N-terminal residues of human dyskerin are required for interactions with telomerase RNA that prevent RNA degradation

MacNeil, Deanna; Lambert-Lanteigne, Patrick; Autexier, Chantal

doi:10.1093/nar/gkz233

Cited by 21 publications

(27 citation statements)

References 63 publications

(115 reference statements)

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“…Accordingly, placement of the archaeal H/ACA RNP structure into the human telomerase holoenzyme cryo-EM map suggests that telomeropathy mutations within dyskerin cluster at the interface between two dyskerin molecules and the telomerase RNA (26). Consistent with defects in dyskerin-TR binding, patient cells harboring mutations in either the N-terminus or the PUA domain have reduced steady-state levels of TR and a reduction in telomerase activity that can be rescued through overexpression of wild type TR (78)(79)(80). Taken together, pathogenic mutations in dyskerin prevent stable assembly of the H/ACA RNP with TR, thereby impairing TR stability and resulting in a reduction in telomerase activity that is causative of the telomere shortening seen in TBDs.…”

Section: Trmentioning

confidence: 97%

Molecular mechanisms of telomere biology disorders

Grill

Nandakumar

2021

Journal of Biological Chemistry

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Genetic mutations that affect telomerase function or telomere maintenance result in a variety of diseases collectively called telomeropathies. This wide spectrum of disorders, which include dyskeratosis congenita (DC), pulmonary fibrosis (PF) and aplastic anemia (AA), is characterized by severely short telomeres, often resulting in hematopoietic stem cell failure in the most severe cases. Recent work has focused on understanding the molecular basis of these diseases. Mutations in the catalytic TERT and TR subunits of telomerase compromise activity, while others, such as those found in the telomeric protein TPP1, reduce the recruitment of telomerase to the telomere. Mutant telomerase-associated proteins TCAB1 and dyskerin, and the telomerase RNA maturation component PARN, affect the maturation and stability of telomerase. In contrast, disease-associated mutations in either CTC1 or RTEL1 are more broadly associated with telomere replication defects. Yet even with the recent surge in studies decoding the mechanisms underlying these diseases, a significant proportion of DC mutations remain uncharacterized or poorly understood. Here we review the current understanding of the molecular basis of telomeropathies and highlight experimental data that illustrate how genetic mutations drive telomere shortening and dysfunction in these patients. This review connects insights from both clinical and molecular studies to create a comprehensive view of the underlying mechanisms that drive these diseases. Through this, we emphasize recent advances in therapeutics and pin-point disease-associated variants that remain poorly defined in their mechanism of action. Finally, we suggest future avenues of research that will deepen our understanding of telomere biology and telomere-related disease.

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Section: Trmentioning

confidence: 97%

Molecular mechanisms of telomere biology disorders

Grill

Nandakumar

2021

Journal of Biological Chemistry

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“…The plasmid pcDNA3.1-FLAG-dyskerin WT from the lab of Dr. François Dragon was used to generate point mutations or truncations via site directed mutagenesis, as previously described (16,46). Specifically, primers (Table S1) Transfection of siRNA was performed with Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) according to the manufacturer's user protocol.…”

Section: Plasmids Cell Culture and Transfectionsmentioning

confidence: 99%

“…Permeabilized cells were then washed with PBS before blocking in 5% BSA-PBS for 1 hour at room temperature. Cells were probed for FLAG-dyskerin with rabbit anti-FLAG (Sigma-Aldrich FLAG-dyskerin and hTR, and was modified based on a protocol that has been previously described for another hTR-interacting protein (88), as well as used for FLAG-tagged dyskerin (16). Briefly, cells were first lysed in low salt buffer (25mM HEPES-KCl pH7.9, 5mM KCl, 0.5mM MgCl 2 , 0.5% NP-40, 1X protease inhibitor cocktail from Roche, 20mM Nethylmaleimide, and 40U/ml RNAseOut) for 10min on ice.…”

Section: Immunofluorescencementioning

confidence: 99%

“…The mature H/ACA complex assembles with noncoding (nc)RNA members of the H/ACA family, such as small nucleolar (sno)RNAs and small Cajal body specific (sca)RNAs that provide target pseudouridine synthesis specificity to dyskerin, the pseudouridine synthase of the H/ACA complex. The H/ACA motif is also a conserved biogenesis domain in telomerase RNAs of metazoans (11), including the human telomerase RNA (hTR) which relies on the H/ACA complex proteins for stability, processing, and function (12)(13)(14)(15)(16).…”

Section: Introductionmentioning

confidence: 99%

“…We previously demonstrated that dyskerin is a SUMOylation target of SUMO1 and SUMO2/3 isoforms, and that substituting either of two N-terminal X-DC-implicated lysine residues to arginine reduces the proportion of SUMOylated dyskerin in cells, leading to reductions in hTR, reduced telomerase activity, and accelerated telomere shortening (46). We have since shown that these two X-DC residues impact the dyskerin-hTR interaction, though the SUMO dependence of this interaction was not investigated (16).…”

Section: Introductionmentioning

confidence: 99%

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SUMOylation- and GAR1-dependent regulation of dyskerin nuclear and subnuclear localization

MacNeil

Lambert-Lanteigne

Qin

et al. 2020

Preprint

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SummaryDyskerin, a telomerase-associated protein and H/ACA ribonucleoprotein complex component plays an essential role in human telomerase assembly and activity. The nuclear and subnuclear compartmentalization of dyskerin and the H/ACA complex is an important though incompletely understood aspect of H/ACA ribonucleoprotein function. The posttranslational modification, SUMOylation, targets a wide variety of proteins, including numerous RNA-binding proteins, and most identified targets reported to date localize to the nucleus. Four SUMOylation sites were previously identified in the C-terminal Nuclear/Nucleolar Localization Signal (N/NoLS) of dyskerin, each located within one of two lysine-rich clusters. We found that a cytoplasmic localized C-terminal truncation variant of dyskerin lacking most of the C-terminal N/NoLS and both lysine-rich clusters represents an under-SUMOylated variant of dyskerin compared to wildtype dyskerin. We demonstrate that mimicking constitutive SUMOylation of dyskerin using a SUMO3-fusion construct can drive nuclear accumulation of this variant, and that the SUMO site K467 in this N/NoLS is particularly important for the subnuclear localization of dyskerin to the nucleolus in a mature H/ACA complex assembly- and SUMO-dependent manner. We also characterize a novel SUMO-interacting motif in the mature H/ACA complex component GAR1 that mediates the interaction between dyskerin and GAR1. Mislocalization of dyskerin, either in the cytoplasm or excluded from the nucleolus, disrupts dyskerin function and leads to reduced interaction of dyskerin with the telomerase RNA. These data indicate a role for dyskerin C-terminal N/NoLS SUMOylation in regulating the nuclear and subnuclear localization of dyskerin, which is essential for dyskerin function as both a telomerase-associated protein and as an H/ACA ribonucleoprotein involved in rRNA and snRNA biogenesis.

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Dual Inhibition of DKC1 and MEK1/2 Synergistically Restrains the Growth of Colorectal Cancer Cells

et al. 2021

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Colorectal cancer, one of the most commonly diagnosed cancers worldwide, is often accompanied by uncontrolled proliferation of tumor cells. Dyskerin pseudouridine synthase 1 (DKC1), screened using the genome-wide RNAi strategy, is a previously unidentified key regulator that promotes colorectal cancer cell proliferation. Enforced expression of DKC1, but not its catalytically inactive mutant D125A, accelerates cell growth in vitro and in vivo. DKC1 knockdown or its inhibitor pyrazofurin attenuates cell proliferation. Proteomics, RNA immunoprecipitation (RIP)-seq, and RNA decay analyses reveal that DKC1 binds to and stabilizes the mRNA of several ribosomal proteins (RPs), including RPL10A, RPL22L1, RPL34, and RPS3. DKC1 depletion significantly accelerates mRNA decay of these RPs, which mediates the oncogenic function of DKC1. Interestingly, these DKC1-regulated RPs also interact with HRAS and suppress the RAS/RAF/MEK/ERK pathway. Pyrazofurin and trametinib combination synergistically restrains colorectal cancer cell growth in vitro and in vivo. Furthermore, DKC1 is markedly upregulated in colorectal cancer tissues compared to adjacent normal tissues. Colorectal cancer patients with higher DKC1 expression has consistently poorer overall survival and progression-free survival outcomes. Taken together, these data suggest that DKC1 is an essential gene and candidate therapeutic target for colorectal cancer.

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N-terminal residues of human dyskerin are required for interactions with telomerase RNA that prevent RNA degradation

Cited by 21 publications

References 63 publications

Molecular mechanisms of telomere biology disorders

Molecular mechanisms of telomere biology disorders

SUMOylation- and GAR1-dependent regulation of dyskerin nuclear and subnuclear localization

Dual Inhibition of DKC1 and MEK1/2 Synergistically Restrains the Growth of Colorectal Cancer Cells

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