RESUMEN
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
In the cytoplasm of mammalian cells, either cap-binding proteins 80 and 20 (CBP80/20) or eukaryotic translation initiation factor (eIF) 4E can direct the initiation of translation. Although the recruitment of ribosomes to mRNAs during eIF4E-dependent translation (ET) is well characterized, the molecular mechanism for CBP80/20-dependent translation (CT) remains obscure. Here, we show that CBP80/20-dependent translation initiation factor (CTIF), which has been shown to be preferentially involved in CT but not ET, specifically interacts with eIF3g, a component of the eIF3 complex involved in ribosome recruitment. By interacting with eIF3g, CTIF serves as an adaptor protein to bridge the CBP80/20 and the eIF3 complex, leading to efficient ribosome recruitment during CT. Accordingly, down-regulation of CTIF using a small interfering RNA causes a redistribution of CBP80 from polysome fractions to subpolysome fractions, without significant consequence to eIF4E distribution. In addition, down-regulation of eIF3g inhibits the efficiency of nonsense-mediated mRNA decay, which is tightly coupled to CT but not to ET. Moreover, the artificial tethering of CTIF to an intercistronic region of dicistronic mRNA results in translation of the downstream cistron in an eIF3-dependent manner. These findings support the idea that CT mechanistically differs from ET.
Asunto(s)
Factor 3 de Iniciación Eucariótica/metabolismo , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/genética , Secuencia de Bases , Far-Western Blotting , Línea Celular , Cartilla de ADN , Humanos , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Técnicas del Sistema de Dos HíbridosRESUMEN
Human transforming growth factor-beta receptor type 2 (TGFbetaR2) mRNA harboring a premature translation termination codon (PTC) generated by frameshift mutation is targeted for nonsense-mediated translational repression (NMTR), rather than nonsense-mediated mRNA decay (NMD). Here we show that exon junction complex (EJC) downstream of a PTC plays an inhibitory role in translation of TGFbetaR2 mRNA. Translational repression by core EJC components occurs after formation of 80S ribosome complex, which is demonstrated using different types of internal ribosome entry sites (IRESes). Our findings implicate EJCs or core EJC components as negative regulators of translation.
Asunto(s)
Codón sin Sentido/genética , Exones/genética , Biosíntesis de Proteínas/genética , Proteínas Serina-Treonina Quinasas/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Empalme Alternativo , Western Blotting , Factor 4A Eucariótico de Iniciación/genética , Factor 4A Eucariótico de Iniciación/metabolismo , Células HeLa , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Helicasas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribosomas/genética , Ribosomas/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , TransfecciónRESUMEN
Nonsense-mediated mRNA decay (NMD) is one of the mRNA surveillance mechanisms, which eliminates aberrant mRNAs harboring premature termination codons. NMD targets only mRNAs bound by the nuclear cap-binding protein complex CBP80/20 which directs the pioneer round of translation. Here we demonstrate that NMD occurs efficiently during prolonged hypoxia in which steady-state translation is drastically inhibited. Accordingly, CBP80 remains in the nucleus, and processing bodies are unaffected with regard to their abundance and number under prolonged hypoxic conditions. These results indicate that mRNAs enter the pioneer round of translation during prolonged hypoxia.
Asunto(s)
Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Biosíntesis de Proteínas/genética , Estabilidad del ARN , ARN Mensajero/metabolismo , Hipoxia de la Célula/genética , Núcleo Celular/química , Núcleo Celular/metabolismo , Células HeLa , Humanos , Complejo Proteico Nuclear de Unión a la Caperuza/análisisRESUMEN
The pioneer round of translation plays a role in translation initiation of newly spliced and exon junction complex (EJC)-bound mRNAs. Nuclear cap-binding protein complex CBP80/20 binds to those mRNAs at the 5'-end, recruiting translation initiation complex. As a consequence of the pioneer round of translation, the bound EJCs are dissociated from mRNAs and CBP80/20 is replaced by the cytoplasmic cap-binding protein eIF4E. Steady-state translation directed by eIF4E allows for an immediate and rapid response to changes in physiological conditions. Here, we show that nonsense-mediated mRNA decay (NMD), which restricts only to the pioneer round of translation but not to steady-state translation, efficiently occurs even during serum starvation, in which steady-state translation is drastically abolished. Accordingly, CBP80 remains in the nucleus and processing bodies are unaffected in their abundance and number in serum-starved conditions. These results suggest that mRNAs enter the pioneer round of translation during serum starvation and are targeted for NMD if they contain premature termination codons.
