The Structural Dynamics of Translation.

Accurate protein synthesis (translation) relies on translation factors that rectify ribosome fluctuations into a unidirectional process. Understanding this process requires structural characterization of the ribosome and translation-factor dynamics. In the 2000s, crystallographic studies determined high-resolution structures of ribosomes stalled with translation factors, providing a starting point for visualizing translation. Recent progress in single-particle cryogenic electron microscopy (cryo-EM) has enabled near-atomic resolution of numerous structures sampled in heterogeneous complexes (ensembles). Ensemble and time-resolved cryo-EM have now revealed unprecedented views of ribosome transitions in the three principal stages of translation: initiation, elongation, and termination. This review focuses on how translation factors help achieve high accuracy and efficiency of translation by monitoring distinct ribosome conformations and by differentially shifting the equilibria of ribosome rearrangements for cognate and near-cognate substrates.

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.

Alternative translation initiation produces synaptic organizer proteoforms with distinct localization and functions.

While many mRNAs contain more than one translation initiation site (TIS), the functions of most alternative TISs and their corresponding protein isoforms (proteoforms) remain undetermined. Here, we showed that alternative usage of CUG and AUG TISs in neuronal pentraxin receptor (NPR) mRNA produced two proteoforms, of which the ratio was regulated by RNA secondary structure and neuronal activity. Downstream AUG initiation truncated the N-terminal transmembrane domain and produced a secreted NPR proteoform sufficient in promoting synaptic clustering of AMPA-type glutamate receptors. Mutations that altered the ratio of NPR proteoforms reduced AMPA receptors in parvalbumin-positive interneurons and affected learning behaviors in mice. In addition to NPR, upstream AUU-initiated N-terminal extension of C1q-like synaptic organizers anchored these otherwise secreted factors to the membrane. Together, these results uncovered the plasticity of N-terminal signal sequences regulated by alternative TIS usage as a potentially widespread mechanism in diversifying protein localization and functions.

ALKBH1-Mediated tRNA Demethylation Regulates Translation.

tRNA is a central component of protein synthesis and the cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact its function. Here, we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N1-methyladenosine (m1A) in tRNAs. The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and decreased usage of tRNAs in protein synthesis. This process is dynamic and responds to glucose availability to affect translation. Our results uncover reversible methylation of tRNA as a new mechanism of post-transcriptional gene expression regulation.

The Hsp90 molecular chaperone governs client proteins by targeting intrinsically disordered regions.

Molecular chaperones govern proteome health to support cell homeostasis. An essential eukaryotic component of the chaperone system is Hsp90. Using a chemical-biology approach, we characterized the features driving the Hsp90 physical interactome. We found that Hsp90 associated with ∼20% of the yeast proteome using its three domains to preferentially target intrinsically disordered regions (IDRs) of client proteins. Hsp90 selectively utilized an IDR to regulate client activity as well as maintained IDR-protein health by preventing the transition to stress granules or P-bodies at physiological temperatures. We also discovered that Hsp90 controls the fidelity of ribosome initiation that triggers a heat shock response when disrupted. Our study provides insights into how this abundant molecular chaperone supports a dynamic and healthy native protein landscape.

In vitro reconstitution of SARS-CoV-2 Nsp1-induced mRNA cleavage reveals the key roles of the N-terminal domain of Nsp1 and the RRM domain of eIF3g.

SARS CoV-2 nonstructural protein 1 (Nsp1) is the major pathogenesis factor that inhibits host translation using a dual strategy of impairing initiation and inducing endonucleolytic cleavage of cellular mRNAs. To investigate the mechanism of cleavage, we reconstituted it in vitro on ß-globin, EMCV IRES, and CrPV IRES mRNAs that use unrelated initiation mechanisms. In all instances, cleavage required Nsp1 and only canonical translational components (40S subunits and initiation factors), arguing against involvement of a putative cellular RNA endonuclease. Requirements for initiation factors differed for these mRNAs, reflecting their requirements for ribosomal attachment. Cleavage of CrPV IRES mRNA was supported by a minimal set of components consisting of 40S subunits and eIF3g's RRM domain. The cleavage site was located in the coding region 18 nt downstream from the mRNA entrance, indicating that cleavage occurs on the solvent side of the 40S subunit. Mutational analysis identified a positively charged surface on Nsp1's N-terminal domain (NTD) and a surface above the mRNA-binding channel on eIF3g's RRM domain that contain residues essential for cleavage. These residues were required for cleavage on all three mRNAs, highlighting general roles of the Nsp1 NTD and eIF3g's RRM domain in cleavage per se, irrespective of the mode of ribosomal attachment.

NMD is required for timely cell fate transitions by fine-tuning gene expression and regulating translation.

Cell fate transitions depend on balanced rewiring of transcription and translation programs to mediate ordered developmental progression. Components of the nonsense-mediated mRNA decay (NMD) pathway have been implicated in regulating embryonic stem cell (ESC) differentiation, but the exact mechanism is unclear. Here we show that NMD controls expression levels of the translation initiation factor Eif4a2 and its premature termination codon-encoding isoform (Eif4a2PTC ). NMD deficiency leads to translation of the truncated eIF4A2PTC protein. eIF4A2PTC elicits increased mTORC1 activity and translation rates and causes differentiation delays. This establishes a previously unknown feedback loop between NMD and translation initiation. Furthermore, our results show a clear hierarchy in the severity of target deregulation and differentiation phenotypes between NMD effector KOs (Smg5 KO > Smg6 KO > Smg7 KO), which highlights heterodimer-independent functions for SMG5 and SMG7. Together, our findings expose an intricate link between mRNA homeostasis and mTORC1 activity that must be maintained for normal dynamics of cell state transitions.

The oncomicropeptide APPLE promotes hematopoietic malignancy by enhancing translation initiation.

Initiation is the rate-limiting step in translation, and its dysregulation is vital for carcinogenesis, including hematopoietic malignancy. Thus, discovery of novel translation initiation regulators may provide promising therapeutic targets. Here, combining Ribo-seq, mass spectrometry, and RNA-seq datasets, we discovered an oncomicropeptide, APPLE (a peptide located in ER), encoded by a non-coding RNA transcript in acute myeloid leukemia (AML). APPLE is overexpressed in various subtypes of AML and confers a poor prognosis. The micropeptide is enriched in ribosomes and regulates the initiation step to enhance translation and to maintain high rates of oncoprotein synthesis. Mechanically, APPLE promotes PABPC1-eIF4G interaction and facilitates mRNA circularization and eIF4F initiation complex assembly to support a specific pro-cancer translation program. Targeting APPLE exhibited broad anti-cancer effects in vitro and in vivo. This study not only reports a previously unknown function of micropeptides but also provides new opportunities for targeting the translation machinery in cancer cells.

Global phosphoproteomics pinpoints uncharted Gcn2-mediated mechanisms of translational control.

The conserved Gcn2 protein kinase mediates cellular adaptations to amino acid limitation through translational control of gene expression that is exclusively executed by phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α). Using quantitative phosphoproteomics, however, we discovered that Gcn2 targets auxiliary effectors to modulate translation. Accordingly, Gcn2 also phosphorylates the ß-subunit of the trimeric eIF2 G protein complex to promote its association with eIF5, which prevents spontaneous nucleotide exchange on eIF2 and thereby restricts the recycling of the initiator methionyl-tRNA-bound eIF2-GDP ternary complex in amino-acid-starved cells. This mechanism contributes to the inhibition of translation initiation in parallel to the sequestration of the nucleotide exchange factor eIF2B by phosphorylated eIF2α. Gcn2 further phosphorylates Gcn20 to antagonize, in an inhibitory feedback loop, the formation of the Gcn2-stimulatory Gcn1-Gcn20 complex. Thus, Gcn2 plays a substantially more intricate role in controlling translation initiation than hitherto appreciated.

eIF3 Associates with 80S Ribosomes to Promote Translation Elongation, Mitochondrial Homeostasis, and Muscle Health.

eIF3, a multi-subunit complex with numerous functions in canonical translation initiation, is known to interact with 40S and 60S ribosomal proteins and translation elongation factors, but a direct involvement in translation elongation has never been demonstrated. We found that eIF3 deficiency reduced early ribosomal elongation speed between codons 25 and 75 on a set of ∼2,700 mRNAs encoding proteins associated with mitochondrial and membrane functions, resulting in defective synthesis of their encoded proteins. To promote elongation, eIF3 interacts with 80S ribosomes translating the first ∼60 codons and serves to recruit protein quality-control factors, functions required for normal mitochondrial physiology. Accordingly, eIF3e+/- mice accumulate defective mitochondria in skeletal muscle and show a progressive decline in muscle strength. Hence, eIF3 interacts with 80S ribosomes to enhance, at the level of early elongation, the synthesis of proteins with membrane-associated functions, an activity that is critical for mitochondrial physiology and muscle health.

Selective 40S Footprinting Reveals Cap-Tethered Ribosome Scanning in Human Cells.

Translation regulation occurs largely during the initiation phase. Here, we develop selective 40S footprinting to visualize initiating 40S ribosomes on endogenous mRNAs in vivo. This reveals the positions on mRNAs where initiation factors join the ribosome to act and where they leave. We discover that in most human cells, most scanning ribosomes remain attached to the 5' cap. Consequently, only one ribosome scans a 5' UTR at a time, and 5' UTR length affects translation efficiency. We discover that eukaryotic initiation factor 3B (eIF3B,) eIF4G1, and eIF4E remain bound to 80S ribosomes as they begin translating, with a decay half-length of ∼12 codons. Hence, ribosomes retain these initiation factors while translating short upstream open reading frames (uORFs), providing an explanation for how ribosomes can reinitiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo.

Autism-Misregulated eIF4G Microexons Control Synaptic Translation and Higher Order Cognitive Functions.

Microexons represent the most highly conserved class of alternative splicing, yet their functions are poorly understood. Here, we focus on closely related neuronal microexons overlapping prion-like domains in the translation initiation factors, eIF4G1 and eIF4G3, the splicing of which is activity dependent and frequently disrupted in autism. CRISPR-Cas9 deletion of these microexons selectively upregulates synaptic proteins that control neuronal activity and plasticity and further triggers a gene expression program mirroring that of activated neurons. Mice lacking the Eif4g1 microexon display social behavior, learning, and memory deficits, accompanied by altered hippocampal synaptic plasticity. We provide evidence that the eIF4G microexons function as a translational brake by causing ribosome stalling, through their propensity to promote the coalescence of cytoplasmic granule components associated with translation repression, including the fragile X mental retardation protein FMRP. The results thus reveal an autism-disrupted mechanism by which alternative splicing specializes neuronal translation to control higher order cognitive functioning.

The ASC-1 complex promotes translation initiation by scanning ribosomes.

Translation initiates when the eIF4F complex binds the 5' mRNA cap, followed by 5' untranslated region scanning for the start codon by scanning ribosomes. Here, we demonstrate that the ASC-1 complex (ASCC), which was previously shown to promote the dissociation of colliding 80S ribosomes, associates with scanning ribosomes to regulate translation initiation. Selective translation complex profiling (TCP-seq) analysis revealed that ASCC3, a helicase domain-containing subunit of ASCC, localizes predominantly to the 5' untranslated region of mRNAs. Ribo-seq, TCP-seq, and luciferase reporter analyses showed that ASCC3 knockdown impairs 43S preinitiation complex loading and scanning dynamics, thereby reducing translation efficiency. Whereas eIF4A, an RNA helicase in the eIF4F complex, is important for global translation, ASCC was found to regulate the scanning process for a specific subset of transcripts. Our results have thus revealed that ASCC is required not only for dissociation of colliding 80S ribosomes but also for efficient translation initiation by scanning ribosomes at a subset of transcripts.

eIF3 mRNA selectivity profiling reveals eIF3k as a cancer-relevant regulator of ribosome content.

eIF3, whose subunits are frequently overexpressed in cancer, regulates mRNA translation from initiation to termination, but mRNA-selective functions of individual subunits remain poorly defined. Using multiomic profiling upon acute depletion of eIF3 subunits, we observed that while eIF3a, b, e, and f markedly differed in their impact on eIF3 holo-complex formation and translation, they were each required for cancer cell proliferation and tumor growth. Remarkably, eIF3k showed the opposite pattern with depletion promoting global translation, cell proliferation, tumor growth, and stress resistance through repressing the synthesis of ribosomal proteins, especially RPS15A. Whereas ectopic expression of RPS15A mimicked the anabolic effects of eIF3k depletion, disruption of eIF3 binding to the 5'-UTR of RSP15A mRNA negated them. eIF3k and eIF3l are selectively downregulated in response to endoplasmic reticulum and oxidative stress. Supported by mathematical modeling, our data uncover eIF3k-l as a mRNA-specific module which, through controlling RPS15A translation, serves as a rheostat of ribosome content, possibly to secure spare translational capacity that can be mobilized during stress.

Retapamulin-Assisted Ribosome Profiling Reveals the Alternative Bacterial Proteome.

The use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding the cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes, but strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at internal in-frame and out-of-frame start sites, can be functionally important and contribute to the "alternative" bacterial proteome. The internal start sites may also play regulatory roles in gene expression.

Computational inference of eIF4F complex function and structure in human cancers.

The canonical eukaryotic initiation factor 4F (eIF4F) complex, composed of eIF4G1, eIF4A1, and the cap-binding protein eIF4E, plays a crucial role in cap-dependent translation initiation in eukaryotic cells. An alternative cap-independent initiation can occur, involving only eIF4G1 and eIF4A1 through internal ribosome entry sites (IRESs). This mechanism is considered complementary to cap-dependent initiation, particularly in tumors under stress conditions. However, the selection and molecular mechanism of specific translation initiation remains poorly understood in human cancers. Thus, we analyzed gene copy number variations (CNVs) in TCGA tumor samples and found frequent amplification of genes involved in translation initiation. Copy number gains in EIF4G1 and EIF3E frequently co-occur across human cancers. Additionally, EIF4G1 expression strongly correlates with genes from cancer cell survival pathways including cell cycle and lipogenesis, in tumors with EIF4G1 amplification or duplication. Furthermore, we revealed that eIF4G1 and eIF4A1 protein levels strongly co-regulate with ribosomal subunits, eIF2, and eIF3 complexes, while eIF4E co-regulates with 4E-BP1, ubiquitination, and ESCRT proteins. Utilizing Alphafold predictions, we modeled the eIF4F structure with and without eIF4E binding. For cap-dependent initiation, our modeling reveals extensive interactions between the N-terminal eIF4E-binding domain of eIF4G1 and eIF4E. Furthermore, the eIF4G1 HEAT-2 domain positions eIF4E near the eIF4A1 N-terminal domain (NTD), resulting in the collaborative enclosure of the RNA binding cavity within eIF4A1. In contrast, during cap-independent initiation, the HEAT-2 domain directly binds the eIF4A1-NTD, leading to a stronger interaction between eIF4G1 and eIF4A1, thus closing the mRNA binding cavity without the involvement of eIF4E.

Ribosome queuing enables non-AUG translation to be resistant to multiple protein synthesis inhibitors.

Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation may be regulated differently from canonical translation is poorly understood. Here, we used start codon-specific reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of multiple non-AUG-encoded reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA is resistant to cycloheximide (CHX), a translation inhibitor that severely slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes can lead to queuing/stacking of scanning preinitiation complexes (PICs), preferentially enhancing recognition of weak non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. We further found that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence but not those targeting newly initiated ribosomes. Together, these data indicate that ribosome queuing enables mRNAs with poor initiation context-namely, those with non-AUG start codons-to be resistant to pharmacological translation inhibitors at concentrations that robustly inhibit global translation.

Molecular architecture of 40S translation initiation complexes on the hepatitis C virus IRES.

Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Biochemical studies revealed that direct binding of the IRES to the 40S ribosomal subunit places the initiation codon into the P site, where it base pairs with eIF2-bound Met-tRNAiMet forming a 48S initiation complex. Subsequently, eIF5 and eIF5B mediate subunit joining, yielding an elongation-competent 80S ribosome. Initiation can also proceed without eIF2, in which case Met-tRNAiMet is recruited directly by eIF5B. However, the structures of initiation complexes assembled on the HCV IRES, the transitions between different states, and the accompanying conformational changes have remained unknown. To fill these gaps, we now obtained cryo-EM structures of IRES initiation complexes, at resolutions up to 3.5 Å, that cover all major stages from the initial ribosomal association, through eIF2-containing 48S initiation complexes, to eIF5B-containing complexes immediately prior to subunit joining. These structures provide insights into the dynamic network of 40S/IRES contacts, highlight the role of IRES domain II, and reveal conformational changes that occur during the transition from eIF2- to eIF5B-containing 48S complexes and prepare them for subunit joining.

The TIA1 RNA-Binding Protein Family Regulates EIF2AK2-Mediated Stress Response and Cell Cycle Progression.

TIA1 and TIAL1 encode a family of U-rich element mRNA-binding proteins ubiquitously expressed and conserved in metazoans. Using PAR-CLIP, we determined that both proteins bind target sites with identical specificity in 3' UTRs and introns proximal to 5' as well as 3' splice sites. Double knockout (DKO) of TIA1 and TIAL1 increased target mRNA abundance proportional to the number of binding sites and also caused accumulation of aberrantly spliced mRNAs, most of which are subject to nonsense-mediated decay. Loss of PRKRA by mis-splicing triggered the activation of the double-stranded RNA (dsRNA)-activated protein kinase EIF2AK2/PKR and stress granule formation. Ectopic expression of PRKRA cDNA or knockout of EIF2AK2 in DKO cells rescued this phenotype. Perturbation of maturation and/or stability of additional targets further compromised cell cycle progression. Our study reveals the essential contributions of the TIA1 protein family to the fidelity of mRNA maturation, translation, and RNA-stress-sensing pathways in human cells.

Structural analysis of noncanonical translation initiation complexes.

Translation initiation is a highly regulated, multi-step process that is critical for efficient and accurate protein synthesis. In bacteria, initiation begins when mRNA, initiation factors, and a dedicated initiator fMet-tRNAfMet bind the small (30S) ribosomal subunit. Specific binding of fMet-tRNAfMet in the peptidyl (P) site is mediated by the inspection of the fMet moiety by initiation factor IF2 and of three conserved G-C base pairs in the tRNA anticodon stem by the 30S head domain. Tandem A-minor interactions form between 16S ribosomal RNA nucleotides A1339 and G1338 and tRNA base pairs G30-C40 and G29-C41, respectively. Swapping the G30-C40 pair of tRNAfMet with C-G (called tRNAfMet M1) reduces discrimination against the noncanonical start codon CUG in vitro, suggesting crosstalk between the gripping of the anticodon stem and recognition of the start codon. Here, we solved electron cryomicroscopy structures of Escherichia coli 70S initiation complexes containing the fMet-tRNAfMet M1 variant paired to the noncanonical CUG start codon, in the presence or absence of IF2 and the non-hydrolyzable GTP analog GDPCP, alongside structures of 70S initiation complexes containing this tRNAfMet variant paired to the canonical bacterial start codons AUG, GUG, and UUG. We find that the M1 mutation weakens A-minor interactions between tRNAfMet and 16S nucleotides A1339 and G1338, with IF2 strengthening the interaction of G1338 with the tRNA minor groove. These structures suggest how even slight changes to the recognition of the fMet-tRNAfMet anticodon stem by the ribosome can impact the start codon selection.