RESUMEN
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.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo L , Desarrollo de Músculos , Regiones Promotoras Genéticas , ARN Largo no Codificante , Transcripción Genética , Humanos , Ribonucleoproteína Heterogénea-Nuclear Grupo L/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo L/metabolismo , Desarrollo de Músculos/genética , Fibras Musculares Esqueléticas/metabolismo , Mioblastos/citología , Mioblastos/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Transcripción Genética/genética , Silenciador del Gen , Transporte de Proteínas/genéticaRESUMEN
We have identified chemical probes that simultaneously inhibit cancer cell progression and an immune checkpoint. Using the computational Site Identification by Ligand Competitive Saturation (SILCS) technology, structural biology and cell-based assays, we identify small molecules that directly and selectively bind to the RNA Recognition Motif (RRM) of hnRNP A18, a regulator of protein translation in cancer cells. hnRNP A18 recognizes a specific RNA signature motif in the 3'UTR of transcripts associated with cancer cell progression (Trx, VEGF, RPA) and, as shown here, a tumor immune checkpoint (CTLA-4). Post-transcriptional regulation of immune checkpoints is a potential therapeutic strategy that remains to be exploited. The probes target hnRNP A18 RRM in vitro and in cells as evaluated by cellular target engagement. As single agents, the probes specifically disrupt hnRNP A18-RNA interactions, downregulate Trx and CTLA-4 protein levels and inhibit proliferation of several cancer cell lines without affecting the viability of normal epithelial cells. These first-in-class chemical probes will greatly facilitate the elucidation of the underexplored biological function of RNA Binding Proteins (RBPs) in cancer cells, including their effects on proliferation and immune checkpoint activation.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Unión al ARN/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Antígeno CTLA-4/genética , Antígeno CTLA-4/metabolismo , Línea Celular Tumoral , Humanos , Ligandos , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Resonancia Magnética Nuclear Biomolecular , Biosíntesis de Proteínas , ARN/metabolismo , Motivo de Reconocimiento de ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismoRESUMEN
Eukaryotic gene expression is tightly regulated post-transcriptionally by RNA-binding proteins (RBPs) and microRNAs. The RBP AU-rich-binding factor 1 (AUF1) isoform p37 was found to have high affinity for the microRNA let-7b in vitro (Kd = â¼ 6 nM) in cells. Ribonucleoprotein immunoprecipitation, in vitro association, and single-molecule-binding analyses revealed that AUF1 promoted let-7b loading onto Argonaute 2 (AGO2), the catalytic component of the RNA-induced silencing complex (RISC). In turn, AGO2-let-7 triggered target mRNA decay. Our findings uncover a novel mechanism by which AUF1 binding and transfer of microRNA let-7 to AGO2 facilitates let-7-elicited gene silencing.
Asunto(s)
Proteínas Argonautas/metabolismo , Silenciador del Gen/fisiología , Ribonucleoproteína Heterogénea-Nuclear Grupo D/metabolismo , MicroARNs/metabolismo , Animales , Células Cultivadas , Células HeLa , Ribonucleoproteína Nuclear Heterogénea D0 , Ribonucleoproteína Heterogénea-Nuclear Grupo D/genética , Humanos , Ratones , Unión Proteica , Estabilidad del ARN/fisiologíaRESUMEN
Cleavage and polyadenylation specificity factor 30 (CPSF30) is a key protein involved in pre-mRNA processing. CPSF30 contains five Cys3His domains (annotated as "zinc-finger" domains). Using inductively coupled plasma mass spectrometry, X-ray absorption spectroscopy, and UV-visible spectroscopy, we report that CPSF30 is isolated with iron, in addition to zinc. Iron is present in CPSF30 as a 2Fe-2S cluster and uses one of the Cys3His domains; 2Fe-2S clusters with a Cys3His ligand set are rare and notably have also been identified in MitoNEET, a protein that was also annotated as a zinc finger. These findings support a role for iron in some zinc-finger proteins. Using electrophoretic mobility shift assays and fluorescence anisotropy, we report that CPSF30 selectively recognizes the AU-rich hexamer (AAUAAA) sequence present in pre-mRNA, providing the first molecular-based evidence to our knowledge for CPSF30/RNA binding. Removal of zinc, or both zinc and iron, abrogates binding, whereas removal of just iron significantly lessens binding. From these data we propose a model for RNA recognition that involves a metal-dependent cooperative binding mechanism.
Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/química , Hierro/química , Señales de Poliadenilación de ARN 3'/genética , ARN Mensajero/química , Azufre/química , Factores de Escisión y Poliadenilación de ARNm/química , Sitios de Unión , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Simulación por Computador , Humanos , Modelos Químicos , Poliadenilación/genética , Unión Proteica , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
Pyrrolo[3,2-d]pyrimidines have been studied for many years as potential lead compounds for the development of antiproliferative agents. Much of the focus has been on modifications to the pyrimidine ring, with enzymatic recognition often modulated by C2 and C4 substituents. In contrast, this work focuses on the N5 of the pyrrole ring by means of a series of novel N5-substituted pyrrolo[3,2-d]pyrimidines. The compounds were screened against the NCI-60 Human Tumor Cell Line panel, and the results were analyzed using the COMPARE algorithm to elucidate potential mechanisms of action. COMPARE analysis returned strong correlation to known DNA alkylators and groove binders, corroborating the hypothesis that these pyrrolo[3,2-d]pyrimidines act as DNA or RNA alkylators. In addition, N5 substitution reduced the EC50 against CCRF-CEM leukemia cells by up to 7-fold, indicating that this position is of interest in the development of antiproliferative lead compounds based on the pyrrolo[3,2-d]pyrimidine scaffold.
Asunto(s)
Antineoplásicos/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Pirimidinas/síntesis química , Pirimidinas/química , Pirroles/síntesis química , Pirroles/química , Relación Estructura-ActividadRESUMEN
Hsp70 is a protein chaperone that prevents protein aggregation and aids protein folding by binding to hydrophobic peptide domains through a reversible mechanism directed by an ATPase cycle. However, Hsp70 also binds U-rich RNA including some AU-rich elements (AREs) that regulate the decay kinetics of select mRNAs and has recently been shown to bind and stabilize some ARE-containing transcripts in cells. Previous studies indicated that both the ATP- and peptide-binding domains of Hsp70 contributed to the stability of Hsp70-RNA complexes and that ATP might inhibit RNA recruitment. This suggested the possibility that RNA binding by Hsp70 might mimic features of its peptide-directed chaperone activities. Here, using purified, cofactor-free preparations of recombinant human Hsp70 and quantitative biochemical approaches, we found that high-affinity RNA binding requires at least 30 nucleotides of RNA sequence but is independent of Hsp70's nucleotide-bound status, ATPase activity, or peptide-binding roles. Furthermore, although both the ATP- and peptide-binding domains of Hsp70 could form complexes with an ARE sequence from VEGFA mRNA in vitro, only the peptide-binding domain could recover cellular VEGFA mRNA in ribonucleoprotein immunoprecipitations. Finally, Hsp70-directed stabilization of VEGFA mRNA in cells was mediated exclusively by the protein's peptide-binding domain. Together, these findings indicate that the RNA-binding and mRNA-stabilizing functions of Hsp70 are independent of its protein chaperone cycle but also provide potential mechanical explanations for several well-established and recently discovered cytoprotective and RNA-based Hsp70 functions.
Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , ARN Mensajero/metabolismo , Ribonucleoproteínas/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Elementos Ricos en Adenilato y Uridilato , Regulación Alostérica , Sitios de Unión , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/genética , Células HeLa , Humanos , Inmunoprecipitación , Cinética , Mutación , Oligopéptidos/genética , Oligopéptidos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , ARN/antagonistas & inhibidores , ARN/metabolismo , Interferencia de ARN , Estabilidad del ARN , ARN Mensajero/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/química , Factor A de Crecimiento Endotelial Vascular/química , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Skeletal muscle contains long multinucleated and contractile structures known as muscle fibers, which arise from the fusion of myoblasts into multinucleated myotubes during myogenesis. The myogenic regulatory factor (MRF) MYF5 is the earliest to be expressed during myogenesis and functions as a transcription factor in muscle progenitor cells (satellite cells) and myocytes. In mouse C2C12 myocytes, MYF5 is implicated in the initial steps of myoblast differentiation into myotubes. Here, using ribonucleoprotein immunoprecipitation (RIP) analysis, we discovered a novel function for MYF5 as an RNA-binding protein which associated with a subset of myoblast mRNAs. One prominent MYF5 target was Ccnd1 mRNA, which encodes the key cell cycle regulator CCND1 (Cyclin D1). Biotin-RNA pulldown, UV-crosslinking and gel shift experiments indicated that MYF5 was capable of binding the 3' untranslated region (UTR) and the coding region (CR) of Ccnd1 mRNA. Silencing MYF5 expression in proliferating myoblasts revealed that MYF5 promoted CCND1 translation and modestly increased transcription of Ccnd1 mRNA. Accordingly, overexpressing MYF5 in C2C12 cells upregulated CCND1 expression while silencing MYF5 reduced myoblast proliferation as well as differentiation of myoblasts into myotubes. Moreover, MYF5 silencing reduced myogenesis, while ectopically restoring CCND1 abundance partially rescued the decrease in myogenesis seen after MYF5 silencing. We propose that MYF5 enhances early myogenesis in part by coordinately elevating Ccnd1 transcription and Ccnd1 mRNA translation.
Asunto(s)
Ciclina D1/genética , Desarrollo de Músculos/genética , Factor 5 Regulador Miogénico/genética , ARN Mensajero/genética , Animales , Diferenciación Celular , Línea Celular , Proliferación Celular , Ciclina D1/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ratones , Análisis por Micromatrices , Mioblastos , Factor 5 Regulador Miogénico/metabolismo , Unión Proteica , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de SeñalRESUMEN
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme best known for its role in glycolysis. However, extra-glycolytic functions of GAPDH have been described, including regulation of protein expression via RNA binding. GAPDH binds to numerous adenine-uridine rich elements (AREs) from various mRNA 3'-untranslated regions in vitro and in vivo despite its lack of a canonical RNA binding motif. How GAPDH binds to these AREs is still unknown. Here we discovered that GAPDH binds with high affinity to the core ARE from tumor necrosis factor-α mRNA via a two-step binding mechanism. We demonstrate that a mutation at the GAPDH dimer interface impairs formation of the second RNA-GAPDH complex and leads to changes in the RNA structure. We investigated the effect of this interfacial mutation on GAPDH oligomerization by crystallography, small-angle x-ray scattering, nano-electrospray ionization native mass spectrometry, and hydrogen-deuterium exchange mass spectrometry. We show that the mutation does not significantly affect GAPDH tetramerization as previously proposed. Instead, the mutation promotes short-range and long-range dynamic changes in regions located at the dimer and tetramer interface and in the NAD(+) binding site. These dynamic changes are localized along the P axis of the GAPDH tetramer, suggesting that this region is important for RNA binding. Based on our results, we propose a model for sequential GAPDH binding to RNA via residues located at the dimer and tetramer interfaces.
Asunto(s)
Gliceraldehído-3-Fosfato Deshidrogenasas/química , ARN/química , Regiones no Traducidas 3' , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Anisotropía , Sitios de Unión , ADN Complementario/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Glucólisis , Humanos , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación , Péptidos/química , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Dispersión de Radiación , Homología de Secuencia de Aminoácido , Espectrometría de Masa por Ionización de Electrospray , Factor de Necrosis Tumoral alfa/metabolismo , Rayos XRESUMEN
Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins bind to AU-rich regions in target mRNAs, leading to their deadenylation and decay. Family members in Saccharomyces cerevisiae influence iron metabolism, whereas the single protein expressed in Schizosaccharomyces pombe, Zfs1, regulates cell-cell interactions. In the human pathogen Candida albicans, deep sequencing of mutants lacking the orthologous protein, Zfs1, revealed significant increases (> 1.5-fold) in 156 transcripts. Of these, 113 (72%) contained at least one predicted TTP family member binding site in their 3'UTR, compared with only 3 of 56 (5%) down-regulated transcripts. The zfs1Δ/Δ mutant was resistant to 3-amino-1,2,4-triazole, perhaps because of increased expression of the potential target transcript encoded by HIS3. Sequences of the proteins encoded by the putative Zfs1 targets were highly conserved among other species within the fungal CTG clade, while the predicted Zfs1 binding sites in these mRNAs often 'disappeared' with increasing evolutionary distance from the parental species. C. albicansâ Zfs1 bound to the ideal mammalian TTP binding site with high affinity, and Zfs1 was associated with target transcripts after co-immunoprecipitation. Thus, the biochemical activities of these proteins in fungi are highly conserved, but Zfs1-like proteins may target different transcripts in each species.
Asunto(s)
Candida albicans/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Procesamiento Postranscripcional del ARN , Tristetraprolina/genética , Tristetraprolina/metabolismo , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Sitios de Unión , Biopelículas/crecimiento & desarrollo , Candida albicans/crecimiento & desarrollo , Candida albicans/metabolismo , Secuencia Conservada , Regulación hacia Abajo/genética , Proteínas Fúngicas/química , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunoprecipitación , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Estructura Terciaria de Proteína , Estabilidad del ARN , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alineación de Secuencia , Tristetraprolina/química , Regulación hacia ArribaRESUMEN
The cellular response to mitogens is tightly regulated via transcriptional and post-transcriptional mechanisms to rapidly induce genes that promote proliferation and efficiently attenuate their expression to prevent malignant growth. RNase L is an endoribonuclease that mediates diverse antiproliferative activities, and tristetraprolin (TTP) is a mitogen-induced RNA-binding protein that directs the decay of proliferation-stimulatory mRNAs. In light of their roles as endogenous proliferative constraints, we examined the mechanisms and functional interactions of RNase L and TTP to attenuate a mitogenic response. Mitogen stimulation of RNase L-deficient cells significantly increased TTP transcription and the induction of other mitogen-induced mRNAs. This regulation corresponded with elevated expression of serum-response factor (SRF), a master regulator of mitogen-induced transcription. RNase L destabilized the SRF transcript and formed a complex with SRF mRNA in cells providing a mechanism by which RNase L down-regulates SRF-induced genes. TTP and RNase L proteins interacted in cells suggesting that RNase L is directed to cleave TTP-bound RNAs as a mechanism of substrate specificity. Consistent with their concerted function in RNA turnover, the absence of either RNase L or TTP stabilized SRF mRNA, and a subset of established TTP targets was also regulated by RNase L. RNase L deficiency enhanced mitogen-induced proliferation demonstrating its functional role in limiting the mitogenic response. Our findings support a model of feedback regulation in which RNase L and TTP target SRF mRNA and SRF-induced transcripts. Accordingly, meta-analysis revealed an enrichment of RNase L and TTP targets among SRF-regulated genes suggesting that the RNase L/TTP axis represents a viable target to inhibit SRF-driven proliferation in neoplastic diseases.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Endorribonucleasas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Mitógenos/farmacología , Estabilidad del ARN/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Animales , Proliferación Celular/fisiología , Regulación de la Expresión Génica/fisiología , Células HEK293 , Células HeLa , Humanos , Ratones , Modelos Biológicos , Estabilidad del ARN/fisiología , Transcripción Genética/fisiología , Tristetraprolina/metabolismoRESUMEN
Members of the mammalian tristetraprolin family of CCCH tandem zinc finger proteins can bind to certain AU-rich elements (AREs) in mRNAs, leading to their deadenylation and destabilization. Mammals express three or four members of this family, but Drosophila melanogaster and other insects appear to contain a single gene, Tis11. We found that recombinant Drosophila Tis11 protein could bind to ARE-containing RNA oligonucleotides with low nanomolar affinity. Remarkably, co-expression in mammalian cells with "target" RNAs demonstrated that Tis11 could promote destabilization of ARE-containing mRNAs and that this was partially dependent on a conserved C-terminal sequence resembling the mammalian NOT1 binding domain. Drosophila Tis11 promoted both deadenylation and decay of a target transcript in this heterologous cell system. We used chromosome deletion/duplication and P element insertion to produce two types of Tis11 deficiency in adult flies, both of which were viable and fertile. To address the hypothesis that Tis11 deficiency would lead to the abnormal accumulation of potential target transcripts, we analyzed gene expression in adult flies by deep mRNA sequencing. We identified 69 transcripts from 56 genes that were significantly up-regulated more than 1.5-fold in both types of Tis11-deficient flies. Ten of the up-regulated transcripts encoded probable proteases, but many other functional classes of proteins were represented. Many of the up-regulated transcripts contained potential binding sites for tristetraprolin family member proteins that were conserved in other Drosophila species. Tis11 is thus an ARE-binding, mRNA-destabilizing protein that may play a role in post-transcriptional gene expression in Drosophila and other insects.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación de la Expresión Génica , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Elementos Ricos en Adenilato y Uridilato/genética , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Sondas de Oligonucleótidos/química , Sondas de Oligonucleótidos/genética , Sondas de Oligonucleótidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Estabilidad del ARN/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN/métodos , Homología de Secuencia de AminoácidoRESUMEN
Halogenated thieno[3,2-d]pyrimidines exhibit antiproliferative activity against a variety of cancer cell models, such as the mouse lymphocytic leukemia cell line L1210 in which they induce apoptosis independent of cell cycle arrest. Here we assessed these activities on MDA-MB-231 cells, a well-established model of aggressive, metastatic breast cancer. While 2,4-dichloro[3,2-d]pyrimidine was less toxic to MDA-MB-231 cells than previously observed in the L1210 model, flow cytometry analysis showed that MDA-MB-231 cell death involved arrest at the G2/M stage of the cell cycle. Conversely, the introduction of bromine at C7 of the 2,4-dichloro[3,2-d]pyrimidine eliminated cell type-dependent differences in cytotoxicity or cell cycle status. Together, these data indicate that a substituent at C7 can profoundly modify the cytotoxic mechanism of halogenated thieno[3,2-d]pyrimidines in a cell type-specific manner.
Asunto(s)
Antineoplásicos/química , Bromuros/química , Pirimidinas/química , Antineoplásicos/síntesis química , Antineoplásicos/toxicidad , Neoplasias de la Mama/patología , Línea Celular Tumoral , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Pirimidinas/síntesis química , Pirimidinas/toxicidadRESUMEN
In vitro evaluation of the halogenated pyrrolo[3,2-d]pyrimidines identified antiproliferative activities in compounds 1 and 2 against four different cancer cell lines. Upon screening of a series of pyrrolo[3,2-d]pyrimidines, the 2,4-Cl compound 1 was found to exhibit antiproliferative activity at low micromolar concentrations. Introduction of iodine at C7 resulted in significant enhancement of potency by reducing the IC50 into sub-micromolar levels, thereby suggesting the importance of a halogen at C7. This finding was further supported by an increased antiproliferative effect for 4 as compared to 3. Cell-cycle and apoptosis studies conducted on the two potent compounds 1 and 2 showed differences in their cytotoxic mechanisms in triple negative breast cancer MDA-MB-231 cells, wherein compound 1 induced cells to accumulate at the G2/M stage with little evidence of apoptotic death. In contrast, compound 2 robustly induced apoptosis with concomitant G2/M cell cycle arrest in this cell model.
Asunto(s)
Antineoplásicos/química , Pirimidinas/química , Pirroles/química , Antineoplásicos/síntesis química , Antineoplásicos/toxicidad , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Halogenación , Humanos , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Pirimidinas/síntesis química , Pirimidinas/toxicidad , Pirroles/síntesis química , Pirroles/toxicidad , Relación Estructura-ActividadRESUMEN
The RNA-binding protein AUF1 binds AU-rich elements in 3'-untranslated regions to regulate mRNA degradation and/or translation. Many of these mRNAs are predicted microRNA targets as well. An emerging theme in post-transcriptional control of gene expression is that RNA-binding proteins and microRNAs co-regulate mRNAs. Recent experiments and bioinformatic analyses suggest this type of co-regulation may be widespread across the transcriptome. Here, we identified mRNA targets of AUF1 from a complex pool of cellular mRNAs and examined a subset of these mRNAs to explore the links between RNA binding and mRNA degradation for both AUF1 and Argonaute 2 (AGO2), which is an essential effector of microRNA-induced gene silencing. Depending on the specific mRNA examined, AUF1 and AGO2 binding is proportional/cooperative, reciprocal/competitive or independent. For most mRNAs in which AUF1 affects their decay rates, mRNA degradation requires AGO2. Thus, AUF1 and AGO2 present mRNA-specific allosteric binding relationships for co-regulation of mRNA degradation.
Asunto(s)
Proteínas Argonautas/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo D/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Regiones no Traducidas 3' , Células HeLa , Ribonucleoproteína Nuclear Heterogénea D0 , Humanos , Células K562RESUMEN
AU-rich element RNA-binding protein 1 (AUF1) regulates the stability and/or translational efficiency of diverse mRNA targets, including many encoding products controlling the cell cycle, apoptosis, and inflammation by associating with AU-rich elements residing in their 3'-untranslated regions. Previous biochemical studies showed that optimal AUF1 binding requires 33-34 nucleotides with a strong preference for U-rich RNA despite observations that few AUF1-associated cellular mRNAs contain such extended U-rich domains. Using the smallest AUF1 isoform (p37(AUF1)) as a model, we employed fluorescence anisotropy-based approaches to define thermodynamic parameters describing AUF1 ribonucleoprotein (RNP) complex formation across a panel of RNA substrates. These data demonstrated that 15 nucleotides of AU-rich sequence were sufficient to nucleate high affinity p37(AUF1) RNP complexes within a larger RNA context. In particular, p37(AUF1) binding to short AU-rich RNA targets was significantly stabilized by interactions with a 3'-purine residue and largely base-independent but non-ionic contacts 5' of the AU-rich site. RNP stabilization by the upstream RNA domain was associated with an enhanced negative change in heat capacity consistent with conformational changes in protein and/or RNA components, and fluorescence resonance energy transfer-based assays demonstrated that these contacts were required for p37(AUF1) to remodel local RNA structure. Finally, reporter mRNAs containing minimal high affinity p37(AUF1) target sequences associated with AUF1 and were destabilized in a p37(AUF1)-dependent manner in cells. These findings provide a mechanistic explanation for the diverse population of AUF1 target mRNAs but also suggest how AUF1 binding could regulate protein and/or microRNA binding events at adjacent sites.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo D/química , ARN Mensajero/química , Sitio Alostérico , Sitios de Unión , Núcleo Celular/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteína Nuclear Heterogénea D0 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/química , Humanos , Conformación de Ácido Nucleico , Unión Proteica , Isoformas de Proteínas , ARN Mensajero/metabolismo , TermodinámicaRESUMEN
KLF4 is a member of the Kruppel-like factor family of transcriptional regulators. KLF4 has been shown to be required for normal terminal differentiation of keratinocytes, but the molecular mechanism whereby KLF4 regulates genes associated with the differentiation process has not been studied. In the present study, we explore the impact of KLF4 on expression of involucrin, a gene that is specifically expressed in differentiated keratinocytes. KLF4 overexpression and knockdown studies show that involucrin mRNA and protein level correlates directly with KLF4 level. Moreover, studies of mutant KLF4 proteins indicate that transcriptionally inactive forms do not increase involucrin expression. PKCδ is a regulator of keratinocyte differentiation that increases expression of differentiation-associated target genes, including involucrin. Overexpression of KLF4 augments the PKCδ-dependent increase in involucrin expression, whereas KLF4 knockdown attenuates this response. The KLF4 induction of human involucrin (hINV) promoter activity is mediated via KLF4 binding to a GC-rich element located in the hINV promoter distal regulatory region, a region of the promoter required for in vivo involucrin expression. Mutation of the GC-rich element, an adjacent AP1 factor binding site, or both sites severely attenuates the response. Moreover, loss of KLF4 in an epidermal equivalent model of differentiation results in loss of hINV expression. These studies suggest that KLF4 is part of a multiprotein complex that interacts that the hINV promoter distal regulatory region to drive differentiation-dependent hINV gene expression in epidermis.
Asunto(s)
Diferenciación Celular , Queratinocitos/fisiología , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteína Quinasa C-delta/metabolismo , Precursores de Proteínas/genética , Células Cultivadas , Inmunoprecipitación de Cromatina , Secuencia Rica en GC , Humanos , Factor 4 Similar a Kruppel , Regiones Promotoras Genéticas , Precursores de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Elementos de Respuesta , Transcripción Genética , Activación TranscripcionalRESUMEN
AUF1 is a family of four proteins generated by alternative pre-mRNA splicing that form high affinity complexes with AU-rich, mRNA-destabilizing sequences located within the 3' untranslated regions of many labile mRNAs. While AUF1 binding is most frequently associated with accelerated mRNA decay, emerging examples have demonstrated roles as a mRNA stabilizer or even translational regulator for specific transcripts. In this review, we summarize recent advances in our understanding of mRNA recognition by AUF1 and the biochemical and functional consequences of these interactions. In addition, unique properties of individual AUF1 isoforms and the roles of these proteins in modulating expression of genes associated with inflammatory, neoplastic, and cardiac diseases are discussed. Finally, we describe mechanisms that regulate AUF1 expression in cells, and current knowledge of regulatory switches that modulate the cellular levels and/or activities of AUF1 isoforms through distinct protein post-translational modifications. This article is part of a Special Issue entitled: RNA Decay mechanisms.
Asunto(s)
Elementos Ricos en Adenilato y Uridilato/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo D/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , Empalme Alternativo/genética , Regulación de la Expresión Génica , Ribonucleoproteína Nuclear Heterogénea D0 , Humanos , Procesamiento Proteico-Postraduccional , Precursores del ARN/genéticaRESUMEN
The in vitro evaluation of thieno[3,2-d]pyrimidines identified halogenated compounds 1 and 2 with antiproliferative activity against three different cancer cell lines. A structure activity relationship study indicated the necessity of the chlorine at the C4-position for biological activity. The two most active compounds 1 and 2 were found to induce apoptosis in the leukemia L1210 cell line. Additionally, the compounds were screened against a variety of other microbial targets and as a result, selective activity against several fungi was also observed. The synthesis and preliminary biological results are reported herein.
Asunto(s)
Antibacterianos/farmacología , Antifúngicos/farmacología , Antineoplásicos/farmacología , Bacillus subtilis/efectos de los fármacos , Hongos/efectos de los fármacos , Pirimidinas/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Antifúngicos/síntesis química , Antifúngicos/química , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Gene expression networks associated with placebo effects are understudied; in this study, we identified transcriptomic profiles associated with placebo responsivity. Participants suffering from chronic pain underwent a verbal suggestion and conditioning paradigm with individually tailored thermal painful stimulations to elicit conditioned placebo effects. Participants reported pain intensity on a visual analog scale (VAS) anchored from zero = no pain to 100 = maximum imaginable pain. RNA was extracted from venous blood and RNA sequencing and validation tests were performed to identify differentially expressed genes (DEGs) associated with placebo effects, controlling for sex and level of pain. Unbiased enrichment analyses were performed to identify biological processes associated with placebo effects. Of the 10,700 protein-coding genes that passed quality control filters, 667 were found to be associated with placebo effects (FDR <0.05). Most genes (97%) upregulated were associated with larger placebo effects. The 17 top transcriptome-wide significant genes were further validated via RT-qPCR in an independent cohort of chronic pain participants. Six of them (CCDC85B, FBXL15, HAGH, PI3, SELENOM, and TNFRSF4) showed positive and significant (P < 0.05) correlation with placebo effects in the cohort. The overall DEGs were highly enriched in regulation of expression of SLITs and ROBOs (R-HSA-9010553, FDR = 1.26e-33), metabolism of RNA (R-HSA-8953854, FDR = 1.34e-30), Huntington's disease (hsa05016, FDR = 9.84e-31), and ribosome biogenesis (GO:0042254, FDR = 2.67e-15); alternations in these pathways might jeopardize the proneness to elicit placebo effects. Future studies are needed to replicate this finding and better understand the unique molecular dynamics of people who are more or less affected by pain and placebo.
Asunto(s)
Dolor Crónico , Efecto Placebo , Transcriptoma , Humanos , Dolor Crónico/genética , Dolor Crónico/tratamiento farmacológico , Masculino , Femenino , Adulto , Persona de Mediana Edad , Dimensión del Dolor/métodos , Perfilación de la Expresión Génica/métodosRESUMEN
Tristetraprolin (TTP) is the prototype of a family of CCCH tandem zinc finger proteins that can bind to AU-rich elements in mRNAs and promote their decay. TTP binds to mRNA through its central tandem zinc finger domain; it then promotes mRNA deadenylation, considered to be the rate-limiting step in eukaryotic mRNA decay. We found that TTP and its related family members could bind to certain isoforms of another AU-rich element-binding protein, HNRNPD/AUF1, as well as a related protein, laAUF1. The interaction domain within AUF1p45 appeared to be a C-terminal "GY" region, and the interaction domain within TTP was the tandem zinc finger domain. Surprisingly, binding of AUF1p45 to TTP occurred even with TTP mutants that lacked RNA binding activity. In cell extracts, binding of AUF1p45 to TTP potentiated TTP binding to ARE-containing RNA probes, as determined by RNA gel shift assays; AUF1p45 did not bind to the RNA probes under these conditions. Using purified, recombinant proteins and a synthetic RNA target in FRET assays, we demonstrated that AUF1p45, but not AUF1p37, increased TTP binding affinity for RNA â¼5-fold. These data suggest that certain isoforms of AUF1 can serve as "co-activators" of TTP family protein binding to RNA. The results raise interesting questions about the ability of AUF1 isoforms to regulate the mRNA binding and decay-promoting activities of TTP and its family members as well as the ability of AUF1 proteins to serve as possible physical links between TTP and other mRNA decay proteins and structures.