RESUMO
Long noncoding RNAs (lncRNAs) are >200 nt RNA transcripts without protein-coding potential. LncRNAs can be categorized into intergenic, intronic, bidirectional, sense, and antisense lncRNAs based on the genomic localization to nearby protein-coding genes. The current CRISPR-based lncRNA knockout strategy works efficiently for lncRNAs distant from the protein-coding gene, whereas it causes genomic perturbance inevitably due to technical limitations. In this study, we introduce a novel lncRNA knockout strategy, BESST, by deleting the genomic DNA fragment from the branch point to the 3' splicing site in the last intron of the target lncRNA. The BESST knockout exhibited comparable or superior repressive efficiency to RNA silencing or conventional promoter-exon1 deletion. Significantly, the BESST knockout strategy minimized the intervention of adjacent/overlap protein-coding genes by removing an average of â¼130 bp from genomic DNA. Our data also found that the BESST knockout strategy causes lncRNA nuclear retention, resulting in decapping and deadenylation of the lncRNA poly(A) tail. Further study revealed that PABPN1 is essential for the BESST-mediated decay and subsequent poly(A) deadenylation and decapping. Together, the BESST knockout strategy provides a versatile tool for investigating gene function by generating knockout cells or animals with high specificity and efficiency.
Assuntos
Técnicas de Inativação de Genes , Genoma , Genômica , RNA Longo não Codificante , Animais , Éxons/genética , Técnicas de Inativação de Genes/métodos , Técnicas de Inativação de Genes/normas , Genoma/genética , Poli A/genética , Poli A/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , Regiões Promotoras Genéticas/genética , RNA Longo não Codificante/genéticaRESUMO
The accumulation of intracellular ß-amyloid peptide (Aß) is important pathological characteristic of Alzheimer's disease (AD). However, the exact underlying molecular mechanism remains to be elucidated. Here, we reported that Nuclear Paraspeckle Assembly Transcript 1 (NEAT1), a long n on-coding RNA, exhibits repressed expression in the early stage of AD and its down-regulation declines neuroglial cell mediating Aß clearance via inhibiting expression of endocytosis-related genes. We find that NEAT1 is associated with P300/CBP complex and its inhibition affects H3K27 acetylation (H3K27Ac) and H3K27 crotonylation (H3K27Cro) located nearby to the transcription start site of many genes, including endocytosis-related genes. Interestingly, NEAT1 inhibition down-regulates H3K27Ac but up-regulates H3K27Cro through repression of acetyl-CoA generation. NEAT1 also mediates the binding between STAT3 and H3K27Ac but not H3K27Cro. Therefore, the decrease of H3K27Ac and/or the increase of H3K27Cro declines expression of multiple related genes. Collectively, this study first reveals the different roles of H3K27Ac and H3K27Cro in regulation of gene expression and provides the insight of the epigenetic regulatory mechanism of NEAT1 in gene expression and AD pathology.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , RNA Longo não Codificante/metabolismo , Acetilcoenzima A/metabolismo , Acetilação/efeitos dos fármacos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/farmacologia , Animais , Caveolina 2/antagonistas & inibidores , Caveolina 2/genética , Caveolina 2/metabolismo , Modelos Animais de Doenças , Epigênese Genética , Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Camundongos , Camundongos Transgênicos , Neuroglia/citologia , Neuroglia/metabolismo , Fragmentos de Peptídeos/farmacologia , Interferência de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator de Crescimento Transformador beta2/antagonistas & inibidores , Fator de Crescimento Transformador beta2/genética , Fator de Crescimento Transformador beta2/metabolismo , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Nuclear paraspeckle assembly transcript 1 (NEAT1) is the crucial structural platform of paraspeckles, which is one type of nuclear bodies. As a stress-induced lncRNA, the expression of NEAT1 increases in response to viral infection, but little is known about the role of NEAT1 or paraspeckles in the replication of herpes simplex virus-1 (HSV-1). Here, we demonstrate that HSV-1 infection increases NEAT1 expression and paraspeckle formation in a STAT3-dependent manner. NEAT1 and other paraspeckle protein components, P54nrb and PSPC1, can associate with HSV-1 genomic DNA. By binding with STAT3, PSPC1 is required for the recruitment of STAT3 to paraspeckles and facilitates the interaction between STAT3 and viral gene promoters, finally increasing viral gene expression and viral replication. Furthermore, thermosensitive gel containing NEAT1 siRNA or STAT3 siRNA effectively healed the skin lesions caused by HSV-1 infection in mice. Our results provide insight into the roles of lncRNAs in the epigenetic control of viral genes and into the function of paraspeckles.
Assuntos
Genes Virais , Herpesvirus Humano 1/fisiologia , RNA Longo não Codificante/metabolismo , Transcrição Gênica , Replicação Viral/genética , Animais , Sequência de Bases , Proteínas de Ligação a DNA , Regulação Viral da Expressão Gênica , Células HeLa , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Humanos , Corpos de Inclusão Intranuclear/metabolismo , Camundongos , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição de Octâmero/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
Nuclear paraspeckles assembly transcript 1 (NEAT1) is a well-known long noncoding RNA (lncRNA) with various functions in different physiological and pathological processes. Notably, aberrant NEAT1 expression is implicated in the pathogenesis of various neurodegenerative diseases, including Alzheimer's disease (AD). However, the molecular mechanism of NEAT1 in AD remains poorly understood. In this study, we investigated that NEAT1 regulated microtubules (MTs) polymerization via FZD3/GSK3ß/p-tau pathway. Downregulation of NEAT1 inhibited Frizzled Class Receptor 3 (FZD3) transcription activity by suppressing H3K27 acetylation (H3K27Ac) at the FZD3 promoter. Our data also demonstrated that P300, an important histone acetyltransferases (HAT), recruited by NEAT1 to bind to FZD3 promoter and mediated its transcription via regulating histone acetylation. In addition, according to immunofluorescence staining of MTs, metformin, a medicine for the treatment of diabetes mellitus, rescued the reduced length of neurites detected in NEAT1 silencing cells. We suspected that metformin may play a neuroprotective role in early AD by increasing NEAT1 expression and through FZD3/GSK3ß/p-tau pathway. Collectively, NEAT1 regulates microtubule stabilization via FZD3/GSK3ß/P-tau pathway and influences FZD3 transcription activity in the epigenetic way.
Assuntos
Doença de Alzheimer/enzimologia , Receptores Frizzled/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Hipocampo/enzimologia , Microtúbulos/enzimologia , Neurônios/enzimologia , RNA Longo não Codificante/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/genética , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Receptores Frizzled/genética , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Humanos , Metformina/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microtúbulos/efeitos dos fármacos , Microtúbulos/genética , Microtúbulos/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Fosforilação , Presenilina-1/genética , RNA Longo não Codificante/genética , Transdução de SinaisRESUMO
Long non-coding RNAs (lncRNAs) are found not only in mammals but also in other organisms, including viruses. Recent findings suggest that lncRNAs play various regulatory roles in multiple major biological and pathological processes. During viral life cycles, lncRNAs are involved in a series of steps, including enhancing viral gene expression, promoting viral replication and genome packaging, boosting virion release, maintaining viral latency and assisting viral transformation; additionally, lncRNAs antagonize host antiviral innate immune responses. In contrast to proteins that function in viral infection, lncRNAs are expected to be novel targets for the modulation of all types of biochemical processes due to their broad characteristics and profound influence. This review highlights our current understanding of the regulatory roles of lncRNAs during viral infection processes with an emphasis on the potential usefulness of lncRNAs as a target for viral intervention strategies, which could have therapeutic implications for the application of a clinical approach for the treatment of viral diseases.