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1.
Gene ; 733: 144384, 2020 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-31978508

RESUMO

BACKGROUND: Globally, hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death. Recently, many studies have demonstrated that small nucleolar RNA (snoRNA) was closely related to HCC. OBJECTIVE: To explore whether snoRNA can be used as a molecular target for HCC. METHODS: The PubMed, Embase, and Cochrane databases were searched for the published literatures related to snoRNA and HCC until August 12, 2019. After identification, screening, and verification, this study finally included 26 studies correlating small nucleolar RNA host gene (SNHG) and HCC, and 8 studies correlating snoRNA and HCC. Based on the collation of the relevant literature, the correlation network diagram between snoRNAs and HCC was constructed. RESULTS: The SNHGs, such as SNHG1, SNHG6, SNHG16, and SNHG20 can play varied roles in HCC through different regulatory mechanisms. These SNHGs can promote and inhibit tumorigenesis. SNORD76 can promote the proliferation of tumor tissues and cells in vitro through different pathways. SnoU2_19 and SNORD76 can function through the same pathway. SNHG3, SNHG20, SNHG6, SNORD76, and snoRA47 can modulate epithelial-mesenchymal transition (EMT) to regulate the development of HCC cell or tissue. SNHG16, SNORD76, and SnoU2_19 can regulate the development of HCC through Wnt/ß-catenin signaling pathway. CONCLUSION: snoRNA can regulate the occurrence of HCC by modulating multiple molecular signaling pathways. Hence, snoRNA can be a potential molecular target for HCC.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Terapia de Alvo Molecular , RNA Nucleolar Pequeno/antagonistas & inibidores , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Prognóstico , RNA Nucleolar Pequeno/genética
2.
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
3.
Mol Biol Evol ; 37(1): 149-166, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31553476

RESUMO

Small nucleolar RNAs (snoRNAs) function primarily as guide RNAs for posttranscriptional modification of rRNAs and spliceosomal snRNAs, both of which are functionally important and evolutionarily conserved molecules. It is commonly believed that snoRNAs and the modifications they mediate are highly conserved across species. However, most relevant data on snoRNA annotation and RNA modification are limited to studies on human and yeast. Here, we used RNA-sequencing data from the giant oocyte nucleus of the frog Xenopus tropicalis to annotate a nearly complete set of snoRNAs. We compared the frog data with snoRNA sets from human and other vertebrate genomes, including mammals, birds, reptiles, and fish. We identified many Xenopus-specific (or nonhuman) snoRNAs and Xenopus-specific domains in snoRNAs from conserved RNA families. We predicted that some of these nonhuman snoRNAs and domains mediate modifications at unexpected positions in rRNAs and snRNAs. These modifications were mapped as predicted when RNA modification assays were applied to RNA from nine vertebrate species: frogs X. tropicalis and X. laevis, newt Notophthalmus viridescens, axolotl Ambystoma mexicanum, whiptail lizard Aspidoscelis neomexicana, zebrafish Danio rerio, chicken, mouse, and human. This analysis revealed that only a subset of RNA modifications is evolutionarily conserved and that modification patterns may vary even between closely related species. We speculate that each functional domain in snoRNAs (half of an snoRNA) may evolve independently and shuffle between different snoRNAs.


Assuntos
RNA Nucleolar Pequeno/genética , Xenopus/genética , Animais , Genoma , Humanos , Anotação de Sequência Molecular , Mutação Puntual
4.
Nat Med ; 25(12): 1873-1884, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31806906

RESUMO

Herpes simplex virus-1 (HSV-1) encephalitis (HSE) is typically sporadic. Inborn errors of TLR3- and DBR1-mediated central nervous system cell-intrinsic immunity can account for forebrain and brainstem HSE, respectively. We report five unrelated patients with forebrain HSE, each heterozygous for one of four rare variants of SNORA31, encoding a small nucleolar RNA of the H/ACA class that are predicted to direct the isomerization of uridine residues to pseudouridine in small nuclear RNA and ribosomal RNA. We show that CRISPR/Cas9-introduced bi- and monoallelic SNORA31 deletions render human pluripotent stem cell (hPSC)-derived cortical neurons susceptible to HSV-1. Accordingly, SNORA31-mutated patient hPSC-derived cortical neurons are susceptible to HSV-1, like those from TLR3- or STAT1-deficient patients. Exogenous interferon (IFN)-ß renders SNORA31- and TLR3- but not STAT1-mutated neurons resistant to HSV-1. Finally, transcriptome analysis of SNORA31-mutated neurons revealed normal responses to TLR3 and IFN-α/ß stimulation but abnormal responses to HSV-1. Human SNORA31 thus controls central nervous system neuron-intrinsic immunity to HSV-1 by a distinctive mechanism.


Assuntos
Encefalite por Herpes Simples/genética , Herpesvirus Humano 1/genética , Neurônios/imunologia , RNA Nucleolar Pequeno/genética , Adulto , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/virologia , Pré-Escolar , Encefalite por Herpes Simples/imunologia , Encefalite por Herpes Simples/patologia , Encefalite por Herpes Simples/virologia , Feminino , Predisposição Genética para Doença , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/patogenicidade , Humanos , Imunidade/genética , Lactente , Masculino , Metagenoma/genética , Metagenoma/imunologia , Pessoa de Meia-Idade , Neurônios/virologia , RNA Nucleolar Pequeno/imunologia
5.
PLoS Genet ; 15(11): e1008469, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31721758

RESUMO

RNA molecules generated by ribonuclease cleavage sometimes harbor a 2',3'-cyclic phosphate (cP) at their 3'-ends. Those cP-containing RNAs (cP-RNAs) form a hidden layer of transcriptome because standard RNA-seq cannot capture them as a result of cP's prevention of an adapter ligation reaction. Here we provide genome-wide analyses of short cP-RNA transcriptome across multiple mouse tissues. Using cP-RNA-seq that can exclusively sequence cP-RNAs, we identified numerous novel cP-RNA species which are mainly derived from cytoplasmic tRNAs, mRNAs, and rRNAs. Determination of the processing sites of substrate RNAs for cP-RNA generation revealed highly-specific RNA cleavage events between cytidine and adenosine in cP-RNA biogenesis. cP-RNAs were not evenly derived from the overall region of substrate RNAs but rather from specific sites, implying that cP-RNAs are not from random degradation but are produced through a regulated biogenesis pathway. The identified cP-RNAs were abundantly accumulated in mouse tissues, and the expression levels of cP-RNAs showed age-dependent reduction. These analyses of cP-RNA transcriptome unravel a novel, abundant class of non-coding RNAs whose expression could have physiological roles.


Assuntos
Envelhecimento/genética , Sequência de Bases/genética , RNA/genética , Transcriptoma/genética , Envelhecimento/patologia , Animais , Regulação da Expressão Gênica/genética , Genômica , Humanos , Camundongos , Fosfatos/química , Fosfatos/metabolismo , RNA/química , Clivagem do RNA/genética , RNA Ribossômico/genética , RNA Nucleolar Pequeno/genética , RNA de Transferência/genética , RNA não Traduzido/genética , Análise de Sequência de RNA
6.
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
7.
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
8.
Nat Neurosci ; 22(9): 1521-1532, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31455884

RESUMO

One fundamental but understudied mechanism of gene regulation in disease is allele-specific expression (ASE), the preferential expression of one allele. We leveraged RNA-sequencing data from human brain to assess ASE in autism spectrum disorder (ASD). When ASE is observed in ASD, the allele with lower population frequency (minor allele) is preferentially more highly expressed than the major allele, opposite to the canonical pattern. Importantly, genes showing ASE in ASD are enriched in those downregulated in ASD postmortem brains and in genes harboring de novo mutations in ASD. Two regions, 14q32 and 15q11, containing all known orphan C/D box small nucleolar RNAs (snoRNAs), are particularly enriched in shifts to higher minor allele expression. We demonstrate that this allele shifting enhances snoRNA-targeted splicing changes in ASD-related target genes in idiopathic ASD and 15q11-q13 duplication syndrome. Together, these results implicate allelic imbalance and dysregulation of orphan C/D box snoRNAs in ASD pathogenesis.


Assuntos
Desequilíbrio Alélico/genética , Transtorno do Espectro Autista/genética , Encéfalo , Transcriptoma/genética , Alelos , Perfilação da Expressão Gênica , Humanos , RNA Nucleolar Pequeno/genética
9.
Mol Biol Rep ; 46(5): 4717-4724, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31270759

RESUMO

High rates of misdiagnosis and delayed intervention in neonatal PWS are leading to poor prognoses. To determine the clinical and image characteristics of newborns with Prader-Willi syndrome (PWS). A total of 102 cases of newborns definitively diagnosed with PWS at the Children's Hospital of Fudan University from 02/2014 to 12/2017 were retrospectively analyzed. We analyzed the modulated voxel-based morphology (VBM) of gray matter in PWS by T2 weighted imaging. Of 102 cases, 75 (73.5%) have paternal deletion of 15q11.2-q13, whereas 27 (26.5%) have maternal uniparental disomy (UPD). Of the 75 deletion cases, 75 (100%) week crying, 71 (94.7%) hypotonia, 70 (93.3%) poor feeding, 46 (61.3%) hypopigmentation, 43 (57.3%) male cryptorchidism, 10 (13.3%) female labia minora, 48 (64%) characteristic facial features. Of 27 UPD cases, 27 (100%) week crying and hypotonia, 25 (92.6%) hypophagia, 20 (74.1%) male cryptorchidism, 1 (3.7%) female labia minora, 19 (70.4%) characteristic facial features, 12 (44.4%) hypopigmentation. The modulated VBM analysis shows that the middle frontal gyrus, orbitofrontal cortex (middle), and inferior frontal gyrus are the most variable brain regions that determine the endo-phenotype difference between the two genotypes. Hypotonia, hypophagia, and maldevelopment of sexual organs are general characteristics of newborns with PWS in Chinese population. In UPD cases, the proportions of premature newborns, elderly parturient women and congenital malformations were higher than for paternal deletion cases. The differences in the gray matter volume of these three regions between the two genotypes may explain the differences in maladaptive behaviors and emotions.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Estudos de Associação Genética , Genótipo , Fenótipo , Síndrome de Prader-Willi/diagnóstico , Síndrome de Prader-Willi/genética , China , Deleção Cromossômica , Metilação de DNA , Feminino , Estudos de Associação Genética/métodos , Testes Genéticos , Substância Cinzenta/diagnóstico por imagem , Humanos , Recém-Nascido , Imagem por Ressonância Magnética , Masculino , Neuroimagem , RNA Nucleolar Pequeno/genética , Dissomia Uniparental
10.
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
11.
RNA ; 25(9): 1192-1201, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31239298

RESUMO

Protein kinase RNA-activated (PKR) is an interferon-inducible kinase that is potently activated by long double-stranded RNA (dsRNA). In a previous study, we found that snoRNAs exhibit increased association with PKR in response to metabolic stress. While it was unclear if snoRNAs also activated PKR in cells, activation in vitro was observed. snoRNAs do not exhibit the double-stranded character typically required for activation of PKR, but some studies suggest such RNAs can activate PKR if triphosphorylated at the 5' terminus, or if they are able to form intermolecular dimers. To interrogate the mechanism of PKR activation by snoRNAs in vitro we focused on SNORD113. Using multiple methods for defining the 5'-phosphorylation state, we find that activation of PKR by SNORD113 does not require a 5'-triphosphate. Gel purification from a native gel followed by analysis using analytical ultracentrifugation showed that dimerization was also not responsible for activation. We isolated distinct conformers of SNORD113 from a native polyacrylamide gel and tracked the activating species to dsRNA formed from antisense RNA synthesized during in vitro transcription with T7 RNA polymerase. Similar studies with additional snoRNAs and small RNAs showed the generality of our results. Our studies suggest that a 5' triphosphate is not an activating ligand for PKR, and emphasize the insidious nature of antisense contamination.


Assuntos
Ativação Enzimática/genética , Polifosfatos/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Dimerização , Humanos , Ligantes , Fosforilação/genética , Ligação Proteica/genética , RNA de Cadeia Dupla/genética , RNA Nucleolar Pequeno/genética , Transcrição Genética/genética , Ultracentrifugação/métodos , Proteínas Virais/metabolismo
12.
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
13.
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
14.
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
15.
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
16.
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
17.
Int J Mol Sci ; 20(9)2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31052553

RESUMO

Small nucleolar RNAs (snoRNAs) are a new type of functional small RNAs involved in the chemical modifications of rRNAs, tRNAs, and small nuclear RNAs. It is reported that they play important roles in tumorigenesis via various regulatory modes. snoRNAs can both participate in the regulation of methylation and pseudouridylation and regulate the expression pattern of their host genes. This research investigated the expression pattern of snoRNAs in eight major cancer types in TCGA via several machine learning algorithms. The expression levels of snoRNAs were first analyzed by a powerful feature selection method, Monte Carlo feature selection (MCFS). A feature list and some informative features were accessed. Then, the incremental feature selection (IFS) was applied to the feature list to extract optimal features/snoRNAs, which can make the support vector machine (SVM) yield best performance. The discriminative snoRNAs included HBII-52-14, HBII-336, SNORD123, HBII-85-29, HBII-420, U3, HBI-43, SNORD116, SNORA73B, SCARNA4, HBII-85-20, etc., on which the SVM can provide a Matthew's correlation coefficient (MCC) of 0.881 for predicting these eight cancer types. On the other hand, the informative features were fed into the Johnson reducer and repeated incremental pruning to produce error reduction (RIPPER) algorithms to generate classification rules, which can clearly show different snoRNAs expression patterns in different cancer types. The analysis results indicated that extracted discriminative snoRNAs can be important for identifying cancer samples in different types and the expression pattern of snoRNAs in different cancer types can be partly uncovered by quantitative recognition rules.


Assuntos
Regulação Neoplásica da Expressão Gênica , Aprendizado de Máquina , Neoplasias/genética , RNA Nucleolar Pequeno/genética , Algoritmos , Humanos , Método de Monte Carlo , Máquina de Vetores de Suporte
18.
Biochimie ; 164: 17-21, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31078583

RESUMO

Small nucleolar RNAs are generally involved in the modification of target ribosomal RNAs. Other possible functions recently emerged with the discovery of the novel class of snoRNA-derived RNAs or sdRNAs. Since then, additional data has revealed the involvement of both snoRNAs and sdRNAs in tumorigenesis. After briefly introducing snoRNA families and functions, this mini-review summarises recently acquired knowledge on snoRNA-related mechanisms associated with cancer. Despite the rapid increase in the number of studies, exactly how snoRNAs lead to cancer remains unclear. However, exciting new research is paving the way for future diagnostic or therapeutic strategies.


Assuntos
Carcinogênese/genética , Neoplasias/genética , RNA Nucleolar Pequeno/genética , Humanos , MicroRNAs/genética , RNA Ribossômico/genética
19.
Nucleic Acids Res ; 47(9): 4431-4441, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30937442

RESUMO

The repertoire of small noncoding RNAs (sncRNAs), particularly miRNAs, in animals is considered to be evolutionarily conserved. Studies on sncRNAs are often largely based on homology-based information, relying on genomic sequence similarity and excluding actual expression data. To obtain information on sncRNA expression (including miRNAs, snoRNAs, YRNAs and tRNAs), we performed low-input-volume next-generation sequencing of 500 pg of RNA from 21 animals at two German zoological gardens. Notably, none of the species under investigation were previously annotated in any miRNA reference database. Sequencing was performed on blood cells as they are amongst the most accessible, stable and abundant sources of the different sncRNA classes. We evaluated and compared the composition and nature of sncRNAs across the different species by computational approaches. While the distribution of sncRNAs in the different RNA classes varied significantly, general evolutionary patterns were maintained. In particular, miRNA sequences and expression were found to be even more conserved than previously assumed. To make the results available for other researchers, all data, including expression profiles at the species and family levels, and different tools for viewing, filtering and searching the data are freely available in the online resource ASRA (Animal sncRNA Atlas) at https://www.ccb.uni-saarland.de/asra/.


Assuntos
Animais de Zoológico/genética , Ácidos Nucleicos Livres/genética , Biologia Computacional , Pequeno RNA não Traduzido/genética , Animais , Ácidos Nucleicos Livres/classificação , Genoma/genética , Alemanha , MicroRNAs/genética , RNA Nucleolar Pequeno/genética , Pequeno RNA não Traduzido/classificação , RNA de Transferência/genética
20.
BMC Mol Biol ; 20(1): 12, 2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-30971200

RESUMO

BACKGROUND: Small nucleolar RNAs (snoRNAs) function in guiding 2'-O-methylation and pseudouridylation of ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs). In recent years, more and more snoRNAs have been found to play novel roles in mRNA regulation, such as pre-mRNA splicing or RNA editing. In our previous study, we found a silkworm C/D box snoRNA Bm-15 can interact with Notch receptor gene in vitro. To further study the function of Bm-15, we cloned its homolog Sf-15 from Spodoptera frugiperda and investigate the function of Sf-15 in Sf9 cells. RESULTS: We showed that knocking down of Sf-15 can inhibit the proliferation, then induce apoptosis of insect S. frugiperda Sf9 cells, but the results were reversed when Sf-15 was overexpressed. De novo sequencing of transcriptome of Sf9 cells showed that the expression of 21 apoptosis-related genes were increased upon Sf-15 repression. Further analysis showed that a Ca2+-induced cell death pathway gene Cn (PPP3C, the serine/threonine-protein phosphatase 2B catalytic subunit), was significantly increased upon Sf-15 depression but decreased when Sf-15 was overexpressed, which indicated that Cn might be a potential target of Sf-15. CONCLUSIONS: We conclude that C/D box snoRNA Sf-15 can participate in apoptosis through regulating the expression of Ca2+-induced cell death pathway gene Cn in Sf9 cells. This is the first time that we found snoRNAs exhibiting dual functions in insect, which reveals a novel layer of ncRNA modulation in cell growth and death.


Assuntos
Apoptose/genética , Proliferação de Células/genética , RNA Nucleolar Pequeno/genética , Spodoptera/genética , Animais , Perfilação da Expressão Gênica , Células Sf9
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