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G-quadruplexes offer a conserved structural motif for NONO recruitment to NEAT1 architectural lncRNA.

ABSTRACT: The long non-coding RNA NEAT1 serves as a scaffold for the assembly of paraspeckles, membraneless nuclear organelles involved in gene regulation. Paraspeckle assembly requires NEAT1 recruitment of the RNA-binding protein NONO, however the NEAT1 elements responsible for recruitment are unknown. Herein we present evidence that previously unrecognized structural features of NEAT1 serve an important role in these interactions. Led by the initial observation that NONO preferentially binds the G-quadruplex conformation of G-rich C9orf72 repeat RNA, we find that G-quadruplex motifs are abundant and conserved features of NEAT1. Furthermore, we determine that NONO binds NEAT1 G-quadruplexes with structural specificity and provide evidence that G-quadruplex motifs mediate NONO-NEAT1 association, with NONO binding sites on NEAT1 corresponding largely to G-quadruplex motifs, and treatment with a G-quadruplex-disrupting small molecule causing dissociation of native NONO-NEAT1 complexes. Together, these findings position G-quadruplexes as a primary candidate for the NONO-recruiting elements of NEAT1 and provide a framework for further investigation into the role of G-quadruplexes in paraspeckle formation and function.

SUBMITTER: Simko EAJ

PROVIDER: S-EPMC7367201 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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G-quadruplexes offer a conserved structural motif for NONO recruitment to NEAT1 architectural lncRNA.

Simko Eric A J EAJ Liu Honghe H Zhang Tao T Velasquez Adan A Teli Shraddha S Haeusler Aaron R AR Wang Jiou J

Nucleic acids research 20200701 13

The long non-coding RNA NEAT1 serves as a scaffold for the assembly of paraspeckles, membraneless nuclear organelles involved in gene regulation. Paraspeckle assembly requires NEAT1 recruitment of the RNA-binding protein NONO, however the NEAT1 elements responsible for recruitment are unknown. Herein we present evidence that previously unrecognized structural features of NEAT1 serve an important role in these interactions. Led by the initial observation that NONO preferentially binds the G-quadr ...[more]

PMID: 32496517

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Project description:BackgroundInflammatory bowel disease (IBD) is a chronic nonspecific intestinal disease. Our previous work showed that long non-coding RNA (LncRNA) nuclear enriched abundant transcript 1 (NEAT1) plays an important role in IBD. In the current study, we aimed to explore the underlying mechanism by which NEAT1 participates in the development of the disease.MethodsReal-time quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to detect the expression of NEAT1 and tumor necrosis factor superfamily member 1B (TNFRSF1B) in clinical specimens and dextran sulfate sodium (DSS) colitis mice. Inflammatory cell models were established by stimulating human normal intestinal epithelial cell line NCM460 and human colon cancer cell line HT-29 with tumor necrosis factor alpha (TNF-α). Expressions of inflammatory cytokines such as interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) were detected by enzyme-linked immunosorbent assay (ELISA) or RT-qPCR, TNFRSF1B, NF-κB p65 and p-NF-κB p65 followed by the knockdown or overexpression of NEAT1 and TNFRSF1B were analyzed by western blotting, and the regulatory effects of NEAT1 on TNFRSF1B were detected by RNA pull-down experiments and RNA-decay assay. The translocation of NF-κB p65 to the nucleus was detected by immunofluorescence.ResultsIn patients' specimens and DSS colitis mouse models, NEAT1 and TNFRSF1B expression were up-regulated compared with the control group. TNF-α stimulation increased NEAT1 and TNFRSF1B expression and activated NF-κB signaling pathway by increasing the translocation of NF-κB p65 to the nucleus. In the presence of TNF-α stimulation, NEAT1 knockdown reduces the expression of TNFRSF1B and the translocation of NF-κB p65, thereby relieves cell inflammation. These effects can be reversed by the overexpression of TNFRSF1B.In addition, NEAT1 is involved in inflammatory response by up-regulating the mRNA levels of TNFRSF1B, and knocking down NEAT1 can alleviate inflammation by down-regulating TNFRSF1B. Moreover, NEAT1 co-precipitates TNFRSF1B mRNA in RNA-pulldown assay, and the presence of NEAT1 stabilizes the mRNA of TNFRSF1B.ConclusionsOur results showed that LncRNA NEAT1 promotes NF-κB p65 translocation and mediates intestinal inflammation by regulating TNFRSF1B.

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G-quadruplexes offer a conserved structural motif for NONO recruitment to NEAT1 architectural lncRNA.

Publications

G-quadruplexes offer a conserved structural motif for NONO recruitment to NEAT1 architectural lncRNA.

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