RESUMO
eIF5-mimic protein (5MP) is a translational regulatory protein that binds the small ribosomal subunit and modulates its activity. 5MP is proposed to reprogram non-AUG translation rates for oncogenes in cancer, but its role in controlling non-AUG initiated synthesis of deleterious repeat-peptide products, such as FMRpolyG observed in fragile-X-associated tremor ataxia syndrome (FXTAS), is unknown. Here, we show that 5MP can suppress both general and repeat-associated non-AUG (RAN) translation by a common mechanism in a manner dependent on its interaction with eIF3. Essentially, 5MP displaces eIF5 through the eIF3c subunit within the preinitiation complex (PIC), thereby increasing the accuracy of initiation. In Drosophila, 5MP/Kra represses neuronal toxicity and enhances the lifespan in an FXTAS disease model. These results implicate 5MP in protecting cells from unwanted byproducts of non-AUG translation in neurodegeneration.
Assuntos
Códon de Iniciação/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Iniciação 3 em Eucariotos/metabolismo , Biossíntese de Proteínas/genética , Expansão das Repetições de Trinucleotídeos/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antígenos de Diferenciação/metabolismo , Proteínas de Ligação a DNA/química , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 3 em Eucariotos/química , Células HEK293 , Humanos , Masculino , Modelos Biológicos , Modelos Moleculares , Mutação/genética , Iniciação Traducional da Cadeia Peptídica , Ligação Proteica , Domínios Proteicos , Receptores Imunológicos/metabolismoRESUMO
In the human genome, translation initiation from non-AUG codons plays an important role in various gene regulation programs. However, mechanisms regulating the non-AUG initiation rate remain poorly understood. Here, we show that the non-AUG initiation rate is nearly consistent under a fixed nucleotide context in various human and insect cells. Yet, it ranges from <1% to nearly 100% compared to AUG translation, depending on surrounding sequences, including Kozak, and possibly additional nucleotide contexts. Mechanistically, this range of non-AUG initiation is controlled in part, by the eIF5-mimic protein (5MP). 5MP represses non-AUG translation by competing with eIF5 for the Met-tRNAi-binding factor eIF2. Consistently, eIF5 increases, whereas 5MP decreases translation of NAT1/EIF4G2/DAP5, whose sole start codon is GUG. By modulating eIF5 and 5MP1 expression in combination with ribosome profiling we identified a handful of previously unknown non-AUG initiation sites, some of which serve as the exclusive start codons. If the initiation rate for these codons is low, then an AUG-initiated downstream ORF prevents the generation of shorter, AUG-initiated isoforms. We propose that the homeostasis of the non-AUG translatome is maintained through balanced expression of eIF5 and 5MP.
Assuntos
Códon de Iniciação/genética , Proteínas de Ligação a DNA/genética , Fator de Iniciação 5 em Eucariotos/genética , Genoma Humano , Animais , Ligação Competitiva , Linhagem Celular , Linhagem Celular Tumoral , Códon de Iniciação/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 5 em Eucariotos/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Homeostase/genética , Humanos , Ligação Proteica , Biossíntese de Proteínas/genética , Ribossomos/genética , Ribossomos/metabolismoRESUMO
During eukaryotic translation initiation, eIF3 binds the solvent-accessible side of the 40S ribosome and recruits the gate-keeper protein eIF1 and eIF5 to the decoding center. This is largely mediated by the N-terminal domain (NTD) of eIF3c, which can be divided into three parts: 3c0, 3c1, and 3c2. The N-terminal part, 3c0, binds eIF5 strongly but only weakly to the ribosome-binding surface of eIF1, whereas 3c1 and 3c2 form a stoichiometric complex with eIF1. 3c1 contacts eIF1 through Arg-53 and Leu-96, while 3c2 faces 40S protein uS15/S13, to anchor eIF1 to the scanning pre-initiation complex (PIC). We propose that the 3c0:eIF1 interaction diminishes eIF1 binding to the 40S, whereas 3c0:eIF5 interaction stabilizes the scanning PIC by precluding this inhibitory interaction. Upon start codon recognition, interactions involving eIF5, and ultimately 3c0:eIF1 association, facilitate eIF1 release. Our results reveal intricate molecular interactions within the PIC, programmed for rapid scanning-arrest at the start codon.