Validating the translation initiation of uORFs.

<p>(A) The proportions of canonical uORFs (beginning with AUG, red) and the other 60 kinds of hypothetical uORFs (each beginning with a distinct non-stop-codon triplet, blue) that are bound with ribosomes in at least 11 out of 12 samples (only uORFs with mRNA RPKM ≥ 1 and TE ≥ 0.5 were considered). (B) Signal-to-noise ratio at different cutoffs of minimum TE_uORF. For each cutoff of minimum TE_uORF, the proportion of canonical uORFs that are bound with ribosomes at this cutoff is divided by those for the other 60 kinds of hypothetical uORFs. The average of those ratios is used as signal-to-noise ratio at this cutoff. (C) The relative mRNA and RPF coverage around cAUGs in S2 cells. For each codon downstream (or triplet upstream) the cAUG of a gene (x-axis), the sequencing coverage was calculated, and the relative coverage of that codon (triplet) was calculated by normalization with the median coverage of CDS of this gene. And then, the relative sequencing coverage was averaged across all the genes (y-axis). Red and blue lines represent mRNA-Seq and Ribo-Seq, respectively, of S2 cells treated with DMSO for 30 min. The green line represents Ribo-Seq of S2 cells treated with harringtonine for 30 min. (D) The relative mRNA and RPF coverage around uAUGs in S2 cells. The data normalization and line colors are the same as those in (C). (E) The proportions of CDSs and uORFs (y-axis) with start codons that showed peaks in Ribo-Seq of S2 cells treated with harringtonine for 30 min. CDSs and uORFs with RPF RPKM > 10 in Ribo-Seq of S2 cells are stratified into 10 bins of equal size based on increasing RPF RPKM (x-axis). Spearman’s rank correlation between the proportion of CDS (or uORFs) with peaks of ribosome occupancy, and the median RPKM of CDSs (or uORFs) in each bin, is displayed in the plot. The raw data for panels (A-E) can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003903#pbio.2003903.s067" target="_blank">S1 Data</a>. cAUG, AUG start codon of CDS; CDS, coding DNA sequence; DMSO, dimethyl sulfoxide; RPF, ribosome-protected mRNA fragment; RPKM, reads per kilobase of transcript per million mapped reads; TE, translational efficiency; uAUG, AUG start codon of uORF; uORF, upstream open reading frame