RESUMEN
The RNA-binding ARS2 protein is centrally involved in both early RNA polymerase II (RNAPII) transcription termination and transcript decay. Despite its essential nature, the mechanisms by which ARS2 enacts these functions have remained unclear. Here, we show that a conserved basic domain of ARS2 binds a corresponding acidic-rich, short linear motif (SLiM) in the transcription restriction factor ZC3H4. This interaction recruits ZC3H4 to chromatin to elicit RNAPII termination, independent of other early termination pathways defined by the cleavage and polyadenylation (CPA) and Integrator (INT) complexes. We find that ZC3H4, in turn, forms a direct connection to the nuclear exosome targeting (NEXT) complex, hereby facilitating rapid degradation of the nascent RNA. Hence, ARS2 instructs the coupled transcription termination and degradation of the transcript onto which it is bound. This contrasts with ARS2 function at CPA-instructed termination sites where the protein exclusively partakes in RNA suppression via post-transcriptional decay.
Asunto(s)
Proteínas Nucleares , Transcripción Genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Estabilidad del ARN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , ARNRESUMEN
In mammalian cells, spurious transcription results in a vast repertoire of unproductive non-coding RNAs, whose deleterious accumulation is prevented by rapid decay. The nuclear exosome targeting (NEXT) complex plays a central role in directing non-functional transcripts to exosome-mediated degradation, but the structural and molecular mechanisms remain enigmatic. Here, we elucidated the architecture of the human NEXT complex, showing that it exists as a dimer of MTR4-ZCCHC8-RBM7 heterotrimers. Dimerization preconfigures the major MTR4-binding region of ZCCHC8 and arranges the two MTR4 helicases opposite to each other, with each protomer able to function on many types of RNAs. In the inactive state of the complex, the 3' end of an RNA substrate is enclosed in the MTR4 helicase channel by a ZCCHC8 C-terminal gatekeeping domain. The architecture of a NEXT-exosome assembly points to the molecular and regulatory mechanisms with which the NEXT complex guides RNA substrates to the exosome.
Asunto(s)
Exosomas , ARN , Núcleo Celular/genética , Núcleo Celular/metabolismo , ARN Helicasas DEAD-box/metabolismo , ADN Helicasas/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Exosomas/genética , Exosomas/metabolismo , Humanos , Unión Proteica , ARN/genética , ARN/metabolismo , ARN Helicasas/metabolismo , Estabilidad del ARN/genéticaRESUMEN
The RNA exosome is a versatile ribonuclease. In the nucleoplasm of mammalian cells, it is assisted by its adaptors the nuclear exosome targeting (NEXT) complex and the poly(A) exosome targeting (PAXT) connection. Via its association with the ARS2 and ZC3H18 proteins, NEXT/exosome is recruited to capped and short unadenylated transcripts. Conversely, PAXT/exosome is considered to target longer and adenylated substrates via their poly(A) tails. Here, mutational analysis of the core PAXT component ZFC3H1 uncovers a separate branch of the PAXT pathway, which targets short adenylated RNAs and relies on a direct ARS2-ZFC3H1 interaction. We further demonstrate that similar acidic-rich short linear motifs of ZFC3H1 and ZC3H18 compete for a common ARS2 epitope. Consequently, while promoting NEXT function, ZC3H18 antagonizes PAXT activity. We suggest that this organization of RNA decay complexes provides co-activation of NEXT and PAXT at loci with abundant production of short exosome substrates.
Asunto(s)
ARN Nuclear , Proteínas de Unión al ARN , Animales , Núcleo Celular/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma/metabolismo , Mamíferos , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Nuclear/genética , Proteínas de Unión al ARN/genéticaRESUMEN
The TRIM-NHL protein Meiotic P26 (Mei-P26) acts as a regulator of cell fate in Drosophila Its activity is critical for ovarian germline stem cell maintenance, differentiation of oocytes, and spermatogenesis. Mei-P26 functions as a post-transcriptional regulator of gene expression; however, the molecular details of how its NHL domain selectively recognizes and regulates its mRNA targets have remained elusive. Here, we present the crystal structure of the Mei-P26 NHL domain at 1.6 Å resolution and identify key amino acids that confer substrate specificity and distinguish Mei-P26 from closely related TRIM-NHL proteins. Furthermore, we identify mRNA targets of Mei-P26 in cultured Drosophila cells and show that Mei-P26 can act as either a repressor or activator of gene expression on different RNA targets. Our work reveals the molecular basis of RNA recognition by Mei-P26 and the fundamental functional differences between otherwise very similar TRIM-NHL proteins.
Asunto(s)
Proteínas de Drosophila , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Masculino , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismoRESUMEN
Intellectual disability (ID) and autism spectrum disorder (ASD) are the most common neurodevelopmental disorders and are characterized by substantial impairment in intellectual and adaptive functioning, with their genetic and molecular basis remaining largely unknown. Here, we identify biallelic variants in the gene encoding one of the Elongator complex subunits, ELP2, in patients with ID and ASD. Modelling the variants in mice recapitulates the patient features, with brain imaging and tractography analysis revealing microcephaly, loss of white matter tract integrity and an aberrant functional connectome. We show that the Elp2 mutations negatively impact the activity of the complex and its function in translation via tRNA modification. Further, we elucidate that the mutations perturb protein homeostasis leading to impaired neurogenesis, myelin loss and neurodegeneration. Collectively, our data demonstrate an unexpected role for tRNA modification in the pathogenesis of monogenic ID and ASD and define Elp2 as a key regulator of brain development.
Asunto(s)
Trastorno del Espectro Autista/genética , Discapacidad Intelectual/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación , Trastornos del Neurodesarrollo/genética , Transcriptoma/genética , Animales , Trastorno del Espectro Autista/metabolismo , Trastorno del Espectro Autista/fisiopatología , Modelos Animales de Enfermedad , Epigénesis Genética , Aseo Animal/fisiología , Humanos , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/fisiopatología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Trastornos del Neurodesarrollo/metabolismo , Trastornos del Neurodesarrollo/fisiopatología , Fenotipo , Células Sf9 , SpodopteraRESUMEN
It has been shown that the metabotropic glutamate receptor subtype 5 (mGluR5) is functionally associated with the NMDA subtype of the glutamate receptor family (NMDA receptors). These two receptors colocalize in brain regions associated with schizophrenia. Although the role of the NMDA receptor in cognitive and negative symptoms of schizophrenia is well studied, information about the role of mGluR5 receptors in schizophrenia is sparse. In our work, we show that subchronic administration of ketamine, a well-studied, non-competitive antagonist of NMDA receptors, caused cognitive deficits in rats as shown by testing novel object recognition (NOR). Moreover, we reveal that subchronic administration of ketamine increased the mRNA and protein expression levels of mGluR5 receptors in regions CA1 and CA3 of the dorsal part of the hippocampus, both of which are strongly associated with the formation of visual memory, which is tested via NOR. We postulate that increased expression of mGluR5 receptors in the dorsal part of the hippocampus may reflect compensatory changes to imbalanced glutamate neurotransmission associated with the hypoactivation of NMDA receptors.
Asunto(s)
Receptor del Glutamato Metabotropico 5/metabolismo , Esquizofrenia/metabolismo , Animales , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/patología , Región CA3 Hipocampal/metabolismo , Región CA3 Hipocampal/patología , Modelos Animales de Enfermedad , Conducta Exploratoria/fisiología , Ketamina , Masculino , Parvalbúminas/metabolismo , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Receptores de Dopamina D2/metabolismo , Reconocimiento en Psicología/fisiología , Esquizofrenia/patologíaRESUMEN
Cerebellar ataxias are severe neurodegenerative disorders with an early onset and progressive and inexorable course of the disease. Here, we report a single point mutation in the gene encoding Elongator complex subunit 6 causing Purkinje neuron degeneration and an ataxia-like phenotype in the mutant wobbly mouse. This mutation destabilizes the complex and compromises its function in translation regulation, leading to protein misfolding, proteotoxic stress, and eventual neuronal death. In addition, we show that substantial microgliosis is triggered by the NLRP3 inflammasome pathway in the cerebellum and that blocking NLRP3 function in vivo significantly delays neuronal degeneration and the onset of ataxia in mutant animals. Our data provide a mechanistic insight into the pathophysiology of a cerebellar ataxia caused by an Elongator mutation, substantiating the increasing body of evidence that alterations of this complex are broadly implicated in the onset of a number of diverse neurological disorders.