Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation.

How the eukaryotic 43S preinitiation complex scans along the 5' untranslated region (5' UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A. Once engaged, 43S scanning occurs rapidly and directionally at ∼100 nucleotides per second, independent of multiple cycles of ATP hydrolysis by RNA helicases post ribosomal loading. Scanning ribosomes can proceed through RNA secondary structures, but 5' UTR hairpin sequences near start codons drive scanning ribosomes at start codons backward in the 5' direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5' UTR structures and upstream near-cognate start codons.

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.

PRRC2 proteins impact translation initiation by promoting leaky scanning.

Roughly half of animal mRNAs contain upstream open reading frames (uORFs). These uORFs can represent an impediment to translation of the main ORF since ribosomes usually bind the mRNA cap at the 5' end and then scan for ORFs in a 5'-to-3' fashion. One way for ribosomes to bypass uORFs is via leaky scanning, whereby the ribosome disregards the uORF start codon. Hence leaky scanning is an important instance of post-transcriptional regulation that affects gene expression. Few molecular factors regulating or facilitating this process are known. Here we show that the PRRC2 proteins PRRC2A, PRRC2B and PRRC2C impact translation initiation. We find that they bind eukaryotic translation initiation factors and preinitiation complexes, and are enriched on ribosomes translating mRNAs with uORFs. We find that PRRC2 proteins promote leaky scanning past translation start codons, thereby promoting translation of mRNAs containing uORFs. Since PRRC2 proteins have been associated with cancer, this provides a mechanistic starting point for understanding their physiological and pathophysiological roles.

Increased levels of eIF2A inhibit translation by sequestering 40S ribosomal subunits.

eIF2A was the first eukaryotic initiator tRNA carrier discovered but its exact function has remained enigmatic. Uncharacteristic of translation initiation factors, eIF2A is reported to be non-cytosolic in multiple human cancer cell lines. Attempts to study eIF2A mechanistically have been limited by the inability to achieve high yield of soluble recombinant protein. Here, we developed a purification paradigm that yields ∼360-fold and ∼6000-fold more recombinant human eIF2A from Escherichia coli and insect cells, respectively, than previous reports. Using a mammalian in vitro translation system, we found that increased levels of recombinant human eIF2A inhibit translation of multiple reporter mRNAs, including those that are translated by cognate and near-cognate start codons, and does so prior to start codon recognition. eIF2A also inhibited translation directed by all four types of cap-independent viral IRESs, including the CrPV IGR IRES that does not require initiation factors or initiator tRNA, suggesting excess eIF2A sequesters 40S subunits. Supplementation with additional 40S subunits prevented eIF2A-mediated inhibition and pull-down assays demonstrated direct binding between recombinant eIF2A and purified 40S subunits. These data support a model that eIF2A must be kept away from the translation machinery to avoid sequestering 40S ribosomal subunits.

Translational fidelity screens in mammalian cells reveal eIF3 and eIF4G2 as regulators of start codon selectivity.

The translation initiation machinery and the ribosome orchestrate a highly dynamic scanning process to distinguish proper start codons from surrounding nucleotide sequences. Here, we performed genome-wide CRISPRi screens in human K562 cells to systematically identify modulators of the frequency of translation initiation at near-cognate start codons. We observed that depletion of any eIF3 core subunit promoted near-cognate start codon usage, though sensitivity thresholds of each subunit to sgRNA-mediated depletion varied considerably. Double sgRNA depletion experiments suggested that enhanced near-cognate usage in eIF3D depleted cells required canonical eIF4E cap-binding and was not driven by eIF2A or eIF2D-dependent leucine tRNA initiation. We further characterized the effects of eIF3D depletion and found that the N-terminus of eIF3D was strictly required for accurate start codon selection, whereas disruption of the cap-binding properties of eIF3D had no effect. Lastly, depletion of eIF3D activated TNFα signaling via NF-κB and the interferon gamma response. Similar transcriptional profiles were observed upon knockdown of eIF1A and eIF4G2, which also promoted near-cognate start codon usage, suggesting that enhanced near-cognate usage could potentially contribute to NF-κB activation. Our study thus provides new avenues to study the mechanisms and consequences of alternative start codon usage.

Selective translational control of cellular and viral mRNAs by RPS3 mRNA binding.

RPS3, a universal core component of the 40S ribosomal subunit, interacts with mRNA at the entry channel. Whether RPS3 mRNA-binding contributes to specific mRNA translation and ribosome specialization in mammalian cells is unknown. Here we mutated RPS3 mRNA-contacting residues R116, R146 and K148 and report their impact on cellular and viral translation. R116D weakened cap-proximal initiation and promoted leaky scanning, while R146D had the opposite effect. Additionally, R146D and K148D displayed contrasting effects on start-codon fidelity. Translatome analysis uncovered common differentially translated genes of which the downregulated set bears long 5'UTR and weak AUG context, suggesting a stabilizing role during scanning and AUG selection. We identified an RPS3-dependent regulatory sequence (RPS3RS) in the sub-genomic 5'UTR of SARS-CoV-2 consisting of a CUG initiation codon and a downstream element that is also the viral transcription regulatory sequence (TRS). Furthermore, RPS3 mRNA-binding residues are essential for SARS-CoV-2 NSP1-mediated inhibition of host translation and for its ribosomal binding. Intriguingly, NSP1-induced mRNA degradation was also reduced in R116D cells, indicating that mRNA decay occurs in the ribosome context. Thus, RPS3 mRNA-binding residues have multiple translation regulatory functions and are exploited by SARS-CoV-2 in various ways to influence host and viral mRNA translation and stability.

Modeling the ribosomal small subunit dynamic in Saccharomyces cerevisiae based on TCP-seq data.

Translation Complex Profile Sequencing (TCP-seq), a protocol that was developed and implemented on Saccharomyces cerevisiae, provides the footprints of the small subunit (SSU) of the ribosome (with additional factors) across the entire transcriptome of the analyzed organism. In this study, based on the TCP-seq data, we developed for the first-time a predictive model of the SSU density and analyzed the effect of transcript features on the dynamics of the SSU scan in the 5'UTR. Among others, our model is based on novel tools for detecting complex statistical relations tailored to TCP-seq. We quantitatively estimated the effect of several important features, including the context of the upstream AUG, the upstream ORF length and the mRNA folding strength. Specifically, we suggest that around 50% of the variance related to the read counts (RC) distribution near a start codon can be attributed to the AUG context score. We provide the first large scale direct quantitative evidence that shows that indeed AUG context affects the small sub-unit movement. In addition, we suggest that strong folding may cause the detachment of the SSU from the mRNA. We also identified a number of novel sequence motifs that can affect the SSU scan; some of these motifs affect transcription factors and RNA binding proteins. The results presented in this study provide a better understanding of the biophysical aspects related to the SSU scan along the 5'UTR and of translation initiation in S. cerevisiae, a fundamental step toward a comprehensive modeling of initiation.

Conformational rearrangements upon start codon recognition in human 48S translation initiation complex.

Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2-GTP-Met-tRNAiMet and eIF3. The 'open' 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The 'closed' form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

Pentatricopeptide repeat protein CNS1 regulates maize mitochondrial complex III assembly and seed development.

Mitochondrial function relies on the assembly of electron transport chain complexes, which requires coordination between proteins encoded by the mitochondrion and those of the nucleus. Here, we cloned a maize (Zea mays) cytochrome c maturation FN stabilizer1 (CNS1) and found it encodes a pentatricopeptide repeat (PPR) protein. Members of the PPR family are widely distributed in plants and are associated with RNA metabolism in organelles. P-type PPR proteins play essential roles in stabilizing the 3'-end of RNA in mitochondria; whether a similar process exists for stabilizing the 5'-terminus of mitochondrial RNA remains unclear. The kernels of cns1 exhibited arrested embryo and endosperm development, whereas neither conventional splicing deficiency nor RNA editing difference in mitochondrial genes was observed. Instead, most of the ccmFN transcripts isolated from cns1 mutant plants were 5'-truncated and therefore lacked the start codon. Biochemical and molecular data demonstrated that CNS1 is a P-type PPR protein encoded by nuclear DNA and that it localizes to the mitochondrion. Also, one binding site of CNS1 located upstream of the start codon in the ccmFN transcript. Moreover, abnormal mitochondrial morphology and dramatic upregulation of alternative oxidase genes were observed in the mutant. Together, these results indicate that CNS1 is essential for reaching a suitable level of intact ccmFN transcripts through binding to the 5'-UTR of the RNAs and maintaining 5'-integrity, which is crucial for sustaining mitochondrial complex III function to ensure mitochondrial biogenesis and seed development in maize.

Multifaceted deregulation of gene expression and protein synthesis with age.

Protein synthesis represents a major metabolic activity of the cell. However, how it is affected by aging and how this in turn impacts cell function remains largely unexplored. To address this question, herein we characterized age-related changes in both the transcriptome and translatome of mouse tissues over the entire life span. We showed that the transcriptome changes govern those in the translatome and are associated with altered expression of genes involved in inflammation, extracellular matrix, and lipid metabolism. We also identified genes that may serve as candidate biomarkers of aging. At the translational level, we uncovered sustained down-regulation of a set of 5'-terminal oligopyrimidine (5'-TOP) transcripts encoding protein synthesis and ribosome biogenesis machinery and regulated by the mTOR pathway. For many of them, ribosome occupancy dropped twofold or even more. Moreover, with age, ribosome coverage gradually decreased in the vicinity of start codons and increased near stop codons, revealing complex age-related changes in the translation process. Taken together, our results reveal systematic and multidimensional deregulation of protein synthesis, showing how this major cellular process declines with age.

The CYCLOPS Response Element in the NIN Promoter Is Important but Not Essential for Infection Thread Formation During Lotus japonicus-Rhizobia Symbiosis.

The establishment of the legume-rhizobia symbiosis, termed the root-nodule symbiosis (RNS), requires elaborate interactions at the molecular level. The host plant-derived transcription factor NODULE INCEPTION (NIN) is known to be crucial for RNS, regulating associated processes such as alteration of root hair morphology, infection thread formation, and cell division during nodulation. This emphasizes the importance of the precise spatiotemporal regulation of NIN expression for the establishment of RNS; however, the detailed role of NIN promoter sequences in this process remains unclear. The daphne mutant, a nin mutant allele containing a chromosomal translocation approximately 7 kb upstream of the start codon, does not form nodules but does form infection threads, indicating that the region within 7 kb of the NIN start codon contributes to NIN expression during infection thread formation. CYCLOPS binds to a CYCLOPS response element (CYC-RE) in the NIN promoter, and cyclops mutants are defective in infection thread formation. Here, we performed complementation analysis in nin mutants, using various truncated forms of the NIN promoter, and found that the CYC-RE is important for infection thread formation. Additionally, the CYC-RE deletion mutant, generated through CRISPR/Cas9 technology, displayed a significant reduction in infection thread formation, indicating that the CYC-RE is important for the fine-tuning of NIN expression during this process. However, the fact that infection thread formation is not completely abolished in the CYC-RE deletion mutant suggests that cis and trans factors other than CYCLOPS and the CYC-RE may cooperatively regulate NIN expression for the induction of infection thread formation. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Free energy landscape of RNA binding dynamics in start codon recognition by eukaryotic ribosomal pre-initiation complex.

Specific interaction between the start codon, 5'-AUG-3', and the anticodon, 5'-CAU-3', ensures accurate initiation of translation. Recent studies show that several near-cognate start codons (e.g. GUG and CUG) can play a role in initiating translation in eukaryotes. However, the mechanism allowing initiation through mismatched base-pairs at the ribosomal decoding site is still unclear at an atomic level. In this work, we propose an extended simulation-based method to evaluate free energy profiles, through computing the distance between each base-pair of the triplet interactions involved in recognition of start codons in eukaryotic translation pre-initiation complex. Our method provides not only the free energy penalty for mismatched start codons relative to the AUG start codon, but also the preferred pathways of transitions between bound and unbound states, which has not been described by previous studies. To verify the method, the binding dynamics of cognate (AUG) and near-cognate start codons (CUG and GUG) were simulated. Evaluated free energy profiles agree with experimentally observed changes in initiation frequencies from respective codons. This work proposes for the first time how a G:U mismatch at the first position of codon (GUG)-anticodon base-pairs destabilizes the accommodation in the initiating eukaryotic ribosome and how initiation at a CUG codon is nearly as strong as, or sometimes stronger than, that at a GUG codon. Our method is expected to be applied to study the affinity changes for various mismatched base-pairs.

Dynamics of ribosome scanning and recycling revealed by translation complex profiling.

Regulation of messenger RNA translation is central to eukaryotic gene expression control. Regulatory inputs are specified by them RNA untranslated regions (UTRs) and often target translation initiation. Initiation involves binding of the 40S ribosomal small subunit (SSU) and associated eukaryotic initiation factors (eIFs)near the mRNA 5' cap; the SSU then scans in the 3' direction until it detects the start codon and is joined by the 60S ribosomal large subunit (LSU) to form the 80S ribosome. Scanning and other dynamic aspects of the initiation model have remained as conjectures because methods to trap early intermediates were lacking. Here we uncover the dynamics of the complete translation cycle in live yeast cells using translation complex profile sequencing (TCP-seq), a method developed from the ribosome profiling approach. We document scanning by observing SSU footprints along 5' UTRs. Scanning SSU have 5'-extended footprints (up to~75 nucleotides), indicative of additional interactions with mRNA emerging from the exit channel, promoting forward movement. We visualized changes in initiation complex conformation as SSU footprints coalesced into three major sizes at start codons (19, 29 and 37 nucleotides). These share the same 5' start site but differ at the 3' end, reflecting successive changes at the entry channel from an open to a closed state following start codon recognition. We also observe SSU 'lingering' at stop codons after LSU departure. Our results underpin mechanistic models of translation initiation and termination, built on decades of biochemical and structural investigation, with direct genome-wide in vivo evidence. Our approach captures ribosomal complexes at all phases of translation and will aid in studying translation dynamics in diverse cellular contexts. Dysregulation of translation is common in disease and, for example, SSU scanning is a target of anti-cancer drug development. TCP-seq will prove useful in discerning differences in mRNA-specific initiation in pathologies and their response to treatment.

Improving the prediction accuracy of protein abundance in Escherichia coli using mRNA accessibility.

RNA secondary structure around translation initiation sites strongly affects the abundance of expressed proteins in Escherichia coli. However, detailed secondary structural features governing protein abundance remain elusive. Recent advances in high-throughput DNA synthesis and experimental systems enable us to obtain large amounts of data. Here, we evaluated six types of structural features using two large-scale datasets. We found that accessibility, which is the probability that a given region around the start codon has no base-paired nucleotides, showed the highest correlation with protein abundance in both datasets. Accessibility showed a significantly higher correlation (Spearman's ρ = 0.709) than the widely used minimum free energy (0.554) in one of the datasets. Interestingly, accessibility showed the highest correlation only when it was calculated by a log-linear model, indicating that the RNA structural model and how to utilize it are important. Furthermore, by combining the accessibility and activity of the Shine-Dalgarno sequence, we devised a method for predicting protein abundance more accurately than existing methods. We inferred that the log-linear model has a broader probabilistic distribution than the widely used Turner energy model, which contributed to more accurate quantification of ribosome accessibility to translation initiation sites.

Quantitative global studies reveal differential translational control by start codon context across the fungal kingdom.

Eukaryotic protein synthesis generally initiates at a start codon defined by an AUG and its surrounding Kozak sequence context, but the quantitative importance of this context in different species is unclear. We tested this concept in two pathogenic Cryptococcus yeast species by genome-wide mapping of translation and of mRNA 5' and 3' ends. We observed thousands of AUG-initiated upstream open reading frames (uORFs) that are a major contributor to translation repression. uORF use depends on the Kozak sequence context of its start codon, and uORFs with strong contexts promote nonsense-mediated mRNA decay. Transcript leaders in Cryptococcus and other fungi are substantially longer and more AUG-dense than in Saccharomyces. Numerous Cryptococcus mRNAs encode predicted dual-localized proteins, including many aminoacyl-tRNA synthetases, in which a leaky AUG start codon is followed by a strong Kozak context in-frame AUG, separated by mitochondrial-targeting sequence. Analysis of other fungal species shows that such dual-localization is also predicted to be common in the ascomycete mould, Neurospora crassa. Kozak-controlled regulation is correlated with insertions in translational initiation factors in fidelity-determining regions that contact the initiator tRNA. Thus, start codon context is a signal that quantitatively programs both the expression and the structures of proteins in diverse fungi.

Elusive Trans-Acting Factors Which Operate with Type I (Poliovirus-like) IRES Elements.

The phenomenon of internal initiation of translation was discovered in 1988 on poliovirus mRNA. The prototypic cis-acting element in the 5' untranslated region (5'UTR) of poliovirus mRNA, which is able to direct initiation at an internal start codon without the involvement of a cap structure, has been called an IRES (Internal Ribosome Entry Site or Segment). Despite its early discovery, poliovirus and other related IRES elements of type I are poorly characterized, and it is not yet clear which host proteins (a.k.a. IRES trans-acting factors, ITAFs) are required for their full activity in vivo. Here we discuss recent and old results devoted to type I IRESes and provide evidence that Poly(rC) binding protein 2 (PCBP2), Glycyl-tRNA synthetase (GARS), and Cold Shock Domain Containing E1 (CSDE1, also known as UNR) are major regulators of type I IRES activity.

A viral RNA motif involved in signaling the initiation of translation on non-AUG codons.

Noncanonical translation, and particularly initiation on non-AUG codons, are frequently used by viral and cellular mRNAs during virus infection and disease. The Sindbis virus (SINV) subgenomic mRNA (sgRNA) constitutes a unique model system to analyze the translation of a capped viral mRNA without the participation of several initiation factors. Moreover, sgRNA can initiate translation even when the AUG initiation codon is replaced by other codons. Using SINV replicons, we examined the efficacy of different codons in place of AUG to direct the synthesis of the SINV capsid protein. The substitution of AUG by CUG was particularly efficient in promoting the incorporation of leucine or methionine in similar percentages at the amino terminus of the capsid protein. Additionally, valine could initiate translation when the AUG is replaced by GUG. The ability of sgRNA to initiate translation on non-AUG codons was dependent on the integrity of a downstream stable hairpin (DSH) structure located in the coding region. The structural requirements of this hairpin to signal the initiation site on the sgRNA were examined in detail. Of interest, a virus bearing CUG in place of AUG in the sgRNA was able to infect cells and synthesize significant amounts of capsid protein. This virus infects the human haploid cell line HAP1 and the double knockout variant that lacks eIF2A and eIF2D. Collectively, these findings indicate that leucine-tRNA or valine-tRNA can participate in the initiation of translation of sgRNA by a mechanism dependent on the DSH. This mechanism does not involve the action of eIF2, eIF2A, or eIF2D.

Comprehensive profiling of translation initiation in influenza virus infected cells.

Translation can initiate at alternate, non-canonical start codons in response to stressful stimuli in mammalian cells. Recent studies suggest that viral infection and anti-viral responses alter sites of translation initiation, and in some cases, lead to production of novel immune epitopes. Here we systematically investigate the extent and impact of alternate translation initiation in cells infected with influenza virus. We perform evolutionary analyses that suggest selection against non-canonical initiation at CUG codons in influenza virus lineages that have adapted to mammalian hosts. We then use ribosome profiling with the initiation inhibitor lactimidomycin to experimentally delineate translation initiation sites in a human lung epithelial cell line infected with influenza virus. We identify several candidate sites of alternate initiation in influenza mRNAs, all of which occur at AUG codons that are downstream of canonical initiation codons. One of these candidate downstream start sites truncates 14 amino acids from the N-terminus of the N1 neuraminidase protein, resulting in loss of its cytoplasmic tail and a portion of the transmembrane domain. This truncated neuraminidase protein is expressed on the cell surface during influenza virus infection, is enzymatically active, and is conserved in most N1 viral lineages. We do not detect globally higher levels of alternate translation initiation on host transcripts upon influenza infection or during the anti-viral response, but the subset of host transcripts induced by the anti-viral response is enriched for alternate initiation sites. Together, our results systematically map the landscape of translation initiation during influenza virus infection, and shed light on the evolutionary forces shaping this landscape.

Non-AUG start codons: Expanding and regulating the small and alternative ORFeome.

Recent ribosome profiling and proteomic studies have revealed the presence of thousands of novel coding sequences, referred to as small open reading frames (sORFs), in prokaryotic and eukaryotic genomes. These genes have defied discovery via traditional genomic tools not only because they tend to be shorter than standard gene annotation length cutoffs, but also because they are, as a class, enriched in sequence properties previously assumed to be unusual, including non-AUG start codons. In this review, we summarize what is currently known about the incidence, efficiency, and mechanism of non-AUG start codon usage in prokaryotes and eukaryotes, and provide examples of regulatory and functional sORFs that initiate at non-AUG codons. While only a handful of non-AUG-initiated novel genes have been characterized in detail to date, their participation in important biological processes suggests that an improved understanding of this class of genes is needed.

Ribosome assembly defects subvert initiation Factor3 mediated scrutiny of bona fide start signal.

In bacteria, the assembly factors tightly orchestrate the maturation of ribosomes whose competency for protein synthesis is validated by translation machinery at various stages of translation cycle. However, what transpires to the quality control measures when the ribosomes are produced with assembly defects remains enigmatic. In Escherichia coli, we show that 30S ribosomes that harbour assembly defects due to the lack of assembly factors such as RbfA and KsgA display suboptimal initiation codon recognition and bypass the critical codon-anticodon proofreading steps during translation initiation. These premature ribosomes on entering the translation cycle compromise the fidelity of decoding that gives rise to errors during initiation and elongation. We show that the assembly defects compromise the binding of initiation factor 3 (IF3), which in turn appears to license the rapid transition of 30S (pre) initiation complex to 70S initiation complex by tempering the validation of codon-anticodon interaction during translation initiation. This suggests that the premature ribosomes harbouring the assembly defects subvert the IF3 mediated proofreading of cognate initiation codon to enter the translation cycle.