RESUMO
The seat of higher-order cognitive abilities in mammals, the neocortex, is a complex structure, organized in several layers. The different subtypes of principal neurons are distributed in precise ratios and at specific positions in these layers and are generated by the same neural progenitor cells (NPCs), steered by a spatially and temporally specified combination of molecular cues that are incompletely understood. Recently, we discovered that an alternatively spliced isoform of the TrkC receptor lacking the kinase domain, TrkC-T1, is a determinant of the corticofugal projection neuron (CFuPN) fate. Here, we show that the finely tuned balance between TrkC-T1 and the better known, kinase domain-containing isoform, TrkC-TK+, is cell type-specific in the developing cortex and established through the antagonistic actions of two RNA-binding proteins, Srsf1 and Elavl1. Moreover, our data show that Srsf1 promotes the CFuPN fate and Elavl1 promotes the callosal projection neuron (CPN) fate in vivo via regulating the distinct ratios of TrkC-T1 to TrkC-TK+. Taken together, we connect spatio-temporal expression of Srsf1 and Elavl1 in the developing neocortex with the regulation of TrkC alternative splicing and transcript stability and neuronal fate choice, thus adding to the mechanistic and functional understanding of alternative splicing in vivo.
Assuntos
Neocórtex , Receptor trkC , Animais , Processamento Alternativo , Mamíferos/metabolismo , Neocórtex/metabolismo , Neurônios/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor trkC/química , Receptor trkC/genética , Receptor trkC/metabolismo , Camundongos , Linhagem Celular TumoralRESUMO
Non-temperature-induced effects of radiofrequency electromagnetic fields (RF) have been controversial for decades. Here, we established measurement techniques to prove their existence by investigating energy deposition in tumor cells under RF exposure and upon adding amplitude modulation (AM) (AMRF). Using a preclinical device LabEHY-200 with a novel in vitro applicator, we analyzed the power deposition and system parameters for five human colorectal cancer cell lines and measured the apoptosis rates in vitro and tumor growth inhibition in vivo in comparison to water bath heating. We showed enhanced anticancer effects of RF and AMRF in vitro and in vivo and verified the non-temperature-induced origin of the effects. Furthermore, apoptotic enhancement by AM was correlated with cell membrane stiffness. Our findings not only provide a strategy to significantly enhance non-temperature-induced anticancer cell effects in vitro and in vivo but also provide a perspective for a potentially more effective tumor therapy.
RESUMO
Overexpression of MYC is a genuine cancer driver in lymphomas and related to poor prognosis. However, therapeutic targeting of the transcription factor MYC remains challenging. Here, we show that inhibition of the histone deacetylase 6 (HDAC6) using the HDAC6 inhibitor Marbostat-100 (M-100) reduces oncogenic MYC levels and prevents lymphomagenesis in a mouse model of MYC-induced aggressive B-cell lymphoma. M-100 specifically alters protein-protein interactions by switching the acetylation state of HDAC6 substrates, such as tubulin. Tubulin facilitates nuclear import of MYC, and MYC-dependent B-cell lymphoma cells rely on continuous import of MYC due to its high turn-over. Acetylation of tubulin impairs this mechanism and enables proteasomal degradation of MYC. M-100 targets almost exclusively B-cell lymphoma cells with high levels of MYC whereas non-tumor cells are not affected. M-100 induces massive apoptosis in human and murine MYC-overexpressing B-cell lymphoma cells. We identified the heat-shock protein DNAJA3 as an interactor of tubulin in an acetylation-dependent manner and overexpression of DNAJA3 resulted in a pronounced degradation of MYC. We propose a mechanism by which DNAJA3 associates with hyperacetylated tubulin in the cytoplasm to control MYC turnover. Taken together, our data demonstrate a beneficial role of HDAC6 inhibition in MYC-dependent B-cell lymphoma.
Assuntos
Linfoma de Células B , Tubulina (Proteína) , Acetilação , Animais , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/metabolismo , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/genética , Camundongos , Fatores de Transcrição/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
RNA-binding proteins regulate mRNA processing and translation and are often aberrantly expressed in cancer. The RNA-binding motif protein 6, RBM6, is a known alternative splicing factor that harbors tumor suppressor activity and is frequently mutated in human cancer. Here, we identify RBM6 as a novel regulator of homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Mechanistically, we show that RBM6 regulates alternative splicing-coupled nonstop-decay of a positive HR regulator, Fe65/APBB1. RBM6 knockdown leads to a severe reduction in Fe65 protein levels and consequently impairs HR of DSBs. Accordingly, RBM6-deficient cancer cells are vulnerable to ATM and PARP inhibition and show remarkable sensitivity to cisplatin. Concordantly, cisplatin administration inhibits the growth of breast tumor devoid of RBM6 in mouse xenograft model. Furthermore, we observe that RBM6 protein is significantly lost in metastatic breast tumors compared with primary tumors, thus suggesting RBM6 as a potential therapeutic target of advanced breast cancer. Collectively, our results elucidate the link between the multifaceted roles of RBM6 in regulating alternative splicing and HR of DSBs that may contribute to tumorigenesis, and pave the way for new avenues of therapy for RBM6-deficient tumors.
Assuntos
Quebras de DNA de Cadeia Dupla , Resistencia a Medicamentos Antineoplásicos , Recombinação Homóloga , Proteínas de Ligação a RNA/metabolismo , Animais , Antineoplásicos/uso terapêutico , Antineoplásicos/toxicidade , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular , Cisplatino/uso terapêutico , Cisplatino/toxicidade , Feminino , Células HCT116 , Humanos , Células MCF-7 , Neoplasias Mamárias Experimentais/tratamento farmacológico , Camundongos , Camundongos SCID , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/genética , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
BACKGROUND & AIMS: Currently, only a few genetic variants explain the heritability of fatty liver disease. Quantitative trait loci (QTL) analysis of mouse strains has identified the susceptibility locus Ltg/NZO (liver triglycerides from New Zealand obese [NZO] alleles) on chromosome 18 as associating with increased hepatic triglycerides. Herein, we aimed to identify genomic variants responsible for this association. METHODS: Recombinant congenic mice carrying 5.3 Mbp of Ltg/NZO were fed a high-fat diet and characterized for liver fat. Bioinformatic analysis, mRNA profiles and electrophoretic mobility shift assays were performed to identify genes responsible for the Ltg/NZO phenotype. Candidate genes were manipulated in vivo by injecting specific microRNAs into C57BL/6 mice. Pulldown coupled with mass spectrometry-based proteomics and immunoprecipitation were performed to identify interaction partners of IFGGA2. RESULTS: Through positional cloning, we identified 2 immunity-related GTPases (Ifgga2, Ifgga4) that prevent hepatic lipid storage. Expression of both murine genes and the human orthologue IRGM was significantly lower in fatty livers. Accordingly, liver-specific suppression of either Ifgga2 or Ifgga4 led to a 3-4-fold greater increase in hepatic fat content. In the liver of low-fat diet-fed mice, IFGGA2 localized to endosomes/lysosomes, while on a high-fat diet it associated with lipid droplets. Pulldown experiments and proteomics identified the lipase ATGL as a binding partner of IFGGA2 which was confirmed by co-immunoprecipitation. Both proteins partially co-localized with the autophagic marker LC3B. Ifgga2 suppression in hepatocytes reduced the amount of LC3B-II, whereas overexpression of Ifgga2 increased the association of LC3B with lipid droplets and decreased triglyceride storage. CONCLUSION: IFGGA2 interacts with ATGL and protects against hepatic steatosis, most likely by enhancing the binding of LC3B to lipid droplets. LAY SUMMARY: The genetic basis of non-alcoholic fatty liver disease remains incompletely defined. Herein, we identified members of the immunity-related GTPase family in mice and humans that act as regulators of hepatic fat accumulation, with links to autophagy. Overexpression of the gene Ifgga2 was shown to reduce hepatic lipid storage and could be a therapeutic target for the treatment of fatty liver disease.
Assuntos
Fígado Gorduroso/genética , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Lipase/genética , Metabolismo dos Lipídeos/genética , Proteínas Associadas aos Microtúbulos/genética , Animais , Autofagia , Modelos Animais de Doenças , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Feminino , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/biossíntese , Células Hep G2 , Hepatócitos/patologia , Humanos , Lipase/biossíntese , Lipase/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/biossíntese , Fenótipo , RNA/genéticaRESUMO
Recent work has associated point mutations in both zinc fingers (ZnF) of the spliceosome component U2AF35 with malignant transformation. However, surprisingly little is known about the functionality of the U2AF35 ZnF domains in general. Here we have analysed key functionalities of the ZnF domains of mammalian U2AF35 and its paralog U2AF26. Both ZnFs are required for splicing regulation, whereas only ZnF2 controls protein stability and contributes to the interaction with U2AF65. These features are confirmed in a naturally occurring splice variant of U2AF26 lacking ZnF2, that is strongly induced upon activation of primary mouse T cells and localized in the cytoplasm. Using Ribo-Seq in a model T cell line we provide evidence for a role of U2AF26 in activating cytoplasmic steps in gene expression, notably translation. Consistently, an MS2 tethering assay shows that cytoplasmic U2AF26/35 increase translation when localized to the 5'UTR of a model mRNA. This regulation is partially dependent on ZnF1 thus providing a connection between a core splicing factor, the ZnF domains and the regulation of translation. Altogether, our work reveals unexpected functions of U2AF26/35 and their ZnF domains, thereby contributing to a better understanding of their role and regulation in mammalian cells.
Assuntos
Regulação da Expressão Gênica , Biossíntese de Proteínas , Fator de Processamento U2AF/metabolismo , Dedos de Zinco , Animais , Células HEK293 , Células HeLa , Humanos , Camundongos , Ligação Proteica , Splicing de RNA , Estabilidade de RNA , Fator de Processamento U2AF/químicaRESUMO
T cells are nodal players in the adaptive immune response against pathogens and malignant cells. Alternative splicing plays a crucial role in T cell activation, which is analyzed mainly at later time points upon stimulation. Here we have discovered a 2-h time window early after stimulation where optimal splicing efficiency or, more generally, gene expression efficiency is crucial for successful T cell activation. Reducing the splicing efficiency at 4 to 6 h poststimulation significantly impaired murine T cell activation, which was dependent on the expression dynamics of the Egr1-Nab2-interleukin-2 (IL-2) pathway. This time window overlaps the time of peak IL-2 de novo transcription, which, we suggest, represents a permissive time window in which decreased splicing (or transcription) efficiency reduces mature IL-2 production, thereby hampering murine T cell activation. Notably, the distinct expression kinetics of the Egr1-Nab2-IL-2 pathway between mouse and human render human T cells refractory to this vulnerability. We propose that the rational temporal modulation of splicing or transcription during peak de novo expression of key effectors can be used to fine-tune stimulation-dependent biological outcomes. Our data also show that critical consideration is required when extrapolating mouse data to the human system in basic and translational research.
Assuntos
Processamento Alternativo , Interleucina-2/genética , Linfócitos T/citologia , Animais , Proteína 1 de Resposta de Crescimento Precoce/genética , Regulação da Expressão Gênica , Humanos , Ativação Linfocitária , Camundongos , Proteínas Repressoras/genética , Transdução de Sinais , Especificidade da Espécie , Linfócitos T/imunologia , Fatores de Tempo , Pesquisa Translacional BiomédicaRESUMO
The gene encoding the kinase Mnk2 (MKNK2) is alternatively spliced to produce two isoforms-Mnk2a and Mnk2b. We previously showed that Mnk2a is downregulated in several types of cancer and acts as a tumor suppressor by activation of the p38-MAPK stress pathway, inducing apoptosis. Moreover, Mnk2a overexpression suppressed Ras-induced transformation in culture and in vivo. In contrast, the Mnk2b isoform acts as a pro-oncogenic factor. In this study, we designed modified-RNA antisense oligonucleotides and screened for those that specifically induce a strong switch in alternative splicing of the MKNK2 gene (splice switching oligonucleotides or SSOs), elevating the tumor suppressive isoform Mnk2a at the expense of the pro-oncogenic isoform Mnk2b. Induction of Mnk2a by SSOs in glioblastoma cells activated the p38-MAPK pathway, inhibited the oncogenic properties of the cells, re-sensitized the cells to chemotherapy and inhibited glioblastoma development in vivo. Moreover, inhibition of p38-MAPK partially rescued glioblastoma cells suggesting that most of the anti-oncogenic activity of the SSO is mediated by activation of this pathway. These results suggest that manipulation of MKNK2 alternative splicing by SSOs is a novel approach to inhibit glioblastoma tumorigenesis.
Assuntos
Neoplasias Encefálicas/terapia , Glioblastoma/terapia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Oligonucleotídeos/genética , Proteínas Serina-Treonina Quinases/genética , Processamento Alternativo , Animais , Apoptose , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Genes Supressores de Tumor , Glioblastoma/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Oligonucleotídeos Antissenso , Fosforilação , Isoformas de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Splicing de RNA , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Recent work has identified cancer-associated U2AF35 missense mutations in two zinc-finger (ZnF) domains, but little is known about Q157R/P substitutions within the second ZnF. Surprisingly, we find that the c.470A>G mutation not only leads to the Q157R substitution, but also creates an alternative 5' splice site (ss) resulting in the deletion of four amino acids (Q157Rdel). Q157P, Q157R, and Q157Rdel control alternative splicing of distinct groups of exons in cell culture and in human patients, suggesting that missplicing of different targets may contribute to cellular aberrations. Our data emphasize the importance to explore missense mutations beyond altered protein sequence.
Assuntos
Mutação de Sentido Incorreto , Neoplasias/genética , Sítios de Splice de RNA , Splicing de RNA , Fator de Processamento U2AF/genética , Sequência de Bases , Ciclo Celular , Proliferação de Células , Células HeLa , Humanos , Neoplasias/patologiaRESUMO
The core body temperature of all mammals oscillates with the time of the day. However, direct molecular consequences of small, physiological changes in body temperature remain largely elusive. Here we show that body temperature cycles drive rhythmic SR protein phosphorylation to control an alternative splicing (AS) program. A temperature change of 1°C is sufficient to induce a concerted splicing switch in a large group of functionally related genes, rendering this splicing-based thermometer much more sensitive than previously described temperature-sensing mechanisms. AS of two exons in the 5' UTR of the TATA-box binding protein (Tbp) highlights the general impact of this mechanism, as it results in rhythmic TBP protein levels with implications for global gene expression in vivo. Together our data establish body temperature-driven AS as a core clock-independent oscillator in mammalian peripheral clocks.
Assuntos
Processamento Alternativo , Regulação da Temperatura Corporal , Relógios Circadianos , Ritmo Circadiano , Proteína de Ligação a TATA-Box/metabolismo , Regiões 5' não Traduzidas , Animais , Linhagem Celular Tumoral , Éxons , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Fosforilação , Interferência de RNA , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismo , Proteína de Ligação a TATA-Box/genética , Fatores de Tempo , TransfecçãoRESUMO
The cellular response to genotoxic stress is mediated by a well-characterized network of DNA surveillance pathways. The contribution of post-transcriptional gene regulatory networks to the DNA damage response (DDR) has not been extensively studied. Here, we systematically identified RNA-binding proteins differentially interacting with polyadenylated transcripts upon exposure of human breast carcinoma cells to ionizing radiation (IR). Interestingly, more than 260 proteins, including many nucleolar proteins, showed increased binding to poly(A)+ RNA in IR-exposed cells. The functional analysis of DDX54, a candidate genotoxic stress responsive RNA helicase, revealed that this protein is an immediate-to-early DDR regulator required for the splicing efficacy of its target IR-induced pre-mRNAs. Upon IR exposure, DDX54 acts by increased interaction with a well-defined class of pre-mRNAs that harbor introns with weak acceptor splice sites, as well as by protein-protein contacts within components of U2 snRNP and spliceosomal B complex, resulting in lower intron retention and higher processing rates of its target transcripts. Because DDX54 promotes survival after exposure to IR, its expression and/or mutation rate may impact DDR-related pathologies. Our work indicates the relevance of many uncharacterized RBPs potentially involved in the DDR.
Assuntos
Neoplasias da Mama/genética , RNA Helicases DEAD-box/genética , Dano ao DNA , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Proteínas de Ligação a RNA/genética , Transcriptoma , Neoplasias da Mama/patologia , Reparo do DNA , Feminino , Redes Reguladoras de Genes , Humanos , Poliadenilação , Splicing de RNA , RNA Mensageiro , Radiação Ionizante , Células Tumorais CultivadasRESUMO
The proliferation and survival of hematopoietic stem cells (HSCs) has to be strictly coordinated to ensure the timely production of all blood cells. Here we report that the splice factor and RNA binding protein hnRNP L (heterogeneous nuclear ribonucleoprotein L) is required for hematopoiesis, since its genetic ablation in mice reduces almost all blood cell lineages and causes premature death of the animals. In agreement with this, we observed that hnRNP L deficient HSCs lack both the ability to self-renew and foster hematopoietic differentiation in transplanted hosts. They also display mitochondrial dysfunction, elevated levels of γH2AX, are Annexin V positive and incorporate propidium iodide indicating that they undergo cell death. Lin(-)c-Kit(+) fetal liver cells from hnRNP L deficient mice show high p53 protein levels and up-regulation of p53 target genes. In addition, cells lacking hnRNP L up-regulated the expression of the death receptors TrailR2 and CD95/Fas and show Caspase-3, Caspase-8 and Parp cleavage. Treatment with the pan-caspase inhibitor Z-VAD-fmk, but not the deletion of p53, restored cell survival in hnRNP L deficient cells. Our data suggest that hnRNP L is critical for the survival and functional integrity of HSCs by restricting the activation of caspase-dependent death receptor pathways.
Assuntos
Sobrevivência Celular/fisiologia , Células-Tronco Hematopoéticas/citologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/fisiologia , Animais , Apoptose/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/genética , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Estresse Fisiológico , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Many aspects of human physiology and behavior display rhythmicity with a period of approximately 24 h. Rhythmic changes are controlled by an endogenous time keeper, the circadian clock, and include sleep-wake cycles, physical and mental performance capability, blood pressure, and body temperature. Consequently, many diseases, such as metabolic, sleep, autoimmune and mental disorders and cancer, are connected to the circadian rhythm. The development of therapies that take circadian biology into account is thus a promising strategy to improve treatments of diverse disorders, ranging from allergic syndromes to cancer. Circadian alteration of body functions and behavior are, at the molecular level, controlled and mediated by widespread changes in gene expression that happen in anticipation of predictably changing requirements during the day. At the core of the molecular clockwork is a well-studied transcription-translation negative feedback loop. However, evidence is emerging that additional post-transcriptional, RNA-based mechanisms are required to maintain proper clock function. Here, we will discuss recent work implicating regulated mRNA stability, translation and alternative splicing in the control of the mammalian circadian clock, and its role in health and disease.
Assuntos
Transtornos Cronobiológicos/genética , Relógios Circadianos/genética , Processamento Pós-Transcricional do RNA , Animais , Humanos , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
MALT1 channels proximal T-cell receptor (TCR) signalling to downstream signalling pathways. With MALT1A and MALT1B two conserved splice variants exist and we demonstrate here that MALT1 alternative splicing supports optimal T-cell activation. Inclusion of exon7 in MALT1A facilitates the recruitment of TRAF6, which augments MALT1 scaffolding function, but not protease activity. Naive CD4(+) T cells express almost exclusively MALT1B and MALT1A expression is induced by TCR stimulation. We identify hnRNP U as a suppressor of exon7 inclusion. Whereas selective depletion of MALT1A impairs T-cell signalling and activation, downregulation of hnRNP U enhances MALT1A expression and T-cell activation. Thus, TCR-induced alternative splicing augments MALT1 scaffolding to enhance downstream signalling and to promote optimal T-cell activation.
Assuntos
Processamento Alternativo/genética , Linfócitos T CD4-Positivos/imunologia , Caspases/genética , Ativação Linfocitária/imunologia , Proteínas de Neoplasias/genética , Transdução de Sinais , Animais , Caspases/metabolismo , Regulação para Baixo , Ativação Enzimática , Éxons/genética , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Humanos , Interleucina-2/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células Jurkat , Camundongos Endogâmicos C57BL , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Células Th17/imunologia , Regulação para CimaRESUMO
The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an â¼ 500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6â¢U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation.
Assuntos
Modelos Moleculares , RNA Helicases/química , RNA Helicases/metabolismo , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Spliceossomos/enzimologia , Adenosina Trifosfatases/metabolismo , Chaetomium/enzimologia , Chaetomium/genética , Cristalização , Humanos , Ligação Proteica , Dobramento de Proteína , Processamento de Proteína , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , RNA Helicases/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/química , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Spliceossomos/genéticaRESUMO
The circadian clock drives daily rhythms in gene expression to control metabolism, behavior, and physiology; while the underlying transcriptional feedback loops are well defined, the impact of alternative splicing on circadian biology remains poorly understood. Here we describe a robust circadian and light-inducible splicing switch that changes the reading frame of the mouse mRNA encoding U2-auxiliary-factor 26 (U2AF26). This results in translation far into the 3' UTR, generating a C terminus with homology to the Drosophila clock regulator TIMELESS. This new U2AF26 variant destabilizes PERIOD1 protein, and U2AF26-deficient mice show nearly arrhythmic PERIOD1 protein levels and broad defects in circadian mRNA expression in peripheral clocks. At the behavioral level, these mice display increased phase advance adaptation following experimental jet lag. These data suggest light-induced U2af26 alternative splicing to be a buffering mechanism that limits PERIOD1 induction, thus stabilizing the circadian clock against abnormal changes in light:dark conditions.
Assuntos
Processamento Alternativo , Relógios Circadianos , Ritmo Circadiano , Mutação da Fase de Leitura , Proteínas Circadianas Period/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Animais , Comportamento Animal/efeitos da radiação , Encéfalo/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Relógios Circadianos/genética , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Fígado/metabolismo , Camundongos , Camundongos Transgênicos , Células NIH 3T3 , Estabilidade Proteica , RNA Mensageiro/genética , Fator de Processamento U2AFRESUMO
The noncanonical nuclear factor κB (ncNFκB) pathway regulates the expression of chemokines required for secondary lymphoid organ formation and thus plays a pivotal role in adaptive immunity. Whereas ncNFκB signaling has been well described in stromal cells and B cells, its role and regulation in T cells remain largely unexplored. ncNFκB activity critically depends on the upstream NFκB-inducing kinase (NIK). NIK expression is negatively regulated by the full-length isoform of TNF receptor-associated factor 3 (Traf3) as formation of a NIK-Traf3-Traf2 complex targets NIK for degradation. Here we show that T cell-specific and activation-dependent alternative splicing generates a Traf3 isoform lacking exon 8 (Traf3DE8) that, in contrast to the full-length protein, activates ncNFκB signaling. Traf3DE8 disrupts the NIK-Traf3-Traf2 complex and allows accumulation of NIK to initiate ncNFκB signaling in activated T cells. ncNFκB activity results in expression of several chemokines, among them B cell chemoattractant (CxCL13), both in a model T cell line and in primary human CD4(+) T cells. Because CxCL13 plays an important role in B cell migration and activation, our data suggest an involvement and provide a mechanistic basis for Traf3 alternative splicing and ncNFκB activation in contributing to T cell-dependent adaptive immunity.
Assuntos
Imunidade Adaptativa/fisiologia , Processamento Alternativo/fisiologia , Linfócitos T CD4-Positivos/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Fator 3 Associado a Receptor de TNF/biossíntese , Animais , Linfócitos T CD4-Positivos/imunologia , Quimiocina CXCL13/genética , Quimiocina CXCL13/imunologia , Quimiocina CXCL13/metabolismo , Células HeLa , Humanos , NF-kappa B/genética , NF-kappa B/imunologia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Fator 2 Associado a Receptor de TNF/biossíntese , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/imunologia , Fator 3 Associado a Receptor de TNF/genética , Fator 3 Associado a Receptor de TNF/imunologia , Quinase Induzida por NF-kappaBRESUMO
The regulation of posttranscriptional modifications of pre-mRNA by alternative splicing is important for cellular function, development, and immunity. The receptor tyrosine phosphatase CD45, which is expressed on all hematopoietic cells, is known for its role in the development and activation of T cells. CD45 is known to be alternatively spliced, a process that is partially regulated by heterogeneous nuclear ribonucleoprotein (hnRNP) L. To investigate the role of hnRNP L further, we have generated conditional hnRNP L knockout mice and found that LckCre-mediated deletion of hnRNP L results in a decreased thymic cellularity caused by a partial block at the transition stage between double-negative 4 and double-positive cells. In addition, hnRNP L(-/-) thymocytes express aberrant levels of the CD45RA splice isoforms and show high levels of phosphorylated Lck at the activator tyrosine Y394, but lack phosphorylation of the inhibitory tyrosine Y505. This indicated an increased basal Lck activity and correlated with higher proliferation rates of double-negative 4 cells in hnRNP L(-/-) mice. Deletion of hnRNP L also blocked the migration and egress of single-positive thymocytes to peripheral lymphoid organs in response to sphingosine-1-phosphate and the chemokines CCL21 and CXCL12 very likely as a result of aberrant splicing of genes encoding GTPase regulators and proteins affecting cytoskeletal organization. Our results indicate that hnRNP L regulates T cell differentiation and migration by regulating pre-TCR and chemokine receptor signaling.
Assuntos
Processamento Alternativo/imunologia , Diferenciação Celular/imunologia , Movimento Celular/imunologia , Proliferação de Células , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/genética , Células-Tronco/imunologia , Subpopulações de Linfócitos T/imunologia , Timo/imunologia , Processamento Alternativo/genética , Animais , Diferenciação Celular/genética , Movimento Celular/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco/citologia , Células-Tronco/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/metabolismo , Timo/citologia , Timo/metabolismoRESUMO
Even with the availability of tissue-restricted knockouts, studying the role of essential, multifunctional proteins in vivo is often complicated by the complete loss of cell viability. Here we make use of a knock-down approach to investigate the function of the essential protein PSF (polypyrimidine tract binding protein-associated splicing factor) in thymocytes. PSF is an RNA- and DNA-binding protein that has roles in all aspects of RNA biogenesis from transcription to stability. We show that a 50% reduction in expression of PSF in the thymus leads to an increase in apoptosis and a corresponding decrease in thymic cellularity. Using microarrays we find that thymocytes depleted of PSF show reduced expression of several histone genes relative to wildtype littermates likely due to reduced mRNA stability. Remarkably, reduced expression of a specific one of these histone genes (H2AE from histone cluster 1) is sufficient to induce apoptosis of a cultured T cell line. Therefore, we conclude that PSF contributes to the stability of a subset of histone genes and that loss of H2AE expression in the PSF-deficient thymocytes uniquely contributes to an increase in thymic apoptosis.
Assuntos
Apoptose/genética , Histonas/genética , Proteínas de Ligação a RNA/metabolismo , Timócitos/fisiologia , Animais , Sobrevivência Celular/genética , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Transgênicos , Fator de Processamento Associado a PTB , Estabilidade de RNA/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Linfócitos T/citologia , Linfócitos T/metabolismo , Linfócitos T/fisiologia , Timócitos/citologia , Timócitos/metabolismoRESUMO
Signal-induced alternative splicing of the CD45 gene in human T cells is essential for proper immune function. Skipping of the CD45 variable exons is controlled, in large part, by the recruitment of PSF to the pre-mRNA substrate upon T cell activation; however, the signaling cascade leading to exon exclusion has remained elusive. Here we demonstrate that in resting T cells PSF is directly phosphorylated by GSK3, thus promoting interaction of PSF with TRAP150, which prevents PSF from binding CD45 pre-mRNA. Upon T cell activation, reduced GSK3 activity leads to reduced PSF phosphorylation, releasing PSF from TRAP150 and allowing it to bind CD45 splicing regulatory elements and repress exon inclusion. Our data place two players, GSK3 and TRAP150, in the complex network that regulates CD45 alternative splicing and demonstrate a paradigm for signal transduction from the cell surface to the RNA processing machinery through the multifunctional protein PSF.