Depletion of NEAT1 lncRNA attenuates nucleolar stress by releasing sequestered P54nrb and PSF to facilitate c-Myc translation

Shen, Wen‐Hui; Liang, Xiaojing; Sun, Hong; Hoyos, Cheryl Li De; Crooke, Stanley T.

doi:10.1371/journal.pone.0173494

Cited by 24 publications

(26 citation statements)

References 30 publications

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“…Nucleolar clearance of RNase H1 was also observed in cells treated with another RNAP I inhibitor, CX5461. CX5461 disrupts the SL1-rDNA interaction and induces nucleolar segregation ( 48 , 49 ). A 6-h exposure of HeLa cells to 250 nM CX5461 reduced levels of 47S pre-rRNA by 75% relative to untreated cells (Figure 1E ) and induced nucleolar clearance of RNase H1 (Figure 1F ).…”

Section: Resultsmentioning

confidence: 99%

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Dynamic nucleoplasmic and nucleolar localization of mammalian RNase H1 in response to RNAP I transcriptional R-loops

Shen

Sun

Hoyos

et al. 2017

Nucleic Acids Research

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An R-loop is a DNA:RNA hybrid formed during transcription when a DNA duplex is invaded by a nascent RNA transcript. R-loops accumulate in nucleoli during RNA polymerase I (RNAP I) transcription. Here, we report that mammalian RNase H1 enriches in nucleoli and co-localizes with R-loops in cultured human cells. Co-migration of RNase H1 and R-loops from nucleoli to perinucleolar ring structures was observed upon inhibition of RNAP I transcription. Treatment with camptothecin which transiently stabilized nucleolar R-loops recruited RNase H1 to the nucleoli. It has been reported that the absence of Topoisomerase and RNase H activity in Escherichia coli or Saccharomyces cerevisiae caused R-loop accumulation along rDNA. We found that the distribution of RNase H1 and Top1 along rDNA coincided at sites where R-loops accumulated in mammalian cells. Loss of either RNase H1 or Top1 caused R-loop accumulation, and the accumulation of R-loops was exacerbated when both proteins were depleted. Importantly, we observed that protein levels of Top1 were negatively correlated with the abundance of RNase H1. We conclude that Top1 and RNase H1 are partially functionally redundant in mammalian cells to suppress RNAP I transcription-associate R-loops.

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Section: Resultsmentioning

confidence: 99%

“…Importantly, p54nrb and PSF are involved directly in DSB repair through the non-homologous end joining (NHEJ) pathway ( 69 ). Simultaneous depletion of p54nrb and PSF induce p53-dependent apoptotic cell death ( 48 ).…”

Section: Resultsmentioning

confidence: 99%

Dynamic nucleoplasmic and nucleolar localization of mammalian RNase H1 in response to RNAP I transcriptional R-loops

Shen

Sun

Hoyos

et al. 2017

Nucleic Acids Research

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“…DBHS proteins are involved in many important cellular processes including transcription, splicing, polyadenylation, DNA repair, translation and apoptosis ( 24–29 ). Cellular levels of DBHS proteins are highly correlated with cell survival ( 28 , 30 ). As a result, degradation of DBHS proteins upon binding to the 2′ F PS-ASO may at least partially contribute to the observed cytotoxic effects.…”

Section: Introductionmentioning

confidence: 99%

Acute hepatotoxicity of 2′ fluoro-modified 5–10–5 gapmer phosphorothioate oligonucleotides in mice correlates with intracellular protein binding and the loss of DBHS proteins

Shen

Hoyos

Sun

et al. 2018

Nucleic Acids Research

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We reported previously that a 2′ fluoro-modified (2′ F) phosphorothioate (PS) antisense oligonucleotides (ASOs) with 5–10–5 gapmer configuration interacted with proteins from Drosophila behavior/human splicing (DBHS) family with higher affinity than PS-ASOs modified with 2′-O-(2-methoxyethyl) (2′ MOE) or 2′,4′-constrained 2′-O-ethyl (cEt) did. Rapid degradation of these proteins and cytotoxicity were observed in cells treated with 2′ F PS-ASO. Here, we report that 2′ F gapmer PS-ASOs of different sequences caused reduction in levels of DBHS proteins and hepatotoxicity in mice. 2′ F PS-ASOs induced activation of the P53 pathway and downregulation of metabolic pathways. Altered levels of RNA and protein markers for hepatotoxicity, liver necrosis, and apoptosis were observed as early as 24 to 48 hours after a single administration of the 2′ F PS-ASO. The observed effects were not likely due to the hybridization-dependent RNase H1 cleavage of on- or potential off-target RNAs, or due to potential toxicity of 2′ F nucleoside metabolites. Instead, we found that 2′ F PS-ASO associated with more intra-cellular proteins including proteins from DBHS family. Our results suggest that protein-binding correlates positively with the 2′ F modification-dependent loss of DBHS proteins and the toxicity of gapmer 2′ F PS-ASO in vivo.

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“…One function of paraspeckles is the subnuclear sequestration of proteins, including NONO, thereby lowering nucleoplasmic levels and inhibiting the downstream target genes of these proteins [27,47,48]. To determine NONO sequestration, we quantified the NONO immunofluorescence signal in the experiments with NEAT1_2 FISH (Fig 3E).…”

Section: Resultsmentioning

confidence: 99%

PolyA-modulating antisense oligonucleotides reveal opposing functions for long non-coding RNA NEAT1 isoforms in neuroblastoma

Naveed

Cooper

et al. 2020

Preprint

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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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Depletion of NEAT1 lncRNA attenuates nucleolar stress by releasing sequestered P54nrb and PSF to facilitate c-Myc translation

Cited by 24 publications

References 30 publications

Dynamic nucleoplasmic and nucleolar localization of mammalian RNase H1 in response to RNAP I transcriptional R-loops

Dynamic nucleoplasmic and nucleolar localization of mammalian RNase H1 in response to RNAP I transcriptional R-loops

Acute hepatotoxicity of 2′ fluoro-modified 5–10–5 gapmer phosphorothioate oligonucleotides in mice correlates with intracellular protein binding and the loss of DBHS proteins

PolyA-modulating antisense oligonucleotides reveal opposing functions for long non-coding RNA NEAT1 isoforms in neuroblastoma

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