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1.
Plant Mol Biol ; 105(1-2): 177-192, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33025522

RESUMO

KEY MESSAGE: Rice is an important crop in the world. However, little is known about rice mRNA deadenylation, which is an important regulation step of gene expression at the post-transcriptional level. The CCR4-NOT1 complex contains two key components, CCR4 and CAF1, which are the main cytoplasmic deadenylases in eukaryotic cells. Expression of OsCAF1B was tightly coupled with low-temperature exposure. In the present study, we investigated the function of OsCAF1B in rice by characterizing the molecular and physiological responses to cold stress in OsCAF1B overexpression lines and dominant-negative mutant lines. Our results demonstrate that OsCAF1B plays an important role in growth and development of rice seedlings at low temperatures. Rice is a tropical and subtropical crop that is sensitive to low temperature, and activates a complex gene regulatory network in response to cold stress. Poly(A) tail shortening, also termed deadenylation, is the rate-limiting step of mRNA degradation in eukaryotic cells. CCR4-associated factor 1 (CAF1) proteins are important enzymes for catalysis of mRNA deadenylation in eukaryotes. In the present study, the role of a rice cold-induced CAF1, OsCAF1B, in adaptation of rice plants to low-temperature stress was investigated. Expression of OsCAF1B was closely linked with low-temperature exposure. The increased survival percentage and reduced electrolyte leakage exhibited by OsCAF1B overexpression transgenic lines subjected to cold stress indicate that OsCAF1B plays a positive role in rice growth under low ambient temperature. The enhancement of cold tolerance by OsCAF1B in transgenic rice seedlings involved OsCAF1B deadenylase gene expression, and was associated with elevated expression of late-response cold-related transcription factor genes. In addition, the expression level of OsCAF1B was higher in a cold-tolerant japonica rice cultivar than in a cold-sensitive indica rice cultivar. The results reveal a hitherto undiscovered function of OsCAF1B deadenylase gene expression, which is required for adaptation to cold stress in rice.


Assuntos
Resposta ao Choque Frio/fisiologia , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Plântula/metabolismo , Temperatura Baixa , Resposta ao Choque Frio/genética , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio , Peroxidação de Lipídeos , Oryza/genética , Fenótipo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Estabilidade de RNA , Plântula/genética , Temperatura , Transcriptoma
2.
Sci Rep ; 10(1): 19683, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-33184471

RESUMO

Rbfox proteins regulate alternative splicing, mRNA stability and translation. These proteins are involved in neurogenesis and have been associated with various neurological conditions. Here, we analyzed Rbfox2 expression in adult and developing mouse retinas and the effect of its downregulation on visual function and retinal transcriptome. In adult rodents, Rbfox2 is expressed in all retinal ganglion cell (RGC) subtypes, horizontal cells, as well as GABAergic amacrine cells (ACs). Among GABAergic AC subtypes, Rbfox2 was colocalized with cholinergic starburst ACs, NPY (neuropeptide Y)- and EBF1 (early B-cell factor 1)-positive ACs. In differentiating retinal cells, Rbfox2 expression was observed as early as E12 and, unlike Rbfox1, which changes its subcellular localization from cytoplasmic to predominantly nuclear at around P0, Rbfox2 remains nuclear throughout retinal development. Rbfox2 knockout in adult animals had no detectable effect on retinal gross morphology. However, the visual cliff test revealed a significant abnormality in the depth perception of Rbfox2-deficient animals. Gene set enrichment analysis identified genes regulating the RNA metabolic process as a top enriched class of genes in Rbfox2-deficient retinas. Pathway analysis of the top 100 differentially expressed genes has identified Rbfox2-regulated genes associated with circadian rhythm and entrainment, glutamatergic/cholinergic/dopaminergic synaptic function, calcium and PI3K-AKT signaling.


Assuntos
Fatores de Processamento de RNA/metabolismo , Retina/metabolismo , Transcriptoma , Visão Ocular/fisiologia , Animais , Ritmo Circadiano , Percepção de Profundidade , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Pupila/fisiologia , Fatores de Processamento de RNA/genética , RNA-Seq , Retina/crescimento & desenvolvimento
3.
Nat Commun ; 11(1): 5621, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33159082

RESUMO

Interactions between U2AF homology motifs (UHMs) and U2AF ligand motifs (ULMs) play a crucial role in early spliceosome assembly in eukaryotic gene regulation. UHM-ULM interactions mediate heterodimerization of the constitutive splicing factors U2AF65 and U2AF35 and between other splicing factors that regulate spliceosome assembly at the 3' splice site, where UHM domains of alternative splicing factors, such as SPF45 and PUF60, contribute to alternative splicing regulation. Here, we performed high-throughput screening using fluorescence polarization assays with hit validation by NMR and identified phenothiazines as general inhibitors of UHM-ULM interactions. NMR studies show that these compounds occupy the tryptophan binding pocket of UHM domains. Co-crystal structures of the inhibitors with the PUF60 UHM domain and medicinal chemistry provide structure-activity-relationships and reveal functional groups important for binding. These inhibitors inhibit early spliceosome assembly on pre-mRNA substrates in vitro. Our data show that spliceosome assembly can be inhibited by targeting UHM-ULM interactions by small molecules, thus extending the toolkit of splicing modulators for structural and biochemical studies of the spliceosome and splicing regulation.


Assuntos
Fenotiazinas/química , Fenotiazinas/farmacologia , Spliceossomos/efeitos dos fármacos , Spliceossomos/metabolismo , Processamento Alternativo , Humanos , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Spliceossomos/genética , Fator de Processamento U2AF/química , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismo
4.
PLoS Genet ; 16(10): e1009103, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33052901

RESUMO

G protein-coupled receptors (GPCRs) allow cells to respond to chemical and sensory stimuli through generation of second messengers, such as cyclic AMP (cAMP), which in turn mediate a myriad of processes, including cell survival, proliferation, and differentiation. In order to gain deeper insights into the complex biology and physiology of these key cellular pathways, it is critical to be able to globally map the molecular factors that shape cascade function. Yet, to this date, efforts to systematically identify regulators of GPCR/cAMP signaling have been lacking. Here, we combined genome-wide screening based on CRISPR interference with a novel sortable transcriptional reporter that provides robust readout for cAMP signaling, and carried out a functional screen for regulators of the pathway. Due to the sortable nature of the platform, we were able to assay regulators with strong and moderate phenotypes by analyzing sgRNA distribution among three fractions with distinct reporter expression. We identified 45 regulators with strong and 50 regulators with moderate phenotypes not previously known to be involved in cAMP signaling. In follow-up experiments, we validated the functional effects of seven newly discovered mediators (NUP93, PRIM1, RUVBL1, PKMYT1, TP53, SF3A2, and HRAS), and showed that they control distinct steps of the pathway. Thus, our study provides proof of principle that the screening platform can be applied successfully to identify bona fide regulators of GPCR/second messenger cascades in an unbiased and high-throughput manner, and illuminates the remarkable functional diversity among GPCR regulators.


Assuntos
Sistemas CRISPR-Cas/genética , Proliferação de Células/genética , AMP Cíclico/genética , Receptores Acoplados a Proteínas-G/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Transporte/genética , Diferenciação Celular/genética , Células Cultivadas , DNA Helicases/genética , DNA Primase/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Processamento de RNA/genética , Transdução de Sinais/genética , Proteína Supressora de Tumor p53/genética
5.
Nat Struct Mol Biol ; 27(10): 901-912, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32807990

RESUMO

The Rbfox family of splicing factors regulate alternative splicing during animal development and in disease, impacting thousands of exons in the maturing brain, heart and muscle. Rbfox proteins have long been known to bind to the RNA sequence GCAUG with high affinity and specificity, but just half of Rbfox binding sites contain a GCAUG motif in vivo. We incubated recombinant RBFOX2 with over 60,000 mouse and human transcriptomic sequences to reveal substantial binding to several moderate-affinity, non-GCAYG sites at a physiologically relevant range of RBFOX2 concentrations. We find that these 'secondary motifs' bind Rbfox robustly in cells and that several together can exert regulation comparable to GCAUG in a trichromatic splicing reporter assay. Furthermore, secondary motifs regulate RNA splicing in neuronal development and in neuronal subtypes where cellular Rbfox concentrations are highest, enabling a second wave of splicing changes as Rbfox levels increase.


Assuntos
Neurônios/fisiologia , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Proteínas Repressoras/metabolismo , Regiões 3' não Traduzidas , Motivos de Aminoácidos , Sítios de Ligação , Antígeno CD47/genética , Antígeno CD47/metabolismo , Diferenciação Celular , Expressão Gênica , Células Hep G2 , Humanos , Neurônios/citologia , Processamento de RNA , Fatores de Processamento de RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Análise de Sequência de RNA
6.
Cancer Sci ; 111(10): 3665-3678, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32745318

RESUMO

Androgen receptor (AR) signaling is considered to be crucial for the pathogenesis of hepatocellular carcinoma (HCC) with obvious sexual dimorphism. Pre-mRNA processing factor 6 (PRPF6) was identified as a coactivator of AR. However, the molecular mechanism underlying the modulation function of PRPF6 on AR-mediated transcriptional activity in HCC needs to be further clarified. In this study, we analyzed data from The Cancer Genome Atlas to show that PRPF6 is highly expressed in HCC. . Our data indicated that PRPF6 interacts with AR/AR splice variants (AR-Vs) and upregulates AR/AR splice variant 7-mediated transcriptional activity even without dihydrotestosterone treatment. We observed that AR is obviously induced by androgen treatment and is mainly expressed in the nucleus in HCC-derived cell lines. Moreover, overexpression of PRPF6 enhances AR expression accompanied with the increase of AR-Vs expression. We provided evidence that PRPF6 participates in upregulating AR self-transcription. PRPF6 facilitates the recruitment of AR to the androgen responsive element region of the AR gene. Finally, PRPF6 depletion inhibits cell proliferation in HCC cells and mouse xenografts. Taken together, our results suggest that PRPF6 as a splicing factor enhances AR self-transcription, thereby coactivating oncogenic AR/AR-Vs actions in HCC.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Fatores de Processamento de RNA/genética , Receptores Androgênicos/genética , Fatores de Transcrição/genética , Animais , Carcinogênese/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Di-Hidrotestosterona/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Xenoenxertos , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Transdução de Sinais , Ativação Transcricional/genética
7.
Nucleic Acids Res ; 48(18): 10329-10341, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32663306

RESUMO

The recently characterized mammalian writer (methyltransferase) and eraser (demethylase) of the DNA N6-methyladenine (N6mA) methyl mark act on single-stranded (ss) and transiently-unpaired DNA. As YTH domain-containing proteins bind N6mA-containing RNA in mammalian cells, we investigated whether mammalian YTH domains are also methyl mark readers of N6mA DNA. Here, we show that the YTH domain of YTHDC1 (known to localize in the nucleus) binds ssDNA containing N6mA, with a 10 nM dissociation constant. This binding is stronger by a factor of 5 than in an RNA context, tested under the same conditions. However, the YTH domains of YTHDF2 and YTHDF1 (predominantly cytoplasmic) exhibited the opposite effect with ∼1.5-2נstronger binding to ssRNA containing N6mA than to the corresponding DNA. We determined two structures of the YTH domain of YTHDC1 in complex with N6mA-containing ssDNA, which illustrated that YTHDC1 binds the methylated adenine in a single-stranded region flanked by duplexed DNA. We discuss the hypothesis that the writer-reader-eraser of N6mA-containining ssDNA is associated with maintaining genome stability. Structural comparison of YTH and SRA domains (the latter a DNA 5-methylcytosine reader) revealed them to be diverse members of a larger family of DNA/RNA modification readers, apparently having originated from bacterial modification-dependent restriction enzymes.


Assuntos
Adenina/química , Complexos Multiproteicos/química , Proteínas do Tecido Nervoso/química , Conformação Proteica , Fatores de Processamento de RNA/química , DNA/química , DNA/genética , DNA/ultraestrutura , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Histona Desmetilases/genética , Humanos , Metilação , Metiltransferases/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/ultraestrutura , Domínios Proteicos/genética , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/ultraestrutura , Proteínas de Ligação a RNA/genética
8.
Mol Cell ; 79(3): 425-442.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32615088

RESUMO

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.


Assuntos
Adenosina/análogos & derivados , Neoplasias de Cabeça e Pescoço/genética , Metiltransferases/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adenosina/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Bleomicina/farmacologia , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Células HEK293 , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas do Tecido Nervoso/metabolismo , Hibridização de Ácido Nucleico , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Am J Psychiatry ; 177(9): 855-866, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32600152

RESUMO

OBJECTIVE: Attention deficit hyperactivity disorder (ADHD) is a highly heritable psychiatric disorder. The objective of this study was to define ADHD-associated candidate genes and their associated molecular modules and biological themes, based on the analysis of rare genetic variants. METHODS: The authors combined data from 11 published copy number variation studies in 6,176 individuals with ADHD and 25,026 control subjects and prioritized genes by applying an integrative strategy based on criteria including recurrence in individuals with ADHD, absence in control subjects, complete coverage in copy number gains, and presence in the minimal region common to overlapping copy number variants (CNVs), as well as on protein-protein interactions and information from cross-species genotype-phenotype annotation. RESULTS: The authors localized 2,241 eligible genes in the 1,532 reported CNVs, of which they classified 432 as high-priority ADHD candidate genes. The high-priority ADHD candidate genes were significantly coexpressed in the brain. A network of 66 genes was supported by ADHD-relevant phenotypes in the cross-species database. Four significantly interconnected protein modules were found among the high-priority ADHD genes. A total of 26 genes were observed across all applied bioinformatic methods. Lookup in the latest genome-wide association study for ADHD showed that among those 26 genes, POLR3C and RBFOX1 were also supported by common genetic variants. CONCLUSIONS: Integration of a stringent filtering procedure in CNV studies with suitable bioinformatics approaches can identify ADHD candidate genes at increased levels of credibility. The authors' analytic pipeline provides additional insight into the molecular mechanisms underlying ADHD and allows prioritization of genes for functional validation in validated model organisms.


Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Encéfalo/metabolismo , Perfilação da Expressão Gênica/métodos , RNA Polimerase III , Fatores de Processamento de RNA , Transtorno do Deficit de Atenção com Hiperatividade/genética , Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Variações do Número de Cópias de DNA/fisiologia , Bases de Dados Genéticas , Estudos de Associação Genética/métodos , Predisposição Genética para Doença , Humanos , Mapeamento de Interação de Proteínas/métodos , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo
10.
PLoS One ; 15(6): e0234062, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32497093

RESUMO

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most lethal and malignant tumours worldwide. New therapeutic targets for HCC are urgently needed. CYCLOPS (copy number alterations yielding cancer liabilities owing to partial loss) genes have been noted to be associated with cancer-targeted therapies. Therefore, we intended to explore the effects of the CYCLOPS gene RBM17 on HCC oncogenesis to determine if it could be further used for targeted therapy. METHODS: We collected data on 12 types of cancer from the Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) queries for comparison with adjacent non-tumour tissues. RBM17 expression levels, clinicopathological factors and survival times were analysed. RNAseq data were downloaded from the Encyclopaedia of DNA Elements database for molecular mechanism exploration. Two representative HCC cell models were built to observe the proliferation capacity of HCC cells when RBM17 expression was inhibited by shRBM17. Cell cycle progression and apoptosis were also examined to investigate the pathogenesis of RBM17. RESULTS: Based on 6,136 clinical samples, RBM17 was markedly overexpressed in most cancers, especially HCC. Moreover, data from 442 patients revealed that high RBM17 expression levels were related to a worse prognosis. Overexpression of RBM17 was related to the iCluster1 molecular subgroup, TNM stage, and histologic grade. Pathway analysis of RNAseq data suggested that RBM17 was involved in mitosis. Further investigation revealed that the proliferation rates of HepG2 (P = 0.003) and SMMC-7721 (P = 0.030) cells were significantly reduced when RBM17 was knocked down. In addition, RBM17 knockdown also arrested the progression of the cell cycle, causing cells to halt at the G2/M phase. Increased apoptosis rates were also found in vitro. CONCLUSION: These results suggest that RBM17 is a potential therapeutic target for HCC treatment.


Assuntos
Carcinoma Hepatocelular/genética , Variações do Número de Cópias de DNA , Neoplasias Hepáticas/genética , Fatores de Processamento de RNA/genética , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/patologia , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/patologia , Prognóstico , Fatores de Processamento de RNA/deficiência
11.
Am J Otolaryngol ; 41(4): 102547, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32474328

RESUMO

BACKGROUND: N6-Methyladenosine (m6A) is a ubiquitous RNA modification with vital roles in various cancers, but little is known about its role in papillary thyroid carcinoma (PTC), a common endocrine malignancy. METHODS: In this study, an m6A RNA methylation regulator-based biomarker signature was developed for the effective prediction of prognosis in patients with PTC. The gene expression profiles of m6A RNA methylation regulators and the corresponding clinical information was downloaded from The Cancer Genome Atlas (TCGA). Differentially expressed m6A RNA methylation regulators between tumor and normal control samples, and correlation expression levels, clinical parameters, and outcomes were evaluated. And a prognostic signature was built using a PTC cohort from TCGA. RESULTS: The expression level of HNRNPC was remarkably upregulated in tumor samples, while WTAP, RBM15, YTHDC2, YTHDC1, FTO, METTL14, METTL3, ALKBH5, KIAA1429, YTHDF1, and ZC3H13 were significantly downregulated in the cancer specimens compared with those in control samples. A three-gene prognostic signature comprising RBM15, KIAA1429, and FTO could predict overall survival in patients with PTC. In addition, the prognostic signature-based risk score was identified as an independent prognostic indicator for PTC. CONCLUSIONS: We established a robust m6A RNA methylation regulator-based molecular signature for predicting prognosis in patients with PTC with high accuracy; this signature might provide important guidance for therapeutic strategies.


Assuntos
Regulação Neoplásica da Expressão Gênica , Expressão Gênica , Metiltransferases/genética , Metiltransferases/metabolismo , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Adulto , Idoso , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Feminino , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Humanos , Masculino , Metilação , Pessoa de Meia-Idade , Prognóstico , RNA Helicases/genética , RNA Helicases/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Taxa de Sobrevida , Câncer Papilífero da Tireoide/mortalidade , Neoplasias da Glândula Tireoide/mortalidade , Regulação para Cima/genética
12.
Gene ; 754: 144839, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32504654

RESUMO

Wilms tumor is the most frequently occurring pediatric renal malignancy. Wilms tumor suppressor-1-associated protein (WTAP) is a vital component of N6-methyltransferase complex involved in tumorigenesis. However, the roles of WTAP gene single nucleotide polymorphisms (SNPs) in Wilms tumor risk have not been clarified to date. We successfully genotyped three WTAP gene SNPs using TaqMan assay in 405 Wilms tumor patients and 1197 cancer-free controls of Chinese children. Odds ratios (ORs) and 95% confidence intervals (CIs) were applied to determine the effects of WTAP gene SNPs on Wilms tumor risk. Carriers of the rs1853259 G variant are less susceptible to developing Wilms tumor, with an adjusted OR of 0.78 (AG vs. AA: 95% CI = 0.61-0.995, P = 0.046). Single locus analysis of rs9457712 G > A and rs7766006 G > T, as well as the combined analysis of risk genotypes, failed to unveil an association with Wilms tumor risk, respectively. Stratified analysis of the three SNPs and their combined risk effects showed more significant relationships with Wilms tumor risk under certain subgroups. In all, we found weak evidence of the association between WTAP gene SNPs and the risk of Wilms tumor. Further replication studies with greater sample size and different ethnicities are necessary to verify our findings.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Proteínas de Ciclo Celular/genética , Transformação Celular Neoplásica/genética , Neoplasias Renais/genética , Polimorfismo de Nucleotídeo Único , Fatores de Processamento de RNA/genética , Tumor de Wilms/genética , Estudos de Casos e Controles , Transformação Celular Neoplásica/patologia , China/epidemiologia , Feminino , Seguimentos , Predisposição Genética para Doença , Genótipo , Humanos , Lactente , Neoplasias Renais/epidemiologia , Neoplasias Renais/patologia , Masculino , Prognóstico , Tumor de Wilms/epidemiologia , Tumor de Wilms/patologia
13.
Neuron ; 107(3): 496-508.e6, 2020 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-32526197

RESUMO

Here, we perform a genome-wide screen for variants that regulate the expression of gene co-expression modules in the aging human brain; we discover and replicate such variants in the TMEM106B and RBFOX1 loci. The TMEM106B haplotype is known to influence the accumulation of TAR DNA-binding protein 43 kDa (TDP-43) proteinopathy, and the haplotype's large-scale transcriptomic effects include the dysregulation of lysosomal genes and alterations in synaptic gene splicing that are also seen in the pathophysiology of TDP-43 proteinopathy. Further, a variant near GRN, another TDP-43 proteinopathy susceptibility gene, shows concordant effects with the TMEM106B haplotype. Leveraging neuropathology data from the same participants, we also show that TMEM106B and APOE-amyloid-ß effects converge to alter myelination and lysosomal gene expression, which then contributes to TDP-43 accumulation. These results advance our mechanistic understanding of the TMEM106B TDP-43 risk haplotype and uncover a transcriptional program that mediates the converging effects of APOE-amyloid-ß and TMEM106B on TDP-43 aggregation in older adults.


Assuntos
Envelhecimento/genética , Encéfalo/metabolismo , Regulação da Expressão Gênica/genética , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Progranulinas/genética , Fatores de Processamento de RNA/genética , Proteinopatias TDP-43/genética , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/psicologia , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Apolipoproteínas E/genética , Encéfalo/patologia , Estudos de Coortes , Proteínas de Ligação a DNA/metabolismo , Feminino , Haplótipos , Humanos , Lisossomos , Masculino , Bainha de Mielina , Locos de Características Quantitativas , Proteinopatias TDP-43/psicologia
14.
Nat Commun ; 11(1): 2973, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532987

RESUMO

Alternative splicing allows expression of mRNA isoforms from a single gene, expanding the diversity of the proteome. Its prevalence in normal biological and disease processes warrant precise tools for modulation. Here we report the engineering of CRISPR Artificial Splicing Factors (CASFx) based on RNA-targeting CRISPR-Cas systems. We show that simultaneous exon inclusion and exclusion can be induced at distinct targets by differential positioning of CASFx. We also create inducible CASFx (iCASFx) using the FKBP-FRB chemical-inducible dimerization domain, allowing small molecule control of alternative splicing. Finally, we demonstrate the activation of SMN2 exon 7 splicing in spinal muscular atrophy (SMA) patient fibroblasts, suggesting a potential application of the CASFx system.


Assuntos
Processamento Alternativo , Sistemas CRISPR-Cas/genética , Éxons/genética , Fatores de Processamento de RNA/genética , RNA/genética , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , RNA/metabolismo , Isoformas de RNA/genética , Isoformas de RNA/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
15.
Anticancer Res ; 40(5): 2941-2946, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366446

RESUMO

BACKGROUND/AIM: The spliceosome pathway, including Splicing Factor 3b Subunit 4 (SF3B4), plays an important role in carcinogenesis and progression in various cancers; however, the clinical relevance of SF3B4 in esophageal squamous cell carcinoma (ESCC) remains unknown. PATIENTS AND METHODS: SF3B4 expression was evaluated by real-time reverse transcription polymerase chain reaction in 80 ESCC patients. In order to explore the mechanism of SF3B4 in ESCC, the mRNA expression and copy number of SF3B4 were obtained from TCGA and we also implemented gene set enrichment analysis (GSEA). RESULTS: The high SF3B4 expression group (n=33) showed significantly more lymphatic permeation and poorer prognosis than the low SF3B4 expression group (n=47). GSEA revealed that high SF3B4 expression was correlated with genes associated with the transcription factor E2F and the G2/M checkpoint. SF3B4 expression was positively correlated with SF3B4 DNA copy number. CONCLUSION: Over-expression of SF3B4 may play a crucial role in the lymphatic progression of ESCC.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas do Esôfago/genética , Fatores de Processamento de RNA/genética , Carcinoma de Células Escamosas do Esôfago/mortalidade , Carcinoma de Células Escamosas do Esôfago/patologia , Feminino , Humanos , Masculino , Prognóstico , Análise de Sobrevida
16.
Leukemia ; 34(10): 2621-2634, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32358566

RESUMO

Previous studies demonstrated that splicing factor mutations are recurrent events in hematopoietic malignancies with both clinical and functional implications. However, their aberrant splicing patterns in acute myeloid leukemia remain largely unexplored. In this study, we characterized mutations in SRSF2, U2AF1, and SF3B1, the most commonly mutated splicing factors. In our clinical analysis of 2678 patients, splicing factor mutations showed inferior relapse-free and overall survival, however, these mutations did not represent independent prognostic markers. RNA-sequencing of 246 and independent validation in 177 patients revealed an isoform expression profile which is highly characteristic for each individual mutation, with several isoforms showing a strong dysregulation. By establishing a custom differential splice junction usage pipeline, we accurately detected aberrant splicing in splicing factor mutated samples. A large proportion of differentially used junctions were novel, including several junctions in leukemia-associated genes. In SRSF2(P95H) mutants, we further explored the possibility of a cascading effect through the dysregulation of the splicing pathway. Furthermore, we observed a validated impact on overall survival for two junctions overused in SRSF2(P95H) mutants. We conclude that splicing factor mutations do not represent independent prognostic markers. However, they do have genome-wide consequences on gene splicing leading to dysregulated isoform expression of several genes.


Assuntos
Leucemia Mieloide Aguda/genética , Mutação , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Processamento de RNA , Fatores de Processamento de Serina-Arginina/genética , Fator de Processamento U2AF/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
17.
Nat Commun ; 11(1): 2506, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427851

RESUMO

The genetic basis and corresponding clinical relevance of prolactinomas remain poorly understood. Here, we perform whole genome sequencing (WGS) on 21 patients with prolactinomas to detect somatic mutations and then validate the mutations with digital polymerase chain reaction (PCR) analysis of tissue samples from 227 prolactinomas. We identify the same hotspot somatic mutation in splicing factor 3 subunit B1 (SF3B1R625H) in 19.8% of prolactinomas. These patients with mutant prolactinomas display higher prolactin (PRL) levels (p = 0.02) and shorter progression-free survival (PFS) (p = 0.02) compared to patients without the mutation. Moreover, we identify that the SF3B1R625H mutation causes aberrant splicing of estrogen related receptor gamma (ESRRG), which results in stronger binding of pituitary-specific positive transcription factor 1 (Pit-1), leading to excessive PRL secretion. Thus our study validates an important mutation and elucidates a potential mechanism underlying the pathogenesis of prolactinomas that may lead to the development of targeted therapeutics.


Assuntos
Fosfoproteínas/genética , Prolactinoma/genética , Fatores de Processamento de RNA/genética , Adulto , Feminino , Humanos , Masculino , Mutação , Fosfoproteínas/metabolismo , Intervalo Livre de Progressão , Prolactina/genética , Prolactina/metabolismo , Prolactinoma/metabolismo , Prolactinoma/mortalidade , Fatores de Processamento de RNA/metabolismo , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Fator de Transcrição Pit-1/genética , Fator de Transcrição Pit-1/metabolismo , Adulto Jovem
18.
Proc Natl Acad Sci U S A ; 117(19): 10305-10312, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32332164

RESUMO

The gene encoding the core spliceosomal protein SF3B1 is the most frequently mutated gene encoding a splicing factor in a variety of hematologic malignancies and solid tumors. SF3B1 mutations induce use of cryptic 3' splice sites (3'ss), and these splicing errors contribute to tumorigenesis. However, it is unclear how widespread this type of cryptic 3'ss usage is in cancers and what is the full spectrum of genetic mutations that cause such missplicing. To address this issue, we performed an unbiased pan-cancer analysis to identify genetic alterations that lead to the same aberrant splicing as observed with SF3B1 mutations. This analysis identified multiple mutations in another spliceosomal gene, SUGP1, that correlated with significant usage of cryptic 3'ss known to be utilized in mutant SF3B1 expressing cells. Remarkably, this is consistent with recent biochemical studies that identified a defective interaction between mutant SF3B1 and SUGP1 as the molecular defect responsible for cryptic 3'ss usage. Experimental validation revealed that five different SUGP1 mutations completely or partially recapitulated the 3'ss defects. Our analysis suggests that SUGP1 mutations in cancers can induce missplicing identical or similar to that observed in mutant SF3B1 cancers.


Assuntos
Biologia Computacional/métodos , Mutação , Neoplasias/genética , Fosfoproteínas/genética , Sítios de Splice de RNA , Fatores de Processamento de RNA/genética , Processamento de RNA , Análise Mutacional de DNA , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/patologia , Spliceossomos
19.
PLoS One ; 15(4): e0229315, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32320410

RESUMO

Mutations in the splicing machinery have been implicated in a number of human diseases. Most notably, the U2 small nuclear ribonucleoprotein (snRNP) component SF3b1 has been found to be frequently mutated in blood cancers such as myelodysplastic syndromes (MDS). SF3b1 is a highly conserved HEAT repeat (HR)-containing protein and most of these blood cancer mutations cluster in a hot spot located in HR4-8. Recently, a second mutational hotspot has been identified in SF3b1 located in HR9-12 and is associated with acute myeloid leukemias, bladder urothelial carcinomas, and uterine corpus endometrial carcinomas. The consequences of these mutations on SF3b1 functions during splicing have not yet been tested. We incorporated the corresponding mutations into the yeast homolog of SF3b1 and tested their impact on splicing. We find that all of these HR9-12 mutations can support splicing in yeast, and this suggests that none of them are loss of function alleles in humans. The Hsh155V502F mutation alters splicing of several pre-mRNA reporters containing weak branch sites as well as a genetic interaction with Prp2 and physical interactions with Prp5 and Prp3. The ability of a single allele of Hsh155 to perturb interactions with multiple factors functioning at different stages of the splicing reaction suggests that some SF3b1-mutant disease phenotypes may have a complex origin on the spliceosome.


Assuntos
Mutação/genética , Fosfoproteínas/genética , Precursores de RNA/genética , Fatores de Processamento de RNA/genética , Processamento de RNA/genética , Sequências Repetitivas de Aminoácidos , Ribonucleoproteína Nuclear Pequena U2/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência Consenso/genética , Epistasia Genética , Humanos , Fosfoproteínas/química , Ligação Proteica , Fatores de Processamento de RNA/química , Ribonucleoproteína Nuclear Pequena U2/química , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química
20.
RNA ; 26(8): 996-1005, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32312846

RESUMO

The decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1 kb). Splicing reporter studies have shown that decoy splice sites are essential for activity, suggesting that decoys act by engaging intron-terminal splice sites and competing with cross-intron interactions required for intron excision. The decoy model predicts that antisense oligonucleotides may be able to block decoy splice sites in endogenous pre-mRNA, thereby reducing IR and increasing productive gene expression. Indeed, we now demonstrate that targeting a decoy 5' splice site in the O-GlcNAc transferase (OGT) gene reduced IR from ∼80% to ∼20% in primary human erythroblasts, accompanied by increases in spliced OGT RNA and OGT protein expression. The remaining OGT IR was refractory to antisense treatment and might be mediated by independent mechanism(s). In contrast, other retained introns were strongly dependent on decoy function, since antisense targeting of decoy 5' splice sites greatly reduced (SNRNP70) or nearly eliminated (SF3B1) IR in two widely expressed splicing factors, and also greatly reduced IR in transcripts encoding the erythroid-specific structural protein, α-spectrin (SPTA1). These results show that modulating decoy exon function can dramatically alter IR and suggest that dynamic regulation of decoy exons could be a mechanism to fine-tune gene expression post-transcriptionally in many cell types.


Assuntos
Eritroblastos/fisiologia , Éxons/genética , Oligonucleotídeos Antissenso/genética , Processamento Alternativo/genética , Células Cultivadas , Humanos , Íntrons/genética , N-Acetilglucosaminiltransferases/genética , Precursores de RNA/genética , Sítios de Splice de RNA/genética , Fatores de Processamento de RNA/genética
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