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2.
Nature ; 579(7798): 291-296, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32103174

RESUMO

The DNA-dependent protein kinase (DNA-PK), which comprises the KU heterodimer and a catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor1. KU binds to DNA ends, initiates cNHEJ, and recruits and activates DNA-PKcs. KU also binds to RNA, but the relevance of this interaction in mammals is unclear. Here we use mouse models to show that DNA-PK has an unexpected role in the biogenesis of ribosomal RNA (rRNA) and in haematopoiesis. The expression of kinase-dead DNA-PKcs abrogates cNHEJ2. However, most mice that both expressed kinase-dead DNA-PKcs and lacked the tumour suppressor TP53 developed myeloid disease, whereas all other previously characterized mice deficient in both cNHEJ and TP53 expression succumbed to pro-B cell lymphoma3. DNA-PK autophosphorylates DNA-PKcs, which is its best characterized substrate. Blocking the phosphorylation of DNA-PKcs at the T2609 cluster, but not the S2056 cluster, led to KU-dependent defects in 18S rRNA processing, compromised global protein synthesis in haematopoietic cells and caused bone marrow failure in mice. KU drives the assembly of DNA-PKcs on a wide range of cellular RNAs, including the U3 small nucleolar RNA, which is essential for processing of 18S rRNA4. U3 activates purified DNA-PK and triggers phosphorylation of DNA-PKcs at T2609. DNA-PK, but not other cNHEJ factors, resides in nucleoli in an rRNA-dependent manner and is co-purified with the small subunit processome. Together our data show that DNA-PK has RNA-dependent, cNHEJ-independent functions during ribosome biogenesis that require the kinase activity of DNA-PKcs and its phosphorylation at the T2609 cluster.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Hematopoese/genética , Autoantígeno Ku/metabolismo , Linfoma/enzimologia , Linfoma/fisiopatologia , RNA Ribossômico 18S/metabolismo , Proteínas de Ligação ao Cálcio/genética , Domínio Catalítico/fisiologia , Reparo do DNA/genética , Ativação Enzimática/genética , Células HeLa , Humanos , Linfoma/genética , Modelos Animais , Mutação , Fosforilação , Ligação Proteica , Biossíntese de Proteínas/genética , RNA Ribossômico 18S/genética , RNA Nucleolar Pequeno/metabolismo
3.
Nat Commun ; 11(1): 987, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080190

RESUMO

Longevity is influenced by genetic and environmental factors, but the underlying mechanisms remain elusive. Here, we functionally characterise a Drosophila small nucleolar RNA (snoRNA), named jouvence whose loss of function reduces lifespan. The genomic region of jouvence rescues the longevity in mutant, while its overexpression in wild-type increases lifespan. Jouvence is required in enterocytes. In mutant, the epithelium of the gut presents more hyperplasia, while the overexpression of jouvence prevents it. Molecularly, the mutant lack pseudouridylation on 18S and 28S-rRNA, a function rescued by targeted expression of jouvence in the gut. A transcriptomic analysis performed from the gut reveals that several genes are either up- or down-regulated, while restoring the mRNA level of two genes (ninaD or CG6296) rescue the longevity. Since snoRNAs are structurally and functionally well conserved throughout evolution, we identified putative jouvence orthologue in mammals including humans, suggesting that its function in longevity could be conserved.


Assuntos
Drosophila melanogaster/genética , Longevidade/genética , RNA Nucleolar Pequeno/genética , Animais , Animais Geneticamente Modificados , Sequência Conservada , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Enterócitos/metabolismo , Evolução Molecular , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Genoma de Inseto , Humanos , Mucosa Intestinal/metabolismo , Mutação com Perda de Função , Masculino , Camundongos , Conformação de Ácido Nucleico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/metabolismo , Receptores Imunológicos/genética
4.
Nat Commun ; 11(1): 168, 2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31924754

RESUMO

Variations in transcription start site (TSS) selection reflect diversity of preinitiation complexes and can impact on post-transcriptional RNA fates. Most metazoan polymerase II-transcribed genes carry canonical initiation with pyrimidine/purine (YR) dinucleotide, while translation machinery-associated genes carry polypyrimidine initiator (5'-TOP or TCT). By addressing the developmental regulation of TSS selection in zebrafish we uncovered a class of dual-initiation promoters in thousands of genes, including snoRNA host genes. 5'-TOP/TCT initiation is intertwined with canonical initiation and used divergently in hundreds of dual-initiation promoters during maternal to zygotic transition. Dual-initiation in snoRNA host genes selectively generates host and snoRNA with often different spatio-temporal expression. Dual-initiation promoters are pervasive in human and fruit fly, reflecting evolutionary conservation. We propose that dual-initiation on shared promoters represents a composite promoter architecture, which can function both coordinately and divergently to diversify RNAs.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Regiões Promotoras Genéticas/genética , Sítio de Iniciação de Transcrição , Transcrição Genética , Animais , Sequência de Bases , Drosophila/genética , Drosophila/crescimento & desenvolvimento , Humanos , RNA/genética , RNA/fisiologia , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/fisiologia , RNA não Traduzido/genética , RNA não Traduzido/fisiologia , Elementos Reguladores de Transcrição , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Zigoto
5.
Nucleic Acids Res ; 48(5): 2271-2286, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31980822

RESUMO

The study of RNA expression is the fastest growing area of genomic research. However, despite the dramatic increase in the number of sequenced transcriptomes, we still do not have accurate estimates of the number and expression levels of non-coding RNA genes. Non-coding transcripts are often overlooked due to incomplete genome annotation. In this study, we use annotation-independent detection of RNA reads generated using a reverse transcriptase with low structure bias to identify non-coding RNA. Transcripts between 20 and 500 nucleotides were filtered and crosschecked with non-coding RNA annotations revealing 111 non-annotated non-coding RNAs expressed in different cell lines and tissues. Inspecting the sequence and structural features of these transcripts indicated that 60% of these transcripts correspond to new snoRNA and tRNA-like genes. The identified genes exhibited features of their respective families in terms of structure, expression, conservation and response to depletion of interacting proteins. Together, our data reveal a new group of RNA that are difficult to detect using standard gene prediction and RNA sequencing techniques, suggesting that reliance on actual gene annotation and sequencing techniques distorts the perceived architecture of the human transcriptome.


Assuntos
Anotação de Sequência Molecular/métodos , RNA Mensageiro/genética , RNA Nucleolar Pequeno/genética , RNA de Transferência/genética , RNA não Traduzido/genética , Transcriptoma , Animais , Pareamento de Bases , Sequência de Bases , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Conformação de Ácido Nucleico , Filogenia , RNA Mensageiro/classificação , RNA Mensageiro/metabolismo , RNA Nucleolar Pequeno/classificação , RNA Nucleolar Pequeno/metabolismo , RNA de Transferência/classificação , RNA de Transferência/metabolismo , RNA não Traduzido/classificação , RNA não Traduzido/metabolismo , Análise de Sequência de RNA , Sequenciamento Completo do Exoma
6.
Nucleic Acids Res ; 48(7): 3848-3868, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-31996908

RESUMO

U3 snoRNA and the associated Rrp9/U3-55K protein are essential for 18S rRNA production by the SSU-processome complex. U3 and Rrp9 are required for early pre-rRNA cleavages at sites A0, A1 and A2, but the mechanism remains unclear. Substitution of Arg 289 in Rrp9 to Ala (R289A) specifically reduced cleavage at sites A1 and A2. Surprisingly, R289 is located on the surface of the Rrp9 ß-propeller structure opposite to U3 snoRNA. To understand this, we first characterized the protein-protein interaction network of Rrp9 within the SSU-processome. This identified a direct interaction between the Rrp9 ß-propeller domain and Rrp36, the strength of which was reduced by the R289A substitution, implicating this interaction in the observed processing phenotype. The Rrp9 R289A mutation also showed strong synergistic negative interactions with mutations in U3 that destabilize the U3/pre-rRNA base-pair interactions or reduce the length of their linking segments. We propose that the Rrp9 ß-propeller and U3/pre-rRNA binding cooperate in the structure or stability of the SSU-processome. Additionally, our analysis of U3 variants gave insights into the function of individual segments of the 5'-terminal 72-nt sequence of U3. We interpret these data in the light of recently reported SSU-processome structures.


Assuntos
Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico 18S/metabolismo , RNA Nucleolar Pequeno/química , Ribonucleoproteínas Nucleolares Pequenas/química , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , RNA Nucleolar Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
Nat Commun ; 10(1): 3401, 2019 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363086

RESUMO

Epitranscriptomic modifications of mRNA are important regulators of gene expression. While internal 2'-O-methylation (Nm) has been discovered on mRNA, questions remain about its origin and function in cells and organisms. Here, we show that internal Nm modification can be guided by small nucleolar RNAs (snoRNAs), and that these Nm sites can regulate mRNA and protein expression. Specifically, two box C/D snoRNAs (SNORDs) and the 2'-O-methyltransferase fibrillarin lead to Nm modification in the protein-coding region of peroxidasin (Pxdn). The presence of Nm modification increases Pxdn mRNA expression but inhibits its translation, regulating PXDN protein expression and enzyme activity both in vitro and in vivo. Our findings support a model in which snoRNA-guided Nm modifications of mRNA can regulate physiologic gene expression by altering mRNA levels and tuning protein translation.


Assuntos
Proteínas da Matriz Extracelular/genética , Peroxidase/genética , RNA Mensageiro/genética , RNA Nucleolar Pequeno/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Metilação , Metiltransferases/metabolismo , Peroxidase/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Nucleolar Pequeno/metabolismo
8.
Biomed Pharmacother ; 118: 109266, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31401397

RESUMO

BACKGROUND: Gallbladder cancer (GBC) ranks fifth in the most common malignancy of the gastrointestinal tract worldwide. It is reported many small nucleolar RNAs (SNORNs) could regulate the progression of GBC. To identify potential therapeutic targets for GBC, we conducted microarray analysis in GBC tissues and adjacent normal tissues. We found that SNORA21 was downregulated most in gallbladder tumor samples. Therefore, this research aimed to investigate the role of SNORA21 during the tumorigenesis of GBC. METHODS: The differential expression of SNORNs between GBC tissues and para-carcinoma tissues were examined by microarray analysis and that were confirmed by qRT-PCR. Cell proliferation was tested by CCK-8 and immunofluorescence. Cell apoptosis and cell cycle in GBC were detected by flow cytometry. Expression of proteins in GBC cells was measured by Western-blot. Transwell assay was used for testing the cell migration and invasion. Xenograft tumor model was established to verify the effect of SNORA21 overexpression on GBC in vivo. RESULTS: The results revealed that SNORA21 overexpression inhibited the proliferation, migration and invasion of GBC cells. Moreover, overexpression of SNORA21 significantly increased the expression of E-cadherin and decreased the levels of N-cadherin and vimentin. Meanwhile, overexpression of SNORA21 significantly induced apoptosis and G1 arrest of GBC cells. Finally, SNORA21 overexpression significantly suppressed the growth of gallbladder tumors in vivo. CONCLUSION: Overexpression of SNORA21 significantly suppressed the tumorigenesis of GBC in vitro and in vivo, which may serve as a potential novel target for the treatment of GBC.


Assuntos
Carcinogênese/genética , Neoplasias da Vesícula Biliar/genética , RNA Nucleolar Pequeno/metabolismo , Idoso , Animais , Apoptose/genética , Carcinogênese/patologia , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Neoplasias da Vesícula Biliar/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Invasividade Neoplásica , RNA Nucleolar Pequeno/genética , Regulação para Cima/genética
9.
Mol Cell ; 75(6): 1270-1285.e14, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31351877

RESUMO

PARP inhibitors (PARPi) prevent cancer cell growth by inducing synthetic lethality with DNA repair defects (e.g., in BRCA1/2 mutant cells). We have identified an alternative pathway for PARPi-mediated growth control in BRCA1/2-intact breast cancer cells involving rDNA transcription and ribosome biogenesis. PARP-1 binds to snoRNAs, which stimulate PARP-1 catalytic activity in the nucleolus independent of DNA damage. Activated PARP-1 ADP-ribosylates DDX21, an RNA helicase that localizes to nucleoli and promotes rDNA transcription when ADP-ribosylated. Treatment with PARPi or mutation of the ADP-ribosylation sites reduces DDX21 nucleolar localization, rDNA transcription, ribosome biogenesis, protein translation, and cell growth. The salient features of this pathway are evident in xenografts in mice and human breast cancer patient samples. Elevated levels of PARP-1 and nucleolar DDX21 are associated with cancer-related outcomes. Our studies provide a mechanistic rationale for efficacy of PARPi in cancer cells lacking defects in DNA repair whose growth is inhibited by PARPi.


Assuntos
Neoplasias da Mama/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas de Neoplasias/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , RNA Neoplásico/metabolismo , RNA Nucleolar Pequeno/metabolismo , Ribossomos/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , RNA Helicases DEAD-box/genética , Reparo do DNA , Feminino , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/genética , Poli(ADP-Ribose) Polimerase-1/genética , RNA Neoplásico/genética , RNA Nucleolar Pequeno/genética , Ribossomos/genética
10.
Nucleic Acids Res ; 47(14): 7548-7563, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31188444

RESUMO

Ribosome biogenesis is an essential process in all living cells, which entails countless highly sequential and dynamic structural reorganization events. These include formation of dozens RNA helices through Watson-Crick base-pairing within ribosomal RNAs (rRNAs) and between rRNAs and small nucleolar RNAs (snoRNAs), transient association of hundreds of proteinaceous assembly factors to nascent precursor (pre-)ribosomes, and stable assembly of ribosomal proteins. Unsurprisingly, the largest group of ribosome assembly factors are energy-consuming proteins (NTPases) including 25 RNA helicases in budding yeast. Among these, the DEAH-box Dhr1 is essential to displace the box C/D snoRNA U3 from the pre-rRNAs where it is bound in order to prevent premature formation of the central pseudoknot, a dramatic irreversible long-range interaction essential to the overall folding of the small ribosomal subunit. Here, we report the crystal structure of the Dhr1 helicase module, revealing the presence of a remarkable carboxyl-terminal domain essential for Dhr1 function in ribosome biogenesis in vivo and important for its interaction with its coactivator Utp14 in vitro. Furthermore, we report the functional consequences on ribosome biogenesis of DHX37 (human Dhr1) mutations found in patients suffering from microcephaly and other neurological diseases.


Assuntos
RNA Helicases DEAD-box/química , Domínios Proteicos , Subunidades Ribossômicas Menores/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Pareamento de Bases , Sítios de Ligação/genética , Cristalografia por Raios X , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Modelos Moleculares , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
11.
Genes Dev ; 33(13-14): 741-746, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171702

RESUMO

Site-specific 2'-O-ribose methylation of mammalian rRNAs and RNA polymerase II-synthesized spliceosomal small nuclear RNAs (snRNAs) is mediated by small nucleolar and small Cajal body (CB)-specific box C/D ribonucleoprotein particles (RNPs) in the nucleolus and the nucleoplasmic CBs, respectively. Here, we demonstrate that 2'-O-methylation of the C34 wobble cytidine of human elongator tRNAMet(CAT) is achieved by collaboration of a nucleolar and a CB-specific box C/D RNP carrying the SNORD97 and SCARNA97 box C/D 2'-O-methylation guide RNAs. Methylation of C34 prevents site-specific cleavage of tRNAMet(CAT) by the stress-induced endoribonuclease angiogenin, implicating box C/D guide RNPs in controlling stress-responsive production of putative regulatory tRNA fragments.


Assuntos
Nucléolo Celular/metabolismo , Corpos Enovelados/metabolismo , Citidina/metabolismo , RNA de Transferência/metabolismo , Ribonucleoproteínas/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Células HeLa , Humanos , Metilação , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , RNA de Transferência/genética , Ribonuclease Pancreático/metabolismo , Ribonucleoproteínas/genética , Estresse Fisiológico
12.
Nat Protoc ; 14(7): 2036-2068, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175345

RESUMO

Chromatin in higher eukaryotic nuclei is extensively bound by various RNA species. We recently developed a method for in situ capture of global RNA interactions with DNA by deep sequencing (GRID-seq) of fixed permeabilized nuclei that allows identification of the entire repertoire of chromatin-associated RNAs in an unbiased manner. The experimental design of GRID-seq is related to those of two recently published strategies (MARGI (mapping RNA-genome interactions) and ChAR-seq (chromatin-associated RNA sequencing)), which also use a bivalent linker to ligate RNA and DNA in proximity. Importantly, however, GRID-seq also implements a combined experimental and computational approach to control nonspecific RNA-DNA interactions that are likely to occur during library construction, which is critical for accurate interpretation of detected RNA-DNA interactions. GRID-seq typically finds both coding and non-coding RNAs (ncRNAs) that interact with tissue-specific promoters and enhancers, especially super-enhancers, from which a global promoter-enhancer connectivity map can be deduced. Here, we provide a detailed protocol for GRID-seq that includes nuclei preparation, chromatin fragmentation, RNA and DNA in situ ligation with a bivalent linker, PCR amplification and high-throughput sequencing. To further enhance the utility of GRID-seq, we include a pipeline for data analysis, called GridTools, into which key steps such as background correction and inference of genomic element proximity are integrated. For researchers experienced in molecular biology with minimal bioinformatics skills, the protocol typically takes 4-5 d from cell fixation to library construction and 2-3 d for data processing.


Assuntos
Cromatina/genética , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/genética , Animais , Núcleo Celular/genética , Cromatina/química , Cromatina/metabolismo , Drosophila/genética , Biblioteca Gênica , Camundongos , Camundongos Endogâmicos , Células-Tronco Embrionárias Murinas/fisiologia , Regiões Promotoras Genéticas , RNA/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Fluxo de Trabalho
13.
Nucleic Acids Res ; 47(14): 7633-7647, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31147702

RESUMO

The parasite Trypanosoma brucei, the causative agent of sleeping sickness, cycles between an insect and a mammalian host. Here, we investigated the presence of pseudouridines (Ψs) on the spliceosomal small nuclear RNAs (snRNAs), which may enable growth at the very different temperatures characterizing the two hosts. To this end, we performed the first high-throughput mapping of spliceosomal snRNA Ψs by small RNA Ψ-seq. The analysis revealed 42 Ψs on T. brucei snRNAs, which is the highest number reported so far. We show that a trypanosome protein analogous to human protein WDR79, is essential for guiding Ψ on snRNAs but not on rRNAs. snoRNA species implicated in snRNA pseudouridylation were identified by a genome-wide approach based on ligation of RNAs following in vivo UV cross-linking. snRNA Ψs are guided by single hairpin snoRNAs, also implicated in rRNA modification. Depletion of such guiding snoRNA by RNAi compromised the guided modification on snRNA and reduced parasite growth at elevated temperatures. We further demonstrate that Ψ strengthens U4/U6 RNA-RNA and U2B"/U2A' proteins-U2 snRNA interaction at elevated temperatures. The existence of single hairpin RNAs that modify both the spliceosome and ribosome RNAs is unique for these parasites, and may be related to their ability to cycle between their two hosts that differ in temperature.


Assuntos
Proteínas de Protozoários/metabolismo , Pseudouridina/metabolismo , RNA Nuclear Pequeno/metabolismo , RNA Nucleolar Pequeno/metabolismo , Spliceossomos/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Sequência de Bases , Humanos , Ligação Proteica , Proteínas de Protozoários/genética , Pseudouridina/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Nuclear Pequeno/genética , RNA Nucleolar Pequeno/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/genética , Trypanosoma brucei brucei/genética
14.
Biomed Pharmacother ; 116: 108995, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31121484

RESUMO

Gefitinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), has been widely used as a first-line agent in EGFR-mutant non-small cell lung cancer (NSCLC). Nevertheless, the development of chemoresistance ultimately limited the curative effect of anti-cancer drugs. The present study aims to investigate the functions of SNHG14 in gefitinib resistance and gain insight into the underlying molecular mechanisms. In the present study, we found that SNHG14 expression was elevated and miR-206-3p expression was decreased in gefitinib-resistant NSCLC tumor tissues and cells. Functionally, SNHG14 overexpression increased gefitinib resistance by promoting cell viability, lowering apoptosis and enhancing colony forming ability, while SNHG14 knockdown reduced gefitinib resistance in NSCLC cells. Mechanistically, SNHG14 induced ABCB1 expression via interaction with miR-206-3p. Moreover, depletion of SNHG14 enhanced the sensitivity of NSCLC cells to gefitinib in vivo. Together, SNHG14 confers gefitinib resistance in NSCLC by regulating miR-206-3p/ABCB1 pathway, contributing to a better understanding of SNHG14 in acquired resistance and elucidating a candidate target to improve treatment response of NSCLC patients.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Gefitinibe/uso terapêutico , Neoplasias Pulmonares/genética , MicroRNAs/metabolismo , RNA Nucleolar Pequeno/metabolismo , Regulação para Cima/genética , Regiões 3' não Traduzidas/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Sequência de Bases , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Feminino , Gefitinibe/farmacologia , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Neoplasias Pulmonares/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , RNA Nucleolar Pequeno/genética
15.
J Hum Genet ; 64(7): 647-652, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30988409

RESUMO

Prader-Willi syndrome (PWS) is a well-known imprinting disorder arising from a loss of paternally imprinted gene(s) at 15q11.2-q13. We report a typical PWS patient with a balanced reciprocal translocation, 46, XY, t(15;19)(q11.2;q13.3). After Illumina whole-genome sequencing, we used BreakDancer-1.45 software to predict candidate breakpoints and manually investigated via the Integrated Genome Viewer. Breakpoint PCR followed by Sanger sequencing determined the t(15;19) breakpoints. We investigated the expression of upstream/centromeric and downstream/telomeric genes of the 15q11.2 breakpoint by reverse transcriptase PCR, using total RNA extracted from the patient's lymphoblasts. Of note, the expression of paternally expressed genes PWAR6, SNORD109A/B, SNORD116, IPW, and PWAR1, downstream of the breakpoint, was abolished. Interestingly, the breakpoint did not destroy protein coding genes or individual snoRNAs. These results indicate that these genes may play a major role in the PWS phenotype.


Assuntos
Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 19/genética , Síndrome de Prader-Willi/genética , RNA Longo não Codificante/genética , Translocação Genética , Adolescente , Pontos de Quebra do Cromossomo , Cromossomos Humanos Par 15/metabolismo , Cromossomos Humanos Par 19/metabolismo , Impressão Genômica/genética , Humanos , Masculino , Fenótipo , Síndrome de Prader-Willi/diagnóstico , Síndrome de Prader-Willi/metabolismo , RNA Nucleolar Pequeno/biossíntese , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Sequenciamento Completo do Genoma
16.
Genetics ; 212(1): 153-174, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30902808

RESUMO

RNA helicases are a class of enzymes that unwind RNA duplexes in vitro but whose cellular functions are largely enigmatic. Here, we provide evidence that the DEAD-box protein Dbp2 remodels RNA-protein complex (RNP) structure to facilitate efficient termination of transcription in Saccharomyces cerevisiae via the Nrd1-Nab3-Sen1 (NNS) complex. First, we find that loss of DBP2 results in RNA polymerase II accumulation at the 3' ends of small nucleolar RNAs and a subset of mRNAs. In addition, Dbp2 associates with RNA sequence motifs and regions bound by Nrd1 and can promote its recruitment to NNS-targeted regions. Using Structure-seq, we find altered RNA/RNP structures in dbp2∆ cells that correlate with inefficient termination. We also show a positive correlation between the stability of structures in the 3' ends and a requirement for Dbp2 in termination. Taken together, these studies provide a role for RNA remodeling by Dbp2 and further suggests a mechanism whereby RNA structure is exploited for gene regulation.


Assuntos
RNA Helicases DEAD-box/metabolismo , RNA Mensageiro/metabolismo , RNA Nucleolar Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Terminação da Transcrição Genética , DNA Helicases/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Nucleares/metabolismo , RNA Helicases/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Análise de Sequência de RNA
17.
Am J Physiol Gastrointest Liver Physiol ; 316(4): G539-G550, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30735452

RESUMO

Hepatic fibrosis is chronic liver damage with many causes that has a relatively high death rate. The current study showed that long noncoding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5), microRNA-23a (miR-23a), and phosphatase and tensin homolog (PTEN) play important roles in the pathological process of hepatic fibrosis but have a relatively unclear regulatory mechanism. This study aimed to investigate the roles of lncRNA GAS5, miR-23a, and PTEN in the pathological process of hepatic fibrosis and hepatic stellate cell (HSC) activation. We used carbon tetrachloride (CCl4) intraperitoneal injections to establish a rat hepatic fibrosis model and exogenous transforming growth factor-ß1 to establish an HSC activation model. Quantitative RT-PCR, Western blot, dual-luciferase reporter system, and RNA pull-down assays were used to investigate which microRNAs and lncRNAs participate in the process of hepatic fibrosis and HSC activation. miR-23a expression increased significantly in hepatic fibrosis tissues and activated HSCs. miR-23a interaction with and degradation of PTEN further influenced the downstream signaling pathway phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin/Snail (PI3K/Akt/mTOR/Snail), causing E-cadherin expression levels to decrease and α-smooth muscle actin and collagen I expression levels to increase. lncRNA GAS5 can be used as a sponge platform for miR-23a to decrease miR-23a expression levels competitively. We revealed the role of the lncRNA GAS5/miR-23a/PTEN/PI3K/Akt/mTOR/Snail signaling pathway in hepatic fibrosis, providing molecular targets for the treatment of hepatic fibrosis. NEW & NOTEWORTHY This is the first study revealing that microRNA-23a (miR-23a) promotes hepatic fibrosis through the phosphatase and tensin homolog/phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin/Snail signaling pathway, and long noncoding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) can act as a sponge platform for miR-23a. Therefore, lncRNA GAS5/miR-23a may bring molecular targets for hepatic fibrosis therapy.


Assuntos
Proteínas de Transporte/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Nucleolar Pequeno/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Descoberta de Drogas , Células Estreladas do Fígado/efeitos dos fármacos , MicroRNAs/genética , RNA Longo não Codificante/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia
18.
RNA ; 25(4): 496-506, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30700579

RESUMO

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins (C/D snoRNPs) is guided by conserved trans-acting factors that act collectively to assemble the core proteins SNU13/Snu13, NOP58/Nop58, NOP56/Nop56, FBL/Nop1, and box C/D small nucleolar RNAs (C/D snoRNAs), in human and in yeast, respectively. This finely elaborated process involves the sequential interplay of snoRNP-related proteins and RNA through the formation of transient pre-RNP complexes. BCD1/Bcd1 protein is essential for yeast cell growth and for the specific accumulation of box C/D snoRNAs. In this work, chromatin, RNA, and protein immunoprecipitation assays revealed the ordered loading of several snoRNP-related proteins on immature and mature snoRNA species. Our results identify Bcd1p as an assembly factor of C/D snoRNP biogenesis that is likely recruited cotranscriptionally and that directs the loading of the core protein Nop58 on RNA.


Assuntos
Fator 6 Semelhante a Kruppel/genética , Proteínas Nucleares/genética , RNA Nucleolar Pequeno/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleolares Pequenas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sítios de Ligação , Cromatina/química , Cromatina/metabolismo , Humanos , Fator 6 Semelhante a Kruppel/metabolismo , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Biossíntese de Proteínas , RNA Nucleolar Pequeno/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Genética
19.
Mol Biol Rep ; 46(2): 2059-2066, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30725348

RESUMO

Derived from rosaceous plant seed, amygdalin belongs to aromatic cyanogenic glycoside group, and its anticancer effects have been supported by mounting evidence. In this study, we objected to investigate amygdalin effect on two antiapoptotic genes (Survivin, XIAP) and two lncRNAs (GAS5, MALAT1) in human cancer cells (A549, MCF7, AGS). Employing RT-qPCR analysis, we compared the mRNA levels of the genes related to apoptosis in A549, MCF7, and AGS cancer cells between amygdalin-treated (24, 48 and 72 h) and un-treated groups. RNA was extracted from both cell groups and then cDNAs were synthesized. The changes in the gene expression levels were specified using ΔΔCt method. RT-qPCR analysis has revealed that the expression of Survivin, XIAP, GAS5 and MALAT1 in amygdala-treated cancer cells were significantly different, compared to the un-treated cells. However, these expressions were different depending on the treatment time. According to the results, amygdalin significantly inhibited the expression level of Survivin, and XIAP genes in treated via untreated group. Our findings suggest that amygdalin might have an anticancer effect due to the various gene expressions in A549, MCF7, and AGS human cancer cells, showing it's potential as a natural therapeutic anticancer drug.


Assuntos
Amigdalina/farmacologia , Survivina/efeitos dos fármacos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/efeitos dos fármacos , Células A549/efeitos dos fármacos , Amigdalina/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7/efeitos dos fármacos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Survivina/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética
20.
RNA ; 25(1): 17-22, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30301832

RESUMO

Posttranscriptional modifications of rRNA occur in the nucleolus where rRNA modification guide RNAs, or snoRNAs, concentrate. On the other hand, scaRNAs, the modification guide RNAs for spliceosomal snRNAs, concentrate in the Cajal body (CB). It is generally assumed, therefore, that snRNAs must accumulate in CBs to be modified by scaRNAs. Here we demonstrate that the evidence for the latter postulate is not consistent. In the nucleus, scaRNA localization is not limited to CBs. Furthermore, canonical scaRNAs can modify rRNAs. We suggest that the conventional view that scaRNAs function only in the CB needs revision.


Assuntos
Corpos Enovelados/metabolismo , RNA Guia/química , RNA Guia/metabolismo , RNA Nucleolar Pequeno/química , RNA Nucleolar Pequeno/metabolismo , Animais , Sequência de Bases , Células HeLa , Humanos , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA , RNA Guia/genética , RNA Ribossômico/química , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , RNA Nucleolar Pequeno/genética , Spliceossomos/genética , Spliceossomos/metabolismo , Xenopus/genética , Xenopus/metabolismo
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