The Experts below are selected from a list of 591 Experts worldwide ranked by ideXlab platform
Tetsuro Hirose - One of the best experts on this subject based on the ideXlab platform.
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triblock copolymer micelle model of spherical Paraspeckles
bioRxiv, 2020Co-Authors: Tetsuya Yamamoto, Toshio Yamazaki, Tetsuro HiroseAbstract:Paraspeckles are nuclear bodies composed of architectural RNA (arcRNA) and RNA-binding proteins. In the wild type, the blocks at the two terminal regions of arcRNAs compose the shell of Paraspeckles and the middle region between the two terminal blocks composes the core, analogous to micelles of ABC triblock copolymers. We here use an extension of the theory of polymer micelles to predict the structure and size of Paraspeckles as one decreases the length of one of the terminal blocks of arcRNA by CRISPR/Cas9, assuming that Paraspeckles are spherical. Our theory predicts that when the length of the edited terminal blocks is larger than a critical value, Paraspeckles show discontinuous transitions between the structure in which all the edited terminal blocks are localized in the shell and the structure in which all the edited terminal blocks are localized in the core at a threshold value of the transcription rate of arcRNA. In contrast, when the length of the edited terminal blocks is smaller than the critical value, the population of edited terminal blocks in the shell decreases continuously as one increases the transcription rate of arcRNA. The size of Paraspeckles increases as one decreases the length of the edited terminal blocks. These predictions are consistent with our experiments.
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molecular dissection of nuclear Paraspeckles towards understanding the emerging world of the rnp milieu
Open Biology, 2018Co-Authors: Shinichi Nakagawa, Tomohiro Yamazaki, Tetsuro HiroseAbstract:Paraspeckles are nuclear bodies built on an architectural long noncoding RNA, NEAT1, and a series of studies have revealed their molecular components, fine internal structures and cellular and phys...
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Paraspeckles modulate the intranuclear distribution of Paraspeckle associated ctn rna
Scientific Reports, 2016Co-Authors: Aparna Anantharaman, Tetsuro Hirose, Shinichi Nakagawa, Mahdieh Jadaliha, Vidisha Tripathi, Michael F Jantsch, Supriya G Prasanth, Kannanganattu V PrasanthAbstract:Paraspeckles are sub-nuclear domains that are nucleated by long noncoding RNA Neat1. While interaction of protein components of Paraspeckles and Neat1 is understood, there is limited information on the interaction of non-structural RNA components with Paraspeckles. Here, by varying Paraspeckle number and size, we investigate how Paraspeckles influence the nuclear organization of their non-structural RNA component Ctn RNA. Our results show that Ctn RNA remains nuclear-retained in the absence of intact Paraspeckles, suggesting that they do not regulate nuclear retention of Ctn RNA. In the absence of Neat1, Ctn RNA continues to interact with Paraspeckle protein NonO to form residual nuclear foci. In addition, in the absence of Neat1-nucleated Paraspeckles, a subset of Ctn RNA localizes to the perinucleolar regions. Concomitant with increase in number of Paraspeckles, transcriptional reactivation resulted in increased number of Paraspeckle-localized Ctn RNA foci. Similar to Neat1, proteasome inhibition altered the localization of Ctn RNA, where it formed enlarged Paraspeckle-like foci. Super-resolution structured illumination microscopic analyses revealed that in Paraspeckles, Ctn RNA partially co-localized with Neat1, and displayed a more heterogeneous intra-Paraspeckle localization. Collectively, these results show that while Paraspeckles do not influence nuclear retention of Ctn RNA, they modulate its intranuclear compartmentalization.
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structural super resolution microscopy analysis of Paraspeckle nuclear body organization
Journal of Cell Biology, 2016Co-Authors: Jason A West, Tetsuro Hirose, Mari Mito, Satoshi Kurosaka, Toru Takumi, Chiharu Tanegashima, Takeshi Chujo, Kaori Yanaka, Robert E Kingston, Charles S BondAbstract:Paraspeckles are nuclear bodies built on the long noncoding RNA Neat1, which regulates a variety of physiological processes including cancer progression and corpus luteum formation. To obtain further insight into the molecular basis of the function of Paraspeckles, we performed fine structural analyses of these nuclear bodies using structural illumination microscopy. Notably, Paraspeckle proteins are found within different layers along the radially arranged bundles of Neat1 transcripts, forming a characteristic core-shell spheroidal structure. In cells lacking the RNA binding protein Fus, Paraspeckle spheroids are disassembled into smaller particles containing Neat1, which are diffusely distributed in the nucleoplasm. Sequencing analysis of RNAs purified from Paraspeckles revealed that AG-rich transcripts associate with Neat1, which are distributed along the shell of the Paraspeckle spheroids. We propose that Paraspeckles sequester core components inside the spheroids, whereas the outer surface associates with other components in the nucleoplasm to fulfill their function.
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the building process of the functional Paraspeckle with long non coding rnas
Frontiers in Bioscience, 2015Co-Authors: Tomohiro Yamazaki, Tetsuro HiroseAbstract:Nuclei of mammalian cells are highly organized and composed of distinct subnuclear structures termed nuclear bodies. Paraspeckles are subnuclear structures that form around the long non-coding RNA (lncRNA) nuclear Paraspeckle assembly transcript 1 (NEAT1) together with numerous RNA-binding proteins, many of which contain an RNA-binding domain and a prion-like domain and are related to specific neurodegenerative diseases. Paraspeckle formation proceeds in conjunction with NEAT1 lncRNA biogenesis and involves the cooperation of multiple Paraspeckle-localized RNA-binding proteins. NEAT1 lncRNA likely sequesters these RNA-binding proteins in Paraspeckle structures, which act as molecular sponges at which the nucleoplasmic activities of the sequestered proteins are modulated. This review presents, firstly, current knowledge regarding factors involved in the formation and function of Paraspeckles. Secondly, the intracellular functions of all identified Paraspeckle proteins, which are potentially controlled through their sequestration in Paraspeckles, are categorized. Thirdly, recently identified nuclear bodies containing putative architectural lncRNAs are described and similarities among the architectures of lncRNA-dependent nuclear bodies are discussed.
Archa H. Fox - One of the best experts on this subject based on the ideXlab platform.
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the role of g quadruplex dna in Paraspeckle formation in cancer
Biochimie, 2021Co-Authors: Uditi Bhatt, Charles S Bond, Amy L Kretzmann, Aurore Guedin, Simon Kobelke, Cameron W Evans, Laurence H Hurley, Jeanlouis Mergny, Swaminathan K Iyer, Archa H. FoxAbstract:Paraspeckles are RNA-protein structures within the nucleus of mammalian cells, capable of orchestrating various biochemical processes. An overexpression of the architectural component of Paraspeckles, a long non-coding RNA called NEAT1 (Nuclear Enriched Abundant Transcript 1), has been linked to a variety of cancers and is often associated with poor patient prognosis. Thus, there is an accumulating interest in the role of Paraspeckles in carcinogenesis, however there is a limited understanding of how NEAT1 expression is regulated. Here, we demonstrate that both nuclear G-quadruplex (G4) and Paraspeckle formation are significantly increased in a human breast cancer cell line compared to non-tumorigenic breast cells. Moreover, we identified and characterized G4-forming sequences within the NEAT1 promoter and demonstrate stabilization of G4 DNA with a G4-stabilizing small molecule results in a significant alteration in both Paraspeckle formation and NEAT1 expression. This G4-mediated alteration of NEAT1 at both the transcriptional and post-transcriptional levels was evident in U2OS osteosarcoma cells, MCF-7 breast adenocarcinoma and MDA-MB-231 triple negative breast cancer cells.
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Paraspeckle nuclear condensates global sensors of cell stress
BioEssays, 2021Co-Authors: Finn Mccluggage, Archa H. FoxAbstract:Paraspeckles are nuclear condensates, or membranelees organelles, that are built on the long noncoding RNA, NEAT1, and have been linked to many diseases. Although originally described as constitutive structures, here, in reviewing this field, we develop the hypothesis that cells increase Paraspeckle abundance as part of a general stress response, to aid pro-survival pathways. Paraspeckles increase in many scenarios: when cells transform from one state to another, become infected with viruses and bacteria, begin to degenerate, under inflammation, in aging, and in cancer. Cells increase Paraspeckles by increasing transcription of NEAT1 and adjusting its RNA processing. These increases in NEAT1 are driven by numerous stress-sensing signaling pathways, including signaling to mitochondria and stress granules, revealing crosstalk between the cytoplasm and nucleoplasm in the stress response. Thus, Paraspeckles are an important piece of the puzzle in cellular homeostasis, and could be considered RNA-scaffolded nuclear equivalents of dynamic stress-induced structures that form in the cytoplasm. We speculate that, in general, cells rely on phase-separated Paraspeckles to transiently tweak gene regulation in times of cellular flux.
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NEAT1 polyA-modulating antisense oligonucleotides reveal opposing functions for both long non-coding RNA isoforms in neuroblastoma
Cellular and Molecular Life Sciences, 2020Co-Authors: Alina Naveed, Alysia Hubbard, Tao Liu, Steve D Wilton, S Fletcher, Jack A. Cooper, Jingwei Chen, Archa H. FoxAbstract:Many long non-coding RNAs (lncRNA) are highly dysregulated in cancer and are emerging as therapeutic targets. One example is NEAT1, which consists of two overlapping lncRNA isoforms, NEAT1_1 (3.7 kb) and NEAT1_2 (23 kb), that are functionally distinct. The longer NEAT1_2 is responsible for scaffolding gene-regulatory nuclear bodies termed Paraspeckles, whereas NEAT1_1 is involved in Paraspeckle-independent function. The NEAT1 isoform ratio is dependent on the efficient cleavage and polyadenylation of NEAT1_1 at the expense of NEAT1_2. Here, we developed a targeted antisense oligonucleotide (ASO) approach to sterically block NEAT1_1 polyadenylation processing, achieving upregulation of NEAT1_2 and abundant Paraspeckles. We have applied these ASOs to cells of the heterogeneous infant cancer, neuroblastoma, as we found higher NEAT1_1:NEAT1_2 ratio and lack of Paraspeckles in high-risk neuroblastoma cells. These ASOs decrease NEAT1_1 levels, increase NEAT1_2/Paraspeckles and concomitantly reduce cell viability in high-risk neuroblastoma specifically. In contrast, overexpression of NEAT1_1 has the opposite effect, increasing cell proliferation. Transcriptomic analyses of high-risk neuroblastoma cells with altered NEAT1 ratios and increased Paraspeckle abundance after ASO treatment showed an upregulation of differentiation pathways, as opposed to the usual aggressive neuroblastic phenotype. Thus, we have developed potential anti-cancer ASO drugs that can transiently increase growth-inhibiting NEAT1_2 RNA at the expense of growth-promoting NEAT1_1 RNA. These ASOs, unlike others that degrade lncRNAs, provide insights into the importance of altering lncRNA polyadenylation events to suppress tumorigenesis as a strategy to combat cancer.
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polya modulating antisense oligonucleotides reveal opposing functions for long non coding rna neat1 isoforms in neuroblastoma
bioRxiv, 2020Co-Authors: Alina Naveed, John Cooper, Alysia Hubbard, Tao Liu, Steve D Wilton, S Fletcher, Archa H. FoxAbstract:Many long non-coding RNAs (lncRNA) are highly dysregulated in cancer and are emerging as therapeutic targets. One example is NEAT1, which consists of two overlapping lncRNA isoforms, NEAT1_1 (3.7kb) and NEAT1_2 (23kb), that are functionally distinct. The longer NEAT1_2 is responsible for scaffolding gene-regulatory nuclear bodies termed Paraspeckles, whereas NEAT1_1 is involved in Paraspeckle-independent function. The NEAT1 isoform ratio is dependent on the efficient cleavage and polyadenylation of NEAT1_1 at the expense of NEAT1_2. Here we developed a targeted antisense oligonucleotide (ASO) approach to sterically block NEAT1_1 polyadenylation processing, achieving upregulation of NEAT1_2 and abundant Paraspeckles. We have applied these ASOs to cells of the heterogeneous infant cancer, neuroblastoma, as we found higher NEAT1_1:NEAT1_2 ratio and lack of Paraspeckles in high-risk neuroblastoma cells. These ASOs decrease NEAT1_1 levels, increase NEAT1_2/Paraspeckles and concomitantly reduce cell viability in high-risk neuroblastoma specifically. In contrast, overexpression of NEAT1_1 has the opposite effect, increasing cell-proliferation. Transcriptomic analyses of high-risk neuroblastoma cells with altered NEAT1 ratios and increased Paraspeckle abundance after ASO treatment showed an upregulation of differentiation pathways, as opposed to the usual aggressive neuroblastic phenotype. Thus, we have developed potential anti-cancer ASO drugs that can transiently increase growth-inhibiting NEAT1_2 RNA at the expense of growth-promoting NEAT1_1 RNA. These ASOs, unlike others that degrade lncRNAs, provide insights into the importance of altering lncRNA polyadenylation events to suppress tumorigenesis as a strategy to combat cancer.
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ALS-linked FUS mutations confer loss and gain of function in the nucleus by promoting excessive formation of dysfunctional Paraspeckles
'Springer Science and Business Media LLC', 2019Co-Authors: Lucy Skelt, Antonietta Notaro, Robin J. Highley, Archa H. Fox, Vincenzo La Bella, Vladimir L. Buchman, Tatyana A. ShelkovnikovaAbstract:Abstract Mutations in the FUS gene cause amyotrophic lateral sclerosis (ALS-FUS). Mutant FUS is known to confer cytoplasmic gain of function but its effects in the nucleus are less understood. FUS is an essential component of Paraspeckles, subnuclear bodies assembled on a lncRNA NEAT1. Paraspeckles may play a protective role specifically in degenerating spinal motor neurons. However it is still unknown how endogenous levels of mutant FUS would affect NEAT1/Paraspeckles. Using novel cell lines with the FUS gene modified by CRISPR/Cas9 and human patient fibroblasts, we found that endogenous levels of mutant FUS cause accumulation of NEAT1 isoforms and Paraspeckles. However, despite only mild cytoplasmic mislocalisation of FUS, Paraspeckle integrity is compromised in these cells, as confirmed by reduced interaction of mutant FUS with core Paraspeckle proteins NONO and SFPQ and increased NEAT1 extractability. This results in NEAT1 localisation outside Paraspeckles, especially prominent under conditions of Paraspeckle-inducing stress. Consistently, Paraspeckle-dependent microRNA production, a readout for functionality of Paraspeckles, is impaired in cells expressing mutant FUS. In line with the cellular data, we observed Paraspeckle hyper-assembly in spinal neurons of ALS-FUS patients. Therefore, despite largely preserving its nuclear localisation, mutant FUS leads to loss (dysfunctional Paraspeckles) and gain (excess of free NEAT1) of function in the nucleus. Perturbed fine structure and functionality of Paraspeckles accompanied by accumulation of non-Paraspeckle NEAT1 may contribute to the disease severity in ALS-FUS
Vladimir L. Buchman - One of the best experts on this subject based on the ideXlab platform.
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ALS-linked FUS mutations confer loss and gain of function in the nucleus by promoting excessive formation of dysfunctional Paraspeckles
'Springer Science and Business Media LLC', 2019Co-Authors: Lucy Skelt, Antonietta Notaro, Robin J. Highley, Archa H. Fox, Vincenzo La Bella, Vladimir L. Buchman, Tatyana A. ShelkovnikovaAbstract:Abstract Mutations in the FUS gene cause amyotrophic lateral sclerosis (ALS-FUS). Mutant FUS is known to confer cytoplasmic gain of function but its effects in the nucleus are less understood. FUS is an essential component of Paraspeckles, subnuclear bodies assembled on a lncRNA NEAT1. Paraspeckles may play a protective role specifically in degenerating spinal motor neurons. However it is still unknown how endogenous levels of mutant FUS would affect NEAT1/Paraspeckles. Using novel cell lines with the FUS gene modified by CRISPR/Cas9 and human patient fibroblasts, we found that endogenous levels of mutant FUS cause accumulation of NEAT1 isoforms and Paraspeckles. However, despite only mild cytoplasmic mislocalisation of FUS, Paraspeckle integrity is compromised in these cells, as confirmed by reduced interaction of mutant FUS with core Paraspeckle proteins NONO and SFPQ and increased NEAT1 extractability. This results in NEAT1 localisation outside Paraspeckles, especially prominent under conditions of Paraspeckle-inducing stress. Consistently, Paraspeckle-dependent microRNA production, a readout for functionality of Paraspeckles, is impaired in cells expressing mutant FUS. In line with the cellular data, we observed Paraspeckle hyper-assembly in spinal neurons of ALS-FUS patients. Therefore, despite largely preserving its nuclear localisation, mutant FUS leads to loss (dysfunctional Paraspeckles) and gain (excess of free NEAT1) of function in the nucleus. Perturbed fine structure and functionality of Paraspeckles accompanied by accumulation of non-Paraspeckle NEAT1 may contribute to the disease severity in ALS-FUS
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protective Paraspeckle hyper assembly downstream of tdp 43 loss of function in amyotrophic lateral sclerosis
Molecular Neurodegeneration, 2018Co-Authors: Tatyana A. Shelkovnikova, Vladimir L. Buchman, Michail S Kukharsky, Pasquale Dimasi, Svetlana Alexeeva, Osman Shabir, Paul R HeathAbstract:Paraspeckles are subnuclear bodies assembled on a long non-coding RNA (lncRNA) NEAT1. Their enhanced formation in spinal neurons of sporadic amyotrophic lateral sclerosis (ALS) patients has been reported but underlying mechanisms are unknown. The majority of ALS cases are characterized by TDP-43 proteinopathy. In current study we aimed to establish whether and how TDP-43 pathology may augment Paraspeckle assembly. Paraspeckle formation in human samples was analysed by RNA-FISH and laser capture microdissection followed by qRT-PCR. Mechanistic studies were performed in stable cell lines, mouse primary neurons and human embryonic stem cell-derived neurons. Loss and gain of function for TDP-43 and other microRNA pathway factors were modelled by siRNA-mediated knockdown and protein overexpression. We show that de novo Paraspeckle assembly in spinal neurons and glial cells is a hallmark of both sporadic and familial ALS with TDP-43 pathology. Mechanistically, loss of TDP-43 but not its cytoplasmic accumulation or aggregation augments Paraspeckle assembly in cultured cells. TDP-43 is a component of the microRNA machinery, and recently, Paraspeckles have been shown to regulate pri-miRNA processing. Consistently, downregulation of core protein components of the miRNA pathway also promotes Paraspeckle assembly. In addition, depletion of these proteins or TDP-43 results in accumulation of endogenous dsRNA and activation of type I interferon response which also stimulates Paraspeckle formation. We demonstrate that human or mouse neurons in vitro lack Paraspeckles, but a synthetic dsRNA is able to trigger their de novo formation. Finally, Paraspeckles are protective in cells with compromised microRNA/dsRNA metabolism, and their assembly can be promoted by a small-molecule microRNA enhancer. Our study establishes possible mechanisms behind Paraspeckle hyper-assembly in ALS and suggests their utility as therapeutic targets in ALS and other diseases with abnormal metabolism of microRNA and dsRNA.
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calcium responsive transactivator crest protein shares a set of structural and functional traits with other proteins associated with amyotrophic lateral sclerosis
Molecular Neurodegeneration, 2015Co-Authors: Haiyan An, Vladimir L. Buchman, Michail S Kukharsky, Annamaria Quintiero, Taisei Matsumoto, Koji Matsukawa, Tadafumi Hashimoto, Takeshi Iwatsubo, Tatyana A. ShelkovnikovaAbstract:Background Mutations in calcium-responsive transactivator (CREST) encoding gene have been recently linked to ALS. Similar to several proteins implicated in ALS, CREST contains a prion-like domain and was reported to be a component of Paraspeckles.
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compromised Paraspeckle formation as a pathogenic factor in fusopathies
Human Molecular Genetics, 2014Co-Authors: Tatyana A. Shelkovnikova, Vladimir L. Buchman, Hannah K Robinson, Claire Troakes, Natalia NinkinaAbstract:Paraspeckles are nuclear bodies formed by a set of specialized proteins assembled on the long non-coding RNA NEAT1; they have a role in nuclear retention of hyperedited transcripts and are associated with response to cellular stress. Fused in sarcoma (FUS) protein, linked to a number of neurodegenerative disorders, is an essential Paraspeckle component. We have shown that its recruitment to these nuclear structures is mediated by the N-terminal region and requires prion-like activity. FUS interacts with p54nrb/NONO, a major constituent of Paraspeckles, in an RNA-dependent manner and responds in the same way as other Paraspeckle proteins to alterations in cellular homeostasis such as changes in transcription rates or levels of protein methylation. FUS also regulates NEAT1 levels and Paraspeckle formation in cultured cells, and FUS deficiency leads to loss of Paraspeckles. Pathological gain-of-function FUS mutations might be expected to affect Paraspeckle function in human diseases because mislocalized amyotrophic lateral sclerosis (ALS)-linked FUS variants sequester other Paraspeckle proteins into aggregates formed in cultured cells and into neuronal inclusions in a transgenic mouse model of FUSopathy. Furthermore, we detected abundant p54nrb/NONO-positive inclusions in motor neurons of patients with familial forms of ALS caused by FUS mutations, but not in other ALS cases. Our results suggest that both loss and gain of FUS function can trigger disruption of Paraspeckle assembly, which may impair protective responses in neurons and thereby contribute to the pathogenesis of FUSopathies.
Shinichi Nakagawa - One of the best experts on this subject based on the ideXlab platform.
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molecular dissection of nuclear Paraspeckles towards understanding the emerging world of the rnp milieu
Open Biology, 2018Co-Authors: Shinichi Nakagawa, Tomohiro Yamazaki, Tetsuro HiroseAbstract:Paraspeckles are nuclear bodies built on an architectural long noncoding RNA, NEAT1, and a series of studies have revealed their molecular components, fine internal structures and cellular and phys...
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Paraspeckles modulate the intranuclear distribution of Paraspeckle associated ctn rna
Scientific Reports, 2016Co-Authors: Aparna Anantharaman, Tetsuro Hirose, Shinichi Nakagawa, Mahdieh Jadaliha, Vidisha Tripathi, Michael F Jantsch, Supriya G Prasanth, Kannanganattu V PrasanthAbstract:Paraspeckles are sub-nuclear domains that are nucleated by long noncoding RNA Neat1. While interaction of protein components of Paraspeckles and Neat1 is understood, there is limited information on the interaction of non-structural RNA components with Paraspeckles. Here, by varying Paraspeckle number and size, we investigate how Paraspeckles influence the nuclear organization of their non-structural RNA component Ctn RNA. Our results show that Ctn RNA remains nuclear-retained in the absence of intact Paraspeckles, suggesting that they do not regulate nuclear retention of Ctn RNA. In the absence of Neat1, Ctn RNA continues to interact with Paraspeckle protein NonO to form residual nuclear foci. In addition, in the absence of Neat1-nucleated Paraspeckles, a subset of Ctn RNA localizes to the perinucleolar regions. Concomitant with increase in number of Paraspeckles, transcriptional reactivation resulted in increased number of Paraspeckle-localized Ctn RNA foci. Similar to Neat1, proteasome inhibition altered the localization of Ctn RNA, where it formed enlarged Paraspeckle-like foci. Super-resolution structured illumination microscopic analyses revealed that in Paraspeckles, Ctn RNA partially co-localized with Neat1, and displayed a more heterogeneous intra-Paraspeckle localization. Collectively, these results show that while Paraspeckles do not influence nuclear retention of Ctn RNA, they modulate its intranuclear compartmentalization.
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the long noncoding rna neat1 is required for mammary gland development and lactation
RNA, 2014Co-Authors: Laura Standaert, Tetsuro Hirose, Carmen Adriaens, Enrico Radaelli, Alexandra Van Keymeulen, Cedric Blanpain, Shinichi Nakagawa, Jeanchristophe MarineAbstract:The lncRNA Neat1 is an essential architectural component of Paraspeckle nuclear bodies. Although cell-based studies identified Neat1-Paraspeckles as key regulators of gene expression through retention of hyperdited mRNAs and/or transcription factors, it is unclear under which specific physiological conditions Paraspeckles are formed in vivo and whether they have any biological relevance. Herein, we show that Paraspeckles are assembled in luminal epithelial cells during mammary gland development. Importantly, genetic ablation of Neat1 results in aberrant mammary gland morphogenesis and lactation defects. We provide evidence that the lactation defect is caused by a decreased ability of Neat1-mutant cells to sustain high rates of proliferation during lobular-alveolar development. This study is the first to assign an important biological function to the lncRNA Neat1 and to link it to the presence of Paraspeckles nuclear bodies in vivo.
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neat1 long noncoding rna regulates transcription via protein sequestration within subnuclear bodies
Molecular Biology of the Cell, 2014Co-Authors: Tetsuro Hirose, Shinichi Nakagawa, Archa H. Fox, Giorgio Virnicchi, Akie Tanigawa, Takao Naganuma, Hiroshi Kimura, Takahide Yokoi, Marianne Benard, Gerard PierronAbstract:Paraspeckles are subnuclear structures formed around nuclear Paraspeckle assembly transcript 1 (NEAT1)/MENe/β long noncoding RNA (lncRNA). Here we show that Paraspeckles become dramatically enlarged after proteasome inhibition. This enlargement is mainly caused by NEAT1 transcriptional up-regulation rather than accumulation of undegraded Paraspeckle proteins. Of interest, however, using immuno-electron microscopy, we find that key Paraspeckle proteins become effectively depleted from the nucleoplasm by 50% when Paraspeckle assembly is enhanced, suggesting a sequestration mechanism. We also perform microarrays from NEAT1-knockdown cells and find that NEAT1 represses transcription of several genes, including the RNA-specific adenosine deaminase B2 (ADARB2) gene. In contrast, the NEAT1-binding Paraspeckle protein splicing factor proline/glutamine-rich (SFPQ) is required for ADARB2 transcription. This leads us to hypothesize that ADARB2 expression is controlled by NEAT1-dependent sequestration of SFPQ. Accordingly, we find that ADARB2 expression is strongly reduced upon enhanced SFPQ sequestration by proteasome inhibition, with concomitant reduction in SFPQ binding to the ADARB2 promoter. Finally, NEAT1(-/-) fibroblasts are more sensitive to proteasome inhibition, which triggers cell death, suggesting that Paraspeckles/NEAT1 attenuates the cell death pathway. These data further confirm that Paraspeckles are stress-responsive nuclear bodies and provide a model in which induced NEAT1 controls target gene transcription by protein sequestration into Paraspeckles.
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neat1 long noncoding rna regulates transcription via protein sequestration within subnuclear bodies
Molecular Biology of the Cell, 2014Co-Authors: Tetsuro Hirose, Shinichi Nakagawa, Giorgio Virnicchi, Akie Tanigawa, Takao Naganuma, Hiroshi Kimura, Takahide Yokoi, Marianne Benard, Ruohan Li, Gerard PierronAbstract:Paraspeckles are subnuclear structures formed around NEAT1 lncRNA. Paraspeckles became enlarged after proteasome inhibition caused by NEAT1 transcriptional activation, leading to protein sequestrat...
Tatyana A. Shelkovnikova - One of the best experts on this subject based on the ideXlab platform.
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stress granules regulate stress induced Paraspeckle assembly
Journal of Cell Biology, 2019Co-Authors: Jing Tong Tan, Tatyana A. ShelkovnikovaAbstract:Eukaryotic cells contain a variety of RNA-protein macrocomplexes termed RNP granules. Different types of granules share multiple protein components; however, the crosstalk between spatially separated granules remains unaddressed. Paraspeckles and stress granules (SGs) are prototypical RNP granules localized exclusively in the nucleus and cytoplasm, respectively. Both granules are implicated in human diseases, such as amyotrophic lateral sclerosis. We characterized the composition of affinity-purified Paraspeckle-like structures and found a significant overlap between the proteomes of Paraspeckles and SGs. We further show that Paraspeckle hyperassembly is typical for cells subjected to SG-inducing stresses. Using chemical and genetic disruption of SGs, we demonstrate that formation of microscopically visible SGs is required to trigger and maintain stress-induced Paraspeckle assembly. Mechanistically, SGs may sequester negative regulators of Paraspeckle formation, such as UBAP2L, alleviating their inhibitory effect on Paraspeckles. Our study reveals a novel function for SGs as positive regulators of nuclear RNP granule assembly and suggests a role for disturbed SG-Paraspeckle crosstalk in human disease.
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Stress granules regulate Paraspeckles: RNP granule continuum at work.
Cell stress, 2019Co-Authors: Tatyana A. ShelkovnikovaAbstract:Eukaryotic cells contain several types of RNA-protein membraneless macro-complexes – ribonucleoprotein (RNP) granules that form by liquid-liquid phase separation. These structures represent biochemical microreactors for a variety of cellular processes and also act as highly accurate sensors of changes in the cellular environment. RNP granules share multiple protein components, however, the connection between spatially separated granules remains surprisingly understudied. Paraspeckles are constitutive nuclear RNP granules whose numbers significantly increase in stressed cells. Our recent work using affinity-purified Paraspeckles revealed that another type of RNP granule, cytoplasmic stress granule (SG), acts as an important regulator of stress-induced Paraspeckle assembly. Our study demonstrates that despite their residency in different cellular compartments, the two RNP granules are closely connected. This study suggests that nuclear and cytoplasmic RNP granules are integral parts of the intracellular “RNP granule continuum” and that rapid exchange of protein components within this continuum is important for the temporal control of cellular stress responses. It also suggests that cells can tolerate and efficiently handle a certain level of phase separation, which is reflected in the existence of “bursts”, or “waves”, of RNP granule formation. Our study triggers a number of important questions related to the mechanisms controlling the flow of RNP granule components within the continuum and to the possibility of targeting these mechanisms in human disease.
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ALS-linked FUS mutations confer loss and gain of function in the nucleus by promoting excessive formation of dysfunctional Paraspeckles
'Springer Science and Business Media LLC', 2019Co-Authors: Lucy Skelt, Antonietta Notaro, Robin J. Highley, Archa H. Fox, Vincenzo La Bella, Vladimir L. Buchman, Tatyana A. ShelkovnikovaAbstract:Abstract Mutations in the FUS gene cause amyotrophic lateral sclerosis (ALS-FUS). Mutant FUS is known to confer cytoplasmic gain of function but its effects in the nucleus are less understood. FUS is an essential component of Paraspeckles, subnuclear bodies assembled on a lncRNA NEAT1. Paraspeckles may play a protective role specifically in degenerating spinal motor neurons. However it is still unknown how endogenous levels of mutant FUS would affect NEAT1/Paraspeckles. Using novel cell lines with the FUS gene modified by CRISPR/Cas9 and human patient fibroblasts, we found that endogenous levels of mutant FUS cause accumulation of NEAT1 isoforms and Paraspeckles. However, despite only mild cytoplasmic mislocalisation of FUS, Paraspeckle integrity is compromised in these cells, as confirmed by reduced interaction of mutant FUS with core Paraspeckle proteins NONO and SFPQ and increased NEAT1 extractability. This results in NEAT1 localisation outside Paraspeckles, especially prominent under conditions of Paraspeckle-inducing stress. Consistently, Paraspeckle-dependent microRNA production, a readout for functionality of Paraspeckles, is impaired in cells expressing mutant FUS. In line with the cellular data, we observed Paraspeckle hyper-assembly in spinal neurons of ALS-FUS patients. Therefore, despite largely preserving its nuclear localisation, mutant FUS leads to loss (dysfunctional Paraspeckles) and gain (excess of free NEAT1) of function in the nucleus. Perturbed fine structure and functionality of Paraspeckles accompanied by accumulation of non-Paraspeckle NEAT1 may contribute to the disease severity in ALS-FUS
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protective Paraspeckle hyper assembly downstream of tdp 43 loss of function in amyotrophic lateral sclerosis
Molecular Neurodegeneration, 2018Co-Authors: Tatyana A. Shelkovnikova, Vladimir L. Buchman, Michail S Kukharsky, Pasquale Dimasi, Svetlana Alexeeva, Osman Shabir, Paul R HeathAbstract:Paraspeckles are subnuclear bodies assembled on a long non-coding RNA (lncRNA) NEAT1. Their enhanced formation in spinal neurons of sporadic amyotrophic lateral sclerosis (ALS) patients has been reported but underlying mechanisms are unknown. The majority of ALS cases are characterized by TDP-43 proteinopathy. In current study we aimed to establish whether and how TDP-43 pathology may augment Paraspeckle assembly. Paraspeckle formation in human samples was analysed by RNA-FISH and laser capture microdissection followed by qRT-PCR. Mechanistic studies were performed in stable cell lines, mouse primary neurons and human embryonic stem cell-derived neurons. Loss and gain of function for TDP-43 and other microRNA pathway factors were modelled by siRNA-mediated knockdown and protein overexpression. We show that de novo Paraspeckle assembly in spinal neurons and glial cells is a hallmark of both sporadic and familial ALS with TDP-43 pathology. Mechanistically, loss of TDP-43 but not its cytoplasmic accumulation or aggregation augments Paraspeckle assembly in cultured cells. TDP-43 is a component of the microRNA machinery, and recently, Paraspeckles have been shown to regulate pri-miRNA processing. Consistently, downregulation of core protein components of the miRNA pathway also promotes Paraspeckle assembly. In addition, depletion of these proteins or TDP-43 results in accumulation of endogenous dsRNA and activation of type I interferon response which also stimulates Paraspeckle formation. We demonstrate that human or mouse neurons in vitro lack Paraspeckles, but a synthetic dsRNA is able to trigger their de novo formation. Finally, Paraspeckles are protective in cells with compromised microRNA/dsRNA metabolism, and their assembly can be promoted by a small-molecule microRNA enhancer. Our study establishes possible mechanisms behind Paraspeckle hyper-assembly in ALS and suggests their utility as therapeutic targets in ALS and other diseases with abnormal metabolism of microRNA and dsRNA.
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NEAT1 and Paraspeckles in neurodegenerative diseases: A missing lnc found?
KeAi, 2018Co-Authors: Non G. Williams, Tatyana A. ShelkovnikovaAbstract:Neurodegenerative diseases are among the most common causes of disability worldwide. Although neurodegenerative diseases are heterogeneous in both their clinical features and the underlying physiology, they are all characterised by progressive loss of specific neuronal populations. Recent experimental evidence suggests that long non-coding RNAs (lncRNAs) play important roles in the CNS in health and disease. Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) is an abundant, ubiquitously expressed lncRNA, which forms a scaffold for a specific RNA granule in the nucleus, or nuclear body, the Paraspeckle. Paraspeckles act as molecular hubs for cellular processes commonly affected by neurodegeneration. Transcriptomic analyses of the diseased human tissue have revealed altered NEAT1 levels in the CNS in major neurodegenerative disorders as well as in some disease models. Although it is clear that changes in NEAT1 expression (and in some cases, Paraspeckle assembly) accompany neuronal damage, our understanding of NEAT1 contribution to the disease pathogenesis is still rudimentary. In this review, we have summarised the available knowledge on NEAT1 involvement in the molecular processes linked to neurodegeneration and on NEAT1 dysregulation in this type of disease, with a special focus on amyotrophic lateral sclerosis. The goal of this review is to attract the attention of researchers in the field of neurodegeneration to NEAT1 and Paraspeckles. Keywords: NEAT1, Paraspeckle, lncRNA, Neurodegeneration, Amyotrophic lateral sclerosi
