Publications by authors named "Ji-Long Liu"

128 Publications

A New Method for Studying RNA-binding Proteins on Specific RNAs.

Bio Protoc 2021 May 20;11(10):e4022. Epub 2021 May 20.

School of Life Science and Technology, ShanghaiTech University, Shanghai, China.

Proximity-based protein labeling has been developed to identify protein-nucleic acid interactions. We have reported a novel method termed CRUIS (CRISPR-based RNA-United Interacting System), which captures RNA-protein interactions in living cells by combining the RNA-binding capacity of CRISPR/Cas13 and the proximity-tagging activity of PUP-IT. Enzymatically deactivated Cas13a (dCas13a) is fused to the proximity labeling enzyme PafA. In the presence of a guide RNA, dCas13a binds specific target RNA region, while the fused PafA mediates the labeling of biotin-tagged Pup on proximal proteins. The labeled proteins can be enriched by streptavidin pull-down and identified by mass spectrometry. Here we describe the general procedure for capturing RNA-protein interactions using this method.
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http://dx.doi.org/10.21769/BioProtoc.4022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8187123PMC
May 2021

CTPS forms the cytoophidium in zebrafish.

Exp Cell Res 2021 Jun 12;405(2):112684. Epub 2021 Jun 12.

School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China; Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, United Kingdom. Electronic address:

Cytidine triphosphate synthase (CTPS) catalyzes the rate-limiting step of de novo CTP biosynthesis. An intracellular structure of CTPS, the cytoophidium, has been found in many organisms including prokaryotes and eukaryotes. Formation of the cytoophidium has been suggested to regulate the activity and stability of CTPS and may participate in certain physiological events. Herein, we demonstrate that both CTPS1a and CTPS1b in zebrafish are able to form the cytoophidium in cultured cells. A point mutation, H355A, abrogates cytoophidium assembly of zebrafish CTPS1a and CTPS1b. In addition, we show the presence of CTPS cytoophidia in multiple tissues of larval and adult fish under normal conditions, while treatment with a CTPS inhibitor 6-diazo-5-oxo-l-norleucine (DON) can induce more cytoophidia in some tissues. Our findings reveal that forming the CTPS cytoophidium is a natural phenomenon of zebrafish and provide valuable information for future research on the physiological importance of this intracellular structure in vertebrates.
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http://dx.doi.org/10.1016/j.yexcr.2021.112684DOI Listing
June 2021

Cell-Cell Communication at the Embryo Implantation Site of Mouse Uterus Revealed by Single-Cell Analysis.

Int J Mol Sci 2021 May 13;22(10). Epub 2021 May 13.

Guangdong Laboratory for Lingnan Modern Agriculture, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China.

As a crucial step for human reproduction, embryo implantation is a low-efficiency process. Despite rapid advances in recent years, the molecular mechanism underlying embryo implantation remains poorly understood. Here, we used the mouse as an animal model and generated a single-cell transcriptomic atlas of embryo implantation sites. By analyzing inter-implantation sites of the uterus as control, we were able to identify global gene expression changes associated with embryo implantation in each cell type. Additionally, we predicted signaling interactions between uterine luminal epithelial cells and mural trophectoderm of blastocysts, which represent the key mechanism of embryo implantation. We also predicted signaling interactions between uterine epithelial-stromal crosstalk at implantation sites, which are crucial for post-implantation development. Our data provide a valuable resource for deciphering the molecular mechanism underlying embryo implantation.
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http://dx.doi.org/10.3390/ijms22105177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8153605PMC
May 2021

IMPDH forms the cytoophidium in zebrafish.

Dev Biol 2021 May 25;478:89-101. Epub 2021 May 25.

School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. Electronic address:

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo guanine nucleotide biosynthesis. Its activity is negatively regulated by the binding of GTP. IMPDH can form a membraneless subcellular structure termed the cytoophidium in response to certain changes in the metabolic status of the cell. The polymeric form of IMPDH, which is the subunit of the cytoophidium, has been shown to be more resistant to the inhibition by GTP at physiological concentrations, implying a functional correlation between cytoophidium formation and the upregulation of GTP biosynthesis. Herein we demonstrate that zebrafish IMPDH1b and IMPDH2 isoforms can assemble abundant cytoophidium in most of cultured cells under stimuli, while zebrafish IMPDH1a shows distinctive properties of forming the cytoophidium in different cell types. Point mutations that disrupt cytoophidium structure in mammalian models also prevent the aggregation of zebrafish IMPDHs. In addition, we discover the presence of the IMPDH cytoophidium in various tissues of larval and adult fish under normal growth conditions. Our results reveal that polymerization and cytoophidium assembly of IMPDH can be a regulatory machinery conserved among vertebrates, and with specific physiological purposes.
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http://dx.doi.org/10.1016/j.ydbio.2021.05.017DOI Listing
May 2021

CTPS and IMPDH form cytoophidia in developmental thymocytes.

Exp Cell Res 2021 Aug 20;405(1):112662. Epub 2021 May 20.

Institute of Biotechnology, National Taiwan University, Taipei, 106, Taiwan; Agricultural Biotechnology Research Center, Academia Sinica, Taipei, 115, Taiwan; Animal Resource Center, National Taiwan University, Taipei, 106, Taiwan. Electronic address:

The cytoophidium, a filamentous structure formed by metabolic enzymes, has emerged as a novel regulatory machinery for certain proteins. The rate-limiting enzymes of de novo CTP and GTP synthesis, cytidine triphosphate synthase (CTPS) and inosine monophosphate dehydrogenase (IMPDH), are the most characterized cytoophidium-forming enzymes in mammalian models. Although the assembly of CTPS cytoophidia has been demonstrated in various organisms including multiple human cancers, a systemic survey for the presence of CTPS cytoophidia in mammalian tissues in normal physiological conditions has not yet been reported. Herein, we examine major organs of adult mouse and observe that CTPS cytoophidia are displayed by a specific thymocyte population ranging between DN3 to early DP stages. Most of these cytoophidium-presenting cells have both CTPS and IMPDH cytoophidia and undergo rapid cell proliferation. In addition, we show that cytoophidium formation is associated with active glycolytic metabolism as the cytoophidium-presenting cells exhibit higher levels of c-Myc, phospho-Akt and PFK. Inhibition of glycolysis with 2DG, however, disrupts most of cytoophidium structures and impairs cell proliferation. Our findings not only indicate that the regulation of CTPS and IMPDH cytoophidia are correlated with the metabolic switch triggered by pre-TCR signaling, but also suggest physiological roles of the cytoophidium in thymocyte development.
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http://dx.doi.org/10.1016/j.yexcr.2021.112662DOI Listing
August 2021

Histone transcription regulator Slm9 is required for cytoophidium biogenesis.

Exp Cell Res 2021 Jun 1;403(1):112582. Epub 2021 Apr 1.

School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. Electronic address:

The cytoophidium, a subcellular structure composed of CTP synthase, can be observed during the division of Schizosaccharomyces pombe. Cytoophidium formation changes periodically with the cell cycle of yeast cells. Here, we find that histone chaperone Slm9 is required for the integrity of cytoophidia in fission yeast. When the slm9 gene is knocked out, we observe that morphological characteristics, the abundance of cytoophidia and the division of the yeast cells are significantly affected. Fragmented cytoophidia occur in slm9 mutant cells, a phenomenon rarely observed in wild-type cells. Our study reveals a potential link between a chromosomal regulatory factor and cytoophidium biogenesis.
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http://dx.doi.org/10.1016/j.yexcr.2021.112582DOI Listing
June 2021

Polarised maintenance of cytoophidia in Drosophila follicle epithelia.

Exp Cell Res 2021 May 16;402(2):112564. Epub 2021 Mar 16.

School of Life Science and Technology, ShanghaiTech University, Shanghai, China; MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, United Kingdom. Electronic address:

The metabolic enzyme CTP synthase (CTPS) can form filamentous structures named cytoophidia in numerous types of cells, including follicle cells. However, the regulation of cytoophidium assembly remains elusive. The apicobasal polarity, a defining characteristic of Drosophila follicle epithelium, is established and regulated by a variety of membrane domains. Here we show that CTPS can form cytoophidia in Drosophila epithelial follicle cells. Cytoophidia localise to the basolateral side of follicle cells. If apical polarity regulators are knocked down, cytoophidia become unstable and distribute abnormally. Knockdown of basolateral polarity regulators has no significant effect on cytoophidia, even though the polarity is disturbed. Our results indicate that cytoophidia are maintained via polarised distribution on the basolateral side of Drosophila follicle epithelia, which is primarily achieved through the apical polarity regulators.
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http://dx.doi.org/10.1016/j.yexcr.2021.112564DOI Listing
May 2021

Highly effective proximate labeling in Drosophila.

G3 (Bethesda) 2021 05;11(5)

School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.

The protein-protein interaction (PPI) is a basic strategy for life to operate. The analysis of PPIs in multicellular organisms is very important but extremely challenging because PPIs are particularly dynamic and variable among different development stages, tissues, cells, and even organelles. Therefore, understanding PPI needs a good resolution of time and space. More importantly, understanding in vivo PPI needs to be realized in situ. Proximity-based biotinylation combined with mass spectrometry (MS) has emerged as a powerful approach to study PPI networks and protein subcellular compartmentation. TurboID, the newly engineered promiscuous ligase, has been reported to label proximate proteins effectively in various species. In Drosophila, we systematically apply TurboID-mediated biotinylation in a wide range of developmental stages and tissues, and demonstrate the feasibility of TurboID-mediated labeling system in desired cell types. For a proof-of-principle, we use the TurboID-mediated biotinylation coupled with MS to distinguish CTP synthase with or without the ability to form filamentous cytoophidia, retrieving two distinct sets of proximate proteomes. Therefore, this makes it possible to map PPIs in vivo and in situ at a defined spatiotemporal resolution, and demonstrates a referable resource for cytoophidium proteome in Drosophila.
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http://dx.doi.org/10.1093/g3journal/jkab077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104946PMC
May 2021

ASNS disruption shortens CTPS cytoophidia in Saccharomyces cerevisiae.

G3 (Bethesda) 2021 01;11(1)

School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China.

Asparagine synthetase (ASNS) and CTP synthase (CTPS) are two metabolic enzymes that catalyze the biosynthesis of asparagine and CTP, respectively. Both CTPS and ASNS have been identified to form cytoophidia in Saccharomyces cerevisiae. Glutamine is a common substrate for both these enzymes, and they play an important role in glutamine homeostasis. Here, we find that the ASNS cytoophidia are shorter than the CTPS cytoophidia, and that disruption of ASNS shortens the length of CTPS cytoophidia. However, the deletion of CTPS has no effect on the formation and length of ASNS cytoophidia, or on the ASNS protein level. We also find that Asn1 overexpression induces the formation of a multi-dot structure in diauxic phase which suggests that the increased protein level may trigger cytoophidia formation. Collectively, our results reveal a connection between ASNS cytoophidia and CTPS cytoophidia.
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http://dx.doi.org/10.1093/g3journal/jkaa060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8022725PMC
January 2021

Effects of Survival Motor Neuron Protein on Germ Cell Development in Mouse and Human.

Int J Mol Sci 2021 Jan 11;22(2). Epub 2021 Jan 11.

Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan.

Survival motor neuron (SMN) is ubiquitously expressed in many cell types and its encoding gene, survival motor neuron 1 gene (SMN1), is highly conserved in various species. SMN is involved in the assembly of RNA spliceosomes, which are important for pre-mRNA splicing. A severe neurogenic disease, spinal muscular atrophy (SMA), is caused by the loss or mutation of SMN1 that specifically occurred in humans. We previously reported that SMN plays roles in stem cell biology in addition to its roles in neuron development. In this study, we investigated whether SMN can improve the propagation of spermatogonia stem cells (SSCs) and facilitate the spermatogenesis process. In in vitro culture, SSCs obtained from SMA model mice showed decreased growth rate accompanied by significantly reduced expression of spermatogonia marker promyelocytic leukemia zinc finger (PLZF) compared to those from heterozygous and wild-type littermates; whereas SMN overexpressed SSCs showed enhanced cell proliferation and improved potency. In vivo, the superior ability of homing and complete performance in differentiating progeny was shown in SMN overexpressed SSCs in host seminiferous tubule of transplant experiments compared to control groups. To gain insights into the roles of SMN in clinical infertility, we derived human induced pluripotent stem cells (hiPSCs) from azoospermia patients (AZ-hiPSCs) and from healthy control (ct-hiPSCs). Despite the otherwise comparable levels of hallmark iPCS markers, lower expression level of was found in AZ-hiPSCs compared with control hiPSCs during in vitro primordial germ cell like cells (PGCLCs) differentiation. On the other hand, overexpressing in AZ-hiPSCs led to increased level of pluripotent markers such as OCT4 and KLF4 during PGCLC differentiation. Our work reveal novel roles of SMN in mammalian spermatogenesis and suggest new therapeutic targets for azoospermia treatment.
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http://dx.doi.org/10.3390/ijms22020661DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827477PMC
January 2021

Hypoosmolality impedes cytoophidium integrity during nitrogen starvation.

Yeast 2021 Apr 28;38(4):276-289. Epub 2021 Jan 28.

School of Life Science and Technology, ShanghaiTech University, Shanghai, China.

CTP synthase (CTPS) cytoophidia have been found in many species over domains of life in the past 10 years, implying the evolutionary conservation of these structures. However, there are differences in cytoophidia between species. The difference in CTPS cytoophidium properties between budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) inspires this research. We study the effects of culture environment on cytoophidia in S. cerevisiae by switching to the optimal medium for S. pombe. S. cerevisiae CTPS cytoophidium fragmentation and pseudohyphae formation are observed after treatment with S. pombe medium YES instead of S. cerevisiae medium YPD. By modifying the level of each ingredient of the media, we find that hypoosmolality impedes cytoophidium integrity during nitrogen starvation. Our study demonstrates the relationship between cytoophidium integrity and environmental stress, supporting the role of cytoophidia in stress resistance.
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http://dx.doi.org/10.1002/yea.3542DOI Listing
April 2021

Cold shock induces novel nuclear bodies in Xenopus oocytes.

Exp Cell Res 2021 01 18;398(1):112386. Epub 2020 Nov 18.

Department of Embryology, Carnegie Institution for Science, Baltimore, MD, 21218, USA. Electronic address:

Here we describe novel spherical structures that are induced by cold shock on the lampbrush chromosomes (LBCs) of Xenopus laevis oocytes. We call these structures cold bodies or C-bodies. C-bodies are distributed symmetrically on homologous LBCs, with a pattern similar to that of 5S rDNA. Neither active transcription nor translation is necessary for their formation. Similar protrusions occur on the edges of some nucleoli. Endogenous LBCs as well as those derived from injected sperm form C-bodies under cold shock conditions. The function of C-bodies is unknown.
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http://dx.doi.org/10.1016/j.yexcr.2020.112386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7771896PMC
January 2021

The atlas of cytoophidia in Drosophila larvae.

J Genet Genomics 2020 06 27;47(6):321-331. Epub 2020 Jun 27.

School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. Electronic address:

In 2010, cytidine 5'-triphosphate synthase (CTPS) was reported to form the filamentous or serpentine structure in Drosophila, which we termed the cytoophidium. In the last decade, CTPS filaments/cytoophidia have been found in bacteria, budding yeast, human cells, mice, fission yeast, plants, and archaea, indicating that this mechanism is highly conserved in evolution. In addition to CTPS, other metabolic enzymes have been identified to have the characteristics of forming cytoophidia or similar advanced structures, demonstrating that this is a basic strategy of cells. Nevertheless, our understanding of the physiological function of the cytoophidium remains incomplete and elusive. Here, we took the larva of Drosophila melanogaster as a model to systematically describe the localization and distribution of cytoophidia in different tissues during larval development. We found that the distribution pattern of CTPS cytoophidia is dynamic and heterogenic in larval tissues. Our study provides a road map for further understanding of the function and regulatory mechanism of cytoophidia.
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http://dx.doi.org/10.1016/j.jgg.2020.06.004DOI Listing
June 2020

Long-Term Imaging and Dynamic Analysis of Cytoophidia in Yeast.

Methods Mol Biol 2021 ;2196:235-244

School of Life Science and Technology, ShanghaiTech University, Shanghai, China.

Live-cell imaging is widely used by researchers to study cellular dynamics and obtain a deep understanding of cell biological processes. Keeping cells in the proper growing environment and immobilizing the cells are essential for the imaging of live yeast cells. Here we describe a protocol for monitoring cytoophidia in Saccharomyces cerevisiae and Schizosaccharomyces pombe using inverted confocal fluorescence microscopy. This protocol includes yeast culture, sample preparation, fluorescence imaging, and data analysis.
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http://dx.doi.org/10.1007/978-1-0716-0868-5_19DOI Listing
March 2021

Comparative Analysis of Mouse Decidualization Models at the Molecular Level.

Genes (Basel) 2020 08 13;11(8). Epub 2020 Aug 13.

Guangdong Laboratory for Lingnan Modern Agriculture, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, Guangdong, China.

The mouse is widely used to study decidualization and there are three well-established mouse models of decidualization, namely natural pregnancy decidualization (NPD), artificial decidualization (AD), and in vitro decidualization (IVD). However, the extent of similarity and difference between these models at the molecular level remains largely unknown. Here, we performed a comparative analysis using the RNA-seq approach. In the NPD model, which is thought to be the golden standard of mouse decidualization, we found a total of 5277 differentially expressed genes, with 3158 genes being up-regulated and 2119 genes being down-regulated. A total of 4294 differentially expressed genes were identified in the AD model: 1127 up-regulated genes and 3167 down-regulated genes. In comparison to NPD, 1977 genes were consistently expressed, whereas only 217 genes were inconsistently expressed, indicating that AD is a reliable model for mouse decidualization. In the IVD model, RNA-seq analysis revealed that 513 genes were up-regulated and 988 genes were down-regulated. Compared to NPD, 310 genes were consistently expressed, whereas 456 genes were inconsistently expressed. Moreover, although the decidualization marker Prl8a2 (prolactin family 8 subfamily a member 2) was up-regulated, the widely-used marker Alpl (alkaline phosphatase liver/bone/kidney) was down-regulated in the IVD model. Therefore, we suggest that the IVD model should be optimized to mimic NPD at the transcriptomic level. Our study contributes to an increase in the knowledge about mouse models of decidualization.
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http://dx.doi.org/10.3390/genes11080935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7465532PMC
August 2020

Differential Histone Distribution Patterns in Induced Asymmetrically Dividing Mouse Embryonic Stem Cells.

Cell Rep 2020 08;32(6):108003

Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA. Electronic address:

Wnt3a-coated beads can induce asymmetric divisions of mouse embryonic stem cells (mESCs), resulting in one self-renewed mESC and one differentiating epiblast stem cell. This provides an opportunity for studying histone inheritance pattern at a single-cell resolution in cell culture. Here, we report that mESCs with Wnt3a-bead induction display nonoverlapping preexisting (old) versus newly synthesized (new) histone H3 patterns, but mESCs without Wnt3a beads have largely overlapping patterns. Furthermore, H4K20me2/3, an old histone-enriched modification, displays a higher instance of asymmetric distribution on chromatin fibers from Wnt3a-induced mESCs than those from non-induced mESCs. These locally distinct distributions between old and new histones have both cellular specificity in Wnt3a-induced mESCs and molecular specificity for histones H3 and H4. Given that post-translational modifications at H3 and H4 carry the major histone modifications, our findings provide a mammalian cell culture system to study histone inheritance for maintaining stem cell fate and for resetting it during differentiation.
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http://dx.doi.org/10.1016/j.celrep.2020.108003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962874PMC
August 2020

The Pre-Implantation Embryo Induces Uterine Inflammatory Reaction in Mice.

Reprod Sci 2021 01 10;28(1):60-68. Epub 2020 Jul 10.

College of Veterinary Medicine, South China Agricultural University, No.483 Wushan Road, Tianhe District, Guangzhou, 510642, China.

It has been well established that uterine function during the peri-implantation period is precisely regulated by ovarian estrogen and progesterone. The embryo enters the uterine cavity before implantation. However, the impact of pre-implantation embryo on uterine function is largely unknown. In the present study, we performed RNA-seq analysis of mouse uterus on day 4 morning of natural pregnancy (with embryos in the uterus) and pseudo-pregnancy (without embryos in the uterus). We found that 146 genes were upregulated, and 77 genes were downregulated by the pre-implantation embryo. Gene ontology and gene network analysis highlighted the activation of inflammatory reaction in the uterus. By examining the promoter region of differentially expressed genes, we found that NF-kappaB was a causal transcription factor. Finally, we validated 4 inflammation-related genes by quantitative RT-PCR. These 4 genes are likely the main mediators of the inflammatory reaction in the uterus triggered by the pre-implantation embryo. Our data indicated that the pre-implantation embryo causes uterine inflammatory reaction, which in turn might contribute to the establishment of uterine receptivity and embryo implantation.
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http://dx.doi.org/10.1007/s43032-020-00259-7DOI Listing
January 2021

CTP synthase forms cytoophidia in archaea.

J Genet Genomics 2020 04 20;47(4):213-223. Epub 2020 Apr 20.

School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. Electronic address:

CTP synthase (CTPS) is an important metabolic enzyme that catalyzes the rate-limiting reaction of nucleotide CTP de novo synthesis. Since 2010, a series of studies have demonstrated that CTPS can form filamentous structures in bacteria and eukaryotes, which are termed cytoophidia. However, it is unknown whether cytoophidia exist in the third domain of life, archaea. Using Haloarcula hispanica as a model system, here we demonstrate that CTPS forms distinct intracellular compartments in archaea. Under stimulated emission depletion microscopy, we find that the structures of H. hispanica CTPS are elongated, similar to cytoophidia in bacteria and eukaryotes. When Haloarcula cells are cultured in low-salt medium, the occurrence of cytoophidia increases dramatically. In addition, treatment of H. hispanica with a glutamine analog or overexpression of CTPS can promote cytoophidium assembly. Our study reveals that CTPS can form cytoophidia in all three domains of life, suggesting that forming cytoophidia is an ancient property of CTPS.
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http://dx.doi.org/10.1016/j.jgg.2020.03.004DOI Listing
April 2020

The proline synthesis enzyme P5CS forms cytoophidia in Drosophila.

J Genet Genomics 2020 03 19;47(3):131-143. Epub 2020 Mar 19.

School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China; MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, United Kingdom. Electronic address:

Compartmentation of enzymes via filamentation has arisen as a mechanism for the regulation of metabolism. In 2010, three groups independently reported that CTP synthase (CTPS) can assemble into a filamentous structure termed the cytoophidium. In searching for CTPS-interacting proteins, here we perform a yeast two-hybrid screening of Drosophila proteins and identify a putative CTPS-interacting protein, △-pyrroline-5-carboxylate synthase (P5CS). Using the Drosophila follicle cell as the in vivo model, we confirm that P5CS forms cytoophidia, which are associated with CTPS cytoophidia. Overexpression of P5CS increases the length of CTPS cytoophidia. Conversely, filamentation of CTPS affects the morphology of P5CS cytoophidia. Finally, in vitro analyses confirm the filament-forming property of P5CS. Our work links CTPS with P5CS, two enzymes involved in the rate-limiting steps in pyrimidine and proline biosynthesis, respectively.
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http://dx.doi.org/10.1016/j.jgg.2020.02.005DOI Listing
March 2020

SNAP29 mediates the assembly of histidine-induced CTP synthase filaments in proximity to the cytokeratin network.

J Cell Sci 2020 05 11;133(9). Epub 2020 May 11.

Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan

Under metabolic stress, cellular components can assemble into distinct membraneless organelles for adaptation. One such example is cytidine 5'-triphosphate synthase (CTPS, for which there are CTPS1 and CTPS2 forms in mammals), which forms filamentous structures under glutamine deprivation. We have previously demonstrated that histidine (His)-mediated methylation regulates the formation of CTPS filaments to suppress enzymatic activity and preserve the CTPS protein under glutamine deprivation, which promotes cancer cell growth after stress alleviation. However, it remains unclear where and how these enigmatic structures are assembled. Using CTPS-APEX2-mediated proximity labeling, we found that synaptosome-associated protein 29 (SNAP29) regulates the spatiotemporal filament assembly of CTPS along the cytokeratin network in a keratin 8 (KRT8)-dependent manner. Knockdown of SNAP29 interfered with assembly and relaxed the filament-induced suppression of CTPS enzymatic activity. Furthermore, APEX2 proximity labeling of keratin 18 (KRT18) revealed a spatiotemporal association of SNAP29 with cytokeratin in response to stress. Super-resolution imaging suggests that during CTPS filament formation, SNAP29 interacts with CTPS along the cytokeratin network. This study links the cytokeratin network to the regulation of metabolism by compartmentalization of metabolic enzymes during nutrient deprivation.
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http://dx.doi.org/10.1242/jcs.240200DOI Listing
May 2020

Capturing RNA-protein interaction via CRUIS.

Nucleic Acids Res 2020 05;48(9):e52

School of Life Science and Technology, ShanghaiTech University, Shanghai, China.

No RNA is completely naked from birth to death. RNAs function with and are regulated by a range of proteins that bind to them. Therefore, the development of innovative methods for studying RNA-protein interactions is very important. Here, we developed a new tool, the CRISPR-based RNA-United Interacting System (CRUIS), which captures RNA-protein interactions in living cells by combining the power of CRISPR and PUP-IT, a novel proximity targeting system. In CRUIS, dCas13a is used as a tracker to target specific RNAs, while proximity enzyme PafA is fused to dCas13a to label the surrounding RNA-binding proteins, which are then identified by mass spectrometry. To identify the efficiency of CRUIS, we employed NORAD (Noncoding RNA activated by DNA damage) as a target, and the results show that a similar interactome profile of NORAD can be obtained as by using CLIP (crosslinking and immunoprecipitation)-based methods. Importantly, several novel NORAD RNA-binding proteins were also identified by CRUIS. The use of CRUIS facilitates the study of RNA-protein interactions in their natural environment, and provides new insights into RNA biology.
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http://dx.doi.org/10.1093/nar/gkaa143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7229851PMC
May 2020

Survival Motor Neuron Protein Participates in Mouse Germ Cell Development and Spermatogonium Maintenance.

Int J Mol Sci 2020 Jan 25;21(3). Epub 2020 Jan 25.

Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan.

The defective human survival motor neuron 1 (SMN1) gene leads to spinal muscular atrophy (SMA), the most common genetic cause of infant mortality. We previously reported that loss of SMN results in rapid differentiation of Drosophila germline stem cells and mouse embryonic stem cells (ESCs), indicating that SMN also plays important roles in germ cell development and stem cell biology. Here, we show that in healthy mice, SMN is highly expressed in the gonadal tissues, prepubertal spermatogonia, and adult spermatocytes, whereas low SMN expression is found in differentiated spermatid and sperm. In SMA-like mice, the growth of testis tissues is retarded, accompanied with gamete development abnormalities and loss of the spermatogonia-specific marker. Consistently, knockdown of Smn1 in spermatogonial stem cells (SSCs) leads to a compromised regeneration capacity in vitro and in vivo in transplantation experiments. In SMA-like mice, apoptosis and accumulation of the R-loop structure were significantly elevated, indicating that SMN plays a critical role in the survival of male germ cells. The present work demonstrates that SMN, in addition to its critical roles in neuronal development, participates in mouse germ cell and spermatogonium maintenance.
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http://dx.doi.org/10.3390/ijms21030794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037566PMC
January 2020

Drosophila CTP synthase can form distinct substrate- and product-bound filaments.

J Genet Genomics 2019 11 29;46(11):537-545. Epub 2019 Nov 29.

School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. Electronic address:

Intracellular compartmentation is a key strategy for the functioning of a cell. In 2010, several studies revealed that the metabolic enzyme CTP synthase (CTPS) can form filamentous structures termed cytoophidia in prokaryotic and eukaryotic cells. However, recent structural studies showed that CTPS only forms inactive product-bound filaments in bacteria while forming active substrate-bound filaments in eukaryotic cells. In this study, using negative staining and cryo-electron microscopy, we demonstrate that Drosophila CTPS, whether in substrate-bound or product-bound form, can form filaments. Our results challenge the previous model and indicate that substrate-bound and product-bound filaments can coexist in the same species. We speculate that the ability to switch between active and inactive cytoophidia in the same cells provides an additional layer of metabolic regulation.
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http://dx.doi.org/10.1016/j.jgg.2019.11.006DOI Listing
November 2019

Temperature-sensitive cytoophidium assembly in Schizosaccharomyces pombe.

J Genet Genomics 2019 09 24;46(9):423-432. Epub 2019 Sep 24.

MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, United Kingdom; School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. Electronic address:

The metabolic enzyme CTP synthase (CTPS) is able to compartmentalize into filaments, termed cytoophidia, in a variety of organisms including bacteria, budding yeast, fission yeast, fruit flies and mammals. A previous study in budding yeast shows that the filament-forming process of CTPS is not sensitive to temperature shift. Here we study CTPS filamentation in the fission yeast Schizosaccharomyces pombe. To our surprise, we find that both the length and the occurrence of cytoophidia in S. pombe decrease upon cold shock or heat shock. The temperature-dependent changes of cytoophidia are fast and reversible. Taking advantage of yeast genetics, we demonstrate that heat-shock proteins are required for cytoophidium assembly in S. pombe. Temperature sensitivity of cytoophidia makes S. pombe an attractive model system for future investigations of this novel membraneless organelle.
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http://dx.doi.org/10.1016/j.jgg.2019.09.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868507PMC
September 2019

The TOR pathway modulates cytoophidium formation in .

J Biol Chem 2019 10 19;294(40):14686-14703. Epub 2019 Aug 19.

School of Life Sciences and Technology, ShanghaiTech University, 201210 Shanghai, China

CTP synthase (CTPS) has been demonstrated to form evolutionarily-conserved filamentous structures termed cytoophidia whose exact cellular functions remain unclear, but they may play a role in intracellular compartmentalization. We have previously shown that the mammalian target of rapamycin complex 1 (mTORC1)-S6K1 pathway mediates cytoophidium assembly in mammalian cells. Here, using the fission yeast as a model of a unicellular eukaryote, we demonstrate that the target of rapamycin (TOR)-signaling pathway regulates cytoophidium formation (from the CTPS ortholog Cts1) also in Conducting a systematic analysis of all viable single TOR subunit-knockout mutants and of several major downstream effector proteins, we found that Cts1 cytoophidia are significantly shortened and often dissociate when TOR is defective. We also found that the activities of the downstream effector kinases of the TORC1 pathway, Sck1, Sck2, and Psk1 S6, as well as of the S6K/AGC kinase Gad8, the major downstream effector kinase of the TORC2 pathway, are necessary for proper cytoophidium filament formation. Interestingly, we observed that the Crf1 transcriptional corepressor for ribosomal genes is a strong effector of Cts1 filamentation. Our findings connect TOR signaling, a major pathway required for cell growth, with the compartmentalization of the essential nucleotide synthesis enzyme CTPS, and we uncover differences in the regulation of its filamentation among higher multicellular and unicellular eukaryotic systems.
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http://dx.doi.org/10.1074/jbc.RA119.009913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779450PMC
October 2019

Forming cytoophidia prolongs the half-life of CTP synthase.

Authors:
Zhe Sun Ji-Long Liu

Cell Discov 2019 18;5:32. Epub 2019 Jun 18.

1School of Life Science and Technology, ShanghaiTech University, 230 Haike Road, 201210 Shanghai, China.

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http://dx.doi.org/10.1038/s41421-019-0098-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6579761PMC
June 2019

Endometrial carcinoma may favor partial, but not complete, loss of the TGF-β signaling pathway.

Proc Natl Acad Sci U S A 2019 05;116(19):9164-9165

College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China.

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http://dx.doi.org/10.1073/pnas.1903646116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511018PMC
May 2019

Identification of Gene Expression Changes Associated With Uterine Receptivity in Mice.

Front Physiol 2019 14;10:125. Epub 2019 Feb 14.

College of Veterinary Medicine, South China Agricultural University, Guangzhou, China.

The mouse is a widely used animal model for studying human reproduction. Although global gene expression changes associated with human uterine receptivity have been determined by independent groups, the same studies in the mouse are scarce. The extent of similarities/differences between mice and humans on uterine receptivity at the molecular level remains to be determined. In the present study, we analyzed global gene expression changes in receptive uterus on day 4 of pregnancy compared to non-receptive uterus on day 3 of pregnancy in mice. A total of 541 differentially expressed genes were identified, of which 316 genes were up-regulated and 225 genes were down-regulated in receptive uterus compared to non-receptive uterus. Gene ontology and gene network analysis highlighted the activation of inflammatory response in the receptive uterus. By analyzing the promoter sequences of differentially expressed genes, we identified 12 causal transcription factors. Through connectivity map (CMap) analysis, we revealed several compounds with potential anti-receptivity activity. Finally, we performed a cross-species comparison against human uterine receptivity from a published dataset. Our study provides a valuable resource for understanding the molecular mechanism underlying uterine receptivity in mice.
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http://dx.doi.org/10.3389/fphys.2019.00125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413723PMC
February 2019

mTOR-S6K1 pathway mediates cytoophidium assembly.

Authors:
Zhe Sun Ji-Long Liu

J Genet Genomics 2019 02 31;46(2):65-74. Epub 2019 Jan 31.

School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China; MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, OX1 3PT, United Kingdom. Electronic address:

CTP synthase (CTPS), the rate-limiting enzyme in de novo CTP biosynthesis, has been demonstrated to assemble into evolutionarily conserved filamentous structures, termed cytoophidia, in Drosophila, bacteria, yeast and mammalian cells. However, the regulation and function of the cytoophidium remain elusive. Here, we provide evidence that the mechanistic target of rapamycin (mTOR) pathway controls cytoophidium assembly in mammalian and Drosophila cells. In mammalian cells, we find that inhibition of mTOR pathway attenuates cytoophidium formation. Moreover, CTPS cytoophidium assembly appears to be dependent on the mTOR complex 1 (mTORC1) mainly. In addition, knockdown of the mTORC1 downstream target S6K1 can inhibit cytoophidium formation, while overexpression of the constitutively active S6K1 reverses mTOR knockdown-induced cytoophidium disassembly. Finally, reducing mTOR protein expression results in a decrease of the length of cytoophidium in Drosophila follicle cells. Therefore, our study connects CTPS cytoophidium formation with the mTOR signaling pathway.
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http://dx.doi.org/10.1016/j.jgg.2018.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459811PMC
February 2019

Cytoophidia respond to nutrient stress in Drosophila.

Exp Cell Res 2019 03 13;376(2):159-167. Epub 2019 Feb 13.

School of Life Science and Technology, ShanghaiTech University, 230 Haike Road, 201210 Shanghai, China; MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3PT, United Kingdom. Electronic address:

CTP synthase (CTPsyn) is a metabolic enzyme essential for the de novo synthesis of CTP the nucleotide. CTPsyn can be compartmented into filamentous structures named cytoophidia. Cytoophidia are conserved in a wide range of species and are highly abundant in Drosophila ovaries. Here we report that cytoophidia elongate upon nutrient deprivation, CTPsyn overexpression or heat shock in Drosophila ovaries. We also show that the curvature of cytoophidia changes during apoptosis. Moreover, cytoophidia can be transported from nurse cells to the oocyte via ring canals. Our study demonstrates that cytoophidia can respond to stress and are very dynamic in Drosophila ovaries.
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http://dx.doi.org/10.1016/j.yexcr.2019.02.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6403103PMC
March 2019