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The Journal of Biological Chemistry Aug 2023Paraspeckles (PS) are nuclear structures scaffolded by the long noncoding RNA NEAT1 and protein components such as NONO and SFPQ. We previously found that the...
Paraspeckles (PS) are nuclear structures scaffolded by the long noncoding RNA NEAT1 and protein components such as NONO and SFPQ. We previously found that the upregulation of RNA N6-methyl-adenosine (mA) demethylase ALKBH5 facilitates hypoxia-induced paraspeckle assembly through erasing mA marks on NEAT1, thus stabilizing it. However, it remains unclear how these processes are spatiotemporally coordinated. Here we discover that ALKBH5 specifically binds to proteins in PS and forms phase-separated droplets that are incorporated into PS through its C-terminal intrinsically disordered region (cIDR). Upon exposure to hypoxia, rapid ALKBH5 condensation in PS induces mA demethylation of NEAT1, which further facilitates PS formation before the upregulation of ALKBH5 expression. In cells expressing ALKBH5 lacking cIDR, PS fail to be formed in response to hypoxia, accompanied with insufficient mA demethylation of NEAT1 and its destabilization. We also demonstrate that ALKBH5-cIDR is indispensable for hypoxia-induced effects such as cancer cell invasion. Therefore, our study has identified the role of ALKBH5 in phase separation as the molecular basis of the positive feedback loop for PS formation between ALKBH5 incorporation into PS and NEAT1 stabilization.
Topics: Humans; AlkB Homolog 5, RNA Demethylase; Hypoxia; Paraspeckles; RNA, Long Noncoding; Transcriptional Activation; Up-Regulation
PubMed: 37474102
DOI: 10.1016/j.jbc.2023.105071 -
Functional & Integrative Genomics Jul 2023We investigated the role of miR-150-5p in osteoarthritic (OA) chondrocytes, as well as the possible regulatory role of long non-coding RNAs (lncRNAs) in miR-150-5p...
We investigated the role of miR-150-5p in osteoarthritic (OA) chondrocytes, as well as the possible regulatory role of long non-coding RNAs (lncRNAs) in miR-150-5p expression. TargetScan, StarBase, DIANA-LncBase, and Open Targets databases were used to predict miR-150-5p target genes, lncRNAs/miR-150-5p interactions, and OA-related genes. Protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). Gene ontology (GO) and pathway analysis were performed using Enrichr database. A publicly available RNA-seq dataset was retrieved to identify differentially expressed lncRNAs in damaged vs intact cartilage. We re-analyzed the retrieved RNA-seq data and revealed 177 differentially expressed lncRNAs in damage vs intact cartilage, including Nuclear Paraspeckle Assembly Transcript 1(NEAT1). MiR-150-5p, NEAT1, b-catenin, matrix metallopeptidase 13 (MMP-13), and ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS-5) expressions were assessed by reverse transcription-quantitative PCR (RT-qPCR) and western blot assay. Knockout and transfection experiments were conducted to investigate the role of NEAT1/miR-150-5p/b-catenin in cartilage degradation. Bioinformatics analysis revealed that b-catenin was an OA-related miR-150-5p target. MiR-150-5p overexpression in OA chondrocytes resulted in decreased expression of b-catenin, as well as MMP-13 and ADAMTS-5, both being Wnt/b-catenin downstream target genes. NEAT1/miR-150-5p interaction was predicted by bioinformatics analysis, while NEAT1 knockout led to increased expression of miR-150-5p in OA chondrocytes. Moreover, inhibition of miR-150-5p reversed the repressive effects of NEAT1 silencing in b-catenin expression in OA chondrocytes. Our results support a possible catabolic role of NEAT1/miR-150-5p interaction in OA progression by regulating b-catenin expression.
Topics: MicroRNAs; Chondrocytes; Down-Regulation; Catenins; RNA, Long Noncoding; Matrix Metalloproteinase 13; Apoptosis; Cell Proliferation
PubMed: 37468759
DOI: 10.1007/s10142-023-01139-4 -
Nucleic Acids Research Aug 2023Phase-separated membraneless organelles often contain RNAs that exhibit unusual semi-extractability using the conventional RNA extraction method, and can be efficiently...
Phase-separated membraneless organelles often contain RNAs that exhibit unusual semi-extractability using the conventional RNA extraction method, and can be efficiently retrieved by needle shearing or heating during RNA extraction. Semi-extractable RNAs are promising resources for understanding RNA-centric phase separation. However, limited assessments have been performed to systematically identify and characterize semi-extractable RNAs. In this study, 1074 semi-extractable RNAs, including ASAP1, DANT2, EXT1, FTX, IGF1R, LIMS1, NEAT1, PHF21A, PVT1, SCMH1, STRG.3024.1, TBL1X, TCF7L2, TVP23C-CDRT4, UBE2E2, ZCCHC7, ZFAND3 and ZSWIM6, which exhibited consistent semi-extractability were identified across five human cell lines. By integrating publicly available datasets, we found that semi-extractable RNAs tend to be distributed in the nuclear compartments but are dissociated from the chromatin. Long and repeat-containing semi-extractable RNAs act as hubs to provide global RNA-RNA interactions. Semi-extractable RNAs were divided into four groups based on their k-mer content. The NEAT1 group preferred to interact with paraspeckle proteins, such as FUS and NONO, implying that RNAs in this group are potential candidates of architectural RNAs that constitute nuclear bodies.
Topics: Humans; Cell Line; Cell Nucleus; Chromatin; DNA-Binding Proteins; RNA; RNA, Long Noncoding
PubMed: 37463833
DOI: 10.1093/nar/gkad567 -
Scientific Reports Jul 2023The early events of HIV-1 infection involve the transport of the viral core into the nucleus. This event triggers the translocation of CPSF6 from paraspeckles into...
The early events of HIV-1 infection involve the transport of the viral core into the nucleus. This event triggers the translocation of CPSF6 from paraspeckles into nuclear speckles forming puncta-like structures. Our investigations revealed that neither HIV-1 integration nor reverse transcription is required for the formation of puncta-like structures. Moreover, HIV-1 viruses without viral genome are competent for the induction of CPSF6 puncta-like structures. In agreement with the notion that HIV-1 induced CPSF6 puncta-like structures are biomolecular condensates, we showed that osmotic stress and 1,6-hexanediol induced the disassembly of CPSF6 condensates. Interestingly, replacing the osmotic stress by isotonic media re-assemble CPSF6 condensates in the cytoplasm of the cell. To test whether CPSF6 condensates were important for infection we utilized hypertonic stress, which prevents formation of CPSF6 condensates, during infection. Remarkably, preventing the formation of CPSF6 condensates inhibits the infection of wild type HIV-1 but not of HIV-1 viruses bearing the capsid changes N74D and A77V, which do not form CPSF6 condensates during infection. We also investigated whether the functional partners of CPSF6 are recruited to the condensates upon infection. Our experiments revealed that CPSF5, but not CPSF7, co-localized with CPSF6 upon HIV-1 infection. We found condensates containing CPSF6/CPSF5 in human T cells and human primary macrophages upon HIV-1 infection. Additionally, we observed that the integration cofactor LEDGF/p75 changes distribution upon HIV-1 infection and surrounds the CPSF6/CPSF5 condensates. Overall, our work demonstrated that CPSF6 and CPSF5 are forming biomolecular condensates that are important for infection of wild type HIV-1 viruses.
Topics: Humans; Biomolecular Condensates; Capsid; Capsid Proteins; Cell Nucleus; HIV Infections; HIV Seropositivity; HIV-1; mRNA Cleavage and Polyadenylation Factors; Virus Replication
PubMed: 37414787
DOI: 10.1038/s41598-023-37364-x -
Drug Metabolism and Disposition: the... Oct 2023Human pregnane X receptor (PXR) is a major nuclear receptor that upregulates the expression of drug-metabolizing enzymes such as CYP3A4. In our recent study, it was...
Human pregnane X receptor (PXR) is a major nuclear receptor that upregulates the expression of drug-metabolizing enzymes such as CYP3A4. In our recent study, it was revealed that PXR interacts with DAZ-associated protein 1 (DAZAP1), which is an essential component of the paraspeckle, a membraneless nuclear body, and the interaction was disassociated by rifampicin, a ligand of PXR. The purpose of this study was to clarify the roles of paraspeckles in PXR-mediated transcriptional regulation. Immunoprecipitation assays using PXR-overexpressing HepG2 (ShP51) cells revealed that PXR interacts with not only DAZAP1 but also NEAT1_2, a long noncoding RNA included in the paraspeckle, and that the interaction between PXR and NEAT1_2 was disassociated by rifampicin. These results suggest that PXR is trapped in paraspeckles and that the activation of PXR by its ligands facilitates its disassociation from paraspeckles. Induction of CYP3A4 by rifampicin was significantly enhanced by the knockdown of NEAT1_2 or DAZAP1 in ShP51 cells and their parental HepG2 cells. A luciferase assay using a plasmid containing the PXR response elements of CYP3A4 revealed that the increased CYP3A4 induction by siNEAT1_2 or siDAZAP1 was due to the increased transactivation by PXR. These results suggest that paraspeckles play a role in trapping nuclear PXR in the absence of the ligand to negatively regulate transactivation of its downstream gene. Collectively, this is the first study to demonstrate that the paraspeckle components NEAT1_2 and DAZAP1 negatively regulate CYP3A4 induction by PXR. SIGNIFICANCE STATEMENT: This study revealed that PXR interacts with paraspeckle components NEAT1_2 and DAZAP1 to suppress CYP3A4 induction by PXR, and the interaction is dissociated by PXR ligands. This finding provides a novel concept that paraspeckles formed by liquid-liquid phase separation potentially affect drug metabolism via negative regulation of PXR function.
Topics: Humans; Cytochrome P-450 CYP3A; Ligands; Paraspeckles; Pregnane X Receptor; Receptors, Steroid; Rifampin; RNA-Binding Proteins
PubMed: 37349114
DOI: 10.1124/dmd.122.001065 -
Epigenetics Dec 2023Increasing evidence has uncovered the essential roles of long noncoding RNAs (lncRNAs) in biological and pathological functions of dendritic cells (DCs) among patients...
Increasing evidence has uncovered the essential roles of long noncoding RNAs (lncRNAs) in biological and pathological functions of dendritic cells (DCs) among patients with systemic lupus erythematosus (SLE). However, whether lncRNA nuclear paraspeckle assembly transcript 1 () could modulate DCs, especially in the inflammation of SLE, remains largely unknown. Fifteen SLE patients and fifteen age-matched healthy controls were included, and their monocyte-derived dendritic cells (moDCs) were cultured in vitro. Our research identified that the expression of was significantly increased in moDCs of SLE patients and positively correlated with disease activity. Interleukin 6 (IL-6) from both plasma and secreted supernatants of moDCs was also elevated in the SLE group. In addition, regulation of in moDCs by transfection could lead to the corresponding change in IL-6 generation. While for , a micro-RNA that can bind with the 3' UTR region of and , it may serve as a negative modulator since its overexpression could result in the reduction of IL-6 levels and vice versa. Additionally, the elevation in expression could increase the secretion of IL-6 by specifically binding to , reducing the negative modulatory effects of on the target gene, which suggested that elevated expression could function as the competing endogenous RNA (ceRNA). In conclusion, our findings indicate that can efficiently sponge to upregulate expression and secretion of IL-6 in moDCs, suggesting that the axis may be involved in the development of SLE disease.
Topics: Humans; Dendritic Cells; DNA Methylation; Interleukin-6; Lupus Erythematosus, Systemic; MicroRNAs; Monocytes; RNA, Long Noncoding
PubMed: 37343193
DOI: 10.1080/15592294.2023.2226492 -
Journal of Cell Communication and... Sep 2023Gynecologic cancers are a worldwide problem among women. Recently, molecular targeted therapy opened up an avenue for cancer diagnosis and treatment. Long non-coding... (Review)
Review
Gynecologic cancers are a worldwide problem among women. Recently, molecular targeted therapy opened up an avenue for cancer diagnosis and treatment. Long non-coding RNAs (lncRNAs) are RNA molecules (> 200 nt) that are not translated into protein, and interact with DNA, RNA, and proteins. LncRNAs were found to play pivotal roles in cancer tumorigenesis and progression. Nuclear paraspeckle assembly transcript 1 (NEAT1) is a lncRNA that mediates cell proliferation, migration, and EMT in gynecologic cancers by targeting several miRNAs/mRNA axes. Therefore, NEAT1 may function as a potent biomarker for the prediction and treatment of breast, ovarian, cervical, and endometrial cancers. In this narrative review, we summarized various NEAT1-related signaling pathways that are critical in gynecologic cancers. Long non-coding RNA (lncRNA) by targeting various signaling pathways involved in its target genes can regulate the occurrence of gynecologic cancers.
PubMed: 37310654
DOI: 10.1007/s12079-023-00746-x -
Seminars in Cell & Developmental Biology Mar 2024In recent years, there has been an emphasis on the role of phase-separated biomolecular condensates, especially stress granules, in neurodegenerative diseases such as... (Review)
Review
In recent years, there has been an emphasis on the role of phase-separated biomolecular condensates, especially stress granules, in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). This is largely due to several ALS-associated mutations occurring in genes involved in stress granule assembly and observations that pathological inclusions detected in ALS patient neurons contain stress granule proteins, including the ALS-linked proteins TDP-43 and FUS. However, protein components of stress granules are also found in numerous other phase-separated biomolecular condensates under physiological conditions which are inadequately discussed in the context of ALS. In this review, we look beyond stress granules and describe the roles of TDP-43 and FUS in physiological condensates occurring in the nucleus and neurites, such as the nucleolus, Cajal bodies, paraspeckles and neuronal RNA transport granules. We also discuss the consequences of ALS-linked mutations in TDP-43 and FUS on their ability to phase separate into these stress-independent biomolecular condensates and perform their respective functions. Importantly, biomolecular condensates sequester multiple overlapping protein and RNA components, and their dysregulation could contribute to the observed pleiotropic effects of both sporadic and familial ALS on RNA metabolism.
Topics: Humans; Amyotrophic Lateral Sclerosis; Biomolecular Condensates; DNA-Binding Proteins; Neurodegenerative Diseases; Mutation; RNA
PubMed: 37268555
DOI: 10.1016/j.semcdb.2023.05.006 -
Human Pathology Aug 2023Prostate cancer (PCa) remains the most commonly diagnosed cancer in men worldwide and is still the second leading cause of cancer-related death. One major cause of PCa...
Prostate cancer (PCa) remains the most commonly diagnosed cancer in men worldwide and is still the second leading cause of cancer-related death. One major cause of PCa development is epigenetic aberration, including histone modification. We have previously demonstrated that Lysine Demethylase 5C (KDM5C) plays an essential role in the development of PCa and drives PCa progression by promoting epithelial-mesenchymal transition. Epigenetic regulators often work in concert, for example, to regulate transcription. We identified Paraspeckle Component 1 (PSPC1) as an interacting protein of KDM5C, suggesting that these proteins might function together in PCa. Here, we systematically investigate the expression patterns of KDM5C and PSPC1 in 2 independent prostate cohorts (432 and 205 prostate tumors in total for PSPC1 and KDM5C, respectively) by immunohistochemistry. We demonstrate that the expression of PSPC1 correlates with that of KDM5C. In addition, PSPC1 is up-regulated in primary and metastatic PCa. Elevated PSPC1 expression correlates with a higher-grade group and an advanced T-stage. Patients with high PSPC1 expression have a worse biochemical recurrence-free survival. In addition, PSPC1 expression is an independent prognostic parameter. Our data indicate that KDM5C and PSPC1 are involved in PCa progression, and therapeutic inhibition of KDM5C and PSPC1 by selective compounds might be a promising approach for the treatment of PCa.
Topics: Male; Humans; Prostatic Neoplasms; Prostate; Epithelial-Mesenchymal Transition; RNA-Binding Proteins; Histone Demethylases
PubMed: 37209920
DOI: 10.1016/j.humpath.2023.05.007 -
International Ophthalmology Sep 2023Oxidative stress plays a significant role in cataract development. It causes the apoptosis of lens epithelial cells (LECs), resulting in lens opacification and...
Long non-coding RNA nuclear paraspeckle assembly transcript 1 downregulation protects lens epithelial cells from oxidative stress-induced apoptosis by regulating the microRNA-124-3p/death-associated protein kinase 1 axis in age-related cataract.
Oxidative stress plays a significant role in cataract development. It causes the apoptosis of lens epithelial cells (LECs), resulting in lens opacification and accelerating cataract progression. Long non-coding RNAs (lncRNAs) and microRNAs have been linked to cataract development. Notably, lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) is involved in LEC apoptosis and cataract formation. However, the molecular mechanism by which NEAT1 causes age-related cataracts remains unknown. In this study, LECs (SRA01/04) were exposed to 200 μM HO2 to generate an in vitro cataract model. The apoptosis and viability of cells were determined using flow cytometry and 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assays, respectively. Additionally, western blotting and quantitative polymerase chain reaction were used to determine the miRNA and lncRNA expression levels. When LECs were treated with hydrogen peroxide, lncRNA NEAT1 expression levels were significantly upregulated, which contributed to LEC apoptosis. Notably, lncRNA NEAT1 suppressed the expression of miR-124-3p, a critical regulator of apoptosis, whereas NEAT1 inhibition increased miR-124-3p expression and alleviated apoptosis. However, this effect was reversed when miR1243p expression was inhibited. Additionally, the miR1243p mimic effectively inhibited the death-associated protein kinase 1 (DAPK1) expression and apoptosis of LECs, while the DAPK1 mimic reversed these effects. In conclusion, our findings indicate that the lncRNA NEAT1/miR-124-3p/DAPK1 signaling loop is involved in the regulation of LEC apoptosis induced by oxidative stress, which can be exploited to develop potential treatment strategies for age-related cataracts.
Topics: Humans; RNA, Long Noncoding; Down-Regulation; Death-Associated Protein Kinases; Paraspeckles; MicroRNAs; Cataract; Epithelial Cells; Oxidative Stress; Apoptosis
PubMed: 37191928
DOI: 10.1007/s10792-023-02749-4