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  1. Article ; Online: Global mapping of RNA G-quadruplexes (G4-RNAs) using G4RP-seq.

    Yang, Sunny Y / Monchaud, David / Wong, Judy M Y

    Nature protocols

    2022  Volume 17, Issue 3, Page(s) 870–889

    Abstract: Guanine-rich RNAs can fold into four-stranded structures, termed G-quadruplexes (G4-RNAs), and participate in a wide range of biological processes. Here we describe in detail a G4-RNA-specific precipitation (G4RP) protocol, which enables the ... ...

    Abstract Guanine-rich RNAs can fold into four-stranded structures, termed G-quadruplexes (G4-RNAs), and participate in a wide range of biological processes. Here we describe in detail a G4-RNA-specific precipitation (G4RP) protocol, which enables the transcriptomic profiling of G4-RNAs. The G4RP protocol consists of a chemical cross-linking step, followed by affinity capture with a G4-specific probe, BioTASQ. G4RP can be coupled with sequencing to capture a comprehensive global snapshot of folded G4-RNAs. This method can also be used to profile induced changes (i.e., through G4 ligand treatments) within the G4-RNA transcriptome. The entire protocol can be completed in 1-2 weeks and can be scaled up or down depending on the specific experimental goals. In addition to the protocol details, we also provide here a guide for optimization in different laboratory setups.
    MeSH term(s) G-Quadruplexes ; Ligands ; RNA/chemistry ; RNA/genetics ; Transcriptome
    Chemical Substances Ligands ; RNA (63231-63-0)
    Language English
    Publishing date 2022-02-09
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Review
    ZDB-ID 2244966-8
    ISSN 1750-2799 ; 1754-2189
    ISSN (online) 1750-2799
    ISSN 1754-2189
    DOI 10.1038/s41596-021-00671-6
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  2. Article: Telomerase Biogenesis and Activities from the Perspective of Its Direct Interacting Partners.

    Nguyen, Kathryn T T T / Wong, Judy M Y

    Cancers

    2020  Volume 12, Issue 6

    Abstract: Telomerase reverse transcriptase (TERT)-the catalytic subunit of telomerase-is reactivated in up to 90% of all human cancers. TERT is observed in heterogenous populations of protein complexes, which are dynamically regulated in a cell type- and cell ... ...

    Abstract Telomerase reverse transcriptase (TERT)-the catalytic subunit of telomerase-is reactivated in up to 90% of all human cancers. TERT is observed in heterogenous populations of protein complexes, which are dynamically regulated in a cell type- and cell cycle-specific manner. Over the past two decades, in vitro protein-protein interaction detection methods have discovered a number of endogenous TERT binding partners in human cells that are responsible for the biogenesis and functionalization of the telomerase holoenzyme, including the processes of TERT trafficking between subcellular compartments, assembly into telomerase, and catalytic action at telomeres. Additionally, TERT have been found to interact with protein species with no known telomeric functions, suggesting that these complexes may contribute to non-canonical activities of TERT. Here, we survey TERT direct binding partners and discuss their contributions to TERT biogenesis and functions. The goal is to review the comprehensive spectrum of TERT pro-malignant activities, both telomeric and non-telomeric, which may explain the prevalence of its upregulation in cancer.
    Language English
    Publishing date 2020-06-24
    Publishing country Switzerland
    Document type Journal Article ; Review
    ZDB-ID 2527080-1
    ISSN 2072-6694
    ISSN 2072-6694
    DOI 10.3390/cancers12061679
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  3. Article ; Online: Non-canonical Functions of Telomerase Reverse Transcriptase: Emerging Roles and Biological Relevance.

    Thompson, Connor A H / Wong, Judy M Y

    Current topics in medicinal chemistry

    2020  Volume 20, Issue 6, Page(s) 498–507

    Abstract: Increasing evidence from research on telomerase suggests that in addition to its catalytic telomere repeat synthesis activity, telomerase may have other biologically important functions. The canonical roles of telomerase are at the telomere ends where ... ...

    Abstract Increasing evidence from research on telomerase suggests that in addition to its catalytic telomere repeat synthesis activity, telomerase may have other biologically important functions. The canonical roles of telomerase are at the telomere ends where they elongate telomeres and maintain genomic stability and cellular lifespan. The catalytic protein component Telomerase Reverse Transcriptase (TERT) is preferentially expressed at high levels in cancer cells despite the existence of an alternative mechanism for telomere maintenance (alternative lengthening of telomeres or ALT). TERT is also expressed at higher levels than necessary for maintaining functional telomere length, suggesting other possible adaptive functions. Emerging non-canonical roles of TERT include regulation of non-telomeric DNA damage responses, promotion of cell growth and proliferation, acceleration of cell cycle kinetics, and control of mitochondrial integrity following oxidative stress. Non-canonical activities of TERT primarily show cellular protective effects, and nuclear TERT has been shown to protect against cell death following double-stranded DNA damage, independent of its role in telomere length maintenance. TERT has been suggested to act as a chromatin modulator and participate in the transcriptional regulation of gene expression. TERT has also been reported to regulate transcript levels through an RNA-dependent RNA Polymerase (RdRP) activity and produce siRNAs in a Dicer-dependent manner. At the mitochondria, TERT is suggested to protect against oxidative stress-induced mtDNA damage and promote mitochondrial integrity. These extra-telomeric functions of TERT may be advantageous in the context of increased proliferation and metabolic stress often found in rapidly-dividing cancer cells. Understanding the spectrum of non-canonical functions of telomerase may have important implications for the rational design of anti-cancer chemotherapeutic drugs.
    MeSH term(s) Humans ; Telomerase/genetics ; Telomerase/metabolism
    Chemical Substances TERT protein, human (EC 2.7.7.49) ; Telomerase (EC 2.7.7.49)
    Language English
    Publishing date 2020-02-06
    Publishing country United Arab Emirates
    Document type Journal Article ; Review
    ZDB-ID 2064823-6
    ISSN 1873-4294 ; 1568-0266
    ISSN (online) 1873-4294
    ISSN 1568-0266
    DOI 10.2174/1568026620666200131125110
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  4. Article ; Online: G-quadruplexes mark alternative lengthening of telomeres.

    Yang, Sunny Y / Chang, Emily Y C / Lim, Joanne / Kwan, Harwood H / Monchaud, David / Yip, Stephen / Stirling, Peter C / Wong, Judy M Y

    NAR cancer

    2021  Volume 3, Issue 3, Page(s) zcab031

    Abstract: About 10-15% of all human cancer cells employ a telomerase-independent recombination-based telomere maintenance method, known as alternative lengthening of telomere (ALT), of which the full mechanism remains incompletely understood. While implicated in ... ...

    Abstract About 10-15% of all human cancer cells employ a telomerase-independent recombination-based telomere maintenance method, known as alternative lengthening of telomere (ALT), of which the full mechanism remains incompletely understood. While implicated in previous studies as the initiating signals for ALT telomere repair, the prevalence of non-canonical nucleic acid structures in ALT cancers remains unclear. Extending earlier reports, we observe higher levels of DNA/RNA hybrids (R-loops) in ALT-positive (ALT+) compared to telomerase-positive (TERT+) cells. Strikingly, we observe even more pronounced differences for an associated four-stranded nucleic acid structure, G-quadruplex (G4). G4 signals are found at the telomere and are broadly associated with telomere length and accompanied by DNA damage markers. We establish an interdependent relationship between ALT-associated G4s and R-loops and confirm that these two structures can be spatially linked into unique structures, G-loops, at the telomere. Additionally, stabilization of G4s and R-loops cooperatively enhances ALT-activity. However, co-stabilization at higher doses resulted in cytotoxicity in a synergistic manner. Nuclear G4 signals are significantly and reproducibly different between ALT+ and TERT+ low-grade glioma tumours. Together, we present G4 as a novel hallmark of ALT cancers with potential future applications as a convenient biomarker for identifying ALT+ tumours and as therapeutic targets.
    Language English
    Publishing date 2021-07-21
    Publishing country England
    Document type Journal Article
    ISSN 2632-8674
    ISSN (online) 2632-8674
    DOI 10.1093/narcan/zcab031
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  5. Article ; Online: Direct visualization of both DNA and RNA quadruplexes in human cells via an uncommon spectroscopic method.

    Laguerre, Aurélien / Wong, Judy M Y / Monchaud, David

    Scientific reports

    2016  Volume 6, Page(s) 32141

    Abstract: Guanine-rich DNA or RNA sequences can fold into higher-order, four-stranded structures termed quadruplexes that are suspected to play pivotal roles in cellular mechanisms including the control of the genome integrity and gene expression. However, the ... ...

    Abstract Guanine-rich DNA or RNA sequences can fold into higher-order, four-stranded structures termed quadruplexes that are suspected to play pivotal roles in cellular mechanisms including the control of the genome integrity and gene expression. However, the biological relevance of quadruplexes is still a matter of debate owing to the paucity of unbiased evidences of their existence in cells. Recent reports on quadruplex-specific antibodies and small-molecule fluorescent probes help dispel reservations and accumulating evidences now pointing towards the cellular relevance of quadruplexes. To better assess and comprehend their biology, developing new versatile tools to detect both DNA and RNA quadruplexes in cells is essential. We report here a smart fluorescent probe that allows for the simple detection of quadruplexes thanks to an uncommon spectroscopic mechanism known as the red-edge effect (REE). We demonstrate that this effect could open avenues to greatly enhance the ability to visualize both DNA and RNA quadruplexes in human cells, using simple protocols and fluorescence detection facilities.
    Language English
    Publishing date 2016-08-18
    Publishing country England
    Document type Journal Article
    ZDB-ID 2615211-3
    ISSN 2045-2322 ; 2045-2322
    ISSN (online) 2045-2322
    ISSN 2045-2322
    DOI 10.1038/srep32141
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  6. Article: Quantification of Pseudouridine Levels in Cellular RNA Pools with a Modified HPLC-UV Assay.

    Xu, Jialin / Gu, Alice Y / Thumati, Naresh R / Wong, Judy M Y

    Genes

    2017  Volume 8, Issue 9

    Abstract: Pseudouridine (Ψ) is the most abundant post-transcriptionally modified ribonucleoside. Different Ψ modifications correlate with stress responses and are postulated to coordinate the distinct biological responses to a diverse panel of cellular stresses. ... ...

    Abstract Pseudouridine (Ψ) is the most abundant post-transcriptionally modified ribonucleoside. Different Ψ modifications correlate with stress responses and are postulated to coordinate the distinct biological responses to a diverse panel of cellular stresses. With the help of different guide RNAs, the dyskerin complex pseudouridylates ribosomal RNA, small nuclear RNA and selective messenger RNAs. To monitor Ψ levels quantitatively, a previously reported high performance liquid chromatography method coupled with ultraviolet detection (HPLC-UV) was modified to determine total Ψ levels in different cellular RNA fractions. Our method was validated to be accurate and precise within the linear range of 0.06-15.36 pmol/μL and to have absolute Ψ quantification levels as low as 3.07 pmol. Using our optimized HPLC assay, we found that 1.20% and 1.94% of all ribonucleosides in nuclear-enriched RNA and small non-coding RNA pools from the HEK293 cell line, and 1.77% and 0.98% of ribonucleosides in 18S and 28S rRNA isolated from the HeLa cell line, were pseudouridylated. Upon knockdown of dyskerin expression, a consistent and significant reduction in total Ψ levels in nuclear-enriched RNA pools was observed. Our assay provides a fast and accurate quantification method to measure changes in Ψ levels of different RNA pools without sample derivatization.
    Language English
    Publishing date 2017-09-05
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2527218-4
    ISSN 2073-4425
    ISSN 2073-4425
    DOI 10.3390/genes8090219
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  7. Article ; Online: Cellular Detection of G-Quadruplexes by Optical Imaging Methods.

    Amor, Souheila / Yang, Sunny Y / Wong, Judy M Y / Monchaud, David

    Current protocols in cell biology

    2017  Volume 76, Page(s) 4.33.1–4.33.19

    Abstract: G-quadruplexes (G4s) are higher-order nucleic acid structures that fold from guanine (G)-rich DNA and RNA strands. This field of research gains traction as a major chemical biology area since it aims at uncovering many key cellular mechanisms in which ... ...

    Abstract G-quadruplexes (G4s) are higher-order nucleic acid structures that fold from guanine (G)-rich DNA and RNA strands. This field of research gains traction as a major chemical biology area since it aims at uncovering many key cellular mechanisms in which quadruplexes are involved. The wealth of knowledge acquired over the past three decades strongly supports pivotal roles of G4 in the regulation of gene expression at both transcriptional (DNA quadruplexes) and translational levels (RNA quadruplexes). Recent biochemical discoveries uncovered myriad of additional G4 actions: from chromosomal stability to the firing of replication origins, from telomere homeostasis to functional dysregulations underlying genetic diseases (including cancers and neurodegeneration). Here, we listed a repertoire of protocols that we have developed over the past years to visualize quadruplexes in cells. These achievements were made possible thanks to the discovery of a novel family of versatile quadruplex-selective fluorophores, the twice-as-smart quadruplex ligands named TASQ (for template-assembled synthetic G-quartet). The versatility of this probe allows for multiple imaging techniques in both fixed and live cells, including the use of the multiphoton microscopy, confocal microscopy, and real-time fluorescent image collection. © 2017 by John Wiley & Sons, Inc.
    MeSH term(s) Antibodies/metabolism ; Cell Survival ; Fluorescent Dyes/chemistry ; G-Quadruplexes ; Humans ; MCF-7 Cells ; Optical Imaging/methods ; Staining and Labeling
    Chemical Substances Antibodies ; Fluorescent Dyes
    Language English
    Publishing date 2017-09-01
    Publishing country United States
    Document type Journal Article
    ISSN 1934-2616
    ISSN (online) 1934-2616
    DOI 10.1002/cpcb.29
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  8. Article ; Online: Dolutegravir-containing HIV therapy reversibly alters mitochondrial health and morphology in cultured human fibroblasts and peripheral blood mononuclear cells.

    Ajaykumar, Abhinav / Caloren, Loïc C / Povshedna, Tetiana / Hsieh, Anthony Y Y / Zakaria, Aya / Cai, Renying / Smith, Marie-Soleil R / Thompson, Connor A H / Becquart, Pierre / Uday, Prakruti / Pattanshetti, Rutuja / Quandt, Jacqueline A / Wong, Judy M Y / Côté, Hélène C F

    AIDS (London, England)

    2022  Volume 37, Issue 1, Page(s) 19–32

    Abstract: Objectives: Given the success of combination antiretroviral therapy (cART) in treating HIV viremia, drug toxicity remains an area of interest in HIV research. Despite newer integrase strand transfer inhibitors (InSTIs), such as dolutegravir (DTG) and ... ...

    Abstract Objectives: Given the success of combination antiretroviral therapy (cART) in treating HIV viremia, drug toxicity remains an area of interest in HIV research. Despite newer integrase strand transfer inhibitors (InSTIs), such as dolutegravir (DTG) and raltegravir (RAL), having excellent clinical tolerance, there is emerging evidence of off-target effects and toxicities. Although limited in number, recent reports have highlighted the vulnerability of mitochondria to these toxicities. The aim of the present study is to quantify changes in cellular and mitochondrial health following exposure to current cART regimens at pharmacological concentrations. A secondary objective is to determine whether any cART-associated toxicities would be modulated by human telomerase reverse transcriptase (hTERT).
    Methods: We longitudinally evaluated markers of cellular (cell count, apoptosis), and mitochondrial health [mitochondrial reactive oxygen species (mtROS), membrane potential (MMP) and mass (mtMass)] by flow cytometry in WI-38 human fibroblast with differing hTERT expression/localization and peripheral blood mononuclear cells. This was done after 9 days of exposure to, and 6 days following the removal of, seven current cART regimens, including three that contained DTG. Mitochondrial morphology was assessed by florescence microscopy and quantified using a recently developed deep learning-based pipeline.
    Results: Exposure to DTG-containing regimens increased apoptosis, mtROS, mtMass, induced fragmented mitochondrial networks compared with non-DTG-containing regimens, including a RAL-based combination. These effects were unmodulated by telomerase expression. All effects were fully reversible following removal of drug pressure.
    Conclusion: Taken together, our observations indicate that DTG-containing regimens negatively impact cellular and mitochondrial health and may be more toxic to mitochondria, even among the generally well tolerated InSTI-based cART.
    MeSH term(s) Humans ; Leukocytes, Mononuclear ; HIV Infections/drug therapy ; Immune Tolerance ; Fibroblasts
    Language English
    Publishing date 2022-08-23
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 639076-6
    ISSN 1473-5571 ; 0269-9370 ; 1350-2840
    ISSN (online) 1473-5571
    ISSN 0269-9370 ; 1350-2840
    DOI 10.1097/QAD.0000000000003369
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    Zs.A 2228: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (1.OG)
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  9. Article ; Online: Mild catalytic defects of tert rs61748181 polymorphism affect the clinical presentation of chronic obstructive pulmonary disease.

    Xu, Jialin / de Oliveira, Diego Madureira / Trudeau, Matthew A / Yang, Yang / Chin, Jessica J Y / Sin, Don D / Sandford, Andrew J / Wong, Judy M Y

    Scientific reports

    2021  Volume 11, Issue 1, Page(s) 4333

    Abstract: Chronic obstructive pulmonary disease (COPD) is a disorder of accelerated lung aging. Multiple pieces of evidence support that the aging biomarker short telomeres, which can be caused by mutations in telomerase reverse transcriptase (TERT), contribute to ...

    Abstract Chronic obstructive pulmonary disease (COPD) is a disorder of accelerated lung aging. Multiple pieces of evidence support that the aging biomarker short telomeres, which can be caused by mutations in telomerase reverse transcriptase (TERT), contribute to COPD pathogenesis. We hypothesized that short telomere risk-associated single nucleotide polymorphisms (SNPs) in TERT, while not able to drive COPD development, nonetheless modify the disease presentation. We set out to test the SNP carrying status in a longitudinal study of smokers with COPD and found that rapid decline of FEV1 in lung function was associated with the minor allele of rs61748181 (adjusted odds ratio 2.49, p = 0.038). Biochemical evaluation of ex vivo engineered human cell models revealed that primary cells expressing the minor allele of rs61748181 had suboptimal telomere length maintenance due to reduced telomerase catalytic activity, despite having comparable cell growth kinetics as WT-TERT expressing cells. This ex vivo observation translated clinically in that shorter telomeres were found in minor allele carriers in a sub-population of COPD patients with non-declining lung function, over the 5-year period of the longitudinal study. Collectively, our data suggest that functional TERT SNPs with mild catalytic defects are nonetheless implicated in the clinical presentation of COPD.
    MeSH term(s) Adult ; Aged ; Alleles ; Disease Progression ; Enzyme Activation ; Female ; Gene Frequency ; Genetic Association Studies ; Genetic Predisposition to Disease ; Genotype ; Humans ; Male ; Middle Aged ; Phenotype ; Polymorphism, Single Nucleotide ; Pulmonary Disease, Chronic Obstructive/diagnosis ; Pulmonary Disease, Chronic Obstructive/genetics ; Pulmonary Disease, Chronic Obstructive/metabolism ; Respiratory Function Tests ; Severity of Illness Index ; Telomerase/genetics ; Telomerase/metabolism ; Telomere Homeostasis ; Telomere Shortening
    Chemical Substances TERT protein, human (EC 2.7.7.49) ; Telomerase (EC 2.7.7.49)
    Language English
    Publishing date 2021-02-22
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2615211-3
    ISSN 2045-2322 ; 2045-2322
    ISSN (online) 2045-2322
    ISSN 2045-2322
    DOI 10.1038/s41598-021-83686-z
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  10. Article ; Online: Small-molecule affinity capture of DNA/RNA quadruplexes and their identification in vitro and in vivo through the G4RP protocol.

    Renard, Isaline / Grandmougin, Michael / Roux, Apolline / Yang, Sunny Y / Lejault, Pauline / Pirrotta, Marc / Wong, Judy M Y / Monchaud, David

    Nucleic acids research

    2019  Volume 47, Issue 11, Page(s) 5502–5510

    Abstract: Guanine-rich DNA and RNA sequences can fold into higher-order structures known as G-quadruplexes (or G4-DNA and G4-RNA, respectively). The prevalence of the G4 landscapes in the human genome, transcriptome and ncRNAome (non-coding RNA), collectively ... ...

    Abstract Guanine-rich DNA and RNA sequences can fold into higher-order structures known as G-quadruplexes (or G4-DNA and G4-RNA, respectively). The prevalence of the G4 landscapes in the human genome, transcriptome and ncRNAome (non-coding RNA), collectively known as G4ome, is strongly suggestive of biological relevance at multiple levels (gene expression, replication). Small-molecules can be used to track G4s in living cells for the functional characterization of G4s in both normal and disease-associated changes in cell biology. Here, we describe biotinylated biomimetic ligands referred to as BioTASQ and their use as molecular tools that allow for isolating G4s through affinity pull-down protocols. We demonstrate the general applicability of the method by purifying biologically relevant G4s from nucleic acid mixtures in vitro and from human cells through the G4RP-RT-qPCR protocol. Overall, the results presented here represent a step towards the optimization of G4-RNAs identification, a key step in studying G4s in cell biology and human diseases.
    MeSH term(s) Biotinylation ; DNA/chemistry ; G-Quadruplexes ; Genome, Human/genetics ; Humans ; Ligands ; MCF-7 Cells ; RNA/chemistry ; Reverse Transcriptase Polymerase Chain Reaction ; Transcriptome/genetics
    Chemical Substances Ligands ; RNA (63231-63-0) ; DNA (9007-49-2)
    Language English
    Publishing date 2019-03-19
    Publishing country England
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 186809-3
    ISSN 1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
    ISSN (online) 1362-4962 ; 1362-4954
    ISSN 0301-5610 ; 0305-1048
    DOI 10.1093/nar/gkz215
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