Translation initiation: structures, mechanisms and evolution

Translation, the process of mRNA-encoded protein synthesis, requires a complex apparatus, composed of the ribosome, tRNAs and additional protein factors, including aminoacyl tRNA synthetases. The ribosome provides the platform for proper assembly of mRNA, tRNAs and protein factors and carries the peptidyl-transferase activity. It consists of small and large subunits. The ribosomes are ribonucleoprotein particles with a ribosomal RNA core, to which multiple ribosomal proteins are bound. The sequence and structure of ribosomal RNAs, tRNAs, some of the ribosomal proteins and some of the additional protein factors are conserved in all kingdoms, underlying the common origin of the translation apparatus. Translation can be subdivided into several steps: initiation, elongation, termination and recycling. Of these, initiation is the most complex and the most divergent among the different kingdoms of life. A great amount of new structural, biochemical and genetic information on translation initiation has been accumulated in recent years, which led to the realization that initiation also shows a great degree of conservation throughout evolution. In this review, we summarize the available structural and functional data on translation initiation in the context of evolution, drawing parallels between eubacteria, archaea, and eukaryotes. We will start with an overview of the ribosome structure and of translation in general, placing emphasis on factors and processes with relevance to initiation. The major steps in initiation and the factors involved will be described, followed by discussion of the structure and function of the individual initiation factors throughout evolution. We will conclude with a summary of the available information on the kinetic and thermodynamic aspects of translation initiation.     

eIF4E as a molecular wildcard in metazoans RNA metabolism

The evolutionary origin of eukaryotes spurred the transition from prokaryotic-like translation to a more sophisticated, eukaryotic translation. During this process, successive gene duplication of a single, primordial eIF4E gene encoding the mRNA cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) gave rise to a plethora of paralog genes across eukaryotes that underwent further functional diversification in RNA metabolism. The ability to take different roles is due to eIF4E promiscuity in binding many partner proteins, rendering eIF4E a highly versatile and multifunctional player that functions as a molecular wildcard. Thus, in metazoans, eIF4E paralogs are involved in various processes, including messenger RNA (mRNA) processing, export, translation, storage, and decay. Moreover, some paralogs display differential expression in tissues and developmental stages and show variable biochemical properties. In this review, we discuss recent advances shedding light on the functional diversification of eIF4E in metazoans. We emphasise humans and two phylogenetically distant species which have become paradigms for studies on development, namely the fruit fly Drosophila melanogaster and the roundworm Caenorhabditis elegans.     

Role of RNA Structure Motifs in IRES-Dependent Translation Initiation of the Coxsackievirus B3: New Insights for Developing Live-Attenuated Strains for Vaccines and Gene Therapy

Internal ribosome entry site (IRES) elements are highly structured RNA sequences that function to recruit ribosomes for the initiation of translation. In contrast to the canonical cap-binding, the mechanism of IRES-mediated translation initiation is still poorly understood. Translation initiation of the coxsackievirus B3 (CVB3), a causative agent of viral myocarditis, has been shown to be mediated by a highly ordered structure of the 5′ untranslated region (5′UTR), which harbors an IRES. Taking into account that efficient initiation of mRNA translation depends on temporally and spatially orchestrated sequence of RNA–protein and RNA–RNA interactions, and that, at present, little is known about these interactions, we aimed to describe recent advances in our understanding of molecular structures and biochemical functions of the translation initiation process. Thus, this review will explore the IRES elements as important RNA structures and the significance of these structures in providing an alternative mechanism of translation initiation of the CVB3 RNA. Since translation initiation is the first intracellular step during the CVB3 infection cycle, the IRES region provides an ideal target for antiviral therapies. Interestingly, the 5′ and 3′UTRs represent promising candidates for the study of CVB3 cardiovirulence and provide new insights for developing live-attenuated vaccines.     

Inhibition of eukaryotic translation initiation by the marine natural product pateamine A

Translation initiation in eukaryotes is accomplished through the coordinated and orderly action of a large number of proteins, including the eIF4 initiation factors. Herein, we report that pateamine A (PatA), a potent antiproliferative and proapoptotic marine natural product, inhibits cap-dependent eukaryotic translation initiation. PatA bound to and enhanced the intrinsic enzymatic activities of eIF4A, yet it inhibited eIF4A-eIF4G association and promoted the formation of a stable ternary complex between eIF4A and eIF4B. These changes in eIF4A affinity for its partner proteins upon binding to PatA caused the stalling of initiation complexes on mRNA in vitro and induced stress granule formation in vivo. These results suggest that PatA will be a valuable molecular probe for future studies of eukaryotic translation initiation and may serve as a lead compound for the development of anticancer agents.     

Baker's yeast, the new work horse in protein synthesis studies: analyzing eukaryotic translation initiation

The possibility of combining powerful genetic methods with biochemical analysis has made baker's yeast Saccharomyces cerevisiae the organism of choice to study the complex process of translation initiation in eukaryotes. Several new initiation factor genes and interactions between components of the translational machinery that were not predicted by current models have been revealed by genetic analysis of extragenic suppressors of translational initiation mutants. In addition, a yeast cell-free translation system has been developed that allows in vivo phenotypes to be correlated with in vitro biochemical activities. We summarize here the current view of yeast translational initiation obtained by these approaches.     

RNA structure-based ribosome recruitment: lessons from the Dicistroviridae intergenic region IRESes

In eukaryotes, the canonical process of initiating protein synthesis on an mRNA depends on many large protein factors and the modified nucleotide cap on the 5' end of the mRNA. However, certain RNA sequences can bypass the need for these proteins and cap, using an RNA structure-based mechanism called internal initiation of translation. These RNAs are called internal ribosome entry sites (IRESes), and the cap-independent initiation pathway they support is critical for successful infection by many viruses of medical and economic importance. In this review, we briefly describe and compare mechanistic and structural groups of viral IRES RNAs, focusing on those IRESes that are capable of direct ribosome recruitment using specific RNA structures. We then discuss in greater detail some recent advances in our understanding of the intergenic region IRESes of the Dicistroviridae, which use the most streamlined ribosome-recruitment mechanism yet discovered. By combining these findings with knowledge of canonical translation and the behavior of other IRESes, mechanistic models of this RNA structure-based process are emerging.     

The dynamics of supply and demand in mRNA translation

We study the elongation stage of mRNA translation in eukaryotes and find that, in contrast to the assumptions of previous models, both the supply and the demand for tRNA resources are important for determining elongation rates. We find that increasing the initiation rate of translation can lead to the depletion of some species of aa-tRNA, which in turn can lead to slow codons and queueing. Particularly striking "competition" effects are observed in simulations of multiple species of mRNA which are reliant on the same pool of tRNA resources. These simulations are based on a recent model of elongation which we use to study the translation of mRNA sequences from the Saccharomyces cerevisiae genome. This model includes the dynamics of the use and recharging of amino acid tRNA complexes, and we show via Monte Carlo simulation that this has a dramatic effect on the protein production behaviour of the system.     

依赖于5′端非编码区高级结构的真核生物mRNA翻译调控

翻译水平的调控是真核基因表达调控的重要环节.近年来的研究表明,许多真核基因的翻译依赖于RNA 5′端非编码区的结构元件.一些小结构元件,如铁离子反应元件,具有1个茎环结构,由铁离子介导控制转铁蛋白的翻译. 核糖开关通过结合特定代谢分子在2种结构状态下切换,调控可变剪接和翻译起始.另1个高度结构化的mRNA元件是内部核糖体进入位点,通过富集核糖体和起始因子促进基因的表达.本文综述了依赖于小结构元件、内部核糖体进入位点和核糖开关的真核基因翻译起始调控相应的研究成果和研究方法.对于研究的前景以及可能存在的挑战也作出阐述.     

Controlling the mechanical properties of three-dimensional matrices via non-enzymatic collagen glycation

The mechanical properties of the extracellular matrix play an important role in maintaining cellular function and overall tissue homeostasis. Recently, a number of hydrogel systems have been developed to investigate the role of matrix mechanics in mediating cell behavior within three-dimensional environments. However, many of the techniques used to modify the stiffness of the matrix also alter properties that are important to cellular function including matrix density, porosity and binding site frequency, or rely on amorphous synthetic materials. In a recent publication, we described the fabrication, characterization and utilization of collagen gels that have been non-enzymatically glycated in their unpolymerized form to produce matrices of varying stiffness. Using these scaffolds, we showed that the mechanical properties of the resulting collagen gels could be increased 3-fold without significantly altering the collagen fiber architecture. Using these matrices, we found that endothelial cell spreading and outgrowth from multi-cellular spheroids changes as a function of the stiffness of the matrix. Our results demonstrate that non-enzymatic collagen glycation is a tractable technique that can be used to study the role of 3D stiffness in mediating cellular function. This commentary will review some of the current methods that are being used to modulate matrix mechanics and discuss how our recent work using non-enzymatic collagen glycation can contribute to this field.     

Identification of compounds that decrease the fidelity of start codon recognition by the eukaryotic translational machinery

Translation initiation in eukaryotes involves more than a dozen protein factors. Alterations in six factors have been found to reduce the fidelity of start codon recognition by the ribosomal preinitiation complex in yeast, a phenotype referred to as Sui(-). No small molecules are known that affect the fidelity of start codon recognition. Such compounds would be useful tools for probing the molecular mechanics of translation initiation and its regulation. To find compounds with this effect, we set up a high-throughput screen using a dual luciferase assay in S. cerevisiae. Screening of over 55,000 compounds revealed two structurally related molecules that decrease the fidelity of start codon selection by approximately twofold in the dual luciferase assay. This effect was confirmed using additional in vivo assays that monitor translation from non-AUG start codons. Both compounds increase translation of a natural upstream open reading frame previously shown to initiate translation at a UUG. The compounds were also found to exacerbate increased use of UUG as a start codon (Sui(-) phenotype) conferred by haploinsufficiency of wild-type eukaryotic initiation factor (eIF) 1, or by mutation in eIF1. Furthermore, the effects of the compounds are suppressed by overexpressing eIF1, which is known to restore the fidelity of start codon selection in strains harboring Sui(-) mutations in various other initiation factors. Together, these data strongly suggest that the compounds affect the translational machinery itself to reduce the accuracy of selecting AUG as the start codon.     

The essential Drosophila ATP-binding cassette domain protein, pixie, binds the 40 S ribosome in an ATP-dependent manner and is required for translation initiation

The Drosophila gene, pixie, is an essential gene required for normal growth and translation. Pixie is the fly ortholog of human RLI, which was first identified as an RNase L inhibitor, and yeast Rli1p, which has recently been shown to play a role in translation initiation and ribosome biogenesis. These proteins are all soluble ATP-binding cassette proteins with two N-terminal iron-sulfur clusters. Here we demonstrate that Pixie can be isolated from cells in complex with eukaryotic translation initiation factor 3 and ribosomal proteins of the small subunit. In addition, our analysis of polysome profiles reveals that double-stranded RNA interference-mediated depletion of Pixie results in an increase in empty 80 S ribosomes and a corresponding decrease in polysomes. Thus Pixie is required for normal levels of translation initiation. We also find that Pixie associates with the 40 S subunit on sucrose density gradients in an ATP-dependent manner. Our observations are consistent with Pixie playing a catalytic role in the assembly of complexes required for translation initiation. Thus, the function of this soluble ATP-binding cassette domain protein family in translation initiation has been conserved from yeast through to higher eukaryotes.     

Cellular IRES-mediated translation: The war of ITAFs in pathophysiological states

Translation of cellular mRNAs via initiation at internal ribosome entry sites (IRESs) has received increased attention during recent years due to its emerging significance for many physiological and pathological stress conditions in eukaryotic cells. Expression of genes bearing IRES elements in their mRNAs is controlled by multiple molecular mechanisms, with IRES-mediated translation favored under conditions when cap-dependent translation is compromised. In this review, we discuss recent advances in the field and future directions that may bring us closer to understanding the complex mechanisms that guide cellular IRES-mediated expression. We present examples in which the competitive action of IRES-transacting factors (ITAFs) plays a pivotal role in IRES-mediated translation and thereby controls cell-fate decisions leading to either pro-survival stress adaptation or cell death.Key words: translation initiation, IRES, canonical initiation factors, ITAFs, stress response, eIF2, angiogenesis, mitosis, nutrient-signaling, hyperosmolar stress     

Cap-dependent translation initiation in eukaryotes is regulated by a molecular mimic of eIF4G.

eIF4G uses a conserved Tyr-X-X-X-X-Leu-phi segment (where X is variable and phi is hydrophobic) to recognize eIF4E during cap-dependent translation initiation in eukaryotes. High-resolution X-ray crystallography and complementary biophysical methods have revealed that this eIF4E recognition motif undergoes a disorder-to-order transition, adopting an L-shaped, extended chain/alpha-helical conformation when it interacts with a phylogenetically invariant portion of the convex surface of eIF4E. Inhibitors of translation initiation known as eIF4E-binding proteins (4E-BPs) contain similar eIF4E recognition motifs. These molecules are molecular mimics of eIF4G, which act by occupying the same binding site on the convex dorsum of eIF4E and blocking assembly of the translation machinery. The implications of our results for translation initiation are discussed in detail, and a molecular mechanism for relief of translation inhibition following phosphorylation of the 4E-BPs is proposed.     

Kinetic analysis of late steps of eukaryotic translation initiation

Little is known about the molecular mechanics of the late events of translation initiation in eukaryotes. We present a kinetic dissection of the transition from a preinitiation complex after start codon recognition to the final 80S initiation complex. The resulting framework reveals that eukaryotic initiation factor (eIF)5B actually accelerates the rate of ribosomal subunit joining, and this acceleration is influenced by the conformation of the GTPase active site of the factor mediated by the bound nucleotide. eIF1A accelerates joining through its C-terminal interaction with eIF5B, and eIF1A release from the initiating ribosome, which occurs only after subunit joining, is accelerated by GTP hydrolysis by eIF5B. Following subunit joining, GTP hydrolysis by eIF5B alters the conformation of the final initiation complex and clears a path to promote rapid release of eIF1A. Our data, coupled with previous work, indicate that eIF1A is present on the ribosome throughout the entire initiation process and plays key roles at every stage.