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International Journal of Molecular... Dec 2023Poly (ADP-ribose) polymerase (PARP) inhibitors are effective against -mutated cancers through synthetic lethality. Unfortunately, most cases ultimately develop acquired...
Poly (ADP-ribose) polymerase (PARP) inhibitors are effective against -mutated cancers through synthetic lethality. Unfortunately, most cases ultimately develop acquired resistance. Therefore, enhancing PARP inhibitor sensitivity and preventing resistance in those cells are an unmet clinical need. Here, we investigated the ability of paraspeckle component 1 (), as an additional synthetic lethal partner with , to enhance olaparib sensitivity in preclinical models of -mutated breast and ovarian cancers. In vitro, the combined olaparib and small interfering RNA (siRNA) exhibited synergistic anti-proliferative activity in -mutated breast and ovarian cancer cells. The combination therapy also demonstrated synergistic tumor inhibition in a xenograft mouse model. Mechanistically, olaparib monotherapy increased the expressions of p-ATM and DNA-PKcs, suggesting the activation of a DNA repair pathway, whereas combining siRNA with olaparib decreased the expressions of p-ATM and DNA-PKcs again. As such, the combination increased the formation of γH2AX foci, indicating stronger DNA double-strand breaks. Subsequently, these DNA-damaged cells escaped G2/M checkpoint activation, as indicated by the suppression of p-cdc25C (Ser216) and p-cdc2 (Tyr15) after combination treatment. Finally, these cells entered mitosis, which induced increased apoptosis. Thus, this proves that inhibition enhances olaparib sensitivity by targeting DNA damage response in our preclinical model. The combination of olaparib and inhibition merits further clinical investigation to enhance PARP inhibitor efficacy.
Topics: Animals; Antineoplastic Agents; Poly(ADP-ribose) Polymerase Inhibitors; Ovarian Neoplasms; Humans; Female; Mice; Breast Neoplasms; Cell Line, Tumor; RNA-Binding Proteins; BRCA1 Protein; BRCA2 Protein; RNA, Small Interfering
PubMed: 38069409
DOI: 10.3390/ijms242317086 -
Journal of Molecular Biology Dec 2023Demixing of proteins and nucleic acids into condensed liquid phases is rapidly emerging as a ubiquitous mechanism underlying the complex spatiotemporal organisation of...
Demixing of proteins and nucleic acids into condensed liquid phases is rapidly emerging as a ubiquitous mechanism underlying the complex spatiotemporal organisation of molecules within the cell. Long disordered regions of low sequence complexity (LCRs) are a common feature of proteins that form liquid-like microscopic biomolecular condensates. In particular, RNA-binding proteins with prion-like regions have emerged as key drivers of liquid demixing to form condensates such as nucleoli, paraspeckles and stress granules. Splicing factor proline- and glutamine-rich (SFPQ) is an RNA- and DNA-binding protein essential for DNA repair and paraspeckle formation. SFPQ contains two LCRs of different length and composition. Here, we show that the shorter C-terminal LCR of SFPQ is the main region responsible for the condensation of SFPQ in vitro and in the cell nucleus. In contrast, we find that the longer N-terminal prion-like LCR of SFPQ attenuates condensation of the full-length protein, suggesting a more regulatory role in preventing aberrant condensate formation in the cell. The compositions of these respective LCRs are discussed with reference to current literature. Our data add nuance to the emerging understanding of biomolecular condensation, by providing the first example of a common multifunctional nucleic acid-binding protein with an extensive prion-like region that serves to regulate rather than drive condensate formation.
Topics: Biomolecular Condensates; RNA-Binding Proteins; DNA-Binding Proteins; RNA; Prions
PubMed: 37952770
DOI: 10.1016/j.jmb.2023.168364 -
Experimental and Therapeutic Medicine Sep 2023Alzheimer's disease (AD) is the most common type of dementia and is a serious social and medical problem threatening human health. The present study investigated the...
Alzheimer's disease (AD) is the most common type of dementia and is a serious social and medical problem threatening human health. The present study investigated the effect and underlying action mechanism of triptolide (Tri) on AD progression. Reverse transcription-quantitative PCR and western blotting analysis were used to determine the changes in RNA expression and levels of NF-κB signaling pathway proteins before and after lipopolysaccharide (LPS) induction. Nucleocytoplasmic separation experiments determined the intracellular localization of long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1). A dual-luciferase assay was used to analyze the binding between NEAT1 and microRNA (miRNA/miR)-361 or tumor necrosis factor receptor-associated factor 2 (TRAF2) and miR-361-3p and RNA pull-down was used to analyze the binding between NEAT1 and miR-361-3p. Cell Counting Kit-8, flow cytometry and ELISA were used to detect the effects of interaction between Tri and NEAT1/miR-361-3p/TRAF2 on cell viability, apoptosis and inflammatory factor levels, respectively. The results showed that LPS-mediated human microglial clone 3 cell line (HMC3) viability decreased and apoptosis and inflammatory factors (IL-1β, IL-6, IL-18 and TNF-α) increased. Tri inhibited LPS-mediated effects in a dose-dependent manner by downregulating NEAT1 expression. NEAT1 is highly expressed in the cytoplasm and reduces the transcription and translation of downstream TRAF2 by acting as a competitive endogenous RNA that adsorbs miR-361-3p. LPS-mediated HMC3 cell injury, inflammation and activation of NF-κB signaling were partially reversed in presence of Tri. The miR-361-3p mimic promoted the Tri effect and overexpression of (ov)-NEAT1 partially reversed the Tri-miR-361-3p combined effect. The effects of ov-NEAT1 were partially attenuated by small interfering (si)-TRAF2. Overall, Tri inhibited the LPS-induced decrease in viability, increase in apoptosis and inflammation and activation of NF-κB signaling in HMC3 cells. Tri regulation affected the NEAT1/miR-361-3p/TRAF2 axis. These findings suggested a potential therapeutic role for Tri in the clinical management of AD by modulating this molecular axis.
PubMed: 37614428
DOI: 10.3892/etm.2023.12139 -
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 -
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 -
Immunity, Inflammation and Disease Jun 2023Parkinson's disease (PD) is the second most frequent neurodegenerative disease. The aim of our study is to explore the role and the regulatory mechanism of long...
PURPOSE
Parkinson's disease (PD) is the second most frequent neurodegenerative disease. The aim of our study is to explore the role and the regulatory mechanism of long noncoding RNA (lncRNA) NEAT1 in MPP -induced pyroptosis in a cell model of PD.
MATERIALS AND METHODS
MPP -treated SH-SY5Y cells were used as an in vitro model of dopaminergic neurons for PD. Expression levels of miR-5047 and YAF2 mRNA were determined through qRT-PCR. TUNEL staining was carried out to analyze neuronal apoptosis. Luciferase activity assay was accomplished to analyze the combination of miR-5047 with NEAT1 or YAF2 3'-UTR region. Besides, concentrations of IL-1β and IL-18 in supernatant samples were analyzed by using ELISA assay. Expression level of proteins were examined through Western blot.
RESULTS
NEAT1 and YAF2 expression were increased, while miR-5047 expression was declined in the SH-SY5Y cells treated with MPP . NEAT1 was a positively regulator to SH-SY5Y cells pyroptosis induced by MPP . In addition, YAF2 was a downstream target of miR-5047. NEAT1 promoted YAF2 expression via inhibiting miR-5047. Importantly, the promotion of NEAT1 to SH-SY5Y cells pyroptosis induced by MPP was rescued by miR-5047 mimic transfection or YAF2 downregulation.
CONCLUSION
In conclusion, NEAT1 was increased in MPP -induced SH-SY5Y cells, and it promoted MPP -induced pyroptosis through facilitating YAF2 expression by sponging miR-5047.
Topics: Humans; MicroRNAs; Muscle Proteins; Neuroblastoma; Neurodegenerative Diseases; Parkinson Disease; Pyroptosis; Repressor Proteins; RNA, Long Noncoding
PubMed: 37382256
DOI: 10.1002/iid3.817 -
Nature Communications Jun 2023N6-methyladenosine (mA) modification plays important roles in bioprocesses and diseases. AlkB homolog 5 (ALKBH5) is one of two mA demethylases. Here, we reveal that...
N6-methyladenosine (mA) modification plays important roles in bioprocesses and diseases. AlkB homolog 5 (ALKBH5) is one of two mA demethylases. Here, we reveal that ALKBH5 is acetylated at lysine 235 (K235) by lysine acetyltransferase 8 and deacetylated by histone deacetylase 7. K235 acetylation strengthens the mA demethylation activity of ALKBH5 by increasing its recognition of mA on mRNA. RNA-binding protein paraspeckle component 1 (PSCP1) is a regulatory subunit of ALKBH5 and preferentially interacts with K235-acetylated ALKBH5 to recruit and facilitate the recognition of mA mRNA by ALKBH5, thereby promoting mA erasure. Mitogenic signals promote ALKBH5 K235 acetylation. K235 acetylation of ALKBH5 is upregulated in cancers and promotes tumorigenesis. Thus, our findings reveal that the mA demethylation activity of ALKBH5 is orchestrated by its K235 acetylation and regulatory subunit PSPC1 and that K235 acetylation is necessary for the mA demethylase activity and oncogenic roles of ALKBH5.
Topics: Humans; Acetylation; RNA, Messenger; Carcinogenesis; Cell Transformation, Neoplastic; AlkB Homolog 5, RNA Demethylase; Demethylation; RNA-Binding Proteins
PubMed: 37369679
DOI: 10.1038/s41467-023-39414-4 -
Nature Communications Jun 2023The transcription factor ΔNp63 regulates epithelial stem cell function and maintains the integrity of stratified epithelial tissues by acting as transcriptional...
The transcription factor ΔNp63 regulates epithelial stem cell function and maintains the integrity of stratified epithelial tissues by acting as transcriptional repressor or activator towards a distinct subset of protein-coding genes and microRNAs. However, our knowledge of the functional link between ∆Np63 transcriptional activity and long non-coding RNAs (lncRNAs) expression is quite limited. Here, we show that in proliferating human keratinocytes ∆Np63 represses the expression of the lncRNA NEAT1 by recruiting the histone deacetylase HDAC1 to the proximal promoter of NEAT1 genomic locus. Upon induction of differentiation, ∆Np63 down-regulation is associated by a marked increase of NEAT1 RNA levels, resulting in an increased assembly of paraspeckles foci both in vitro and in human skin tissues. RNA-seq analysis associated with global DNA binding profile (ChIRP-seq) revealed that NEAT1 associates with the promoter of key epithelial transcription factors sustaining their expression during epidermal differentiation. These molecular events might explain the inability of NEAT1-depleted keratinocytes to undergo the proper formation of epidermal layers. Collectively, these data uncover the lncRNA NEAT1 as an additional player of the intricate network orchestrating epidermal morphogenesis.
Topics: Humans; Cell Differentiation; Down-Regulation; Gene Expression Regulation; MicroRNAs; RNA, Long Noncoding; Keratinocytes
PubMed: 37365156
DOI: 10.1038/s41467-023-39011-5 -
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