Impact of highly translated uORFS on coding sequence translation. a SPECtre identified uORFs were filtered to include only uORFs that have significant coverage in all four Non-Diff and RA-Diff libraries; these are considered highly translated. b Average TE values for cORFs and oORFs in the Non-Diff (left) and RA-Diff (right) conditions. c Conservation analysis of annotated 5′ leaders in all three reading frames (orange), annotated CDS regions over all three frames (grey), predicted AUG-initiated uORFs (dark blue), and predicted non-AUG uORFs (light blue). d Average GC nucleotide content is shown for 5′ leader regions (orange), CDSs (grey), AUG uORFs (dark blue), and non-AUG uORFs (light blue). For oORFs, only the 5′ leader region of the oORF is included. 5′ leaders are significantly more GC rich than both AUG uORFs and non-AUG uORFs, p = 5.72e-12 and 1.54e-07, respectively. Non-AUG uORFs are significantly more GC rich than CDSs and AUG uORFs, p = 7.92e-18 and 2.16e-06. e Average TE for AUG uORFs and non-AUG uORFs reveals no difference between the two subtypes. f Empirical cumulative distribution of TE in all CDSs (black) versus CDSs from transcripts with two subsets of uORFs: those with an AUG initiation site (red) and those with a non-AUG initiation site (Blue). g Empirical cumulative distribution of TE in all CDSs (black) versus CDSs from transcripts with two subsets of non-AUG uORFs: those in the highest quartile of conservation (Conserved, red) and those in the lowest quartile of conservation (Non-Conserved, Blue). Distributions are significantly different with p-values annotated on graph. h GC content of non-AUG uORFs grouped by conservation. All versus conserved: p = 5.89e-08, all versus non-conserved: p = 2.54e-06, conserved versus non-conserved: p = 1.62e-18.