RESUMO
The long non-coding RNA EPR is expressed in epithelial tissues, binds to chromatin and controls distinct biological activities in mouse mammary gland cells. Because of its high expression in the intestine, in this study we have generated a colon-specific conditional targeted deletion (EPR cKO) to evaluate EPR in vivo functions in mice. EPR cKO mice display epithelium hyperproliferation, impaired mucus production and secretion, as well as inflammatory infiltration in the proximal portion of the large intestine. RNA sequencing analysis reveals a rearrangement of the colon crypt transcriptome with strong reduction of goblet cell-specific factors including those involved in the synthesis, assembly, transport and control of mucus proteins. Further, colon mucosa integrity and permeability are impaired in EPR cKO mice, and this results in higher susceptibility to dextran sodium sulfate (DSS)-induced colitis and tumor formation. Human EPR is down-regulated in human cancer cell lines as well as in human cancers, and overexpression of EPR in a colon cancer cell line results in enhanced expression of pro-apoptotic genes. Mechanistically, we show that EPR directly interacts with select genes involved in mucus metabolism whose expression is reduced in EPR cKO mice and that EPR deletion causes tridimensional chromatin organization changes.
Assuntos
Transformação Celular Neoplásica , Inflamação , Muco , RNA Longo não Codificante , Animais , Humanos , Camundongos , Transformação Celular Neoplásica/imunologia , Colo/metabolismo , Modelos Animais de Doenças , Inflamação/imunologia , Mucosa Intestinal/metabolismo , Camundongos Endogâmicos C57BL , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
EPR is a long non-coding RNA (lncRNA) that controls cell proliferation in mammary gland cells by regulating gene transcription. Here, we report on Mettl7a1 as a direct target of EPR. We show that EPR induces Mettl7a1 transcription by rewiring three-dimensional chromatin interactions at the Mettl7a1 locus. Our data indicate that METTL7A1 contributes to EPR-dependent inhibition of TGF-ß signaling. METTL7A1 is absent in tumorigenic murine mammary gland cells and its human ortholog (METTL7A) is downregulated in breast cancers. Importantly, re-expression of METTL7A1 in 4T1 tumorigenic cells attenuates their transformation potential, with the putative methyltransferase activity of METTL7A1 being dispensable for its biological functions. We found that METTL7A1 localizes in the cytoplasm whereby it interacts with factors implicated in the early steps of mRNA translation, associates with ribosomes, and affects the levels of target proteins without altering mRNA abundance. Overall, our data indicates that METTL7A1-a transcriptional target of EPR-modulates translation of select transcripts.
Assuntos
Neoplasias da Mama , Metiltransferases/metabolismo , RNA Longo não Codificante , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células , Cromatina/genética , Feminino , Humanos , Camundongos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Ribossomos/metabolismoRESUMO
Long non-coding RNAs are emerging as essential regulators of gene expression, but their role in normal and neoplastic B cells is still largely uncharacterized. Here, we report on the expression pattern of the LINC00152 in normal B cells and Chronic Lymphocytic Leukemia B cell clones. Higher LINC00152 levels were consistently observed in memory B cell populations when compared to naïve B cells in the normal tissues analyzed [peripheral blood (PB), tonsils, and spleen]. In addition, independent stimulation via Immunoglobulins (IG), CD40, or Toll-like Receptor 9 (TLR9) upregulated LINC00152 in PB B cells. The expression of LINC00152 in a cohort of 107 early stage Binet A CLL patients was highly variable and did not correlate with known prognostic markers or clinical evolution. TLR9 stimulation, but not CD40 or IG challenge, was able to upregulate LINC00152 expression in CLL cells. In addition, LINC00152 silencing in CLL cell lines expressing LINC00152 failed to induce significant cell survival or apoptosis changes. These data suggest that, in normal B cells, the expression of LINC00152 is regulated by immunomodulatory signals, which are only partially effective in CLL cells. However, LINC00152 does not appear to contribute to CLL cell expansion and/or survival in a cohort of newly diagnosed CLL patients.
Assuntos
Biomarcadores Tumorais/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Tonsila Palatina/metabolismo , RNA Longo não Codificante/metabolismo , Baço/metabolismo , Biomarcadores Tumorais/genética , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Prognóstico , Estudos Prospectivos , RNA Longo não Codificante/genética , Taxa de SobrevidaRESUMO
Long non-coding RNAs (lncRNAs) can affect multiple layers of gene expression to control crucial cellular functions. We have previously demonstrated that the lncRNA EPR, by controlling gene expression at different levels, affects cell proliferation and migration in cultured mammary gland cells and impairs breast tumor formation in an orthotopic transplant model in mice. Here, we used ChIRP-Seq to identify EPR binding sites on chromatin of NMuMG mammary gland cells overexpressing EPR and identified its trans binding sites in the genome. Then, with the purpose of relating EPR/chromatin interactions to the reshaping of the epitranscriptome landscape, we profiled histone activation marks at promoter/enhancer regions by ChIP-Seq. Finally, we integrated data derived from ChIRP-Seq, ChIP-Seq as well as RNA-Seq in a comprehensive analysis and we selected a group of bona fide direct transcriptional targets of EPR. Among them, we identified a subset of EPR targets whose expression is controlled by TGF-ß with one of them-Arrdc3-being able to modulate Epithelial to Mesenchymal Transition. This experimental framework allowed us to correlate lncRNA/chromatin interactions with the real outcome of gene expression and to start defining the gene network regulated by EPR as a component of the TGF-ß pathway.
Assuntos
Arrestinas/genética , Neoplasias da Mama/genética , RNA Longo não Codificante/genética , Fator de Crescimento Transformador beta/genética , Animais , Sítios de Ligação/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Cromatina/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Transcriptoma/genéticaRESUMO
Resveratrol (RESV) is a natural polyphenolic compound endowed with anti-inflammatory, anti-proliferative, as well as pro-apoptotic activities that make it a potential anti-tumor compound. Here we show that RESV counteracts the TGF-ß-induced Epithelial to Mesenchymal Transition (EMT) phenotype in mammary gland cells and affects the alternative exon usage of pre-mRNAs that encode crucial factors in adhesion and migration -including CD44, ENAH, and FGFR2- in a panel of immortalized and transformed mammary gland cells. RESV causes a shift from the mesenchymal-specific forms of these factors to the respective epithelial forms and increases the expression of the RNA-binding proteins KHSRP and hnRNPA1. From a mechanistic point of view, we show that the combined silencing of KHSRP and hnRNPA1 prevents the RESV-dependent inclusion of the epithelial-type exons in the Cd44 pre-mRNA. Our findings support an unexpected regulatory mechanism where RESV limits EMT by controlling gene expression at post-transcriptional level.
Assuntos
Processamento Alternativo/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Glândulas Mamárias Animais/metabolismo , Proteínas de Ligação a RNA/metabolismo , Estilbenos/farmacologia , Animais , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Feminino , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Camundongos , Proteínas dos Microfilamentos , Proteínas de Ligação a RNA/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , ResveratrolRESUMO
Epithelial-to-mesenchymal transition (EMT) confers several traits to cancer cells that are required for malignant progression. Here, we report that miR-27b-3p-mediated silencing of the single-strand RNA binding protein KHSRP is required for transforming growth factor ß (TGF-ß)-induced EMT in mammary gland cells. Sustained KHSRP expression limits TGF-ß-dependent induction of EMT factors and cell migration, whereas its knockdown in untreated cells mimics TGF-ß-induced EMT. Genome-wide sequencing analyses revealed that KHSRP controls (1) levels of mature miR-192-5p, a microRNA that targets a group of EMT factors, and (2) alternative splicing of a cohort of pre-mRNAs related to cell adhesion and motility including Cd44 and Fgfr2. KHSRP belongs to a ribonucleoprotein complex that includes hnRNPA1, and the two proteins cooperate in promoting epithelial-type exon usage of select pre-mRNAs. Thus, TGF-ß-induced KHSRP silencing is central in a pathway leading to gene-expression changes that contribute to the cellular changes linked to EMT.
Assuntos
Processamento Alternativo/genética , Transição Epitelial-Mesenquimal/genética , MicroRNAs/genética , Interferência de RNA/fisiologia , Proteínas de Ligação a RNA/genética , Transativadores/genética , Fator de Crescimento Transformador beta/genética , Animais , Adesão Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Receptores de Hialuronatos/genética , Glândulas Mamárias Animais , Camundongos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genéticaRESUMO
The single-stranded nucleic acid-binding protein KHSRP (KH-type splicing regulatory protein) modulates RNA life and gene expression at various levels. KHSRP controls important cellular functions as different as proliferation, differentiation, metabolism, and response to infectious agents. We summarize and discuss experimental evidence providing a potential link between changes in KHSRP expression/function and human diseases including neuromuscular disorders, obesity, type II diabetes, and cancer.
Assuntos
Diferenciação Celular , Suscetibilidade a Doenças , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Imunomodulação , Metabolismo dos Lipídeos , Desenvolvimento Muscular , Músculos/fisiologia , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/metabolismo , Neurônios/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Transdução de Sinais , Transativadores/química , Transativadores/genética , Viroses/imunologia , Viroses/metabolismo , Viroses/virologiaRESUMO
HuR promotes myogenesis by stabilizing the MyoD, myogenin and p21 mRNAs during the fusion of muscle cells to form myotubes. Here we show that HuR, via a novel mRNA destabilizing activity, promotes the early steps of myogenesis by reducing the expression of the cell cycle promoter nucleophosmin (NPM). Depletion of HuR stabilizes the NPM mRNA, increases NPM protein levels and inhibits myogenesis, while its overexpression elicits the opposite effects. NPM mRNA destabilization involves the association of HuR with the decay factor KSRP as well as the ribonuclease PARN and the exosome. The C terminus of HuR mediates the formation of the HuR-KSRP complex and is sufficient for maintaining a low level of the NPM mRNA as well as promoting the commitment of muscle cells to myogenesis. We therefore propose a model whereby the downregulation of the NPM mRNA, mediated by HuR, KSRP and its associated ribonucleases, is required for proper myogenesis.
Assuntos
Proteínas ELAV/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Desenvolvimento Muscular/genética , Fibras Musculares Esqueléticas/metabolismo , Proteínas Nucleares/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Animais , Humanos , Camundongos , Proteína MyoD/genética , Miogenina/genética , Proteínas Nucleares/metabolismo , Nucleofosmina , Regiões Promotoras Genéticas , Estabilidade de RNARESUMO
The single-strand-RNA binding protein KSRP is able to negatively regulate gene expression operating with at least two distinct and integrated postranscriptional mechanisms: (i) by promoting decay of unstable mRNAs and (ii) by favoring maturation from precursors of select microRNAs (miRNAs) including the prototypical tumor suppressor let-7. Studies performed in primary and cultured cells as well as in mice proved that the ability of KSRP to integrate different levels of gene expression is required for proper immune response, lipid metabolism, cell-fate decisions, tissue regeneration, and DNA damage response.
Assuntos
Proteínas de Ligação a RNA/fisiologia , Transativadores/fisiologia , Animais , Diferenciação Celular , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica , Genes MHC da Classe II , Humanos , Metabolismo dos Lipídeos , MicroRNAs/genética , MicroRNAs/metabolismo , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
White adipose tissue (WAT) releases fatty acids from stored triacylglycerol for an energy source. Here, we report that targeted deletion of KH-type splicing regulatory protein (KSRP), an RNA-binding protein that regulates gene expression at multiple levels, enhances lipolysis in epididymal WAT (eWAT) because of the upregulation of genes promoting lipolytic activity. Expression of microRNA 145 (miR-145) is decreased because of impaired primary miR-145 processing in Ksrp(-/-) eWAT. We show that miR-145 directly targets and represses Foxo1 and Cgi58, activators of lipolytic activity, and forced expression of miR-145 attenuates lipolysis. This study reveals a novel in vivo function of KSRP in controlling adipose lipolysis through posttranscriptional regulation of miR-145 expression.
Assuntos
Tecido Adiposo Branco/metabolismo , Lipólise/genética , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Adiposidade , Animais , Diferenciação Celular , Tamanho Celular , Regulação para Baixo/genética , Epididimo/metabolismo , Ácidos Graxos/metabolismo , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Tamanho do Órgão , Oxirredução , Processamento Pós-Transcricional do RNA/genética , Termogênese/genética , Transativadores/deficiência , Triglicerídeos/metabolismoRESUMO
Understanding the molecular mechanisms that control the balance between multipotency and differentiation is of great importance to elucidate the genesis of both developmental disorders and cell transformation events. To investigate the role of the RNA binding protein KSRP in controlling neural differentiation, we used the P19 embryonal carcinoma cell line that is able to differentiate into neuron-like cells under appropriate culture conditions. We have recently reported that KSRP controls the differentiative fate of multipotent mesenchymal cells owing to its ability to promote decay of unstable transcripts and to favor maturation of selected micro-RNAs (miRNAs) from precursors. Here we report that KSRP silencing in P19 cells favors neural differentiation increasing the expression of neuronal markers. Further, the expression of two master transcriptional regulators of neurogenesis, ASCL1 and JMJD3, was enhanced while the maturation of miR-200 family members from precursors was impaired in KSRP knockdown cells. These molecular changes can contribute to the reshaping of P19 cells transcriptome that follows KSRP silencing. Our data suggests that KSRP function is required to maintain P19 cells in a multipotent undifferentiated state and that its inactivation can orient cells towards neural differentiation.
Assuntos
Inativação Gênica , Neurogênese/genética , Proteínas de Ligação a RNA/genética , Transativadores/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Histona Desmetilases com o Domínio Jumonji/metabolismo , Células-Tronco Mesenquimais/citologia , Camundongos , MicroRNAs/metabolismo , Precursores de RNA/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Teratocarcinoma , Transativadores/metabolismo , Transcrição Gênica , TranscriptomaRESUMO
Let-7 is an important tumor-suppressive microRNA (miRNA) that acts as an on-off switch for cellular differentiation and regulates the expression of a set of human oncogenes. Binding of the human KSRP protein to let-7 miRNA precursors positively regulates their processing to mature let-7, thereby contributing to control of cell proliferation, apoptosis and differentiation. Here we analyze the molecular basis for KSRP-let-7 precursor selectivity and show how the third KH domain of the protein recognizes a G-rich sequence in the pre-let-7 terminal loop and dominates the interaction. The structure of the KH3-RNA complex explains the protein recognition of this noncanonical KH target sequence, and we demonstrate that the specificity of this binding is crucial for the functional interaction between the protein and the miRNA precursor.
Assuntos
MicroRNAs/biossíntese , Proteínas de Ligação a RNA/fisiologia , Transativadores/fisiologia , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear BiomolecularRESUMO
Gene silencing mediated by either microRNAs (miRNAs) or Adenylate/uridylate-rich elements Mediated mRNA Degradation (AMD) is a powerful way to post-transcriptionally modulate gene expression. We and others have reported that the RNA-binding protein KSRP favors the biogenesis of select miRNAs (including let-7 family) and activates AMD promoting the decay of inherently labile mRNAs. Different layers of interplay between miRNA- and AMD-mediated gene silencing have been proposed in cultured cells, but the relationship between the two pathways in living organisms is still elusive. We conditionally deleted Dicer in mouse pituitary from embryonic day (E) 9.5 through Cre-mediated recombination. In situ hybridization, immunohistochemistry, and quantitative reverse transcriptase-PCR revealed that Dicer is essential for pituitary morphogenesis and correct expression of hormones. Strikingly, αGSU (alpha glycoprotein subunit, common to three pituitary hormones) was absent in Dicer-deleted pituitaries. αGSU mRNA is unstable and its half-life increases during pituitary development. A transcriptome-wide analysis of microdissected E12.5 pituitaries revealed a significant increment of KSRP expression in conditional Dicer-deleted mice. We found that KSRP directly binds to αGSU mRNA, promoting its rapid decay; and, during pituitary development, αGSU expression displays an inverse temporal relationship to KSRP. Further, let-7b/c downregulated KSRP expression, promoting the degradation of its mRNA by directly binding to the 3'UTR. Therefore, we propose a model in which let-7b/c and KSRP operate within a negative feedback loop. Starting from E12.5, KSRP induces the maturation of let-7b/c that, in turn, post-transcriptionally downregulates the expression of KSRP itself. This event leads to stabilization of αGSU mRNA, which ultimately enhances the steady-state expression levels. We have identified a post-transcriptional regulatory network active during mouse pituitary development in which the expression of the hormone αGSU is increased by let7b/c through downregulation of KSRP. Our study unveils a functional crosstalk between miRNA- and AMD-dependent gene regulation during mammalian organogenesis events.
Assuntos
MicroRNAs/genética , Organogênese/genética , Hipófise , RNA Mensageiro , Proteínas de Ligação a RNA/genética , Transativadores/genética , Animais , RNA Helicases DEAD-box/genética , Desenvolvimento Embrionário/genética , Retroalimentação Fisiológica , Regulação da Expressão Gênica no Desenvolvimento , Subunidade alfa de Hormônios Glicoproteicos/genética , Subunidade alfa de Hormônios Glicoproteicos/metabolismo , Células HEK293 , Células HeLa , Humanos , Camundongos , MicroRNAs/metabolismo , Células NIH 3T3 , Hipófise/embriologia , Hipófise/crescimento & desenvolvimento , Hipófise/metabolismo , Hormônios Hipofisários/genética , Hormônios Hipofisários/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonuclease III/genética , Transativadores/metabolismoRESUMO
Human papillomavirus (HPV) infection is a risk factor for the development of cervical intraepithelial neoplasia (CIN). The incidence of certain cancers such as HPV-associated CIN is higher among dialysis patients than in the general population. In the literature there are few studies on the prevalence of HPV infection among dialyzed women and almost all of these studies concerned women with positive Pap smears. We enrolled 73 hemodialyzed women attending our center from January 2009 to December 2010; 29 denied informed consent and 44 underwent Pap tests and cervical curettage for HPV (mean age 62 ± 15 years). We found HPV positivity in 6 women (prevalence 13.6%). The prevalence of CIN in our sample was also 13.6% (6/44), 83.3% of which HPV related. Since cervical curettage for HPV is a cheap and easy to perform test with high specificity and sensitivity, we believe it is worthwhile including it in the pre-transplant workup of such women to lower the incidence of CIN in dialyzed patients and transplant recipients.
Assuntos
Infecções por Papillomavirus/epidemiologia , Diálise Renal , Displasia do Colo do Útero/epidemiologia , Displasia do Colo do Útero/virologia , Neoplasias do Colo do Útero/epidemiologia , Neoplasias do Colo do Útero/virologia , Adolescente , Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Prevalência , Adulto JovemRESUMO
microRNAs (miRNAs) are small noncodingRNAs that down-regulate gene expression by reducing stability and/or translation of target mRNAs. In animals, miRNAs arise from sequential processing of hairpin primary transcripts by two RNAse III domain-containing enzymes, namely Drosha and Dicer, to generate a mature form of about 22 nucleotides. In this chapter we discuss our latest findings indicating that KSRP is an integral component of both Drosha and Dicer complexes. KSRP binds to the terminal loop sequence of a subset of miRNA precursors promoting their maturation. Our data indicate that the terminal loop is a pivotal structure where activators of miRNA processing as well as repressors of miRNA processing act in a coordinated way to convert cellular signals into changes in miRNA expression processing. This uncovers a new level of complexity of miRNA mechanisms for gene expression regulation.
Assuntos
MicroRNAs/fisiologia , Precursores de RNA/fisiologia , Proteínas de Ligação a RNA/fisiologia , Transativadores/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Proteínas Correpressoras/fisiologia , Humanos , Neoplasias/etiologiaRESUMO
BACKGROUND: Parathyroid hormone (PTH) gene expression is regulated post-transcriptionally through the binding of the trans-acting proteins AU rich binding factor 1 (AUF1), Upstream of N-ras (Unr) and KH-type splicing regulatory protein (KSRP) to an AU rich element (ARE) in PTH mRNA 3'-UTR. AUF1 and Unr stabilize PTH mRNA while KSRP, recruiting the exoribonucleolytic complex exosome, promotes PTH mRNA decay. RESULTS: PTH mRNA is cleaved by the endoribonuclease polysomal ribonuclease 1 (PMR1) in an ARE-dependent manner. Moreover, PMR1 co-immunoprecipitates with PTH mRNA, the exosome and KSRP. Knock-down of either exosome components or KSRP by siRNAs prevents PMR1-mediated cleavage of PTH mRNA. CONCLUSION: PTH mRNA is a target for the endonuclease PMR1. The PMR1 mediated decrease in PTH mRNA levels involves the PTH mRNA 3'-UTR ARE, KSRP and the exosome. This represents an unanticipated mechanism by which the decay of an ARE-containing mRNA is facilitated by KSRP and is dependent on both the exosome and an endoribonuclease.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Exossomos/metabolismo , Hormônio Paratireóideo/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Regiões 3' não Traduzidas , Linhagem Celular , Humanos , Hormônio Paratireóideo/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/metabolismo , TransfecçãoRESUMO
Consistent with the role of microRNAs (miRNAs) in down-regulating gene expression by reducing the translation and/or stability of target messenger RNAs, the levels of specific miRNAs are important for correct embryonic development and have been linked to several forms of cancer. However, the regulatory mechanisms by which primary miRNAs (pri-miRNAs) are processed first to precursor miRNAs (pre-miRNAs) and then to mature miRNAs by the multiprotein Drosha and Dicer complexes, respectively, remain largely unknown. The KH-type splicing regulatory protein (KSRP, also known as KHSRP) interacts with single-strand AU-rich-element-containing mRNAs and is a key mediator of mRNA decay. Here we show in mammalian cells that KSRP also serves as a component of both Drosha and Dicer complexes and regulates the biogenesis of a subset of miRNAs. KSRP binds with high affinity to the terminal loop of the target miRNA precursors and promotes their maturation. This mechanism is required for specific changes in target mRNA expression that affect specific biological programs, including proliferation, apoptosis and differentiation. These findings reveal an unexpected mechanism that links KSRP to the machinery regulating maturation of a cohort of miRNAs that, in addition to its role in promoting mRNA decay, independently serves to integrate specific regulatory programs of protein expression.
Assuntos
MicroRNAs/biossíntese , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Ribonuclease III/química , Ribonuclease III/metabolismoRESUMO
The importance of post-transcriptional mechanisms for the regulation of the homoeostasis of the immune system and the response to challenge by microorganisms is becoming increasingly appreciated. We investigated the contribution of microRNAs (miRNAs) to macrophage activation induced by lipopolysaccharide (LPS). We first observed that Dicer knockout in bone marrow-derived macrophages (BMDMs) increases the LPS-induced expression of some inflammation mediators. miRNA microarray analysis in BMDMs revealed that LPS significantly induces the expression of a single miRNA, miR-155, and this induction depends on enhanced miR-155 maturation from its precursors. The single-strand RNA-binding protein KH-type splicing regulatory protein (KSRP) binds to the terminal loop of miR-155 precursors and promotes their maturation. Both inhibition of miR-155 and KSRP knockdown enhance the LPS-induced expression of select inflammation mediators, and the effect of KSRP knockdown is reverted by mature miR-155. Our studies unveil the existence of an LPS-dependent post-transcriptional regulation of miR-155 biogenesis. Once induced, miR-155 finely tunes the expression of select inflammation mediators in response to LPS.
Assuntos
Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , MicroRNAs/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Animais , RNA Helicases DEAD-box/deficiência , Endorribonucleases/deficiência , Mediadores da Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos , Processamento Pós-Transcricional do RNA , Ribonuclease III , Ativação TranscricionalRESUMO
BACKGROUND: KSRP is a AU-rich element (ARE) binding protein that causes decay of select sets of transcripts in different cell types. We have recently described that phosphatidylinositol 3-kinase/AKT (PI3K-AKT) activation induces stabilization and accumulation of the labile beta-catenin mRNA through an impairment of KSRP function. RESULTS: Aim of this study was to identify additional KSRP targets whose stability and steady-state levels are enhanced by PI3K-AKT activation. First, through microarray analyses of the AU-rich transcriptome in pituitary alphaT3-1 cells, we identified 34 ARE-containing transcripts upregulated in cells expressing a constitutively active form of AKT1. In parallel, by an affinity chromatography-based technique followed by microarray analyses, 12 mRNAs target of KSRP, additional to beta-catenin, were identified. Among them, seven mRNAs were upregulated in cells expressing activated AKT1. Both steady-state levels and stability of these new KSRP targets were consistently increased by either KSRP knock-down or PI3K-AKT activation. CONCLUSION: Our study identified a set of transcripts that are targets of KSRP and whose expression is increased by PI3K-AKT activation. These mRNAs encode RNA binding proteins, signaling molecules and a replication-independent histone. The increased expression of these gene products upon PI3K-AKT activation could play a role in the cellular events initiated by this signaling pathway.
Assuntos
Perfilação da Expressão Gênica , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Animais , Células Cultivadas , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Proteínas de Ligação a RNA/genética , Ratos , Proteínas Recombinantes/genética , Transdução de Sinais/fisiologia , Transativadores/genética , Transfecção , Regulação para CimaRESUMO
Adenine-uridine rich elements (AREs) play an important role in modulating mRNA stability, being the target site of many ARE-binding proteins (AUBPs) that are involved in the decay process. Three 26-mer 2'-O-methyl oligoribonucleotides (ORNs) homologous to the core region of ARE of bcl2 mRNA have been studied for decoy-aptamer activity in UV cross-linking assays. Sense-oriented ORNs competed with the ARE motif for the interaction with both destabilizing and stabilizing AUBPs in cell-free systems and in cell lines. Moreover, ORNs induced mRNA stabilization and up-regulated both Bcl2 mRNA and protein levels in the cells. Bcl2 ORNs stabilized other ARE-containing transcripts and up-regulated their expression. These results indicate that Bcl2 ORNs compete for AUBP-ARE interactions independently of ARE class and suggest that in the cell, the default labile status of ARE-containing mRNAs depends on the combined interaction of such transcripts with destabilizing AUBPs.