viability in a mannose-binding dependence manner. The antifungal activity of AFP1 is conferred by its interaction with the PMT-dependent mannosylated chitin deacetylases (CDAs) and interfering with the conversion of chitin. Our finding that AFP1 targets CDAs from pathogenic fungi and nonpathogenic budding yeast suggests a potential application of the CRRSP in combating fungal diseases and reducing threats posed by the fungal kingdom.

microbiology↗

bioRxiv2019

Identification and genetic validation of drug-comorbidity interactions in type 2 diabetes using data-driven disease trajectory analysis

Given various risk profiles, manifestations, comorbidities, and outcomes for individuals with Type 2 diabetes mellitus (T2D), a one-size-fits-all approach towards treatment and management is inadequate, and there is a clear need for personalized medications to reduce rates of complications and comorbidities. Here, we leveraged comprehensive Electronic Medical Records (EMR) to identify associations between T2D comorbidities and medications and evaluated their biological determinants using EMR-linked genetic information. We discovered clinically novel associations supported by the previous laboratory studies; e.g. 5-hydroxytryptamine 3 receptor antagonists, ondansetron and granisetron, are protective against cognitive disorders, and clopidogrel is protective against retinopathy. Furthermore, potentially novel associations were validated by genetic analysis; e.g. association between gabapentin and cognitive disorders was supported via variants of its target genes, GRIN1 and CACNA2D2. These results of the current study open the door for optimizing treatment combinations to mitigate risks and for individualizing therapy based on T2D subtype risk profiles.

bioinformatics↗

bioRxiv2019

Germline RNA helicases couple RNA binding to P granule assembly at nuclear periphery

P granules are phase-separated liquid droplets that play important roles in the maintenance of the germ cell fate in C. elegans. The localization and formation of P granules are highly dynamic, but mechanisms that regulate such processes remain poorly understood. Here we show that germline RNA helicases (GLHs) control the formation and disassembly of germ granules through their binding and release of RNAs, respectively. In addition, the FGG repeats in the GLHs promote the formation of germ granules at the perinucleus. Proteomic analyses of a mutation that traps RNA-bound GLH-1 complex revealed transient interactions of GLH-1 with several Argonautes and RNA binding proteins. Finally, we found that defects in perinuclear P granule formation correlate with the fertility defects observed in various GLH mutants. Together, our results highlight the versatile roles of RNA helicases in controlling the formation of liquid droplets in space and time.

cell biology↗