RESUMEN
Eukaryotic initiation factor 4G (eIF4G) plays a central role in translation initiation through its interactions with the cap-binding protein eIF4E. This interaction is a major drug target for repressing translation and is naturally regulated by 4E-binding proteins (4E-BPs). 4E-BPs and eIF4G compete for binding to the eIF4E dorsal surface via a shared canonical 4E-binding motif, but also contain auxiliary eIF4E-binding sequences, which were assumed to contact non-overlapping eIF4E surfaces. However, it is unknown how metazoan eIF4G auxiliary sequences bind eIF4E. Here, we describe crystal structures of human and Drosophila melanogaster eIF4E-eIF4G complexes, which unexpectedly reveal that the eIF4G auxiliary sequences bind to the lateral surface of eIF4E, using a similar mode to that of 4E-BPs. Our studies provide a molecular model of the eIF4E-eIF4G complex, shed light on the competition mechanism of 4E-BPs, and enable the rational design of selective eIF4G inhibitors to dampen dysregulated translation in disease.
Asunto(s)
Drosophila melanogaster/metabolismo , Factor 4E Eucariótico de Iniciación/química , Factor 4G Eucariótico de Iniciación/química , Iniciación de la Cadena Peptídica Traduccional , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Drosophila melanogaster/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/genética , Factor 4G Eucariótico de Iniciación/metabolismo , Expresión Génica , Humanos , Cinética , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TermodinámicaRESUMEN
The eIF4E-binding proteins (4E-BPs) are a diverse class of translation regulators that share a canonical eIF4E-binding motif (4E-BM) with eIF4G. Consequently, they compete with eIF4G for binding to eIF4E, thereby inhibiting translation initiation. Mextli (Mxt) is an unusual 4E-BP that promotes translation by also interacting with eIF3. Here we present the crystal structures of the eIF4E-binding regions of the Drosophila melanogaster (Dm) and Caenorhabditis elegans (Ce) Mxt proteins in complex with eIF4E in the cap-bound and cap-free states. The structures reveal unexpected evolutionary plasticity in the eIF4E-binding mode, with a classical bipartite interface for Ce Mxt and a novel tripartite interface for Dm Mxt. Both interfaces comprise a canonical helix and a noncanonical helix that engage the dorsal and lateral surfaces of eIF4E, respectively. Remarkably, Dm Mxt contains a C-terminal auxiliary helix that lies anti-parallel to the canonical helix on the eIF4E dorsal surface. In contrast to the eIF4G and Ce Mxt complexes, the Dm eIF4E-Mxt complexes are resistant to competition by bipartite 4E-BPs, suggesting that Dm Mxt can bind eIF4E when eIF4G binding is inhibited. Our results uncovered unexpected diversity in the binding modes of 4E-BPs, resulting in eIF4E complexes that display differential sensitivity to 4E-BP regulation.
Asunto(s)
Proteínas de Caenorhabditis elegans/química , Proteínas de Drosophila/química , Regulación de la Expresión Génica/fisiología , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas/fisiología , Animales , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/química , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Evolución Molecular , Variación Genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Estructura Terciaria de Proteína , Reproducibilidad de los ResultadosRESUMEN
The interaction of the eukaryotic initiation factor 4G (eIF4G) with the cap-binding protein eIF4E initiates cap-dependent translation and is regulated by the 4E-binding proteins (4E-BPs), which compete with eIF4G to repress translation. Metazoan eIF4G and 4E-BPs interact with eIF4E via canonical and non-canonical motifs that bind to the dorsal and lateral surface of eIF4E in a bipartite recognition mode. However, previous studies pointed to mechanistic differences in how fungi and metazoans regulate protein synthesis. We present crystal structures of the yeast eIF4E bound to two yeast 4E-BPs, p20 and Eap1p, as well as crystal structures of a fungal eIF4E-eIF4G complex. We demonstrate that the core principles of molecular recognition of eIF4E are in fact highly conserved among translational activators and repressors in eukaryotes. Finally, we reveal that highly specialized structural motifs do exist and serve to modulate the affinity of protein-protein interactions that regulate cap-dependent translation initiation in fungi.
Asunto(s)
Factor 4E Eucariótico de Iniciación/química , Factor 4G Eucariótico de Iniciación/química , Regulación Fúngica de la Expresión Génica , Iniciación de la Cadena Peptídica Traduccional , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Factores de Transcripción/química , Secuencias de Aminoácidos , Unión Competitiva , Chaetomium/genética , Secuencia Conservada , Cristalografía por Rayos X , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Evolución Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Análogos de Caperuza de ARN/metabolismo , Proteínas Recombinantes/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Dispersión del Ángulo Pequeño , Alineación de Secuencia , Especificidad de la Especie , Relación Estructura-Actividad , Factores de Transcripción/metabolismoRESUMEN
Half of mammalian transcripts contain short upstream open reading frames (uORFs) that potentially regulate translation of the downstream coding sequence (CDS). The molecular mechanisms governing these events remain poorly understood. Here, we find that the non-canonical initiation factor Death-associated protein 5 (DAP5 or eIF4G2) is required for translation initiation on select transcripts. Using ribosome profiling and luciferase-based reporters coupled with mutational analysis we show that DAP5-mediated translation occurs on messenger RNAs (mRNAs) with long, structure-prone 5' leader sequences and persistent uORF translation. These mRNAs preferentially code for signalling factors such as kinases and phosphatases. We also report that cap/eIF4F- and eIF4A-dependent recruitment of DAP5 to the mRNA facilitates main CDS, but not uORF, translation suggesting a role for DAP5 in translation re-initiation. Our study reveals important mechanistic insights into how a non-canonical translation initiation factor involved in stem cell fate shapes the synthesis of specific signalling factors.
Asunto(s)
Factores de Iniciación de Péptidos , Sistemas de Lectura Abierta/genética , ARN Mensajero/genéticaRESUMEN
Current models of mRNA turnover indicate that cytoplasmic degradation is coupled with translation. However, our understanding of the molecular events that coordinate ribosome transit with the mRNA decay machinery is still limited. Here, we show that 4EHP-GIGYF1/2 complexes trigger co-translational mRNA decay. Human cells lacking these proteins accumulate mRNAs with prominent ribosome pausing. They include, among others, transcripts encoding secretory and membrane-bound proteins or tubulin subunits. In addition, 4EHP-GIGYF1/2 complexes fail to reduce mRNA levels in the absence of ribosome stalling or upon disruption of their interaction with the cap structure, DDX6, and ZNF598. We further find that co-translational binding of GIGYF1/2 to the mRNA marks transcripts with perturbed elongation to decay. Our studies reveal how a repressor complex linked to neurological disorders minimizes the protein output of a subset of mRNAs.