RESUMEN
The activation of T helper 17 signaling plays a critical role in psoriasis pathogenesis, and systemically-administered IL-17 inhibitors are highly effective therapy for moderate-to-severe disease. We generated topically-delivered gene-regulating nanoconstructs, comprised of spherically-arrayed antisense DNA (liposomal spherical nucleic acids [L-SNAs]), which are able to penetrate human skin to knock down cutaneous gene targets. Topically-applied L-SNAs targeting the gene encoding the mouse IL-17A receptor (Il17ra) reversed the development of psoriasis clinically, histologically, and transcriptionally in imiquimod-treated psoriasis-like mouse skin. Il17ra L-SNAs reduced the modified PASI by 74% versus controls and decreased epidermal thickness by 56%. Il17ra L-SNA reduced Il17ra protein expression by 75% and significantly decreased the mRNA expression of psoriasis markers, including Defb4, Il17c, S100a7, Pi3, Krt16, and Tnfa versus scrambled spherical nucleic acid (Scr SNA) controls. A human IL17RA L-SNA penetrates 3-dimensional cultures and normal human explants to knock down IL17RA mRNA by 63% and 66%, respectively. After topical application to psoriatic 3-dimensional rafts, anti-human IL17RA L-SNAs reduced the expression of IL17RA (by 72%) and the IL-17-induced genes IL17C (by 85%), DEFB4 (by 83%), TNFA (by 77%), and PI3 (by 65%) versus scrambled L-SNA and vehicle controls (all P < 0.001). Taken together, these data suggest that targeted suppression of IL17RA is a promising new topical treatment strategy for psoriasis.
Asunto(s)
ADN sin Sentido/administración & dosificación , Nanosferas/administración & dosificación , Psoriasis/tratamiento farmacológico , ARN Mensajero/efectos de los fármacos , Receptores de Interleucina-17/antagonistas & inhibidores , Administración Cutánea , Animales , Biomarcadores/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Imiquimod/inmunología , Queratinocitos , Liposomas , Ratones , Cultivo Primario de Células , Psoriasis/inducido químicamente , Psoriasis/diagnóstico , Psoriasis/inmunología , ARN Mensajero/inmunología , ARN Mensajero/metabolismo , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/inmunología , Índice de Severidad de la Enfermedad , Piel/citología , Piel/efectos de los fármacos , Piel/inmunología , Piel/patologíaRESUMEN
Fragile X syndrome, the most common inherited form of intellectual disability, is caused by the CGG trinucleotide expansion in the 5'-untranslated region of the Fmr1 gene on the X chromosome, which silences the expression of the fragile X mental retardation protein (FMRP). FMRP has been shown to bind to a G-rich region within the PSD-95 mRNA, which encodes for the postsynaptic density protein 95, and together with microRNA-125a to mediate the reversible inhibition of the PSD-95 mRNA translation in neurons. The miR-125a binding site within the PSD-95 mRNA 3'-untranslated region (UTR) is embedded in a G-rich region bound by FMRP, which we have previously demonstrated folds into two parallel G-quadruplex structures. The FMRP regulation of PSD-95 mRNA translation is complex, being mediated by its phosphorylation. While the requirement for FMRP in the regulation of PSD-95 mRNA translation is clearly established, the exact mechanism by which this is achieved is not known. In this study, we have shown that both unphosphorylated FMRP and its phosphomimic FMRP S500D bind to the PSD-95 mRNA G-quadruplexes with high affinity, whereas only FMRP S500D binds to miR-125a. These results point towards a mechanism by which, depending on its phosphorylation status, FMRP acts as a switch that potentially controls the stability of the complex formed by the miR-125a-guided RNA induced silencing complex (RISC) and PSD-95 mRNA.
Asunto(s)
Homólogo 4 de la Proteína Discs Large/biosíntesis , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , MicroARNs/metabolismo , ARN Mensajero/metabolismo , Sustitución de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Homólogo 4 de la Proteína Discs Large/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/química , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , G-Cuádruplex , Humanos , MicroARNs/química , MicroARNs/genética , Modelos Moleculares , Fosforilación , Unión Proteica , Biosíntesis de Proteínas , ARN Mensajero/química , ARN Mensajero/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Fragile X mental retardation protein (FMRP) is a multifunctional RNA-binding protein with crucial roles in neuronal development and function. Efforts aimed at elucidating how FMRP target mRNAs are selected have produced divergent sets of target mRNA and putative FMRP-bound motifs, and a clear understanding of FMRP's binding determinants has been lacking. To clarify FMRP's binding to its target mRNAs, we produced a shared dataset of FMRP consensus binding sequences (FCBS), which were reproducibly identified in two published FMRP CLIP sequencing datasets. This comparative dataset revealed that of the various sequence and structural motifs that have been proposed to specify FMRP binding, the short sequence motifs TGGA and GAC were corroborated, and a novel TAY motif was identified. In addition, the distribution of the FCBS set demonstrates that FMRP preferentially binds to the coding region of its targets but also revealed binding along 3' UTRs in a subset of target mRNAs. Beyond probing these putative motifs, the FCBS dataset of reproducibly identified FMRP binding sites is a valuable tool for investigating FMRP targets and function.
Asunto(s)
Secuencia de Consenso , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Secuencia de Bases , Sitios de Unión , Codón/genética , Metilación de ADN/genética , Bases de Datos de Proteínas , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/química , G-Cuádruplex , Células HEK293 , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Motivos de Nucleótidos/genética , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Fragile X syndrome is a common cause of intellectual disability and autism spectrum disorder. The gene underlying the disorder, fragile X mental retardation 1 (FMR1), is silenced in most cases by a CGG-repeat expansion mutation in the 5' untranslated region (UTR). Recently, we identified a variant located in the 3'UTR of FMR1 enriched among developmentally delayed males with normal repeat lengths. A patient-derived cell line revealed reduced levels of endogenous fragile X mental retardation protein (FMRP), and a reporter containing a patient 3'UTR caused a decrease in expression. A control reporter expressed in cultured mouse cortical neurons showed an expected increase following synaptic stimulation that was absent when expressing the patient reporter, suggesting an impaired response to neuronal activity. Mobility-shift assays using a control RNA detected an RNA-protein interaction that is lost with the patient RNA, and HuR was subsequently identified as an associated protein. Cross-linking immunoprecipitation experiments identified the locus as an in vivo target of HuR, supporting our in vitro findings. These data suggest that the disrupted interaction of HuR impairs activity-dependent translation of FMRP, which may hinder synaptic plasticity in a clinically significant fashion.
Asunto(s)
Regiones no Traducidas 3'/genética , Proteína 1 Similar a ELAV/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Neuronas/metabolismo , Biosíntesis de Proteínas , Alelos , Animales , Secuencia de Bases , Biotinilación , Células Cultivadas , Dendritas/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Genes Reporteros , Sitios Genéticos , Humanos , Luciferasas/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Unión Proteica , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Glutamato/metabolismo , Alineación de Secuencia , Transducción de Señal/genética , Sinapsis/metabolismo , Espectrometría de Masas en TándemRESUMEN
Fragile X syndrome, a common cause of intellectual disability and a well-known cause of autism spectrum disorder, is the result of loss or dysfunction of fragile X mental retardation protein (FMRP), a highly selective RNA-binding protein and translation regulator. A major research priority has been the identification of the mRNA targets of FMRP, particularly as recent studies suggest an excess of FMRP targets among genes implicated in idiopathic autism and schizophrenia. Several large-scale studies have attempted to identify mRNAs bound by FMRP through several methods, each generating a list of putative target genes, leading to distinct hypotheses by which FMRP recognizes its targets; namely, by RNA structure or sequence. However, no in depth analyses have been performed to identify the level of consensus among the studies. Here, we analyze four large FMRP target datasets to generate high-confidence consensus lists, and examine all datasets for sequence elements within the target RNAs to validate reported FMRP binding motifs (GACR, ACUK and WGGA). We found GACR to be highly enriched in FMRP datasets, while ACUK was not. The WGGA pattern was modestly enriched in several, but not all datasets. The previous association between FMRP and G-quadruplexes prompted the analysis of the distribution of WGGA in the target genes. Consistent with the requirements for G-quadruplex formation, we observed highly clustered WGGA motifs in FMRP targets compared with other genes, implicating both RNA structure and sequence in the recognition motif of FMRP. In addition, we generate a list of the top 40 FMRP targets associated with FXS-related phenotypes.
Asunto(s)
Secuencias de Aminoácidos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , G-Cuádruplex , ARN Mensajero/química , Secuencia de Aminoácidos , Sitios de Unión , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/química , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Síndrome del Cromosoma X Frágil/genética , Humanos , ARN Mensajero/metabolismoRESUMEN
The interferon-induced enzymes 2'-5'-oligoadenylate synthetase (OAS) and RNase L are key components of innate immunity involved in sensory and effector functions following viral infections. Upon binding target RNA, OAS is activated to produce 2'-5'-linked oligoadenylates (2-5A) that activate RNase L, which then cleaves single-stranded self and non-self RNA. Modified nucleosides that are present in cellular transcripts have been shown to suppress activation of several RNA sensors. Here, we demonstrate that in vitro transcribed, unmodified RNA activates OAS, induces RNase L-mediated ribosomal RNA (rRNA) cleavage and is rapidly cleaved by RNase L. In contrast, RNA containing modified nucleosides activates OAS less efficiently and induces limited rRNA cleavage. Nucleoside modifications also make RNA resistant to cleavage by RNase L. Examining translation in RNase L(-/-) cells and mice confirmed that RNase L activity reduces translation of unmodified mRNA, which is not observed with modified mRNA. Additionally, mRNA containing the nucleoside modification pseudouridine is translated longer and has an extended half-life. The observation that modified nucleosides in RNA reduce 2-5A pathway activation joins OAS and RNase L to the list of RNA sensors and effectors whose functions are limited when RNA is modified, confirming the role of nucleoside modifications in suppressing immune recognition of RNA.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/metabolismo , Endorribonucleasas/metabolismo , Nucleósidos/fisiología , ARN/química , Animales , Células Cultivadas , Semivida , Humanos , Ratones , Biosíntesis de Proteínas , Seudouridina/metabolismo , ARN/metabolismo , ARN Ribosómico/metabolismo , Uridina/fisiologíaRESUMEN
Previous studies have shown that the translation level of in vitro transcribed messenger RNA (mRNA) is enhanced when its uridines are replaced with pseudouridines; however, the reason for this enhancement has not been identified. Here, we demonstrate that in vitro transcripts containing uridine activate RNA-dependent protein kinase (PKR), which then phosphorylates translation initiation factor 2-alpha (eIF-2α), and inhibits translation. In contrast, in vitro transcribed mRNAs containing pseudouridine activate PKR to a lesser degree, and translation of pseudouridine-containing mRNAs is not repressed. RNA pull-down assays demonstrate that mRNA containing uridine is bound by PKR more efficiently than mRNA with pseudouridine. Finally, the role of PKR is validated by showing that pseudouridine- and uridine-containing RNAs were translated equally in PKR knockout cells. These results indicate that the enhanced translation of mRNAs containing pseudouridine, compared to those containing uridine, is mediated by decreased activation of PKR.