An approach combining fluorescence-activated cell sorting and high-throughput DNA sequencing
(FACS-seq) was employed to determine the efficiency of start codon recognition for all possible
translation initiation sites (TIS) utilizing AUG start codons. Using FACS-seq, we measured
translation from a genetic reporter library representing all 65,536 possible TIS sequences spanning
the −6 to +5 positions. We found that the motif RYMRMVAUGGC enhanced start codon
recognition and translation efficiency. However, dinucleotide interactions, which cannot be conveyed
by a single motif, were also important for modeling TIS efficiency. Our dataset combined with
modeling allowed us to predict genome-wide translation initiation efficiency for all mRNA
transcripts. Additionally, we screened somatic TIS mutations associated with tumorigenesis to
identify candidate driver mutations consistent with known tumor expression patterns. Finally, we
implemented a quantitative leaky scanning model to predict alternative initiation sites that produce
truncated protein isoforms and compared predictions with ribosome footprint profiling data. The
comprehensive analysis of the TIS sequence space enables quantitative predictions of translation
initiation based on genome sequence.
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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