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1.
Mol Cell ; 69(3): 412-425.e6, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29395063

RESUMO

Mutations in several general pre-mRNA splicing factors have been linked to myelodysplastic syndromes (MDSs) and solid tumors. These mutations have generally been assumed to cause disease by the resultant splicing defects, but different mutations appear to induce distinct splicing defects, raising the possibility that an alternative common mechanism is involved. Here we report a chain of events triggered by multiple splicing factor mutations, especially high-risk alleles in SRSF2 and U2AF1, including elevated R-loops, replication stress, and activation of the ataxia telangiectasia and Rad3-related protein (ATR)-Chk1 pathway. We further demonstrate that enhanced R-loops, opposite to the expectation from gained RNA binding with mutant SRSF2, result from impaired transcription pause release because the mutant protein loses its ability to extract the RNA polymerase II (Pol II) C-terminal domain (CTD) kinase-the positive transcription elongation factor complex (P-TEFb)-from the 7SK complex. Enhanced R-loops are linked to compromised proliferation of bone-marrow-derived blood progenitors, which can be partially rescued by RNase H overexpression, suggesting a direct contribution of augmented R-loops to the MDS phenotype.


Assuntos
Sequência de Bases/genética , Síndromes Mielodisplásicas/genética , Fatores de Processamento de RNA/genética , Pontos de Checagem do Ciclo Celular/genética , Células HEK293 , Humanos , Mutação , Proteínas Nucleares/genética , Fosfoproteínas/genética , Splicing de RNA/genética , Fatores de Processamento de RNA/metabolismo , Ribonucleoproteínas/genética , Fatores de Processamento de Serina-Arginina/genética , Fator de Processamento U2AF/genética
2.
Mol Cell ; 62(6): 875-889, 2016 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-27211866

RESUMO

Increasing evidence suggests that diverse RNA binding proteins (RBPs) interact with regulatory RNAs to regulate transcription. RBFox2 is a well-characterized pre-mRNA splicing regulator, but we now encounter an unexpected paradigm where depletion of this RBP induces widespread increase in nascent RNA production in diverse cell types. Chromatin immunoprecipitation sequencing (ChIP-seq) reveals extensive interaction of RBFox2 with chromatin in a nascent RNA-dependent manner. Bayesian network analysis connects RBFox2 to Polycomb complex 2 (PRC2) and H3K27me3, and biochemical experiments demonstrate the ability of RBFox2 to directly interact with PRC2. Strikingly, RBFox2 inactivation eradicates PRC2 targeting on the majority of bivalent gene promoters and leads to transcriptional de-repression. Together, these findings uncover a mechanism underlying the enigmatic association of PRC2 with numerous active genes, highlight the importance of gene body sequences to gauge transcriptional output, and suggest nascent RNAs as critical signals for transcriptional feedback control to maintain homeostatic gene expression in mammalian genomes.


Assuntos
Genoma , Miócitos Cardíacos/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Fatores de Processamento de RNA/metabolismo , RNA/metabolismo , Transcrição Gênica , Animais , Teorema de Bayes , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Genótipo , Células HEK293 , Histonas/metabolismo , Humanos , Camundongos Knockout , Modelos Genéticos , Fenótipo , Complexo Repressor Polycomb 2/genética , Regiões Promotoras Genéticas , Ligação Proteica , RNA/genética , Interferência de RNA , Fatores de Processamento de RNA/deficiência , Fatores de Processamento de RNA/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transfecção
3.
Genes Dev ; 29(19): 2054-66, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26443849

RESUMO

Alternative splicing is prevalent among genes encoding signaling molecules; however, the functional consequence of differential isoform expression remains largely unknown. Here we demonstrate that, in response to T-cell activation, the Jun kinase (JNK) kinase MAP kinase kinase 7 (MKK7) is alternatively spliced to favor an isoform that lacks exon 2. This isoform restores a JNK-docking site within MKK7 that is disrupted in the larger isoform. Consistently, we show that skipping of MKK7 exon 2 enhances JNK pathway activity, as indicated by c-Jun phosphorylation and up-regulation of TNF-α. Moreover, this splicing event is itself dependent on JNK signaling. Thus, MKK7 alternative splicing represents a positive feedback loop through which JNK promotes its own signaling. We further show that repression of MKK7 exon 2 is dependent on the presence of flanking sequences and the JNK-induced expression of the RNA-binding protein CELF2, which binds to these regulatory elements. Finally, we found that ∼25% of T-cell receptor-mediated alternative splicing events are dependent on JNK signaling. Strikingly, these JNK-dependent events are also significantly enriched for responsiveness to CELF2. Together, our data demonstrate a widespread role for the JNK-CELF2 axis in controlling splicing during T-cell activation, including a specific role in propagating JNK signaling.


Assuntos
Processamento Alternativo/genética , Proteínas CELF/metabolismo , Regulação da Expressão Gênica , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase 7/genética , Proteínas do Tecido Nervoso/metabolismo , Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Retroalimentação Fisiológica/fisiologia , Humanos , Células Jurkat , MAP Quinase Quinase 7/metabolismo , Estabilidade de RNA/genética , Transdução de Sinais/genética , Linfócitos T/citologia
4.
Biochem Biophys Res Commun ; 632: 92-99, 2022 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-36206599

RESUMO

BACKGROUND: Mesenchymal stem cells-derived exosomes (MSCs-Exo) were able to exert neuroprotective effects in brain injury after ischemic stroke (IS). In addition, exosomes containing microRNAs (miRNAs) can be transported to recipient cells to mediate intercellular communication. It has been shown that the level of miR-145 was significantly downregulated in brain tissues of rats subjected to middle cerebral artery occlusion (MCAO). However, the role of MSCs-derived exosomal miR-145 in IS progression remains largely unknown. METHODS: Microglial BV2 cell exposed to oxygen-glucose deprivation/reperfusion (OGD/R) was applied to mimic cerebral ischemia/reperfusion (I/R) injury conditions in vitro. In addition, a rat model of MCAO was established to induce I/R injury. Meanwhile, exosomes were isolated from miR-145-transfected bone marrow MSCs, and then these isolated exosomes were used to treat OGD/R-stimulated BV-2 cell and rats subject to MCAO/R. RESULTS: In this study, we found that miR-145 could be transferred from MSCs to BV2 cells via exosomes. In addition, exosomal miR-145-derived from MSCs was able to shift microglia polarization toward anti-inflammatory M2 phenotype in OGD/R-stimulated BV2 cells. Moreover, exosomal miR-145 markedly suppressed the apoptosis, cell cycle arrest and oxidative stress in OGD/R-treated BV2 cells. Additionally, exosomal miR-145 notably decreased the expression of FOXO1 in BV2 cell exposed to OGD/R and in brain tissues of MCAO rats. Furthermore, exosomal miR-145 remarkably decreased infarct area in MCAO rats. CONCLUSION: Collectively, exosomal miR-145-derived from MSCs was able to attenuate cerebral I/R injury through downregulation of FOXO1. These studies may serve as a potential approach for treating of cerebral I/R injury.


Assuntos
Lesões Encefálicas , Exossomos , Proteína Forkhead Box O1 , Células-Tronco Mesenquimais , MicroRNAs , Fármacos Neuroprotetores , Traumatismo por Reperfusão , Animais , Ratos , Medula Óssea/metabolismo , Lesões Encefálicas/metabolismo , Regulação para Baixo , Exossomos/genética , Exossomos/metabolismo , Glucose/metabolismo , Infarto da Artéria Cerebral Média/genética , Infarto da Artéria Cerebral Média/terapia , Infarto da Artéria Cerebral Média/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fármacos Neuroprotetores/metabolismo , Oxigênio/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/terapia , Proteína Forkhead Box O1/genética
5.
Mol Cell ; 56(2): 298-310, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25263594

RESUMO

BS69 (also called ZMYND11) contains tandemly arranged PHD, BROMO, and PWWP domains, which are chromatin recognition modalities. Here, we show that BS69 selectively recognizes histone variant H3.3 lysine 36 trimethylation (H3.3K36me3) via its chromatin-binding domains. We further identify BS69 association with RNA splicing regulators, including the U5 snRNP components of the spliceosome, such as EFTUD2. Remarkably, RNA sequencing shows that BS69 mainly regulates intron retention (IR), which is the least understood RNA alternative splicing event in mammalian cells. Biochemical and genetic experiments demonstrate that BS69 promotes IR by antagonizing EFTUD2 through physical interactions. We further show that regulation of IR by BS69 also depends on its binding to H3K36me3-decorated chromatin. Taken together, our study identifies an H3.3K36me3-specific reader and a regulator of IR and reveals that BS69 connects histone H3.3K36me3 to regulated RNA splicing, providing significant, important insights into chromatin regulation of pre-mRNA processing.


Assuntos
Processamento Alternativo , Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Precursores de RNA/genética , RNA Mensageiro/genética , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cromatina/genética , Proteínas Correpressoras , Metilação de DNA/genética , Proteínas de Ligação a DNA , Células HeLa , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Humanos , Íntrons/genética , Lisina/genética , Lisina/metabolismo , Fatores de Alongamento de Peptídeos/antagonistas & inibidores , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Processamento Pós-Transcricional do RNA/genética , RNA Interferente Pequeno , Ribonucleoproteína Nuclear Pequena U5/antagonistas & inibidores , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Análise de Sequência de RNA , Spliceossomos/genética
6.
Genes Dev ; 28(23): 2663-76, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25452276

RESUMO

Alternative splicing modulates expression of most human genes. The kinetic model of cotranscriptional splicing suggests that slow elongation expands and that fast elongation compresses the "window of opportunity" for recognition of upstream splice sites, thereby increasing or decreasing inclusion of alternative exons. We tested the model using RNA polymerase II mutants that change average elongation rates genome-wide. Slow and fast elongation affected constitutive and alternative splicing, frequently altering exon inclusion and intron retention in ways not predicted by the model. Cassette exons included by slow and excluded by fast elongation (type I) have weaker splice sites, shorter flanking introns, and distinct sequence motifs relative to "slow-excluded" and "fast-included" exons (type II). Many rate-sensitive exons are misspliced in tumors. Unexpectedly, slow and fast elongation often both increased or both decreased inclusion of a particular exon or retained intron. These results suggest that an optimal rate of transcriptional elongation is required for normal cotranscriptional pre-mRNA splicing.


Assuntos
RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Elongação da Transcrição Genética/fisiologia , Éxons/genética , Células HEK293 , Humanos , Íntrons/genética , Mutação , RNA Polimerase II/genética , Precursores de RNA/genética
7.
Mol Cell ; 50(2): 223-35, 2013 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-23562324

RESUMO

SR proteins are well-characterized RNA binding proteins that promote exon inclusion by binding to exonic splicing enhancers (ESEs). However, it has been unclear whether regulatory rules deduced on model genes apply generally to activities of SR proteins in the cell. Here, we report global analyses of two prototypical SR proteins, SRSF1 (SF2/ASF) and SRSF2 (SC35), using splicing-sensitive arrays and CLIP-seq on mouse embryo fibroblasts (MEFs). Unexpectedly, we find that these SR proteins promote both inclusion and skipping of exons in vivo, but their binding patterns do not explain such opposite responses. Further analyses reveal that loss of one SR protein is accompanied by coordinated loss or compensatory gain in the interaction of other SR proteins at the affected exons. Therefore, specific effects on regulated splicing by one SR protein actually depend on a complex set of relationships with multiple other SR proteins in mammalian genomes.


Assuntos
Processamento Alternativo , Genoma , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Sequência Consenso , Éxons , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Íntrons , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Ligação Proteica , Splicing de RNA , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas/genética , Análise de Sequência de RNA , Fatores de Processamento de Serina-Arginina , Transcriptoma
9.
EMBO J ; 35(10): 1077-97, 2016 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-26951610

RESUMO

FUS is an RNA-binding protein involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cytoplasmic FUS-containing aggregates are often associated with concomitant loss of nuclear FUS Whether loss of nuclear FUS function, gain of a cytoplasmic function, or a combination of both lead to neurodegeneration remains elusive. To address this question, we generated knockin mice expressing mislocalized cytoplasmic FUS and complete FUS knockout mice. Both mouse models display similar perinatal lethality with respiratory insufficiency, reduced body weight and length, and largely similar alterations in gene expression and mRNA splicing patterns, indicating that mislocalized FUS results in loss of its normal function. However, FUS knockin mice, but not FUS knockout mice, display reduced motor neuron numbers at birth, associated with enhanced motor neuron apoptosis, which can be rescued by cell-specific CRE-mediated expression of wild-type FUS within motor neurons. Together, our findings indicate that cytoplasmic FUS mislocalization not only leads to nuclear loss of function, but also triggers motor neuron death through a toxic gain of function within motor neurons.


Assuntos
Neurônios Motores/metabolismo , Proteína FUS de Ligação a RNA/genética , Animais , Encéfalo/metabolismo , Citoplasma/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Proteína FUS de Ligação a RNA/metabolismo , Medula Espinal/metabolismo
10.
Mol Cell ; 47(3): 422-33, 2012 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-22727668

RESUMO

Pre-mRNA splicing is regulated by developmental and environmental cues, but little is known about how specific signals are transduced in mammalian cells to regulate this critical gene expression step. Here, we report massive reprogramming of alternative splicing in response to EGF signaling. By blocking individual branches in EGF signaling, we found that Akt activation plays a major role, while other branches, such as the JAK/STAT and ERK pathways, make minor contributions to EGF-induced splicing. Activated Akt next branches to SR protein-specific kinases, rather than mTOR, by inducing SRPK autophosphorylation that switches the splicing kinases from Hsp70- to Hsp90-containing complexes. This leads to enhanced SRPK nuclear translocation and SR protein phosphorylation. These findings reveal a major signal transduction pathway for regulated splicing and place SRPKs in a central position in the pathway, consistent with their reputed roles in a large number of human cancers.


Assuntos
Processamento Alternativo/fisiologia , Fator de Crescimento Epidérmico/fisiologia , Sistema de Sinalização das MAP Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Núcleo Celular/enzimologia , Núcleo Celular/genética , Células HEK293 , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética
12.
Nucleic Acids Res ; 45(6): 3503-3518, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-27899633

RESUMO

JMJD6, a jumonji C (Jmj C) domain-containing protein demethylase and hydroxylase, has been implicated in an array of biological processes. It has been shown that JMJD6 interacts with and hydroxylates multiple serine/arginine-rich (SR) proteins and SR related proteins, including U2AF65, all of which are known to function in alternative splicing regulation. However, whether JMJD6 is widely involved in alternative splicing and the molecular mechanism underlying JMJD6-regulated alternative splicing have remained incompletely understood. Here, by using RASL-Seq, we investigated the functional impact of RNA-dependent interaction between JMJD6 and U2AF65, revealing that JMJD6 and U2AF65 co-regulated a large number of alternative splicing events. We further demonstrated the JMJD6 function in alternative splicing in jmjd6 knockout mice. Mechanistically, we showed that the enzymatic activity of JMJD6 was required for a subset of JMJD6-regulated splicing, and JMJD6-mediated lysine hydroxylation of U2AF65 could account for, at least partially, their co-regulated alternative splicing events, suggesting both JMJD6 enzymatic activity-dependent and independent control of alternative splicing. These findings reveal an intimate link between JMJD6 and U2AF65 in alternative splicing regulation, which has important implications in development and disease processes.


Assuntos
Processamento Alternativo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Fator de Processamento U2AF/metabolismo , Animais , Células HEK293 , Humanos , Hidroxilação , Lisina/metabolismo , Camundongos , Camundongos Knockout , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fator de Processamento U2AF/química
13.
RNA ; 22(10): 1535-49, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27492256

RESUMO

Myelodysplastic syndromes (MDS) are heterogeneous myeloid disorders with prevalent mutations in several splicing factors, but the splicing programs linked to specific mutations or MDS in general remain to be systematically defined. We applied RASL-seq, a sensitive and cost-effective platform, to interrogate 5502 annotated splicing events in 169 samples from MDS patients or healthy individuals. We found that splicing signatures associated with normal hematopoietic lineages are largely related to cell signaling and differentiation programs, whereas MDS-linked signatures are primarily involved in cell cycle control and DNA damage responses. Despite the shared roles of affected splicing factors in the 3' splice site definition, mutations in U2AF1, SRSF2, and SF3B1 affect divergent splicing programs, and interestingly, the affected genes fall into converging cancer-related pathways. A risk score derived from 11 splicing events appears to be independently associated with an MDS prognosis and AML transformation, suggesting potential clinical relevance of altered splicing patterns in MDS.


Assuntos
Mutação , Síndromes Mielodisplásicas/genética , Fosfoproteínas/genética , Sítios de Splice de RNA , Fatores de Processamento de RNA/genética , Fatores de Processamento de Serina-Arginina/genética , Fator de Processamento U2AF/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/patologia , Splicing de RNA
14.
Proc Natl Acad Sci U S A ; 112(17): E2139-48, 2015 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-25870297

RESUMO

Studies in several cell types have highlighted dramatic and diverse changes in mRNA processing that occur upon cellular stimulation. However, the mechanisms and pathways that lead to regulated changes in mRNA processing remain poorly understood. Here we demonstrate that expression of the splicing factor CELF2 (CUGBP, Elav-like family member 2) is regulated in response to T-cell signaling through combined increases in transcription and mRNA stability. Transcriptional induction occurs within 6 h of stimulation and is dependent on activation of NF-κB. Subsequently, there is an increase in the stability of the CELF2 mRNA that correlates with a change in CELF2 3'UTR length and contributes to the total signal-induced enhancement of CELF2 expression. Importantly, we uncover dozens of splicing events in cultured T cells whose changes upon stimulation are dependent on CELF2 expression, and provide evidence that CELF2 controls a similar proportion of splicing events during human thymic T-cell development. Taken together, these findings expand the physiologic impact of CELF2 beyond that previously documented in developing neuronal and muscle cells to T-cell development and function, identify unappreciated instances of alternative splicing in the human thymus, and uncover novel mechanisms for CELF2 regulation that may broadly impact CELF2 expression across diverse cell types.


Assuntos
Regiões 3' não Traduzidas/fisiologia , Processamento Alternativo/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Estabilidade de RNA/fisiologia , Proteínas de Ligação a RNA/biossíntese , Transdução de Sinais/fisiologia , Linfócitos T/metabolismo , Proteínas CELF , Humanos , Células Jurkat , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA/genética , Linfócitos T/citologia
15.
RNA ; 21(12): 2053-66, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26437669

RESUMO

HnRNP L is a ubiquitous splicing-regulatory protein that is critical for the development and function of mammalian T cells. Previous work has identified a few targets of hnRNP L-dependent alternative splicing in T cells and has described transcriptome-wide association of hnRNP L with RNA. However, a comprehensive analysis of the impact of hnRNP L on mRNA expression remains lacking. Here we use next-generation sequencing to identify transcriptome changes upon depletion of hnRNP L in a model T-cell line. We demonstrate that hnRNP L primarily regulates cassette-type alternative splicing, with minimal impact of hnRNP L depletion on transcript abundance, intron retention, or other modes of alternative splicing. Strikingly, we find that binding of hnRNP L within or flanking an exon largely correlates with exon repression by hnRNP L. In contrast, exons that are enhanced by hnRNP L generally lack proximal hnRNP L binding. Notably, these hnRNP L-enhanced exons share sequence and context features that correlate with poor nucleosome positioning, suggesting that hnRNP may enhance inclusion of a subset of exons via a cotranscriptional or epigenetic mechanism. Our data demonstrate that hnRNP L controls inclusion of a broad spectrum of alternative cassette exons in T cells and suggest both direct RNA regulation as well as indirect mechanisms sensitive to the epigenetic landscape.


Assuntos
Epigênese Genética , RNA Mensageiro/metabolismo , Ribonucleoproteínas/fisiologia , Processamento Alternativo , Éxons , Humanos , Células Jurkat , Nucleossomos/metabolismo , RNA Mensageiro/genética , Transcriptoma
16.
Nature ; 474(7351): 390-4, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21572438

RESUMO

Mammalian genomes are populated with thousands of transcriptional enhancers that orchestrate cell-type-specific gene expression programs, but how those enhancers are exploited to institute alternative, signal-dependent transcriptional responses remains poorly understood. Here we present evidence that cell-lineage-specific factors, such as FoxA1, can simultaneously facilitate and restrict key regulated transcription factors, exemplified by the androgen receptor (AR), to act on structurally and functionally distinct classes of enhancer. Consequently, FoxA1 downregulation, an unfavourable prognostic sign in certain advanced prostate tumours, triggers dramatic reprogramming of the hormonal response by causing a massive switch in AR binding to a distinct cohort of pre-established enhancers. These enhancers are functional, as evidenced by the production of enhancer-templated non-coding RNA (eRNA) based on global nuclear run-on sequencing (GRO-seq) analysis, with a unique class apparently requiring no nucleosome remodelling to induce specific enhancer-promoter looping and gene activation. GRO-seq data also suggest that liganded AR induces both transcription initiation and elongation. Together, these findings reveal a large repository of active enhancers that can be dynamically tuned to elicit alternative gene expression programs, which may underlie many sequential gene expression events in development, cell differentiation and disease progression.


Assuntos
Elementos Facilitadores Genéticos/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , RNA não Traduzido/genética , Receptores Androgênicos/metabolismo , Transcrição Gênica/genética , Sequência de Bases , Linhagem Celular Tumoral , Linhagem da Célula , Di-Hidrotestosterona/farmacologia , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Genoma Humano/genética , Células HEK293 , Fator 3-alfa Nuclear de Hepatócito/deficiência , Fator 3-alfa Nuclear de Hepatócito/genética , Histonas/metabolismo , Humanos , Calicreínas , Masculino , Antígeno Prostático Específico , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
17.
Nucleic Acids Res ; 43(18): 9006-16, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26261210

RESUMO

PSF (a.k.a. SFPQ) is a ubiquitously expressed, essential nuclear protein with important roles in DNA damage repair and RNA biogenesis. In stimulated T cells, PSF binds to and suppresses the inclusion of CD45 exon 4 in the final mRNA; however, in resting cells, TRAP150 binds PSF and prevents access to the CD45 RNA, though the mechanism for this inhibition has remained unclear. Here, we show that TRAP150 binds a region encompassing the RNA recognition motifs (RRMs) of PSF using a previously uncharacterized, 70 residue region we have termed the PSF-interacting domain (PID). TRAP150's PID directly inhibits the interaction of PSF RRMs with RNA, which is mediated through RRM2. However, interaction of PSF with TRAP150 does not appear to inhibit the dimerization of PSF with other Drosophila Behavior, Human Splicing (DBHS) proteins, which is also dependent on RRM2. Finally, we use RASL-Seq to identify ∼40 T cell splicing events sensitive to PSF knockdown, and show that for the majority of these, PSF's effect is antagonized by TRAP150. Together these data suggest a model in which TRAP150 interacts with dimeric PSF to block access of RNA to RRM2, thereby regulating the activity of PSF toward a broad set of splicing events in T cells.


Assuntos
Processamento Alternativo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/química , Fator de Processamento Associado a PTB , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , RNA/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Linfócitos T , Fatores de Transcrição/química
18.
Biochem J ; 466(2): 311-22, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25529026

RESUMO

The alternative splicing of human genes is dependent on SR proteins, a family of essential splicing factors whose name derives from a signature C-terminal domain rich in arginine-serine dipeptide repeats (RS domains). Although the SRPKs (SR-specific protein kinases) phosphorylate these repeats, RS domains also contain prolines with flanking serines that are phosphorylated by a second family of protein kinases known as the CLKs (Cdc2-like kinases). The role of specific serine-proline phosphorylation within the RS domain has been difficult to assign since CLKs also phosphorylate arginine-serine dipeptides and, thus, display overlapping residue specificities with the SRPKs. In the present study, we address the effects of discrete serine-proline phosphorylation on the conformation and cellular function of the SR protein SRSF1 (SR protein splicing factor 1). Using chemical tagging and dephosphorylation experiments, we show that modification of serine-proline dipeptides broadly amplifies the conformational ensemble of SRSF1. The induction of these new structural forms triggers SRSF1 mobilization in the nucleus and alters its binding mechanism to an exonic splicing enhancer in precursor mRNA. These physical events correlate with changes in the alternative splicing of over 100 human genes based on a global splicing assay. Overall, these studies draw a direct causal relationship between a specific type of chemical modification in an SR protein and the regulation of alternative gene splicing programmes.


Assuntos
Processamento Alternativo , Proteínas Nucleares/química , Prolina/química , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Precursores de RNA/metabolismo , Proteínas de Ligação a RNA/química , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Sequência Conservada , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Cinética , Dados de Sequência Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Prolina/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Proteínas Tirosina Quinases/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Serina/química , Serina/metabolismo , Fatores de Processamento de Serina-Arginina , Especificidade por Substrato
19.
Proc Natl Acad Sci U S A ; 110(47): E4530-9, 2013 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-24170860

RESUMO

Expanded hexanucleotide repeats in the chromosome 9 open reading frame 72 (C9orf72) gene are the most common genetic cause of ALS and frontotemporal degeneration (FTD). Here, we identify nuclear RNA foci containing the hexanucleotide expansion (GGGGCC) in patient cells, including white blood cells, fibroblasts, glia, and multiple neuronal cell types (spinal motor, cortical, hippocampal, and cerebellar neurons). RNA foci are not present in sporadic ALS, familial ALS/FTD caused by other mutations (SOD1, TDP-43, or tau), Parkinson disease, or nonneurological controls. Antisense oligonucleotides (ASOs) are identified that reduce GGGGCC-containing nuclear foci without altering overall C9orf72 RNA levels. By contrast, siRNAs fail to reduce nuclear RNA foci despite marked reduction in overall C9orf72 RNAs. Sustained ASO-mediated lowering of C9orf72 RNAs throughout the CNS of mice is demonstrated to be well tolerated, producing no behavioral or pathological features characteristic of ALS/FTD and only limited RNA expression alterations. Genome-wide RNA profiling identifies an RNA signature in fibroblasts from patients with C9orf72 expansion. ASOs targeting sense strand repeat-containing RNAs do not correct this signature, a failure that may be explained, at least in part, by discovery of abundant RNA foci with C9orf72 repeats transcribed in the antisense (GGCCCC) direction, which are not affected by sense strand-targeting ASOs. Taken together, these findings support a therapeutic approach by ASO administration to reduce hexanucleotide repeat-containing RNAs and raise the potential importance of targeting expanded RNAs transcribed in both directions.


Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Expansão das Repetições de DNA/genética , Degeneração Lobar Frontotemporal/tratamento farmacológico , Terapia Genética/métodos , Oligonucleotídeos Antissenso/farmacologia , Proteínas/genética , Esclerose Lateral Amiotrófica/genética , Animais , Southern Blotting , Proteína C9orf72 , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , Primers do DNA/genética , Fibroblastos/metabolismo , Degeneração Lobar Frontotemporal/genética , Genótipo , Hibridização in Situ Fluorescente , Camundongos , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA
20.
Proc Natl Acad Sci U S A ; 109(12): 4609-14, 2012 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-22396588

RESUMO

The advent of powerful genomics technologies has uncovered many fundamental aspects of biology, including the mechanisms of cancer; however, it has not been appropriately matched by the development of global approaches to discover new medicines against human diseases. Here we describe a unique high-throughput screening strategy by high-throughput sequencing, referred to as HTS(2), to meet this challenge. This technology enables large-scale and quantitative analysis of gene matrices associated with specific disease phenotypes, therefore allowing screening for small molecules that can specifically intervene with disease-linked gene-expression events. By initially applying this multitarget strategy to the pressing problem of hormone-refractory prostate cancer, which tends to be accelerated by the current antiandrogen therapy, we identify Peruvoside, a cardiac glycoside, which can potently inhibit both androgen-sensitive and -resistant prostate cancer cells without triggering severe cytotoxicity. We further show that, despite transcriptional reprogramming in prostate cancer cells at different disease stages, the compound can effectively block androgen receptor-dependent gene expression by inducing rapid androgen receptor degradation via the proteasome pathway. These findings establish a genomics-based phenotypic screening approach capable of quickly connecting pathways of phenotypic response to the molecular mechanism of drug action, thus offering a unique pathway-centric strategy for drug discovery.


Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Androgênios/química , Antígenos/química , Apoptose , Cardenolídeos/farmacologia , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Desenho de Fármacos , Genômica , Glicosídeos/química , Humanos , Fenótipo , Complexo de Endopeptidases do Proteassoma/química , Interferência de RNA
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