RESUMEN
Translation of messenger ribonucleic acids (mRNAs) encoding integral membrane proteins or secreted proteins occurs on the surface of the endoplasmic reticulum (ER). When a nascent signal peptide is synthesized from the mRNAs, the ribosome-nascent chain complex (RNC) is recognized by the signal recognition particle (SRP) and then transported to the surface of the ER. The appropriate targeting of the RNC-SRP complex to the ER is monitored by a quality control pathway, a nuclear cap-binding complex (CBC)-ensured translational repression of RNC-SRP (CENTRE). In this study, using ribosome profiling of CBC-associated and eukaryotic translation initiation factor 4E-associated mRNAs, we reveal that, at the transcriptomic level, CENTRE is in charge of the translational repression of the CBC-RNC-SRP until the complex is specifically transported to the ER. We also find that CENTRE inhibits the nonsense-mediated mRNA decay (NMD) of mRNAs within the CBC-RNC-SRP. The NMD occurs only after the CBC-RNC-SRP is targeted to the ER and after eukaryotic translation initiation factor 4E replaces CBC. Our data indicate dual surveillance for properly targeting mRNAs encoding integral membrane or secretory proteins to the ER. CENTRE blocks gene expression at the translation level before the CBC-RNC-SRP delivery to the ER, and NMD monitors mRNA quality after its delivery to the ER.
Asunto(s)
Retículo Endoplásmico , Degradación de ARNm Mediada por Codón sin Sentido , ARN Mensajero , Partícula de Reconocimiento de Señal , Retículo Endoplásmico/metabolismo , ARN Mensajero/metabolismo , ARN Mensajero/genética , Humanos , Partícula de Reconocimiento de Señal/metabolismo , Partícula de Reconocimiento de Señal/genética , Señales de Clasificación de Proteína/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Células HeLa , Ribosomas/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Biosíntesis de ProteínasRESUMEN
An RNA structure or modified RNA sequences can provide a platform for ribosome loading and internal translation initiation. The functional significance of internal translation has recently been highlighted by the discovery that a subset of circular RNAs (circRNAs) is internally translated. However, the molecular mechanisms underlying the internal initiation of translation in circRNAs remain unclear. Here, we identify eIF3g (a subunit of eIF3 complex) as a binding partner of eIF4A3, a core component of the exon-junction complex (EJC) that is deposited onto spliced mRNAs and plays multiple roles in the regulation of gene expression. The direct interaction between eIF4A3-eIF3g serves as a molecular linker between the eIF4A3 and eIF3 complex, thereby facilitating internal ribosomal entry. Protein synthesis from in vitro-synthesized circRNA demonstrates eIF4A3-driven internal translation, which relies on the eIF4A3-eIF3g interaction. Furthermore, our transcriptome-wide analysis shows that efficient polysomal association of endogenous circRNAs requires eIF4A3. Notably, a subset of endogenous circRNAs can express a full-length intact protein, such as ß-catenin, in an eIF4A3-dependent manner. Collectively, our results expand the understanding of the protein-coding potential of the human transcriptome, including circRNAs.
Asunto(s)
Factor 3 de Iniciación Eucariótica , Factor 4A Eucariótico de Iniciación , ARN Circular , Humanos , Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Proteínas , Ribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Mitochondria are energy-generating organelles and mitochondrial biogenesis is stimulated to meet energy requirements in response to extracellular stimuli, including exercise. However, the mechanisms underlying mitochondrial biogenesis remain unknown. Here, we demonstrate that transcriptional coactivator with PDZ-binding motif (TAZ) stimulates mitochondrial biogenesis in skeletal muscle. In muscle-specific TAZ-knockout (mKO) mice, mitochondrial biogenesis, respiratory metabolism, and exercise ability were decreased compared to wild-type mice. Mechanistically, TAZ stimulates the translation of mitochondrial transcription factor A via Ras homolog enriched in brain (Rheb)/Rheb like 1 (Rhebl1)-mTOR axis. TAZ stimulates Rhebl1 expression via TEA domain family transcription factor. Rhebl1 introduction by adeno-associated virus or mTOR activation recovered mitochondrial biogenesis in mKO muscle. Physiologically, mKO mice did not stimulate exercise-induced mitochondrial biogenesis. Collectively, our results suggested that TAZ is a novel stimulator for mitochondrial biogenesis and exercise-induced muscle adaptation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas de Unión al ADN/genética , Mitocondrias Musculares/genética , Proteínas Mitocondriales/genética , Biogénesis de Organelos , Condicionamiento Físico Animal , Factores de Transcripción/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Células Cultivadas , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células HEK293 , Humanos , Ratones Noqueados , Mitocondrias Musculares/metabolismo , Proteínas Mitocondriales/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Mioblastos/citología , Mioblastos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The pioneer (or first) round of translation of newly synthesized mRNAs is largely mediated by a nuclear cap-binding complex (CBC). In a transcriptome-wide analysis of polysome-associated and CBC-bound transcripts, we identify RN7SL1, a noncoding RNA component of a signal recognition particle (SRP), as an interaction partner of the CBC. The direct CBC-SRP interaction safeguards against abnormal expression of polypeptides from a ribosome-nascent chain complex (RNC)-SRP complex until the latter is properly delivered to the endoplasmic reticulum. Failure of this surveillance causes abnormal expression of misfolded proteins at inappropriate intracellular locations, leading to a cytosolic stress response. This surveillance pathway also blocks protein synthesis through RNC-SRP misassembled on an mRNA encoding a mitochondrial protein. Thus, our results reveal a surveillance pathway in which pioneer translation ensures proper targeting of endoplasmic reticulum and mitochondrial proteins.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas Mitocondriales/metabolismo , Biosíntesis de Proteínas , Partícula de Reconocimiento de Señal/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Mitocondriales/genética , Modelos Genéticos , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Polirribosomas/genética , Polirribosomas/metabolismo , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Partícula de Reconocimiento de Señal/genética , Transducción de Señal/genéticaRESUMEN
Argonaute is the primary mediator of metazoan miRNA targeting (MT). Among the currently identified >1,500 human RNA-binding proteins (RBPs), there are only a handful of RBPs known to enhance MT and several others reported to suppress MT, leaving the global impact of RBPs on MT elusive. In this study, we have systematically analyzed transcriptome-wide binding sites for 150 human RBPs and evaluated the quantitative effect of individual RBPs on MT efficacy. In contrast to previous studies, we show that most RBPs significantly affect MT and that all of those MT-regulating RBPs function as MT enhancers rather than suppressors, by making the local secondary structure of the target site accessible to Argonaute. Our findings illuminate the unappreciated regulatory impact of human RBPs on MT, and as these RBPs may play key roles in the gene regulatory network governed by metazoan miRNAs, MT should be understood in the context of co-regulating RBPs.
Asunto(s)
MicroARNs/metabolismo , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3'/genética , Sitios de Unión , Evolución Molecular , Células HeLa , Células Hep G2 , Humanos , MicroARNs/genética , Conformación de Ácido Nucleico , Unión Proteica , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Especificidad por SustratoRESUMEN
COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which infected >200 million people resulting in >4 million deaths. However, temporal landscape of the SARS-CoV-2 translatome and its impact on the human genome remain unexplored. Here, we report a high-resolution atlas of the translatome and transcriptome of SARS-CoV-2 for various time points after infecting human cells. Intriguingly, substantial amount of SARS-CoV-2 translation initiates at a novel translation initiation site (TIS) located in the leader sequence, termed TIS-L. Since TIS-L is included in all the genomic and subgenomic RNAs, the SARS-CoV-2 translatome may be regulated by a sophisticated interplay between TIS-L and downstream TISs. TIS-L functions as a strong translation enhancer for ORF S, and as translation suppressors for most of the other ORFs. Our global temporal atlas provides compelling insight into unique regulation of the SARS-CoV-2 translatome and helps comprehensively evaluate its impact on the human genome.
Asunto(s)
COVID-19/virología , Biosíntesis de Proteínas , SARS-CoV-2/genética , Transcriptoma , Regulación Viral de la Expresión Génica , Genoma Humano , Humanos , Sistemas de Lectura Abierta , ARN Viral/genética , ARN Viral/metabolismo , SARS-CoV-2/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Newly synthesized mRNA is translated during its export through the nuclear pore complex, when its 5'-cap structure is still bound by the nuclear cap-binding complex (CBC), a heterodimer of cap-binding protein (CBP) 80 and CBP20. Despite its critical role in mRNA surveillance, the mechanism by which CBC-dependent translation (CT) is regulated remains unknown. Here, we demonstrate that the CT initiation factor (CTIF) is tethered in a translationally incompetent manner to the perinuclear region by the DEAD-box helicase 19B (DDX19B). DDX19B hands over CTIF to CBP80, which is associated with the 5'-cap of a newly exported mRNA. The resulting CBP80-CTIF complex then initiates CT in the perinuclear region. We also show that impeding the interaction between CTIF and DDX19B leads to uncontrolled CT throughout the cytosol, consequently dysregulating nonsense-mediated mRNA decay. Altogether, our data provide molecular evidence supporting the importance of tight control of local translation in the perinuclear region.
Asunto(s)
ARN Helicasas DEAD-box/genética , Factores Eucarióticos de Iniciación/genética , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Biosíntesis de Proteínas , Citoplasma/genética , Células HeLa , Humanos , Degradación de ARNm Mediada por Codón sin Sentido/genética , Mapas de Interacción de Proteínas/genética , Proteínas de Unión a Caperuzas de ARN/genética , ARN Mensajero/genéticaRESUMEN
Selective recognition and elimination of misfolded polypeptides are crucial for protein homeostasis. When the ubiquitin-proteasome system is impaired, misfolded polypeptides tend to form small cytosolic aggregates and are transported to the aggresome and eventually eliminated by the autophagy pathway. Despite the importance of this process, the regulation of aggresome formation remains poorly understood. Here, we identify TRIM28/TIF1ß/KAP1 (tripartite motif containing 28) as a negative regulator of aggresome formation. Direct interaction between TRIM28 and CTIF (cap binding complex dependent translation initiation factor) leads to inefficient aggresomal targeting of misfolded polypeptides. We also find that either treatment of cells with poly I:C or infection of the cells by influenza A viruses triggers the phosphorylation of TRIM28 at S473 in a way that depends on double-stranded RNA-activated protein kinase. The phosphorylation promotes association of TRIM28 with CTIF, inhibits aggresome formation, and consequently suppresses viral proliferation. Collectively, our data provide compelling evidence that TRIM28 is a negative regulator of aggresome formation.Abbreviations: BAG3: BCL2-associated athanogene 3; CTIF: CBC-dependent translation initiation factor; CED: CTIF-EEF1A1-DCTN1; DCTN1: dynactin subunit 1; EEF1A1: eukaryotic translation elongation factor 1 alpha 1; EIF2AK2: eukaryotic translation initiation factor 2 alpha kinase 2; HDAC6: histone deacetylase 6; IAV: influenza A virus; IP: immunoprecipitation; PLA: proximity ligation assay; polypeptidyl-puro: polypeptidyl-puromycin; qRT-PCR: quantitative reverse-transcription PCR; siRNA: small interfering RNA.
Asunto(s)
Autofagia , Virus de la Influenza A , Cuerpos de Inclusión/metabolismo , Virus de la Influenza A/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismoRESUMEN
The SH3 and multiple ankyrin repeat domains 3 (Shank3) protein is a core organizer of the macromolecular complex in excitatory postsynapses, and its defects cause numerous synaptopathies, including autism spectrum disorders. Although the function of Shank3 as a postsynaptic scaffold is adequately established, other potential mechanisms through which Shank3 broadly modulates the postsynaptic proteome remain relatively unexplored. In our previous quantitative proteomic analysis, six up-regulated ribosomal proteins were identified in the striatal synaptosome of Shank3-overexpressing transgenic (TG) mice. In the present study, we validated the increased levels of RPLP1 and RPL36A in synaptosome, but not in whole lysate, of the TG striatum. Moreover, protein synthesis and extracellular signaling-regulated kinase (ERK) activity were enhanced in the TG striatal synaptosome. To understand the potential contribution of increased protein synthesis to the proteomic change in the TG striatal synaptosome, we performed RNA-sequencing analyses on both whole synaptosomal and synaptic polysome-enriched fractions. Comparative analyses showed a positive correlation only between the polysome-associated transcriptome and up-regulated proteome in the TG striatal synaptosome. Our findings suggest a novel mechanism through which Shank3 may remodel the postsynaptic proteome by regulating synaptic protein synthesis, whose dysfunction can be implicated in SHANK3-associated synaptopathies.
Asunto(s)
Cuerpo Estriado/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Biosíntesis de Proteínas , Proteínas Ribosómicas/metabolismo , Sinaptosomas/metabolismo , Animales , Sistema de Señalización de MAP Quinasas , Ratones Transgénicos , Receptores de Dopamina D1/metabolismoRESUMEN
Nonsense-mediated mRNA decay (NMD) typifies an mRNA surveillance pathway. Because NMD necessitates a translation event to recognize a premature termination codon on mRNAs, truncated misfolded polypeptides (NMD-polypeptides) could potentially be generated from NMD substrates as byproducts. Here, we show that when the ubiquitin-proteasome system is overwhelmed, various misfolded polypeptides including NMD-polypeptides accumulate in the aggresome: a perinuclear nonmembranous compartment eventually cleared by autophagy. Hyperphosphorylation of the key NMD factor UPF1 is required for selective targeting of the misfolded polypeptide aggregates toward the aggresome via the CTIF-eEF1A1-DCTN1 complex: the aggresome-targeting cellular machinery. Visualization at a single-particle level reveals that UPF1 increases the frequency and fidelity of movement of CTIF aggregates toward the aggresome. Furthermore, the apoptosis induced by proteotoxic stresses is suppressed by UPF1 hyperphosphorylation. Altogether, our data provide evidence that UPF1 functions in the regulation of a protein surveillance as well as an mRNA quality control.
Asunto(s)
Degradación de ARNm Mediada por Codón sin Sentido , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Helicasas/metabolismo , ARN Mensajero/metabolismo , Transactivadores/metabolismo , Respuesta de Proteína Desplegada/genética , Autofagia , Codón sin Sentido , Complejo Dinactina/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Células HEK293 , Células HeLa , Humanos , Imagen Molecular , Factor 1 de Elongación Peptídica/metabolismo , Fosforilación , Agregado de Proteínas/genética , Imagen Individual de Molécula , Ubiquitina/metabolismoRESUMEN
Newly synthesized mRNAs are exported from the nucleus to cytoplasm with a 5'-cap structure bound by the nuclear cap-binding complex (CBC). During or after export, the CBC should be properly replaced by cytoplasmic cap-binding protein eIF4E for efficient protein synthesis. Nonetheless, little is known about how the replacement takes place. Here, we show that double-stranded RNA-binding protein staufen1 (STAU1) promotes efficient replacement by facilitating an association between the CBC-importin α complex and importin ß. Our transcriptome-wide analyses and artificial tethering experiments also reveal that the replacement occurs more efficiently when an mRNA associates with STAU1. This event is inhibited by a key nonsense-mediated mRNA decay factor, UPF1, which directly interacts with STAU1. Furthermore, we find that cellular apoptosis that is induced by ionizing radiation is accompanied by inhibition of the replacement via increased association between STAU1 and hyperphosphorylated UPF1. Altogether, our data highlight the functional importance of STAU1 and UPF1 in the course of the replacement of the CBC by eIF4E, adding a previously unappreciated layer of post-transcriptional gene regulation.
Asunto(s)
Proteínas del Citoesqueleto/genética , Factor 4E Eucariótico de Iniciación/genética , Biosíntesis de Proteínas/genética , ARN Helicasas/genética , Proteínas de Unión al ARN/genética , Transactivadores/genética , Regiones no Traducidas 5' , Núcleo Celular/genética , Citoplasma/genética , Citoplasma/metabolismo , Humanos , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Proteínas de Unión a Caperuzas de ARN/genética , Estabilidad del ARN/genética , ARN Mensajero/genéticaRESUMEN
We have systematically studied the physiological responses elicited by amino acids from the principal taste organ of the Drosophila head. Although the detection and coding of sugars and bitter compounds have been examined extensively in this organism, little attention has been paid to the physiology of amino acid taste. We find that one class of sensilla, the S sensilla, yield the strongest responses to amino acids, although these responses were much weaker than the most robust responses to sugar or bitter compounds. S sensilla are heterogeneous in their amino acid responses and amino acids differ in the responses they elicit from individual sensilla. Tryptophan elicited relatively strong responses from S sensilla and these responses were eliminated when bitter-sensing neurons were ablated. Although tryptophan yielded little if any response in a behavioral paradigm, phenylalanine elicited a relatively strong response in the same paradigm and had a different physiological profile, supporting the notion that different amino acids are differentially encoded by the repertoire of taste neurons.
Asunto(s)
Aminoácidos , Drosophila/fisiología , Sensilos/fisiología , Gusto/fisiología , AnimalesRESUMEN
Many neurodegenerative disorders feature the presence of misfolded polypeptide-containing intracellular inclusion bodies biochemically and morphologically analogous to cellular aggresomes. However, it is largely unknown how misfolded polypeptides form aggresomes and are eventually cleared by the aggresome-macroautophagy/autophagy pathway, so-called aggrephagy. Our recent study revealed that when the ubiquitin-proteasome system is impaired, the accumulated misfolded polypeptides are selectively recognized and transported to the aggresome by a CED complex. This complex is composed of CTIF, originally identified as a specific factor for nuclear cap-binding protein complex (a heterodimer of NCBP1/CBP80 and NCBP2/CBP20)-dependent translation (CT), and its associated factors EEF1A1 and DCTN1. Aggresomal targeting of a misfolded polypeptide via the CED complex is accompanied by CTIF release from the CT complex and thereby inhibits CT efficiency. Therefore, our study provides new mechanistic insights into the crosstalk between translational inhibition and aggresome formation under the influence of a misfolded polypeptide.
Asunto(s)
Autofagia , Cuerpos de Inclusión , Péptidos , Complejo de la Endopetidasa Proteasomal , Pliegue de ProteínaRESUMEN
Misfolded polypeptides are rapidly cleared from cells via the ubiquitin-proteasome system (UPS). However, when the UPS is impaired, misfolded polypeptides form small cytoplasmic aggregates, which are sequestered into an aggresome and ultimately degraded by aggrephagy. Despite the relevance of the aggresome to neurodegenerative proteinopathies, the molecular mechanisms underlying aggresome formation remain unclear. Here we show that the CTIF-eEF1A1-DCTN1 (CED) complex functions in the surveillance of either pre-existing or newly synthesized polypeptides by linking two molecular events: selective recognition and aggresomal targeting of misfolded polypeptides. These events are accompanied by CTIF sequestration into the aggresome, preventing the additional synthesis of misfolded polypeptides from mRNAs bound by nuclear cap-binding complex. These events render cells more resistant to apoptosis induced by proteotoxic stresses. Collectively, our data provide compelling evidence for a previously unappreciated protein surveillance pathway and a regulatory gene expression network for coping with misfolded polypeptides.
Asunto(s)
Apoptosis , Cuerpos de Inclusión/química , Factor 1 de Elongación Peptídica/química , Péptidos/química , Complejo de la Endopetidasa Proteasomal/química , Pliegue de Proteína , Autofagia , Transporte Biológico , Citoplasma/química , Regulación hacia Abajo , Células HEK293 , Células HeLa , Humanos , Unión Proteica , Desnaturalización Proteica , Transporte de Proteínas , Ubiquitina/químicaRESUMEN
Glucocorticoid (GC) receptor (GR) has been shown recently to bind a subset of mRNAs and elicit rapid mRNA degradation. However, the molecular details of GR-mediated mRNA decay (GMD) remain unclear. Here, we demonstrate that GMD triggers rapid degradation of target mRNAs in a translation-independent and exon junction complex-independent manner, confirming that GMD is mechanistically distinct from nonsense-mediated mRNA decay (NMD). Efficient GMD requires PNRC2 (proline-rich nuclear receptor coregulatory protein 2) binding, helicase ability, and ATM-mediated phosphorylation of UPF1 (upstream frameshift 1). We also identify two GMD-specific factors: an RNA-binding protein, YBX1 (Y-box-binding protein 1), and an endoribonuclease, HRSP12 (heat-responsive protein 12). In particular, using HRSP12 variants, which are known to disrupt trimerization of HRSP12, we show that HRSP12 plays an essential role in the formation of a functionally active GMD complex. Moreover, we determine the hierarchical recruitment of GMD factors to target mRNAs. Finally, our genome-wide analysis shows that GMD targets a variety of transcripts, implicating roles in a wide range of cellular processes, including immune responses.
Asunto(s)
Monocitos/metabolismo , Estabilidad del ARN/fisiología , Receptores de Glucocorticoides/metabolismo , Adenosina Trifosfatasas/metabolismo , Quimiocina CCL2/metabolismo , Quimiotaxis/genética , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/metabolismo , Humanos , Monocitos/enzimología , Monocitos/inmunología , Fosforilación , Polimerizacion , ARN Helicasas , Estabilidad del ARN/genética , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Ribonucleasas/metabolismo , Transactivadores/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismoRESUMEN
Glucocorticoid receptor (GR), which was originally known to function as a nuclear receptor, plays a role in rapid mRNA degradation by acting as an RNA-binding protein. The mechanism by which this process occurs remains unknown. Here, we demonstrate that GR, preloaded onto the 5'UTR of a target mRNA, recruits UPF1 through proline-rich nuclear receptor coregulatory protein 2 (PNRC2) in a ligand-dependent manner, so as to elicit rapid mRNA degradation. We call this process GR-mediated mRNA decay (GMD). Although GMD, nonsense-mediated mRNA decay (NMD), and staufen-mediated mRNA decay (SMD) share upstream frameshift 1 (UPF1) and PNRC2, we find that GMD is mechanistically distinct from NMD and SMD. We also identify de novo cellular GMD substrates using microarray analysis. Intriguingly, GMD functions in the chemotaxis of human monocytes by targeting chemokine (C-C motif) ligand 2 (CCL2) mRNA. Thus, our data provide molecular evidence of a posttranscriptional role of the well-studied nuclear hormone receptor, GR, which is traditionally considered a transcription factor.
Asunto(s)
Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Glucocorticoides/metabolismo , Transactivadores/metabolismo , Quimiocina CCL2/metabolismo , Quimiotaxis , Genes Reporteros , Células HEK293 , Células HeLa , Humanos , Ligandos , Monocitos/metabolismo , Degradación de ARNm Mediada por Codón sin Sentido , Unión Proteica , ARN Helicasas , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Many insect vectors of disease detect their hosts through olfactory cues, and thus it is of great interest to understand better how odors are encoded. However, little is known about the molecular underpinnings that support the unique function of coeloconic sensilla, an ancient and conserved class of sensilla that detect amines and acids, including components of human odor that are cues for many insect vectors. Here, we generate antennal transcriptome databases both for wild type Drosophila and for a mutant that lacks coeloconic sensilla. We use these resources to identify genes whose expression is highly enriched in coeloconic sensilla, including many genes not previously implicated in olfaction. Among them, we identify an ammonium transporter gene that is essential for ammonia responses in a class of coeloconic olfactory receptor neurons (ORNs), but is not required for responses to other odorants. Surprisingly, the transporter is not expressed in ORNs, but rather in neighboring auxiliary cells. Thus, our data reveal an unexpected non-cell autonomous role for a component that is essential to the olfactory response to ammonia. The defective response observed in a Drosophila mutant of this gene is rescued by its Anopheles ortholog, and orthologs are found in virtually all insect species examined, suggesting that its role is conserved. Taken together, our results provide a quantitative analysis of gene expression in the primary olfactory organ of Drosophila, identify molecular components of an ancient class of olfactory sensilla, and reveal that auxiliary cells, and not simply ORNs, play an essential role in the coding of an odor that is a critical host cue for many insect vectors of human disease.
Asunto(s)
Amoníaco/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Drosophila/genética , Neuronas Receptoras Olfatorias/metabolismo , Olfato/genética , Transcriptoma/genética , Animales , Drosophila/genética , Drosophila/fisiología , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Insectos Vectores/genética , Mutación , Odorantes/análisis , Sensilos/metabolismo , Olfato/fisiologíaRESUMEN
It has long been considered that intron-containing (spliced) mRNAs are translationally more active than intronless mRNAs (identical mRNA not produced by splicing). The splicing-dependent translational enhancement is mediated, in part, by the exon junction complex (EJC). Nonetheless, the molecular mechanism by which each EJC component contributes to the translational enhancement remains unclear. Here, we demonstrate the previously unappreciated role of eukaryotic translation initiation factor 4AIII (eIF4AIII), a component of EJC, in the translation of mRNAs bound by the nuclear cap-binding complex (CBC), a heterodimer of cap-binding protein 80 (CBP80) and CBP20. eIF4AIII is recruited to the 5'-end of mRNAs bound by the CBC by direct interaction with the CBC-dependent translation initiation factor (CTIF); this recruitment of eIF4AIII is independent of the presence of introns (deposited EJCs after splicing). Polysome fractionation, tethering experiments, and in vitro reconstitution experiments using recombinant proteins show that eIF4AIII promotes efficient unwinding of secondary structures in 5'UTR, and consequently enhances CBC-dependent translation in vivo and in vitro. Therefore, our data provide evidence that eIF4AIII is a specific translation initiation factor for CBC-dependent translation.
Asunto(s)
Regiones no Traducidas 5'/genética , Núcleo Celular/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Caperuzas de ARN/metabolismo , Replicación del ADN , Regulación hacia Abajo , Factores Eucarióticos de Iniciación/metabolismo , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Intrones/genética , Modelos Biológicos , Unión Proteica , Empalme del ARN/genética , Estabilidad del ARN/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
The organization of neurons and the maintenance of that arrangement are critical to brain function. Failure of these processes in humans can lead to severe birth defects, mental retardation, and epilepsy. Several kinesins have been shown to play important roles in cell migration in vertebrate systems, but few upstream and downstream pathway members have been identified. Here, we utilize the genetic model organism Caenorhabditis elegans to elucidate the pathway by which the C. elegans Kinesin-1 Heavy Chain (KHC)/KIF5 ortholog UNC-116 functions to maintain neuronal cell body position in the PHB sensory neurons. We find that UNC-116/KHC acts in part with the cell and axon migration molecules UNC-6/Netrin and UNC-40/DCC in this process, but in parallel to SAX-3/Robo. We have also identified several potential adaptor, cargo, and regulatory proteins that may provide insight into the mechanism of UNC-116/KHC's function in this process. These include the cargo receptor UNC-33/CRMP2, the cargo adaptor protein UNC-76/FEZ and its regulator UNC-51/ULK, the cargo molecule UNC-69/SCOCO, and the actin regulators UNC-44/Ankyrin and UNC-34/Enabled. These genes also act in cell migration and axon outgrowth; however, many proteins that function in these processes do not affect PHB position. Our findings suggest an active posterior cell migration mediated by UNC-116/KHC occurs throughout development to maintain proper PHB cell body position and define a new pathway that mediates maintenance of neuronal cell body position.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Moléculas de Adhesión Celular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cinesinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/metabolismo , Actinas/metabolismo , Animales , Factores de Crecimiento de Fibroblastos/metabolismo , Ganglios de Invertebrados/citología , Ganglios de Invertebrados/metabolismo , Proteínas Motoras Moleculares/metabolismo , Mutación/genética , Netrinas , Prohibitinas , Receptores Inmunológicos/metabolismo , Vía de Señalización Wnt , Proteínas RoundaboutRESUMEN
BACKGROUND: An essential stage of neural development involves the assembly of neural circuits via formation of inter-neuronal connections. Early steps in neural circuit formation, including cell migration, axon guidance, and the localization of synaptic components, are well described. However, upon reaching their target region, most neurites still contact many potential partners. In order to assemble functional circuits, it is critical that within this group of cells, neurons identify and form connections only with their appropriate partners, a process we call synaptic partner recognition (SPR). To understand how SPR is mediated, we previously developed a genetically encoded fluorescent trans-synaptic marker called NLG-1 GRASP, which labels synaptic contacts between individual neurons of interest in dense cellular environments in the genetic model organism Caenorhabditis elegans. RESULTS: Here, we describe the first use of NLG-1 GRASP technology, to identify SPR genes that function in this critical process. The NLG-1 GRASP system allows us to assess synaptogenesis between PHB sensory neurons and AVA interneurons instantly in live animals, making genetic analysis feasible. Additionally, we employ a behavioral assay to specifically test PHB sensory circuit function. Utilizing this approach, we reveal a new role for the secreted UNC-6/Netrin ligand and its transmembrane receptor UNC-40/Deleted in colorectal cancer (DCC) in SPR. Synapses between PHB and AVA are severely reduced in unc-6 and unc-40 animals despite normal axon guidance and subcellular localization of synaptic components. Additionally, behavioral defects indicate a complete disruption of PHB circuit function in unc-40 mutants. Our data indicate that UNC-40 and UNC-6 function in PHB and AVA, respectively, to specify SPR. Strikingly, overexpression of UNC-6 in postsynaptic neurons is sufficient to promote increased PHB-AVA synaptogenesis and to potentiate the behavioral response beyond wild-type levels. Furthermore, an artificially membrane-tethered UNC-6 expressed in the postsynaptic neurons promotes SPR, consistent with a short-range signal between adjacent synaptic partners. CONCLUSIONS: These results indicate that the conserved UNC-6/Netrin-UNC-40/DCC ligand-receptor pair has a previously unknown function, acting in a juxtacrine manner to specify recognition of individual postsynaptic neurons. Furthermore, they illustrate the potential of this new approach, combining NLG-1 GRASP and behavioral analysis, in gene discovery and characterization.