Biology subjects

Lee, C.-M.

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

At least 19 recordsLinked to original sources

bioRxiv2026

Telmisartan alters the transcriptomes of cancer associated fibroblasts and fibrocytes in a solid tumour model

Cancer associated fibroblasts (CAFs) are the main contributors to fibrosis in solid tumours. Fibrosis can lead to increased tumoural stiffness, immune cell exclusion, and poor response to cancer therapies. However, therapeutically depleting CAFs has been shown to increase tumour progression and metastasis in some pre-clinical tumour models. Targeted approaches to inhibit CAF activity and decrease fibrosis without abrogating CAFs altogether may improve the effect of existing cancer therapies. We have previously shown that the blood pressure medication telmisartan decreases tumoural collagen, resulting in improved vascular perfusion, decreased hypoxia, and improved radiation treatment efficacy. To understand how telmisartan influences CAF populations in solid tumours, we conducted single cell RNA sequencing analyses of host cells isolated from tumours in mice treated with telmisartan. Telmisartan modified the transcriptomes of CAF populations, promoting the expression of chemokine signaling while decreasing the expression of extracellular matrix components in myCAFs. Telmisartan decreased expression of collagen transcripts in myCAFs and fibrocytes, indicating that telmisartan does not remove myCAFs but changes the myCAF transcriptome to become less fibrillar. Our work provides further support for using telmisartan to manipulate CAF populations in the solid tumour microenvironment to decrease collagen deposition and render tumours more responsive to current cancer therapies.

cancer biology↗

bioRxiv2026

Dodecagon light-sheet fluorescence microscopy for large-volume imaging without striping artifacts

Light-sheet fluorescence microscopy (LSFM) has revolutionized biological imaging by enabling high spatial and temporal resolution with minimal photodamage. However, conventional LSFM techniques often suffer from striping artifacts in the resulting images due to light scattering and absorption within samples, leading to uneven illumination that negatively impacts the accuracy of subsequent image analyses. To address this limitation, we introduce dodecagon light-sheet fluorescence microscopy (dodecaLSFM), a novel approach that maximizes angular diversity to achieve homogeneous illumination and suppress striping artifacts. dodecaLSFM employs diffraction optics and cylindrical lenses to generate twelve light sheets, providing 360{degrees} omnidirectional illumination that significantly enhances illumination uniformity compared to traditional mSPIM, mDSLM, and ultramicroscopy systems, which use only one or two illumination planes. We demonstrate the effectiveness of dodecaLSFM by achieving high-resolution imaging of whole mouse brain vasculature following tissue clearing, allowing precise morphometric analysis of vascular networks without striping artifacts. Furthermore, we show that combining dodecaLSFM with expansion microscopy (ExM) enables whole-organ 3D imaging at cellular resolution. This novel approach provides an advanced, scalable solution for large-volume imaging, facilitating detailed structural and functional studies across diverse biological applications.

bioengineering↗

bioRxiv2025

Utilization of Arabidopsis E3 ubiquitin decoys high-throughput yeast screen platform to dissect the ubiquitin-mediated circadian clock regulation

Protein ubiquitination, mediated by E3 ubiquitin ligases, is a critical regulatory mechanism of eukaryotic cellular processes, including circadian clock function. However, identifying E3-substrate pairs remains technically challenging due to substrate instability and the genetic redundancy of E3s. To overcome these limitations, we developed a high-throughput yeast two-hybrid E3 decoy screening platform, enabling systematic mapping of E3-substrate interactions. Using a library of 283 Arabidopsis F-box and U-box E3 decoys, we screened 21 core circadian clock regulators and identified 77 potential E3-substrate interaction pairs involving 56 E3s and 16 clock proteins. Focusing on high-confidence hits, we demonstrated that PUB18 physically interacts with the central clock regulators LHY and JMJD5 and promotes their ubiquitination in planta. Genetic analyses further revealed that PUB18 and its homolog PUB19 function redundantly in circadian clock regulation. This study establishes the E3 decoy yeast two-hybrid platform as a versatile and scalable tool for dissecting ubiquitination networks in broad biological processes.

plant biology↗

bioRxiv2025

Shared Alteration of Whole-Brain Connectivity and Olfactory Deficits in Multiple Autism Mouse Models

Autism spectrum disorder (ASD) is a disconnection condition influenced by both heterogeneous genetic and environmental factors, yet it remains unclear whether common connectivity deficits exist. Here, we demonstrate that different ASD-linked mutations lead to distinct circuit abnormalities but share deficits in the piriform cortex and olfactory discrimination. Using advanced artificial intelligence, we developed a whole-brain mapping platform to analyze the distribution of the Thy1-YFP projection neurons in three ASD mouse models (Tbr1+/-, Nf1+/-, Vcp+/R95G). Our analysis revealed changes in axonal patterns and neuronal distribution, indicating deficits in projection neuron differentiation and maintenance. Notably, the piriform cortex consistently exhibited reduced YFP+ cells and signals and impaired functionality across all models. Visual and somatosensory cortices were also affected, but the patterns varied. These findings highlight that the sensory regions, especially the piriform cortex, are susceptible to ASD-related mutations, strengthening the notion that different sensory experiences are common in ASD. HighlightsO_LIAI-powered whole-mouse brain quantification accelerates neural connectivity study. C_LIO_LIAutism-linked mutations lead to various circuit abnormalities of Thy1-YFP neurons. C_LIO_LIMultiple sensory regions all exhibit circuit deficits among the three autism models. C_LIO_LIAbnormalities of olfaction and piriform cortex circuits are common in autism models. C_LI

neuroscience↗

bioRxiv2024

Arabidopsis EID1 E3 ubiquitin ligase regulates acquired thermotolerance by modulating HSBP translocation

Climate change is causing a rapid increase in global average temperatures and more frequent heatwaves, posing serious threats to agricultural production and global biodiversity. In response to heat stress (HS), plants can develop acquired thermotolerance (AT) by initiating a heat shock response (HSR) after mild HS priming, thereby enhancing their ability to withstand subsequent later lethal HS events. Central to this process are the HEAT SHOCK FACTORs (HSFs), which form trimeric complexes and activate the expression of HEAT SHOCK PROTEINs (HSPs) and other HSFs to maintain proper protein and cellular functionality. After heat stress subsides, the HSFs activities can be modulated to attenuate the negative effects of HSR during the heat. The SHOCK FACTOR BINDING PROTEIN (HSBP) is a conserved microprotein that plays a prominent role in modulating HSF activities. HSBP can translocate from the cytoplasm into the nucleus during heat stress to directly interact with HSFs and prevent the formation of HSF timers. However, the mechanism that regulates the HSBP cytoplasmic-nuclear shuttling remains unclear. Here, we identified an F-box E3 ubiquitin ligase, EMPFINDLICHER IM DUNKELROTEN LICHT 1 (EID1), whose mutant form shows reduced thermotolerance in AT. We showed that EID1 interacts with HSBP to modulate HSBP cytoplasm-nuclear localization during heat stress, possibly through modulating the K41 of HSBP. The decreased thermotolerance in the eid1 mutant can be explained by alterations of some HSPs expression caused by the mis-localization of HSBP. This finding provided a novel example of E3 ubiquitin-mediated regulation of heat stress in plants.

plant biology↗

bioRxiv2024

MOS4-Associated Complex subunits 3A and 3B modulate FLM splicing to repress photoperiod-dependent floral transition

Plants adjust their flowering time by integrating environmental cues through complex regulatory networks. RNA splicing plays a crucial role in modulating gene expression in response to flowering signals. The MOS4-associated complex (MAC), consisting of the evolutionarily conserved E3 ubiquitin ligases MAC3A and MAC3B, is pivotal in splicing regulation. However, their involvement in floral transition remained unclear. This study observed that mac3a/mac3b mutants flowered significantly earlier under short-day (SD) conditions, a phenotype absent under long-day (LD) conditions. This early flowering correlated with upregulation of FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) compared to wild-type plants. Transcriptomic analysis revealed alterations in transcript levels and splicing profiles of key floral regulators across different flowering pathways. Further investigation identified the thermosensory flowering regulator FLOWERING LOCUS M (FLM) as being influenced by MAC3A and MAC3B. Subsequently, we found that MAC3A and MAC3B exhibited higher expression and were associated with FLM transcripts to modulate their splicing in SD. This study elucidates how the MAC complex, through RNA splicing regulation, integrates environmental signals to modulate flowering, unveiling a new layer of complexity in flowering pathways crosstalk under non-inductive photoperiods.

plant biology↗

bioRxiv2023

Chitinase 3-like-1 (CHI3L1) Inhibits Innate Anti-Tumor and Tissue Remodeling ImmuneResponses by Regulating CD47-SIRPα and CD24-Siglec10-Mediated Phagocytosis

Innate immune responses such as phagocytosis are critically linked to the generation of adaptive immune responses against the neoantigens in cancer and the efferocytosis that is essential for homeostasis in diseases characterized by lung injury, inflammation, and remodeling as in Chronic Obstructive Pulmonary Disease (COPD). Chitinase 3-like-1 (CHI3L1) is induced in many cancers where it inhibits adaptive immune responses by stimulating immune checkpoint molecules (ICPs) and portends a poor prognosis. CHI3L1 is also induced in COPD where it regulates epithelial cell death. Here we demonstrate that pulmonary melanoma metastasis inhibits macrophage phagocytosis by stimulating the CD47-SIRP and CD24-Siglec10 phagocytosis checkpoint pathways while inhibiting macrophage "eat me" signals from calreticulin and HMGB1. We also demonstrate that these effects on macrophage phagocytosis are mediated by CHI3L1 stimulation of the SHP-1 and SHP-2 phosphatases and the inhibition of the accumulation and phosphorylation of cytoskeleton-regulating non-muscle myosin IIa. This inhibition of innate immune responses like phagocytosis provides a mechanistic explanation for the ability of CHI3L1 to stimulate ICPs and inhibit adaptive immune responses in cancer and diseases like COPD. The ability of CHI3L1 to simultaneously inhibit innate immune responses, stimulate ICPs, inhibit T cell co-stimulation, and regulate a number of other oncogenic and inflammation pathways suggest that CHI3L1-targeted therapeutics are promising interventions in cancer, COPD and other disorders.

immunology↗

bioRxiv2023

Chitinase 3-like-1 (CHI3L1) in the Pathogenesis of Epidermal Growth Factor Receptor Mutant Non-Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancers. In NSCLC, 10-20% of Caucasian patients and 30-50% of Asian patients have tumors with activating mutations in the Epidermal Growth Factor Receptor (EGFR). A high percentage of these patients exhibit favorable responses to treatment with tyrosine kinase inhibitors (TKI). Unfortunately, a majority of these patients develop therapeutic resistance with progression free survival lasting 9-18 months. The mechanisms that underlie the tumorigenic effects of EGFR and the ability of NSCLC to develop resistance to TKI therapies, however, are poorly understood. Here we demonstrate that CHI3L1 is produced by EGFR activation of normal epithelial cells, transformed epithelial cells with wild type EGFR and cells with cancer-associated, activating EGFR mutations. We also demonstrate that CHI3L1 auto-induces itself and feeds back to stimulate EGFR and its ligands. Highly specific antibodies against CHI3L1 (anti-CHI3L1/FRG) and TKI, individually and in combination, abrogated the effects of EGFR activation on CHI3L1 and the ability of CHI3L1 to stimulate the EGFR axis. Anti-CHI3L1 also interacted with osimertinib to reverse TKI therapeutic resistance and induce tumor cell death and inhibit pulmonary metastasis while stimulating tumor suppressor genes including KEAP1. CHI3L1 is a downstream target of EGFR that feeds back to stimulate and activate the EGFR axis. Anti-CHI3L1 is an exciting potential therapeutic for EGFR mutant NSCLC, alone and in combination with osimertinib or other TKIs.

cancer biology↗

bioRxiv2022

CHI3L1 Enhances Melanoma Lung Metastasis via Regulation of T cell Co-stimulators and CTLA-4/B7 Axis

ICOS/ICOSL and CD28/B7-1/B7-2 are T cell co-stimulators and CTLA-4 is an immune checkpoint inhibitor that play critical roles in the pathogenesis of neoplasia. Chitinase 3-like-1 (CHI3L1) is induced in many cancers where it portends a poor prognosis and contributes to tumor metastasis. Here we demonstrate that CHI3L1 inhibits the expression of ICOS, ICOSL and CD28 while stimulating CTLA-4 and the B7 moieties in melanoma lung metastasis. We also demonstrate that RIG-like helicase innate immune activation augments T cell co-stimulation, inhibits CTLA-4 and suppresses pulmonary metastasis. At least additive antitumor responses were seen in melanoma lung metastasis treated with anti-CTLA-4 and anti-CHI3L1 antibodies in combination. Synergistic cytotoxic T cell-induced tumor cell death and the heightened induction of the tumor suppressor PTEN were seen in co-cultures of T and tumor cells treated with bispecific antibodies that target both CHI3L1 and CTLA-4. Thus, CHI3L1 contributes to pulmonary metastasis by inhibiting T cell co-stimulation and stimulating CTLA-4. The simultaneous targeting of CHI3L1 and the CTLA-4 axis with individual and, more powerfully with bispecific antibodies, represent promising therapeutic strategies for pulmonary metastasis.

immunology↗

bioRxiv2021

Macrophages break interneuromast cell quiescence by intervening the inhibition of Schwann cells in zebrafish lateral line

In the zebrafish lateral line system, interneuromast cells (INCs) between neuromasts are normally kept quiescent by underlying Schwann cells (SWCs). Upon severe injuries that cause the complete loss of an entire neuromast, INCs can occasionally differentiate into neuromasts but how they escape from the inhibition by SWCs is still unclear. Using a genetic/chemical method to specifically ablate a neuromast, we found a small portion of larvae can regenerate a new neuromast, but the regeneration was hindered by inhibiting macrophages. By in toto imaging, we further discovered heterogeneities in macrophage behavior and distribution along lateral line. We witnessed the crawling of macrophages in between injured lateral line and SWCs during regeneration and also in between the second primordium and the first mature lateral line during development. It implies that macrophages may physically separate and alleviate the inhibition from pLLn and SWCs to break the quiescence of INCs during regeneration and development in the zebrafish lateral line.

developmental biology↗

bioRxiv2021

Kasugamycin is a novel chitinase 1 inhibitor with strong antifibrotic effects on pulmonary fibrosis

RationalePulmonary fibrosis is a devastating lung disease with few therapeutic options. Chitinase 1 (CHIT1), an 18 glycosyl hydrolase family member, contributes to the pathogenesis of pulmonary fibrosis through regulation of Transforming Growth Factor (TGF)-{beta} signaling and effector function. Therefore, CHIT1 is a potential therapeutic target of pulmonary fibrosis. ObjectivesThis study aimed to identify and characterize a druggable CHIT1 inhibitor with strong antifibrotic activity and minimal toxicity for therapeutic application to pulmonary fibrosis. MethodsExtensive screening of small molecule libraries identified the aminoglycoside antibiotic Kasugamycin as a potent CHIT1 inhibitor. Measurements and Main ResultsElevated levels of CHIT1 were detected in the lungs of patients with pulmonary fibrosis. In vivo bleomycin- and TGF-{beta}-stimulated murine models of pulmonary fibrosis, Kasugamycin showed impressive anti-fibrotic effects in both preventive and therapeutic conditions. In vitro studies also demonstrated that Kasugamycin inhibits fibrotic macrophage activation, fibroblast proliferation and myofibroblast transformation. Null mutation of transforming growth factor beta associated protein 1 (TGFBRAP1), a recently identified CHIT1 interacting signaling molecule, phenocopied antifibrotic effects of Kasugamycin in in vivo lungs and in vitro fibroblasts responses. Kasugamycin inhibits physical association between CHIT1 and TGFBRAP1, suggesting that antifibrotic effect of Kasugamycin is mediated through regulation of TGFBRAP1, at least in part. ConclusionsThese studies demonstrate that Kasugamycin is a novel CHIT1 inhibitor with strong antifibrotic effect that can be further developed as an effective and safe therapeutic drug for pulmonary fibrosis.

immunology↗

bioRxiv2021

Chitinase 3-like-1 Stimulates PD-L1 and Other Immune Checkpoint Inhibitors

PD-1 and its ligand PD-L1 are major mediators of tumor-induced immunosuppression. Chitinase 3-like-1 (Chi3l1) is induced in many cancers where it portends a poor prognosis and contributes to tumor metastasis. Here we demonstrate that Chi3l1 regulates the expression of PD-L1, PD-L2, PD-1 and LAG3 in melanoma lung metastasis. Chi3l1 stimulates macrophage PD-L1 expression and mediates optimal IFN-{gamma}-stimulated PD-L1 expression via IL-13R2. We also demonstrate that RIG-like helicase innate immune activation suppresses Chi3l1, PD-L1, LAG3 and pulmonary metastasis. At least additive antitumor responses were seen in metastasis models treated simultaneously with individual antibodies against PD-1 and Chi3l1. At least additive cytotoxic T cell-induced tumor cell death was also seen in co-cultures of T and tumor cells treated with antibodies that target Chi3l1 and PD-1. Thus, Chi3l1 contributes to pulmonary metastasis by stimulating the PD1-PD-L1 axis and other checkpoint molecules. The simultaneous targeting of Chi3l1 and the PD-1-PD-L1 axis, represents a promising therapeutic strategy for pulmonary metastasis.

cancer biology↗

bioRxiv2021

Chitinase 3-like-1 is a Therapeutic Target That Mediates the Effects of Aging in COVID-19

COVID-19 is caused by the SARS-CoV-2 (SC2) virus and is more prevalent and severe in the elderly and patients with comorbid diseases (CM). Because chitinase 3-like-1 (CHI3L1) is induced during aging and CM, the relationships between CHI3L1 and SC2 were investigated. Here we demonstrate that CHI3L1 is a potent stimulator of the SC2 receptor ACE2 and viral spike protein priming proteases (SPP), that ACE2 and SPP are induced during aging and that anti-CHI3L1, kasugamycin and inhibitors of phosphorylation, abrogate these ACE2- and SPP-inductive events. Human studies also demonstrated that the levels of circulating CHI3L1 are increased in the elderly and patients with CM where they correlate with COVID-19 severity. These studies demonstrate that CHI3L1 is a potent stimulator of ACE2 and SPP; that this induction is a major mechanism contributing to the effects of aging during SC2 infection and that CHI3L1 coopts the CHI3L1 axis to augment SC2 infection. CHI3L1 plays a critical role in the pathogenesis of and is an attractive therapeutic target in COVID-19.

immunology↗

bioRxiv2020

Natural Killer cell activation, reduced ACE2, TMPRSS2, cytokines G-CSF, M-CSF and SARS-CoV-2-S pseudovirus infectivity by MEK inhibitor treatment of human cells

COVID-19 affects vulnerable populations including elderly individuals and patients with cancer. Natural Killer (NK) cells and innate-immune TRAIL suppress transformed and virally-infected cells. ACE2, and TMPRSS2 protease promote SARS-CoV-2 infectivity, while inflammatory cytokines IL-6, or G-CSF worsen COVID-19 severity. We show MEK inhibitors (MEKi) VS-6766, trametinib and selumetinib reduce ACE2 expression in human cells. In some human cells, remdesivir increases ACE2-promoter luciferase-reporter expression, ACE2 mRNA and protein, and ACE2 expression is attenuated by MEKi. In serum-deprived and stimulated cells treated with remdesivir and MEKi we observed correlations between pRB, pERK, and ACE2 expression further supporting role of proliferative state and MAPK pathway in ACE2 regulation. We show elevated cytokines in COVID-19-(+) patient plasma (N=9) versus control (N=11). TMPRSS2, inflammatory cytokines G-CSF, M-CSF, IL-1, IL-6 and MCP-1 are suppressed by MEKi alone or with remdesivir. We observed MEKi stimulation of NK-cell killing of target-cells, without suppressing TRAIL-mediated cytotoxicity. Pseudotyped SARS-CoV-2 virus with a lentiviral core and SARS-CoV-2 D614 or G614 SPIKE (S) protein on its envelope infected human bronchial epithelial cells, small airway epithelial cells, or lung cancer cells and MEKi suppressed infectivity of the pseudovirus. We show a drug class-effect with MEKi to stimulate NK cells, inhibit inflammatory cytokines and block host-factors for SARS-CoV-2 infection leading also to suppression of SARS-CoV-2-S pseudovirus infection of human cells. MEKi may attenuate SARS-CoV-2 infection to allow immune responses and antiviral agents to control disease progression.

cell biology↗

bioRxiv2020

Genome-Enabled Discovery of Anthraquinone Biosynthesis in Senna tora

Senna tora is a widely used medicinal plant. Its health benefits have been attributed to the large quantity of anthraquinones, but how they are made in plants remains a mystery. To identify the genes responsible for plant anthraquinone biosynthesis, we sequenced and annotated the genome of S. tora at the chromosome level with contig N50 and super-scaffold N50 of 4.03 Mb and 41.7 Mb. Comparison among related plant species showed that a chalcone synthase-like (CHS-L) gene family has lineage-specifically and rapidly expanded in S. tora. Combining genomics, transcriptomics, metabolomics, and biochemistry, we identified a CHS-L responsible for biosynthesis of anthraquinones, the first example in plants. The S. tora reference genome will accelerate the discovery of biologically active anthraquinone biosynthesis pathways in medicinal plants. One Sentence SummaryThe chromosome-scale reference genome of a medicinal plant Senna tora, transcriptomics, metabolomics, and biochemical analysis provide new insights into anthraquinone biosynthesis in plants.

plant biology↗

bioRxiv2019

De novo transcriptome sequence of Senna tora provides insights into anthraquinone biosynthesis

Senna tora is an annual herb with rich source of anthraquinones that have tremendous pharmacological properties. However, there is little mention of genetic information for this species, especially regarding the biosynthetic pathways of anthraquinones. To understand the key genes and regulatory mechanism of anthraquinone biosynthesis pathways, we performed spatial and temporal transcriptome sequencing of S. tora using short RNA sequencing (RNA-Seq) and long-read isoform sequencing (Iso-Seq) technologies, and generated two unigene sets composed of 118,635 and 39,364, respectively. A comprehensive functional annotation and classification with multiple public databases identified array of genes involved in major secondary metabolite biosynthesis pathways and important transcription factor (TF) families (MYB, MYB-related, AP2/ERF, C2C2-YABBY, and bHLH). Differential expression analysis indicated that the expression level of genes involved in anthraquinone biosynthetic pathway regulates differently depending on the degree of tissues and seeds development. Furthermore, we identified that the amount of anthraquinone compounds were greater in late seeds than early ones. In conclusion, these results provide a rich resource for understanding the anthraquinone metabolism in S. tora.

plant biology↗

bioRxiv2019

Single cell transcriptomic analysis revealed long-lasting adverse effects of prenatal tamoxifen administration on neurogenesis in prenatal and adult brains

CreER/LoxP system has enabled precise gene manipulation in distinct cell subpopulations at any specific time point upon tamoxifen (TAM) administration. This system is widely accepted to track neural lineages and study gene functions. We have observed prenatal TAM treatment caused high rate of delayed delivery and mortality of pups. These substances could promote undesired results, leading to data misinterpretation. Here, we report that TAM administration during early stages of cortical neurogenesis promoted precocious neural differentiation, while inhibited neural progenitor cell (NPC) proliferation. The TAM-induced inhibition of NPC proliferation led to deficits in cortical neurogenesis, dendritic morphogenesis, and cortical patterning in neonatal and postnatal offspring. Mechanistically, single cell RNA sequencing (scRNA-seq) analysis combined with in vivo and in vitro assays showed TAM could exert these drastic effects mainly through dysregulating the expression of Dmrta2 and Wnt8b. In adult mice, administration of TAM significantly attenuated NPC proliferation in both the subventricular zone and the dentate gyrus. This study revealed the cellular and molecular mechanisms for the adverse effects of prenatal tamoxifen administration on corticogenesis, suggesting that tamoxifen-induced CreER/LoxP system may not be suitable for neural lineage tracing and genetic manipulation studies in both embryonic and adult brains. SignificantFor the first time, our study revealed the molecular mechanisms underlying tamoxifen activities on cortical development. This study also clearly showed that care must be taken when using tamoxifen-induced CreER/LoxP system for neural lineage tracing and genetic manipulation studies.

neuroscience↗

bioRxiv2018

Decoys provide a scalable platform for the genetic analysis of plant E3 ubiquitin ligases that regulate circadian clock function

The circadian clock in all eukaryotes relies on the regulated degradation of clock proteins to maintain 24-hour rhythmicity. Despite this knowledge, we know very few of the components that mediate degradation of proteins to control clock function. This is likely due to high levels of gene duplication and functional redundancy within plant E3 ubiquitin ligase gene families. In order to overcome this issue and discover E3 ubiquitin ligases that control circadian clock function, we generated a library of transgenic Arabidopsis lines expressing dominant-negative \"decoy\" E3 ubiquitin ligases. We determined their effects on the plant circadian clock and identified dozens of new potential regulators of circadian clock function. To demonstrate the potency of the decoy screening methodology to overcome genetic redundancy and identify bona fide clock regulators, we performed follow-up studies on PUB59 and PUB60. Using knock-out studies, we show that they redundantly control circadian clock period by regulating gene splicing. Furthermore, we confirm that they are part of a conserved protein complex that mediates splicing in eukaryotes. This work demonstrates the viability of E3 ubiquitin ligase decoys as a scalable screening platform to overcome traditional genetic challenges and discover E3 ubiquitin ligases that regulate plant developmental processes.

plant biology↗