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1.
Life Sci Alliance ; 7(8)2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38843934

RESUMO

RNA-binding proteins are frequently deregulated in cancer and emerge as effectors of the DNA damage response (DDR). The non-POU domain-containing octamer-binding protein NONO/p54nrb is a multifunctional RNA-binding protein that not only modulates the production and processing of mRNA, but also promotes the repair of DNA double-strand breaks (DSBs). Here, we investigate the impact of Nono deletion in the murine KP (KRas G12D , Trp53 -/- ) cell-based lung cancer model. We show that the deletion of Nono impairs the response to DNA damage induced by the topoisomerase II inhibitor etoposide or the radiomimetic drug bleomycin. Nono-deficient KP (KPN) cells display hyperactivation of DSB signalling and high levels of DSBs. The defects in the DDR are accompanied by reduced RNA polymerase II promoter occupancy, impaired nascent RNA synthesis, and attenuated induction of the DDR factor growth arrest and DNA damage-inducible beta (Gadd45b). Our data characterise Gadd45b as a putative Nono-dependent effector of the DDR and suggest that Nono mediates a genome-protective crosstalk of the DDR with the RNA metabolism via induction of Gadd45b.


Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Ligação a RNA , Animais , Camundongos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Quebras de DNA de Cadeia Dupla , Antígenos de Diferenciação/metabolismo , Antígenos de Diferenciação/genética , Bleomicina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Etoposídeo/farmacologia , Transdução de Sinais , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , RNA Polimerase II/metabolismo , Humanos , Proteínas GADD45
2.
Nucleic Acids Res ; 52(6): 3050-3068, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38224452

RESUMO

RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localization to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localization is poorly understood. Here we show that the topoisomerase II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-inducible antisense intergenic non-coding RNA (asincRNA) in human cancer cells. Such transcripts originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in an RNA recognition motif 1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signalling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.


Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Humanos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Etoposídeo/farmacologia , Precursores de RNA/metabolismo , Fatores de Transcrição/metabolismo , DNA , Proteínas de Ligação a RNA/metabolismo
3.
Front Mol Biosci ; 9: 914873, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35733943

RESUMO

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 in vivo proximity labeling, we demonstrate that NONO-APEX2-HA is a stably expressed fusion protein that mimics endogenous NONO in terms of expression, localisation and bona fide interactors. We propose that in vivo 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.

4.
Int J Mol Sci ; 23(8)2022 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-35457249

RESUMO

The nuclear paraspeckle assembly transcript 1 (NEAT1) locus encodes two long non-coding (lnc)RNA isoforms that are upregulated in many tumours and dynamically expressed in response to stress. NEAT1 transcripts form ribonucleoprotein complexes with numerous RNA-binding proteins (RBPs) to assemble paraspeckles and modulate the localisation and activity of gene regulatory enzymes as well as a subset of messenger (m)RNA transcripts. The investigation of the dynamic composition of NEAT1-associated proteins and mRNAs is critical to understand the function of NEAT1. Interestingly, a growing number of biochemical and genetic tools to assess NEAT1 interactomes has been reported. Here, we discuss the Hybridisation Proximity (HyPro) labeling technique in the context of NEAT1. HyPro labeling is a recently developed method to detect spatially ordered interactions of RNA-containing nuclear compartments in cultured human cells. After introducing NEAT1 and paraspeckles, we describe the advantages of the HyPro technology in the context of other methods to study RNA interactomes, and review the key findings in mapping NEAT1-associated RNA transcripts and protein binding partners. We further discuss the limitations and potential improvements of HyPro labeling, and conclude by delineating its applicability in paraspeckles-related cancer research.


Assuntos
RNA Longo não Codificante , Núcleo Celular/genética , Núcleo Celular/metabolismo , Regulação da Expressão Gênica , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
5.
Noncoding RNA ; 7(3)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34287370

RESUMO

Gene expression is an essential process for cellular growth, proliferation, and differentiation. The transcription of protein-coding genes and non-coding loci depends on RNA polymerases. Interestingly, numerous loci encode long non-coding (lnc)RNA transcripts that are transcribed by RNA polymerase II (RNAPII) and fine-tune the RNA metabolism. The nucleolus is a prime example of how different lncRNA species concomitantly regulate gene expression by facilitating the production and processing of ribosomal (r)RNA for ribosome biogenesis. Here, we summarise the current findings on how RNAPII influences nucleolar structure and function. We describe how RNAPII-dependent lncRNA can both promote nucleolar integrity and inhibit ribosomal (r)RNA synthesis by modulating the availability of rRNA synthesis factors in trans. Surprisingly, some lncRNA transcripts can directly originate from nucleolar loci and function in cis. The nucleolar intergenic spacer (IGS), for example, encodes nucleolar transcripts that counteract spurious rRNA synthesis in unperturbed cells. In response to DNA damage, RNAPII-dependent lncRNA originates directly at broken ribosomal (r)DNA loci and is processed into small ncRNA, possibly to modulate DNA repair. Thus, lncRNA-mediated regulation of nucleolar biology occurs by several modes of action and is more direct than anticipated, pointing to an intimate crosstalk of RNA metabolic events.

6.
Artigo em Inglês | MEDLINE | ID: mdl-31932468

RESUMO

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor that is frequently down-modulated in human cancer. PTEN inhibits the phosphatidylinositol 3-phosphate kinase (PI3K)/AKT pathway through its lipid phosphatase activity. Multiple PI3K/AKT-independent actions of PTEN, protein-phosphatase activities and functions within the nucleus have also been described. PTEN, therefore, regulates many cellular processes including cell proliferation, survival, genomic integrity, polarity, migration, and invasion. Even a modest decrease in the functional dose of PTEN may promote cancer development. Understanding the molecular and cellular mechanisms that regulate PTEN protein levels and function, and how these may go awry in cancer contexts, is, therefore, key to fully understanding the role of PTEN in tumorigenesis. Here, we discuss current knowledge on posttranslational control and conformational plasticity of PTEN, as well as therapeutic possibilities toward reestablishment of PTEN tumor-suppressor activity in cancer.


Assuntos
Transformação Celular Neoplásica/genética , Neoplasias/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proliferação de Células/genética , Genes Supressores de Tumor , Humanos , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Transdução de Sinais/fisiologia
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