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Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context.

ABSTRACT: Eukaryotic translation initiation involves preinitiation ribosomal complex 5'-to-3' directional probing of mRNA for codons suitable for starting protein synthesis. The recognition of codons as starts depends on the codon identity and on its immediate nucleotide context known as Kozak context. When the context is weak (i.e., nonoptimal), leaky scanning takes place during which a fraction of ribosomes continues the mRNA probing. We explored the relationship between the context of AUG codons annotated as starts of protein-coding sequences and the next AUG codon occurrence. We found that AUG codons downstream from weak starts occur in the same frame more frequently than downstream from strong starts. We suggest that evolutionary selection on in-frame AUGs downstream from weak start codons is driven by the advantage of the reduction of wasteful out-of-frame product synthesis and also by the advantage of producing multiple proteoforms from certain mRNAs. We confirmed translation initiation downstream from weak start codons using ribosome profiling data. We also tested translation of alternative start codons in 10 specific human genes using reporter constructs. In all tested cases, initiation at downstream start codons was more productive than at the annotated ones. In most cases, optimization of Kozak context did not completely abolish downstream initiation, and in the specific example of CMPK1 mRNA, the optimized start remained unproductive. Collectively, our work reveals previously uncharacterized forces shaping the evolution of protein-coding genes and points to the plurality of translation initiation and the existence of sequence features influencing start codon selection, other than Kozak context.

SUBMITTER: Benitez-Cantos MS

PROVIDER: S-EPMC7397870 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context.

Benitez-Cantos Maria S MS Yordanova Martina M MM O'Connor Patrick B F PBF Zhdanov Alexander V AV Kovalchuk Sergey I SI Papkovsky Dmitri B DB Andreev Dmitry E DE Baranov Pavel V PV

Genome research 20200715 7

Eukaryotic translation initiation involves preinitiation ribosomal complex 5'-to-3' directional probing of mRNA for codons suitable for starting protein synthesis. The recognition of codons as starts depends on the codon identity and on its immediate nucleotide context known as Kozak context. When the context is weak (i.e., nonoptimal), leaky scanning takes place during which a fraction of ribosomes continues the mRNA probing. We explored the relationship between the context of AUG codons annota ...[more]

PMID: 32669370

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Project description:Recent studies have shown that haloarchaea employ leaderless and Shine-Dalgarno (SD)-less mechanisms for translation initiation of leaderless transcripts with a 5' untranslated region (5' UTR) of <10 nucleotides (nt) and leadered transcripts with a 5' UTR of ?10 nt, respectively. However, whether the two mechanisms can operate on the same naturally occurring haloarchaeal transcript carrying multiple potential start codons is unknown. In this study, the transcript of the sptA gene (encoding an extracellular serine protease of Natrinema sp. strain J7-2) was experimentally determined and found to contain two potential in-frame AUG codons (AUG(1) and AUG(2)) located 5 and 29 nt, respectively, downstream of the transcription start site. Mutational analysis revealed that both AUGs can function as the translation start codon for production of active SptA, although AUG(1) is more efficient than AUG(2) for translation initiation. Insertion of a stable stem-loop structure between the two AUGs completely abolished initiation at AUG(1) but did not affect initiation at AUG(2), indicating that AUG(2)-initiated translation does not involve ribosome scanning from the 5' end of the transcript. Furthermore, the efficiency of AUG(2)-initiated translation was not influenced by an upstream SD-like sequence. In addition, both AUG(1) and AUG(2) contribute to transcript stability, probably by recruiting ribosomes to protect the transcript against degradation. These data suggest that depending on which of two in-frame start codons is used, the sptA transcript can act as either a leaderless or a leadered transcript for SptA production in haloarchaea.In eukaryotes and bacteria, alternative translation start sites contribute to proteome complexity and can be used as a functional mechanism to increase translation efficiency. However, little is known about alternative translation initiation in archaea. Our results demonstrate that leaderless and SD-less mechanisms can be used for translation initiation of the sptA transcript from two in-frame start codons, raising the possibility that in haloarchaea, alternative translation initiation on one transcript functions to increase translation efficiency and/or contribute to proteome complexity.

Project description:Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential protein that is the target for translational regulation in many cellular processes and viral systems. It has been shown to function in both cap-dependent and cap-independent translation initiation by recruiting the 40S ribosomal subunit to the mRNA cap structure or internal ribosome entry site (IRES) element, respectively. Interestingly eIF4GI mRNA itself has been reported to contain an IRES element in its 5' end that facilitates eIF4GI protein synthesis via a cap-independent mechanism. In HeLa cells, eIF4GI exists as several isoforms that differ in their migration in sodium dodecyl sulfate (SDS) gels; however, the nature of these isoforms was unclear. Here, we report a new cDNA clone for eIF4GI that extends the 5' sequence 340 nucleotides beyond the previously published sequence. The new extended sequence of eIF4GI is located on chromosome 3, within two additional exons immediately upstream of the previously published eIF4GI sequence. When mRNA transcribed from this cDNA clone was translated in vitro, five eIF4GI polypeptides were generated that comigrated in SDS-polyacrylamide gels with the five isoforms of native eIF4GI. Furthermore, translation of eIF4GI-enhanced green fluorescent protein fusion constructs in vitro or in vivo generated five isoforms of fusion polypeptides, suggesting that multiple isoforms of eIF4GI are generated by alternative translation initiation in vitro and in vivo. Mutation of two of the five in-frame AUG residues in the eIF4GI cDNA sequence resulted in loss of corresponding polypeptides after translation in vitro, confirming alternate use of AUGs as the source of the multiple polypeptides. The 5' untranslated region of eIF4GI mRNA also contains an out-of-frame open reading frame (ORF) that may down-regulate expression of eIF4GI. Further, data are presented to suggest that a proposed IRES embedded in the eIF4GI ORF is able to catalyze synthesis of multiple eIF4GI isoforms as well. Our data suggest that expression of the eIF4GI isoforms is partly controlled by a complex translation strategy involving both cap-dependent and cap-independent mechanisms.

Dataset Information

Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context.

Publications

Translation initiation downstream from annotated start codons in human mRNAs coevolves with the Kozak context.

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