In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 109, No. 37 ( 2012-09-11)
Abstract:
Comprehensive cataloging of global TIS and associated ORFs is only the beginning in unveiling the role of translational control in gene expression. A systematic, high-throughput method like GTI-seq opens an avenue to identifying different translational products from the same transcript. GTI-seq is readily applicable to broad fields of fundamental biology. For instance, applications of GTI-seq in different tissues will facilitate the elucidation of the tissue-specific translational control. The illumination of altered TIS selection under different growth conditions will set the stage for future investigation of translational reprogramming during organismal development as well as in human diseases such as cancer. If beneficial to fitness, the biological function of alternative initiators could be maintained across species. Analysis of a mouse embryonic fibroblast cell line identified TIS positions across the mouse transcriptome, including uTIS and dTIS. The TIS features of human and mouse cells are remarkably similar. For example, uTIS codons predominantly use non-AUG, in particular CUG. Furthermore, about half the mouse transcripts possess multiple initiators. We also analyzed the conservation of individual alternative TIS positions on each transcript. For highly similar genes, 85% of the uTIS and 60% of dTIS positions are conserved. Remarkably, genes with low sequence similarity also display high TIS conservation. The evolutionary conservation of those TIS positions is a strong indication of the functional significance of alternative translation in the regulation of gene expression. Using the single-nucleotide–resolution TIS map, we assessed the underlying principles that specify start codon use in vivo. Our GTI-seq analysis supports the linear scanning mechanism. First, the uTIS context largely influenced the frequency of aTIS initiation. Second, the stringency of an aTIS codon negatively influenced the dTIS efficiency. Third, the by-pass potential at the first AUG correlated inversely with the strength of its sequence context. We also compared aTIS initiation in transcripts bearing separated or overlapping upstream ORFs (uORFs). Our results suggest that the reinitiation generally is less efficient than leaky scanning, as is consistent with the negative role of uORFs in the translation of primary ORFs. We first applied GTI-seq to a human cell line, HEK293, to achieve high-resolution mapping of TIS positions across the entire transcriptome. From ∼10,000 transcripts with detectable TIS peaks, we identified 16,863 TIS sites. Codon sequence analysis revealed that AUG represented more than half the TIS codons. GTI-seq also identified a significant proportion of TIS codons that differed from AUG by a single nucleotide, in particular CUG (16%). Remarkably, nearly half the transcripts (49.6%) contained multiple TIS sites, suggesting that alternative translation occurs even under physiological conditions. In addition to validating initiation at the start codons that have been previously annotated (aTIS), GTI-seq revealed that 27% and 50% of the transcripts contain downstream (dTIS) and upstream TIS sites (uTIS), respectively. Although dTIS codons are mainly AUG, a significant number of uTIS codons are non-AUG, most frequently CUG. We experimentally validated different translational products initiated from alternative start codons. We developed GTI-seq by using two related translation inhibitors to differentiate ribosome initiation from elongation effectively. Although cycloheximide (CHX) freezes all translating ribosomes, the translation inhibitor lactimidomycin (LTM) acts preferentially on the initiating ribosome but not on the elongating ribosome ( Fig. P1 ). Compared with harringtonine, LTM allows high-resolution mapping of global TIS positions. First, LTM binds to the 80S ribosome already assembled at the initiation codon and permits the formation of the first peptide bond ( 5 ). Thus, the LTM-associated RPF more likely represents physiological TIS positions. Second, LTM occupies the empty exit (E)-site of initiating ribosomes and thus completely blocks the translocation. This action allows TIS identification at single-nucleotide resolution. Third, because of the similarities in their structures and ribosomal binding sites, LTM and CHX can be applied side-by-side to assess initiation and elongation for the same transcript. With its high signal-to-noise ratio, GTI-seq identifies TIS sites directly with simpler computational analysis. Ribosome profiling, based on deep sequencing of ribosome-protected mRNA fragments (RPF), is a powerful technique for defining ribosome positions on the entire transcriptome ( 3 ). An initiation-specific translation inhibitor, harringtonine, depletes elongating ribosomes from mRNAs, thereby halting ribosomes at initiation codons by an unknown mechanism ( 4 ). This approach uncovered an unexpected abundance of alternative TIS codons, in particular non-AUG codons in the 5′ UTR of the mRNA. However, whether the harringtonine-marked TIS codons are truly authentic remains to be confirmed. Furthermore, harringtonine is imperfect in freezing the ribosome at the initiation site, making it difficult to distinguish alternative initiators for different reading frames. Protein synthesis is the final step in gene expression. Selection of the proper translation initiation site (TIS) on mRNAs is crucial for accurate protein synthesis. In eukaryotes, ribosomal scanning most often selects the first initiation codon (AUG) that the ribosome encounters to initiate protein synthesis ( 1 ). However, a surprising variety of potential translation start sites downstream and upstream of the first AUG have been uncovered ( 2 ). Because alternative start codons cannot be predicted reliably by sequence analysis, experimental approaches for identifying genome-wide TIS are required urgently. Here, we present an approach, Global Translation Initiation Sequencing (GTI-seq), that uncovers hidden coding potential of transcripts and offers a greater understanding of the complexity of translation initiation.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.1207846109
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2012
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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