Protein synthesis is a fundamental biological mechanism bringing the DNA-encoded genetic information into life by its translation into molecular effectors - proteins. The initiation phase of translation is one of the key points of gene regulation in eukaryotes, playing a role in processes from neuronal function to development. Indeed, the importance of the study of protein synthesis is increasing with the growing list of genetic diseases caused by mutations that affect mRNA translation. To grasp how this regulation is achieved or altered in the latter case, we must first understand the molecular details of all underlying processes of the translational cycle with the main focus put on its initiation. In this review I discuss recent advances in our comprehension of the molecular basis of particular initiation reactions set into the context of how and where individual eIFs bind to the small ribosomal subunit in the pre-initiation complex. I also summarize our current knowledge on how eukaryotic initiation factor eIF3 controls gene expression in the gene-specific manner via reinitiation.

• MeSH
• Eukaryota metabolism   MeSH
• Eukaryotic Initiation Factors metabolism   MeSH
• Peptide Chain Initiation, Translational genetics   MeSH
• Humans MeSH
• Models, Molecular MeSH
• Ribosomes metabolism   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Research Support, N.I.H., Extramural MeSH

Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ss) in addition to 80S ribosomes (80Ss), revealed that mammalian and yeast 40Ss distribute similarly across 5'TRs, indicating considerable evolutionary conservation. We further developed yeast and human selective TCP-seq (Sel-TCP-seq), enabling selection of 40Ss and 80Ss associated with immuno-targeted factors. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5' UTRs with scanning 40Ss to successively dissociate upon AUG recognition; notably, a proportion of eIF3 lingers on during the initial elongation cycles. Highlighting Sel-TCP-seq versatility, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.

• MeSH
• 5' Untranslated Regions MeSH
• Eukaryotic Initiation Factor-2 genetics   metabolism   MeSH
• Eukaryotic Initiation Factor-3 genetics   metabolism   MeSH
• HEK293 Cells MeSH
• Peptide Initiation Factors genetics   metabolism   MeSH
• Codon, Initiator MeSH
• Humans MeSH
• Ribosome Subunits, Small, Eukaryotic genetics   metabolism   MeSH
• Multiprotein Complexes genetics   metabolism   MeSH
• Protein Biosynthesis * MeSH
• Ribosomes genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Activating Transcription Factor 4 genetics   metabolism   MeSH
• Basic-Leucine Zipper Transcription Factors genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In eukaryotes, for a protein to be synthesized, the 40 S subunit has to first scan the 5'-UTR of the mRNA until it has encountered the AUG start codon. Several initiation factors that ensure high fidelity of AUG recognition were identified previously, including eIF1A, eIF1, eIF2, and eIF5. In addition, eIF3 was proposed to coordinate their functions in this process as well as to promote their initial binding to 40 S subunits. Here we subjected several previously identified segments of the N-terminal domain (NTD) of the eIF3c/Nip1 subunit, which mediates eIF3 binding to eIF1 and eIF5, to semirandom mutagenesis to investigate the molecular mechanism of eIF3 involvement in these reactions. Three major classes of mutant substitutions or internal deletions were isolated that affect either the assembly of preinitiation complexes (PICs), scanning for AUG, or both. We show that eIF5 binds to the extreme c/Nip1-NTD (residues 1-45) and that impairing this interaction predominantly affects the PIC formation. eIF1 interacts with the region (60-137) that immediately follows, and altering this contact deregulates AUG recognition. Together, our data indicate that binding of eIF1 to the c/Nip1-NTD is equally important for its initial recruitment to PICs and for its proper functioning in selecting the translational start site.

• MeSH
• Eukaryotic Initiation Factor-3 genetics   metabolism   MeSH
• Peptide Chain Initiation, Translational physiology   MeSH
• Codon, Initiator genetics   metabolism   MeSH
• Ribosome Subunits, Small, Eukaryotic genetics   metabolism   MeSH
• Multiprotein Complexes genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed β-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo. Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1(G107R) but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs.

• MeSH
• Eukaryotic Initiation Factor-1 genetics   MeSH
• Eukaryotic Initiation Factor-3 chemistry   genetics   metabolism   MeSH
• Phenotype MeSH
• Gene Dosage MeSH
• Peptide Chain Initiation, Translational MeSH
• Codon, Initiator MeSH
• Crystallography, X-Ray MeSH
• Yeasts genetics   growth & development   MeSH
• Ribosome Subunits, Small, Eukaryotic metabolism   MeSH
• Models, Molecular MeSH
• Mutation MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Translation initiation in the hepatitis C virus (HCV) occurs through a cap-independent mechanism that involves an internal ribosome entry site (IRES) capable of interacting with and utilizing the eukaryotic translational machinery. In this review, we focus on the structural configuration of the different HCV IRES domains and the impact of IRES primary sequence variations on secondary structure conservation and function. In some cases, multiple mutations, even those scattered across different domains, led to restoration of the translational activity of the HCV IRES, although the individual occurrences of these mutations were found to be deleterious. We propose that such observation may be attributed to probable long-range inter- and/or intra-domain functional interactions. The precise functioning of the HCV IRES requires the specific interaction of its domains with ribosomal subunits and a subset of eukaryotic translation initiation factors (eIFs). The structural conformation, sequence preservation and variability, and translational machinery association with the HCV IRES regions are also thoroughly discussed, along with other factors that can affect and influence the formation of translation initiation complexes.

• MeSH
• Models, Biological MeSH
• Eukaryotic Initiation Factors metabolism   MeSH
• Hepacivirus physiology   MeSH
• Peptide Chain Initiation, Translational * MeSH
• Nucleic Acid Conformation * MeSH
• Models, Molecular MeSH
• Ribosomes metabolism   MeSH
• RNA, Viral chemistry   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

For many years initiation and termination of mRNA translation have been studied separately. However, a direct link between these 2 isolated stages has been suggested by the fact that some initiation factors also control termination and can even promote ribosome recycling; i.e. the last stage where post-terminating 80S ribosomes are split to start a new round of initiation. Notably, it is now firmly established that, among other factors, ribosomal recycling critically requires the NTPase ABCE1. However, several earlier reports have proposed that ABCE1 also somehow participates in the initiation complex assembly. Based on an extended analysis of our recently published late-stage 48S initiation complex from rabbit, here we provide new mechanistic insights into this putative role of ABCE1 in initiation. This point of view represents the first structural evidence in which the regulatory role of the recycling factor ABCE1 in initiation is discussed and establishes a corner stone for elucidating the interplay between ABCE1 and several initiation factors during the transit from ribosomal recycling to formation of the elongation competent 80S initiation complex.

• MeSH
• ATP-Binding Cassette Transporters chemistry   metabolism   MeSH
• Peptide Elongation Factors MeSH
• Hydrolysis MeSH
• Peptide Chain Initiation, Translational * MeSH
• Peptide Initiation Factors metabolism   MeSH
• Rabbits MeSH
• Models, Molecular MeSH
• Nucleosides chemistry   MeSH
• Ribosomes metabolism   MeSH
• Peptide Chain Termination, Translational MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Rabbits MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation.

• MeSH
• Peptide Chain Elongation, Translational * MeSH
• Eukaryotic Initiation Factor-4E metabolism   MeSH
• Eukaryotic Initiation Factor-4G metabolism   MeSH
• Peptide Chain Initiation, Translational * MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Multiprotein Complexes metabolism   MeSH
• Poly A MeSH
• Polyribosomes metabolism   MeSH
• Selenium-Binding Proteins metabolism   MeSH
• Protein Biosynthesis MeSH
• Ribosomes metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

Programmed stop codon readthrough is a post-transcription regulatory mechanism specifically increasing proteome diversity by creating a pool of C-terminally extended proteins. During this process, the stop codon is decoded as a sense codon by a near-cognate tRNA, which programs the ribosome to continue elongation. The efficiency of competition for the stop codon between release factors (eRFs) and near-cognate tRNAs is largely dependent on its nucleotide context; however, the molecular mechanism underlying this process is unknown. Here, we show that it is the translation initiation (not termination) factor, namely eIF3, which critically promotes programmed readthrough on all three stop codons. In order to do so, eIF3 must associate with pre-termination complexes where it interferes with the eRF1 decoding of the third/wobble position of the stop codon set in the unfavorable termination context, thus allowing incorporation of near-cognate tRNAs with a mismatch at the same position. We clearly demonstrate that efficient readthrough is enabled by near-cognate tRNAs with a mismatch only at the third/wobble position. Importantly, the eIF3 role in programmed readthrough is conserved between yeast and humans.

• MeSH
• RNA, Transfer, Amino Acyl metabolism   MeSH
• Peptide Chain Elongation, Translational * MeSH
• Eukaryotic Initiation Factor-3 metabolism   MeSH
• HeLa Cells MeSH
• Peptide Chain Initiation, Translational MeSH
• Yeasts genetics   MeSH
• Humans MeSH
• Paromomycin pharmacology   MeSH
• Gene Expression Regulation MeSH
• Ribosomes drug effects   metabolism   MeSH
• Codon, Terminator * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Translation is divided into initiation, elongation, termination and ribosome recycling. Earlier work implicated several eukaryotic initiation factors (eIFs) in ribosomal recycling in vitro. Here, we uncover roles for HCR1 and eIF3 in translation termination in vivo. A substantial proportion of eIF3, HCR1 and eukaryotic release factor 3 (eRF3) but not eIF5 (a well-defined "initiation-specific" binding partner of eIF3) specifically co-sediments with 80S couples isolated from RNase-treated heavy polysomes in an eRF1-dependent manner, indicating the presence of eIF3 and HCR1 on terminating ribosomes. eIF3 and HCR1 also occur in ribosome- and RNA-free complexes with both eRFs and the recycling factor ABCE1/RLI1. Several eIF3 mutations reduce rates of stop codon read-through and genetically interact with mutant eRFs. In contrast, a slow growing deletion of hcr1 increases read-through and accumulates eRF3 in heavy polysomes in a manner suppressible by overexpressed ABCE1/RLI1. Based on these and other findings we propose that upon stop codon recognition, HCR1 promotes eRF3·GDP ejection from the post-termination complexes to allow binding of its interacting partner ABCE1/RLI1. Furthermore, the fact that high dosage of ABCE1/RLI1 fully suppresses the slow growth phenotype of hcr1Δ as well as its termination but not initiation defects implies that the termination function of HCR1 is more critical for optimal proliferation than its function in translation initiation. Based on these and other observations we suggest that the assignment of HCR1 as a bona fide eIF3 subunit should be reconsidered. Together our work characterizes novel roles of eIF3 and HCR1 in stop codon recognition, defining a communication bridge between the initiation and termination/recycling phases of translation.

• MeSH
• ATP-Binding Cassette Transporters genetics   MeSH
• Eukaryotic Initiation Factor-3 genetics   MeSH
• Peptide Initiation Factors genetics   MeSH
• Mutation MeSH
• Protein Biosynthesis * MeSH
• Saccharomyces cerevisiae Proteins genetics   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Amino Acid Sequence MeSH
• Peptide Chain Termination, Translational * MeSH
• Codon, Terminator genetics   MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The main role of the translation initiation factor 3 (eIF3) is to orchestrate formation of 43S-48S preinitiation complexes (PICs). Until now, most of our knowledge on eIF3 functional contribution to regulation of gene expression comes from yeast studies. Hence, here we developed several novel in vivo assays to monitor the integrity of the 13-subunit human eIF3 complex, defects in assembly of 43S PICs, efficiency of mRNA recruitment, and postassembly events such as AUG recognition. We knocked down expression of the PCI domain-containing eIF3c and eIF3a subunits and of eIF3j in human HeLa and HEK293 cells and analyzed the functional consequences. Whereas eIF3j downregulation had barely any effect and eIF3a knockdown disintegrated the entire eIF3 complex, eIF3c knockdown produced a separate assembly of the a, b, g, and i subunits (closely resembling the yeast evolutionary conserved eIF3 core), which preserved relatively high 40S binding affinity and an ability to promote mRNA recruitment to 40S subunits and displayed defects in AUG recognition. Both eIF3c and eIF3a knockdowns also severely reduced protein but not mRNA levels of many other eIF3 subunits and indeed shut off translation. We propose that eIF3a and eIF3c control abundance and assembly of the entire eIF3 and thus represent its crucial scaffolding elements critically required for formation of PICs.

• MeSH
• Cell Line MeSH
• Eukaryotic Initiation Factor-3 genetics   metabolism   MeSH
• HEK293 Cells MeSH
• HeLa Cells MeSH
• Peptide Chain Initiation, Translational genetics   MeSH
• Humans MeSH
• RNA, Small Interfering MeSH
• Cell Proliferation MeSH
• RNA-Binding Proteins genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Ribosomal Proteins genetics   metabolism   MeSH
• RNA Interference MeSH
• RNA, Ribosomal genetics   MeSH
• Protein Binding genetics   physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH