Unveiling Genetic Diversity Among Bacterial Isolates Using SCoT Markers.

This study aimed to unveil the genetic diversity among 47 bacterial isolates from various species using start codon targeted (SCoT) markers. Six SCoT primers yielded 219 reproducible bands, with 89.04% exhibiting polymorphism. The amplification process generated 28 to 50 fragments per primer, with an average of 36.50. Genetic diversity was quantified using polymorphic information content (PIC) values ranging from 0.11 to 0.14, with SCoT32 showing the highest PIC (0.14) and SCoT23 the lowest (0.11). The resolving power (RP) index, used to assess primer discriminatory power, varied significantly, with SCoT23 demonstrating the highest RP (6.00) and SCoT29 the lowest (4.51). Comparative analysis with conventional markers like M13 and (GTG)5 revealed that certain SCoT primers exhibited superior PIC values, which indicates enhanced utility for interspecies differentiation. The high discrimination level achieved by SCoT primers underscores their effectiveness in genetic differentiation and biodiversity assessment within bacterial populations. This research highlights SCoT markers as powerful tools for microbial genetic studies, which offers valuable insights into bacterial diversity and provides a robust methodological framework for future investigations aimed at elucidating genetic variation and improving species identification. The application of SCoT markers represents a significant advancement in molecular techniques for bacterial characterization and phylogenetic analysis, demonstrating their potential to enhance our understanding of microbial genetics and evolution.

Assessment of genetic diversity by phenological traits, field performance, and Start Codon Targeted (SCoT) polymorphism marker of seventeen soybean genotypes (Glycine max L.).

The Egyptian-farmed soybeans have a wide range of genetic diversity which is most important in plant improvement programs in order to develop new higher yielding soybean genotypes. The present study is designed to determine the genetic variability among seventeen genotypes of cultivated soybean (Glycine max L.) by examining the phenotypic level at the seedling stage, field performance over two years 2022/2023 and genetically using Start Codon Targeted (SCoT) markers. Results indicated that the SCoT markers, 100 seed weight, and tip angle (TA) traits were positively correlated with H2L12, DR 101, H15L5, and H117 genotypes. In addition, the number of branches per plant and plant height were associated with H113, H32, Crowford, H129, and D7512035. Furthermore, the length of the first internode (LFI), root width (RW), root length (RL), and shoot length (SL) were more associated with Giza 111, NC105, and Hutcheson. The hierarchical cluster analysis (HCA) and its associated heatmap explored the differences among the genotypes. It showed that all examined parameters were clustered into four distinct clusters. The obtained results showed that genotypes NC105, H30, D75_12035, and H2L12 have promising phenological and morphological traits besides tracking the inheritance of nearby genes surrounding the ATG translation start codon since they are in a monoclades. The obtained results will help the breeder plan appropriate selection strategies for improving seed yield in soybeans through hybridization from divergent clusters.

Evaluation of genetic diversity and population structure of Annamocarya sinensis using SCoT markers.

Annamocarya sinensis (Dode) Leroy, a relict plant from the Tertiary period, is a member of Annamocarya genus in the Juglandaceae family. Despite its wide distribution in Guangxi Province, the habitats of this species had become fragmented and isolated, causing it facing deterioration. For protecting this endangered species, it is crucial to understand its status in the wild and genetic diversity. In this study, 216 A. sinensis accessions from 18 populations in Guangxi were examined using Start Codon Target Polymorphism (SCoT) markers for PCR amplification, genetic diversity, and population structure analysis. Out of the 20 SCoT primers used, 222 sites were amplified, with 185 being polymorphic (PPB of 83.33%). Polymorphic information content values ranged from 0.4380 to 0.4999, Nei's genetic diversity index ranging from 0.1573 to 0.2503, and Shannon diversity index ranged from 0.1583 to 0.3812. Through AMOVA analysis, the total genetic diversity and genetic diversity within populations was calculated out as 0.3271 and 0.1542 respectively, the genetic differentiation coefficient between populations was 0.5286, with a gene flow 0.4458. Cluster analysis categorized A. sinensis germplasm into three groups, while population structure analysis divided all accessions into three ancestral sources with 19.91% showing mixed ancestral origins. No significant correlation was observed between genetic and geographical distance on the Mentel test (r = 0.07348, p = 0.7468). Overall, A. sinensis displays a relatively rich genetic diversity at the species level, albeit with a fairly uniform genetic background and high genetic differentiation. This study provides a crucial basis for the conservation and innovative use of A. sinensis germplasm resources.

Structural basis of AUC codon discrimination during translation initiation in yeast.

In eukaryotic translation initiation, the 48S preinitiation complex (PIC) scans the 5' untranslated region of mRNAs to search for the cognate start codon (AUG) with assistance from various eukaryotic initiation factors (eIFs). Cognate start codon recognition is precise, rejecting near-cognate codons with a single base difference. However, the structural basis of discrimination of near-cognate start codons was not known. We have captured multiple yeast 48S PICs with a near-cognate AUC codon at the P-site, revealing that the AUC codon induces instability in the codon-anticodon at the P-site, leading to a disordered N-terminal tail of eIF1A. Following eIF1 dissociation, the N-terminal domain of eIF5 fails to occupy the vacant eIF1 position, and eIF2ß becomes flexible. Consequently, 48S with an AUC codon is less favourable for initiation. Furthermore, we observe hitherto unreported metastable states of the eIF2-GTP-Met-tRNAMet ternary complex, where the eIF2ß helix-turn-helix domain may facilitate eIF5 association by preventing eIF1 rebinding to 48S PIC. Finally, a swivelled head conformation of 48S PIC appears crucial for discriminating incorrect and selection of the correct codon-anticodon pair during translation initiation.

Non-canonical start codons confer context-dependent advantages in carbohydrate utilization for commensal E. coli in the murine gut.

Resource competition is a driver of gut microbiota composition. Bacteria can outcompete metabolically similar rivals through the limitation of shared growth-fuelling nutrients. The mechanisms underlying this remain unclear for bacteria with identical sets of metabolic genes. Here we analysed the lactose utilization operon in the murine commensal Escherichia coli 8178. Using in vitro and in vivo approaches, we showed that translation of the lactose utilization repressor gene lacI from its native non-canonical GTG start codon increases the basal expression of the lactose utilization cluster, enhancing adaptation to lactose consumption. Consequently, a strain carrying the wild type lacI GTG start codon outperformed the lacI ATG start codon mutant in the mouse intestine. This advantage was attenuated upon limiting host lactose intake through diet shift or altering the mutant frequency, emphasizing the context-dependent effect of a single nucleotide change on the bacterial fitness of a common member of the gut microbiota. Coupled with a genomic analysis highlighting the selection of non-ATG start codons in sugar utilization regulator genes across the Enterobacteriaceae family, our data exposed an unsuspected function of non-canonical start codons in metabolic competition.

Evidence for widespread translation of 5' untranslated regions.

Ribosome profiling experiments support the translation of a range of novel human open reading frames. By contrast, most peptides from large-scale proteomics experiments derive from just one source, 5' untranslated regions. Across the human genome we find evidence for 192 translated upstream regions, most of which would produce protein isoforms with extended N-terminal ends. Almost all of these N-terminal extensions are from highly abundant genes, which suggests that the novel regions we detect are just the tip of the iceberg. These upstream regions have characteristics that are not typical of coding exons. Their GC-content is remarkably high, even higher than 5' regions in other genes, and a large majority have non-canonical start codons. Although some novel upstream regions have cross-species conservation - five have orthologues in invertebrates for example - the reading frames of two thirds are not conserved beyond simians. These non-conserved regions also have no evidence of purifying selection, which suggests that much of this translation is not functional. In addition, non-conserved upstream regions have significantly more peptides in cancer cell lines than would be expected, a strong indication that an aberrant or noisy translation initiation process may play an important role in translation from upstream regions.

Start codon variant in LAG3 is associated with decreased LAG-3 expression and increased risk of autoimmune thyroid disease.

Autoimmune thyroid disease (AITD) is a common autoimmune disease. In a GWAS meta-analysis of 110,945 cases and 1,084,290 controls, 290 sequence variants at 225 loci are associated with AITD. Of these variants, 115 are previously unreported. Multiomics analysis yields 235 candidate genes outside the MHC-region and the findings highlight the importance of genes involved in T-cell regulation. A rare 5'-UTR variant (rs781745126-T, MAF = 0.13% in Iceland) in LAG3 has the largest effect (OR = 3.42, P = 2.2 × 10-16) and generates a novel start codon for an open reading frame upstream of the canonical protein translation initiation site. rs781745126-T reduces mRNA and surface expression of the inhibitory immune checkpoint LAG-3 co-receptor on activated lymphocyte subsets and halves LAG-3 levels in plasma among heterozygotes. All three homozygous carriers of rs781745126-T have AITD, of whom one also has two other T-cell mediated diseases, that is vitiligo and type 1 diabetes. rs781745126-T associates nominally with vitiligo (OR = 5.1, P = 6.5 × 10-3) but not with type 1 diabetes. Thus, the effect of rs781745126-T is akin to drugs that inhibit LAG-3, which unleash immune responses and can have thyroid dysfunction and vitiligo as adverse events. This illustrates how a multiomics approach can reveal potential drug targets and safety concerns.

Possible involvement of three-stemmed pseudoknots in regulating translational initiation in human mRNAs.

RNA pseudoknots play a crucial role in various cellular functions. Established pseudoknots show significant variation in both size and structural complexity. Specifically, three-stemmed pseudoknots are characterized by an additional stem-loop embedded in their structure. Recent findings highlight these pseudoknots as bacterial riboswitches and potent stimulators for programmed ribosomal frameshifting in RNA viruses like SARS-CoV2. To investigate the possible presence of functional three-stemmed pseudoknots in human mRNAs, we employed in-house developed computational methods to detect such structures within a dataset comprising 21,780 full-length human mRNA sequences. Numerous three-stemmed pseudoknots were identified. A selected set of 14 potential instances are presented, in which the start codon of the mRNA is found in close proximity either upstream, downstream, or within the identified three-stemmed pseudoknot. These pseudoknots likely play a role in translational initiation regulation. The probability of their existence gains support from their ranking as the most stable pseudoknot identified in the entire mRNA sequence, structural conservation across homologous mRNAs, stereochemical feasibility as demonstrated by structural modeling, and classification as members of the CPK-1 pseudoknot family, which includes many well-established pseudoknots. Furthermore, in four of the mRNAs, two or three closely spaced or tandem three-stemmed pseudoknots were identified. These findings suggest the frequent occurrence of three-stemmed pseudoknots in human mRNAs. A stepwise co-transcriptional folding mechanism is proposed for the formation of a three-stemmed pseudoknot structure. Our results not only provide fresh insights into the structures and functions of pseudoknots but also unveil the potential to target pseudoknots for treating human diseases.

Induced genetic diversity through mutagenesis in wheat gene pool and significant use of SCoT markers to underpin key agronomic traits.

BACKGROUND: This research explores the efficacy of mutagenesis, specifically using sodium azide (SA) and hydrazine hydrate (HZ) treatments, to introduce genetic diversity and enhance traits in three wheat (Triticum aestivum L.) genotypes. The experiment entails subjecting the seeds to different doses of SA and HZ and cultivating them in the field for two consecutive generations: M1 (first generation) and M2 (second generation). We then employed selective breeding techniques with Start Codon Targeted (SCoT) markers to select traits within the wheat gene pool. Also, the correlation between SCoT markers and specific agronomic traits provides insights into the genetic mechanisms underlying mutagenesis-induced changes in wheat. RESULTS: In the study, eleven genotypes were derived from parent varieties Sids1, Sids12, and Giza 168, and eight mutant genotypes were selected from the M1 generation and further cultivated to establish the M2 generation. The results revealed that various morphological and agronomical characteristics, such as plant height, spikes per plant, spike length, spikelet per spike, grains per spikelet, and 100-grain weight, showed increases in different genotypes from M1 to M2. SCoT markers were employed to assess genetic diversity among the eleven genotypes. The bioinformatics analysis identified a correlation between SCoT markers and the transcription factors ABSCISIC ACID INSENSITIVE3 (ABI3) and VIVIPAROUS1 (VP1), crucial for plant development, growth, and stress adaptation. A comprehensive examination of genetic distance and the function identification of gene-associated SCoT markers may provide valuable insights into the mechanisms by which SA and HZ act as mutagens, enhancing wheat agronomic qualities. CONCLUSIONS: This study demonstrates the effective use of SA and HZ treatments to induce gene diversity through mutagenesis in the wheat gene pool, resulting in the enhancement of agronomic traits, as revealed by SCoT markers. The significant improvements in morphological and agronomical characteristics highlight the potential of mutagenesis techniques for crop improvement. These findings offer valuable information for breeders to develop effective breeding programs to enhance wheat quality and resilience through increased genetic diversity.

From DNA to lytic proteins: transcription and translation of the bacteriophage T5 holin/endolysin operon.

The analysis of transcriptional activity of the bacteriophage T5 hol/endo operon conducted in the paper revealed a strong constitutive promoter recognized by E. coli RNA polymerase and a transcription initiation point of the operon. It was also shown that the only translational start codon for holin was a non-canonical TTG. Translation initiation regions (TIRs) of both genes of the operon (hol and endo) were further analyzed using chimeric constructs, in which parts of the hol/endo regulatory regions were fused with the gene of a reporter protein (EGFP). It was found that TIR of hol was 20 times less effective than that of endo. As it turned out, the level of EGFP production was influenced by the composition of the constructs and the type of the hol start codon. Apparently, the translational suppression of holin's accumulation and posttranslational activation of endolysin by Ca2+ are the main factors ensuring the proper timing of the host cell lysis by bacteriophage T5. The approach based on the use of chimeric constructs proposed in the paper can be recommended for studying other native or artificial operons of any complexity: analyzing the impacts of separate DNA regions, as well as their coupled effect, on the processes of transcription and translation of recombinant protein(s).

Alternative translation initiation by ribosomal leaky scanning produces multiple isoforms of the Pif1 helicase.

In budding yeast, the integrity of both the nuclear and mitochondrial genomes relies on dual-targeted isoforms of the conserved Pif1 helicase, generated by alternative translation initiation (ATI) of PIF1 mRNA from two consecutive AUG codons flanking a mitochondrial targeting signal. Here, we demonstrate that ribosomal leaky scanning is the specific ATI mechanism that produces not only these, but also novel, previously uncharacterized Pif1 isoforms. Both in-frame, downstream AUGs as well as near-cognate start codons contribute to the generation of these alternative isoforms. This has crucial implications for the rational design of genuine separation-of-function alleles and provides an explanation for the suboptimal behaviour of the widely employed mitochondrial- (pif1-m1) and nuclear-deficient (pif1-m2) alleles, with mutations in the first or second AUG codon, respectively. We have taken advantage of this refined model to develop improved versions of these alleles, which will serve as valuable tools to elucidate novel functions of this helicase and to disambiguate previously described genetic interactions of PIF1 in the context of nuclear and mitochondrial genome stability.

TIR predictor and optimizer: Web-tools for accurate prediction of translation initiation rate and precision gene design in Saccharomyces cerevisiae.

Translation initiation is the primary determinant of the rate of protein production. The variation in the rate with which this step occurs can cause up to three orders of magnitude differences in cellular protein levels. Several mRNA features, including mRNA stability in proximity to the start codon, coding sequence length, and presence of specific motifs in the mRNA molecule, have been shown to influence the translation initiation rate. These molecular factors acting at different strengths allow precise control of in vivo translation initiation rate and thus the rate of protein synthesis. However, despite the paramount importance of translation initiation rate in protein synthesis, accurate prediction of the absolute values of initiation rate remains a challenge. In fact, as of now, there is no available model for predicting the initiation rate in Saccharomyces cerevisiae. To address this, we train a machine learning model for predicting the in vivo initiation rate in S. cerevisiae transcripts. The model is trained using a diverse set of mRNA transcripts, enabling the comparison of initiation rates across different transcripts. Our model exhibited excellent accuracy in predicting the translation initiation rate and demonstrated its effectiveness with both endogenous and exogenous transcripts. Then, by combining the machine learning model with the Monte-Carlo search algorithm, we have also devised a method to optimize the nucleotide sequence of any gene to achieve a specific target initiation rate. The machine learning model we've developed for predicting translation initiation rates, along with the gene optimization method, are deployed as a web server. Both web servers are accessible for free at the following link: ajeetsharmalab.com/TIRPredictor. Thus, this research advances our fundamental understanding of translation initiation processes, with direct applications in biotechnology.

Fndc5 is translated from an upstream ATG start codon and cleaved to produce irisin myokine precursor protein in humans and mice.

Witmer et al. provide genomic and molecular evidence to demonstrate that Fndc5 (irisin myokine precursor protein) is translated in humans from an overlooked upstream ATG codon.

JUN mRNA translation regulation is mediated by multiple 5' UTR and start codon features.

Regulation of mRNA translation by eukaryotic initiation factors (eIFs) is crucial for cell survival. In humans, eIF3 stimulates translation of the JUN mRNA which encodes the transcription factor JUN, an oncogenic transcription factor involved in cell cycle progression, apoptosis, and cell proliferation. Previous studies revealed that eIF3 activates translation of the JUN mRNA by interacting with a stem loop in the 5' untranslated region (5' UTR) and with the 5' -7-methylguanosine cap structure. In addition to its interaction site with eIF3, the JUN 5' UTR is nearly one kilobase in length, and has a high degree of secondary structure, high GC content, and an upstream start codon (uAUG). This motivated us to explore the complexity of JUN mRNA translation regulation in human cells. Here we find that JUN translation is regulated in a sequence and structure-dependent manner in regions adjacent to the eIF3-interacting site in the JUN 5' UTR. Furthermore, we identify contributions of an additional initiation factor, eIF4A, in JUN regulation. We show that enhancing the interaction of eIF4A with JUN by using the compound Rocaglamide A (RocA) represses JUN translation. We also find that both the upstream AUG (uAUG) and the main AUG (mAUG) contribute to JUN translation and that they are conserved throughout vertebrates. Our results reveal additional layers of regulation for JUN translation and show the potential of JUN as a model transcript for understanding multiple interacting modes of translation regulation.

Upstream open reading frame-introducing variants in patients with primary familial brain calcification.

More than 50% of patients with primary familial brain calcification (PFBC), a rare neurological disorder, remain genetically unexplained. While some causative genes are yet to be identified, variants in non-coding regions of known genes may represent a source of missed diagnoses. We hypothesized that 5'-Untranslated Region (UTR) variants introducing an AUG codon may initiate mRNA translation and result in a loss of function in some of the PFBC genes. After reannotation of exome sequencing data of 113 unrelated PFBC probands, we identified two upstream AUG-introducing variants in the 5'UTR of PDGFB. One, NM_002608.4:c.-373C>G, segregated with PFBC in the family. It was predicted to create an upstream open reading frame (ORF). The other one, NM_002608.4:c.-318C>T, was found in a simplex case. It was predicted to result in an ORF overlapping the natural ORF with a frameshift. In a GFP reporter assay, both variants were associated with a dramatic decrease in GFP levels, and, after restoring the reading frame with the GFP sequence, the c.-318C>T variant was associated with a strong initiation of translation as measured by western blotting. Overall, we found upstream AUG-introducing variants in the 5'UTR of PDGFB in 2/113 (1.7%) undiagnosed PFBC cases. Such variants thus represent a source of putative pathogenic variants.

CdsA, a CDP-diacylglycerol synthase involved in phospholipid and glycolipid MPIase biosynthesis, possesses multiple initiation codons.

CdsA is a CDP-diacylglycerol synthase essential for phospholipid and glycolipid MPIase biosynthesis, and therefore for growth. The initiation codon of CdsA has been assigned as "TTG," while methionine at the 37th codon was reported to be an initiation codon in the original report. Since a vector containing the open reading frame starting with "TTG" under a controllable promoter complemented the cdsA knockout, "TTG" could function as an initiation codon. However, no evidence supporting that this "TTG" is the sole initiation codon has been reported. We determined the initiation codon by examining the ability of mutants around the N-terminal region to complement cdsA mutants. Even if the "TTG" was substituted with a stop codon, the clear complementation was observed. Moreover, the clones with multiple mutations of stop codons complemented the cdsA mutant up to the 37th codon, indicating that cdsA possesses multiple codons that can function as initiation codons. We constructed an experimental system in which the chromosomal expression of cdsA can be analyzed. By means of this system, we found that the cdsA mutant with substitution of "TTG" with a stop codon is fully functional. Thus, we concluded that CdsA contains multiple initiation codons.

Unraveling the plasticity of translation initiation in prokaryotes: Beyond the invariant Shine-Dalgarno sequence.

Translation initiation in prokaryotes is mainly defined, although not exclusively, by the interaction between the anti-Shine-Dalgarno sequence (antiSD), located at the 3'-terminus of the 16S ribosomal RNA, and a complementary sequence, the ribosome binding site, or Shine-Dalgarno (SD), located upstream of the start codon in prokaryotic mRNAs. The antiSD has a conserved 5'-CCUCC-3' core, but inter-species variations have been found regarding the participation of flanking bases in binding. These variations have been described for certain bacteria and, to a lesser extent, for some archaea. To further analyze these variations, we conducted binding-energy prediction analyses on over 6,400 genomic sequences from both domains. We identified 15 groups of antiSD variants that could be associated with the organisms' phylogenetic origin. Additionally, our findings revealed that certain organisms exhibit variations in the core itself. Importantly, an unaltered core is not necessarily required for the interaction between the 3'-terminus of the rRNA and the region preceding the AUG of the mRNA. In our study, we classified organisms into four distinct categories: i) those possessing a conserved core and demonstrating binding; ii) those with a conserved core but lacking evidence of binding; iii) those exhibiting binding in the absence of a conserved core; and iv) those lacking both a conserved core and evidence of binding. Our results demonstrate the flexibility of organisms in evolving different sequences involved in translation initiation beyond the traditional Shine-Dalgarno sequence. These findings are discussed in terms of the evolution of translation initiation in prokaryotic organisms.

Yeast eIF2A has a minimal role in translation initiation and uORF-mediated translational control in vivo.

Initiating translation of most eukaryotic mRNAs depends on recruitment of methionyl initiator tRNA (Met-tRNAi) in a ternary complex (TC) with GTP-bound eukaryotic initiation factor 2 (eIF2) to the small (40S) ribosomal subunit, forming a 43S preinitiation complex (PIC) that attaches to the mRNA and scans the 5'-untranslated region (5' UTR) for an AUG start codon. Previous studies have implicated mammalian eIF2A in GTP-independent binding of Met-tRNAi to the 40S subunit and its recruitment to specialized mRNAs that do not require scanning, and in initiation at non-AUG start codons, when eIF2 function is attenuated by phosphorylation of its α-subunit during stress. The role of eIF2A in translation in vivo is poorly understood however, and it was unknown whether the conserved ortholog in budding yeast can functionally substitute for eIF2. We performed ribosome profiling of a yeast deletion mutant lacking eIF2A and isogenic wild-type (WT) cells in the presence or absence of eIF2α phosphorylation induced by starvation for amino acids isoleucine and valine. Whereas starvation of WT confers changes in translational efficiencies (TEs) of hundreds of mRNAs, the eIF2AΔ mutation conferred no significant TE reductions for any mRNAs in non-starved cells, and it reduced the TEs of only a small number of transcripts in starved cells containing phosphorylated eIF2α. We found no evidence that eliminating eIF2A altered the translation of mRNAs containing putative internal ribosome entry site (IRES) elements, or harboring uORFs initiated by AUG or near-cognate start codons, in non-starved or starved cells. Thus, very few mRNAs (possibly only one) appear to employ eIF2A for Met-tRNAi recruitment in yeast cells, even when eIF2 function is attenuated by stress.

N6-methyladenosine in 5' UTR does not promote translation initiation.

The most abundant N6-methyladenosine (m6A) modification on mRNAs is installed non-stoichiometrically across transcripts, with 5' untranslated regions (5' UTRs) being the least conductive. 5' UTRs are essential for translation initiation, yet the molecular mechanisms orchestrated by m6A remain poorly understood. Here, we combined structural, biochemical, and single-molecule approaches and show that at the most common position, a single m6A does not affect translation yields, the kinetics of translation initiation complex assembly, or start codon recognition both under permissive growth and following exposure to oxidative stress. Cryoelectron microscopy (cryo-EM) structures of the late preinitiation complex reveal that m6A purine ring established stacking interactions with an arginine side chain of the initiation factor eIF2α, although with only a marginal energy contribution, as estimated computationally. These findings provide molecular insights into m6A interactions with the initiation complex and suggest that the subtle stabilization is unlikely to affect the translation dynamics under homeostatic conditions or stress.

The molecular basis of translation initiation and its regulation in eukaryotes.

The regulation of gene expression is fundamental for life. Whereas the role of transcriptional regulation of gene expression has been studied for several decades, it has been clear over the past two decades that post-transcriptional regulation of gene expression, of which translation regulation is a major part, can be equally important. Translation can be divided into four main stages: initiation, elongation, termination and ribosome recycling. Translation is controlled mainly during its initiation, a process which culminates in a ribosome positioned with an initiator tRNA over the start codon and, thus, ready to begin elongation of the protein chain. mRNA translation has emerged as a powerful tool for the development of innovative therapies, yet the detailed mechanisms underlying the complex process of initiation remain unclear. Recent studies in yeast and mammals have started to shed light on some previously unclear aspects of this process. In this Review, we discuss the current state of knowledge on eukaryotic translation initiation and its regulation in health and disease. Specifically, we focus on recent advances in understanding the processes involved in assembling the 43S pre-initiation complex and its recruitment by the cap-binding complex eukaryotic translation initiation factor 4F (eIF4F) at the 5' end of mRNA. In addition, we discuss recent insights into ribosome scanning along the 5' untranslated region of mRNA and selection of the start codon, which culminates in joining of the 60S large subunit and formation of the 80S initiation complex.