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Translation Initiation Site Profiling Reveals Widespread Synthesis of Non-AUG-Initiated Protein Isoforms in Yeast.

ABSTRACT: Genomic analyses in budding yeast have helped define the foundational principles of eukaryotic gene expression. However, in the absence of empirical methods for defining coding regions, these analyses have historically excluded specific classes of possible coding regions, such as those initiating at non-AUG start codons. Here, we applied an experimental approach to globally annotate translation initiation sites in yeast and identified 149 genes with alternative N-terminally extended protein isoforms initiating from near-cognate codons upstream of annotated AUG start codons. These isoforms are produced in concert with canonical isoforms and translated with high specificity, resulting from initiation at only a small subset of possible start codons. The non-AUG initiation driving their production is enriched during meiosis and induced by low eIF5A, which is seen in this context. These findings reveal widespread production of non-canonical protein isoforms and unexpected complexity to the rules by which even a simple eukaryotic genome is decoded.

SUBMITTER: Eisenberg AR

PROVIDER: S-EPMC7508262 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Translation initiation site profiling reveals widespread synthesis of non-AUG-initiated protein isoforms in yeast

Project description:Genomic analyses in budding yeast have helped to define the foundational principles of eukaryotic gene expression, but have systematically excluded specific classes of potential coding regions, including those with non-AUG start codons. Without methods to define coding regions empirically, the prevalence of these non-canonical coding regions has been impossible to assess. Here, we applied an experimental approach to globally annotate translation initiation sites in yeast and identified a class of 149 genes that encode N-terminally extended alternate protein isoforms that result from translation initiation at non-AUG codons upstream of the annotated AUG start codon. These alternate isoforms are produced in concert with canonical isoforms and are translated with a high degree of specificity, resulting from initiation at only a small subset of possible start codons in 5’ leader regions. Their translation is enriched during meiosis, and is induced by low eIF5A levels, which are observed in this context. These findings reveal widespread production of non-canonical protein isoforms and, more generally, show unexpected complexity to the rules by which the budding yeast genome is decoded.

2020-07-24 | GSE150375 | GEO

Translation initiation site profiling reveals widespread synthesis of non-AUG-initiated protein isoforms in yeast

Project description:Translation initiation site profiling reveals widespread synthesis of non-AUG-initiated protein isoforms in yeast

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DDX3X and specific initiation factors modulate FMR1 repeat-associated non-AUG-initiated translation.

Project description:A CGG trinucleotide repeat expansion in the 5' UTR of FMR1 causes the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). This repeat supports a non-canonical mode of protein synthesis known as repeat-associated, non-AUG (RAN) translation. The mechanism underlying RAN translation at CGG repeats remains unclear. To identify modifiers of RAN translation and potential therapeutic targets, we performed a candidate-based screen of eukaryotic initiation factors and RNA helicases in cell-based assays and a Drosophila melanogaster model of FXTAS. We identified multiple modifiers of toxicity and RAN translation from an expanded CGG repeat in the context of the FMR1 5'UTR. These include the DEAD-box RNA helicase belle/DDX3X, the helicase accessory factors EIF4B/4H, and the start codon selectivity factors EIF1 and EIF5. Disrupting belle/DDX3X selectively inhibited FMR1 RAN translation in Drosophila in vivo and cultured human cells, and mitigated repeat-induced toxicity in Drosophila and primary rodent neurons. These findings implicate RNA secondary structure and start codon fidelity as critical elements mediating FMR1 RAN translation and identify potential targets for treating repeat-associated neurodegeneration.

| S-EPMC6726903 | biostudies-literature

Non-AUG Translation Initiation Generates Peroxisomal Isoforms of 6-Phosphogluconate Dehydrogenase in Fungi.

Project description:Proteins destined for transport to specific organelles usually contain targeting information, which are embedded in their sequence. Many enzymes are required in more than one cellular compartment and different molecular mechanisms are used to achieve dual localization. Here we report a cryptic type 2 peroxisomal targeting signal encoded in the 5' untranslated region of fungal genes coding for 6-phosphogluconate dehydrogenase (PGD), a key enzyme of the oxidative pentose phosphate pathway. The conservation of the cryptic PTS2 motif suggests a biological function. We observed that translation from a non-AUG start codon generates an N-terminally extended peroxisomal isoform of Ustilago maydis PGD. Non-canonical initiation occurred at the sequence AGG AUU, consisting of two near-cognate start codons in tandem. Taken together, our data reveal non-AUG translation initiation as an additional mechanism to achieve the dual localization of a protein required both in the cytosol and the peroxisomes.

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Yeast 18 S rRNA is directly involved in the ribosomal response to stringent AUG selection during translation initiation.

Project description:In eukaryotes, the 40 S ribosomal subunit serves as the platform of initiation factor assembly, to place itself precisely on the AUG start codon. Structural arrangement of the 18 S rRNA determines the overall shape of the 40 S subunit. Here, we present genetic evaluation of yeast 18 S rRNA function using 10 point mutations altering the polysome profile. All the mutants reduce the abundance of the mutant 40 S, making it limiting for translation initiation. Two of the isolated mutations, G875A, altering the core of the platform domain that binds eIF1 and eIF2, and A1193U, changing the h31 loop located below the P-site tRNA(i)(Met), show phenotypes indicating defective regulation of AUG selection. Evidence is provided that these mutations reduce the interaction with the components of the preinitiation complex, thereby inhibiting its function at different steps. These results indicate that the 18 S rRNA mutations impair the integrity of scanning-competent preinitiation complex, thereby altering the 40 S subunit response to stringent AUG selection. Interestingly, nine of the mutations alter the body/platform domains of 18 S rRNA, potentially affecting the bridges to the 60 S subunit, but they do not change the level of 18 S rRNA intermediates. Based on these results, we also discuss the mechanism of the selective degradation of the mutant 40 S subunits.

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Non-AUG initiation of AGAMOUS mRNA translation in Arabidopsis thaliana.

Project description:The MADS box organ identity gene AGAMOUS (AG) controls several steps during Arabidopsis thaliana flower development. AG cDNA contains an open reading frame that lacks an ATG triplet to function as the translation initiation codon, and the actual amino terminus of the AG protein remains uncharacterized. We have considered the possibility that AG translation can be initiated at a non-AUG codon. Two possible non-AUG initiation codons, CUG and ACG, are present in the 5' region of AG mRNA preceding the highly conserved MADS box sequence. We prepared a series of AG genomic constructs in which these codons are mutated and assayed their activity in phenotypic rescue experiments by introducing them as transgenes into ag mutant plants. Alteration of the CTG codon to render it unsuitable for acting as a translation initiation site does not affect complementation of the ag-3 mutation in transgenic plants. However, a similar mutation of the downstream ACG codon prevents the rescue of the ag-3 mutant phenotype. Conversely, if an ATG is introduced immediately 5' to the disrupted ACG codon, the resulting construct fully complements the ag-3 mutation. The AG protein synthesized in vitro by initiating translation at the ACG position is active in DNA binding and is of the same size as the AG protein detected from floral tissues, whereas AG polypeptides with additional amino-terminal residues do not appear to bind DNA. These results indicate that translation of AG is initiated exclusively at an ACG codon and prove that non-AUG triplets may be efficiently used as the sole translation initiation site in some plant cellular mRNAs.

| S-EPMC84964 | biostudies-literature

Global mapping of translation initiation sites by TIS profiling in budding yeast.

Project description:Translation initiation site (TIS) profiling allows for the genome-wide identification of TISs in vivo by exclusively capturing mRNA fragments within ribosomes that have just completed translation initiation. It leverages translation inhibitors, such as harringtonine and lactimidomycin (LTM), that preferentially capture ribosomes at start codon positions, protecting TIS-derived mRNA fragments from nuclease digestion. Here, we describe a step-by-step protocol for TIS profiling in LTM-treated budding yeast that we developed to identify TISs and open reading frames in vegetative and meiotic cells. For complete details on the use and execution of this protocol, please refer to Eisenberg et al. (2020).

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Conformational changes in the P site and mRNA entry channel evoked by AUG recognition in yeast translation preinitiation complexes.

Project description:The translation preinitiation complex (PIC) is thought to assume an open conformation when scanning the mRNA leader, with AUG recognition evoking a closed conformation and more stable P site interaction of Met-tRNAi; however, physical evidence is lacking that AUG recognition constrains interaction of mRNA with the 40S binding cleft. We compared patterns of hydroxyl radical cleavage of rRNA by Fe(II)-BABE tethered to unique sites in eIF1A in yeast PICs reconstituted with mRNA harboring an AUG or near-cognate (AUC) start codon. rRNA residues in the P site display reduced cleavage in AUG versus AUC PICs; and enhanced cleavage in the AUC complexes was diminished by mutations of scanning enhancer elements of eIF1A that increase near-cognate recognition in vivo. This suggests that accessibility of these rRNA residues is reduced by accommodation of Met-tRNAi in the P site (PIN state) and by their interactions with the anticodon stem of Met-tRNAi. Our cleavage data also provide evidence that AUG recognition evokes dissociation of eIF1 from its 40S binding site, ejection of the eIF1A-CTT from the P-site and rearrangement to a closed conformation of the entry channel with reduced mobility of mRNA.

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Mutations in Caenorhabditis elegans eIF2beta permit translation initiation from non-AUG start codons.

Project description:Recognition of the AUG start codon on mRNAs during translation initiation in eukaryotes occurs in a preinitiation complex that includes small ribosomal subunits and multiple translation initiation factors. The complexity of this process and the lack of appropriate tools have prevented its genetic study in multicellular organisms. Here we describe a genetic system in the nematode Caenorhabditis elegans to study how the AUG start codon is selected. We have generated a sensitive reporter assay that allows for the isolation of mutants with reduced fidelity to recognize the AUG start codon. Two mutants were identified to have dominant missense mutations in iftb-1, which encodes the beta-subunit of eIF2 (eIF2beta). Both mutations occur in a conserved region located outside of the C2-C2 zinc finger domain where yeast SUI3 mutations are localized in Saccharomyces cerevisiae eIF2beta. C. elegans iftb-1, as well as mutant eIF2betas carrying the equivalent SUI3 mutations, are able to initiate translation at non-AUG codons that retain two potential base-pairing interactions with the anticodon of the initiator methionyl tRNA. These analyses further support the critical role of eIF2beta in start codon selection, and two functional domains within eIF2beta are likely involved, one defined by our C. elegans mutants and the other by the yeast SUI3 mutants.

| S-EPMC2870950 | biostudies-literature

Complete motif analysis of sequence requirements for translation initiation at non-AUG start codons.

Project description:The initiation of mRNA translation from start codons other than AUG was previously believed to be rare and of relatively low impact. More recently, evidence has suggested that as much as half of all translation initiation utilizes non-AUG start codons, codons that deviate from AUG by a single base. Furthermore, non-AUG start codons have been shown to be involved in regulation of expression and disease etiology. Yet the ability to gauge expression based on the sequence of a translation initiation site (start codon and its flanking bases) has been limited. Here we have performed a comprehensive analysis of translation initiation sites that utilize non-AUG start codons. By combining genetic-reporter, cell-sorting, and high-throughput sequencing technologies, we have analyzed the expression associated with all possible variants of the -4 to +4 positions of non-AUG translation initiation site motifs. This complete motif analysis revealed that 1) with the right sequence context, certain non-AUG start codons can generate expression comparable to that of AUG start codons, 2) sequence context affects each non-AUG start codon differently, and 3) initiation at non-AUG start codons is highly sensitive to changes in the flanking sequences. Complete motif analysis has the potential to be a key tool for experimental and diagnostic genomics.

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