RESUMEN
Post-transcriptional regulation by RNA binding proteins can determine gene expression levels and drive changes in cancer cell proteomes. Identifying mechanisms of protein-RNA binding, including preferred sequence motifs bound in vivo, provides insights into protein-RNA networks and how they impact mRNA structure, function, and stability. In this review, we will focus on proteins that bind to AU-rich elements (AREs) in nascent or mature mRNA where they play roles in response to stresses encountered by cancer cells. ARE-binding proteins (ARE-BPs) specifically impact alternative splicing, stability, decay and translation, and formation of RNA-rich biomolecular condensates like cytoplasmic stress granules (SGs). For example, recent findings highlight the role of ARE-BPs - like TIAR and HUR - in chemotherapy resistance and in translational regulation of mRNAs encoding pro-inflammatory cytokines. We will discuss emerging evidence that different modes of ARE-BP activity impact leukaemia and lymphoma development, progression, adaptation to microenvironment and chemotherapy resistance.
Asunto(s)
Resistencia a Antineoplásicos , Neoplasias Hematológicas , Proteínas de Unión al ARN , Humanos , Resistencia a Antineoplásicos/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/metabolismo , Neoplasias Hematológicas/genética , Elementos Ricos en Adenilato y Uridilato , Regulación Neoplásica de la Expresión Génica , Animales , ARN Mensajero/metabolismo , ARN Mensajero/genética , Antineoplásicos/uso terapéutico , Antineoplásicos/farmacología , Estabilidad del ARN , Unión ProteicaRESUMEN
The integrated stress response (ISR) is a key cellular signaling pathway activated by environmental alterations that represses protein synthesis to restore homeostasis. To prevent sustained damage, the ISR is counteracted by the upregulation of growth arrest and DNA damage-inducible 34 (GADD34), a stress-induced regulatory subunit of protein phosphatase 1 that mediates translation reactivation and stress recovery. Here, we uncover a novel ISR regulatory mechanism that post-transcriptionally controls the stability of PPP1R15A mRNA encoding GADD34. We establish that the 3' untranslated region of PPP1R15A mRNA contains an active AU-rich element (ARE) recognized by proteins of the ZFP36 family, promoting its rapid decay under normal conditions and stabilization for efficient expression of GADD34 in response to stress. We identify the tight temporal control of PPP1R15A mRNA turnover as a component of the transient ISR memory, which sets the threshold for cellular responsiveness and mediates adaptation to repeated stress conditions.
Asunto(s)
Regiones no Traducidas 3' , Proteína Fosfatasa 1 , Animales , Humanos , Ratones , Regiones no Traducidas 3'/genética , Adaptación Fisiológica/genética , Elementos Ricos en Adenilato y Uridilato/genética , Células HEK293 , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 1/genética , Estabilidad del ARN/genética , ARN Mensajero/metabolismo , ARN Mensajero/genética , Estrés Fisiológico/genética , Tristetraprolina/metabolismo , Tristetraprolina/genéticaRESUMEN
T cells are key players in our defence against infections and malignancies. When T cells differentiate or become activated, they undergo substantial alterations in gene expression. Even though RNA expression levels are now well documented throughout different stages of T cells, it is not well understood how mRNA expression translates into the protein landscape. By combining paired RNA sequencing and mass spectrometry data of primary human CD8+ T cells, we report that mRNA expression is a poor proxy for the overall protein output, irrespective of the differentiation or activation status. Yet, gene class stratification revealed a function-specific correlation of mRNA with protein expression. This gene class-specific expression pattern associated with differences in gene characteristics such as sequence conservation and untranslated region (UTR) lengths. In addition, the presence of AU-rich elements in the 3'UTR associated with alterations in mRNA and protein abundance T cell activation dependent, gene class-specific manner. In conclusion, our study highlights the role of gene characteristics as a determinant for gene expression in T cells.
Asunto(s)
Elementos Ricos en Adenilato y Uridilato , Activación de Linfocitos , Humanos , ARN Mensajero/metabolismo , Regiones no Traducidas 3'/genética , Activación de Linfocitos/genética , Linfocitos T CD8-positivos/metabolismoRESUMEN
One of the primary mechanisms of post-transcriptional gene regulation is the modulation of RNA stability. We recently discovered that LINC00675, a transcript annotated as a long noncoding RNA (lncRNA), is transcriptionally regulated by FOXA1 and encodes a highly conserved small protein that localizes to the endoplasmic reticulum, hence renamed as FORCP (FOXA1-regulated conserved small protein). Here, we show that the endogenous FORCP transcript is rapidly degraded and rendered unstable as a result of 3'UTR-mediated degradation. Surprisingly, although the FORCP transcript is a canonical nonsense-mediated decay (NMD) and microRNA (miRNA) target, we found that it is not degraded by NMD or miRNAs. Targeted deletion of an evolutionarily conserved region in the FORCP 3'UTR using CRISPR/Cas9 significantly increased the stability of the FORCP transcript. Interestingly, this region requires the presence of an immediate downstream 55-nt-long sequence for transcript stability regulation. Functionally, colorectal cancer cells lacking this conserved region expressed from the endogenous FORCP locus displayed decreased proliferation and clonogenicity. These data demonstrate that the FORCP transcript is destabilized via conserved elements within its 3'UTR and emphasize the need to interrogate the function of a given 3'UTR in its native context.
Asunto(s)
ARN Largo no Codificante , Regiones no Traducidas 3'/genética , Elementos Ricos en Adenilato y Uridilato , Regulación de la Expresión Génica , Estabilidad del ARN/genética , ARN Largo no Codificante/genéticaRESUMEN
Fragile X-related protein-1 (FXR1) gene is highly amplified in patients with ovarian cancer, and this amplification is associated with increased expression of both FXR1 mRNA and protein. FXR1 expression directly associates with the survival and proliferation of cancer cells. Surface sensing of translation (SUnSET) assay demonstrates that FXR1 enhances the overall translation in cancer cells. Reverse-phase protein array (RPPA) reveals that cMYC is the key target of FXR1. Mechanistically, FXR1 binds to the AU-rich elements (ARE) present within the 3' untranslated region (3'UTR) of cMYC and stabilizes its expression. In addition, the RGG domain in FXR1 interacts with eIF4A1 and eIF4E proteins. These two interactions of FXR1 result in the circularization of cMYC mRNA and facilitate the recruitment of eukaryotic translation initiation factors to the translation start site. In brief, we uncover a mechanism by which FXR1 promotes cMYC levels in cancer cells.
Asunto(s)
Factor 4F Eucariótico de Iniciación/metabolismo , Neoplasias Ováricas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3' , Elementos Ricos en Adenilato y Uridilato , Animales , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Factor 4F Eucariótico de Iniciación/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones Desnudos , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Iniciación de la Cadena Peptídica Traduccional , Proteínas Proto-Oncogénicas c-myc/genética , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Transducción de Señal , Carga TumoralRESUMEN
Tristetraprolin (TTP), an RNA-binding protein, controls the stability of RNA by capturing AU-rich elements on their target genes. It has recently been identified that TTP serves as an anti-inflammatory protein by guiding the unstable mRNAs of pro-inflammatory proteins in multiple cells. However, it has not yet been investigated whether TTP affects the inflammatory responses in the hypothalamus. Since hypothalamic inflammation is tightly coupled to the disturbance of energy homeostasis, we designed the current study to investigate whether TTP regulates hypothalamic inflammation and thereby affects energy metabolism by utilizing TTP-deficient mice. We observed that deficiency of TTP led to enhanced hypothalamic inflammation via stimulation of a variety of pro-inflammatory genes. In addition, microglial activation occurred in the hypothalamus, which was accompanied by an enhanced inflammatory response. In line with these molecular and cellular observations, we finally confirmed that deficiency of TTP results in elevated core body temperature and energy expenditure. Taken together, our findings unmask novel roles of hypothalamic TTP on energy metabolism, which is linked to inflammatory responses in hypothalamic microglial cells.
Asunto(s)
Hipertermia/genética , Hipotálamo/patología , Microglía/metabolismo , Tristetraprolina/deficiencia , Elementos Ricos en Adenilato y Uridilato , Animales , Temperatura Corporal , Peso Corporal , Citocinas/metabolismo , Homeostasis , Inflamación , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Estabilidad del ARN , ARN Mensajero/metabolismo , Tristetraprolina/genética , Tristetraprolina/metabolismoRESUMEN
As part of the adaptive immune system, T cells are vital for the eradication of infected and malignantly transformed cells. To perform their protective function, T cells produce effector molecules that are either directly cytotoxic, such as granzymes, perforin, interferon-γ and tumour necrosis factor α, or attract and stimulate (immune) cells, such as interleukin-2. As these molecules can also induce immunopathology, tight control of their production is required. Indeed, inflammatory cytokine production is regulated on multiple levels. Firstly, locus accessibility and transcription factor availability and activity determine the amount of mRNA produced. Secondly, post-transcriptional mechanisms, influencing mRNA splicing/codon usage, stability, decay, localization and translation rate subsequently determine the amount of protein that is produced. In the immune suppressive environments of tumours, T cells gradually lose the capacity to produce effector molecules, resulting in tumour immune escape. Recently, the role of post-transcriptional regulation in fine-tuning T-cell effector function has become more appreciated. Furthermore, several groups have shown that exhausted or dysfunctional T cells from cancer patients or murine models possess mRNA for inflammatory mediators, but fail to produce effector molecules, hinting that post-transcriptional events also play a role in hampering tumour-infiltrating lymphocyte effector function. Here, the post-transcriptional regulatory events governing T-cell cytokine production are reviewed, with a specific focus on the importance of post-transcriptional regulation in anti-tumour responses. Furthermore, potential approaches to circumvent tumour-mediated dampening of T-cell effector function through the (dis)engagement of post-transcriptional events are explored, such as CRISPR/Cas9-mediated genome editing or chimeric antigen receptors.
Asunto(s)
Inmunoterapia/tendencias , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias/inmunología , Procesamiento Postranscripcional del ARN/inmunología , Linfocitos T/inmunología , Elementos Ricos en Adenilato y Uridilato/genética , Animales , Edición Génica , Humanos , Tolerancia Inmunológica , Activación de Linfocitos , Microambiente TumoralRESUMEN
Cancer is caused primarily by genomic alterations resulting in deregulation of gene regulatory circuits in key growth, apoptosis, or DNA repair pathways. Multiple genes associated with the initiation and development of tumors are also regulated at the level of mRNA decay, through the recruitment of RNA-binding proteins to AU-rich elements (AREs) located in their 3'-untranslated regions. One of these ARE-binding proteins, tristetraprolin (TTP; encoded by Zfp36), is consistently dysregulated in many human malignancies. Herein, using regulated overexpression or conditional ablation in the context of cutaneous chemical carcinogenesis, we show that TTP represents a critical regulator of skin tumorigenesis. We provide evidence that TTP controlled both tumor-associated inflammation and key oncogenic pathways in neoplastic epidermal cells. We identify Areg as a direct target of TTP in keratinocytes and show that EGFR signaling potentially contributed to exacerbated tumor formation. Finally, single-cell RNA-Seq analysis indicated that ZFP36 was downregulated in human malignant keratinocytes. We conclude that TTP expression by epidermal cells played a major role in the control of skin tumorigenesis.
Asunto(s)
Carcinogénesis/metabolismo , Queratinocitos/metabolismo , Neoplasias Cutáneas/metabolismo , Piel/metabolismo , Tristetraprolina/metabolismo , Regiones no Traducidas 3' , Elementos Ricos en Adenilato y Uridilato , Animales , Carcinogénesis/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo , Receptores ErbB/metabolismo , Redes Reguladoras de Genes , Humanos , Inflamación/metabolismo , Ratones Endogámicos C57BL , Estabilidad del ARN , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Neoplasias Cutáneas/genéticaRESUMEN
The negative regulator of G-protein signalling 4 (Rgs4) is linked to several neurologic diseases, e.g. schizophrenia, addiction, seizure and pain perception. Consequently, Rgs4 expression is tightly regulated, resulting in high mRNA and protein turnover. The post-transcriptional control of gene expression is mediated via RNA-binding proteins (RBPs) that interact with mRNAs in a combinatorial fashion. Here, we show that in neurons the RBP HuR reduces endogenous Rgs4 expression by destabilizing Rgs4 mRNA. Interestingly, in smooth muscle cells, Rgs4 is stabilized by HuR, indicating tissue-dependent differences in HuR function. Using in vitro RNA-based pulldown experiments, we identify the functional AU-rich element (ARE) within the Rgs4 3'-UTR that is recognized and bound by HuR. Bioinformatic analysis uncovered that this ARE lies within a highly conserved area next to a miR-26 binding site. We find that the neuronal-enriched miR-26 negatively influences Rgs4 expression in neurons. Further, HuR and miR-26 act synergistically in fluorescent reporter assays. Together, our data suggest a regulatory mechanism, in which an RBP selectively destabilizes a target mRNA in cooperation with a miRNA and the RISC machinery.
Asunto(s)
Elementos Ricos en Adenilato y Uridilato , Proteína 1 Similar a ELAV/genética , Regulación de la Expresión Génica , MicroARNs/genética , Neuronas/metabolismo , Proteínas RGS/genética , Complejo Silenciador Inducido por ARN/genética , Regiones no Traducidas 3' , Animales , Sitios de Unión , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Secuencia Conservada , Proteína 1 Similar a ELAV/metabolismo , Embrión de Mamíferos , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , MicroARNs/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Neuronas/citología , Especificidad de Órganos , Cultivo Primario de Células , Unión Proteica , Proteínas RGS/metabolismo , Estabilidad del ARN , Complejo Silenciador Inducido por ARN/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
An AU-rich element (ARE) is RNA element that enhances the rapid decay of mRNA. The RNA binding protein HuR stabilizes ARE-mRNA by exporting it to the cytoplasm. In most of cancer cells, HuR is exported to the cytoplasm and ARE-mRNA is stabilized. In addition, the viral gene product E4orf6 exports HuR to stabilize ARE-mRNA in adenovirus-infected cells and the stabilization is required for full virus replication. Previously we showed the oncolytic activity of E4orf6-deleted adenovirus dl355, which can replicate in cancer cells where ARE-mRNA is stabilized. In this study, we examined whether the further enhancement of HuR export can stimulate the replication and the oncolytic activity of dl355. We found that ethanol treatment promoted the cytoplasmic relocalization of HuR in cancer cells. In addition, the replication efficiency of dl355 increased in ethanol-treated cells, and in response, the cytolytic activity of the virus also increased in vitro and in vivo. Upregulation of a cleaved-PARP level in infected cells mediated by ethanol is suggesting that ethanol activated the apoptosis induced by dl355. IVa2 mRNA, the only ARE-mRNA among transcripts of adenovirus was augmented by ethanol treatment. These data indicate that the enhancement of ARE-mRNA stabilization as a result of ethanol treatment upregulates the oncolytic activity of dl355 and suggests that the combined use of an oncolytic adenovirus and ethanol treatment may be a good strategy for cancer therapy.
Asunto(s)
Adenoviridae/genética , Proteínas E4 de Adenovirus/genética , Proteína 1 Similar a ELAV/metabolismo , Neoplasias/terapia , Viroterapia Oncolítica , Células A549 , Elementos Ricos en Adenilato y Uridilato , Transporte Activo de Núcleo Celular , Adenoviridae/fisiología , Proteínas E4 de Adenovirus/metabolismo , Animales , Línea Celular , Proteína 1 Similar a ELAV/genética , Femenino , Eliminación de Gen , Células HeLa , Humanos , Ratones Endogámicos BALB C , Neoplasias/genética , Neoplasias/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Replicación ViralRESUMEN
Adenylate/uridylate-rich elements (AREs) are the most common cis-regulatory elements in the 3'-untranslated region (UTR) of mRNAs, where they fine-tune turnover by mediating mRNA decay. They increase plasticity and efficacy of mRNA regulation and are recognized by several ARE-specific RNA-binding proteins (RBPs). Typically, AREs are short linear motifs with a high content of complementary A and U nucleotides and often occur in multiple copies. Although thermodynamically rather unstable, the high AU-content might enable transient secondary structure formation and modify mRNA regulation by RBPs. We have recently suggested that the immunoregulatory RBP Roquin recognizes folded AREs as constitutive decay elements (CDEs), resulting in shape-specific ARE-mediated mRNA degradation. However, the structural evidence for a CDE-like recognition of AREs by Roquin is still lacking. We here present structures of CDE-like folded AREs, both in their free and protein-bound form. Moreover, the AREs in the UCP3 3'-UTR are additionally bound by the canonical ARE-binding protein AUF1 in their linear form, adopting an alternative binding-interface compared to the recognition of their CDE structure by Roquin. Strikingly, our findings thus suggest that AREs can be recognized in multiple ways, allowing control over mRNA regulation by adapting distinct conformational states, thus providing differential accessibility to regulatory RBPs.
Asunto(s)
Elementos Ricos en Adenilato y Uridilato , Proteínas de Unión al ARN/química , Ubiquitina-Proteína Ligasas/química , Sitios de Unión , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Motivos de Nucleótidos , Unión Proteica , Proteínas de Unión al ARN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Eukaryotic gene expression can be spatiotemporally tuned at the post-transcriptional level by cis-regulatory elements in mRNA sequences. An important example is the AU-rich element (ARE), which induces mRNA destabilization in a variety of biological contexts in mammals and can also mediate translational control. Regulation is mediated by trans-acting factors that recognize the ARE, such as Tristetraprolin (TTP) and BRF1/ZFP36L1. Although both proteins can destabilize their target mRNAs through the recruitment of the CCR4-NOT deadenylation complex, TTP also directly regulates translation. Whether ZFP36L1 can directly repress translation remains unknown. Here, we used an in vitro translation system derived from mammalian cell lines to address this key mechanistic issue in ARE regulation by ZFP36L1. Functional assays with mutant proteins reveal that ZFP36L1 can repress translation via AU-Rich elements independent of deadenylation. ZFP36L1-mediated translation repression requires interaction between ZFP36L1 and CNOT1, suggesting that it might use a repression mechanism similar to either TPP or miRISC. However, several lines of evidence suggest that the similarity ends there. Unlike, TTP, it does not efficiently interact with either 4E-HP or GIGYF2, suggesting it does not repress translation by recruiting these proteins to the mRNA cap. Moreover, ZFP36L1 could not repress ECMV-IRES driven translation and was resistant to pharmacological eIF4A inhibitor silvestrol, suggesting fundamental differences with miRISC repression via eIF4A. Collectively, our results reveal that ZFP36L1 represses translation directly and suggest that it does so via a novel mechanism distinct from other translational regulators that interact with the CCR4-NOT deadenylase complex.
Asunto(s)
Factor 1 de Respuesta al Butirato/metabolismo , Regulación de la Expresión Génica , Biosíntesis de Proteínas , Factores de Transcripción/metabolismo , Elementos Ricos en Adenilato y Uridilato , Células HEK293 , Humanos , Unión ProteicaRESUMEN
Low grade, chronic inflammation is a critical risk factor for immunologic dysfunction including autoimmune diseases. However, the multiplicity of complex mechanisms and lack of relevant murine models limit our understanding of the precise role of chronic inflammation. To address these hurdles, we took advantage of multi-omics data and a unique murine model with a low but chronic expression of IFN-γ, generated by replacement of the AU-rich element (ARE) in the 3' UTR region of IFN-γ mRNA with random nucleotides. Herein, we demonstrate that low but differential expression of IFN-γ in mice by homozygous or heterozygous ARE replacement triggers distinctive gut microbial alterations, of which alteration is female-biased with autoimmune-associated microbiota. Metabolomics data indicates that gut microbiota-dependent metabolites have more robust sex-differences than microbiome profiling, particularly those involved in fatty acid oxidation and nuclear receptor signaling. More importantly, homozygous ARE-Del mice have dramatic changes in tryptophan metabolism, bile acid and long-chain lipid metabolism, which interact with gut microbiota and nuclear receptor signaling similarly with sex-dependent metabolites. Consistent with these findings, nuclear receptor signaling, encompassing molecules such as PPARs, FXR, and LXRs, was detectable as a top canonical pathway in comparison of blood and tissue-specific gene expression between female homozygous vs heterozygous ARE-Del mice. Further analysis implies that dysregulated autophagy in macrophages is critical for breaking self-tolerance and gut homeostasis, while pathways interact with nuclear receptor signaling to regulate inflammatory responses. Overall, pathway-based integration of multi-omics data provides systemic and cellular insights about how chronic inflammation driven by IFN-γ results in the development of autoimmune diseases with specific etiopathological features.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Disbiosis/inmunología , Inflamación/inmunología , Interferón gamma/metabolismo , Macrófagos/inmunología , Regiones no Traducidas 3'/genética , Elementos Ricos en Adenilato y Uridilato/genética , Animales , Autofagia , Enfermedad Crónica , Femenino , Microbioma Gastrointestinal/inmunología , Interferón gamma/genética , Masculino , Ratones , Ratones Noqueados , Receptores Citoplasmáticos y Nucleares/metabolismo , Sexismo , Transducción de SeñalRESUMEN
Long-lasting CD8+ T cell responses are critical in combatting infections and tumors. The pro-inflammatory cytokine IFN-γ is a key effector molecule herein. We recently showed that in murine T cells the production of IFN-γ is tightly regulated through adenylate uridylate-rich elements (AREs) that are located in the 3' untranslated region (UTR) of the Ifng mRNA molecule. Loss of AREs resulted in prolonged cytokine production in activated T cells and boosted anti-tumoral T cell responses. Here, we investigated whether these findings can be translated to primary human T cells. Utilizing CRISPR-Cas9 technology, we deleted the ARE region from the IFNG 3' UTR in peripheral blood-derived human T cells. Loss of AREs stabilized the IFNG mRNA in T cells and supported a higher proportion of IFN-γ protein-producing T cells. Importantly, combining MART-1 T cell receptor engineering with ARE-Del gene editing showed that this was also true for antigen-specific activation of T cells. MART-1-specific ARE-Del T cells showed higher percentages of IFN-γ producing T cells in response to MART-1 expressing tumor cells. Combined, our study reveals that ARE-mediated posttranscriptional regulation is conserved between murine and human T cells. Furthermore, generating antigen-specific ARE-Del T cells is feasible, a feature that could potentially be used for therapeutical purposes.
Asunto(s)
Elementos Ricos en Adenilato y Uridilato , Linfocitos T CD8-positivos/inmunología , Interferón gamma/inmunología , Sistemas CRISPR-Cas , Línea Celular Tumoral , Femenino , Humanos , Interferón gamma/genética , Antígeno MART-1/genética , Antígeno MART-1/inmunología , Masculino , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Tristetraprolin (TTP) regulates inflammatory and immune responses by destabilizing target mRNAs via binding to their 3'-UTR AREs. We have recently reported that TTP preferentially up-regulates the expression level of innate immunity genes involved in the type I interferon-mediated signaling pathway and viral response in cancer cells. To elucidate the role of TTP-RNA interaction in TTP-mediated upregulation of gene expression, we performed iRIP-seq experiments to obtain the RNA interaction map consisting of direct and indirect binding sites of TTP in HeLa cells. We found substantial TTP binding signals in mRNA regions and the introns. ARE-motif AUUUA is over-represented in TTP binding peaks. Strikingly, AUUUA frequency is high both in 3'UTR and intronic regions, and the intronic peaks were more associated with TTP-regulated genes. Analysis of the over-represented motifs in TTP peaks revealed the high frequencies of UAGG and GUGUG motifs reported for hnRNPA2/B1 and CELF1 respectively in the 3'UTR and introns, and also the UGGAC motif overlapping with the m6A motif GGACU in the CDS regions. We further demonstrated that TTP binds to multiple intronic and exonic sites in the pre-mRNA/mRNA of the transcription factor RelB, correlating with the TTP-upregulated expression of RelB. TTP-up-regulated genes without a TTP binding site, but not those with, are highly enriched in innate immunity pathways and show higher tendency of harboring RelB binding sites in their promoter regions. These findings support a model in which TTP binding of RelB pre-mRNA/mRNA coordinates the RelB upregulation and activation of the innate immunity for antiviral response.
Asunto(s)
Redes Reguladoras de Genes/inmunología , Inmunidad Innata/genética , ARN Mensajero/metabolismo , Factor de Transcripción ReIB/metabolismo , Tristetraprolina/metabolismo , Regiones no Traducidas 3'/genética , Elementos Ricos en Adenilato y Uridilato/genética , Empalme Alternativo/inmunología , Sitios de Unión , Perfilación de la Expresión Génica , Células HeLa , Humanos , Intrones/genética , Regiones Promotoras Genéticas , Precursores del ARN/genética , Precursores del ARN/metabolismo , Estabilidad del ARN , ARN Mensajero/genética , Regulación hacia ArribaRESUMEN
BACKGROUND: Quiescence (G0) is a transient, cell cycle-arrested state. By entering G0, cancer cells survive unfavorable conditions such as chemotherapy and cause relapse. While G0 cells have been studied at the transcriptome level, how post-transcriptional regulation contributes to their chemoresistance remains unknown. RESULTS: We induce chemoresistant and G0 leukemic cells by serum starvation or chemotherapy treatment. To study post-transcriptional regulation in G0 leukemic cells, we systematically analyzed their transcriptome, translatome, and proteome. We find that our resistant G0 cells recapitulate gene expression profiles of in vivo chemoresistant leukemic and G0 models. In G0 cells, canonical translation initiation is inhibited; yet we find that inflammatory genes are highly translated, indicating alternative post-transcriptional regulation. Importantly, AU-rich elements (AREs) are significantly enriched in the upregulated G0 translatome and transcriptome. Mechanistically, we find the stress-responsive p38 MAPK-MK2 signaling pathway stabilizes ARE mRNAs by phosphorylation and inactivation of mRNA decay factor, Tristetraprolin (TTP) in G0. This permits expression of ARE mRNAs that promote chemoresistance. Conversely, inhibition of TTP phosphorylation by p38 MAPK inhibitors and non-phosphorylatable TTP mutant decreases ARE-bearing TNFα and DUSP1 mRNAs and sensitizes leukemic cells to chemotherapy. Furthermore, co-inhibiting p38 MAPK and TNFα prior to or along with chemotherapy substantially reduces chemoresistance in primary leukemic cells ex vivo and in vivo. CONCLUSIONS: These studies uncover post-transcriptional regulation underlying chemoresistance in leukemia. Our data reveal the p38 MAPK-MK2-TTP axis as a key regulator of expression of ARE-bearing mRNAs that promote chemoresistance. By disrupting this pathway, we develop an effective combination therapy against chemosurvival.
Asunto(s)
Elementos Ricos en Adenilato y Uridilato , Resistencia a Antineoplásicos , Procesamiento Postranscripcional del ARN , Tristetraprolina/metabolismo , Animales , Ciclo Celular , Células Cultivadas , Fosfatasa 1 de Especificidad Dual/genética , Fosfatasa 1 de Especificidad Dual/metabolismo , Células Hep G2 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células K562 , Células MCF-7 , Ratones , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteoma/genética , Proteoma/metabolismo , Células THP-1 , Transcriptoma , Tristetraprolina/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Fish interferon (IFN)-mediated antiviral innate immunity is the first line of defense against virus invasion. In the present study, we identify two fish IFN genes (here tentatively named IFNa and IFNc) with different-sized 3' UTRs from clone F strain of gibel carp Carassius auratus gibelio. Carp IFNa has a relatively short 3'UTR without AU-rich elements (AREs) but IFNc has a long one with 9 AREs. Functionally, carp IFNa and IFNc display significantly antiviral potential to viral infection, likely through induction of downstream IFN-stimulated genes (ISGs). Both carp IFN genes are induced by viral infection, poly(I:C) treatment and IRF3/7, which are ascribed to the IFN-sensitive response elements (ISRE) within their promoters. Carp IFN genes are also induced by each other and by themselves, indicating existence of a positive feedback loop in fish IFN-mediated antiviral immune response. Comparative analyses of 3'UTR-mediated expression regulation at mRNA and protein levels show that the ARE-containing 3'UTR of carp IFNc rather than the short 3'UTR of carp IFNa promotes mRNA decay but instead results in high-level protein expression, indicating that 3'UTR of fish IFN mRNAs might be a potential factor for regulation of IFN-mediated antiviral immune response. Considering a fact that a given protein function is largely related to its protein level, these results suggest that both promoter and 3'UTR contribute to the transcription and translation of fish IFN genes, thus shaping their eventually antiviral potential.
Asunto(s)
Regiones no Traducidas 3' , Enfermedades de los Peces/genética , Carpa Dorada/genética , Interferones/genética , Regulación hacia Arriba , Elementos Ricos en Adenilato y Uridilato , Animales , Línea Celular , Enfermedades de los Peces/inmunología , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/veterinaria , Infecciones por Herpesviridae/virología , Especificidad de la EspecieRESUMEN
Ferroptosis is a recently discovered form of programmed cell death, but its regulatory mechanisms remain poorly understood. Here, we show that the RNA-binding protein ZFP36/TTP (ZFP36 ring finger protein) plays a crucial role in regulating ferroptosis in hepatic stellate cells (HSCs). Upon exposure to ferroptosis-inducing compounds, the ubiquitin ligase FBXW7/CDC4 (F-box and WD repeat domain containing 7) decreased ZFP36 protein expression by recognizing SFSGLPS motif. FBXW7 plasmid contributed to classical ferroptotic events, whereas ZFP36 plasmid impaired FBXW7 plasmid-induced HSC ferroptosis. Interestingly, ZFP36 plasmid inhibited macroautophagy/autophagy activation by destabilizing ATG16L1 (autophagy related 16 like 1) mRNA. ATG16L1 plasmid eliminated the inhibitory action of ZFP36 plasmid on ferroptosis, and FBXW7 plasmid enhanced the effect of ATG16L1 plasmid on autophagy. Importantly, ZFP36 plasmid promoted ATG16L1 mRNA decay via binding to the AU-rich elements (AREs) within the 3'-untranslated region. The internal mutation of the ARE region abrogated the ZFP36-mediated ATG16L1 mRNA instability, and prevented ZFP36 plasmid-mediated ferroptosis resistance. In mice, treatment with erastin and sorafenib alleviated murine liver fibrosis by inducing HSC ferroptosis. HSC-specific overexpression of Zfp36 impaired erastin- or sorafenib-induced HSC ferroptosis. Noteworthy, we analyzed the effect of sorafenib on HSC ferroptosis in fibrotic patients with hepatocellular carcinoma receiving sorafenib monotherapy. Attractively, sorafenib monotherapy led to ZFP36 downregulation, ferritinophagy activation, and ferroptosis induction in human HSCs. Overall, these results revealed novel molecular mechanisms and signaling pathways of ferroptosis, and also identified ZFP36-autophagy-dependent ferroptosis as a potential target for the treatment of liver fibrosis. ABBREVIATIONS: ARE: AU-rich elements; ATG: autophagy related; BECN1: beclin 1; CHX: cycloheximide; COL1A1: collagen type I alpha 1 chain; ELAVL1/HuR: ELAV like RNA binding protein 1; FBXW7/CDC4: F-box and WD repeat domain containing 7; FN1: fibronectin 1; FTH1: ferritin heavy chain 1; GPX4/PHGPx: glutathione peroxidase 4; GSH: glutathione; HCC: hepatocellular carcinoma; HSC: hepatic stellate cell; LSEC: liver sinusoidal endothelial cell; MAP1LC3A: microtubule associated protein 1 light chain 3 alpha; MDA: malondialdehyde; NCOA4: nuclear receptor coactivator 4; PTGS2/COX2: prostaglandin-endoperoxide synthase 2; RBP: RNA-binding protein; ROS: reactive oxygen species; SLC7A11/xCT: solute carrier family 7 member 11; SQSTM1/p62: sequestosome 1; TNF: tumor necrosis factor; TP53/p53: tumor protein p53; UTR: untranslated region; ZFP36/TTP: ZFP36 ring finger protein.
Asunto(s)
Autofagia , Ferroptosis , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Transducción de Señal , Tristetraprolina/metabolismo , Elementos Ricos en Adenilato y Uridilato/genética , Adulto , Anciano , Anciano de 80 o más Años , Secuencias de Aminoácidos , Animales , Autofagia/efectos de los fármacos , Proteínas Relacionadas con la Autofagia/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Regulación hacia Abajo/efectos de los fármacos , Femenino , Ferroptosis/efectos de los fármacos , Humanos , Cirrosis Hepática/patología , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Piperazinas/farmacología , Plásmidos/genética , Unión Proteica/efectos de los fármacos , Estabilidad del ARN/efectos de los fármacos , Ratas , Transducción de Señal/efectos de los fármacos , Sorafenib/farmacología , Tristetraprolina/químicaRESUMEN
The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases 3 (PFKFB3) catalyzes the first committed rate-limiting step of glycolysis and is upregulated in cancer cells. The mechanism of PFKFB3 expression upregulation in cancer cells has not been fully elucidated. The PFKFB3 3'-UTR is reported to contain AU-rich elements (AREs) that are important for regulating PFKFB3 mRNA stability. However, the mechanisms by which PFKFB3 mRNA stability is determined by its 3'-UTR are not well known. We demonstrated that tristetraprolin (TTP), an ARE-binding protein, has a critical function regulating PFKFB3 mRNA stability. Our results showed that PFKFB3 mRNA contains three AREs in the 3'-UTR. TTP bound to the 3rd ARE and enhanced the decay of PFKFB3 mRNA. Overexpression of TTP decreased PFKFB3 expression and ATP levels but increased GSH level in cancer cells. Overexpression of PFKFB3 cDNA without the 3'-UTR rescued ATP level and GSH level in TTP-overexpressing cells. Our results suggested that TTP post-transcriptionally downregulated PFKFB3 expression and that overexpression of TTP may contribute to suppression of glycolysis and energy production of cancer cells in part by downregulating PFKFB3 expression.
Asunto(s)
Regulación hacia Abajo , Neoplasias/patología , Fosfofructoquinasa-2/metabolismo , Tristetraprolina/fisiología , Elementos Ricos en Adenilato y Uridilato , Glucólisis , Humanos , Neoplasias/metabolismo , Fosfofructoquinasa-2/genética , Estabilidad del ARN , ARN Mensajero , Transcripción Genética , Tristetraprolina/metabolismo , Células Tumorales CultivadasRESUMEN
AU-rich elements (AREs) comprise one of the most widely studied families of regulatory RNA structures met in RNAs engaged in complex immunological reactions. A multitude of genetic, molecular, holistic and functional studies have been utilized for the analyses of the AREs and their interactions to proteins that bind to them. Data stemming from these studies brought forth a world of RNA-related check-points against infection, chronic inflammation, tumor associated immunity, and autoimmunity; and the interest to capitalize the interactions of AREs for clinical management and therapy. They also provided lessons on the cellular capabilities of post-transcriptional control. Originally thought as transcript-restricted regulators of turnover and translation, ARE-binding proteins do in fact harbor great versatility and interactivity across nuclear and cytoplasmic compartments; and act as functional coordinators of immune-cellular programs. Harnessing these deterministic functions requires extensive knowledge of their synergies or antagonisms at a cell-specific level; but holds great promise since it can provide the efficacy of combinatorial therapies with single agents.