RESUMO
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
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.
Assuntos
Iniciação Traducional da Cadeia Peptídica/fisiologia , Capuzes de RNA/metabolismo , Ribossomos/metabolismo , Animais , Humanos , Capuzes de RNA/genética , Ribossomos/genéticaRESUMO
Protein synthesis is a major energy-consuming process, which is rapidly repressed upon energy stress by AMPK. How energy deficiency affects translation of mRNAs that cope with the stress response is poorly understood. We found that mitochondrial genes remain translationally active upon energy deprivation. Surprisingly, inhibition of translation is partially retained in AMPKα1/AMPKα2 knockout cells. Mitochondrial mRNAs are enriched with TISU, a translation initiator of short 5' UTR, which confers resistance specifically to energy stress. Purified 48S preinitiation complex is sufficient for initiation via TISU AUG, when preceded by a short 5' UTR. eIF1 stimulates TISU but inhibits non-TISU-directed initiation. Remarkably, eIF4GI shares this activity and also interacts with eIF1. Furthermore, eIF4F is released upon 48S formation on TISU. These findings describe a specialized translation tolerance mechanism enabling continuous translation of TISU genes under energy stress and reveal that a key step in start codon selection of short 5' UTR is eIF4F release.
Assuntos
Códon de Iniciação/genética , Fator de Iniciação Eucariótico 4G/genética , Fatores de Iniciação em Eucariotos/genética , Genes Mitocondriais/genética , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Biossíntese de Proteínas/genética , Estresse Fisiológico/genética , Proteínas Quinases Ativadas por AMP/genética , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , RNA Mensageiro/genética , RNA Mitocondrial , Ribossomos/genéticaRESUMO
Translation Initiator of Short 5' UTR (TISU) is a unique regulatory element of both transcription and translation initiation. It is present in a sizable number of genes with basic cellular functions and a very short untranslated region (5' UTR). Here, we investigated translation initiation from short 5' UTR mRNAs with AUG in various contexts. Reducing 5' UTR length to the minimal functional size increases leaky scanning from weak and strong initiators but hardly affects translation initiation and ribosomal binding directed by TISU. Ribosome interaction with TISU mRNA is cap dependent and involves AUG downstream nucleotides that compensate for the absent 5' UTR contacts. Interestingly, eIF1 inhibits cap-proximal AUG selection within weak or strong contexts but not within TISU. Furthermore, TISU-directed translation is unaffected by inhibition of the RNA helicase eIF4A. Thus, TISU directs efficient cap-dependent translation initiation without scanning, a mechanism that would be advantageous when intracellular levels of eIF1 and eIF4A fluctuate.
Assuntos
Iniciação Traducional da Cadeia Peptídica , Sequências Reguladoras de Ácido Ribonucleico , Regiões 5' não Traduzidas , Códon de Iniciação , Fator de Iniciação 1 em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/antagonistas & inibidores , Fatores de Iniciação em Eucariotos/metabolismo , Células HEK293 , Células HeLa , Humanos , Capuzes de RNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Ribossomos/metabolismoRESUMO
BACKGROUND: Diversity in rates of gene expression is essential for basic cell functions and is controlled by a variety of intricate mechanisms. Revealing general mechanisms that control gene expression is important for understanding normal and pathological cell functions and for improving the design of expression systems. Here we analyzed the relationship between general features of genes and their contribution to expression levels. RESULTS: Genes were divided into four groups according to their core promoter type and their characteristics analyzed statistically. Surprisingly we found that small variations in the TATA box are linked to large differences in gene length. Genes containing canonical TATA are generally short whereas long genes are associated with either non-canonical TATA or TATA-less promoters. These differences in gene length are primarily determined by the size and number of introns. Generally, gene expression was found to be tightly correlated with the strength of the TATA-box. However significant reduction in gene expression levels were linked with long TATA-containing genes (canonical and non-canonical) whereas intron length hardly affected the expression of TATA-less genes. Interestingly, features associated with high translation are prevalent in TATA-containing genes suggesting that their protein production is also more efficient. CONCLUSION: Our results suggest that interplay between core promoter type and gene size can generate significant diversity in gene expression.
