RESUMO
The nuclear factor kappa B (NF-κB) family of transcription factors orchestrates signal-induced gene expression in diverse cell types. Cellular responses to NF-κB activation are regulated at the level of cell and signal specificity, as well as differential use of family members (subunit specificity). Here we used time-dependent multi-omics to investigate the selective functions of Rel and RelA, two closely related NF-κB proteins, in primary B lymphocytes activated via the B cell receptor. Despite large numbers of shared binding sites genome wide, Rel and RelA directed kinetically distinct cascades of gene expression in activated B cells. Single-cell RNA sequencing revealed marked heterogeneity of Rel- and RelA-specific responses, and sequential binding of these factors was not a major mechanism of protracted transcription. Moreover, nuclear co-expression of Rel and RelA led to functional antagonism between the factors. By rigorously identifying the target genes of each NF-κB subunit, these studies provide insights into exclusive functions of Rel and RelA in immunity and cancer.
Assuntos
NF-kappa B , Fator de Transcrição RelA , NF-kappa B/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Linfócitos B/metabolismo , Sítios de Ligação , Receptores de Antígenos/metabolismoRESUMO
RNA modifications, including N6-methyladenosine (m6A), critically modulate protein expression programs in a range of cellular processes. Although the transcriptomes of cells undergoing senescence are strongly regulated, the landscape and impact of m6A modifications during senescence are poorly understood. Here, we report a robust m6A modification of PTCHD4 mRNA, encoding Patched Domain-Containing Protein 4, in senescent cells. The METTL3/METTL14 complex was found to incorporate the m6A modification on PTCHD4 mRNA; addition of m6A rendered PTCHD4 mRNA more stable and increased PTCHD4 production. MeRIP RT-qPCR and eCLIP analyses were used to map this m6A modification to the last exon of PTCHD4 mRNA. Further investigation identified IGF2BP1, but not other m6A readers, as responsible for the stabilization and increased abundance of m6A-modified PTCHD4 mRNA. Silencing PTCHD4, a transmembrane protein, enhanced growth arrest and DNA damage in pre-senescent cells and sensitized them to senolysis and apoptosis. Our results indicate that m6A modification of PTCHD4 mRNA increases the production of PTCHD4, a protein associated with senescent cell survival, supporting the notion that regulating m6A modification on specific mRNAs could be exploited to eliminate senescent cells for therapeutic benefit.
Assuntos
Adenosina , Sobrevivência Celular , Senescência Celular , Metiltransferases , RNA Mensageiro , Proteínas de Ligação a RNA , Humanos , Senescência Celular/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Metiltransferases/metabolismo , Metiltransferases/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Sobrevivência Celular/genética , Apoptose/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dano ao DNARESUMO
Senescent cells are beneficial for repairing acute tissue damage, but they are harmful when they accumulate in tissues, as occurs with advancing age. Senescence-associated extracellular vesicles (S-EVs) can mediate cell-to-cell communication and export intracellular content to the microenvironment of aging tissues. Here, we studied the uptake of EVs from senescent cells (S-EVs) and proliferating cells (P-EVs) and found that P-EVs were readily taken up by proliferating cells (fibroblasts and cervical cancer cells) while S-EVs were not. We thus investigated the surface proteome (surfaceome) of P-EVs relative to S-EVs derived from cells that had reached senescence via replicative exhaustion, exposure to ionizing radiation, or treatment with etoposide. We found that relative to P-EVs, S-EVs from all senescence models were enriched in proteins DPP4, ANXA1, ANXA6, S10AB, AT1A1, and EPHB2. Among them, DPP4 was found to selectively prevent uptake by proliferating cells, as ectopic overexpression of DPP4 in HeLa cells rendered DPP4-expressing EVs that were no longer taken up by other proliferating cells. We propose that DPP4 on the surface of S-EVs makes these EVs refractory to internalization by proliferating cells, advancing our knowledge of the impact of senescent cells in aging-associated processes.
Assuntos
Senescência Celular , Vesículas Extracelulares , Humanos , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Células HeLa , Vesículas Extracelulares/metabolismo , EnvelhecimentoRESUMO
Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) modulate gene expression programs in physiology and disease. Here, we report a noncoding RNA regulatory network that modulates myoblast fusion into multinucleated myotubes, a process that occurs during muscle development and muscle regeneration after injury. In early stages of human myogenesis, the levels of lncRNA OIP5-AS1 increased, while the levels of miR-7 decreased. Moreover, OIP5-AS1 bound and induced miR-7 decay via target RNA-directed miRNA decay; accordingly, loss of OIP5-AS1 attenuated, while antagonizing miR-7 accelerated, myotube formation. We found that the OIP5-AS1-mediated miR-7 degradation promoted myoblast fusion, as it derepressed the miR-7 target MYMX mRNA, which encodes the fusogenic protein myomixer (MYMX). Remarkably, an oligonucleotide site blocker interfered with the OIP5-AS1-directed miR-7 degradation, allowing miR-7 to accumulate, lowering MYMX production and suppressing myotube formation. These results highlight a mechanism whereby lncRNA OIP5-AS1-mediated miR-7 decay promotes myotube formation by stimulating a myogenic fusion program.
Assuntos
MicroRNAs , RNA Longo não Codificante , Humanos , RNA Longo não Codificante/genética , MicroRNAs/genética , Desenvolvimento Muscular/genéticaRESUMO
The mammalian transcriptome comprises a vast family of long noncoding (lnc)RNAs implicated in physiologic processes such as myogenesis, through which muscle forms during embryonic development and regenerates in the adult. However, the specific molecular mechanisms by which lncRNAs regulate human myogenesis are poorly understood. Here, we identified a novel muscle-specific lncRNA, lncFAM71E1-2:2 (lncFAM), which increased robustly during early human myogenesis. Overexpression of lncFAM promoted differentiation of human myoblasts into myotubes, while silencing lncFAM suppressed this process. As lncFAM resides in the nucleus, chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS) analysis was employed to identify the molecular mechanisms whereby it might promote myogenesis. Analysis of lncFAM-interacting proteins revealed that lncFAM recruited the RNA-binding protein HNRNPL to the promoter of MYBPC2, in turn increasing MYBPC2 mRNA transcription and enhancing production of the myogenic protein MYBPC2. These results highlight a mechanism whereby a novel ribonucleoprotein complex, lncFAM-HNRNPL, elevates MYBPC2 expression transcriptionally to promote myogenesis.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo L , Desenvolvimento Muscular , Regiões Promotoras Genéticas , RNA Longo não Codificante , Transcrição Gênica , Humanos , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transcrição Gênica/genética , Inativação Gênica , Transporte Proteico/genéticaRESUMO
A major stress response influenced by microRNAs (miRNAs) is senescence, a state of indefinite growth arrest triggered by sublethal cell damage. Here, through bioinformatic analysis and experimental validation, we identified miR-340-5p as a novel miRNA that foments cellular senescence. miR-340-5p was highly abundant in diverse senescence models, and miR-340-5p overexpression in proliferating cells rendered them senescent. Among the target mRNAs, miR-340-5p prominently reduced the levels of LBR mRNA, encoding lamin B receptor (LBR). Loss of LBR by ectopic overexpression of miR-340-5p derepressed heterochromatin in lamina-associated domains, promoting the expression of DNA repetitive elements characteristic of senescence. Importantly, overexpressing miR-340-5p enhanced cellular sensitivity to senolytic compounds, while antagonization of miR-340-5p reduced senescent cell markers and engendered resistance to senolytic-induced cell death. We propose that miR-340-5p can be exploited for removing senescent cells to restore tissue homeostasis and mitigate damage by senescent cells in pathologies of human aging.
Assuntos
Senescência Celular/genética , MicroRNAs/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Linhagem Celular , Proliferação de Células , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Regulação da Expressão Gênica , Heterocromatina , Humanos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptor de Lamina BRESUMO
Mammalian circRNAs can influence different cellular processes by interacting with proteins and other nucleic acids. Here, we used ribonucleoprotein immunoprecipitation (RIP) analysis to identify systematically the circRNAs associated with the cancer-related protein AUF1. Among the circRNAs interacting with AUF1 in HeLa (human cervical carcinoma) cells, we focused on hsa_circ_0032434 (circPCNX), an abundant target of AUF1. Overexpression of circPCNX specifically interfered with the binding of AUF1 to p21 (CDKN1A) mRNA, thereby promoting p21 mRNA stability and elevating the production of p21, a major inhibitor of cell proliferation. Conversely, silencing circPCNX increased AUF1 binding to p21 mRNA, reducing p21 production and promoting cell division. Importantly, eliminating the AUF1-binding region of circPCNX abrogated the rise in p21 levels and rescued proliferation. Therefore, we propose that the interaction of circPCNX with AUF1 selectively prevents AUF1 binding to p21 mRNA, leading to enhanced p21 mRNA stability and p21 protein production, thereby suppressing cell growth.
Assuntos
Proliferação de Células/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Ribonucleoproteína Nuclear Heterogênea D0/metabolismo , RNA Circular/metabolismo , Regiões 3' não Traduzidas , Sítios de Ligação , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células HeLa , Humanos , RNA Circular/química , RNA Mensageiro/metabolismoRESUMO
Deregulation of the translational machinery is emerging as a critical contributor to cancer development. The contribution of microRNAs in translational gene control has been established however; the role of microRNAs in disrupting the cap-dependent translation regulation complex has not been previously described. Here, we established that elevated miR-520c-3p represses global translation, cell proliferation and initiates premature senescence in HeLa and DLBCL cells. Moreover, we demonstrate that miR-520c-3p directly targets translation initiation factor, eIF4GII mRNA and negatively regulates eIF4GII protein synthesis. miR-520c-3p overexpression diminishes cells colony formation and reduces tumor growth in a human xenograft mouse model. Consequently, downregulation of eIF4GII by siRNA decreases translation, cell proliferation and ability to form colonies, as well as induces cellular senescence. In vitro and in vivo findings were further validated in patient samples; DLBCL primary cells demonstrated low miR-520c-3p levels with reciprocally up-regulated eIF4GII protein expression. Our results provide evidence that the tumor suppressor effect of miR-520c-3p is mediated through repression of translation while inducing senescence and that eIF4GII is a key effector of this anti-tumor activity.
Assuntos
Proliferação de Células , Fator de Iniciação Eucariótico 4G/genética , Linfoma Difuso de Grandes Células B/genética , MicroRNAs/genética , Animais , Linhagem Celular Tumoral , Senescência Celular/genética , Regulação para Baixo , Fator de Iniciação Eucariótico 4G/biossíntese , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma Difuso de Grandes Células B/patologia , Camundongos , MicroRNAs/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human diffuse large B-cell lymphomas (DLBCLs) often aberrantly express oncogenes that generally contain complex secondary structures in their 5' untranslated region (UTR). Oncogenes with complex 5'UTRs require enhanced eIF4A RNA helicase activity for translation. PDCD4 inhibits eIF4A, and PDCD4 knockout mice have a high penetrance for B-cell lymphomas. Here, we show that on B-cell receptor (BCR)-mediated p70s6K activation, PDCD4 is degraded, and eIF4A activity is greatly enhanced. We identified a subset of genes involved in BCR signaling, including CARD11, BCL10, and MALT1, that have complex 5'UTRs and encode proteins with short half-lives. Expression of these known oncogenic proteins is enhanced on BCR activation and is attenuated by the eIF4A inhibitor Silvestrol. Antigen-experienced immunoglobulin (Ig)G(+) splenic B cells, from which most DLBCLs are derived, have higher levels of eIF4A cap-binding activity and protein translation than IgM(+) B cells. Our results suggest that eIF4A-mediated enhancement of oncogene translation may be a critical component for lymphoma progression, and specific targeting of eIF4A may be an attractive therapeutic approach in the management of human B-cell lymphomas.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Guanilato Ciclase/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Regiões 5' não Traduzidas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 10 de Linfoma CCL de Células B , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Western Blotting , Proteínas Adaptadoras de Sinalização CARD/genética , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , RNA Helicases DEAD-box/antagonistas & inibidores , RNA Helicases DEAD-box/genética , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Fator de Iniciação 4A em Eucariotos/genética , Guanilato Ciclase/genética , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Pessoa de Meia-Idade , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Triterpenos/farmacologiaRESUMO
BACKGROUND: The mechanistic target of rapamycin, (mTOR) kinase plays a pivotal role in controlling critical cellular growth and survival pathways, and its aberrant induction is implicated in cancer pathogenesis. Therefore, suppression of active mTOR signaling has been of great interest to researchers; several mTOR inhibitors have been discovered to date. Ethanol (EtOH), similar to pharmacologic mTOR inhibitors, has been shown to suppress the mTOR signaling pathway, though in a non-catalytic manner. Despite population studies showing that the consumption of EtOH has a protective effect against hematological malignancies, the mechanisms behind EtOH's modulation of mTOR activity in cells and its downstream consequences are largely unknown. Here we evaluated the effects of EtOH on the mTOR pathway, in comparison to the active-site mTOR inhibitor INK128, and compared translatome analysis of their downstream effects in diffuse large B-cell lymphoma (DLBCL). RESULTS: Treatment of DLBCL cells with EtOH suppressed mTORC1 complex formation while increasing AKT phosphorylation and mTORC2 complex assembly. INK128 completely abrogated AKT phosphorylation without affecting the structure of mTORC1/2 complexes. Accordingly, EtOH less profoundly suppressed cap-dependent translation and global protein synthesis, compared to a remarkable inhibitory effect of INK128 treatment. Importantly, EtOH treatment induced the formation of stress granules, while INK128 suppressed their formation. Microarray analysis of polysomal RNA revealed that although both agents primarily affected cell growth and survival, EtOH and INK128 regulated the synthesis of mostly distinct genes involved in these processes. Though both EtOH and INK128 inhibited cell cycle, proliferation and autophagy, EtOH, in contrast to INK128, did not induce cell apoptosis. CONCLUSION: Given that EtOH, similar to pharmacologic mTOR inhibitors, inhibits mTOR signaling, we systematically explored the effect of EtOH and INK128 on mTOR signal transduction, components of the mTORC1/2 interaction and their downstream effectors in DLBCL malignancy. We found that EtOH partially inhibits mTOR signaling and protein translation, compared to INK128's complete mTOR inhibition. Translatome analysis of mTOR downstream target genes established that differential inhibition of mTOR by EtOH and INK128 distinctly modulates translation of specific subsets of mRNAs involved in cell growth and survival, leading to differential cellular response and survival.
Assuntos
Benzoxazóis/farmacologia , Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Linfoma Difuso de Grandes Células B/metabolismo , Pirimidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , Autofagia/efeitos dos fármacos , Autofagia/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Interactions between NK and dendritic cells (DC) affect maturation and function of both cell populations, including NK killing of DC (editing) that is important for controlling the quality of immune responses. We also know that antigen-stimulated Vγ2Vδ2 T cells costimulate NK cells via 4-1BB to enhance killing of tumor cell lines but we do not know what regulates 4-1BB expression or whether other NK effector functions including DC killing, might also be influenced by NK:γδ T cell cross talk. Here we show that antigen-stimulated γδ T cells costimulate NK through ICOS:ICOSL and this signal increases NK killing of autologous DC. Effects of NK:γδ T cell co-culture, which could be reproduced with soluble ICOS-Fc fusion protein, included increased CD69 and 4-1BB expression, IFN-γ, TNF-α, MIP-1ß, I-309, RANTES and sFasL production, as well as elevated mRNA levels for costimulatory receptors OX40 (TNFRSF4) and GITR (TNFRSF18). Thus, ICOS/ICOSL costimulation of NK by Vγ2Vδ2 T cells had broad effects on NK phenotype and effector functions. The NK γδ T cell cross talk links innate and antigen-specific lymphocyte responses in the control of cytotoxic effector function and dendritic cell killing. This article is protected by copyright. All rights reserved.
RESUMO
Evaporation of sweat on the skin surface is the major mechanism for dissipating heat in humans. The secretory capacity of sweat glands (SWGs) declines during aging, leading to heat intolerance in the elderly, but the mechanisms responsible for this decline are poorly understood. We investigated the molecular changes accompanying SWG aging in mice, where sweat tests confirmed a significant reduction of active SWGs in old mice relative to young mice. We first identified SWG-enriched mRNAs by comparing the skin transcriptome of Eda mutant Tabby male mice, which lack SWGs, with that of wild-type control mice by RNA-sequencing analysis. This comparison revealed 171 mRNAs enriched in SWGs, including 47 mRNAs encoding 'core secretory' proteins such as transcription factors, ion channels, ion transporters, and trans-synaptic signaling proteins. Among these, 28 SWG-enriched mRNAs showed significantly altered abundance in the aged male footpad skin, and 11 of them, including Foxa1, Best2, Chrm3, and Foxc1 mRNAs, were found in the 'core secretory' category. Consistent with the changes in mRNA expression levels, immunohistology revealed that higher numbers of secretory cells from old SWGs express the transcription factor FOXC1, the protein product of Foxc1 mRNA. In sum, our study identified mRNAs enriched in SWGs, including those that encode core secretory proteins, and altered abundance of these mRNAs and proteins with aging in mouse SWGs.
Assuntos
Envelhecimento , Glândulas Sudoríparas , Animais , Glândulas Sudoríparas/metabolismo , Camundongos , Envelhecimento/genética , Envelhecimento/metabolismo , Masculino , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , TranscriptomaRESUMO
Myogenesis is a highly orchestrated process whereby muscle precursor cells, myoblasts, develop into muscle fibers to form skeletal muscle during embryogenesis and regenerate adult muscle. Here, we studied the RNA-binding protein FUS (fused in sarcoma), which has been implicated in muscular and neuromuscular pathologies but is poorly characterized in myogenesis. Given that FUS levels declined in human and mouse models of skeletal myogenesis, and that silencing FUS enhanced myogenesis, we hypothesized that FUS might be a repressor of myogenic differentiation. Interestingly, overexpression of FUS delayed myogenesis, accompanied by slower production of muscle differentiation markers. To identify the mechanisms through which FUS inhibits myogenesis, we uncovered RNA targets of FUS by ribonucleoprotein immunoprecipitation (RIP) followed by RNA-sequencing (RNA-seq) analysis. Stringent selection of the bound transcripts uncovered Tnnt1 mRNA, encoding troponin T1 (TNNT1), as a major effector of FUS influence on myogenesis. We found that in myoblasts, FUS retained Tnnt1 mRNA in the nucleus, preventing TNNT1 expression; however, reduction of FUS during myogenesis or by silencing FUS released Tnnt1 mRNA for export to the cytoplasm, enabling TNNT1 translation and promoting myogenesis. We propose that FUS inhibits myogenesis by suppressing TNNT1 expression through a mechanism of nuclear Tnnt1 mRNA retention.
RESUMO
Cellular senescence, a state of persistent growth arrest, is closely associated with aging and age-related diseases. Deciphering the heterogeneity within senescent cell populations and identifying therapeutic targets are paramount for mitigating senescence-associated pathologies. In this study, proteins on the surface of cells rendered senescent by replicative exhaustion and by exposure to ionizing radiation (IR) were identified using mass spectrometry analysis, and a subset of them was further studied using single-cell CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) analysis. Based on the presence of proteins on the cell surface, we identified two distinct IR-induced senescent cell populations: one characterized by high levels of CD109 and CD112 (cluster 3), the other characterized by high levels of CD112, CD26, CD73, HLA-ABC, CD54, CD49A, and CD44 (cluster 0). We further found that cluster 0 represented proliferating and senescent cells in the G1 phase of the division cycle, and CITE-seq detection of cell surface proteins selectively discerned those in the senescence group. Our study highlights the heterogeneity of senescent cells and underscores the value of cell surface proteins as tools for distinguishing senescent cell programs and subclasses, paving the way for targeted therapeutic strategies in disorders exacerbated by senescence.
RESUMO
Cellular senescence is a dynamic biological process triggered by sublethal cell damage and driven by specific changes in gene expression programs. We recently identified ANKRD1 (ankyrin repeat domain 1) as a protein strongly elevated after triggering senescence in fibroblasts. Here, we set out to investigate the mechanisms driving the elevated production of ANKRD1 in the early stages of senescence. Our results indicated that the rise in ANKRD1 levels after triggering senescence using etoposide (Eto) was the result of moderate increases in transcription and translation, and robust mRNA stabilization. Antisense oligomer (ASO) pulldown followed by mass spectrometry revealed a specific interaction of the RNA-binding protein RBMS1 with ANKRD1 mRNA that was confirmed by ribonucleoprotein immunoprecipitation analysis. RBMS1 abundance decreased in the nucleus and increased in the cytoplasm during Eto-induced senescence; in agreement with the hypothesis that RBMS1 may participate in post-transcriptional stabilization of ANKRD1 mRNA, silencing RBMS1 reduced, while overexpressing RBMS1 enhanced ANKRD1 mRNA half-life after Eto treatment. A segment proximal to the ANKRD1 coding region was identified as binding RBMS1 and conferring RBMS1-dependent increased expression of a heterologous reporter. We propose that RBMS1 increases expression of ANKRD1 during the early stages of senescence by stabilizing ANKRD1 mRNA.
Assuntos
Senescência Celular , Proteínas Nucleares , Estabilidade de RNA , RNA Mensageiro , Proteínas de Ligação a RNA , Proteínas Repressoras , Humanos , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Estabilidade de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Etoposídeo/farmacologia , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Núcleo Celular/metabolismo , Linhagem Celular , Proteínas MuscularesRESUMO
The posttranslational modification of proteins critically influences many biological processes and is a key mechanism that regulates the function of the RNA-binding protein Hu antigen R (HuR), a hub in liver cancer. Here, we show that HuR is SUMOylated in the tumor sections of patients with hepatocellular carcinoma in contrast to the surrounding tissue, as well as in human cell line and mouse models of the disease. SUMOylation of HuR promotes major cancer hallmarks, namely proliferation and invasion, whereas the absence of HuR SUMOylation results in a senescent phenotype with dysfunctional mitochondria and endoplasmic reticulum. Mechanistically, SUMOylation induces a structural rearrangement of the RNA recognition motifs that modulates HuR binding affinity to its target RNAs, further modifying the transcriptomic profile toward hepatic tumor progression. Overall, SUMOylation constitutes a mechanism of HuR regulation that could be potentially exploited as a therapeutic strategy for liver cancer.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Humanos , Camundongos , Carcinoma Hepatocelular/metabolismo , Modelos Animais de Doenças , Proteína Semelhante a ELAV 1/metabolismo , Neoplasias Hepáticas/patologia , RNA/metabolismo , SumoilaçãoRESUMO
Maintenance of genomic stability depends on the DNA damage response, a biologic barrier in early stages of cancer development. Failure of this response results in genomic instability and high predisposition toward lymphoma, as seen in patients with ataxia-telangiectasia mutated (ATM) dysfunction. ATM activates multiple cell-cycle checkpoints and DNA repair after DNA damage, but its influence on posttranscriptional gene expression has not been examined on a global level. We show that ionizing radiation modulates the dynamic association of the RNA-binding protein HuR with target mRNAs in an ATM-dependent manner, potentially coordinating the genotoxic response as an RNA operon. Pharmacologic ATM inhibition and use of ATM-null cells revealed a critical role for ATM in this process. Numerous mRNAs encoding cancer-related proteins were differentially associated with HuR depending on the functional state of ATM, in turn affecting expression of encoded proteins. The findings presented here reveal a previously unidentified role of ATM in controlling gene expression posttranscriptionally. Dysregulation of this DNA damage response RNA operon is probably relevant to lymphoma development in ataxia-telangiectasia persons. These novel RNA regulatory modules and genetic networks provide critical insight into the function of ATM in oncogenesis.
Assuntos
Proteínas de Ciclo Celular/genética , Dano ao DNA , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Linfócitos/metabolismo , Óperon/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Supressoras de Tumor/genética , Antígenos de Superfície/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Reparo do DNA , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Redes Reguladoras de Genes , Humanos , Linfoma/etiologia , Proteínas Mutantes , Ligação Proteica/efeitos da radiação , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Radiação IonizanteRESUMO
The ubiquitously expressed transcription factor TFII-I is a multifunctional protein with pleiotropic roles in gene regulation. TFII-I associated polymorphisms are implicated in Sjögren's syndrome and Lupus in humans and, germline deletion of the Gtf2i gene in mice leads to embryonic lethality. Here we report a unique role for TFII-I in homeostasis of innate properties of B lymphocytes. Loss of Gtf2i in murine B lineage cells leads to an alteration in transcriptome, chromatin landscape and associated transcription factor binding sites, which exhibits myeloid-like features and coincides with enhanced sensitivity to LPS induced gene expression. TFII-I deficient B cells also show increased switching to IgG3, a phenotype associated with inflammation. These results demonstrate a role for TFII-I in maintaining immune homeostasis and provide clues for GTF2I polymorphisms associated with B cell dominated autoimmune diseases in humans.
Assuntos
Síndrome de Sjogren , Fatores de Transcrição TFIII , Fatores de Transcrição TFII , Humanos , Camundongos , Animais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Cromatina , Ligação Proteica , Fatores de Transcrição TFIII/genética , Fatores de Transcrição TFIII/metabolismo , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFII/metabolismoRESUMO
Senescence is a state of enduring growth arrest triggered by sublethal cell damage. Given that senescent cells actively secrete proinflammatory and matrix-remodeling proteins, their accumulation in tissues of older persons has been linked to many diseases of aging. Despite intense interest in identifying robust markers of senescence, the highly heterogeneous and dynamic nature of the senescent phenotype has made this task difficult. Here, we set out to comprehensively analyze the senescent transcriptome of human diploid fibroblasts at the individual-cell scale by performing single-cell RNA-sequencing analysis through two approaches. First, we characterized the different cell states in cultures undergoing senescence triggered by different stresses, and found distinct cell subpopulations that expressed mRNAs encoding proteins with roles in growth arrest, survival, and the secretory phenotype. Second, we characterized the dynamic changes in the transcriptomes of cells as they developed etoposide-induced senescence; by tracking cell transitions across this process, we found two different senescence programs that developed divergently, one in which cells expressed traditional senescence markers such as p16 (CDKN2A) mRNA, and another in which cells expressed long noncoding RNAs and splicing was dysregulated. Finally, we obtained evidence that the proliferation status at the time of senescence initiation affected the path of senescence, as determined based on the expressed RNAs. We propose that a deeper understanding of the transcriptomes during the progression of different senescent cell phenotypes will help develop more effective interventions directed at this detrimental cell population.
Assuntos
Senescência Celular , Transcriptoma , Humanos , Idoso , Idoso de 80 Anos ou mais , Senescência Celular/genética , Envelhecimento/genética , FenótipoRESUMO
Senescent cells release a variety of cytokines, proteases, and growth factors collectively known as the senescence-associated secretory phenotype (SASP). Sustained SASP contributes to a pattern of chronic inflammation associated with aging and implicated in many age-related diseases. Here, we investigated the expression and function of the immunomodulatory cytokine BAFF (B-cell activating factor; encoded by the TNFSF13B gene), a SASP protein, in multiple senescence models. We first characterized BAFF production across different senescence paradigms, including senescent human diploid fibroblasts (WI-38, IMR-90) and monocytic leukemia cells (THP-1), and tissues of mice induced to undergo senescence. We then identified IRF1 (interferon regulatory factor 1) as a transcription factor required for promoting TNFSF13B mRNA transcription in senescence. We discovered that suppressing BAFF production decreased the senescent phenotype of both fibroblasts and monocyte-like cells, reducing IL6 secretion and SA-ß-Gal staining. Importantly, however, the influence of BAFF on the senescence program was cell type-specific: in monocytes, BAFF promoted the early activation of NF-κB and general SASP secretion, while in fibroblasts, BAFF contributed to the production and function of TP53 (p53). We propose that BAFF is elevated across senescence models and is a potential target for senotherapy.