sui1-L96P increases the utilization of near-cognate and upstream AUG start codons. (A–C) Ribosome protected fragments (RPFs) and mRNA reads on the SUI1, GRS1, and ALA1 genes in WT and L96P cells in units of rpb (reads per billion mapped reads), showing schematically the position of the main CDS (pink), the CDS for amino-terminal extensions (green), and the −3 to −1 and +4 context nucleotides surrounding the AUG or near-cognate codon (NCC) (in brick red). ΔRPF and ΔmRNA give the ratios of RPFs and total mRNA fragments, respectively, in L96P versus WT cells. The Integrated Genomics Viewer (Broad Institute) was used to display ribosome/mRNA reads, with the rpb and nt scales indicated in each panel. For these and all other gene browser views, the reads per billion mapped reads (rpb) are plotted; the ribosome-protected-fragments are plotted to their predicted A-site positions, and the mRNA fragments are plotted to the 15th nt from their 5′ ends. (D–F) Scatter plots comparing log2 RRO (relative ribosome occupancy) values in L96P versus WT cells for 2484 5′UTRs (D), 2226 NCC-uORFs (E), and 205 uAUG uORFs (F) from genes with ≥32 RPF reads in the main CDS and ≥2 RPF reads in the 5′UTR or uORF in the averaged reads from four samples, two replicates of WT and two replicates of sui1-L96P. The sets of NCC_uORF and AUG_uORFs showing evidence of translation in current or previous ribosome profiling experiments were described previously (Martin-Marcos et al. 2017). 5′UTRs or uORFs showing significantly changed TE values in L96P versus WT cells of greater than or equal to twofold (at FDR < 0.05) are indicated in red or dark blue, and the numbers (n) of 5′UTRs or uORFs plotted are indicated. Notched box-plots below each scatterplot compare the distributions of log2 RRO values in WT versus L96P cells for the complete sets of 5′UTRs (D), NCC_uORFs (E), or AUG_uORFs (F). In these and other box-plots below, box limits indicate the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots, and nonoverlapping notches indicate that two medians differ with ∼95% confidence. A fraction of outliers was omitted from the plots to expand the y-axis scale. (G–H) Three nucleotide periodicity of RPFs in subsets of the 205 translated AUG_uORFs (G, n = 123) and 2226 NCC_uORFs (H, n = 1394) described in panels F and E, respectively, in which the uORF length is >29 nt and the distance from the uORF start codon to the main CDS AUG is >29 nt. Black and gray lines show the average RPF reads per billion at each position relative to the 5′ end of these groups of uORFs. Reads at first, second, and third reading frames are labeled with red, blue, and green circles, respectively. (An AUG_uORF at YDR133C, YDR133C_52, was excluded because its high expression would dominate the plot.)
