RESUMEN
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
The nonsense-mediated mRNA decay (NMD) pathway is well known as a translation-coupled quality control system that recognizes and degrades aberrant mRNAs with truncated open reading frames (ORF) due to the presence of a premature termination codon (PTC). However, a more general role of NMD in posttranscriptional regulation of gene expression is indicated by transcriptome-wide mRNA profilings that identified a plethora of physiological mRNAs as NMD targets. In this review, we focus on mechanistic aspects of target mRNA identification and degradation in mammalian cells, based on the available biochemical and genetic data, and point out knowledge gaps. Translation termination in a messenger ribonucleoprotein particle (mRNP) environment lacking necessary factors for proper translation termination emerges as a key determinant for subjecting an mRNA to NMD, and we therefore review recent structural and mechanistic insight into translation termination. In addition, the central role of UPF1, its crucial phosphorylation/dephosphorylation cycle and dynamic interactions with other NMD factors are discussed. Moreover, we address the role of exon junction complexes (EJCs) in NMD and summarize the functions of SMG5, SMG6 and SMG7 in promoting mRNA decay through different routes. This article is part of a Special Issue entitled: RNA Decay mechanisms.
Asunto(s)
Degradación de ARNm Mediada por Codón sin Sentido/genética , Estabilidad del ARN/genética , Ribonucleoproteínas/genética , Transactivadores/genética , Regiones no Traducidas 3' , Animales , Mamíferos , Terminación de la Cadena Péptídica Traduccional , Fosforilación , Biosíntesis de Proteínas , ARN HelicasasRESUMEN
Nonsense-mediated mRNA decay (NMD), which is best known for degrading mRNAs with premature termination codons (PTCs), is thought to be triggered by aberrant translation termination at stop codons located in an environment of the mRNP that is devoid of signals necessary for proper termination. In mammals, the cytoplasmic poly(A)-binding protein 1 (PABPC1) has been reported to promote correct termination and therewith antagonize NMD by interacting with the eukaryotic release factors 1 (eRF1) and 3 (eRF3). Using tethering assays in which proteins of interest are recruited as MS2 fusions to a NMD reporter transcript, we show that the three N-terminal RNA recognition motifs (RRMs) of PABPC1 are sufficient to antagonize NMD, while the eRF3-interacting C-terminal domain is dispensable. The RRM1-3 portion of PABPC1 interacts with eukaryotic initiation factor 4G (eIF4G) and tethering of eIF4G to the NMD reporter also suppresses NMD. We identified the interactions of the eIF4G N-terminus with PABPC1 and the eIF4G core domain with eIF3 as two genetically separable features that independently enable tethered eIF4G to inhibit NMD. Collectively, our results reveal a function of PABPC1, eIF4G and eIF3 in translation termination and NMD suppression, and they provide additional evidence for a tight coupling between translation termination and initiation.
Asunto(s)
Factor 4G Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Degradación de ARNm Mediada por Codón sin Sentido , Codón sin Sentido/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Factor 4G Eucariótico de Iniciación/química , Humanos , Proteína I de Unión a Poli(A)/química , Proteína I de Unión a Poli(A)/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína , Proteínas Proto-Oncogénicas c-ets/química , Proteínas Proto-Oncogénicas c-ets/metabolismo , Ribonucleósido Difosfato Reductasa/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Eukaryotic mRNAs with premature translation termination codons (PTCs) are recognized and degraded through a process termed nonsense-mediated mRNA decay (NMD). The evolutionary conservation of the core NMD factors UPF1, UPF2 and UPF3 implies a similar basic mechanism of PTC recognition in all eukaryotes. However, while PTC-containing mRNAs in yeast seem to be available to NMD at each round of translation, mammalian NMD has been reported to be restricted to cap-binding complex (CBC)-bound mRNAs during the pioneer round of translation. Here, we compared decay kinetics of two NMD reporter genes in mRNA fractions bound to either CBC or the eukaryotic initiation factor 4E (eIF4E) in human cells and demonstrate that NMD destabilizes eIF4E-bound transcripts as efficiently as those associated with CBC. These results corroborate an emerging unified model for NMD substrate recognition, according to which NMD can ensue at every aberrant translation termination event.