dCas13-mediated translational repression for accurate gene silencing in mammalian cells.

Current gene silencing tools based on RNA interference (RNAi) or, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)‒Cas13 systems have critical drawbacks, such as off-target effects (RNAi) or collateral mRNA cleavage (CRISPR‒Cas13). Thus, a more specific method of gene knockdown is needed. Here, we develop CRISPRδ, an approach for translational silencing, harnessing catalytically inactive Cas13 proteins (dCas13). Owing to its tight association with mRNA, dCas13 serves as a physical roadblock for scanning ribosomes during translation initiation and does not affect mRNA stability. Guide RNAs covering the start codon lead to the highest efficacy regardless of the translation initiation mechanism: cap-dependent, internal ribosome entry site (IRES)-dependent, or repeat-associated non-AUG (RAN) translation. Strikingly, genome-wide ribosome profiling reveals the ultrahigh gene silencing specificity of CRISPRδ. Moreover, the fusion of a translational repressor to dCas13 further improves the performance. Our method provides a framework for translational repression-based gene silencing in eukaryotes.

Modeling alternative translation initiation sites in plants reveals evolutionarily conserved cis-regulatory codes in eukaryotes.

mRNA translation relies on identifying translation initiation sites (TISs) in mRNAs. Alternative TISs are prevalent across plant transcriptomes, but the mechanisms for their recognition are unclear. Using ribosome profiling and machine learning, we developed models for predicting alternative TISs in the tomato (Solanum lycopersicum). Distinct feature sets were predictive of AUG and nonAUG TISs in 5' untranslated regions and coding sequences, including a novel CU-rich sequence that promoted plant TIS activity, a translational enhancer found across dicots and monocots, and humans and viruses. Our results elucidate the mechanistic and evolutionary basis of TIS recognition, whereby cis-regulatory RNA signatures affect start site selection. The TIS prediction model provides global estimates of TISs to discover neglected protein-coding genes across plant genomes. The prevalence of cis-regulatory signatures across plant species, humans, and viruses suggests their broad and critical roles in reprogramming the translational landscape.

Protein phosphatase 1 regulatory subunit 15 A promotes translation initiation and induces G2M phase arrest during cuproptosis in cancers.

Copper ions play a crucial role as cofactors for essential enzymes in cellular processes. However, when the intracellular concentration of copper ions exceeds the homeostatic threshold, they become toxic to cells. In our study, we demonstrated that elesclomol, as a carrier of copper ions, caused an upregulation of protein phosphatase 1 regulatory subunit 15 A (PPP1R15A), which plays a role in regulating substrate selectivity of protein phosphatase 1 during cuproptosis. Mechanistically, we investigated that PPP1R15A activated translation initiation by dephosphorylating eukaryotic translation initiation factor 2 subunit alpha at the S51 residue through protein phosphatase 1 and phosphorylating eukaryotic translation initiation factor 4E binding protein 1 at the T70 residue. In addition, PPP1R15A reduced H3K4 methylation by altering the phosphorylation of histone methyltransferases, which led to the silencing of MYC and G2M phase arrest.

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.

How Dedicated Ribosomes Translate a Leaderless mRNA.

In bacteriophage λ lysogens, the λcI repressor is encoded by the leaderless transcript (lmRNA) initiated at the λpRM promoter. Translation is enhanced in rpsB mutants deficient in ribosomal protein uS2. Although translation initiation of lmRNA is conserved in bacteria, archaea, and eukaryotes, structural insight of a lmRNA translation initiation complex is missing. Here, we use cryo-EM to solve the structures of the uS2-deficient 70S ribosome of host E. coli mutant rpsB11 and the wild-type 70S complex with λcI lmRNA and fMet-tRNAfMet. Importantly, the uS2-deficient 70S ribosome also lacks protein bS21. The anti-Shine-Dalgarno (aSD) region is structurally supported by bS21, so that the absence of the latter causes the aSD to divert from the normal mRNA exit pathway, easing the exit of lmRNA. A π-stacking interaction between the monitor base A1493 and A(+4) of lmRNA potentially acts as a recognition signal. Coulomb charge flow, along with peristalsis-like dynamics within the mRNA entrance channel due to the increased 30S head rotation caused by the absence of uS2, are likely to facilitate the propagation of lmRNA through the ribosome. These findings lay the groundwork for future research on the mechanism of translation and the co-evolution of lmRNA and mRNA that includes the emergence of a defined ribosome-binding site of the transcript.

The structure of a human translation initiation complex reveals two independent roles for the helicase eIF4A.

Eukaryotic translation initiation involves recruitment of the 43S pre-initiation complex to the 5' end of mRNA by the cap-binding complex eIF4F, forming the 48S translation initiation complex (48S), which then scans along the mRNA until the start codon is recognized. We have previously shown that eIF4F binds near the mRNA exit channel of the 43S, leaving open the question of how mRNA secondary structure is removed as it enters the mRNA channel on the other side of the 40S subunit. Here we report the structure of a human 48S that shows that, in addition to the eIF4A that is part of eIF4F, there is a second eIF4A helicase bound at the mRNA entry site, which could unwind RNA secondary structures as they enter the 48S. The structure also reveals conserved interactions between eIF4F and the 43S, probaby explaining how eIF4F can promote mRNA recruitment in all eukaryotes.

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.

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.

Functional analysis of the AUG initiator codon context reveals novel conserved sequences that disfavor mRNA translation in eukaryotes.

mRNA translation is a fundamental process for life. Selection of the translation initiation site (TIS) is crucial, as it establishes the correct open reading frame for mRNA decoding. Studies in vertebrate mRNAs discovered that a purine at -3 and a G at +4 (where A of the AUG initiator codon is numbered + 1), promote TIS recognition. However, the TIS context in other eukaryotes has been poorly experimentally analyzed. We analyzed in vitro the influence of the -3, -2, -1 and + 4 positions of the TIS context in rabbit, Drosophila, wheat, and yeast. We observed that -3A conferred the best translational efficiency across these species. However, we found variability at the + 4 position for optimal translation. In addition, the Kozak motif that was defined from mammalian cells was only weakly predictive for wheat and essentially non-predictive for yeast. We discovered eight conserved sequences that significantly disfavored translation. Due to the big differences in translational efficiency observed among weak TIS context sequences, we define a novel category that we termed 'barren AUG context sequences (BACS)', which represent sequences disfavoring translation. Analysis of mRNA-ribosomal complexes structures provided insights into the function of BACS. The gene ontology of the BACS-containing mRNAs is presented.

The interplay between cis- and trans-acting factors drives selective mRNA translation initiation in eukaryotes.

Translation initiation consists in the assembly of the small and large ribosomal subunits on the start codon. This important step directly modulates the general proteome in living cells. Recently, genome wide studies revealed unexpected translation initiation events from unsuspected novel open reading frames resulting in the synthesis of a so-called 'dark proteome'. Indeed, the identification of the start codon by the translation machinery is a critical step that defines the translational landscape of the cell. Therefore, translation initiation is a highly regulated process in all organisms. In this review, we focus on the various cis- and trans-acting factors that rule the regulation of translation initiation in eukaryotes. Recent discoveries have shown that the guidance of the translation machinery for the choice of the start codon require sophisticated molecular mechanisms. In particular, the 5'UTR and the coding sequences contain cis-acting elements that trigger the use of AUG codons but also non-AUG codons to initiate protein synthesis. The use of these alternative start codons is also largely influenced by numerous trans-acting elements that drive selective mRNA translation in response to environmental changes.

The distinct translational landscapes of gram-negative Salmonella and gram-positive Listeria.

Translational control in pathogenic bacteria is fundamental to gene expression and affects virulence and other infection phenotypes. We used an enhanced ribosome profiling protocol coupled with parallel transcriptomics to capture accurately the global translatome of two evolutionarily distant pathogenic bacteria-the Gram-negative bacterium Salmonella and the Gram-positive bacterium Listeria. We find that the two bacteria use different mechanisms to translationally regulate protein synthesis. In Salmonella, in addition to the expected correlation between translational efficiency and cis-regulatory features such as Shine-Dalgarno (SD) strength and RNA secondary structure around the initiation codon, our data reveal an effect of the 2nd and 3rd codons, where the presence of tandem lysine codons (AAA-AAA) enhances translation in both Salmonella and E. coli. Strikingly, none of these features are seen in efficiently translated Listeria transcripts. Instead, approximately 20% of efficiently translated Listeria genes exhibit 70 S footprints seven nt upstream of the authentic start codon, suggesting that these genes may be subject to a novel translational initiation mechanism. Our results show that SD strength is not a direct hallmark of translational efficiency in all bacteria. Instead, Listeria has evolved additional mechanisms to control gene expression level that are distinct from those utilised by Salmonella and E. coli.

The Roles of eIF4G2 in Leaky Scanning and Reinitiation on the Human Dual-Coding POLG mRNA.

Upstream open reading frames (uORFs) are a frequent feature of eukaryotic mRNAs. Upstream ORFs govern main ORF translation in a variety of ways, but, in a nutshell, they either filter out scanning ribosomes or allow downstream translation initiation via leaky scanning or reinitiation. Previous reports concurred that eIF4G2, a long-known but insufficiently studied eIF4G1 homologue, can rescue the downstream translation, but disagreed on whether it is leaky scanning or reinitiation that eIF4G2 promotes. Here, we investigated a unique human mRNA that encodes two highly conserved proteins (POLGARF with unknown function and POLG, the catalytic subunit of the mitochondrial DNA polymerase) in overlapping reading frames downstream of a regulatory uORF. We show that the uORF renders the translation of both POLGARF and POLG mRNAs reliant on eIF4G2. Mechanistically, eIF4G2 enhances both leaky scanning and reinitiation, and it appears that ribosomes can acquire eIF4G2 during the early steps of reinitiation. This emphasizes the role of eIF4G2 as a multifunctional scanning guardian that replaces eIF4G1 to facilitate ribosome movement but not ribosome attachment to an mRNA.

Reconstitution of C9orf72 GGGGCC repeat-associated non-AUG translation with purified human translation factors.

Nucleotide repeat expansion of GGGGCC (G4C2) in the non-coding region of C9orf72 is the most common genetic cause underlying amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts harboring this repeat expansion undergo the translation of dipeptide repeats via a non-canonical process known as repeat-associated non-AUG (RAN) translation. In order to ascertain the essential components required for RAN translation, we successfully recapitulated G4C2-RAN translation using an in vitro reconstituted translation system comprising human factors, namely the human PURE system. Our findings conclusively demonstrate that the presence of fundamental translation factors is sufficient to mediate the elongation from the G4C2 repeat. Furthermore, the initiation mechanism proceeded in a 5' cap-dependent manner, independent of eIF2A or eIF2D. In contrast to cell lysate-mediated RAN translation, where longer G4C2 repeats enhanced translation, we discovered that the expansion of the G4C2 repeats inhibited translation elongation using the human PURE system. These results suggest that the repeat RNA itself functions as a repressor of RAN translation. Taken together, our utilization of a reconstituted RAN translation system employing minimal factors represents a distinctive and potent approach for elucidating the intricacies underlying RAN translation mechanism.

Translation initiation at AUG and non-AUG triplets in plants.

In plants and other eukaryotes, precise selection of translation initiation site (TIS) on mRNAs shapes the proteome in response to cellular events or environmental cues. The canonical translation of mRNAs initiates at a 5' proximal AUG codon in a favorable context. However, the coding and non-coding regions of plant genomes contain numerous unannotated alternative AUG and non-AUG TISs. Determining how and why these unexpected and prevalent TISs are activated in plants has emerged as an exciting research area. In this review, we focus on the selection of plant TISs and highlight studies that revealed previously unannotated TISs used in vivo via comparative genomics and genome-wide profiling of ribosome positioning and protein N-terminal ends. The biological signatures of non-AUG TIS-initiated open reading frames (ORFs) in plants are also discussed. We describe what is understood about cis-regulatory RNA elements and trans-acting eukaryotic initiation factors (eIFs) in the site selection for translation initiation by featuring the findings in plants along with supporting findings in non-plant species. The prevalent, unannotated TISs provide a hidden reservoir of ORFs that likely help reshape plant proteomes in response to developmental or environmental cues. These findings underscore the importance of understanding the mechanistic basis of TIS selection to functionally annotate plant genomes, especially for crops with large genomes.

An mTRAN-mRNA interaction mediates mitochondrial translation initiation in plants.

Plant mitochondria represent the largest group of respiring organelles on the planet. Plant mitochondrial messenger RNAs (mRNAs) lack Shine-Dalgarno-like ribosome-binding sites, so it is unknown how plant mitoribosomes recognize mRNA. We show that "mitochondrial translation factors" mTRAN1 and mTRAN2 are land plant-specific proteins, required for normal mitochondrial respiration chain biogenesis. Our studies suggest that mTRANs are noncanonical pentatricopeptide repeat (PPR)-like RNA binding proteins of the mitoribosomal "small" subunit. We identified conserved Adenosine (A)/Uridine (U)-rich motifs in the 5' regions of plant mitochondrial mRNAs. mTRAN1 binds this motif, suggesting that it is a mitoribosome homing factor to identify mRNAs. We demonstrate that mTRANs are likely required for translation of all plant mitochondrial mRNAs. Plant mitochondrial translation initiation thus appears to use a protein-mRNA interaction that is divergent from bacteria or mammalian mitochondria.

Precise genome editing of the Kozak sequence enables bidirectional and quantitative modulation of protein translation to anticipated levels without affecting transcription.

None of the existing approaches for regulating gene expression can bidirectionally and quantitatively fine-tune gene expression to desired levels. Here, on the basis of precise manipulations of the Kozak sequence, which has a remarkable influence on translation initiation, we proposed and validated a novel strategy to directly modify the upstream nucleotides of the translation initiation codon of a given gene to flexibly alter the gene translation level by using base editors and prime editors. When the three nucleotides upstream of the translation initiation codon (named KZ3, part of the Kozak sequence), which exhibits the most significant base preference of the Kozak sequence, were selected as the editing region to alter the translation levels of proteins, we confirmed that each of the 64 KZ3 variants had a different translation efficiency, but all had similar transcription levels. Using the ranked KZ3 variants with different translation efficiencies as predictors, base editor- and prime editor-mediated mutations of KZ3 in the local genome could bidirectionally and quantitatively fine-tune gene translation to the anticipated levels without affecting transcription in vitro and in vivo. Notably, this strategy can be extended to the whole Kozak sequence and applied to all protein-coding genes in all eukaryotes.

N-terminal alanine-rich (NTAR) sequences drive precise start codon selection resulting in elevated translation of multiple proteins including ERK1/2.

We report the discovery of N-terminal alanine-rich sequences, which we term NTARs, that act in concert with their native 5'-untranslated regions to promote selection of the proper start codon. NTARs also facilitate efficient translation initiation while limiting the production of non-functional polypeptides through leaky scanning. We first identified NTARs in the ERK1/2 kinases, which are among the most important signaling molecules in mammals. Analysis of the human proteome reveals that hundreds of proteins possess NTARs, with housekeeping proteins showing a particularly high prevalence. Our data indicate that several of these NTARs act in a manner similar to those found in the ERKs and suggest a mechanism involving some or all of the following features: alanine richness, codon rarity, a repeated amino acid stretch and a nearby second AUG. These features may help slow down the leading ribosome, causing trailing pre-initiation complexes (PICs) to pause near the native AUG, thereby facilitating accurate translation initiation. Amplification of erk genes is frequently observed in cancer, and we show that NTAR-dependent ERK protein levels are a rate-limiting step for signal output. Thus, NTAR-mediated control of translation may reflect a cellular need to precisely control translation of key transcripts such as potential oncogenes. By preventing translation in alternative reading frames, NTAR sequences may be useful in synthetic biology applications, e.g. translation from RNA vaccines.

Initiation of translation is essential for protein synthesis. A crucial step is the correct choice of the start AUG, which leads to the production of the fully functional polypeptide. To date, nucleotide composition next to the AUG has been considered the only determinant of start codon selection. Our work identifies a large family of proteins whose start codon choice is determined by an N-terminal alanine-rich sequence (NTAR) that enables efficient protein translation. Many of these proteins are encoded by housekeeping genes. Among them, the NTARs of the pivotal kinases ERK1 and ERK2 are highly optimized in humans, shaping ERK signal transduction by increasing the kinase quantity. Our findings could be useful for applied biology, especially for mRNA-based therapeutics.

The mRNA translation initiation factor eIF4G1 controls mitochondrial oxidative phosphorylation, axonal morphogenesis, and memory.

mRNA translation initiation plays a critical role in learning and memory. The eIF4F complex, composed of the cap-binding protein eIF4E, ATP-dependent RNA helicase eIF4A, and scaffolding protein eIF4G, is a pivotal factor in the mRNA translation initiation process. eIF4G1, the major paralogue of the three eIF4G family members, is indispensable for development, but its function in learning and memory is unknown. To study the role of eIF4G1 in cognition, we used an eIF4G1 haploinsufficient (eIF4G1-1D) mouse model. The axonal arborization of eIF4G1-1D primary hippocampal neurons was significantly disrupted, and the mice displayed impairment in hippocampus-dependent learning and memory. Translatome analysis showed that the translation of mRNAs encoding proteins of the mitochondrial oxidative phosphorylation (OXPHOS) system was decreased in the eIF4G1-1D brain, and OXPHOS was decreased in eIF4G1-silenced cells. Thus, eIF4G1-mediated mRNA translation is crucial for optimal cognitive function, which is dependent on OXPHOS and neuronal morphogenesis.

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.