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1.
EMBO J ; 42(12): e112858, 2023 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-37140366

RESUMEN

The obligate anaerobic, enteric pathogen Clostridioides difficile persists in the intestinal tract by forming antibiotic-resistant endospores that contribute to relapsing and recurrent infections. Despite the importance of sporulation for C. difficile pathogenesis, environmental cues and molecular mechanisms that regulate sporulation initiation remain ill-defined. Here, by using RIL-seq to globally capture the Hfq-dependent RNA-RNA interactome, we discovered a network of small RNAs that bind to mRNAs encoding sporulation-related genes. We show that two of these small RNAs, SpoX and SpoY, regulate translation of the master regulator of sporulation, Spo0A, in an opposing manner, which ultimately leads to altered sporulation rates. Infection of antibiotic-treated mice with SpoX and SpoY deletion mutants revealed a global effect on gut colonization and intestinal sporulation. Our work uncovers an elaborate RNA-RNA interactome controlling the physiology and virulence of C. difficile and identifies a complex post-transcriptional layer in the regulation of spore formation in this important human pathogen.


Asunto(s)
Clostridioides difficile , Clostridioides , Animales , Humanos , Ratones , Clostridioides/genética , Clostridioides/metabolismo , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Antibacterianos , ARN/metabolismo , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica
2.
Genes Dev ; 32(5-6): 415-429, 2018 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-29535189

RESUMEN

N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes, playing crucial roles in multiple biological processes. m6A is catalyzed by the activity of methyltransferase-like 3 (Mettl3), which depends on additional proteins whose precise functions remain poorly understood. Here we identified Zc3h13 (zinc finger CCCH domain-containing protein 13)/Flacc [Fl(2)d-associated complex component] as a novel interactor of m6A methyltransferase complex components in Drosophila and mice. Like other components of this complex, Flacc controls m6A levels and is involved in sex determination in Drosophila We demonstrate that Flacc promotes m6A deposition by bridging Fl(2)d to the mRNA-binding factor Nito. Altogether, our work advances the molecular understanding of conservation and regulation of the m6A machinery.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/fisiología , Metiltransferasas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Adenosina/metabolismo , Animales , Proteínas de Ciclo Celular , Línea Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Regulación del Desarrollo de la Expresión Génica , Metilación , Ratones , Células Madre Embrionarias de Ratones , Transporte de Proteínas , Precursores del ARN/genética , Empalme del ARN , Factores de Empalme de ARN , Procesos de Determinación del Sexo/genética
3.
EMBO J ; 40(4): e104975, 2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33428246

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/genética
4.
Nucleic Acids Res ; 49(4): e23, 2021 02 26.
Artículo en Inglés | MEDLINE | ID: mdl-33313868

RESUMEN

Methods for the detection of m6A by RNA-Seq technologies are increasingly sought after. We here present NOseq, a method to detect m6A residues in defined amplicons by virtue of their resistance to chemical deamination, effected by nitrous acid. Partial deamination in NOseq affects all exocyclic amino groups present in nucleobases and thus also changes sequence information. The method uses a mapping algorithm specifically adapted to the sequence degeneration caused by deamination events. Thus, m6A sites with partial modification levels of ∼50% were detected in defined amplicons, and this threshold can be lowered to ∼10% by combination with m6A immunoprecipitation. NOseq faithfully detected known m6A sites in human rRNA, and the long non-coding RNA MALAT1, and positively validated several m6A candidate sites, drawn from miCLIP data with an m6A antibody, in the transcriptome of Drosophila melanogaster. Conceptually related to bisulfite sequencing, NOseq presents a novel amplicon-based sequencing approach for the validation of m6A sites in defined sequences.


Asunto(s)
Adenosina/análogos & derivados , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN/química , Análisis de Secuencia de ARN/métodos , Adenosina/análisis , Algoritmos , Animales , Cromatografía Liquida , Desaminación , Drosophila melanogaster/genética , Células HEK293 , Células HeLa , Humanos , ARN Largo no Codificante/química , ARN Mensajero/química , ARN Ribosómico 18S/química , Alineación de Secuencia , Espectrometría de Masas en Tándem
5.
Nature ; 540(7632): 242-247, 2016 12 08.
Artículo en Inglés | MEDLINE | ID: mdl-27919077

RESUMEN

N6-methyladenosine RNA (m6A) is a prevalent messenger RNA modification in vertebrates. Although its functions in the regulation of post-transcriptional gene expression are beginning to be unveiled, the precise roles of m6A during development of complex organisms remain unclear. Here we carry out a comprehensive molecular and physiological characterization of the individual components of the methyltransferase complex, as well as of the YTH domain-containing nuclear reader protein in Drosophila melanogaster. We identify the member of the split ends protein family, Spenito, as a novel bona fide subunit of the methyltransferase complex. We further demonstrate important roles of this complex in neuronal functions and sex determination, and implicate the nuclear YT521-B protein as a main m6A effector in these processes. Altogether, our work substantially extends our knowledge of m6A biology, demonstrating the crucial functions of this modification in fundamental processes within the context of the whole animal.


Asunto(s)
Adenosina/análogos & derivados , Drosophila melanogaster/fisiología , Neuronas/fisiología , Procesos de Determinación del Sexo/fisiología , Adenosina/metabolismo , Empalme Alternativo , Animales , Conducta Animal/fisiología , Proteínas de Drosophila/química , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/enzimología , Femenino , Masculino , Metiltransferasas/química , Metiltransferasas/metabolismo , Sistema Nervioso/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenotipo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas de Unión al ARN/genética , Procesos de Determinación del Sexo/genética
6.
Nucleic Acids Res ; 48(4): 2050-2072, 2020 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-31943105

RESUMEN

2'-O-Methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structure and function with crucial roles in various RNA-mediated processes ranging from RNA silencing, translation, self versus non-self recognition to viral defense mechanisms. Here, we identify two Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1. Genetic knockout studies together with MALDI-TOF mass spectrometry and RiboMethSeq mapping revealed that CG7009 is responsible for methylating the wobble position in tRNAPhe, tRNATrp and tRNALeu, while CG5220 methylates position C32 in the same tRNAs and also targets additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as lifespan reduction, small RNA pathways dysfunction and increased sensitivity to RNA virus infections. Our results provide the first detailed characterization of two TRM7 family members in Drosophila and uncover a molecular link between enzymes catalyzing Nm at specific tRNAs and small RNA-induced gene silencing pathways.


Asunto(s)
Drosophila melanogaster/genética , Silenciador del Gen , ARN de Transferencia/genética , ARNt Metiltransferasas/genética , Animales , Regulación de la Expresión Génica/genética , Humanos , Metilación , Metiltransferasas/genética , Proteínas Nucleares/genética , Interferencia de ARN , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
7.
RNA Biol ; 14(9): 1232-1240, 2017 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-28353398

RESUMEN

RNA modifications are an emerging layer of posttranscriptional gene regulation in eukaryotes. N6-methyladenosine (m6A) is among the most abundant modifications in mRNAs (mRNAs) that was shown to influence many physiological processes from yeast to mammals. Like DNA methylation, m6A in mRNA is dynamically regulated. A conserved methyltransferase complex catalyzes the deposition of the methyl group on adenosine, which can be removed by specific classes of demethylases. Furthermore, YTH-domain containing proteins can recognize this modification to mediate m6A-dependent activities. Here we review the functions and mechanisms of the main m6A players with a particular focus on Drosophila melanogaster.


Asunto(s)
Adenosina/análogos & derivados , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Animales , Regulación de la Expresión Génica , Metilación , Empalme del ARN
8.
Nat Commun ; 12(1): 3778, 2021 06 18.
Artículo en Inglés | MEDLINE | ID: mdl-34145251

RESUMEN

N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex.


Asunto(s)
Adenosina/análogos & derivados , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Metiltransferasas/metabolismo , ARN Mensajero/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Adenosina/metabolismo , Animales , Línea Celular , Drosophila melanogaster , Células HeLa , Humanos , Metilación , Metiltransferasas/genética , Procesamiento Postranscripcional del ARN/genética , Empalme del ARN/genética
9.
Biochim Biophys Acta Gene Regul Mech ; 1862(3): 222-229, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30395944

RESUMEN

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/química
10.
Artículo en Inglés | MEDLINE | ID: mdl-29707539

RESUMEN

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.

11.
Cell Rep ; 19(5): 1056-1067, 2017 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-28467899

RESUMEN

Many RNA-binding proteins (RBPs) regulate both alternative exons and poly(A) site selection. To understand their regulatory principles, we developed expressRNA, a web platform encompassing computational tools for integration of iCLIP and RNA motif analyses with RNA-seq and 3' mRNA sequencing. This reveals at nucleotide resolution the "RNA maps" describing how the RNA binding positions of RBPs relate to their regulatory functions. We use this approach to examine how TDP-43, an RBP involved in several neurodegenerative diseases, binds around its regulated poly(A) sites. Binding close to the poly(A) site generally represses, whereas binding further downstream enhances use of the site, which is similar to TDP-43 binding around regulated exons. Our RNAmotifs2 software also identifies sequence motifs that cluster together with the binding motifs of TDP-43. We conclude that TDP-43 directly regulates diverse types of pre-mRNA processing according to common position-dependent principles.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Poliadenilación , Empalme del ARN , ARN Mensajero/metabolismo , Células HEK293 , Humanos , Unión Proteica , Señales de Poliadenilación de ARN 3' , ARN Mensajero/química , ARN Mensajero/genética
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