RESUMO
Translation initiation of most mRNAs involves m7G-cap binding, ribosomal scanning, and AUG selection. Initiation from an m7G-cap-proximal AUG can be bypassed resulting in leaky scanning, except for mRNAs bearing the translation initiator of short 5' untranslated region (TISU) element. m7G-cap binding is mediated by the eukaryotic initiation factor 4E (eIF4E)-eIF4G1 complex. eIF4G1 also associates with eIF1, and both promote scanning and AUG selection. Understanding of the dynamics and significance of these interactions is lacking. We report that eIF4G1 exists in two complexes, either with eIF4E or with eIF1. Using an eIF1 mutant impaired in eIF4G1 binding, we demonstrate that eIF1-eIF4G1 interaction is important for leaky scanning and for avoiding m7G-cap-proximal initiation. Intriguingly, eIF4E-eIF4G1 antagonizes the scanning promoted by eIF1-eIF4G1 and is required for TISU. In mapping the eIF1-binding site on eIF4G1, we unexpectedly found that eIF4E also binds it indirectly. These findings uncover the RNA features underlying regulation by eIF4E-eIF4G1 and eIF1-eIF4G1 and suggest that 43S ribosome transition from the m7G-cap to scanning involves relocation of eIF4G1 from eIF4E to eIF1.
Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Sítios de Ligação , Fator de Iniciação 1 em Eucariotos/química , Fator de Iniciação 1 em Eucariotos/genética , Fator de Iniciação 1 em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/química , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação Eucariótico 4G/química , Fator de Iniciação Eucariótico 4G/genética , Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/genética , Células HEK293 , Humanos , Modelos Biológicos , Modelos Moleculares , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Iniciação Traducional da Cadeia Peptídica , Domínios e Motivos de Interação entre Proteínas , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Eukaryotic cells evolved highly complex and accurate protein synthesis machinery that is finely tuned by various signaling pathways. Dysregulation of translation is a hallmark of many diseases, including cancer, and thus pharmacological approaches to modulate translation become very promising. While there has been much progress in our understanding of mammalian mRNA-specific translation control, surprisingly, relatively little is known about whether and how the protein components of the translation machinery shape translation of their own mRNAs. Here we analyze mammalian mRNAs encoding components of the translation initiation machinery for potential regulatory features such as 5'TOP motifs, TISU motifs, poor start codon nucleotide context and upstream open reading frames.
Assuntos
Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica , RNA Mensageiro/metabolismo , Regiões 5' não Traduzidas , Animais , Humanos , Mamíferos , Biossíntese de Proteínas , Sequência de Oligopirimidina na Região 5' Terminal do RNARESUMO
Canonical translation initiation involves ribosomal scanning, but short 5' untranslated region (5'UTR) mRNAs are translated in a scanning-independent manner. The extent and mechanism of scanning-independent translation are not fully understood. Here we report that short 5'UTR mRNAs constitute a substantial fraction of the translatome. Short 5'UTR mRNAs are enriched with TISU (translation initiator of short 5'UTR), a 12-nucleotide element directing efficient scanning-independent translation. Comprehensive mutagenesis revealed that each AUG codon-flanking nucleotide of TISU contributes to translational strength, but only a few are important for accuracy. Using site-specific UV cross-linking of ribosomal complexes assembled on TISU mRNA, we demonstrate specific binding of TISU to ribosomal proteins at the E and A sites. We identified RPS3 as the major TISU binding protein in the 48S complex A site. Upon 80S complex formation, RPS3 interaction is weakened and switched to RPS10e (formerly called RPS10). We further demonstrate that TISU is particularly dependent on eukaryotic initiation factor 1A (eIF1A) which interacts with both RPS3 and RPS10e. Our findings suggest that the cap-recruited ribosome specifically binds the TISU nucleotides at the A and E sites in cooperation with eIF1A to promote scanning arrest.
Assuntos
Regiões 5' não Traduzidas , Fator de Iniciação 1 em Eucariotos/metabolismo , Biossíntese de Proteínas , Proteínas Ribossômicas/metabolismo , Animais , Linhagem Celular , Células HEK293 , Células HeLa , Humanos , Camundongos , Ligação Proteica , Mapas de Interação de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Diurnal oscillations of gene expression are a hallmark of rhythmic physiology across most living organisms. Such oscillations are controlled by the interplay between the circadian clock and feeding rhythms. Although rhythmic mRNA accumulation has been extensively studied, comparatively less is known about their transcription and translation. Here, we quantified simultaneously temporal transcription, accumulation, and translation of mouse liver mRNAs under physiological light-dark conditions and ad libitum or night-restricted feeding in WT and brain and muscle Arnt-like 1 (Bmal1)-deficient animals. We found that rhythmic transcription predominantly drives rhythmic mRNA accumulation and translation for a majority of genes. Comparison of wild-type and Bmal1 KO mice shows that circadian clock and feeding rhythms have broad impact on rhythmic gene expression, Bmal1 deletion affecting surprisingly both transcriptional and posttranscriptional levels. Translation efficiency is differentially regulated during the diurnal cycle for genes with 5'-Terminal Oligo Pyrimidine tract (5'-TOP) sequences and for genes involved in mitochondrial activity, many harboring a Translation Initiator of Short 5'-UTR (TISU) motif. The increased translation efficiency of 5'-TOP and TISU genes is mainly driven by feeding rhythms but Bmal1 deletion also affects amplitude and phase of translation, including TISU genes. Together this study emphasizes the complex interconnections between circadian and feeding rhythms at several steps ultimately determining rhythmic gene expression and translation.
Assuntos
Ritmo Circadiano/genética , Comportamento Alimentar , Biossíntese de Proteínas , Transcrição Gênica , Regiões 5' não Traduzidas/genética , Fatores de Transcrição ARNTL/metabolismo , Adenilato Quinase/metabolismo , Animais , Deleção de Genes , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos Knockout , Modelos Genéticos , Complexos Multiproteicos , Motivos de Nucleotídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Ribossomos/metabolismo , Serina-Treonina Quinases TORRESUMO
Eukaryotic translation initiation is an intricate and multi-step process that includes 43S Pre-Initiation Complex (PIC) assembly, attachment of the PIC to the mRNA, scanning, start codon selection and 60S subunit joining. Translation initiation of most mRNAs involves recognition of a 5'end m7G cap and ribosomal scanning in which the 5' UTR is checked for complementarity with the AUG. There is however an increasing number of mRNAs directing translation initiation that deviate from the predominant mechanism. In this review we summarize the canonical translation initiation process and describe non-canonical mechanisms that are cap-dependent but operate without scanning. In particular we focus on several examples of translation initiation driven either by mRNAs with extremely short 5' leaders or by highly complex 5' UTRs that promote ribosome shunting.