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Bi-directional ribosome scanning controls the stringency of start codon selection

ABSTRACT: The fidelity of start codon recognition by ribosomes is paramount during protein synthesis. The current knowledge of eukaryotic translation initiation implies unidirectional 5ʹ→3ʹ migration of the pre-initiation complex (PIC) along the 5ʹ UTR. In probing translation initiation from ultra-short 5ʹ UTR, we report that an AUG triplet near the 5ʹ end can be selected via PIC backsliding. Bi-directional ribosome scanning is supported by competitive selection of closely spaced AUG codons and recognition of two initiation sites flanking an internal ribosome entry site. Transcriptome-wide PIC profiling reveals footprints with an oscillation pattern near the 5ʹ end and start codons. Depleting the RNA helicase eIF4A leads to reduced PIC oscillations and impaired selection of 5ʹ end start codons. Enhancing the ATPase activity of eIF4A promotes nonlinear PIC scanning and stimulates upstream translation initiation. The helicase-mediated PIC conformational switch may provide an operational mechanism that unifies ribosome recruitment, scanning, and start codon selection. Start codon selection is commonly thought to occur through the unidirectional scanning of the mRNA by the 40 S ribosome. Here the authors provide evidence that the pre-initiation complex can backslide on the mRNA to initiate translation at upstream AUG codons.

SUBMITTER: Gu Y

PROVIDER: S-EPMC8593136 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Bi-directional ribosome scanning controls the stringency of start codon selection

Project description:The fidelity of start codon recognition by ribosomes is paramount during protein synthesis. The textbook knowledge of eukaryotic translation initiation depicts 5’→3’ unidirectional migration of the pre-initiation complex (PIC) along the 5’UTR. In probing translation initiation from ultra-short 5’UTR, we report that an AUG triplet near the 5’ end can be selected via PIC backsliding. The bi-directional ribosome scanning is supported by competitive selection of closely spaced AUG codons and recognition of two initiation sites flanking an internal ribosome entry site. Transcriptome-wide PIC profiling reveals footprints with an oscillation pattern near the 5’ end and start codons. Depleting the RNA helicase eIF4A leads to reduced PIC oscillations and impaired selection of 5’ end start codons. Enhancing the ATPase activity of eIF4A promotes nonlinear PIC scanning and stimulates upstream translation initiation. The helicase-mediated PIC conformational switch may provide an operational mechanism that unifies ribosome recruitment, scanning, and start codon selection.

2021-11-24 | GSE176058 | GEO

Bi-directional ribosome scanning controls the stringency of start codon selection

Project description:Bi-directional ribosome scanning controls the stringency of start codon selection

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Stringency of start codon selection modulates autoregulation of translation initiation factor eIF5.

Project description:An AUG in an optimal nucleotide context is the preferred translation initiation site in eukaryotic cells. Interactions among translation initiation factors, including eIF1 and eIF5, govern start codon selection. Experiments described here showed that high intracellular eIF5 levels reduced the stringency of start codon selection in human cells. In contrast, high intracellular eIF1 levels increased stringency. High levels of eIF5 induced translation of inhibitory upstream open reading frames (uORFs) in eIF5 mRNA that initiate with AUG codons in conserved poor contexts. This resulted in reduced translation from the downstream eIF5 start codon, indicating that eIF5 autoregulates its own synthesis. As with eIF1, which is also autoregulated through translation initiation, features contributing to eIF5 autoregulation show deep evolutionary conservation. The results obtained provide the basis for a model in which auto- and cross-regulation of eIF5 and eIF1 translation establish a regulatory feedback loop that would stabilize the stringency of start codon selection.

| S-EPMC3326321 | biostudies-literature

Evolutionarily Conserved Inhibitory uORFs Sensitize Hox mRNA Translation to Start Codon Selection Stringency

Project description:Translation start site selection in eukaryotes is influenced by context nucleotides flanking the AUG codon and by levels of the eukaryotic translation initiation factors eIF1 and eIF5. In a search of human genes, we identified 5 Hox gene paralogs initiated by AUG codons in conserved suboptimal context as well as 13 Hox genes that contain evolutionarily conserved upstream open reading frames (uORFs) that initiate at AUG codons in poor sequence context. We analyzed published CAGE-seq data and generated CAGE-seq data from mRNAs from mouse somites. These data demonstrate that the 5’ leaders of Hox mRNAs of interest contain conserved uORFs, are much shorter than reported, and lack previously proposed IRES elements. We show that the conserved uORFs inhibit Hox reporter expression and that altering the stringency of start codon selection by overexpressing eIF1 or eIF5 modulates the expression of Hox reporters. We also show that modifying ribosome homeostasis by depleting a large ribosomal subunit protein or treating cells with sublethal concentrations of puromycin leads to lower stringency of start codon selection. Thus, altering global translation can confer gene-specific effects through altered start codon selection stringency.

2022-02-21 | GSE184515 | GEO

Translational autoregulation of BZW1 and BZW2 expression by modulating the stringency of start codon selection.

Project description:The efficiency of start codon selection during ribosomal scanning in eukaryotic translation initiation is influenced by the context or flanking nucleotides surrounding the AUG codon. The levels of eukaryotic translation initiation factors 1 (eIF1) and 5 (eIF5) play critical roles in controlling the stringency of translation start site selection. The basic leucine zipper and W2 domain-containing proteins 1 and 2 (BZW1 and BZW2), also known as eIF5-mimic proteins, are paralogous human proteins containing C-terminal HEAT domains that resemble the HEAT domain of eIF5. We show that translation of mRNAs encoding BZW1 and BZW2 homologs in fungi, plants and metazoans is initiated by AUG codons in conserved unfavorable initiation contexts. This conservation is reminiscent of the conserved unfavorable initiation context that enables autoregulation of EIF1. We show that overexpression of BZW1 and BZW2 proteins enhances the stringency of start site selection, and that their poor initiation codons confer autoregulation on BZW1 and BZW2 mRNA translation. We also show that overexpression of these two proteins significantly diminishes the effect of overexpressing eIF5 on stringency of start codon selection, suggesting they antagonize this function of eIF5. These results reveal a surprising role for BZW1 and BZW2 in maintaining homeostatic stringency of start codon selection, and taking into account recent biochemical, genetic and structural insights into eukaryotic initiation, suggest a model for BZW1 and BZW2 function.

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Nuclear release of eIF1 globally increases stringency of start codon selection to preserve mitotic arrest physiology

Project description:Regulated start codon selection has the potential to reshape the proteome through the differential production of uORFs, canonical proteins, and alternative translational isoforms. However, conditions under which start codon selection is altered remain poorly defined. Here, using transcriptome-wide translation initiation site profiling, we reveal a global increase in the stringency of start codon selection during mammalian mitosis. Low-efficiency initiation sites are preferentially repressed in mitosis, resulting in pervasive changes in the translation of thousands of start sites and their corresponding protein products. This increased stringency of start codon selection during mitosis results from increased associations between the 40S ribosome and the key regulator of start codon selection, eIF1. We find that increased eIF1-40S ribosome interactions during mitosis are mediated by the release of a nuclear pool of eIF1 upon nuclear envelope breakdown. Selectively depleting the nuclear pool of eIF1 eliminates the changes to translational stringency during mitosis, resulting in the altered synthesis of thousands of protein isoforms. In addition, preventing mitotic translational rewiring results in substantially increased cell death and decreased mitotic slippage in cells that experience a mitotic delay induced by anti-mitotic chemotherapeutics. Thus, cells globally control translation initiation stringency with critical roles during the mammalian cell cycle to preserve mitotic cell physiology.

2024-10-07 | PXD054183 | Pride

eIF1 Loop 2 interactions with Met-tRNAi control the accuracy of start codon selection by the scanning preinitiation complex.

Project description:The eukaryotic 43S preinitiation complex (PIC), bearing initiator methionyl transfer RNA (Met-tRNAi) in a ternary complex (TC) with eukaryotic initiation factor 2 (eIF2)-GTP, scans the mRNA leader for an AUG codon in favorable context. AUG recognition evokes rearrangement from an open PIC conformation with TC in a "POUT" state to a closed conformation with TC more tightly bound in a "PIN" state. eIF1 binds to the 40S subunit and exerts a dual role of enhancing TC binding to the open PIC conformation while antagonizing the PIN state, necessitating eIF1 dissociation for start codon selection. Structures of reconstituted PICs reveal juxtaposition of eIF1 Loop 2 with the Met-tRNAi D loop in the PIN state and predict a distortion of Loop 2 from its conformation in the open complex to avoid a clash with Met-tRNAi We show that Ala substitutions in Loop 2 increase initiation at both near-cognate UUG codons and AUG codons in poor context. Consistently, the D71A-M74A double substitution stabilizes TC binding to 48S PICs reconstituted with mRNA harboring a UUG start codon, without affecting eIF1 affinity for 40S subunits. Relatively stronger effects were conferred by arginine substitutions; and no Loop 2 substitutions perturbed the rate of TC loading on scanning 40S subunits in vivo. Thus, Loop 2-D loop interactions specifically impede Met-tRNAi accommodation in the PIN state without influencing the POUT mode of TC binding; and Arg substitutions convert the Loop 2-tRNAi clash to an electrostatic attraction that stabilizes PIN and enhances selection of poor start codons in vivo.

| S-EPMC5939108 | biostudies-literature

Why is start codon selection so precise in eukaryotes?

Project description:Translation generally initiates with the AUG codon. While initiation at GUG and UUG is permitted in prokaryotes (Archaea and Bacteria), cases of CUG initiation were recently reported in human cells. The varying stringency in translation initiation between eukaryotic and prokaryotic domains largely stems from a fundamental problem for the ribosome in recognizing a codon at the peptidyl-tRNA binding site. Initiation factors specific to each domain of life evolved to confer stringent initiation by the ribosome. The mechanistic basis for high accuracy in eukaryotic initiation is described based on recent findings concerning the role of the multifactor complex (MFC) in this process. Also discussed are whether non-AUG initiation plays any role in translational control and whether start codon accuracy is regulated in eukaryotes.

| S-EPMC4705826 | biostudies-other

Regulatory elements in eIF1A control the fidelity of start codon selection by modulating tRNA(i)(Met) binding to the ribosome.

Project description:eIF1A is the eukaryotic ortholog of bacterial translation initiation factor IF1, but contains a helical domain and long unstructured N-terminal tail (NTT) and C-terminal tail (CTT) absent in IF1. Here, we identify elements in these accessory regions of eIF1A with dual functions in binding methionyl initiator tRNA (Met-tRNA(i)(Met)) to the ribosome and in selecting AUG codons. A pair of repeats in the eIF1A CTT, dubbed Scanning Enhancer 1 (SE1) and SE2, was found to stimulate recruitment of Met-tRNA(i)(Met) in the ternary complex (TC) with eIF2.GTP and also to block initiation at UUG codons. In contrast, the NTT and segments of the helical domain are required for the elevated UUG initiation occurring in SE mutants, and both regions also impede TC recruitment. Remarkably, mutations in these latter elements, dubbed scanning inhibitors SI1 and SI2, reverse the defects in TC loading and UUG initiation conferred by SE substitutions, showing that the dual functions of SE elements in TC binding and UUG suppression are mechanistically linked. It appears that SE elements enhance TC binding in a conformation conducive to scanning but incompatible with initiation, whereas SI elements destabilize this conformation to enable full accommodation of Met-tRNA(i)(Met) in the P site for AUG selection.

| S-EPMC2802195 | biostudies-literature

Suppression of ribosomal pausing by eIF5A is necessary to maintain the fidelity of start codon selection (Ribosome profiling)

Project description:Sequences within 5' untranslated regions (UTRs) dictate the site and efficiency of translation initiation. In this study, an unbiased screen designed to interrogate the 5' UTR-mediated regulation of the growth-promoting gene MYC unexpectedly revealed the ribosomal pause-relief factor eIF5A as a regulator of translation initiation codon selection. Depletion of eIF5A enhanced upstream translation within 5' UTRs across yeast and human transcriptomes, including on the MYC transcript where this resulted in increased production of an N-terminally extended protein. Furthermore, ribosome profiling experiments established that the function of eIF5A as a suppressor of ribosomal pausing at sites of suboptimal peptide bond formation is conserved in human cells. We present evidence that proximal ribosomal pausing on a transcript triggers enhanced usage of upstream suboptimal or non-canonical initiation codons. Thus, we propose that eIF5A functions not only to maintain efficient translation elongation in eukaryotic cells, but also to maintain the fidelity of translation initiation.

2019-12-03 | GSE132007 | GEO

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