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International Journal of Molecular... Jul 2022NONO and SFPQ are involved in multiple nuclear processes (e.g., pre-mRNA splicing, DNA repair, and transcriptional regulation). These proteins, along with NEAT1, enable...
NONO and SFPQ are involved in multiple nuclear processes (e.g., pre-mRNA splicing, DNA repair, and transcriptional regulation). These proteins, along with NEAT1, enable paraspeckle formation, thus promoting multiple myeloma cell survival. In this paper, we investigate NONO and SFPQ dimer stability, highlighting the hetero- and homodimer structural differences, and model their interactions with RNA, simulating their binding to a polyG probe mimicking NEAT1guanine-rich regions. We demonstrated in silico that NONO::SFPQ heterodimerization is a more favorable process than homodimer formation. We also show that NONO and SFPQ RRM2 subunits are primarily required for protein-protein interactions with the other DBHS protomer. Simulation of RNA binding to NONO and SFPQ, beside validating RRM1 RNP signature importance, highlighted the role of β2 and β4 strand residues for RNA specific recognition. Moreover, we demonstrated the role of the NOPS region and other protomer's RRM2 β2/β3 loop in strengthening the interaction with RNA. Our results, having deepened RNA and DBHS dimer interactions, could contribute to the design of small molecules to modulate the activity of these proteins. RNA-mimetics, able to selectively bind to NONO and/or SFPQ RNA-recognition site, could impair paraspeckle formation, thus representing a first step towards the discovery of drugs for multiple myeloma treatment.
Topics: DNA-Binding Proteins; Dimerization; Humans; Multiple Myeloma; PTB-Associated Splicing Factor; Protein Subunits; RNA; RNA Splicing Factors; RNA-Binding Proteins
PubMed: 35886974
DOI: 10.3390/ijms23147626 -
Cell Reports Jul 2022Hyperosmotic stress as physiologic dysfunction can reduce the cell volume and then redistribute both protein concentration and ionic strength, but its effect on...
Hyperosmotic stress as physiologic dysfunction can reduce the cell volume and then redistribute both protein concentration and ionic strength, but its effect on liquid-liquid phase separation (LLPS) is not well understood. Here, we map the hyperosmotic-stress-induced nuclear LLPS of amyotrophic lateral sclerosis (ALS)-related proteins (fused in sarcoma [FUS], TAR DNA-binding protein 43 [TDP-43]). The dynamic and reversibility of FUS granules are continuable with the increase of hypertonic stimulation time, but those of TDP-43 granules decrease significantly. Strikingly, FUS granules, but not TDP-43 granules, contain essential chaperone Hsp40, which can protect amyloid protein from solid aggregation. Moreover, FUS nuclear granules can co-localize with paraspeckles, but not promyelocytic leukemia (PML) bodies or nuclear speckles, while TDP-43 nuclear granules cannot co-localize with the above nuclear bodies. Together, these results may broaden our understanding of the LLPS of ALS-related proteins in response to cellular stress.
Topics: Amyotrophic Lateral Sclerosis; Cell Nucleus; Humans; RNA-Binding Protein FUS
PubMed: 35858576
DOI: 10.1016/j.celrep.2022.111086 -
Cells Jun 2022Cells possess membraneless ribonucleoprotein (RNP) granules, including stress granules, processing bodies, Cajal bodies, or paraspeckles, that play physiological or... (Review)
Review
Cells possess membraneless ribonucleoprotein (RNP) granules, including stress granules, processing bodies, Cajal bodies, or paraspeckles, that play physiological or pathological roles. RNP granules contain RNA and numerous RNA-binding proteins, transiently formed through the liquid-liquid phase separation. The assembly or disassembly of numerous RNP granules is strongly controlled to maintain their homeostasis and perform their cellular functions properly. Normal RNA granules are reversibly assembled, whereas abnormal RNP granules accumulate and associate with various neurodegenerative diseases. This review summarizes current studies on the physiological or pathological roles of post-translational modifications of various cellular RNP granules and discusses the therapeutic methods in curing diseases related to abnormal RNP granules by autophagy.
Topics: Cytoplasmic Granules; Cytoplasmic Ribonucleoprotein Granules; Protein Processing, Post-Translational; RNA-Binding Proteins; Ribonucleoproteins
PubMed: 35805146
DOI: 10.3390/cells11132063 -
Bioengineered Apr 2022Dysfunction of intestinal epithelial cells (IECs) leads to intestinal epithelial barrier damage and critically involves in the pathogenesis and development of ulcerative...
Inhibition of LncRNA-NEAT1 alleviates intestinal epithelial cells (IECs) dysfunction in ulcerative colitis by maintaining the homeostasis of the glucose metabolism through the miR-410-3p-LDHA axis.
Dysfunction of intestinal epithelial cells (IECs) leads to intestinal epithelial barrier damage and critically involves in the pathogenesis and development of ulcerative colitis (UC). Accumulating studies revealed essential functions of non-coding RNAs in UC. LncRNA NEAT1 (long non-coding RNA nuclear paraspeckle assembly transcript 1) is frequently dysregulated in diverse human diseases. Currently, the precise roles of NEAT1 in the dysfunction of IECs during UC remain unclear. We report NEAT1 was significantly upregulated in IECs from UC patients. In addition, microRNA-410-3p was remarkedly suppressed in IECs from UC patients. Silencing NEAT1 effectively ameliorates the LPS-induced IECs dysfunction. Bioinformatical analysis, RNA pull-down and luciferase assays illustrated that NEAT1 sponged miR-410-3p to downregulate its expression in IECs. Interestingly, the glucose metabolism was obviously elevated in IECs from UC compared with normal colon tissues. Furthermore, NEAT1 promoted and miR-410-3p suppressed glucose metabolism of IECs. We identified lactate dehydrogenase A (LDHA), a glucose metabolism key enzyme, was a direct target of miR-410-3p in IECs. Rescue experiments verified that restoration of miR-410-3p in NEAT1-overexpressing IECs successfully overcame the NEAT1-promoted cell death under LPS treatment by targeting LDHA. In summary, these results unveiled new roles and molecular mechanisms for the NEAT1-mediated IECs dysfunction during the ulcerative colitis.
Topics: Colitis, Ulcerative; Epithelial Cells; Glucose; Homeostasis; Humans; Lactate Dehydrogenase 5; Lipopolysaccharides; MicroRNAs; RNA, Long Noncoding
PubMed: 35735114
DOI: 10.1080/21655979.2022.2037957 -
Frontiers in Molecular Biosciences 2022Cellular stress can induce DNA lesions that threaten the stability of genes. The DNA damage response (DDR) recognises and repairs broken DNA to maintain genome...
Cellular stress can induce DNA lesions that threaten the stability of genes. The DNA damage response (DDR) recognises and repairs broken DNA to maintain genome stability. Intriguingly, components of nuclear paraspeckles like the non-POU domain containing octamer-binding protein (NONO) participate in the repair of DNA double-strand breaks (DSBs). NONO is a multifunctional RNA-binding protein (RBP) that facilitates the retention and editing of messenger (m)RNA as well as pre-mRNA processing. However, the role of NONO in the DDR is poorly understood. Here, we establish a novel human U2OS cell line that expresses NONO fused to the engineered ascorbate peroxidase 2 (U2OS:NONO-APEX2-HA). We show that NONO-APEX2-HA accumulates in the nucleolus in response to DNA damage. Combining viability assays, subcellular localisation studies, coimmunoprecipitation experiments and proximity labeling, we demonstrate that NONO-APEX2-HA is a stably expressed fusion protein that mimics endogenous NONO in terms of expression, localisation and interactors. We propose that proximity labeling in U2OS:NONO-APEX2-HA cells is capable for the assessment of NONO interactomes by downstream assays. U2OS:NONO-APEX2-HA cells will likely be a valuable resource for the investigation of NONO interactome dynamics in response to DNA damage and other stimuli.
PubMed: 35733943
DOI: 10.3389/fmolb.2022.914873 -
The Kaohsiung Journal of Medical... Aug 2022The objective of the present study was to explore the function and mechanism of long noncoding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) in...
The objective of the present study was to explore the function and mechanism of long noncoding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) in pulmonary fibrosis (PF) progression. HPAEpic cells and A549 cells were exposed to hypoxic conditions to establish an in vitro model. Cell apoptosis was detected by TUNEL assay, and inflammatory cytokine levels were detected by ELISA. Gene and protein expression levels were identified by qRT-PCR and Western blot assays, respectively. The interaction among NEAT1, miR-29a, and NFATc3 was identified by dual-luciferase reporter and RNA pull-down assays. In hypoxia-treated cells, hypoxia markers (HIF-1α and HIF-2α), cytokines (TNF-α, IL-1β, and IL-6) and fibrotic markers (α-SMA, collagen I and collagen III) were significantly enhanced. Consistently, the expression levels of NEAT1 and NFATc3 were increased, but miR-29a was decreased in hypoxia-stimulated cells. Knockdown of NEAT1 significantly decreased cell apoptosis and the releases of TNF-α, IL-1β, and IL-6 as well as reduced the levels of α-SMA, collagen I, and collagen III. Moreover, NEAT1 positively regulated NFATc3 expression by directly targeting miR-29a. Functional experiments showed that the anti-apoptotic, anti-inflammatory, and anti-fibrotic effects mediated by NETA1 silencing were impeded by miR-29a inhibition or NFATc3 overexpression in hypoxia-stimulated HPAEpic and A549 cells. Collectively, these data demonstrated that NEAT1 knockdown inhibited hypoxia-induced cell apoptosis, inflammation, and fibrosis by targeting the miR-29a/NFATc3 axis in PF, suggesting that NEAT1 might be a potential therapeutic target for relieving PF progression.
Topics: Alveolar Epithelial Cells; Apoptosis; Fibrosis; Humans; Hypoxia; Inflammation; Interleukin-6; MicroRNAs; NFATC Transcription Factors; RNA, Long Noncoding; Tumor Necrosis Factor-alpha
PubMed: 35708150
DOI: 10.1002/kjm2.12535 -
Technology in Cancer Research &... 2022Long noncoding RNAs have been associated with various types of malignant tumors; however, the specific role of long noncoding RNAs in tumorigenesis still remains...
Long noncoding RNAs have been associated with various types of malignant tumors; however, the specific role of long noncoding RNAs in tumorigenesis still remains unclear in colorectal cancer. Here, we aim to elucidate the role of long noncoding RNA nuclear paraspeckle assembly transcript 1 in the malignant progression of colorectal cancer and investigate its underlying mechanisms. Real-time polymerase chain reaction was used to detect the expression of nuclear paraspeckle assembly transcript 1 in colorectal cancer tissues and cells. Cell Counting Kit-8 assay was used to determine the effect of nuclear paraspeckle assembly transcript 1 in proliferation. Transwell assay was used to explore the role of nuclear paraspeckle assembly transcript 1 in metastasis. Bioinformatics method was used to predict the core nuclear paraspeckle assembly transcript 1 interaction network. Real-time polymerase chain reaction was used to detect nuclear paraspeckle assembly transcript 1 and miR-448 expression levels. Western blotting was used to detect the expression levels of ZEB1. Luciferase assay was used to verify the relationship among nuclear paraspeckle assembly transcript 1, miR-448, and ZEB1. The effect of nuclear paraspeckle assembly transcript 1 on tumor growth was detected by tumorigenesis test in nude mice. Long noncoding RNA-nuclear paraspeckle assembly transcript 1 was up-regulated in colorectal cancer tissues and cells. Knocking down of nuclear paraspeckle assembly transcript 1 can suppress colorectal cancer proliferation and invasion, and caused a reduction of ZEB1 expression and an increase of miR-448 expression. Furthermore, knockdown of nuclear paraspeckle assembly transcript 1 regulated miR-448/ZEB1 axis to inhibit the expression of ZEB1. miR-448 silencing can reverse the effect of nuclear paraspeckle assembly transcript 1 knockdown. Our result demonstrated that long noncoding RNA nuclear paraspeckle assembly transcript 1 promotes proliferation and invasion of colorectal cancer by targeting miR-448 to promote the expression of ZEB1, which may play a significant role in the tumorigenesis of colorectal cancer.
Topics: Animals; Apoptosis; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; MicroRNAs; RNA, Long Noncoding; Zinc Finger E-box-Binding Homeobox 1
PubMed: 35695254
DOI: 10.1177/15330338221085348 -
RNA (New York, N.Y.) Aug 2022Paraspeckles are mammalian-specific nuclear bodies built on the long noncoding RNA The molecular mechanisms of paraspeckle formation have been mainly studied using...
Paraspeckles are mammalian-specific nuclear bodies built on the long noncoding RNA The molecular mechanisms of paraspeckle formation have been mainly studied using human or mouse cells, and it is not known if the same molecular components are involved in the formation of paraspeckles in other mammalian species. We thus investigated the expression pattern of in naked mole-rats (n), which exhibit extreme longevity and lower susceptibility to cancer. In the intestine, n is widely expressed along the entire intestinal epithelium, which is different from the expression of m that is restricted to the cells of the distal tip in mice. Notably, the expression of FUS, a FET family RNA binding protein, essential for the formation of paraspeckles both in humans and mice, was absent in the distal part of the intestinal epithelium in naked mole-rats. Instead, mRNAs of other FET family proteins EWSR1 and TAF15 were expressed in the distal region. Exogenous expression of these proteins in Fus-deficient murine embryonic fibroblast cells rescued the formation of paraspeckles. These observations suggest that n recruits a different set of RNA binding proteins in a cell type-specific manner during the formation of paraspeckles in different organisms.
Topics: Animals; Humans; Intestinal Mucosa; Mice; Mole Rats; Paraspeckles; RNA, Long Noncoding; RNA-Binding Proteins
PubMed: 35654483
DOI: 10.1261/rna.079135.122 -
Nucleic Acids Research Jun 2022UPF3 is a key nonsense-mediated mRNA decay (NMD) factor required for mRNA surveillance and eukaryotic gene expression regulation. UPF3 exists as two paralogs (A and B)...
Structures of nonsense-mediated mRNA decay factors UPF3B and UPF3A in complex with UPF2 reveal molecular basis for competitive binding and for neurodevelopmental disorder-causing mutation.
UPF3 is a key nonsense-mediated mRNA decay (NMD) factor required for mRNA surveillance and eukaryotic gene expression regulation. UPF3 exists as two paralogs (A and B) which are differentially expressed depending on cell type and developmental stage and believed to regulate NMD activity based on cellular requirements. UPF3B mutations cause intellectual disability. The underlying molecular mechanisms remain elusive, as many of the mutations lie in the poorly characterized middle-domain of UPF3B. Here, we show that UPF3A and UPF3B share structural and functional homology to paraspeckle proteins comprising an RNA-recognition motif-like domain (RRM-L), a NONA/paraspeckle-like domain (NOPS-L), and extended α-helical domain. These domains are essential for RNA/ribosome-binding, RNA-induced oligomerization and UPF2 interaction. Structures of UPF2's third middle-domain of eukaryotic initiation factor 4G (MIF4GIII) in complex with either UPF3B or UPF3A reveal unexpectedly intimate binding interfaces. UPF3B's disease-causing mutation Y160D in the NOPS-L domain displaces Y160 from a hydrophobic cleft in UPF2 reducing the binding affinity ∼40-fold compared to wildtype. UPF3A, which is upregulated in patients with the UPF3B-Y160D mutation, binds UPF2 with ∼10-fold higher affinity than UPF3B reliant mainly on NOPS-L residues. Our characterization of RNA- and UPF2-binding by UPF3's middle-domain elucidates its essential role in NMD.
Topics: Binding, Competitive; Humans; Intellectual Disability; Mutation; Nonsense Mediated mRNA Decay; RNA; RNA-Binding Proteins
PubMed: 35640974
DOI: 10.1093/nar/gkac421 -
Molecular & Cellular Proteomics : MCP Jul 2022Protein arginine (R) methylation is a post-translational modification involved in various biological processes, such as RNA splicing, DNA repair, immune response, signal...
Protein arginine (R) methylation is a post-translational modification involved in various biological processes, such as RNA splicing, DNA repair, immune response, signal transduction, and tumor development. Although several advancements were made in the study of this modification by mass spectrometry, researchers still face the problem of a high false discovery rate. We present a dataset of high-quality methylations obtained from several different heavy methyl stable isotope labeling with amino acids in cell culture experiments analyzed with a machine learning-based tool and show that this model allows for improved high-confidence identification of real methyl-peptides. Overall, our results are consistent with the notion that protein R methylation modulates protein-RNA interactions and suggest a role in rewiring protein-protein interactions, for which we provide experimental evidence for a representative case (i.e., NONO [non-POU domain-containing octamer-binding protein]-paraspeckle component 1 [PSPC1]). Upon intersecting our R-methyl-sites dataset with the PhosphoSitePlus phosphorylation dataset, we observed that R methylation correlates differently with S/T-Y phosphorylation in response to various stimuli. Finally, we explored the application of heavy methyl stable isotope labeling with amino acids in cell culture to identify unconventional methylated residues and successfully identified novel histone methylation marks on serine 28 and threonine 32 of H3. The database generated, named ProMetheusDB, is freely accessible at https://bioserver.ieo.it/shiny/app/prometheusdb.
Topics: Amino Acids; Humans; Isotope Labeling; Mass Spectrometry; Methylation; Protein Processing, Post-Translational; Proteome; RNA-Binding Proteins
PubMed: 35577067
DOI: 10.1016/j.mcpro.2022.100243