RESUMEN
The human genome expresses thousands of natural antisense transcripts (NAT) that can regulate epigenetic state, transcription, RNA stability or translation of their overlapping genes1,2. Here we describe MAPT-AS1, a brain-enriched NAT that is conserved in primates and contains an embedded mammalian-wide interspersed repeat (MIR), which represses tau translation by competing for ribosomal RNA pairing with the MAPT mRNA internal ribosome entry site3. MAPT encodes tau, a neuronal intrinsically disordered protein (IDP) that stabilizes axonal microtubules. Hyperphosphorylated, aggregation-prone tau forms the hallmark inclusions of tauopathies4. Mutations in MAPT cause familial frontotemporal dementia, and common variations forming the MAPT H1 haplotype are a significant risk factor in many tauopathies5 and Parkinson's disease. Notably, expression of MAPT-AS1 or minimal essential sequences from MAPT-AS1 (including MIR) reduces-whereas silencing MAPT-AS1 expression increases-neuronal tau levels, and correlate with tau pathology in human brain. Moreover, we identified many additional NATs with embedded MIRs (MIR-NATs), which are overrepresented at coding genes linked to neurodegeneration and/or encoding IDPs, and confirmed MIR-NAT-mediated translational control of one such gene, PLCG1. These results demonstrate a key role for MAPT-AS1 in tauopathies and reveal a potentially broad contribution of MIR-NATs to the tightly controlled translation of IDPs6, with particular relevance for proteostasis in neurodegeneration.
Asunto(s)
Biosíntesis de Proteínas/genética , Proteostasis/genética , ARN sin Sentido/genética , Tauopatías/genética , Tauopatías/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo , Anciano , Animales , Sitios de Unión , Encéfalo/metabolismo , Encéfalo/patología , Estudios de Casos y Controles , Diferenciación Celular , Progresión de la Enfermedad , Femenino , Humanos , Sitios Internos de Entrada al Ribosoma/genética , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Neuronas/metabolismo , Neuronas/patología , Ribosomas/metabolismo , Proteínas tau/biosíntesisRESUMEN
The RNA-binding protein HuD promotes neurogenesis and favors recovery from peripheral axon injury. HuD interacts with many mRNAs, altering both stability and translation efficiency. We generated a nucleotide resolution map of the HuD RNA interactome in motor neuron-like cells, identifying HuD target sites in 1,304 mRNAs, almost exclusively in the 3' UTR. HuD binds many mRNAs encoding mTORC1-responsive ribosomal proteins and translation factors. Altered HuD expression correlates with the translation efficiency of these mRNAs and overall protein synthesis, in a mTORC1-independent fashion. The predominant HuD target is the abundant, small non-coding RNA Y3, amounting to 70% of the HuD interaction signal. Y3 functions as a molecular sponge for HuD, dynamically limiting its recruitment to polysomes and its activity as a translation and neuron differentiation enhancer. These findings uncover an alternative route to the mTORC1 pathway for translational control in motor neurons that is tunable by a small non-coding RNA.
Asunto(s)
Proteína 4 Similar a ELAV/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Neuronas Motoras/fisiología , ARN Pequeño no Traducido/genética , Regiones no Traducidas 3' , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Animales , Línea Celular , Proteína 4 Similar a ELAV/metabolismo , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Neuronas Motoras/metabolismo , Neurogénesis/genética , Polirribosomas/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/metabolismoRESUMEN
Cyclin-dependent kinase 5 regulatory subunit 1 (CDK5R1) encodes p35, a specific activator of cyclin-dependent kinase 5 (CDK5). CDK5 and p35 have a fundamental role in neuronal migration and differentiation during CNS development. Both the CDK5R1 3'-UTR's remarkable size and its conservation during evolution strongly indicate an important role in post-transcriptional regulation. We previously validated different regulatory elements in the 3'-UTR of CDK5R1, which affect transcript stability, p35 levels and cellular migration through the binding with nELAV proteins and miR-103/7 miRNAs. Interestingly, a 138 bp-long region, named C2.1, was identified as the most mRNA destabilizing portion within CDK5R1 3'-UTR. This feature was maintained by a shorter region of 73 bp, characterized by two poly-U stretches. UV-CL experiments showed that this region interacts with protein factors. UV-CLIP assays and pull-down experiments followed by mass spectrometry analysis demonstrated that nELAV and hnRNPA2/B1 proteins bind to the same U-rich element. These RNA-binding proteins (RBPs) were shown to oppositely control CDK5R1 mRNA stability and p35 protein content at post-trascriptional level. While nELAV proteins have a positive regulatory effect, hnRNPA2/B1 has a negative action that is responsible for the mRNA destabilizing activity both of the C2.1 region and of the full-length 3'-UTR. In co-expression experiments of hnRNPA2/B1 and nELAV RBPs we observed an overall decrease of p35 content. We also demonstrated that hnRNPA2/B1 can downregulate nELAV protein content but not vice versa. This study, by providing new insights on the combined action of different regulatory factors, contributes to clarify the complex post-transcriptional control of CDK5R1 gene expression.
Asunto(s)
Regiones no Traducidas 3'/genética , Proteínas ELAV/metabolismo , Regulación Neoplásica de la Expresión Génica , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Proteínas del Tejido Nervioso/genética , Secuencias Reguladoras de Ácido Ribonucleico/genética , Western Blotting , Diferenciación Celular , Proteínas ELAV/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Inmunoprecipitación , Luciferasas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma/genética , Neuroblastoma/metabolismo , Estabilidad del ARN , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Células Tumorales CultivadasRESUMEN
Little is known regarding the post-transcriptional networks that control gene expression in eukaryotes. Additionally, we still need to understand how these networks evolve, and the relative role played in them by their sequence-dependent regulatory factors, non-coding RNAs (ncRNAs) and RNA-binding proteins (RBPs). Here, we used an approach that relied on both phylogenetic sequence sharing and conservation in the whole mapped 3'-untranslated regions (3'-UTRs) of vertebrate species to gain knowledge on core post-transcriptional networks. The identified human hyper conserved elements (HCEs) were predicted to be preferred binding sites for RBPs and not for ncRNAs, namely microRNAs and long ncRNAs. We found that the HCE map identified a well-known network that post-transcriptionally regulates histone mRNAs. We were then able to discover and experimentally confirm a translational network composed of RNA Recognition Motif (RRM)-type RBP mRNAs that are positively controlled by HuR, another RRM-type RBP. HuR shows a preference for these RBP mRNAs bound in stem-loop motifs, confirming its role as a 'regulator of regulators'. Analysis of the transcriptome-wide HCE distribution revealed a profile of prevalently small clusters separated by unconserved intercluster RNA stretches, which predicts the formation of discrete small ribonucleoprotein complexes in the 3'-UTRs.
Asunto(s)
Regiones no Traducidas 3' , Proteínas ELAV/fisiología , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Animales , Secuencia de Bases , Sitios de Unión , Secuencia Conservada , Proteínas ELAV/genética , Proteínas ELAV/metabolismo , Histonas/genética , Humanos , Secuencias Invertidas Repetidas , Células MCF-7 , Biosíntesis de Proteínas , Alineación de Secuencia , VertebradosRESUMEN
Post-transcriptional regulation (PTR) of gene expression is a powerful determinant of cellular phenotypes. The 5' and 3' untranslated regions of the mRNA (UTRs) mediate this role through sequence and secondary structure elements bound by RNA-binding proteins (RBPs) and non-coding RNAs. While functional regions in the 3'UTRs have been extensively studied, the 5'UTRs are still relatively uncharacterized. To fill this gap, we used a computational approach exploiting phylogenetic conservation to identify hyperconserved elements in human 5'UTRs (5'HCEs). Our assumption was that 5'HCEs would represent evolutionarily stable and hence important PTR sites. We identified over 5000 5'HCEs occurring in 10% of human protein-coding genes. These sequence elements are rather short and mostly found in narrowly-spaced clusters. 5'HCEs-containing genes are enriched in essential cellular functions and include 20% of all homeotic genes. Homeotic genes are essential transcriptional regulators, driving body plan and neuromuscular development. However, the role of PTR in their expression is mostly unknown. By integrating computational and experimental approaches we identified RBMX as the initiator RBP of a post-transcriptional cascade regulating many homeotic genes. This work thus establishes 5'HCEs as mediators of essential post-transcriptional regulatory networks.
RESUMEN
Non-coding RNAs (ncRNAs) are known to regulate gene expression at the transcriptional and post-transcriptional levels, chromatin remodeling, and signal transduction. The identification of different species of ncRNAs, microRNAs (miRNAs), circular RNAs (circRNAs), and long ncRNAs (lncRNAs)-and in some cases, their combined regulatory function on specific target genes-may help to elucidate their role in biological processes. NcRNAs' deregulation has an impact on the impairment of physiological programs, driving cells in cancer development. We here carried out a review of literature concerning the implication of ncRNAs on tumor development in neurofibromatosis type 1 (NF1), an inherited tumor predisposition syndrome. A number of miRNAs and a lncRNA has been implicated in NF1-associated tumors, such as malignant peripheral nerve sheath tumors (MPNSTs) and astrocytoma, as well as in the pathognomonic neurofibromas. Some authors reported that the lncRNA ANRIL was deregulated in the blood of NF1 patients with plexiform neurofibromas (PNFs), even if its role should be further elucidated. We here provided original data concerning the association of a specific genotype about ANRIL rs2151280 with the presence of optic gliomas and a mild expression of the NF1 phenotype. We also detected the LOH of ANRIL in different tumors from NF1 patients, supporting the involvement of ANRIL in some NF1-associated tumors. Our results suggest that ANRIL rs2151280 may be a potential diagnostic and prognostic marker, addressing early diagnosis of optic glioma and predicting the phenotype severity in NF1 patients.
Asunto(s)
Neurofibromatosis 1/genética , Glioma del Nervio Óptico/genética , ARN Largo no Codificante/genética , Astrocitoma/complicaciones , Genes de Neurofibromatosis 1 , Genotipo , Humanos , Pérdida de Heterocigocidad , MicroARNs/genética , Neoplasias de la Vaina del Nervio/complicaciones , Neurofibroma/complicaciones , Neurofibroma Plexiforme/complicaciones , Neurofibromatosis 1/complicaciones , Glioma del Nervio Óptico/complicaciones , Glioma del Nervio Óptico/metabolismo , Fenotipo , ARN Largo no Codificante/metabolismo , ARN no Traducido/genética , Transducción de Señal/genéticaRESUMEN
The RBP associated with lethal yellow mutation (RALY) is a member of the heterogeneous nuclear ribonucleoprotein family whose transcriptome and interactome have been recently characterized. RALY binds poly-U rich elements within several RNAs and regulates the expression as well as the stability of specific transcripts. Here we show that RALY binds PRMT1 mRNA and regulates its expression. PRMT1 catalyzes the arginine methylation of Fused in Sarcoma (FUS), an RNA-binding protein that interacts with RALY. We demonstrate that RALY down-regulation decreases protein arginine N-methyltransferase 1 levels, thus reducing FUS methylation. It is known that mutations in the FUS nuclear localization signal (NLS) retain the protein to the cytosol, promote aggregate formation, and are associated with amyotrophic lateral sclerosis. Confirming that inhibiting FUS methylation increases its nuclear import, we report that RALY knockout enhances FUS NLS mutants' nuclear translocation, hence decreasing aggregate formation. Furthermore, we characterize the RNA-dependent interaction of RALY with FUS in motor neurons. We show that mutations in FUS NLS as well as in RALY NLS reciprocally alter their localization and interaction with target mRNAs. These data indicate that RALY's activity is impaired in FUS pathology models, raising the possibility that RALY might modulate disease onset and/or progression.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo C/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Neuronas Motoras/metabolismo , Proteína-Arginina N-Metiltransferasas/genética , Proteína FUS de Unión a ARN/genética , Proteínas Represoras/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Línea Celular Tumoral , Embrión de Mamíferos , Regulación de la Expresión Génica , Células HeLa , Ribonucleoproteína Heterogénea-Nuclear Grupo C/antagonistas & inhibidores , Ribonucleoproteína Heterogénea-Nuclear Grupo C/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metilación , Ratones , Neuronas Motoras/citología , Mutación , Señales de Localización Nuclear , Cultivo Primario de Células , Transporte de Proteínas , Proteína-Arginina N-Metiltransferasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Médula Espinal/citología , Médula Espinal/metabolismoRESUMEN
Genetic alterations impacting ubiquitously expressed proteins involved in RNA metabolism often result in neurodegenerative conditions, with increasing evidence suggesting that translation defects can contribute to disease. Spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of SMN protein, whose role in pathogenesis remains unclear. Here, we identified in vivo and in vitro translation defects that are cell autonomous and SMN dependent. By determining in parallel the in vivo transcriptome and translatome in SMA mice, we observed a robust decrease in translation efficiency arising during early stages of disease. We provide a catalogue of RNAs with altered translation efficiency, identifying ribosome biology and translation as central processes affected by SMN depletion. This was further supported by a decrease in the number of ribosomes in SMA motor neurons in vivo. Overall, our findings suggest ribosome biology as an important, yet largely overlooked, factor in motor neuron degeneration.
Asunto(s)
Atrofia Muscular Espinal/metabolismo , Polirribosomas/metabolismo , Transcriptoma , Animales , Células Cultivadas , Ratones , Neuronas Motoras/metabolismo , Atrofia Muscular Espinal/genética , Biosíntesis de Proteínas , Proteoma/genética , Proteoma/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismoRESUMEN
Polysome fractionation by sucrose density gradient centrifugation followed by analysis of RNA and protein is a technique that allows to understand the changes in translation of individual mRNAs as well as genome-wide effects on the translatome. Here, we describe the polysome profiling technique and RNA as well as protein isolation procedures from sucrose fractions.
Asunto(s)
Biología Molecular/métodos , Polirribosomas/genética , Biosíntesis de Proteínas , Proteínas/aislamiento & purificación , Genoma , Humanos , Polirribosomas/metabolismo , Biosíntesis de Proteínas/genética , Proteínas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Cancer-associated gene expression imbalances are conventionally studied at the genomic, epigenomic and transcriptomic levels. Given the relevance of translational control in determining cell phenotypes, we evaluated the translatome, i.e., the transcriptome engaged in translation, as a descriptor of the effects of genetic instability in cancer. We performed this evaluation in high-risk neuroblastomas, which are characterized by a low frequency of point mutations or known cancer-driving genes and by the presence of several segmental chromosomal aberrations that produce gene-copy imbalances that guide aggressiveness. We thus integrated genome, transcriptome, translatome and miRome profiles in a representative panel of high-risk neuroblastoma cell lines. We identified a number of genes whose genomic imbalance was corrected by compensatory adaptations in translational efficiency. The transcriptomic level of these genes was predictive of poor prognosis in more than half of cases, and the genomic imbalances found in their loci were shared by 27 other tumor types. This homeostatic process is also not limited to copy number-altered genes, as we showed the translational stoichiometric rebalance of histone genes. We suggest that the translational buffering of fluctuations in these dose-sensitive transcripts is a potential driving process of neuroblastoma evolution.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica , Neuroblastoma/genética , Biosíntesis de Proteínas , Línea Celular Tumoral , Hibridación Genómica Comparativa , Variaciones en el Número de Copia de ADN , Amplificación de Genes , Perfilación de la Expresión Génica , Genes myc , Histonas/metabolismo , Humanos , Neuroblastoma/metabolismoRESUMEN
Neuroblastoma is the most common pediatric cancer, arising from the neural crest cells of the sympathetic nervous system. Its most aggressive subtype, characterized by the amplification of the MYCN oncogene, has a dismal prognosis and no effective treatment is available. Understanding the alterations induced by the tumor on the various layers of gene expression is therefore important for a complete characterization of this neuroblastoma subtype and for the discovery of new therapeutic opportunities. Here we describe the profiling of 13 MYCN-amplified neuroblastoma cell lines at the genome (copy number), transcriptome, translatome and miRome levels (GEO series GSE56654, GSE56552 and GSE56655). We provide detailed experimental and data analysis procedures by means of which we derived the results described in [1].
RESUMEN
Centaurin-α2 is a GTPase-activating protein for ARF (ARFGAP) showing a diffuse cytoplasmic localization capable to translocate to membrane, where it binds phosphatidylinositols. Taking into account that Centaurin-α2 can localize in cytoplasm and that its cytoplasmatic function is not well defined, we searched for further interactors by yeast two-hybrid assay to investigate its biological function. We identified a further Centaurin-α2 interacting protein, ß-Tubulin, by yeast two-hybrid assay. The interaction, involving the C-terminal region of ß-Tubulin, has been confirmed by coimmunoprecipitation experiments. After Centaurin-α2 overexpression in HeLa cells and extraction of soluble (αß dimers) and insoluble (microtubules) fractions of Tubulin, we observed that Centaurin-α2 mainly interacts with the polymerized Tubulin fraction, besides colocalizing with microtubules (MTs) in cytoplasm accordingly. Even following the depolimerizing Tubulin treatments Centaurin-α2 remains mainly associated to nocodazole- and cold-resistant MTs. We found an increase of MT stability in transfected HeLa cells, evaluating as marker of stability the level of MT acetylation. In vitro assays using purified Centaurin-α2 and tubulin confirmed that Centaurin-α2 promotes tubulin assembly and increases microtubule stability. The biological effect of Centaurin-α2 overexpression, assessed through the detection of an increased number of mitotic HeLa cells with bipolar spindles and with the correct number of centrosomes in both dividing and not dividing cells, is consistent with the Centaurin-α2 role on MT stabilization. Centaurin-α2 interacts with ß-Tubulin and it mainly associates to MTs, resistant to destabilizing agents, in vitro and in cell. We propose Centaurin-α2 as a new microtubule-associated protein (MAP) increasing MT stability.
Asunto(s)
Proteínas Activadoras de GTPasa/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Acetilación , Células Cultivadas , Proteínas Activadoras de GTPasa/antagonistas & inhibidores , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Multimerización de Proteína/genética , Procesamiento Proteico-Postraduccional/fisiología , Estabilidad Proteica/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Saccharomyces cerevisiae , Tubulina (Proteína)/genéticaRESUMEN
CDK5R1 encodes p35, a specific activator of the serine/threonine kinase CDK5, which plays crucial roles in CNS development and maintenance. CDK5 activity strongly depends on p35 levels and p35/CDK5 misregulation is deleterious for correct CNS function, suggesting that a tightly controlled regulation of CDK5R1 expression is needed for proper CDK5 activity. Accordingly, CDK5R1 expression was demonstrated to be controlled at both transcriptional and post-transcriptional levels, but a possible regulation through microRNAs (miRNAs) has never been investigated. We predicted, within the large CDK5R1 3'UTR several miRNA target sites. Among them, we selected for functional studies miR-103 and miR-107, whose expression has shown a strong inverse correlation with p35 levels in different cell lines. A significant reduction of CDK5R1 mRNA and p35 levels was observed after transfection of SK-N-BE neuroblastoma cells with the miR-103 or miR-107 precursor (pre-miR-103 or pre-miR-107). Conversely, p35 levels significantly increased following transfection of the corresponding antagonists (anti-miR-103 or anti-miR-107). Moreover, the level of CDK5R1 transcript shifts from the polysomal to the subpolysomal mRNA fraction after transfection with pre-miR-107 and, conversely, from the subpolysomal to the polysolmal mRNA fraction after transfection with anti-miR-107, suggesting a direct action on translation efficiency. We demonstrate, by means of luciferase assays, that miR-103 and miR-107 are able to directly interact with the CDK5R1 3'-UTR, in correspondence of a specific target site. Finally, miR-103 and miR-107 overexpression, as well as CDK5R1 silencing, caused a reduction in SK-N-BE migration ability, indicating that these miRNAs affect neuronal migration by modulating CDK5R1 expression. These findings indicate that miR-103 and miR-107 regulate CDK5R1 expression, allowing us to hypothesize that a miRNA-mediated mechanism may influence CDK5 activity and the associated molecular pathways.