RESUMEN
N6-methyladenosine (m6 A) regulates a variety of physiological processes through modulation of RNA metabolism. This modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m6 A alters fly behavior, albeit the underlying molecular mechanism and the role of m6 A during nervous system development have remained elusive. Here we find that impairment of the m6 A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m6 A reader in the nervous system, being required to limit axonal growth. Mechanistically, we show that the m6 A reader Ythdf directly interacts with Fmr1, the fly homolog of Fragile X mental retardation RNA binding protein (FMRP), to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6 A pathway controls development of the nervous system and modulates Fmr1 target transcript selection.
Asunto(s)
Adenosina/análogos & derivados , Axones/fisiología , Proteínas de Drosophila/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Neuronas/citología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Adenosina/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Neuronas/fisiología , ARN Mensajero/genética , Proteínas de Unión al ARN/genéticaRESUMEN
RNA modifications have recently emerged as an important regulatory layer of gene expression. The most prevalent and reversible modification on messenger RNA (mRNA), N6-methyladenosine, regulates most steps of RNA metabolism and its dysregulation has been associated with numerous diseases. Other modifications such as 5-methylcytosine and N1-methyladenosine have also been detected on mRNA but their abundance is lower and still debated. Adenosine to inosine RNA editing is widespread on coding and non-coding RNA and can alter mRNA decoding as well as protect against autoimmune diseases. 2'-O-methylation of the ribose and pseudouridine are widespread on ribosomal and transfer RNA and contribute to proper RNA folding and stability. While the understanding of the individual role of RNA modifications has now reached an unprecedented stage, still little is known about their interplay in the control of gene expression. In this review we discuss the examples where such interplay has been observed and speculate that with the progress of mapping technologies more of those will rapidly accumulate.
Asunto(s)
5-Metilcitosina , ARN , Adenosina/metabolismo , ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismoRESUMEN
PURPOSE: 5-methylcytosine RNA modifications are driven by NSUN methyltransferases. Although variants in NSUN2 and NSUN3 were associated with neurodevelopmental diseases, the physiological role of NSUN6 modifications on transfer RNAs and messenger RNAs remained elusive. METHODS: We combined exome sequencing of consanguineous families with functional characterization to identify a new neurodevelopmental disorder gene. RESULTS: We identified 3 unrelated consanguineous families with deleterious homozygous variants in NSUN6. Two of these variants are predicted to be loss-of-function. One maps to the first exon and is predicted to lead to the absence of NSUN6 via nonsense-mediated decay, whereas we showed that the other maps to the last exon and encodes a protein that does not fold correctly. Likewise, we demonstrated that the missense variant identified in the third family has lost its enzymatic activity and is unable to bind the methyl donor S-adenosyl-L-methionine. The affected individuals present with developmental delay, intellectual disability, motor delay, and behavioral anomalies. Homozygous ablation of the NSUN6 ortholog in Drosophila led to locomotion and learning impairment. CONCLUSION: Our data provide evidence that biallelic pathogenic variants in NSUN6 cause one form of autosomal recessive intellectual disability, establishing another link between RNA modification and cognition.
Asunto(s)
Discapacidad Intelectual , Trastornos del Neurodesarrollo , Humanos , Discapacidad Intelectual/genética , Homocigoto , Trastornos del Neurodesarrollo/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , ARN , Linaje , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/metabolismoRESUMEN
BACKGROUND/AIMS: The tumor suppressor p53 is rarely mutated in gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) but they frequently show a strong expression of negative regulators of p53, rendering these tumors excellent targets for a p53 recovery therapy. Therefore, we analyzed the mechanisms of a p53 recovery therapy on intestinal neuroendocrine tumors in vitro and in vivo. METHODS: By Western blot and immunohistochemistry, we found that in GEP-NEN biopsy material overexpression of MDM2 was present in intestinal NEN. Therefore, we analyzed the effect of a small-molecule inhibitor, nutlin-3a, in p53 wild-type and mutant GEP-NEN cell lines by proliferation assay, flow cytometry, immunofluorescence, Western blot, and by multiplex gene expression analysis. Finally, we analyzed the antitumor effect of nutlin-3a in a xenograft mouse model in vivo. During the study, the tumor volume was determined. RESULTS: The midgut wild-type cell line KRJ-I responded to the treatment with cell cycle arrest and apoptosis. By gene expression analysis, we could demonstrate that nutlins reactivated an antiproliferative p53 response. KRJ-I-derived xenograft tumors showed a significantly decreased tumor growth upon treatment with nutlin-3a in vivo. Furthermore, our data suggest that MDM2 also influences the expression of the oncogene FOXM1 in a p53-independent manner. Subsequently, a combined treatment of nutlin-3a and cisplatin (as chemoresistance model) resulted in synergistically enhanced antiproliferative effects. CONCLUSION: In summary, MDM2 overexpression is a frequent event in p53 wild-type intestinal neuroendocrine neoplasms and therefore recovery of a p53 response might be a novel personalized treatment approach in these tumors.
Asunto(s)
Antineoplásicos/farmacología , Imidazoles/farmacología , Neoplasias Intestinales/patología , Tumores Neuroendocrinos/patología , Piperazinas/farmacología , Adulto , Anciano , Animales , Proteína Forkhead Box M1/antagonistas & inhibidores , Humanos , Ratones , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-mdm2/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
m6A is the most abundant internal modification on mRNA. Recent improvements of high-throughput sequencing techniques enables its detection at the transcriptome level, even at the nucleotide resolution. However most current techniques require large amounts of starting material to detect the modification. Here, we describe a complementary technique of standard meRIP-seq/miCLIP-seq approaches to identify methylated RNA using a low amount of material. We believe this approach can be applied in vivo to identify methylated targets in specific tissues or subpopulations of cells.
Asunto(s)
Biología Computacional/métodos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Clonación Molecular , Edición Génica , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Metilación , Procesamiento Postranscripcional del ARN , ARN Mensajero/aislamiento & purificación , Programas Informáticos , TranscriptomaRESUMEN
The field of RNA modifications, so-called epitranscriptomics, has flourished over the past years owing to improvements of detection methods and the identification of important regulatory players. N6-methyladenosine (m6A) is the most abundant internal modification in messenger (mRNA) and long non-coding (lncRNA), and controls most steps of RNA metabolism. Its physiological roles range from gametogenesis, stem cell differentiation to immunity, neuronal development and functions, while its alterations are associated with cancer development and progression. In this review we focus on the proteins that catalyze formation of m6A (also called writers) on RNA. Interestingly, distinct proteins deposit m6A on different classes of RNA, indicating that specific RNA features dictate recognition mechanisms. Associated factors and post-translational modifications can also alter m6A enzyme activity. A better understanding of the underlying regulation involved in m6A deposition is the first step towards developing tools for cancer therapy and for treatment of other m6A-associated diseases.
Asunto(s)
Adenosina/análogos & derivados , Metiltransferasas/química , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Adenosina/química , Adenosina/metabolismo , Animales , Dominio Catalítico , Regulación del Desarrollo de la Expresión Génica , Humanos , Metiltransferasas/metabolismo , ARN Mensajero/químicaRESUMEN
Analogous to DNA methylation and histone modifications, RNA modifications represent a novel layer of regulation of gene expression. The dynamic nature and increasing number of RNA modifications offer new possibilities to rapidly alter gene expression upon specific environmental changes. Recent lines of evidence indicate that modified RNA molecules and associated complexes regulating and "reading" RNA modifications play key roles in the nervous system of several organisms, controlling both, its development and function. Mutations in several human genes that modify transfer RNA (tRNA) have been linked to neurological disorders, in particular to intellectual disability. Loss of RNA modifications alters the stability of tRNA, resulting in reduced translation efficiency and generation of tRNA fragments, which can interfere with neuronal functions. Modifications present on messenger RNAs (mRNAs) also play important roles during brain development. They contribute to neuronal growth and regeneration as well as to the local regulation of synaptic functions. Hence, potential combinatorial effects of RNA modifications on different classes of RNA may represent a novel code to dynamically fine tune gene expression during brain function. Here we discuss the recent findings demonstrating the impact of modified RNAs on neuronal processes and disorders.
RESUMEN
Several bacterial pathogens produce nucleotidyl cyclase toxins to manipulate eukaryotic host cells. Inside host cells they are activated by endogenous cofactors to produce high levels of cyclic nucleotides (cNMPs). The ExoY toxin from Pseudomonas aeruginosa (PaExoY) and the ExoY-like module (VnExoY) found in the MARTX (Multifunctional-Autoprocessing Repeats-in-ToXin) toxin of Vibrio nigripulchritudo share modest sequence similarity (~38%) but were both recently shown to be activated by actin after their delivery to the eukaryotic host cell. Here, we further characterized the ExoY-like cyclase of V. nigripulchritudo. We show that, in contrast to PaExoY that requires polymerized actin (F-actin) for maximum activation, VnExoY is selectively activated by monomeric actin (G-actin). These two enzymes also display different nucleotide substrate and divalent cation specificities. In vitro in presence of the cation Mg2+, the F-actin activated PaExoY exhibits a promiscuous nucleotidyl cyclase activity with the substrate preference GTP>ATP≥UTP>CTP, while the G-actin activated VnExoY shows a strong preference for ATP as substrate, as it is the case for the well-known calmodulin-activated adenylate cyclase toxins from Bordetella pertussis or Bacillus anthracis. These results suggest that the actin-activated nucleotidyl cyclase virulence factors despite sharing a common activator may actually display a greater variability of biological effects in infected cells than initially anticipated.
Asunto(s)
Citoesqueleto de Actina/genética , Toxina de Adenilato Ciclasa/química , Células Eucariotas/efectos de los fármacos , Pseudomonas aeruginosa/química , Citoesqueleto de Actina/química , Adenosina Trifosfato/química , Toxina de Adenilato Ciclasa/genética , Bacillus anthracis/efectos de los fármacos , Bacillus anthracis/patogenicidad , Proteínas Bacterianas/genética , Bordetella pertussis/efectos de los fármacos , Bordetella pertussis/patogenicidad , Glucosiltransferasas/genética , Interacciones Huésped-Patógeno/genética , Humanos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Especificidad por Sustrato , Toxinas Biológicas/química , Toxinas Biológicas/genética , Vibrio/efectos de los fármacos , Vibrio/genética , Vibrio/patogenicidad , Factores de Virulencia/química , Factores de Virulencia/genéticaRESUMEN
The nucleotidyl cyclase toxin ExoY is one of the virulence factors injected by the Pseudomonas aeruginosa type III secretion system into host cells. Inside cells, it is activated by an unknown eukaryotic cofactor to synthesize various cyclic nucleotide monophosphates. ExoY-like adenylate cyclases are also found in Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) toxins produced by various Gram-negative pathogens. Here we demonstrate that filamentous actin (F-actin) is the hitherto unknown cofactor of ExoY. Association with F-actin stimulates ExoY activity more than 10,000 fold in vitro and results in stabilization of actin filaments. ExoY is recruited to actin filaments in transfected cells and alters F-actin turnover. Actin also activates an ExoY-like adenylate cyclase MARTX effector domain from Vibrio nigripulchritudo. Finally, using a yeast genetic screen, we identify actin mutants that no longer activate ExoY. Our results thus reveal a new sub-group within the class II adenylyl cyclase family, namely actin-activated nucleotidyl cyclase (AA-NC) toxins.